U.S. patent application number 13/112391 was filed with the patent office on 2012-05-10 for compositions and methods for demonstrating secretory immune system regulation of steroid hormone responsive cancer cell growth.
Invention is credited to David A. Sirbasku.
Application Number | 20120115161 13/112391 |
Document ID | / |
Family ID | 27539481 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120115161 |
Kind Code |
A1 |
Sirbasku; David A. |
May 10, 2012 |
COMPOSITIONS AND METHODS FOR DEMONSTRATING SECRETORY IMMUNE SYSTEM
REGULATION OF STEROID HORMONE RESPONSIVE CANCER CELL GROWTH
Abstract
Serum-containing and serum-free immunoglobulin inhibitors of
steroid hormone responsive cancer cell growth are disclosed, along
with their methods of production. Also disclosed are defined cell
culture media, assay protocols, and model systems using the
inhibitors for demonstrating steroid hormone growth effects of
natural and synthetic substances, and other cell culture
applications. The disclosed compositions and methods employing the
immunoglobulin inhibitors are also useful as reagents in research,
and for the diagnosis, treatment and prevention of mucus epithelial
cancers.
Inventors: |
Sirbasku; David A.; (Flower
Mound, TX) |
Family ID: |
27539481 |
Appl. No.: |
13/112391 |
Filed: |
May 20, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09852958 |
May 10, 2001 |
7947275 |
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13112391 |
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60203314 |
May 10, 2000 |
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60208348 |
May 31, 2000 |
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60208111 |
May 31, 2000 |
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60229071 |
Aug 30, 2000 |
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60231273 |
Sep 8, 2000 |
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Current U.S.
Class: |
435/7.1 ; 435/29;
435/408 |
Current CPC
Class: |
A61K 35/16 20130101;
C12N 2501/395 20130101; C07K 16/12 20130101; C12N 5/0631 20130101;
C12N 2503/02 20130101; C12N 2510/04 20130101; A61P 35/00 20180101;
G01N 33/6854 20130101; G01N 2333/71 20130101; C07K 14/70567
20130101; C07K 2317/73 20130101; C07K 2317/52 20130101; C12N
2510/00 20130101; C12N 2500/14 20130101; C12N 2500/90 20130101;
A61K 31/138 20130101; C12N 5/0693 20130101; G01N 33/743 20130101;
C07K 16/00 20130101; A61K 2039/545 20130101; G01N 33/96 20130101;
A61K 2039/505 20130101; A61K 2039/542 20130101; C12N 2501/392
20130101; A61K 39/0008 20130101; G01N 33/5011 20130101; C07K 16/26
20130101; C07K 16/065 20130101; C12N 5/0629 20130101; C12N 2510/02
20130101; C12N 2500/24 20130101; C12N 2500/25 20130101; G01N 33/574
20130101 |
Class at
Publication: |
435/7.1 ;
435/408; 435/29 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C12Q 1/02 20060101 C12Q001/02; C12N 5/09 20100101
C12N005/09 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Research leading to the present invention was supported in
part by the federal government under Grant Nos. DAMD17-94-J-4473,
DAMD17-98-8337 and DAMD17-99-1-9405 awarded by the Defense
Department through the US Army Medical Research and Materiel
Command, Breast Cancer Research Program. The United States
government may have certain rights in the invention.
Claims
1-108. (canceled)
109. A serum composition comprising: at least one secretory
immunoglobulin chosen from the group consisting of IgA, IgM and IgG
in inactived form with respect to the ability to inhibit steroid
hormone-responsive cell proliferation in the absence of steroid
hormone; and steroid hormone-depleted blood plasma or steroid
hormone-depleted blood serum.
110. The serum composition according to claim 109, wherein said at
least one secretory immunoglobulin is inactivated by heating said
composition at about 50-60.degree. C. for a period of time
sufficient to render the inhibitor inactive with respect to the
ability to inhibit steroid hormone responsive cancer cell growth in
vitro.
111. The serum composition according to claim 110, wherein the
period of time is between about 90 minutes and about 30 hours.
112. The serum composition according to claim 109, wherein said
composition comprises steroid hormone-depleted blood serum.
113. The serum composition of claim 112, wherein said steroid
hormone-depleted blood serum is prepared by a method comprising:
obtaining a non-heat-inactivated fresh or frozen serum specimen;
performing a first charcoal-dextran extraction on the specimen at
about 30-37.degree. C. to yield a first extracted serum; and
performing a second 30-37.degree. C. charcoal-dextran extraction on
the first extracted serum to yield a steroid hormone-depleted serum
having steroid hormone reversible activity for inhibiting steroid
hormone-responsive cancer cell growth.
114. The serum composition of claim 113, wherein said first and
second charcoal-dextran extractions are performed at about
34.degree. C.
115. The serum composition according to claim 112, wherein said
steroid hormone-depleted blood serum is prepared by a method
comprising: obtaining a non-heat-inactivated fresh or frozen serum
specimen; and performing an XAD.TM. ion exchange resin extraction
of non-heat-inactivated fresh or frozen serum to provide steroid
hormone-depleted serum having steroid hormone reversible activity
for inhibiting steroid hormone-responsive cancer cell growth.
116. The serum composition of claim 109 for use in a cancer cell
proliferation assay.
117. The serum composition of claim 109, wherein said at least one
secretory immunoglobulin is in inactivated form in the absence of a
reactivating amount of calcium.
118. The negative control serum composition of claim 109, wherein
said immunoglobulin inhibitor is in reactivated form and said
composition comprises calcium ion.
119. A serum composition comprising: at least one secretory
immunoglobulin inhibitor; and steroid hormone-depleted blood plasma
or steroid hormone-depleted blood serum.
120. The serum composition according to claim 119, wherein said
composition comprises steroid hormone-depleted blood serum.
121. The serum composition of claim 120, wherein said steroid
hormone-depleted blood serum is prepared by a method comprising:
obtaining a non-heat-inactivated fresh or frozen serum specimen;
performing a first charcoal-dextran extraction on the specimen at
about 30-37.degree. C. to yield a first extracted serum; and
performing a second 30-37.degree. C. charcoal-dextran extraction on
the first extracted serum to yield a steroid hormone-depleted serum
having steroid hormone reversible activity for inhibiting steroid
hormone-responsive cancer cell growth.
122. The serum composition of claim 121, wherein said first and
second charcoal-dextran extractions are performed at about
34.degree. C.
123. The serum composition according to claim 120, wherein said
steroid hormone-depleted blood serum is prepared by a method
comprising: obtaining a non-heat-inactivated fresh or frozen serum
specimen; and performing an XAD.TM. ion exchange resin extraction
of non-heat-inactivated fresh or frozen serum to provide steroid
hormone-depleted serum having steroid hormone reversible activity
for inhibiting steroid hormone-responsive cancer cell growth.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application Nos. 60/203,314
filed May 10, 2000; 60/208,348 filed May 31, 2000; 60/208,111 filed
May 31, 2000; 60/229,071 filed Aug. 30, 2000 and 60/231,273 filed
Sep. 8, 2000.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention generally relates to the regulation of
steroid hormone responsive cancer cell growth, and more
particularly to compositions and in vitro methods and models for
demonstrating secretory immune system immunoglobulin regulation of
mucosal epithelial cancer cell growth.
[0005] 2. Description of Related Art
Steroid Hormone Responsive Tumor Cell Growth
[0006] In 1896, a physician named Beatson reported in the medical
journal Lancet (Beatson G T (1896) Lancet (Part 1, July 11),
104-107 and Lancet (Part 2, July 18), 162-165) that removal of the
ovaries from breast cancer patients slowed or stopped the growth of
their tumors. As medical science has moved forward, it is now
understood that Dr. Beatson had found that the estrogens made by
the ovaries promoted the growth of breast cancers. In the 1940s and
1950s, work by Professor Charles Huggins (Huggins C B and Hodges C
V (1941) Cancer Res 1, 293-297; Huggins et al. (1941) Arch Surg 43,
209-223) proved that surgical or chemical castration very
substantially reduced the growth of prostate cancers. These results
indicated that testicular androgens were important promoters of the
growth of tumors of this male accessory organ. In subsequent work,
researchers have established that estrogens and androgens act on
breast and prostate cancer cells via receptors within the cell
nucleus (Tsai M-J and O'Malley B W (1994) Annu Rev Biochem 63,
451-486; Evans R E (1988) Science (Wash D.C.) 240, 889-895). In
fact, estrogen receptors are now commonly measured in breast cancer
specimens to assist in decisions regarding the most effective
therapies for each patient, and chemical and surgical castration
are common treatments for prostate cancer. The regulation of
estrogen target tissue cell growth has been a topic of dynamic
experimental interest for several years (Jensen E V and DeSombre E
R (1973) Science (Wash D.C.) 182, 126-134; O'Malley B W and Means A
R (1974) Science (Wash D.C.) 183, 610-620). Today, it is generally
accepted that estrogen interaction with specific nuclear located
DNA binding receptors is necessary to initiate critical cell cycle
events (Dickson R B and Stancel G M (2000) J Natl Cancer Inst
Monogr No 27, 135-145). It is also highly likely that other
non-steroid factors are essential participants in this process
(Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku
D A (1981) Banbury Report 8, 425-443; Dickson R B and Lippman M E
(1987) Endocr Rev 8, 29-43; Soto A M and Sonnenschein C (1987)
Endocr Rev 8, 44-52). Many of these new regulators fall into the
general class of positive acting substances called growth factors
(Gospodarowitz D and Moran J S (1976) Annu Rev Biochem 45, 531-558;
Goustin A S et al. (1986) Cancer Res 46, 1015-1029). Simply stated,
these agents cause cells to undergo cell division and thereby lead
to growth. Because the hallmark of cancer is uncontrolled cell
division, understanding these molecules and how they act is of
vital importance. Other members of this regulatory family include
negative acting agents called growth inhibitors (Knabbe et al.
(1987) Cell 48, 417-428; de Jong J S et al. (1998) J Pathol 184,
44-52). They block cell division, and because of this, are
important targets for new anticancer therapies. A great deal of
study has focused on cellular site(s) of estrogen action, and
various models have been proposed attempting to explain how
estrogen participates with these additional factors to regulate
growth.
[0007] The relative merits of positive versus negative regulation
of cell growth have been debated (Dickson R B and Lippman M E
(1987) Endocr Rev 8, 29-43; Soto A M and Sonnenschein C (1987)
Endocr Rev 8, 44-52). Although the positive direct and positive
indirect models (as defined by Soto A M and Sonnenschein C (1987)
Endocr Rev 8, 44-52) have received the most attention, the concept
of negative regulation has intrinsic appeal because its loss offers
a ready explanation for the uncontrolled replication of cancer
cells. Factors that negatively regulate cell proliferation are now
classified as members of the "tumor suppressor" family (Sager R
(1997) Proc Natl Acad Sci USA 94, 952-955). Defining and
understanding this family of intracellular and extracellular growth
regulators is a primary focus of current cancer research.
[0008] A number of years ago, studies were reported which indicated
that serum-borne inhibitors, later named "estrocolyones," had an
important if not essential role in steroid responsive cell growth
(Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52; Soto A M
et al. (1992) J Steroid Biochem Mol Biol 43, 703-712; Soto A M et
al. (1986) Cancer Res 46, 2271-2275; Soto A M and Sonnenschein C
(1984) Biochem Biophys Res Commun 122, 1097-1103; Schatz R W et al.
(1985) J Cell Physiol 124, 386-390; Soto A M and Sonnenschein C
(1985) J Steroid Biochem 23, 87-94). Estrocolyones appeared to act
as estrogen reversible inhibitors of steroid hormone target tissue
cell growth. Subsequently, the inhibitor has been variously
identified as an unstable M.sub.r 70,000 to 80,000 protein (Soto A
M et al. (1992) J Steroid Biochem Mol Biol 43, 703-712), the intact
serum albumin molecule (Laursen I et al. (1990) Anticancer Res 10,
343-352; Sonnenschein C et al. (1996) J Steroid Biochem Mol Biol
59, 147-154), two domains of serum albumin (Sonnenschein C et al
(1996) J Steroid Biochem Mol Biol 59, 147-154), and the plasma
steroid carrier protein sex hormone binding globulin (SHBG) (Reese
C C et al. (1988) Ann NY Acad Sci 538, 112-121; Fissore F et al.
(1994) Steroids 59, 661-667; Fortunati N et al. (1993) J Steroid
Biochem Mol Biol 45, 435-444). Other investigators also thought it
possible that SHBG, as well as the other major plasma steroid
hormone carrier protein corticosteroid-binding globulin (CBG), were
potential growth regulators independent of their steroid hormone
binding capacity. This conclusion was based on the fact that
specific cellular membrane receptors have been identified for
steroid free CBG and SHBG (Hryb D J et al. (1986) Proc Natl Acad
Sci USA 83, 3253-3256; Hryb D J et al. (1990) J Biol Chem 265,
6048-6054) and that binding of SHBG and CBG to cells caused changes
to cell growth mediators such as cyclic AMP and protein kinase A
(Rosner W (1990) Endocrine Rev 11, 80-91; Fortunati N et al. (1996)
Endocrinology 137, 686-692; Rosner W et al. (1991) J Steroid
Biochem Mol Biol 40, 813-820; Nakhla A M et al. 153, 1012-1018;
Rosner W (1992) J Andrology 13, 101-106).
[0009] Nonetheless, the roles of both albumin and SHBG as estrogen
reversible serum-borne growth regulators have been challenged by
the present Inventor, and others (Soto A M et al. (1992) J Steroid
Biochem Mol Biol 43, 703-712; Damassa D A et al. (1991)
Endocrinology 129, 75-84). In fact, in one report, SHBG stimulated
growth of the androgen responsive ALVA-41 human prostate cancer
cell line (Plymate S R et al. (1991) J Steroid Biochem Mol Biol 40,
833-839). In 1997, Sirbasku et al. reported that nearly pure CBG
and an approximately 85% homogeneous SHBG-like protein were
obtained from horse serum (Sirbasku D A et al. "Serum factor
regulation of estrogen responsive mammary tumor cell growth.
"Proceedings of the 1997 Meeting of the "Department of Defense
Breast Cancer Research Program: An Era of Hope", (Abstract) pp.
739-740, Washington, D.C., Oct. 31-Nov. 4, 1997) by employing a
procedure similar to that described for use with human cord serum
(Fernlund P and Laurell C-B (1981) J Steroid Biochem 14, 545-552).
The Fernlund and Laurell procedure was stated to produce human CBG
and SHBG in pure or very nearly pure states using cortisol-agarose
affinity chromatography at pH 5.5 followed by Phenyl Sepharose.TM.
chromatography at pH 7.4. Under serum-free defined cell culture
conditions, the partially purified SHBG-like fraction obtained by
Sirbasku et al. demonstrated progressive inhibition of cell growth
in a rat mammary tumor cell line (MTW9/PL2) with increasing
concentration of the SHBG-like fraction. Addition of
17.beta.-estradiol (E.sub.2) completely reversed even the maximum
inhibition. Sirbasku et al. found that the active SHBG-like
fraction contained little or no serum albumin as judged by
immunological methods and by standard polyacylamide gel
electrophoresis in the presence of reducing agents and sodium
dodecyl sulfate (SDS-PAGE) (Laemmli U K (1976) Nature (Lond) 227,
680-685). Although the SHBG-like inhibitor displayed certain
immunological similarities to SHBG, it was clearly distinguishable
from SHBG based on physiologic, physical and biochemical analyses.
Despite its first proposal more than fifteen years ago, the
purified estrogen reversible serum-borne inhibitor has yet to be
described. Sirbasku et al., as well as others (Soto A M et al.
(1992) J Steroid Biochem Mol Biol 43, 703-712), has observed that
the estrogen reversible inhibitory activity of serum was very
labile during isolation by conventional protein purification
methods. Other investigators have used a combination of cortisol
affinity chromatorgraphy and an ammonium sulfate precipitation to
isolate a cell growth inhibitor from human serum. These studies
(Tanji M et al. (2000) Anticancer Res. 20, 2779-2783; Tanji M et
al. (2000) Anticancer Res. 20, 2785-2789) describe estrogen
inhibition of MCF-7 human breast carcinoma cells that had been
maintained at least 3 months in serum-free medium, but no
estrogenic effect was observed with normally cultured MCF-7 cells
(i.e., cells not long term conditioned to serum-free medium). An
isolated steroid-binding protein was stated to mediate an
estrogen-dependent inhibition of cell growth. Other serum-borne
inhibitors also have been separated from whole serum by
diethylaminoethyl (DEAE) chromatography (Dell'Aquila M L and
Gaffney E V (1984) J Natl Cancer Inst 73, 397-403). The properties
of these inhibitors have not been defined further nor have they
been shown to act as estrogen-reversible inhibitors.
[0010] Carcinogen-induced rat mammary tumors have been studied
extensively as models for the in vivo role of hormones in the
induction and growth of breast cancer (Welsch C W (1985) Cancer Res
45, 3415-3443). Despite ample evidence of hormone dependence in
vivo, the carcinogen-induced tumors have not yet yielded permanent
tissue culture cell lines that show the same responsiveness to
steroid hormones in in vitro culture. Typically, cultures initiated
from primary tumors very quickly lose hormone responsiveness.
Because of this, the earliest endocrine studies were done with
organ cultures (Welsch C W and Rivera E M (172) Proc Soc Exp Biol
Med 139, 623-626; Lewis D and Hallowes R C (1974) J Endocrinol 62,
225-240; Chan P-C et al. (1976) Proc Soc Exp Biol Med 151, 362-365;
Pasteels J-L et al. (1976) Cancer Res 36, 2162-2170) and short-term
cultures of dissociated cells (Chan P--C et al. (1976) Proc Soc Exp
Biol Med 151, 362-365). Now investigators recognized that those
approaches were inadequate. More recently, cell lines have been
developed from carcinogen-induced rat mammary tumors (Bennett D C
et al. (1978) Cell 15, 283-298; Rudland P S (1987) Cancer Metast
Rev 6, 55-83; Webster M K et al. (1990) J Biol Chem 265, 4831-4838;
Lichtner R B et al. Cancer Res 51, 5943-5950; Lichtner R B et al.
(1995) Oncogene 10, 1823-1832). Although these lines have been
useful for investigations related to breast properties,
investigators have found that in general they do not display
steroid hormone responsiveness in cell culture. To compound the
difficulties, most of these lines could not be evaluated for
hormone responsiveness in vivo because they were derived from
outbred rats. Simply stated, they lack the syngeneic inbred hosts
absolutely required for in vivo transplantation.
[0011] One of the basic tenets of endocrine physiology is that
estrogens and androgens cause coordinate growth of several target
tissues (Clark J H et al. (1992) In: Williams Textbook of
Endocrinology, 8.sup.th Edition, WB Saunders, Philadelphia, pp
35-90). A partial list of estrogen target tissues includes breast,
uterus, cervix, vagina, ovary, pituitary, liver, leukocytes and
kidney. A partial list of androgen target tissues includes the male
reproductive tract (e.g. prostate, epididymus, and testis), kidney,
bladder, liver and muscle. Whatever mechanism is proposed to
explain sex steroid dependent growth, one would expect it to be
applicable to cells from several of the major target tissues.
[0012] The history of attempts to demonstrate steroid hormone
responsive tumor cell growth in culture has led to two important
conclusions. First, demonstration of estrogen and androgen
responsive cell growth in culture required the presence of hormone
deficient/depleted serum. One of the first studies to demonstrate
this requirement was done with human breast cancer cells (Page M J
et al. (1983) Cancer Res 43, 1244-1250). Some notable examples of
demonstration by others of estrogen responsiveness in serum
containing culture include studies with the MCF-7 human breast
cancer cells (Lippman M E et al. (1977) Cancer Res 37, 1901-1907;
Soto A M and Sonnenschein C (1985) J Steroid Biochem 23, 87-94;
Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A, 595-602), the
T47D human breast cancer cells (Chalbos D et al. (1982) J Clin
Endocrinol Metab 55, 276-283; Schatz R W et al. (1985) J Cell
Physiol 124, 386-390; Soto A M et al. (1986) Cancer Res 46,
2271-2275), the ZR-75-1 human breast cancer cells (Darbre P et al.
(1983) Cancer Res 43, 349-355), the GH.sub.4C.sub.1 rat pituitary
tumor cells (Amara J F and Dannies P S (1983) Endocrinology 112,
1141-1143), and the H-301 Syrian hamster kidney tumor cell line
(Soto A M et al. (1988) Cancer Res 48, 3676-3680). Two reports have
proposed that estrogen responsiveness can be observed in serum-free
defined medium with ZR-75-1 cells (Allegra J C and Lippman M E
(1978) Cancer Res 38, 3823-3829; Darbre P D et al. (1984) Cancer
Res 44, 2790-2793). However, in both of those studies, the cells
were first incubated for several days in medium supplemented with
serum before changing to serum-free defined medium conditions. M
Ogasawara and DA Sirbasku previously demonstrated that this
approach leaves a problematic serum factor "memory" with cells
(Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,
911-920). When completely serum-free defined medium conditions were
applied (Barnes D and Sato G (1980) Nature 281, 388-389; Danielpour
D et al. (1988) In Vitro Cell Dev Biol 24, 42-52; Karey K P and
Sirbasku D A (1988) Cancer Res 48, 4083-4092; Ogasawara M and
Sirbasku D A (1988) In Vitro Cell Dev Biol 24, 911-920; Riss T L
and Sirbasku D A (1989) In Vitro Cell Dev Biol 25, 136-142), no
growth effects of estrogens were observed. Comparison of the
observations in serum-free defined medium versus those in medium
with serum led to the second important conclusion. Serum contains a
mediator(s) that is required for steroid hormone responsiveness in
culture. When the mediator is completely purified and defined
chemically, its addition to serum-free defined medium will be
expected to provide unequivocal confirmation of its role in hormone
dependent cell growth.
[0013] The purification of the serum-borne mediator has been a
challenging undertaking. Sirbasku et al. originally proposed that
estrogens per se were not mitogenic, but instead caused the
production of endocrine, paracrine or autocrine "estromedins" that
were themselves the promoters of target tissue cell growth
(Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku
D A (1981) Banbury Report 8, 425-443; Ikeda T et al. (1982) In
Vitro 18, 961-979; Sirbasku D A and Leland F E (1982) Biochemical
Action of Hormones 9, 115-140; Leland F E et al. In: Cold Spring
Harbor Conferences on Cell Proliferation, Volume 9, Books A and B,
Growth of Cells in Hormonally Defined Media, Cold Spring Harbor,
N.Y., pp 741-750). From 1970 through 1984, estrogenic mitogenic
effects were most often not seen in culture. Although some
laboratories were reporting positive results in serum containing
medium, as cited above, others were at the same time recording
negative results using the same or related cell lines (Sirbasku D A
(1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A and
Kirkland W L (1976) Endocrinology 98, 1260-1272; Kirkland W L et
al. (1976) J Natl Cancer Inst 56, 1159-1164; Ikeda T et al. (1982)
In Vitro 18, 961-979; Butler W B et al. (1983) Cancer Res 41,
82-88; Edwards D P et al. (1980) Biochem Biophys Res Commun 93,
804-812; Shafie S M (1980) Science (Wash D.C.) 209, 701-702). Part
of the problem may have been due to culture conditions (Ruedl C et
al. (1990) J Steroid Biochem Mol Biol 37, 195-200; Zugmaier G et
al. (1991) J Cell Physiol 141, 353-361) or possibly caused by
differences that arose because of variations in the properties of
cell lines in different laboratories (Seibert K et al. (1983)
Cancer Res 43, 2223-2239). In addition, there are other more
technical issues that are well known in this field, have been
described in the literature, and which are addressed in more detail
elsewhere herein and in subsequent publications (Moreno-Cuevas J E
and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427;
Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol
36, 428-446; and Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro
Cell Dev Biol 36, 447-464.) Another vital matter has been how
"growth" is defined. Sonnenschein and Soto (Sonnenschein C and Soto
A M (1980) J Natl Cancer Inst 64, 211-215) have addressed this
issue very effectively. To be accepted as valid, sex steroids must
cause significant changes in cellular logarithmic growth rates.
Elucidation of the nature and activity of the estrogen reversible
serum inhibitor(s) continues to be an area of intense experimental
interest.
[0014] As cited above, A M Soto and C Sonnenschein have proposed
that the serum mediator is an estrogen reversible inhibitor they
have named estrocolyone. They have alternately described the
inhibitor as a pituitary factor (Sonnenschein C and Soto A M (1978)
J Steroid Biochem 6, 533-537), .alpha.-fetoprotein (Sonnenschein C
et al. (1980) J Natl Cancer Inst 64, 1141-1146; Sonnenschein C et
al. (1980) J Natl Cancer Inst 64, 1147-1152; Soto A M and
Sonnenschein C (1980) Proc Natl Acad Sci USA 77, 2084-2087), a
serum protein different than human serum albumin (Soto A M et al.
(1992) J Steroid Biochem Mol Biol 43, 703-712), and in a later
reversal of this view, stated that estrocolyone 1 (i.e. the
serum-borne estrogen reversible inhibitor) was human serum albumin
or a combination of two domains of albumin (Sonnenschein C (1996) J
Steroid Biochem Mol Biol 59, 147-154). They have also sought the
inhibitor as an estrogen-binding glycoprotein different than SHBG
using Concanavalin-A chromatography (Reny J-L and Soto A M (1989) J
Clin Endocrinol Metab 68, 938-945). The outcome of this effort did
not identify the inhibitor. The exact chemical nature of the
inhibitor was even further complicated by U.S. Pat. No. 4,859,585
(Sonnenschein) and U.S. Pat. No. 5,135,849 (Soto) describing an
inhibitor that was derived from heat inactivated serum depleted of
its endogenous estrogens and androgens by a 37.5.degree. C.
charcoal-dextran procedure. Alternatively, the inhibitor was
obtained from serum by ammonium sulfate precipitation. This
inhibitor is said to be useful for in vitro testing of substances
of interest for activity as an estrogen or androgen agonist or
antagonist using the MCF-7 cell line grown in Dulbecco's modified
Eagle minimal essential medium supplemented with 5% (v/v) fetal
bovine serum. However, the two above-mentioned U.S. patents do not
address the issues of (i) whether there are one or more inhibitors,
(ii) what is/are the exact chemical composition of the
inhibitor(s), and (iii) what conditions were required to yield the
long term stable product(s) necessary for the commercial
application of the testing methodology described.
Steroid Hormone Receptors
[0015] As the matter stands today, it has not been established
beyond doubt which of the many estrogen receptors and/or variants
is the one that regulates the estrogen induced mitogenic effect. It
is generally assumed that the ER.alpha. is the most likely positive
growth mediator. Estrogens, androgens, progestins, corticosteroids,
mineral steroids, vitamin D, retinoic acid and thyroid hormone
receptors all belong to a family of DNA binding intracellular
receptors that are activated by binding of the appropriate
hormone/ligand (Evans R M (1988) Science (Wash D.C.) 240, 889-895;
Giguere V (1990) Genetic Eng (NY) 12, 183-200; Williams G R and
Franklyn J A (1994) Baillieres Clin Endocrinol Metab 8, 241-266;
Kumar R and Thompson E B (1999) Steroids 64, 310-319; Pemrick S M
et al. (1994) Leukemia 8, 1797-806; Carson-Jurica M A et al.
(1990), Endocr Rev 11, 201-220; Tsai M J and O'Malley B W (1994)
Annu Rev Biochem 63, 451-486; Alberts B et al. (1994) Molecular
Biology of The Cell, 3rd edition, Garland Publishing, New York, pp
729-731). The estrogen receptor described in the citations above is
now designated the classical estrogen receptor alpha (ER.alpha.).
Its role in steroid regulated gene expression has been studied
extensively and often reviewed (Yamamoto K R (1985) Annu Rev Genet.
19, 209-252; Green S and Chambon P (1991) In: Nuclear Hormone
Receptors, Academic Press, New York, pp 15-38; Tsai M-J and
O'Malley B W (1994) Annu Rev Biochem 63, 451-486; McDonnell D P et
al. (1992) Proc Natl Acad Sci USA 89, 10563-10567; Landel C C et
al. (1994) Mol Endocrinol 8, 1407-1419; Landers J P and Spelsberg T
C (1992) Crit Rev Eukary Gene Exp 2, 19-63; Cavailles V et al.
(1994) Proc Natl Acad (Sci USA 91, 10009-10013; Halachmi S et al.
(1994) Science (Wash D.C.) 264, 1455-1458; Brasch K and Ochs R L
(1995) Int rev Cyto 159, 161-194; Hard T and Gustafsson J-.ANG.
(1993) Acc Chem Res 26, 644-650).
[0016] It is noteworthy that estrogen resistance in man is caused
by a mutation in the ER.alpha. (Smith E P et al. N Eng J Med 331,
1056-1061). The most startling fact is that this point mutation
(i.e. cytosine.fwdarw.thymidine) generated a premature stop codon,
but was not lethal. Although many metabolic abnormalities were
noted, development into adulthood was observed without expression
of a functional ER.alpha.. This fact is further strengthened by the
experiments with ER.alpha. gene knockout mice (Couse J F and Korach
K S (1999) Endocr Rev 20, 358-417). The authors state "the list of
unpredictable phenotypes in the .alpha. ERKO (estrogen receptor
knockout) must begin with the observation that generation of an
animal lacking a functional ER .alpha. gene was successful and
produced animals of both sexes that exhibit a life span comparable
to wild-type". Furthermore, in the review of the ERKO results, it
was not possible to conclude that the ER.alpha. regulated estrogen
responsive cell growth. Indeed, functions normally ascribed to the
ER.alpha. seemed unaffected. In fact, only relationships to
development in tissues such as breast seemed best correlated
(Boccchinfuso W P and Korach K S (1997) J Mammary Gland Biol
Neoplasia 2, 323-334). The situation with ERKO mice and ER.beta. is
similar (Couse J F and Korach K S (1999) Endocr Rev 20, 358-417).
The results from ER.beta. knockout suggest an indirect role of this
receptor via stromal tissue (Gustafsson J-.ANG. and Warner M (2000)
J Steroid Biochem Mol Biol 74, 254-248). Certainly a direct growth
role for ER.beta. in breast epithelial cells was not established.
The results available from ERKO do not yet provide confidence that
either the ER.alpha. or the ER.beta. mediate estrogen responsive
cell growth.
[0017] There are other pertinent lines of evidence that relate to
the role of the ER.alpha. and growth. The first is from a study of
transfection of estrogen receptor negative cells with the full
length functional ER.alpha. (Zajchowski D A et al. (1993) Cancer
Res 53, 5004-5011). Those investigators arrived at a remarkable
result. They had expected to regain estrogen responsive growth in
the transfected hormone independent cells. This was definitely not
the case. Instead, addition of E.sub.2 caused cell growth
inhibition. Their results indicated that ER.alpha. was not a
positive mediator, but instead a negative regulator. However,
similarly transfected estrogen responsive cell lines such as MCF-7
and T47D were not E.sub.2 inhibited in those studies.
[0018] More recently, another estrogen receptor has been cloned and
cDNA sequenced from rat prostate and ovary (Kuiper G G et al.
(1996) Proc Natl Acad Sci USA 93, 5925-5930). It has now also been
cloned from mouse (Tremblay G B et al. (1997) Mol Endocinol 11,
353-365) and human (Mosselman S et al. (1996) FEBS Lett 392,
49-53). This new receptor has been named estrogen receptor beta
(ER.beta.). Evidence that ER.beta. is separate from ER.alpha. comes
from the fact that the genes are located on different chromosomes
(Enmark E et al. (1997) 82, 4258-4265). Therefore, ER.beta. is not
simply an alternate splicing product of the ER.alpha. gene.
Furthermore, ER.beta. is distinguishable from ER.alpha. based on
critical differences in the amino acid sequences of functional
domains (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93,
5925-5930; Enmark E et al. (1997) 82, 4258-4265; Dickson R B and
Stancel G M (2000) J Natl Cancer Inst Monogr No. 27, 135-145). For
example, the sequence homology between the two receptors is 97% in
the DNA binding domain, but 59% in the C-terminal ligand-binding
(i.e. steroid hormone-binding) domain, and only 17% in the
N-terminal domain. The En.beta. N-terminal domain is much
abbreviated compared to the ER.alpha. (Enmark E et al (1997) 82,
4258-4265). Rat ER.beta. contains an 18 amino acid insert in the
domain binding the ligand. Despite the significant differences in
structure, ER.alpha. and ER.beta. bind E.sub.2 with the same
affinity (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93,
5925-5930; Dickson R B and Stancel G M (2000) J Natl Cancer Inst
Monogr No. 27, 135-145). In fact, others (Tremblay G B et al.
(1997) Mol Endocrinol 11, 353-365) have stated that ER.beta. has a
slightly lower affinity for E.sub.2 than ER.alpha. (Tremblay G B et
al. (1997) Mol Endocrinol 11, 353-365). Therefore, if either of
these receptors mediates estrogen-induced growth, the steroid
hormone concentrations required for one-half maximum growth (i.e.
ED.sub.50), or for optimum growth (i.e. ED.sub.100), are expected
to be about the same.
[0019] It is thought that ER.alpha. and ER.beta. are functionally
interrelated (Kuiper G G et al. (1998) Endocrinology 139,
4252-4263) and that one role of ER.beta. is to modulate the
transcriptional activity of ER.alpha. (Hall J M and McDonnell D P
(1999) Endocrinology 140, 5566-5578). Clearly however, there are
significant functional differences between ER.alpha. and ER.beta.,
which have been discussed (Gustafsson J-.ANG. (1999) J Endocrinol
163, 379-383). Also, there are functional differences expected
because of the different pattern of steroid hormone binding shown
by ER.beta. (Kuiper G G et al. (1996) Proc Natl Acad Sci USA 93,
5925-5930). For example, ER.beta. binds androgens whereas ER.alpha.
does not. This fact, plus the location of ER.beta. in prostate
indicates a new function that may be androgen related.
[0020] It should also be noted that there have been "estrogen
related receptors" (ERR 1 and 2) or "orphan" receptors identified
that share properties with ER.alpha. but do not have a known
function and do not have a known ligand (Giguere V et al. (1988)
Nature (Lond) 331, 91-94; Gustafsson J-.ANG. (1999) J Endocrinol
163, 379-383). In fact, today, there are more than 70 "orphan"
receptors seeking ligands and functions (Gustafsson J-.ANG. (1999)
Science (Wash D.C.) 284, 1285-1286).
The Secretory Immune System
[0021] Turning now to discussion of a separate body of work from
that described above, as further background for understanding the
present invention, it should be noted that the immunological
function and physiological properties of the body's secretory
immune system have been recognized for many years (Tomasi T B et
al. (1965) J Exp Med 121, 101-124; Brandtzaeg P and Baklien K
(1977) Ciba Foundation Symposium 46, 77-113; Tomasi T B (1970) Ann
Rev Med 21, 281-298; Spiegelberg H L (1974) Adv Immunol 19,
259-294; Tomasi T B (1976) The Immune System of Secretions,
Prentice-Hall, Englewood Cliffs, New Jersey; Mestecky J and McGhee
J R (1987) Adv Immunol 40, 153-245). The major immunoglobulins
secreted as mucosal immune protectors include IgA, IgM and IgG. In
human serum, the percent content of IgG, IgA and IgM are 80, 6 and
13%, respectively. In humans, the major subclasses of IgG are IgG1,
IgG2, IgG3 and IgG4. These are 66, 23, 7 and 4% of the total IgG,
respectively. The relative content of human immunoglobulin
classes/subclasses in adult serum follow the order
IgG1>IgG2>IgA1>IgM>IgG3>IgA2>IgD>IgE
(Spiegelberg H L (1974) Adv Immunol 19, 259-294). When the serum
concentrations of immunoglobulins are compared to those in exocrine
secretion fluids, the relative contents change dramatically
(Brandtzaeg P (1983) Ann NY Acad Sci 409, 353-382; Brandtzaeg P
(1985) Scand J Immunol 22, 111-146). For example in colostrum (a
breast fluid secretion), IgA is 80% of the total immunoglobulins.
IgM is .ltoreq.10% of the total. IgG represents a few percent. In
human colostrum and milk, IgG1 and IgG2 are the major subclasses of
IgG (Kim K et al. (1992) Acta Paediatr 81, 113-118). Clearly,
comparison of serum and mucosal fluid concentrations indicate
selective immunoglobulin secretion.
[0022] Immunoglobulin Function. All human mucus membranes are
protected by the secretory immune system (Hanson L .ANG. and
Brandtzaeg P (1989) In: Immunological Disorders in Infants and
Children, 3rd edition, Stiehm E R, ed, Saunders, Philadelphia, pp
169-172). The primary protector is sIgA that is produced as dimers
and larger polymers. A single joining "J" chain connects IgA
monomers to form the dimers and polymers (Garcia-Pardo A et al.
(1981) J Biol Chem 256, 11734-11738), and connects monomers of IgM
to give pentamers (Niles M J et al. (1995) Proc Natl Acad Sci USA
92, 2884-2888). This critical joining endows these structures with
a very important immunological property. IgA and IgM are known to
bind to bacterial, parasite and viral surface antigens. These
complexes bind to receptors on inflammatory cells leading to
destruction of the pathogen by antibody-dependent cell-mediated
cytotoxicity (Hamilton R G (1997) "Human Immunoglobulins" In:
Handbook of Human Immunology, Leffell M S et al., eds, CRC Press,
Boca Raton, Chapter 3). Dimeric and polymeric sIgA have a high
antigen binding valence that effectively agglutinates/neutralizes
bacteria and virus (Janeway C A Jr et al. (1999) Immunobiology, The
Immune System in Health and Disease, 4.sup.th edition, Garland
Publishing, New York, pp 326-327). Also, sIgA shows little or no
complement activation. This means that it does not cause
inflammatory responses (Johansen F E et al. (2000) Scand J Immunol
52, 240-248). In addition, the fact that IgA exists as two separate
forms is significant (Loomes L M et al (1991) J Immunol Methods
141, 209-218). The IgA1 predominates in the general circulation. In
contrast, IgA2 is often higher in mucosal secretions such as those
from breast, gut, and respiratory epithelium, salivary and tear
glands, the male and female reproductive tracts, and the urinary
tracts of both males and females. This difference in proportions is
important to immune protection of mucosal surfaces. Although the
secretory form of IgA1 is by and large resistant to proteolysis
(Lindh E (1975) J Immunol 114, 284-286), a number of different
bacteria secrete proteolytic enzymes that cleave it into Fab and Fc
fragments (Wann J H et al. (1996) Infect Immun 64, 3967-3974;
Poulsen K et al. (1989) Infect Immun 57, 3097-3105; Gilbert J V et
al. (1988) Infect Immun 56, 1961-1966; Reinholdt J et al. (1993)
Infect Immun 61, 3998-4000; Blake M S and Eastby C (1991) J Immunol
Methods 144, 215-221; Burton J et al. (1988) J Med Chem 31,
1647-1651; Mortensen S B and Kilian M (1984) Infect Immun 45,
550-557; Simpson D A et al. (1988) J Bacteriol 170, 1866-1873;
Blake M S and Swanson J et al. (1978) Infect Immun 22, 350-358;
Labib R S et al. (1978) Biochim Biophys Acta 526, 547-559). In
effect, the bacterial proteinases negate the neutralizing effects
of multivalent sIgA1. In contrast, because of structural
differences (Chintalacharuvu K R and Morrison S L (1996) J Immunol
157, 3443-3449), IgA2 lacks sites required for proteolysis. This
makes IgA2 more resistant to bacterial digest than IgA1 (Hamilton R
G (1997) "Human immunoglobulins" In: Handbook of Human Immunology,
Leffell M S et al., eds, CRC Press, Boca Raton, Chapter 3).
[0023] With regard to IgM, its function is somewhat different. IgM
antibodies serve primarily as efficient agglutinating and cytolytic
agents. They appear early in the response to infection and are
largely confined to the bloodstream. Whether secreted or
plasma-borne, IgM is a highly effective activator of the classical
complement cascade. It is less effective as a neutralizing agent or
an effector of opsinization (i.e. facilitation of phagocytosis of
microorganisms). Nonetheless, IgM complement activation causes
lysis of some bacteria. The effects of the IgG class are more
encompassing. All four subclasses cause neutralization,
opsinization and complement activation to defend against mucosal
microorganisms. IgG1 is an active subclass in this regard (Janeway
C A Jr et al. (1999) Immunobiology, The Immune System in Health and
Disease, 4.sup.th edition, Garland Publishing, New York, pp
326-327).
[0024] Immunoglobulin Structure. It was established that
immunoglobulin A (IgA) represents 5 to 15% of the total plasma
immunoglobulins in humans (Spiegelberg H L (1974) Adv Immunol 19,
259-294). IgA has a typical immunoglobulin four-chain structure
(M.sub.r 160,000) made up of two heavy chains (M.sub.r 55,000) and
two light chains (M.sub.r 23,000) (Fallgreen-Gebauer E et al (1993)
Biol Chem Hoppe-Seyler 374, 1023-1028; Kratzin H et al. (1978)
Hoppe-Seylers Z Physiol Chem 359, 1717-1745; Yang C et al. (1979)
Hoppe-Seylers Z Physiol Chem 360, 1919-1940; Eiffert H et al.
(1984) Hoppe-Seylers Z Physiol Chem 365, 1489-1495). In humans,
there are two subclasses of IgA. These are IgA1 and IgA2 that have
1 and 2 heavy chains, respectively. The IgA2 subclass has been
further subdivided into A.sub.2m(1) and A.sub.2m(2) allotypes
(Mestecky J and Russell M W (1986) Monogr Allergy 19, 277-301;
Morel A et al. (1973) Clin Exp Immunol 13, 521-528). IgA can occur
as monomers, dimers, trimers or multimers E et al. (1996) J Biol
Chem 271, 16300-16309). In plasma, 10% of the total IgA is
polymeric while the remaining 90% is monomeric. Formation of
dimeric or multimeric IgA requires the participation of an
elongated glycoprotein of approximately M.sub.r 15,000, designated
the "J" chain (Mestecky J et al. (1990) Am J Med 88, 411-416;
Mestecky J and McGhee J R (1987) Adv Immunol 40, 153-245; Cann G M
et al. (1982) Proc Natl Acad Sci USA 79, 6656-6660). Structurally,
the J chain is disulfide linked to the penultimate cysteine residue
of heavy chains of two IgA monomers to form a dimeric complex of
approximately M.sub.r 420,000. The general structure of the dimer
has been well described in the literature (Fallgreen-Gebauer E et
al (1993) Biol Chem Hoppe-Seyler 374, 1023-1028). Multimeric forms
of IgA and IgM require only a single J chain to form (Mestecky J
and McGhee J R (1987) Adv Immunol 40, 153-245; Chapus R M and
Koshland M E (1974) Proc Natl Acad Sci USA 71, 657-661; Brewer J W
et al. (1994) J Biol Chem 269, 17338-17348). The structures and
chemical properties of IgA and IgM have been described in detail
(Janeway C A Jr et al. (1996) Immunobiology, The Immune System in
Health and Disease, Second edition, Garland Publishing, New York,
pp 3-32 and pp 8-19).
[0025] Immunoglobulin Production. Dimeric and multimeric IgA and
IgM are secreted by a number of exocrine tissues. IgA is the
predominant secretory immunoglobulin present in colostrum, saliva,
tears, bronchial secretions, nasal mucosa, prostatic fluid, vaginal
secretions, and mucous secretions from the small intestine
(Mestecky J et al. (1987) Adv Immunol 40, 153-245; Goldblum R M, et
al. (1996) In: Stiehm E R, ed, Immunological Disorders in Infants
and Children, 4.sup.th edition, Saunders, Philadelphia, pp 159-199;
Heremans J F (1970) In: Immunoglobulins, Biological Aspects and
Clinical Uses, Merler E, ed, National Academy of Sciences, Wash
D.C. pp 52-73; Tomasi T B Jr (1971) In: Immunology, Current
Knowledge of Basic Concepts in Immunology and their Clinical
Applications, Good R A and Fisher D W, eds, Sinauer Associates,
Stanford, Conn., p 76; Brandtzaeg P (1971) Acta Path Microbiol
Scand 79, 189-203). IgA output exceeds that of all other
immunoglobulins, making it the major antibody produced by the body
daily (Heremans J F (1974) In: The Antigens, Vol 2, Sela M, ed,
Academic Press, New York, pp 365-522; Conley M E et al. (1987) Ann
Intern Med 106, 892-899. IgA is the major immunoglobulin found in
human milk/whey/colostrum (Ammann A J et al. (1966) Soc Exp Biol
Med 122, 1098-1113; Peitersen B et al. (1975) Acta Paediatr Scand
64, 709-717); Woodhouse L et al. (1988) Nutr Res 8, 853-864). IgM
secretion is less abundant but can increase to compensate for
deficiencies in IgA secretion.
[0026] During passage of IgA through the cell, its structure is
modified. A M.sub.r 80,000 fragment of the receptor containing all
five of the extracellular domains becomes covalently attached to
dimeric IgA to form secretory IgA (sIgA) (Fallgreen-Gebauer E et al
(1993) Biol Chem Hoppe-Seyler 374, 1023-1028). The receptor that
mediates the translocation has been interchangeably called the
"poly-Ig receptor" (poly-Ig receptor) or the "secretory component"
(Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,
2309-2315). For the purposes of the present disclosure, however,
the term "poly-Ig receptor" refers to the full length M.sub.r
100,000 transmembrane protein and the term "secretory component"
denotes only the M.sub.r 80,000 extracellular five domains of the
receptor that become covalently attached to IgA in forming the sIgA
structure (Fallgreen-Gebauer E et al (1993) Biol Chem Hoppe-Seyler
374, 1023-1028; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol
22, 2309-2315). Because of the unique structure of sIgA, it is
highly resistant to acid and proteolysis (Lindh E (1975) J Immunol
114, 284-286) and therefore remains intact in secretions to perform
extracellular immunological functions. IgM also binds secretory
component, but not covalently (Lindh E and Bjork I (1976) Eur J
Biochem 62, 271-278). However, IgM is less stabilized because of
its different association with the secretory component, and
therefore has a shorter functional survival time in acidic
secretions (Haneberg B (1974) Scand J Immunol 3, 71-76; Haneberg B
(1974) Scand J Immunol 3, 191-197).
[0027] The secretion mechanism for IgA and IgM are well described.
Conversely, there is a fundamental question surrounding IgG
secretion. There is no "J" chain present in IgG1 and IgG2. From the
known facts of transcytosis/secretion of immunoglobulins (Johansen
F E et al. (2000) Scand J Immunol 52, 240-248), it is unlikely that
IgG secretion is mediated by the poly-Ig receptor. An epithelial
receptor specific for IgG1 has been reported in bovine mammary
gland (Kemler R et al. (1975) Eur J Immunol 5, 603-608).
Apparently, it preferentially transports this class of
immunoglobulins from serum into colostrum. Despite this 1975 report
however, the receptor has not been chemically or structurally
identified nor has the mechanism of transport of IgG monomers been
satisfactorily defined. It is possible that this receptor is a
member of a large group now designated as Fe receptors (Fridman W H
(1991) FASEB J 5, 2684-2690), but there is one study with IgG
showing that of 31 different long-term human carcinoma cell lines
including breast "all lines were found to be consistently Fc
receptor negative" (Kerbel R S et al. (1997) Int J Cancer 20,
673-679). One possible candidate for the epithelial transport of
IgG1 is the neonatal Fc receptor (Raghavan M and Bjorkman P J
(1996) Annu Rev Cell Dev Biol 12, 181-220). However, there is no
indication yet of the presence of this receptor in adult mucosal
tissues.
[0028] Transcytosis Mediating Receptors. J chain-containing IgA is
produced and secreted by plasma B immunocytes located in the lamina
propria just beneath the basement membrane of exocrine cells
(Brandtzaeg P (1985) Scan J Immunol 22, 111-146). The secreted IgA
binds to a M.sub.r 100,000 poly-Ig receptor positioned in the
basolateral surface of most mucosal cells (Heremans J F (1970) In:
Immunoglobulins, Biological Aspects and Clinical Uses, Merler E,
ed, National Academy of Sciences, Wash D.C., pp 52-73; Brandtzaeg P
(1985) Clin Exp Immunol 44, 221-232; Goodman J W (1987) In: Basic
and Clinical Immunology, Stites D P, Stobo J D and Wells J V, eds,
Appleton and Lange, Norwalk, Conn., Chapter 4). The receptor-IgA
complex is next translocated to the apical surface where IgA is
secreted. The binding of dimeric IgA to the poly-Ig receptor is
completely dependent upon the presence of a J chain (Brandtzaeg P
(1985) Scan J Immunol 22, 111-146; Brandtzaeg P and Prydz H (1984)
Nature 311:71-73; Vaerman J-P et al. (1998) Eur J Immunol 28,
171-182). Monomeric IgA will not bind to the receptor. The J chain
requirement for IgM binding to the poly-Ig receptor is also true
for this immunoglobulin (Brandtzaeg P (1985) Scan J Immunol 22,
111-146; Brandtzaeg P (1975) Immunology 29, 559-570; Norderhaug I N
et al. (1999) Crit. Rev Immunol 19, 481-508). Because IgA and IgM
bind to the poly-Ig receptor via their Fc domains, and because of a
repeating Ig-like structure in the extracellular domains, the
poly-Ig receptor classifies as a member of the Fc superfamily of
immungobulin receptors (Kraj{hacek over (c)}i P et al. (1992) Eur J
Immunol 22, 2309-2315; Daeron M (1997) Annu Rev Immunol 15,
203-234).
[0029] The poly-Ig receptor and the secretory component from human
has been cDNA cloned and DNA sequenced (Kraj{hacek over (c)}i P et
al. (1992) Eur J Immunol 22, 2309-2315; Kraj{hacek over (c)}i P et
al. (1995) Adv Exp Med Biol 371A, 617-623; Kraj{hacek over (c)}i P
et al. (1991) Hum Genet. 87, 642-648; Kraj{hacek over (c)}i P et
al. (1989) Biochem Biophys Res Commun 237, 9-20) as has the poly-Ig
receptor from mouse (Kushiro A and Sato T (1997) Gene 204, 277-282;
Piskurich J F et al. (1995) and bovine tissue (Verbeet M P et al.
(1995) Gene 164, 329-333). Altogether, the human poly-Ig receptor
coding sequence encompassed 11 exons. The extracellular five
domains originate from exons 3 (D1), exon 4 (D2) exon 5 (D3 and
D4), exon 6 (D5), exon 7 (the conserved cleavage site to form the
secretory component), exon 8 (the membrane spanning domain), exon 9
(a serine residue required for transcytosis), exon 9 (sequence to
avoid degradation), exon 10, no known function) and exon 11
(sequence contains a threonine residue and the COOH terminus)
(Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,
2309-2315). With the exception of domains 3 and 4 (both from one
exon), the receptor structure follows the rule of one domain/one
exon. The poly-Ig receptor binds IgA and IgM via their Fc domains,
and more particularly, via a specific amino acid sequence
(15.fwdarw.67) of domain 1 (Bakos M-A et al. (1991) J Immunol 147,
3419-3426). Of the other extracellular domains, only D5 is known
for a specific function. It contains the disulfide bonds that
covalently attach to IgA to for sIgA during transcytosis. The role
of this receptor in transcytosis of IgA/IgM has been well studied
with mucosal tissues and epithelial cells in culture (Vaerman J P
et al. (1998) Eur J Immunol 28, 171-182; Fahey J V et al. (1998)
Immunol Invest 27, 167-180; Brandtzaeg P (1997) J Reprod Immunol
36, 23-50; Loman S et at (1997) Am J Physiol 272, L951-L958; Mostov
K E et al. (1995) Cold Spring Harbor Symp Quant Biol 60, 775-781;
Schaerer E et al. (1990) J Cell Biol 110, 987-998).
[0030] During passage of IgA through the cell, its structure is
modified. A M.sub.r 80,000 fragment of the receptor containing all
five of the extracellular domains becomes covalently attached to
dimeric IgA to form secretory IgA (sIgA) (Fallgreen-Gebauer E et al
(1993) Biol Chem Hoppe-Seyler 374, 1023-1028). The receptor that
mediates the translocation has been interchangeably called the
"poly-Ig receptor" (poly-Ig receptor) or the "secretory component"
(Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,
2309-2315). For the purposes of the present disclosure, however,
the term "poly-Ig receptor" refers to the full length M.sub.r
100,000 transmembrane protein and the term "secretory component"
denotes only the M.sub.r 80,000 extracellular five domains of the
receptor that become covalently attached to IgA in forming the sIgA
structure (Fallgreen-Gebauer E et al (1993) Biol Chem Hoppe-Seyler
374, 1023-1028; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol
22, 2309-2315). Because of the unique structure of sIgA, it is
highly resistant to acid and proteolysis (Lindh E (1975) J Immunol
114, 284-286) and therefore remains intact in secretions to perform
extracellular immunological functions. IgM also binds secretory
component, but not covalently (Lindh E and Bjork I (1976) Eur J
Biochem 62, 271-278). However, IgM is less stabilized because of
its different association with the secretory component, and
therefore has a shorter functional survival time in acidic
secretions (Haneberg B (1974) Scand J Immunol 3, 71-76; Haneberg B
(1974) Scand J Immunol 3, 191-197).
[0031] While the secretion mechanism for IgA and IgM are well
described, conversely, a fundamental question surrounds IgG
secretion. There is no "J" chain present in IgG1 and IgG2. From the
known facts of transcytosis/secretion of immunoglobulins (Johansen
F E et al. (2000) Scand J Immunol 52, 240-248), it is unlikely that
IgG secretion is mediated by the poly-Ig receptor. An epithelial
receptor specific for IgG1 has been reported in bovine mammary
gland (Kemler R et al. (1975) Eur J Immunol 5, 603-608).
Apparently, it preferentially transports this class of
immunoglobulins from serum into colostrum. Despite this 1975 report
however, the receptor has not been chemically or structurally
identified nor has the mechanism of transport of IgG monomers been
satisfactorily defined. It is possible that this receptor is a
member of a large group now designated as Fc receptors (Fridman W H
(1991) FASEB J 5, 2684-2690), but there is one study with IgG
showing that of 31 different long-term human carcinoma cell lines
including breast "all lines were found to be consistently Fc
receptor negative" (Kerbel R S et al. (1997) Int J Cancer 20,
673-679). One possible candidate for the epithelial transport of
IgG1 is the neonatal Fc receptor (Raghavan M and Bjorkman P J
(1996) Annu Rev Cell Dev Biol 12, 181-220). However, there is no
indication yet of the presence of this receptor in adult mucosal
tissues.
[0032] Fc receptors are so named because they bind specific heavy
chains (Fc domains). However, before coming to this conclusion, it
should be emphasized strongly that the Fc family represented by
Fc.gamma. (IgG), Fc.alpha. (IgA), and Fc.mu. (IgM) have
traditionally been considered to be located on lymphoid series
cells (Fridman W H (1991) FASEB J 5, 2684-2690; Raghavan M and
Bjorkman P J (1996) Annu Rev Cell Dev Biol 12, 181-220). There is
only limited experimental support for the concept that these
receptors are located on epithelial cells (Tonder O et al. (1976)
Acta Pathol Microbiol Scand 84, 105-111). For the family of
leukocyte IgG receptors, 12 transmembrane or soluble receptor
isoforms are known. These are grouped into three classes
Fc.gamma.R1 (CD64), Fc.gamma. RII (CD32) and Fc.gamma. RIII (CD16)
(Valerius T et al. (1997) Blood 90, 4485-4492). For IgA, there is
one gene that encodes several receptors) (i.e. Fc.alpha.) by
alternate splicing to yield forms from M.sub.r 55,000 to 110,000
(Pleass R J et al. (1996) Biochem J 318, 771-777; van Dijk T B et
al. (1996) Blood 88, 4229-4238; Morton H C et al. (1996)
Immunogenetics 43, 246-247). One of these, Fc.alpha.R1 is
constitutively expressed on monocytes and macrophages and other
leukocytes. It binds IgA1 and IgA2 with about the same affinity.
The receptor for IgM (i.e. Fc.mu.) is less well defined, but still
has been partially characterized as a M.sub.r 60,000 protein
present on activated B cells and other B series cells (Ohno T et
al. (1990) J Exp Med 172, 1165-1175). The Fe superfamily has
another very important aspect pertinent to this disclosure.
Receptors of this family mediate negative effects on cells (Cambier
J C (1997) Proc Natl Acad Sci USA 94, 5993-5995). These receptors
have an intracellular amino acid sequence motif I/VxYxxL described
as an immunoreceptor tyrosine-based inhibitory motif (ITIM) that
signals cell growth shutdown after ligand binding. These signals
have been characterized in the FC.gamma.RIIB1 receptors of human
and mouse (Olcese L et al. (1996) J Immunol 156, 4531-4534). The
hallmark of these ITIM receptors is that they shut off growth
factor dependent growth.
[0033] Although the advances and teachings in the prior art have
indicated that a serum-borne inhibitor of steroid hormone
responsive tumor cell growth exists, until now there has been no
adequate isolation or identification of such an inhibitor, and very
little understanding of its mode of action has been gained. There
is no satisfactory in vitro testing model presently available for
demonstrating steroid hormone responsive cell growth that can be
correlated to the in vivo situation, or for testing drugs, or other
natural or synthetic substances for possible hormone-mimicking or
anti-hormone effects.
SUMMARY OF THE INVENTION
[0034] The compositions, methods and models of the present
invention overcome major shortcomings of the prior art and satisfy
long-felt needs for, among other things, a sensitive way to screen
substances for estrogenic and androgenic effects. It was
discovered, and the embodiments herein demonstrate, that the immune
system plays a major role in the growth of estrogen responsive
breast and androgen responsive prostate cancers, as well as cancers
of other steroid and thyroid hormone responsive mucosal epithelial
tissues. IgA, IgM and certain IgGs provide negative regulation of
steroid hormone responsive mucosal epithelial cancer cell growth,
including breast, prostate, pituitary, kidney and other glandular
cancer cells. For the purposes of this disclosure, "cell growth"
means cell proliferation or an increase in the size of a population
of cells through mitogenesis and cell division rather than an
increase in cytoplasmic volume of an individual cell. Prior to the
present disclosure, no growth regulating role was known for the
secretory immune system, which produces predominantly
immunoglobulin A (IgA) and immunoglobulin M (IgM) and lesser
amounts of immunoglobulin G (IgG). The discovery that IgA and IgM
are the major negative regulators of steroid hormone responsive
cell growth arose out of the Inventor's work directed at purifying
breast cancer regulatory factors from biological fluids, as
described in the following Examples. This discovery and the present
invention constitute a major breakthrough in the understanding of
these cancers, and other glandular/mucosal tissues that secrete or
are bathed by polymeric IgA, secretory IgA (sIgA), IgM and certain
IgGs. For the first time, a direct link has been established
between the secretory immune system (IgA and IgM) and the most
prevalent types of cancer that occur throughout the world. Binding
of IgA and IgM to the poly-immunoglobulin receptor (i.e. poly-Ig
receptor or poly IgR) is an important step in carrying out the
regulatory function of IgA and IgM, and initial indications are
that poly IgR mediates the negative regulation of steroid hormone
dependent cell growth. Application of these scientific
breakthroughs to the detection, diagnosis, prognosis, treatment and
deterrence or prevention of cancer of mucosal epithelial tissues
(e.g., breast, prostate, kidney, pituitary, thyroid and colon) is
described in U.S. patent application Ser. No. ______ (Atty. Dkt.
No. 1944-00800)/PCT/US2001/______ (Atty. Dkt. No. 1944-00801)
entitled "Compositions and Methods for the Diagnosis, Treatment and
Prevention of Steroid Hormone Responsive Cancers," which is hereby
incorporated herein by reference.
[0035] No such serum-derived inhibitor has been previously isolated
or identified that replicates the large magnitude estrogen
reversible inhibitory effects demonstrated in the present
investigations using hormone depleted full serum. The serum-borne
inhibitor(s) are ubiquitous in mammals and lack species
specificity. Their inhibitory activity is completely reversible by
picomolar concentrations of steroid hormones when assayed in the
new in vitro conditions. Moreover, before the surprising discovery
that the serum-borne inhibitor(s) are secretory immunoglobulins,
there has been no previous report that IgA, IgM or IgG play any
role in the negative regulation of steroid hormone responsive (SHR)
mucosal epithelial cell growth, or that binding of IgA and IgM to a
polyimmunoglobulin receptor (poly-Ig receptor) is instrumental in
carrying out such growth regulation. Prior to the present
disclosure, a cell growth related function for the poly-Ig receptor
transcytosis receptor, or a poly IgR-like receptor, has not been
recognized, nor had such a role ever been attributed to an IgG
Fc.gamma. receptor.
[0036] In accordance with certain embodiments of the present
invention, an immunoglobulin inhibitor of in vitro steroid
hormone-responsive steroid hormone responsive cancer cell growth or
proliferation are provided. The cancer cells that are inhibited
from proliferating in in vitro culture come from a cell line that
is also capable of proliferating in vivo when implanted into a
suitable host. This immunoglobulin inhibitor (e.g., one or more of
the secretory immunoglobulins IgA, IgM and certain IgG subtypes) is
a long sought after serum-derived negative regulator of steroid
hormone responsive cancer cell growth that, in impure form, was
previously referred to as a steroid hormone binding globulin like
("SHBG-like") fraction. For the first time it is disclosed that,
surprisingly, certain immunoglobulins exert a steroid hormone
reversible negative regulatory (inhibitory) effect on cancer cell
growth that is distinct from their well-established immune
functions. In the most preferred embodiments, the inhibitor(s)
is/are dimeric IgA (non-sIgA), polymeric IgM, IgG1.kappa. and
IgG2.
[0037] In some embodiments an isolated steroid hormone reversible
inhibitor of steroid hormone-responsive cancer cell growth is
provided, the inhibitor comprising a secretory immunoglobulin, such
as IgA, IgM or IgG.
[0038] In some embodiments a steroid hormone irreversible cell
growth inhibitor composition is provided that comprises at least
one immunoglobulin inhibitor that is active with respect to the
ability to inhibit steroid hormone-responsive cancer cell
proliferation and inactive with respect to steroid hormone
reversibility of the inhibition, and a carrier.
[0039] In some embodiments a method of making a steroid hormone
irreversible cancer cell growth inhibitor composition comprising
exposing an above-described inhibitor composition to calcium
depleted conditions for a defined period of time sufficient to
render the immunoglobulin irreversibly inhibitory of steroid
hormone responsive cancer cell growth in vitro.
[0040] According to still other embodiments of the present
invention an immunoglobulin inhibitor mimicking substance is
provided. In certain embodiments the mimicking substance is
tamoxifen.
[0041] In certain other embodiments of the present invention a
negative control serum is provided which contains steroid hormone
depleted blood plasma or serum and is inactive with respect to the
ability to inhibit steroid hormone-responsive cell proliferation in
the absence of steroid hormone. Some embodiments of the invention
provide a method of making a negative control serum, preferably
comprising heat treatment at about 50-60.degree. C. for about 90
minutes to about 30 hours.
[0042] Also provided in accordance with certain other embodiments
of the present invention is a control serum composition containing
plasma or serum and containing a reactivatible immunoglobulin
inhibitor that is inactive with respect to the ability to inhibit
steroid hormone-responsive cell proliferation in the absence of the
steroid hormone and in the absence of an activating amount of
calcium. In some embodiments the control serum is reactivated and
contains calcium ion.
[0043] These immunoglobulin inhibitors have many immediate and
potential applications as reagents for cell growth assays and
therapeutic agents. For example, they are useful for in vitro
testing of substances for estrogenic effects (or other steroid
hormone-like effects) on steroid hormone responsive cell growth, in
a suitable assay system. They are useful for demonstrating steroid
hormone reversible inhibition or arrest of cancer cell growth in a
variety of in vitro cell culture models employing cancer cell lines
that are capable of in vivo tumor growth when implanted into a
compatible host. The immunoglobulin inhibitors are also useful as
an aid in assessing risk of cancer development or growth in a
mucosal epithelial tissue (i.e., glands and tissues that secrete or
are bathed by secretory immunoglobulins). Some of these tissues are
breast, prostate, oral cavity mucosa, salivary/parotid glands,
esophagus, stomach, small intestine, colon, tear ducts, nasal
passages, liver and bile ducts, bladder, pancreas, adrenals, kidney
tubules, glomeruli, lungs, ovaries, fallopian tube, uterus, cervix,
vagina, and secretory anterior pituitary gland cells. The
immunoglobulin inhibitors are also expected to be useful in the
detection, diagnosis, prognosis, treatment and prevention of
steroid hormone responsive cancers of the mucosal epithelial
tissues.
[0044] In some embodiments of the present invention, a steroid
hormone reversible, steroid hormone responsive cancer cell growth
inhibitor composition is provided that contains at least one of the
above-described immunoglobulin inhibitors together with a carrier,
which preferably includes an inhibitor stabilizing medium. Such a
composition is especially useful for storing and shipping
preparations of the inhibitors without loss of activity. Preferably
the stabilizing medium also contains an activity-stabilizing amount
of calcium ion, a steroid hormone such as (DHT), and a substance
that depresses the freezing point of the composition below about
-20.degree. C. (e.g., glycerol). In some embodiments the
composition contains steroid hormone depleted body fluid such as
blood plasma or serum.
[0045] In some embodiments of the present invention, an
immunoglobulin inhibitor composition containing steroid-hormone
depleted blood plasma or serum is provided. For many in vitro tests
of steroid hormone responsive cancer cell growth, it is especially
desirable to more closely approximate the in vivo condition by
employing serum-containing assay medium instead of completely
serum-free medium. In some embodiments this steroid hormone
depleted serum-based immunoglobulin inhibitor composition is
supplemented or "spiked" with a predetermined amount of certain
inhibitors (e.g., IgA or IgM). Such serum-containing compositions
are especially useful in assaying for estrogen-like cell growth
stimulating effects by a substance of interest. These serum based
compositions will also facilitate identification of substances that
demonstrate a steroidogenic effect (e.g., estrogen-like stimulation
of cell proliferation) in serum-free cell growth assays, but which
do not demonstrate the same estrogenic effect in the presence of
serum (i.e., when tested in a similar cell growth assay medium that
contains serum.) The ability to determine whether a new drug, or
other substance of interest, is likely to be non-estrogenic in vivo
due to the presence or ameliorating effect of serum factors is of
value to the medical profession and the pharmaceutical industry, in
particular.
[0046] Accordingly, certain embodiments of the present invention
provide methods of testing substances of interest, such as drugs or
environmental chemicals, for their steroid hormone-like effects on
cell growth stimulation employing one of the above-described
immunoglobulin inhibitors or serum-based immunoglobulin inhibitor
compositions with an appropriate steroid hormone responsive cell
line and nutrient medium.
[0047] Certain embodiments of the present invention provide methods
of testing substances of interest, such as drugs or environmental
chemicals, to distinguish cytotoxic effects from anti-estrogenic
effects on cell growth. These methods employ one of the
above-described immunoglobulin inhibitors or serum-based
immunoglobulin inhibitor compositions in an appropriate steroid
hormone responsive cell line maintained in a suitable nutrient
medium.
[0048] In still other embodiments of the present invention a
non-inhibitory steroid hormone depleted serum composition is
provided that contains steroid hormone-depleted blood plasma or
serum, similar to certain of the above-described steroid hormone
depleted serum-based immunoglobulin inhibitor compositions, except
in this embodiment it contains either no immunoglobulin
inhibitor(s) or it contains the immunoglobulin inhibitor(s) in
inactive form with respect to ability of the immunoglobulin(s) to
inhibit steroid hormone responsive cell proliferation in serum-free
cell culture in the absence of a cell growth stimulating amount of
steroid hormone. A non-inhibitory steroid hormone depleted serum
composition is useful for many in vitro testing situations
utilizing serum or plasma, in which the presence of steroid
hormones is undesirable. For example, such a serum composition,
prepared from a mature animal source, may be advantageously
substituted for conventional fetal bovine serum to provide the in
vitro growth promoting factors found in serum without introducing
spurious amounts of steroid hormone.
[0049] In some embodiments, a non-inhibitory steroid hormone
depleted serum composition comprises steroid hormone depleted blood
plasma or serum plus an immunoglobulin inhibitor in a reactivatibly
inactive form with respect to ability of the immunoglobulin to
inhibit steroid hormone responsive cell proliferation in a suitable
cell growth assay absent an inhibition-reversing amount of the
steroid hormone. In certain embodiments the non-inhibitory steroid
hormone depleted serum composition contains less than an inhibitor
activating amount of calcium ion. In certain embodiments an active
immunoglobulin inhibitor containing steroid hormone depleted serum
composition is provided that is in reactivated form and contains an
immunoglobulin inhibitor reactivating amount of calcium ion.
[0050] In accordance with certain other embodiments of the
invention, a method of making a steroid hormone-depleted serum
extract comprising a steroid hormone reversible inhibitor of
steroid hormone responsive cell growth is provided. In some
embodiments the method comprises (a) obtaining a
non-heat-inactivated fresh or frozen serum specimen; (b) performing
a first charcoal-dextran extraction on the specimen at about
30-37.degree. C., preferably 34.degree. C., to yield a first
extract; and (c) performing a second 30-37.degree. C., preferably
34.degree. C., charcoal-dextran extraction on the first extract to
yield a substantially steroid hormone-depleted serum extract.
[0051] In another embodiment of the present invention, an
alternative method of making a substantially steroid
hormone-depleted serum extract comprising a steroid hormone
reversible inhibitor of steroid hormone responsive cell growth is
provided. In certain embodiments this method comprises obtaining a
non-heat-inactivated fresh or frozen serum specimen and performing
an XAD.TM.-4 extraction of the specimen.
[0052] In still another embodiment of the invention, a method of
making a purified immunoglobulin inhibitor of steroid hormone
responsive cancer cell growth is provided. This method includes (a)
obtaining a substantially steroid hormone-depleted serum comprising
an inhibitor of steroid hormone responsive cancer cell growth; (b)
loading the depleted serum onto an agarose-based affinity matrix
and eluting a fraction comprising the inhibitor; (c) loading the
fraction onto a phenyl-Sepharose.TM. matrix and eluting a
substantially purified inhibitor pool with a suitable buffer
containing ethylene glycol; and concentrating the pool to yield a
substantially purified inhibitor.
[0053] Certain embodiments of the present invention provide in
vitro assay methods for detecting steroid hormone-like cell growth
stimulation by a substance of interest. In some embodiments, the
assay method comprises maintaining a predetermined population of
steroid hormone-responsive cells in a nutrient medium comprising a
quantity of an immunoglobulin cell growth inhibitor sufficient to
inhibit cell growth in the absence of an inhibition-reversing
amount of the steroid hormone. In some embodiments the medium is
serum-free and the cells themselves are serum free and obtained
from a stable steroid hormone-responsive cell line. The method also
comprises adding a substance of interest to the cells and medium to
yield a test mixture. The test mixture is then incubated for a
predetermined period of time under cell growth promoting
conditions. "Cell growth promoting conditions" refer to general
environmental conditions, other than defined medium components, and
include such things as favorable conditions of gaseous atmosphere,
temperature and pH. For example, cell growth promoting conditions
could include incubation at 37.degree. C. in a humid atmosphere of
5% (v/v) CO.sub.2 and 95% (v/v) air in a defined nutrient medium at
pH 7.4. After incubation for the desired period of time, it is
determined whether the cell population in the test mixture has
measurably increased, an increase indicating a steroid hormone-like
cell growth stimulating effect by the substance of interest. An
assay procedure such as this can be used for in vitro screening of
drugs or other body-affecting substances for unwanted cell growth
stimulating properties as an aid to avoiding undesirable side
effects of such drug or substance in vivo. In certain alternative
embodiments, the assay method includes adding to the nutrient
medium a defined amount of steroid-hormone depleted serum, which
contains the inhibitor(s), and which is obtained from non-heat
inactivated serum.
[0054] In some embodiments of the assay method, in which the
substance of interest contains or is suspected of containing
proteolytic activity, the method includes selecting an
immunoglobulin inhibitor such as IgA2, which resists protease
degradation.
[0055] In some embodiments of the assay method an inactive
inhibitor-containing control serum is substituted for an active
inhibitor-containing serum, to evaluate a substance of interest for
cytotoxicity.
[0056] In certain embodiments, the assay method comprises an assay
procedure similar to the one previously described except that a
defined amount of inactive immunoglobulin cell growth inhibitor
(i.e., incapable of inhibiting steroid hormone responsive cell
growth in the absence of an inhibition-reversing amount of the
steroid hormone) is substituted for the active (inhibitory)
immunoglobulin inhibitor. In some embodiments a test substance is
included in the test mixture. As assay of this type is particularly
useful for determining a maximum (uninhibited) level of steroid
hormone responsive cell growth stimulation by a test substance.
Alternatively, this type of assay can be used to distinguish
cytotoxic effects of a test substance from anti-estrogen activity,
for example.
[0057] In accordance with certain embodiments of the invention, a
method of detecting a steroid hormone antagonistic substance is
provided. The method comprises (a) maintaining a predetermined
population of steroid hormone responsive cancer cells in a nutrient
medium comprising a quantity of immunoglobulin inhibitor sufficient
to inhibit cell growth in the absence of an inhibition-reversing
amount of the steroid hormone, the cells also being steroid hormone
responsive for in vivo proliferation; (b) adding a defined amount
of the substance of interest to the cells and medium; (c) adding to
the cells and medium a defined amount of steroid hormone sufficient
to stimulate cell growth in the presence of the inhibitor and in
the absence of the substance of interest, to yield a test culture;
(d) incubating the test culture for a predetermined period of time
under cell growth promoting conditions; (e) testing the substance
of interest for cytotoxic effects on the cells; and (f) determining
the cell population in the test culture after the predetermined
period of time, a lack of measurable increase in the cell
population not attributable to cytotoxic effects of the substance
indicating a steroid hormone antagonistic effect by the substance
of interest.
[0058] In accordance with certain embodiments of the invention,
cell culture media are provided that comprise a basal nutrient
fluid, such as D-MEM/F12, and are substantially devoid of unbound
Fe(III), i.e., preferably containing less than 1 .mu.M Fe (III),
and more preferably containing 0.15 .mu.M or less. In certain
preferred embodiments, the amount of free, or active Fe (III) in
the medium is less than a cell growth inhibiting concentration. The
media also contain calcium ion, preferably about 0.6 mM to 1.0 M,
and more preferably about 0.6 to 10 mM calcium. In certain
preferred embodiments, the concentration of calcium ion in the
nutrient medium is preferably sufficient to maintain the inhibitory
activity of any immunoglobulin inhibitors present in the media. In
certain embodiments, a cell culture medium that is especially
suited for use in serum-free cell growth studies also includes a Fe
(III) chelating agent, preferably deferoxamine, and a cell
attachment promoting protein, preferably fibronectin. In certain
preferred embodiments the defined composition medium is DDM-2MF,
CAPM, DDM-2A or PCM-9, the compositions of which are set out in the
Examples below. In preferred embodiments, the cell culture media
comprise 100 ng/mL to 10 .mu.g/mL insulin, 0.3-10 nM
triiodothyronine, 2-50 .mu.g/mL diferric transferrin, 5-100 .mu.M
ethanolamine, 0.2-5.0 mg/mL bovine serum albumin (BSA), 5-20 ng/mL
selenium, 2-10 .mu.M deferoxamine. Depending on the requirements of
the selected cells to be cultures, the medium may also contain at
least one of the following components: 1-50 ng/mL EGF, 0.2-20 ng/mL
aFGF, 5-50 .mu.M phosphoethanolamine, 50-500 m/mL linoleic
acid-BSA, 1-50 .mu.g/mL reduced glutathione, 0.5-2.0 mM glutamine,
1-10 ug/mL heparin, and 20-50 .mu.g (per 35-mm diameter culture
dish) human fibronectin. In some embodiments the cell culture
medium also includes steroid hormone depleted serum.
[0059] According to other embodiments of the present invention an
in vitro method of culturing steroid hormone responsive cancer
cells or autonomous cancer cells is provided. The method comprises
(a) maintaining a predetermined population of steroid hormone
responsive cells or steroid hormone-independent cancer cells in a
steroid hormone-free nutrient medium comprising an above-described
cell culture medium and a quantity of immunoglobulin inhibitor
sufficient to inhibit cell growth of steroid hormone responsive
cancer cells in the absence of an inhibition-reversing amount of
the steroid hormone, to provide an incubation mixture, the steroid
hormone responsive cells also being steroid hormone responsive for
proliferation in vivo when implanted into a suitable host, and the
steroid hormone independent cancer cells also being steroid hormone
independent for proliferation in vivo when implanted into a
suitable host; (b) optionally, adding an inhibition-reversing
amount of the steroid hormone to the incubation mixture; (c)
incubating the incubation mixture under cell growth promoting
conditions; (d) optionally, determining the cell population in the
reaction mixture after incubation for a predetermined period of
time.
[0060] According to certain embodiments of the invention an in
vitro method of detecting a cell growth stimulatory or inhibitory
effect of a substance of interest on steroid hormone independent
cancer cells is provided. The method includes (a) maintaining a
predetermined population of steroid hormone independent cancer
cells in a nutrient medium as described above, optionally, devoid
of the steroid hormone, and, optionally, containing a predetermined
quantity of immunoglobulin inhibitor, the steroid hormone
independent cells also being steroid hormone independent for
proliferation in vivo when implanted into a suitable host; (b)
adding a predetermined quantity of the substance of interest to the
cells and medium to yield a test mixture; (c) incubating the test
mixture for a predetermined period of time under cell growth
promoting conditions; (d) optionally, assessing cytotoxicity of the
substance of interest; and (e) determining the cell population in
the test mixture after the incubation for the predetermined period
of time, a measurable increase in the cell population indicating a
cell growth stimulating effect by the substance of interest, and an
absence of increase in the cell population, not attributable to
cytotoxic effects, indicating a cell growth inhibitory effect by
the substance of interest.
[0061] In accordance with still another embodiment of the present
invention, an in vitro method of detecting an immunoglobulin
inhibitor-like cancer cell growth inhibitory effect by a substance
of interest is provided which comprises (a) maintaining a
predetermined population of steroid hormone responsive cancer cells
in a nutrient medium as described above, optionally, devoid of the
steroid hormone, and, optionally, containing a predetermined
quantity of inactivated immunoglobulin inhibitor, the steroid
hormone responsive cells also being steroid hormone responsive for
proliferation in vivo when implanted into a suitable host; (b)
adding a predetermined quantity of the substance of interest to the
cells and medium to yield a test mixture; (c) adding to the test
mixture an amount of the steroid hormone that would be sufficient
to induce cell growth in the absence of an active immunoglobulin
inhibitor; (d) incubating the test mixture for a predetermined
period of time under cell growth promoting conditions; (e)
optionally, assessing cytotoxicity of the substance of interest;
and (f) determining the cell population in the test mixture after
the predetermined period of time, a measurable increase in the cell
population indicating a lack of cell growth inhibitory effect by
the amount of the substance of interest, and no increase in the
cell population, not attributable to a cytotoxic effect, indicating
a cell growth inhibitory effect by the amount of the substance of
interest.
[0062] In accordance with another embodiment, a method of producing
a quantity of a biomolecule, of interest such as a protein, peptide
or polynucleotide. The method includes, in a serum-free nutrient
medium as described above, culturing a population of cells
expressing the biomolecule of interest, harvesting and recovering
the biomolecule from the medium. In certain preferred embodiments
the protein is a monoclonal antibody.
[0063] In accordance with another embodiment, a method of
propagating a virus of interest is provided which comprises
culturing a population of virus infected cells in an
above-described serum-free nutrient medium, harvesting and
recovering viruses from the medium.
[0064] Further provided in accordance with certain embodiments of
the invention is an assay kit for detecting in vitro steroid
hormone reversible steroid hormone-responsive cell growth by a
substance of interest. In some embodiments such a kit comprises a
serum-free defined nutrient cell culture medium substantially free
of unbound Fe(III) and containing calcium ion. The kit also
contains a substantially steroid hormone-depleted serum comprising
a steroid hormone reversible immunoglobulin inhibitor of steroid
hormone responsive cell growth. In certain preferred embodiments
the extract is prepared by either a double charcoal-dextran
extraction method or the XAD-4.TM. extraction method, described
above. In some embodiments the kit also includes a control serum
composition comprising an inactivated immunoglobulin inhibitor. In
some embodiments the kit also includes a population of cultured
steroid hormone responsive cancer cells that are also steroid
hormone responsive for proliferation in vivo, preferably MTW9/PL2
rat mammary tumor cells.
[0065] In alternative embodiments, assay kits for detecting in
vitro steroid hormone reversible steroid hormone-responsive cell
growth by a substance of interest are provided. In certain
embodiments the kit, which is similar to the one described above,
isolated immunoglobulin inhibitors (e.g., IgA, IgM and/or IgG1) are
included in addition to, or instead of, the serum-based inhibitor
composition(s). Use of this kit will be preferred when the user
requires a totally serum-free assay system. In some situations both
the steroid hormone depleted serum-containing and the serum-free
assay systems are employed in order to detect serum factor effects
or to distinguish the influence of serum on detection of cytotoxic
effects of a chemical, for example.
[0066] In some embodiments the kit also contains other components
such as various steroid hormones, or agonists or antagonists
thereof, that may be desired for adding to the medium in particular
test situations.
[0067] In certain embodiments of the invention in vitro assay
methods for detecting an immunoglobulin inhibitor of steroid
hormone responsive cell growth in a sample of interest, such as a
drug or environmental substance, blood serum or another body fluid,
are provided. In some embodiments the method comprises (a)
maintaining a predetermined population of steroid
hormone-responsive culture cells in a nutrient medium, the cells
also being steroid hormone dependent for proliferation in vivo when
implanted into a suitable host; (b) adding a quantity of steroid
hormone to the medium sufficient to stimulate proliferation of the
cells under cell growth promoting culture conditions; (c) adding a
predetermined quantity of the sample of interest to the medium to
yield a test mixture; (d) incubating the test mixture for a
predetermined period of time under cell growth promoting culture
conditions; (e) optionally, testing the sample for cytotoxic
effects on the cells; and (f) determining the cell population in
the test mixture after the predetermined period of time, a
measurable decrease in the cell population not attributable to
cytotoxic effects indicating inhibition by the amount of sample of
steroid hormone responsive cell growth.
[0068] In some embodiments the assay method also includes adding to
the test mixture an amount of the steroid hormone in excess of the
minimum amount necessary to maximally stimulate proliferation of
the cells; and determining the cell population of the test mixture
after the predetermined period of time, a measurable increase in
the cell population indicating reversal by the excess amount of
steroid hormone of steroid hormone responsive cell growth
inhibition.
[0069] In accordance with still other embodiments of the present
invention, in vitro cell culture models for predicting an in vivo
steroid hormone-responsive cancer cell growth effect of a defined
stimulus, such as an estrogen, an anti-estrogen, androgen, or other
steroid hormone, or a steroid hormone mimicking compound, are
provided. In certain embodiments the model includes steroid
hormone-responsive cancer cells maintained in a growth medium
containing a basal nutrient fluid substantially free of unbound Fe
(III), containing calcium ion, and containing an amount of steroid
hormone reversible immunoglobulin inhibitor sufficient to arrest
cancer cell growth in the absence of an inhibition-reversing amount
of the steroid hormone. The cells are also steroid hormone
responsive for proliferation in vivo, when implanted into a
suitable host. The immunoglobulin inhibitor is chosen from among
IgA, IgM and IgG, and combinations thereof. In some embodiments the
nutrient medium is serum free, and in others it contains steroid
hormone depleted blood plasma or serum. In certain embodiments the
steroid hormone responsive culture cells are MTW9/PL2 (rat mammary
cancer), T47D (human breast carcinoma), MCF-7 (human breast
carcinoma), MCF-7A (human breast carcinoma), MCF-7K (human breast
carcinoma), LNCaP (human prostatic carcinoma), ZR-75-1 (human
prostatic carcinoma), H-301 (Syrian hamster kidney tumor), GH.sub.1
or GH.sub.3 (rat pituitary tumor), GH.sub.4C.sub.1 (rat pituitary
tumor), or HT-29 (human colonic cancer).
[0070] In still other embodiments of the present invention an
isolated estrogen receptor gamma (ER.gamma.) is provided. In
certain embodiments the (ER.gamma.) has an estradiol binding
affinity greater than that of estrogen receptor alpha (ER.alpha.)
or estrogen receptor beta (ER.beta.), preferably having a K.sub.d
for E.sub.2 on the order of >10.sup.-9 M. The ER.gamma. also
preferably has specificity for steroid hormone binding in the order
estradiol>>diethylstilbestrol>>testosterone=dihydrotestostero-
ne, and has a molecular weight of approximately 50 kDa.
[0071] In certain embodiments a mediator of estrogen responsive
cell growth comprises ER.gamma., and in certain embodiments a
mediator of estrogen reversal of immunoglobulin inhibition of
estrogen responsive cell growth comprises ER.gamma..
[0072] Also provided by the present invention are methods of
detecting an estrogenic substance. According to certain
embodiments, the method comprises (a) maintaining a predetermined
population of estrogen responsive cancer cells in a steroid
hormone-free nutrient medium comprising a quantity of
immunoglobulin inhibitor sufficient to inhibit cancer cell growth
in the absence of an inhibition-reversing amount of estrogen, the
cells also being estrogen responsive for proliferation in vivo when
implanted into a suitable host; (b) adding a defined amount of the
substance of interest to the cells and medium, to yield a test
culture; (c) incubating the test culture for a predetermined period
of time under cell growth promoting conditions; and (d) determining
the cell population in the test culture after the predetermined
period of time, a measurable increase in the cell population
indicating an estrogen-like cell growth stimulating effect by the
substance of interest. In some embodiment the method also includes
testing the substance of interest for binding to estrogen receptor
gamma and/or testing for cytotoxic effects. In certain embodiments,
the method includes selecting estrogen responsive cancer cells
containing estrogen receptor gamma.
[0073] Also provided by the present invention are methods of
detecting an anti-estrogenic substance, such as an antagonist.
According to certain embodiments, the method comprises (a)
maintaining a predetermined population of estrogen responsive
cancer cells in a nutrient medium comprising a quantity of
immunoglobulin inhibitor sufficient to inhibit cell growth in the
absence of an inhibition-reversing amount of estrogen, the cells
being capable of growing in vivo; (b) adding a defined amount of
the substance of interest to the cells and medium; (c) adding a
defined amount of an estrogen sufficient to stimulate cell growth
in the presence of the inhibitor and in the absence of the
substance of interest to the cells and medium, to yield a test
culture; (d) incubating the test culture for a predetermined period
of time under cell growth promoting conditions; (e) testing the
substance of interest for cytotoxic effects on the cells; and (f)
determining the cell population in the test culture after the
predetermined period of time, a lack of measurable increase in the
cell population not attributable to cytotoxic effects of the
substance indicating a steroid hormone antagonistic effect by the
substance of interest. In some embodiments the method also includes
testing the substance of interest for binding to estrogen receptor
gamma and/or testing for cytotoxic effects. In certain embodiments,
the method includes selecting estrogen responsive cancer cells
containing estrogen receptor gamma.
[0074] Also provided in accordance with the present invention are
methods of identifying an estrogen responsive cell that is capable
of being inhibited or prevented from proliferating by an estrogen
reversible inhibitor of estrogen responsive cell growth. In certain
embodiments the method comprises detecting estrogen receptor gamma
in the cell.
[0075] According to the present invention, methods of inhibiting in
vitro cancer cell growth are provided. In certain embodiments the
method comprises (a) maintaining a predetermined population of
cancer cells in an above-described nutrient medium; (b) adding an
effective amount of an iron compound to the medium, to provide an
incubation mixture comprising unbound Fe (III), preferably at least
about 1 .mu.M Fe (III); (c) incubating the incubation mixture for a
predetermined period of time under cell growth promoting
conditions; and (d) determining the cell population in the
incubation mixture after the predetermined period of time, an
increase in cell population indicating lack of inhibition by the Fe
(III), and the absence of an increase in cell population indicating
inhibition of cell growth by the Fe (III).
[0076] In certain embodiments of the present invention, a method of
killing cancer cells in vitro is provided. In some of those
embodiments a concentration of at least about 10 .mu.M unbound Fe
(III) is maintained in the nutrient medium. Alternatively, extended
arrest of cancer cell growth by an immunoglobulin inhibitor can
also serve to kill steroid hormone responsive cancer cells in
culture.
[0077] Accordingly, in certain embodiments of the present
invention, a method of killing steroid hormone responsive cancer
cells in culture is provided which comprises (a) combining a
predetermined population of steroid hormone responsive cancer cells
with a nutrient medium comprising an above-described cell culture
medium and a quantity of steroid hormone irreversible
immunoglobulin inhibitor sufficient to inhibit cell growth of
steroid hormone responsive cancer cells, to provide an incubation
mixture, the steroid hormone responsive cells also being steroid
hormone responsive for proliferation in vivo when implanted into a
suitable host; (b) incubating the incubation mixture for a
predetermined period of time under cell growth promoting
conditions; and (c) optionally, determining the cell population in
the reaction mixture after the incubation for the predetermined
period of time. In some embodiments the immunoglobulin inhibitor is
irreversibly, or permanently inhibitory (i.e., the inhibitor is
active with respect to the ability to inhibit steroid
hormone-responsive cell proliferation and inactive with respect to
steroid hormone reversibility of the inhibition.)
[0078] Another embodiment of the present invention provides a
method of killing a mixed population of steroid hormone responsive
cancer cells and autonomous cancer cells. The method comprises
contacting the mixed population of cells with an amount of an iron
depleting substance sufficient to substantially deprive the
autonomous cells of Fe (III), and then maintaining the cells in an
iron depleted environment for a sufficient period of time for the
autonomous cells to die. The method also includes contacting the
mixed population of cells with an amount of a Fe (III) containing
substance sufficient to inhibit cell growth and/or kill the steroid
hormone responsive cells, and then maintaining the cells in a Fe
(III)-enhanced environment for a predetermined period of time
sufficient to inhibit cell growth and/or kill the steroid hormone
responsive cancer cells. In certain embodiments, the method also
includes contacting the mixed population of cells with an amount of
immunoglobulin inhibitor sufficient to inhibit proliferation of the
steroid hormone responsive cells.
[0079] Still other embodiments provided by the present invention
are methods of determining the concentration of a steroid hormone
in a defined amount of a body fluid. In certain embodiments the
method comprises assaying the body fluid for binding of steroid
hormone to an immunoglobulin inhibitor of steroid hormone
responsive cancer cell growth.
[0080] These and other embodiments, features and advantages of the
present invention will become apparent with reference to the
following description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] For the detailed descriptions of the preferred embodiments,
reference will now be made to the accompanying figures which
include graphs, charts, and test results:
[0082] FIG. 1. Effect of Temperature on .sup.3H-E.sub.2 Binding to
MTW9/PL2 Cells. The kinetics are shown (SD of triplicates) at 37 C
(closed circles), 23 C (open circles), and at 4 C (open triangles).
Specific binding was determined in phenol red-free D-MEM/F-12. Each
assay sample contained 300,000 CPM and 1.0.times.10 6 cells.
[0083] FIG. 2. Scatchard Analysis and Saturation Binding Analysis
of .sup.3H-E.sub.2 Binding to MTW9/PL2 Cells. Scatchard analysis of
H-E binding (closed circles) was conducted using the traditional
method with labeled-unlabeled mixtures of hormone and DES (100-fold
excess) over the concentration range 37 .mu.M to 5.0 nM H-E. In
both experiments, 5 nM H-E was 300,000 DPM. Each assay sample
contained 1.0.times.10 cells. INSERT: The insert shows a separate
experiment in which the effect of H-E concentration was measured on
specific binding (DPM) after 2 h at 37 C in phenol red-free
D-MEM/F-12.
[0084] FIG. 3. Effect of Unlabeled Competitor Steroids on
.sup.3H-E.sub.2 Binding to MTW9/PL2 Cells. (A) Competition with
Unlabeled Androgens; shows the effects of unlabeled DES (open
triangles), unlabeled DHT (open circles), and unlabeled T (closed
circles). (B) Competition with Unlabeled Progesterone and Cortisol.
shows the effects of unlabeled DES (open triangles), unlabeled
progesterone (open circles), and unlabeled cortisol (closed
circles).
[0085] FIG. 4. Effect of Temperature on .sup.3H-Progesterone
Binding to MTW9/PL2 Cells. The kinetics are shown (SD of
triplicates) at 37 C (closed circles), 23 C (open circles), and at
4 C (open triangles). Specific binding was determined in phenol
red-free D-MEM/F-12. Each assay sample contained 287,000 CPM
H-progesterone
[0086] FIG. 5. Scatchard Analysis and Saturation Binding Analysis
of .sup.3H-Progesterone to MTW9/PL2 Cells. A Scatchard analysis of
3H-progesterone binding (closed circles) was conducted using the
traditional method with labeled-unlabeled mixtures of hormone and
R5020 (100 fold excess) over the concentration range 37 .mu.M to
5.0 nM 3H-progesterone. In both experiments, 5.0 nM 3H-progesterone
was 287,000 CPM. Each assay sample contained 1.0.times.10.sup.6
cells. INSERT: The insert shows a separate experiment in which the
effect of 3H-progesterone concentration was measured on specific
binding (bound dpm) after 2 h at 37.degree. C. in phenol redfree
D-MEM/F-12.
[0087] FIG. 6. Effect of Unlabeled Competitor Steroids on
.sup.3H-Progesterone Binding to MTW9/PL2 Cells. The cells were
incubated at 37 C for 2 h in the presence of 5 nM H-progesterone
(287,000 CPM) alone or in the presence of the labeled hormone plus
the designated fold excess (M) of unlabeled R5020 (open triangles),
unlabeled DHT (open circles), or unlabeled T (closed circles). Each
assay sample contained 1.0.times.10.sup.6 cells.
[0088] FIG. 7. Estrogen Induction of Progesterone Receptors with
MTW9/PL2 Cells. Each specific binding presented is the average of
triplicate incubations.+-.SD (closed circles). INSERT: The insert
shows the effect of E2 concentration in the culture medium for 2 d
prior to the assay of progesterone receptors (open circles).
[0089] FIG. 8. Western immunoblotting Analysis of Androgen,
Progesterone and Estrogen Receptors in MTW9/PL2 cells. Lanes 1
through 8 contain 10 .mu.g of the following cell extract protein:
Lanes 1 and 2, cytosolic extracts of rat and human fibroblasts,
respectively; Lanes 3 and 4, cytosolic and nuclear extracts,
respectively, of MTW9/PL2 Cells; Lanes 5 and 6, cytosolic and
nuclear extracts, respectively, of 47D Cells; Lanes 7 and 8,
cytosolic and nuclear extracts, respectively, of LNCaP cells. Top,
Middle and Bottom Panels are Androgen, Progesterone and Estrogen
receptors, respectively.
[0090] FIG. 9. CDE-horse Serum Effect on MTW9/PL2 Cell Growth.+-.10
nM E.sub.2 for 7 days. (A) Dose-response data expressed as cell
numbers; data expressed as cell number after 7 days Growth with
1.0.times.10 M E (closed circles) and without hormone (open
circles) in medium containing the designated concentrations of
serum. (B) Dose-response data expressed as cell population
doublings (CPD) per 7 days data in (A) expressed as CPO The symbols
indicate the same conditions as (A) except the open triangles show
CPO differences between growth in dishes with and without the
hormone (Difference=estrogenic effect on growth.
[0091] FIG. 10. Restoration of Growth by Addition of 10 nM E.sub.2
on days 0, 2, 4 and 6 After Seeding the MTW9/PL2 cells into Fully
Inhibitory Medium Containing 50% (v/v) of CDE-horse serum.
[0092] FIG. 11. Dose-Response Effects of Steroid Hormones on Growth
of the MTW9/PL2 Cells in Medium Containing 50% (v/v) CDE-horse
Serum.
[0093] FIG. 12. MTW9/PL2 Cell Growth.+-.E.sub.2 in Medium with CDE
Sera from Several Species. (A) CDE-porcine Serum; (B) CDE-pregnant
Human Serum; (C) CDE-adult Rat Serum; (D) CDE-adult Bovine Serum;
(E) CDE-fetal Bovine Serum; (F) CDE-fetal Horse Serum.
[0094] FIG. 13. CDE-horse Serum Effect on GH.sub.4C.sub.1 Cell
Growth.+-.10 nM E.sub.2 for 10 days.
[0095] FIG. 14. CDE-horse Serum Effect on ZR-75-1 Cell Growth.+-.10
nM E.sub.2 for 14 days.
[0096] FIG. 15. CDE-horse Serum Effect on MCF-7A Cell Growth.+-.10
nM E.sub.2 for 10 days.
[0097] FIG. 16. Kinetics of T47D Cell Growth in CDE-horse
Serum.+-.10 nM E.sub.2. (A) Growth Kinetics in 20%
CDE-horse.+-.E.sub.2 versus 10% Fetal Bovine Serum; The growth of
the cells in medium with 20% (v/v) serum with 10 nM E.sub.2 (closed
circles) and without the steroid (open circles). As comparison,
growth is shown in medium containing 10% (v/v) FBS (triangles). (B)
Growth Kinetics in 50% CDE-horse Serum.+-.E.sub.2. T47D cell growth
kinetics in medium with 50% (v/v) serum with E.sub.2 (closed
circles) and without the steroid (open circles).
[0098] FIG. 17. Rodent and Human ER.sup.+ Cell Growth in 50%
CDE-human Serum.+-.E.sub.2. (A) T47D Human Breast Cancer Cells; (B)
LNCaP Human Prostate Cancer Cells; (C) MTW9/PL2 Rat Mammary Tumor
Cells; (D) GH.sub.3 Rat Pituitary Tumor Cells; (E) GH.sub.4C.sub.1
Rat Pituitary Tumor Cells; (F) H301 Syrian Hamster Kidney Tumor
Cells.
[0099] FIG. 18. Dose-Response of Steroid Hormones with T47D Cells
in 50% CDE-horse Serum.
[0100] FIG. 19. Dose-Response of Steroid Hormones with
GH.sub.4C.sub.1 Cells in 50% CDE-horse Serum.
[0101] FIG. 20. Dose-Response of Steroid Hormones with H301 Cells
in 50% CDE-horse Serum.
[0102] FIG. 21. Dose-Response of Steroid Hormones with LNCaP Cells
in 50% CDE-horse Serum.
[0103] FIG. 22. T.sub.3 Growth Effects with GH.sub.3 Cells in
Serum-free Medium (PCM).
[0104] FIG. 23. E.sub.2 Growth Effects with GH.sub.3 Cells in
Serum-free Medium (PCM) Minus E.sub.2.
[0105] FIG. 24. T.sub.3 Growth Effects with Three GH Cell Lines in
2.5% CDE-horse Serum.
[0106] FIG. 25. T.sub.3 Growth Effects with Two GH Cell Lines in
50% CDE-horse Serum.
[0107] FIG. 26. Effect of 56.degree. C. Versus 34.degree. C.
CDE-horse Serum on MTW9/PL2 Cell Growth. FILLED BARS: Estrogenic
effic in 34.degree. C. prepared CDE-serum DARK HATCHED BARS:
56.degree. C. prepared CDE-serum LIGHT SHADED BARS: Charcoal
extracted 34.degree. C. then charcoal extraction at 56.degree. C.
LIGHT HATCHED BARS: Charcoal extracted at 34.degree. C. then
Incubation for 20 min at 56.degree. C. INSERT: Dose-response growth
effects of horse serum extracted at 34.degree. C. followed by
incubation for 20 min at 56.degree. C. Open circles--Growth without
E.sub.2 Closed Circles--Growth with 1.0.times.10.sup.-8 E.sub.2
Triangle--Estrogenic effect
[0108] FIG. 27. Effect of XAD-4 Resin Treated Horse Serum on
MTW9/PL2 Cell Growth.+-.E.sub.2.
[0109] FIG. 28. Effect of XAD-4 Resin Treated Horse Serum on T47D
Cell Growth.+-.E.sub.2.
[0110] FIG. 29. Effect of Phenol Red on Estrogen Responsive MCF-7
Cell Growth. (A) MCF-7A Cell Growth in CDE-horse Serum.+-.Phenol
Red and .+-.E.sub.2; MCF-7A cell growth in phenol red containing
medium with E (closed circles) and without E.sub.2 (closed
triangles), and in phenol red-free medium with E.sub.2 (open
circles) and without E.sub.2 (open triangles). (B) Estrogenic
Effects with MCF-7A Cells.+-.Phenol Red; Estrogenic effects with
MCF-7A cells in medium with phenol red (solid bars) and without
phenol red (shaded bars) were calculated from (A) and defined as
the CPD in medium containing E.sub.2 minus the CPD in medium
without added E.sub.2. (C) MCF-7K Cell Growth in CDE-horse
Serum.+-.Phenol Red and .+-.E.sub.2; MCF-7K cell growth in phenol
red medium with E (closed circles) and without E (closed
triangles), and in phenol red-free medium with E (open circles) and
without E.sub.2 (open triangles). (D) Estrogenic Effects with
MCF-7K Cells.+-.Phenol Red. Estrogenic effects with MCF-7K cells in
medium with phenol red (solid bars) and without phenol red (shaded
bars), calculated from (C).
[0111] FIG. 30. Effect of Phenol Red on Estrogen Responsive T47D
and ZR-75-1 Cell Growth. (A) T47D Cell Growth in CDE-horse
Serum.+-.Phenol Red and .+-.E.sub.2; T47D cell growth in phenol red
containing medium with E.sub.2 (closed circles) and without E.sub.2
(closed triangles), and in phenol red-free medium with E.sub.2
(open circles) and without E.sub.2 (open triangles). (B) Estrogenic
Effects with T47D Cells.+-.Phenol Red; Estrogenic effects with T47D
cells in medium with phenol red (solid bars) and without phenol red
(shaded bars) were calculated from (A) and defined as the CPD in
medium containing E.sub.2 minus the CPD in medium without added
E.sub.2. (C) ZR-75-1 Cell Growth in CDE-horse Serum.+-.Phenol Red
and .+-.E.sub.2; ZR-75-1 cell growth in phenol red medium with
E.sub.2 (closed circles) and without E.sub.2 (closed triangles),
and in phenol red-free medium with E.sub.2 (open circles) and
without E.sub.2 (open triangles). (D) Estrogenic Effects with
ZR-75-1 Cells.+-.Phenol Red. Estrogenic effects with ZR-75-1 cells
in medium with phenol red (solid bars) and without phenol red
(shaded bars), calculated from (C).
[0112] FIG. 31. Effect of Phenol Red on Estrogen Responsive
MTW9/PL2 Cell Growth. (A) MTW9/PL2 Cell Growth in CDE-horse
Serum.+-.Phenol Red and .+-.E.sub.2; MTW9/PL2 growth in phenol red
medium with E.sub.2 (closed circles) and without E.sub.2 (closed
triangles), and in phenol red-free medium with E.sub.2 (open
circles) and without E.sub.2 (open triangles). (B) Estrogenic
Effects with MTW9/PL2 Cells.+-.Phenol Red. Estrogenic effects with
MTW9/PL2 cells in medium with phenol red (solid bars) and without
(shaded bars) were calculated from (A).
[0113] FIG. 32. Dose-Response Effects of Phenol Red versus E.sub.2
with Three ER.sup.+ Cell Lines. (A) Growth Effects of Phenol Red
with MCF-7K, T47D and MTW9/PL2 Cells; (B) Growth Effects of E.sub.2
with MCF-7K, T47D and MTW9/PL2 Cells.
[0114] FIG. 33. Estrogen Induction of Progesterone Receptors by
Phenol Red versus E.sub.2. (A) Induction by E.sub.2 with T47D
Cells. The effects of E.sub.2 at 1.0.times.10.sup.-8 M (closed
circles), 1.0.times.10.sup.10 M (open circles),
1.0.times.10.sup.-12 M (closed triangles), 1.0.times.10.sup.-14 M
(open triangles) and the control without added E.sub.2 (open
squares). (B) Induction by Phenol Red with T47D Cells. The effects
of phenol red at 16 mg/L (closed circles), 8 mg/L (open circles), 4
mg/L (closed triangles), 2 mg/L (open triangles), and the control
without phenol red (open squares).
[0115] FIG. 34. Effects of TGF.beta.1 on Cell Growth in 2.5%
CDE-horse Serum.+-.E.sub.2. (A) MCF-7K Cell Growth. The effect of
the transforming growth inhibitor on human breast MCF-7K cell
growth as measured after 12 d either with 10 nM E.sub.2 (closed
circles) or without the hormone (open circles). The insert shows
conversion of the cell number results to CPD. (B) MTW9/PL2 Cell
Growth. The same experiment with rat mammary MTW9/PL2 cells after 9
d growth.
[0116] FIG. 35. TGF.beta.1 Inhibition of ER.sup.+ Rodent and Human
Cell Line Growth.+-.E.sub.2. (A) Inhibition Data.+-.E.sub.2
Presented in Cell Number. The effect of TFG-bestal on five cell
lines after 10-14 d growth in medium.+-.E.sub.2. The results are
expressed as cell number decreases caused by TGF-beta1. In these
studies, TGF-besta1 was added at 40 ng/ml. Estradiol (.+-.E.sub.2)
indicates either no added E.sub.2 or the sertiod at 10 nM. (B)
Inhibition Data.+-.B.sub.2 Presented in CPD. The CPD decreases
caused by TGF-beta1.+-.E.sub.2 with each of the cell lines shown in
(A).
[0117] FIG. 36. EGF and TGF.alpha. as Substitutes for the Effects
of E.sub.2 in CDE-horse Serum. (A) MCF-7A Cell Growth; (B) MCF-7K
Cell Growth; (C) T47D Cell Growth; (D) ZR-75-1 Cell Growth. The
cells were grown in D-MEM/F-12 supplemented with increasing
concentrations of CDE horse serum. Each line tested was grown in
serum alone (open circles) and in serum plus 50 ng/ml EGF (open
triangles), 50 ng/ml TGF-alpha (closed triangles), or 10 nM E2
without exogenous growth factors (closed circles). (A)-(D) show the
results with the MCF-7A, MCF-7K, T47D, and ZR-75-1 cell lines,
respectively.
[0118] FIG. 37. IGF-I as a Substitute for the Effects of E.sub.2 in
CDE-horse Serum. (A) MCF-7K Cell Growth MCF-7A Cell Growth; (B)
T47D Cell Growth. Breast cancer cells were grown in D-MEM/F-12
supplemented with increasing concentrations of CDE horse serum.
Each cell line tested was grown in serum alone (open circles) and
in serum plus 1.0 ug/ml IGF-1 (triangles), or in serum with 10 nM E
without exogenous growth factors (closed circles). (A)-(C) show the
results with the MCF-7K, MCF-7A and T47D cells, respectively.
Assays were conducted for 12-14 d.
[0119] FIG. 38. Growth of T47D Human Breast Cancer Cells in
Standard and "low-Fe" D-MEM/F-12.
[0120] FIG. 39. Growth of LNCaP Human Prostate Cancer Cells in
Standard and "low-Fe" D-MEM/F-12.
[0121] FIG. 40. Growth of MDCK Dog Kidney Tubule Cells in Standard
and "low-Fe" D-MEM/F-12.
[0122] FIG. 41. Growth of AR.sup.+ LNCaP Cells in CAPM.+-.DHT
versus Growth in D-MEM/F-12 Containing 10% Fetal Bovine Serum.
[0123] FIG. 42 Growth of the AR.sup.- DU145 and AR.sup.- PC3 Cells
in CAPM versus Growth in D-MEM/F-12 Containing 10% Fetal Bovine
Serum.
[0124] FIG. 43. Dose-Response Effects of Individual Components of
CAPM Serum-free Defined Medium on LNCaP Cell Growth.
[0125] FIG. 44. Effects of Deletion of Individual Components from
CAPM Serum-free Medium on LNCaP, DU145 and PC3 Cell
Growth.+-.DHT.
[0126] FIG. 45. Effect of Fe (III) on MCF-7A Cell Growth in DDM-2MF
Serum-free Defined Medium.
[0127] FIG. 46. Effect of Fe (III) on T47D Cell Growth in DDM-2MF
Serum-free Defined Medium.
[0128] FIG. 47. Effect of Fe (III) on LNCaP Cell Growth in CAPM
Plus Apotransferrin.
[0129] FIG. 48. Comparative Effect of Fe (III) on LNCaP, DU145 and
PC3 Cell Growth in CAPM.
[0130] FIG. 49. Growth Restoring Effect of Fe (III) Chelators in
serum-free medium with T47D Cells.
[0131] FIG. 50. Growth Restoring Effect of Fe (III) Chelators in
serum-free medium with LNCaP Cells
[0132] FIG. 51. Comparison of DU145 Cell Growth in "low-Fe" and
"standard" D-MEM/F-12 Based Serum-free Defined Medium CAPM.
[0133] FIG. 52. Comparison of PC3 Cell Growth in "low-Fe" and
"standard" D-MEM/F-12 Based Serum-free Defined Medium CAPM.
[0134] FIG. 53. Growth of the DU145 Cells in CDE-horse
Serum.+-.DHT.
[0135] FIG. 54. Growth of the PC3 Cells in CDE-horse
Serum.+-.DHT.
[0136] FIG. 55. Growth of the ALVA-41 Cells in CDE-horse
Serum.+-.DHT.
[0137] FIG. 56. Comparison of Estrogenic Effects in Serum-free
Defined Medium and in D-MEM/F-12 Medium Supplemented with CDE-Horse
Serum. (A) MCF-7K Cell Growth in Serum-free Defined
Medium.+-.E.sub.2; (B) MCF-7K Cell Growth in D-MEM/F-12 with
CDE-horse Serum.+-.E.sub.2; (C) T47D Cell Growth in Serum-free
Defined Medium.+-.E.sub.2; (D) T47D Cell Growth in D-MEM/F-12 with
CDE-horse Serum.+-.E.sub.2; (E) LNCaP Cell Growth in Serum-free
Defined Medium.+-.E.sub.2; (F) LNCaP Cell Growth in D-MEM/F-12 with
CDE-horse Serum.+-.E.sub.2. The cells were grown in serum-free
defined medium and in D-MEM/F-12 supplemented with increasing
concentrations of CDE horse serum. (A) MCF-7K cell growth was
measured daily in serum-free defined DDM-2MF with 10 nM E.sub.2
(closed circles) and without steroid (open circles) E.sub.2.
Triangles=estrogenic effect. (B) MCF-7K cell growth measured after
12 d in D-MEM-F-12 supplemented with designated concentrations of
serum with) E.sub.2 (closed circles) and without steroid (open
circles). The estrogenic effect is shown by triangles. (C) and (D)
show the same experiments as in (A) and (B), respectively, except
the T47D cells. (E) and (F) show the same experiments as in (A) and
(B), respectively, except with LNCaP cells. In (E) the serum-free
medium was CAPM.
[0138] FIG. 57. Comparison of Estrogenic Effects in Serum-free
Defined Medium and in D-MEM/F-12 Medium Supplemented with CDE-Horse
Serum. Comparison of the effects of estrogen on steroid
hormone-responsive rodent tumor cell growth in serum-free defined
medium and in D-MEM/F-12 supplemented with increasing
concentrations of CDE horse serum. (A) GH.sub.4C.sub.1 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2; GH4C1 rat pituitary tumor
cell growth measured daily in serum-free PCM-9 with E2 (closed
circle) and without E2 (open circles). The estrogenic effect is
shown by triangles. (B) GH.sub.4C.sub.1 Cell Growth in D-MEM/F-12
with CDE-horse Serum.+-.E.sub.2; GH4C1 cell growth measured after 9
d in D-MEM-F-12 supplemented with the designated concentrations of
CDE horse serum with E2 (closed circles) and without E2 (open
circles). The estrogenic effect is shown by triangles. (C) MTW9/PL2
Cell Growth in Serum-free Defined Medium.+-.E.sub.2; (D) MTW9/PL2
Cell Growth in D-MEM/F-12 with CDE-horse Serum.+-.E.sub.2; (C) and
(D) show the same experiments as in (A) and (B) respectively, but
with the MTW9/PL2 rat mammary tumor cells. The serum-free medium in
(D) was DDM-2A. (E) H301 Cell Growth in Serum-free Defined
Medium.+-.E.sub.2; (F) H301 Cell Growth in D-MEM/F-12 with
CDE-horse Serum.+-.E.sub.2. (E) and (F) show the same experiments
as in (A) and (B), respectively, except with the H-301 hamster
kidney tumor cells. In (E) the serum-free medium was CAPM.
[0139] FIG. 58. Effect of CDE-horse Serum on LNCaP Cell Growth in
Serum-free CAPM.+-.E.sub.2 and .+-.DHT.
[0140] FIG. 59. Comparison of the Inhibitor Reversing Effects of
DHT, E.sub.2, and DES on LNCaP Cell Growth in CDE-horse Serum
Containing Medium. (A) Effect of DHT as an Inhibitor Reversing
Steroid; (B) Effect of E.sub.2 as an Inhibitor Reversing Steroid;
(C) Effect of DES as an Inhibitor Reversing Steroid; (D) Effect of
Combinations of DHT, E.sub.2, and DES as Inhibitor Reversing
Steroids.
[0141] FIG. 60. Effect of Tris Buffer (pH 7.4) Dialysis on the
Estrogen Reversible Inhibitor Activity of CDE-horse Serum Assayed
with MTW9/PL2 Cell.+-.E.sub.2.
[0142] FIG. 61. Ultrafiltration of CDE-horse Serum and Assay of the
Filtrate and Retentate with MTW9/PL2 Cells.+-.E.sub.2.
[0143] FIG. 62. 50.degree. C. Treatment of CDE-horse Serum for 30
minutes and Assay with MTW9/PL2 Cells.+-.E.sub.2.
[0144] FIG. 63. Time Course of Heat Treatment of CCDE-horse serum
at 50.degree. C. and Measurement of Estrogenic Effects with
MTW9/PL2 Cells.
[0145] FIG. 64. 50.degree. C. Treatment of CDE-horse Serum for 20
hours and Assay with MTW9/PL2 Cells.+-.E.sub.2.
[0146] FIG. 65. 60.degree. C. Treatment of CDE-horse Serum for 90
minutes and Assay with MTW9/PL2 Cells.+-.E2.
[0147] FIG. 66. Affi-Gel Blue Treatment of CDE-horse Serum and
Assay with MTW9/PL2 Cells.+-.E.sub.2.
[0148] FIG. 67. Effect of 6 M Urea on the Estrogenic Activity of
CDE-horse Serum Assayed with MTW9/PL2 Cells.+-.E.sub.2.
[0149] FIG. 68. ED.sub.50 Estimations for Purification
Quantification of Beginning with Serum. (A) MCF-7K Cells ED.sub.50
of CDE-horse Serum.+-.E.sub.2; (B) ZR-75-1 Cells ED.sub.50 of
CDE-horse Serum.+-.E.sub.2; (C) MTW9/PL2 Cells ED.sub.50 of
CDE-horse Serum.+-.E.sub.2; (D) GH.sub.4C.sub.1 Cells ED.sub.50 of
CDE-horse Serum.+-.E.sub.2.
[0150] FIG. 69. Assay of Estrogenic Activity (ED.sub.50) of
Chromatographic Pools. (A) Ammonium Sulfate Active Fraction; (B)
Affi-Gel BlueGel Albumin Rich Fraction; (C) DEAE Sepharose Pool IV
Active Fraction Assay #1; (D) DEAE Sepharose Pool IV Active
Fraction Assay #2.
[0151] FIG. 70. Assay of Affi-Gel BlueGel By-Pass
Fraction.+-.E.sub.2.
[0152] FIG. 71. DEAE Sepharose Chromatography Elution Profile with
Whole CDE-horse Serum.
[0153] FIG. 72. Phenyl Sepharose Chromatography Elution Profile
with DEAE Sepharose Pool IV.
[0154] FIG. 73. HTP Bio-Gel (hydroxylapatite) Elution Profile with
DEAE Sepharose Pool IV.
[0155] FIG. 74. MTW9/PL2 Cell Assay of CDE-horse Serum Estrogenic
Activity after Dialysis in Tris-HCl, pH 7.4, plus 50 mM
CaCl.sub.2.
[0156] FIG. 75. Effect of Calcium on the 50.degree. C. Heat
Stability of the Estrogenic Activity in Chelex Treated CDE-horse
Serum Assayed with MTW9/PL2 Cells. =Chelex treatment only =CDE
horse serum =Chelex and 1 mM calcium chloride =Chelex and 10 mM
calcium chloride =Chelex and 50 mM calcium chloride
[0157] FIG. 76. Effect of Zn, Mn, Mg and Ca on the 37.degree. C.
Heat Stability of the Estrogenic Activity in Chelex Treated
CDE-horse Serum Assayed with MTW9/PL2 Cells. Chelex treated serum
Chelex treated serum+10 mM Calcium Chelex treated serum+50 uM
Manganese Chelex treated serum+100 uM Magnesium Chelex treated
serum+10 uM Zinc
[0158] FIG. 77. Binding Affinity (K.sub.d) of .sup.3H-DHT to
CDE-horse Serum.
[0159] FIG. 78. Calcium Protection of both the Estrogenic Effect
with MTW9/PL2 Cell and the Binding of .sup.3H-DHT with Chelex
Treated CDE-horse Serum. (A) Estrogenic Effect Protection by
Calcium; (B) Calcium Protection of .sup.3H-DHT Binding.
[0160] FIG. 79. Immunoprecipitation of .sup.3H-DHT Binding and
Estrogenic Activity of CDE-horse Serum by Anti-Human SHBG. (A)
.sup.3H-DHT Binding Reduction; (B) Estrogenic Activity
Reduction.
[0161] FIG. 80. Column Elution Profiles of the Two-step Cortisol
Affinity and Phenyl Sepharose Elution of CA-PA-pool I and
CA-PS-pool II.
[0162] FIG. 81. Identification of the Molecular Forms Present in
Active CA-PS-pool II. (A) SDS-PAGE with Coomassie Blue Staining;
(B) Western Analysis with Anti-human SHBG.
[0163] FIG. 82. CA-PS-pool II Effect on ER.sup.+ Cell Growth in
2.5% CDE-horse Serum.+-.E.sub.2. (A) GH.sub.1 Cells; (B) GH.sub.3
Cells; (C) GH.sub.4C.sub.1 Cells; (D) H301 Cells; (E) MTW9/PL2
Cells; (F) MCF-7K Cells; (G) ZR-75-1 Cells; (H) T47D Cells.
[0164] FIG. 83. Cortisol Affinity Column Depletion of the
Estrogenic Activity in CDE-horse Serum Assayed with ER.sup.+ Cell
Lines.+-.E.sub.2. (A) T47D Cells Pre-Column; (B) T47D Cells
Post-Column; (C) GH.sub.3 Cells Pre-Column; (D) GH.sub.3 Cells
Pre-Column; (E) H301 Cells Pre-Column; (F) H301 Cells
Post-Column.
[0165] FIG. 84. Serum-free Growth of Cells in Four Different
Defined Media.+-.E.sub.2. (A) MTW9/PL2 Cells in DDM-2A; (B) T47D
Cells in DDM-2MF; (C) GH.sub.4C.sub.1 Cells in PCM-9; (D) H301
Cells in CAPM.
[0166] FIG. 85. Effects of CDE-horse Serum on Estrogen
Responsiveness of Three ER.sup.+ Cell Lines Growing in Serum-free
Defined Media. (A) T47D Cells in DDM-2MF; (B) MTW9/PL2 Cells in
DDM-2A; (C) GH.sub.4C.sub.1 Cells in PCM-9.
[0167] FIG. 86. Effects of CA-PS-pool II on the Growth of Eight
ER.sup.+ Cell Lines in Serum-free Defined Medium.+-.E.sub.2.
[0168] FIG. 87. Protein Sequencing Results with CA-PS-Pool II
Peptides and Homology to Human SHBG, Rabbit SHBG and Rat and
Hamster Androgen Binding Protein.
[0169] FIG. 88. Western Analysis of CA-PS-pool I and CA-PS-pool II
with the Antibody Raised to the 54 kDa Band.
[0170] FIG. 89. Effect of the Anti-54 kDa Antiserum on the
Inhibition of MWT9/PL2 Cell Growth by the Isolated Fraction
CS-PS-Pool II.
[0171] FIG. 90. Western Immunoblotting of Commercially Prepared
Horse IgG, IgA and IgM with anti-54 kDa Antiserum.
[0172] FIG. 91. Effect of Horse IgG on MTW9/PL2 Cell Growth in 2.5%
CDE-horse Serum.+-.E.sub.2.
[0173] FIG. 92. Effect of Horse IgM on MTW9/PL2 Cell Growth in 2.5%
CDE-horse Serum.+-.E.sub.2.
[0174] FIG. 93. Effect of Horse IgA on MTW9/PL2 Cell Growth in 2.5%
CDE-horse Serum.+-.E.sub.2.
[0175] FIG. 94. SDS-PAGE with Coomassie Staining and Western
Analysis of Rat Purified "SHBG-like" Proteins. (A) SDS-PAGE of
Purified Rat Preparations; (B) Western Analysis with Anti-rat
IgG.
[0176] FIG. 95. Western Analysis of a Rat Purified "SHBG-like"
Preparation. (A) Western with Anti-rat IgA with Purified IgA
Control; (B) Western with Anti-rat IgG1 with Purified IgG1 Control;
(C) Western with Anti-rat IgM with Purified IgM Control.
[0177] FIG. 96. Protein Sequencing Results with Rat "SHBG-like"
Peptides and Homology to Human SHBG, Rabbit SHBG and Rat and
Hamster Androgen Binding Protein.
[0178] FIG. 97. Comparison of Rat IgG Subclasses. (A) SDS-PAGE with
Coomassie Blue Staining; (B) Western Analysis with Rabbit
Anti-Human SHBG.
[0179] FIG. 98. Effect of Rat IgG on MTW9/PL2 Cell Growth in Medium
with 2.5% CDE-rat Serum.+-.E.sub.2.
[0180] FIG. 99. Effect of Rat IgA on MTW9/PL2 Cell Growth in Medium
with 2.5% CDE-rat Serum.+-.E.sub.2.
[0181] FIG. 100. Effect of Rat IgM on MTW9/PL2 Cell Growth in
Medium with 2.5% CDE-rat Serum.+-.E.sub.2.
[0182] FIG. 101. Mannan Binding Protein Isolation of Human
Plasma/Serum IgM.
[0183] FIG. 102. Jacalin Lectin Purification of Human Plasma/Serum
IgA.
[0184] FIG. 103. Effect of Human IgM on MTW9/PL2 Cell
Growth.+-.E.sub.2 in Serum-free Defined Medium.
[0185] FIG. 104. Comparison of the Effects of Rat and Horse IgA and
IgM on MTW9/PL2 Cell Growth.+-.E.sub.2 in Serum-free Defined Medium
Expressed in Cell Number and CPD.
[0186] FIG. 105. Effect of Rat Myeloma IgA on GH.sub.1 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2.
[0187] FIG. 106. Effect of Human Plasma IgA on GH.sub.1 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2.
[0188] FIG. 107. Effect of Human Plasma IgM on GH.sub.1 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2.
[0189] FIG. 108. Effects of sIgA on GH.sub.1 Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0190] FIG. 109. Model of Mucosal Epithelial Cell Transport of
IgA/IgM.
[0191] FIG. 110. Essential Structures of Human Plasma and Secretory
IgA.
[0192] FIG. 111. Effect of Rat Myeloma IgA on GH.sub.3 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2.
[0193] FIG. 112. Effect of Rat IgM on GH.sub.3 Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0194] FIG. 113. Effect of Human Plasma IgA on GH.sub.3 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2.
[0195] FIG. 114. Effect of Human Plasma IgM on GH.sub.3 Cell Growth
in Serum-free Defined Medium.+-.E.sub.2.
[0196] FIG. 115. Effect of Human Secretory IgA on GH.sub.3 Cell
Growth in Serum-free Defined Medium.+-.E.sub.2.
[0197] FIG. 116. Effect of Rat Myeloma IgA on GH.sub.4C.sub.1Cell
Growth in Serum-free Defined Medium.+-.E.sub.2.
[0198] FIG. 117. Effect of Rat Plasma IgM on GH.sub.4C.sub.1Cell
Growth in Serum-free Defined Medium.+-.E.sub.2.
[0199] FIG. 118. Effect of Human Plasma IgA on GH.sub.4C.sub.1 Cell
Growth in Serum-free Defined Medium.+-.E.sub.2.
[0200] FIG. 119. Effect of Human Plasma IgM on GH.sub.4C.sub.1 Cell
Growth in Serum-free Defined Medium.+-.E.sub.2.
[0201] FIG. 120. Effect of Human Secretory IgA on GH.sub.4C.sub.1
Cell Growth in Serum-free Defined Medium.+-.B.sub.2.
[0202] FIG. 121. Effect of Mouse IgA on H301 Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0203] FIG. 122. Effect of Human IgA on H301 Cell Growth in
Serum-free Defined Medium.+-.E.sub.2 (A) Plasma IgA Effects; (B)
Secretory sIgA Effects.
[0204] FIG. 123. Dose-Response Effects of E.sub.2 on H301 Cell
Growth in Serum-free Defined Medium Containing 40 .mu.g/mL Human
Plasma IgM.
[0205] FIG. 124. Effect of Human IgA on MCF-7A Cell Growth in
Serum-free Defined Medium.+-.E.sub.2. (A) Plasma IgA Effects; (B)
Secretory sIgA Effects.
[0206] FIG. 125. Effect of Human IgA on MCF-7K Cell Growth in
Serum-free Defined Medium.+-.E.sub.2. (A) Plasma IgA Effects; (B)
Secretory sIgA Effects.
[0207] FIG. 126. Effect of Human IgM on MCF-7A Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0208] FIG. 127. Effect of Human IgM on MCF-7K Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0209] FIG. 128. Dose-Response Effects of E.sub.2 on MCF-7K Cell
Growth in Serum-free Defined Medium Containing 40 .mu.g/mL Human
Plasma IgM.
[0210] FIG. 129. Effect of Human IgA on T47D Cell Growth in
Serum-free Defined Medium.+-.E.sub.2. (A) Plasma IgA Effects; (B)
Secretory sIgA Effects.
[0211] FIG. 130. Effect of Human IgM on T47D Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0212] FIG. 131. Dose-Response Effects of E.sub.2 on T47D Cell
Growth in Serum-free Defined Medium Containing 40 .mu.g/mL Human
Plasma IgM.
[0213] FIG. 132. Effect of Human IgA on ZR-75-1 Cell Growth in
Serum-free Defined Medium.+-.E.sub.2. (A) Plasma IgA Effects; (B)
Secretory sIgA Effects.
[0214] FIG. 133. Effect of Human IgM on ZR-75-1 Cell Growth in
Serum-free Defined Medium.+-.E.sub.2.
[0215] FIG. 134. Effect of Human IgM on HT-29 Cell Growth in
Serum-free Defined Medium.+-.T.sub.3.
[0216] FIG. 135. Effect of Human IgA on LNCaP Cell Growth in
Serum-free Defined Medium.+-.E.sub.2. (A) Plasma IgA Effects; (B)
Secretory sIgA Effects.
[0217] FIG. 136. Effects of Human Plasma versus Human Myeloma IgM
on LNCaP Cell Growth in Serum free Defined Medium.+-.DHT.
[0218] FIG. 137. Summary of Estrogenic Effects with Various
ER.sup.+ Cell lines and Different Ig Sources. 1. Human IgM on
MTW9/PL2 Cells=6.36 cpd 2. Mouse IgM on MTW9/PL2 Cells=6.00 cpd 3.
Rat IgM on MTW9/PL2 Cells=5.77 cpd 4. Human IgM on H301 Cells=7.57
cpd 5. Mouse IgM on H301 Cells=7.56 cpd 6. Rat IgM on H301
Cells=6.11 cpd 7. Human IgM on GH1 Cells=4.12 cpd 8. Rat IgM on GH1
Cells=5.83 cpd 9. Human IgM on GH3 Cells=4.09 cpd 10. Human IgM on
GH4 Cells=5.41 cpd 11. Human IgM on MCF-7A Cells=5.01 cpd 12. Human
IgM on MCF-7K Cells=5.89 cpd
[0219] FIG. 138. Effect of Tamoxifen on T47D Cell Growth in
Serum-free Defined Medium
[0220] FIG. 139. Estrogen Reversal of Tamoxifen Inhibition of T47D
cells in Serum-free Defined Medium
[0221] FIG. 140. Effect of Rat Immunoglobulins on Estrogen
Responsive Growth of MTW9/PL2 Cells In Serum-free Defined
Medium.
[0222] FIG. 141. Comparison of the Estrogenic Effects of Human
Immungobulin with T47D Cells in Serum-free Defined Medium.
[0223] FIG. 142. Effect of Human IgG Isotypes on LNCaP Cell Growth
in Serum-free Defined Medium.+-.DHT.
[0224] FIG. 143. Western Detection of the Secretory Component of
Human Milk sIgA.
[0225] FIG. 144. Effect of Anti-Secretory Component on IgM
Inhibition of T47D Cell Growth in Serum-free Defined Medium.
[0226] FIG. 145. Effect of Anti-Secretory Component on pIgA
Inhibition of LNCaP Cell Growth in Serum-free Defined Medium.
[0227] FIG. 146. Western Analysis with Anti-Secretory Component to
Detect the Poly-Ig Receptor in AR.sup.+ and AR.sup.- Prostate
Cancer Cells plus Control Cell Lines.
[0228] FIG. 147. Effect of Human pIgA on DU145 Cell Growth in
Serum-free Defined Medium.+-.DHT.
[0229] FIG. 148. Effect of Human pIgA on PC3 Cell Growth in
Serum-free Defined Medium.+-.DHT.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0230] To facilitate review of the detailed description of
preferred embodiments, a Table of Contents is provided. The titles
used for the various subsections and examples are not intended to
be limiting and are only an aid to locating certain subject matter.
In addition, each Example begins with a short summary of that
Example, which is intended merely to facilitate review and is not
limiting on the disclosure contained in the full Example, and ends
with a Discussion of some conclusions that may be drawn from that
Example.
TABLE-US-00001 Table of Contents Subsection Paragraph No. I.
Introduction 228 II. General Materials and Methods 233 III.
Examples Example 1. Identification of Steroid Hormone Receptors in
MTW9/PL2 Cells 256 Example 2. Three Preparations of Steroid Hormone
Depleted Serum and Examples of 275 Support of Estrogen Responsive
Cell Growth in Culture A. Charcoal-dextran Extraction at 34.degree.
C. 276 B. Charcoal-dextran Extraction at 56.degree. C. 278 C.
Amberlite .TM. XAD .TM.-4 Resin Treatment 279 Example 3. Cancer
Cell Line MTW9/PL2 Exhibits Estrogen Responsiveness 282 in
34.degree. C. Charcoal-dextran Extracted Serum Example 4. Estrogen
Responsive Growth of Additional Rodent and Human Cell Lines In
34.degree. C. 296 Charcoal-dextran Extracted Horse and Human Serum
Example 5. Thyroid Hormone Growth Effects in CDE-Horse Serum
Prepared at 34.degree. C. 310 Example 6. Effect of 56.degree. C.
Versus 34.degree. C. CDE-horse Serum on MTW9/PL2 Cell Growth 313
Example 7. Demonstration of Estrogenic Effects in XAD-4 Resin
Treated Horse Serum 315 Example 8. Testing of Substances for
Estrogenic Activity 317 Example 9. Testing of Substances for
Inhibitor-like Activity 332 Example 10. Serum-free Defined Culture
Medium Formulations 351 Example 11. Serum-free Defined Medium that
Supports Hormone Sensitive and 375 Autonomous Cancer Cell Growth
Example 12: Differential Effects of Fe (III) on the Growth of
Hormone Responsive and 381 Autonomous Human Breast and Human
Prostate Cancer Cells Example 13: Growth in Serum-free Defined
Medium versus Growth in CDE-Serum .+-. E2 391 Example 14: Action of
DES on Human AR + LNCaP Prostate Cancer Cells 400 Example 15:
Preparation of Inhibitor Depleted Serum for Control Studies and 403
Stability Properties of the Inhibitor Example 16: Effects of
Conventional Purification Methods and Properties of the Estrogen
Reversible 413 Serum-borne Inhibitor Example 17: Calcium
Stabilization and Correlation with 3H-DHT Binding and 429
Immunoprecipitation by Antibodies Raised to Human SHBG Example 18:
Cortisol Affinity and Phenyl Sepharose Isolation of the "SHBG-like"
438 Estrogen Reversible Inhibitor from CDE-Horse Serum Example 19:
Serum-free Assay Systems for Measuring Large Magnitude Steroid
Hormone 457 Mitogenic Responses with the Two-Step Purified
Inhibitor Example 20: Chemical and Immunological Properties of the
Partially Purified CA-PS-Pool II 467 Inhibitors and Identification
as IgA and IgM Example 21: Regulation of Steroid Hormone-responsive
and Thyroid Hormone-responsive 485 Cancer Cell Growth in Serum-free
Defined Medium by Secretory and Plasma Forms of IgA and Plasma and
Cell Culture Derived IgM A. MTW9/PL2 Cells - ER+ rat mammary tumor
487 B. GH1, GH3, and GH4C1 Cells - ER+ rat pituitary tumor 489 C.
H301 Cells - ER+ Syrian hamster kidney tumor. 492 D. MCF-7A and
MCF-7K Cells - ER+ human breast cancer. 493 E. T47D Cells - ER+
human breast cancer. 494 F. ZR-75-1 Cells - ER+ human breast
cancer. 495 G. HT-29 Cells - Thyroid hormone responsive human colon
cancer. 496 H. LNCaP Cells - AR+ human prostate cancer. 497 Example
22: Effect of Tamoxifen Antiestrogen in Serum-free Defined Medium.
501 Example 23: IgG1 and IgG2 as an Immunoglobulin Regulators of
Estrogen and 508 Androgen Responsive Cancer Cell Growth. Example
24: Mediation of IgA/IgM Effects by the Poly-Ig Receptor. 514
Example 25: Mediation of IgG1.kappa. Effects by a Fc-like Receptor.
526 Example 26. Immunoglobulin Inhibitors as Tools for Identifying
the Receptors that 529 Mediate the IgA/IgM/IgG Cell Growth
Regulating Effects. Example 27: Conceptual Model for Cascading Loss
of Cell Growth Inhibition in Cancer Cells. 540 Example 28. IgA/IgM
Based Test to Detect Lowered Levels of Steroid Hormone Reversible
550 Cell Growth Inhibitors in Plasma or Body Secretions.
INTRODUCTION
[0231] Extracellular negative regulation is a key control mechanism
of cell proliferation in steroid hormone responsive cancer cells.
Sex steroid hormones (both estrogens and androgens) act to reverse
the effects of a serum-borne inhibitor(s) that normally blocks
target cell proliferation (Moreno-Cuevas J E and Sirbasku D A
(2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D A and
Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446;
Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol
36, 447-464, incorporated herein by reference). As demonstrated in
the Examples that follow, these results were obtained with nine
different estrogen receptor alpha (ER.alpha.) (Kumar V et al.
(1987) Cell 51, 941-951) containing cell lines representing four
target tissues and three species (Sirbasku D A and Moreno-Cuevas J
E (2000) In Vitro Cell Dev Biol 36, 428-446).
[0232] As mentioned in the Background of the Invention, the prior
art fails to adequately address the issues of (i) whether there are
one or more of the serum-derived inhibitors, (ii) what is/are the
exact chemical composition of the inhibitor(s), and (iii) what
conditions were required to yield the long term stable product(s)
necessary for the commercial application of the testing methodology
described. Methods and compositions are presented herein that are
useful for testing and assessment of compounds and mixtures for
estrogenic or androgenic activity as well as others possessing
antiestrogenic and antiandrogenic activities. In the Examples that
follow, cell culture methodology and compositions are described
that permit testing at concentrations lower than was previously
possible using existing methodologies. Moreover, the new in vitro
model assay systems obviate the need to conduct animal testing to
predict in vivo responses. Some practical applications for the
model include protecting the human population from unrecognized
exposure to hormone-like compounds that present health hazards as
well as developing new antihormone compounds to counterbalance
these hazards. The testing of these compounds and mixtures are
preferably conducted in serum-containing medium to mimic the
conditions encountered by a blood borne agent. Testing can
additionally be done under completely serum-free defined medium
conditions to determine direct actions on cells without serum or
non-essential proteins present.
[0233] It has been discovered that the negative regulators of
steroid hormone responsive cancer cell growth (estrogen reversible
inhibitors) in serum are products from the secretory immune system,
i.e., the immunologobulins A (IgA), M (IgM) and IgG1. These
"immunoglobulin inhibitors" act as steroid hormone and thyroid
hormone reversible inhibitors of mucosal cell growth. There has
been no previous identification of secretory immune system
immunoglobulins as regulators of epithelial (mucosal) cell growth,
and this discovery is unique in the cell growth regulation field.
Application of certain of the compositions and methods is expected
to relate to 80% of all human cancers because this high incidence
rate arises from mucosal tissues. There is no previously reported
evidence directly linking the secretory immunoglobulins with
regulation of mucosal cell growth.
[0234] With regard to applicability to several mucosal tissues, it
is recognized that breast and prostate cancers are very similar
diseases. Aside from tissue specific epidemiological and social
factors, breast and prostate cancers have remarkable parallels
(Grody W W et al. (1994) Am J Clin Pathol 102, S1-S67). The
secretory immune system acts as a sex steroid hormone reversible
inhibitor with target tumor cells from both of these cancers. Both
are adenocarcinomas arising from sexually differentiate tissues.
Certainly both cancers are very common in North America and
northern Europe compared to the rest of the world. Both are
strongly influenced by steroid hormones. Both increase in incidence
with age. Both are thought to have at least some genetic component.
Finally, both have very similar patterns of development when
examined histologically.
[0235] These facts also have implications with regard to colon,
uterine and ovarian cancers. These cancers show familial clustering
with breast cancer (Nelson C L et al. (1993) Genet Epidemiol 10,
235-244). The aggregation of colon, ovarian, endometrial and breast
cancer in families has been described as a "cancer family" which
now has the name Lynch Syndrome I and II (Lynch H T et al. (1978)
Cancer 41, 1543-1549). Other studies have shown links between
colorectal cancer and breast cancer (Rozen P et al. (1990) Cancer
Lett 55, 189-194) and colorectal cancer and breast, uterine and
ovarian cancer (Rozen P et al. (1986) Cancer 57, 1235-1239). It is
clear that the incidence of these several mucosal origin cancers
are linked and that this linkage has not been explained.
II. GENERAL MATERIALS AND METHODS
[0236] In the Examples below, which describe representative,
preferred embodiments of the present invention, the following
general materials and methods are employed, except as otherwise
noted in the Examples.
[0237] Cell Culture Medium. The water used to prepare culture media
and all other solutions was purified first by reverse osmosis
followed by passage through a U.S. Filter Corporation system with a
charcoal filter and two mixed bed ion exchangers. The effluent was
distilled using a Bellco glass apparatus with quartz heating
elements. The distilled water was stored in airflow restricted
glass containers. No metal fittings are allowed in contact with the
final purified water. This necessary precaution minimizes
recontamination with metal ions. Standard phenol red containing
Ham's F12-Dulbecco's modified Eagle's medium (D-MEM/F-12), phenol
red-free standard D-MEM/F-12 and a custom-prepared "low-Fe"
D-MEM/F-12 medium were supplied by Gibco-BRL (Catalog No.
11330-032) or Bio.diamond-solid.Whittacker (Catalog No. 12-719,
liquid). The "low-Fe" medium was standard phenol red containing
D-MEM/F-12 from which the usual additions of ferric nitrate and
ferrous sulfate had been omitted (Eby J E et al. (1992) Anal
Biochem 203, 317-325; Eby J E et al. (1993) J Cell Physiol 156,
588-600). This medium was a special formulation purchased from
Gibco-BRL as a powder and prepared in the highly purified water
before 0.2 .mu.m pore filter membrane sterilization. A number of
other stock solutions are required for cell culture in either serum
containing or serum-free defined medium. Descriptions of each
preparation are provided along with specific instructions for their
use. The solutions used were designed to minimize the exogenous
content of steroid hormone and to minimize the Fe (III) content of
the water. Steps are taken for the exclusion of all extraneous
sources of steroid hormones and Fe (III). Exclusion of Fe (III) is
highly preferred, and in most of the totally serum-free
applications, it is considered essential. Wherever possible,
disposable plastic ware or glassware is used to minimize potential
contamination. It is important to note that excess solutions are
preferably discarded after use with each individual cell line to
avoid cross-contamination of cell types (Nelson-Rees W A and
Fladermeyer R R (1977) Science (Wash D.C.) 195, 134-136).
[0238] General Cell Culture--Serum. Adult and fetal horse, adult
pig, adult sheep and adult and fetal bovine serum were obtained
from Gibco-BRL. A mixture of adult male and female rat serum was
purchased from Pel-Freez, Rodgers, Ark. Human serum was purchased
from Bio.diamond-solid.Whittacker. Human plasma was a pool of
samples collected from pregnant females during routine visits to a
local clinic. All serum was stored frozen at -20.degree. C. until
used. Repeated freeze-thaw of serum or plasma is avoided. Before
charcoal extraction, the EDTA was removed by dialysis at 7.degree.
C. for 24 hours against forty volumes of 0.05 M Tris-HCl, pH 7.4,
containing 50 mM CaCl.sub.2. Dialysis was done with Spectropor 1
membranes (Spectrum Medical Industries, molecular weight cut-off
6,000 to 8,000). The clotted material was removed by
centrifugation. This preparation is termed plasma-derived serum.
The serum or plasma was not heat pre-treated, or heat inactivated
prior to use in the methods described below.
[0239] General Cell Culture--Normal Saline. Sterile normal saline
(0.15 M NaCl) was prepared in 10 mL aliquots and stored at room
temperature. Unused portions are discarded at the end of each
experiment. A large supply is sterilized by autoclaving and used to
prepare the solutions described below.
[0240] General Cell Culture--Trypsin/EDTA for Subculture. Sterile
preparations were purchased from Irvine Scientific (Catalog No.
9341) or Bio.diamond-solid.Whittacker (Trypsin-Versene EDTA
Mixture) (Catalog No. 17-161F). This preparation contained 0.5 g/L
trypsin and 0.2 g/L EDTA in Hank's balanced salts solutions with 10
mg/L phenol red. This preparation does not contain Ca or Mg salts
nor does it have NaHCO.sub.3. To trypsinize cells, 1.5 mL of this
preparation was typically used. Aliquots (2 mL) were stored frozen
until used and residual solution discarded at the end of each
experiment or application to a cell line.
[0241] General Cell Culture--Soybean Trypsin Inhibitor (STI). STI
was purchased from Sigma (Catalog No. T9128, Type II-2). An amount
of 1.0 mg of this preparation will inactivate 1.0 mg of trypsin
activity. The solution is prepared as 0.2% (w/v) in normal saline
and sterilized using a 0.2 .mu.m pore diameter filter membranes.
Aliquots of 3.0 mL are stored at -20.degree. C. until used. This
preparation is used to stop the action of trypsin during harvest of
stock cultures for growth assays. STI ensures that all trypsin used
to harvest cells for growth assays is inactivated and therefore
will not damage the protein additions to serum-free defined medium.
Also, use of STI ensures that no extraneous steroid hormones are
introduced after harvest of cells from the stock culture
dishes.
[0242] General Cell Culture--Crude Pancreatic Trypsin for Cell
Counting. This trypsin preparation was used to harvest the cells
for determining cell numbers. The cells are typically grown in
35-mm diameter dishes. This enzyme was purchased from ICN
Biochemicals as the 1-300 porcine pancreatic trypsin preparation
(Catalog No. 103140). A stock solution is typically prepared by
adding the contents of a preweighed bottle of 1.times. Dulbecco's
modified PBS medium without calcium or magnesium to 800 mL of
water. This solution dissolves very gradually with adjustment to pH
7.3 using NaOH. After the solution was clear, 20 g of crude trypsin
was added and this mixture stirred for 30 minutes at room
temperature. The somewhat cloudy solution was diluted to 1000 mL
with water and this volume was stored frozen in bulk overnight at
-20.degree. C. to induce cold related precipitation that typically
occurs when this preparation was frozen and thawed. After thawing
at 37.degree. C. in a water bath, the preparation was filtered
through 0.45 .mu.m pore membranes. This preparation was stored at
-20.degree. C. in useable portions.
[0243] General Cell Culture--EDTA for Cell Counting. The EDTA used
is the disodium and dihydrate salt (Sigma Catalog No. E1644). A
0.29 M solution is prepared by adding 107.9 g to 800 mL of water
with stirring and adjustment to pH 7.2 with NaOH. The volume is
brought to one liter with water and the solution stored at room
temperature. Because this solution is used only at the end of the
experiments, it does not require sterilization.
[0244] General Cell Culture. In TABLE 1 the cell lines used in the
described Examples are listed. The abbreviation "KCC" is the
Karmanos Cancer Center, Cell Line Repository, Detroit, Mich. The
abbreviation "ATCC" is the American Type Culture Collection, Cell
Line Repository, Manassas, Va. Professor Armen Tashjian's address
is Harvard University, Boston, Mass. Dr. William Rosner's address
is Columbia University, New York. Dr. Sirbasku's address is The
University of Texas, Houston, Tex. The superscript designations in
TABLE 1 for each of the cell lines indicate references that verify
that the estrogen and androgen responsive cell lines used in this
study are bona fide hormone responsive based on their tumor forming
characteristics in host animals. Those reports are clear
demonstrations of the reliability of the models used in the present
investigations to study sex hormone dependence in culture.
TABLE-US-00002 TABLE 1 Cell Lines Employed in the Examples.
ER.sup.+ indicates receptor containing/E.sub.2 sensitive CELL LINES
SOURCES REFERENCES/CELL LINE ORIGIN MCF-7K.sup.1 KCC Soule HD et
al. (1973) J Natl Cancer Inst 51, 1409-1416 ER.sup.+ human breast
cancer MCF-7A.sup.1 ATCC Soule HD et al. (1973) J Natl Cancer Inst
51, 1409-1416 ER.sup.+ human breast cancer T47D.sup.2 ATCC Keydar I
et al. (1979) Eur J Cancer 15, 659-670 ER.sup.+ human breast cancer
ZR-75-1.sup.3 ATCC Engle LW et al. (1978) Cancer Res 38, 3352-3364.
ER.sup.+ human breast cancer GH.sub.4C.sub.1.sup.4 Dr. A. Tashjian
Tashjian AH Jr (1979) Methods Enzymol 58, 527-535 ER.sup.+ rat
pituitary tumor GH.sub.3.sup.5 ATCC Tashjian AH Jr (1979) Methods
Enzymol 58, 527-535. ER.sup.+ rat pituitary tumor GH.sub.1 ATCC
Tashjian AH Jr (1979) Methods Enzymol 58, 527-535 ER.sup.+ rat
pituitary tumor MTW9/PL2.sup.6 Dr. D. Sirbasku Danielpour D et al.
(1988) In Vitro Cell Dev Biol 24, 42-52 ER.sup.+ rat mammary tumor
H301.sup.7 Dr. D. Sirbasku Sirbasku DA and Kirkland WL (1976)
Endocrinology 98, 1260-1272 ER.sup.+ Syrian hamster kidney tumor
LNCaP.sup.8 ATCC Horoszewicz JS et al. (1983) Cancer Res 43,
1809-1818 AR.sup.+ human prostatic carcinoma Fibroblasts Dr. D.
Sirbasku Primary cultures of human foreskin and rat ear cartilage;
Eastment CT and Sirbasku DA (1980) In Vitro 16, 694-705 ALVA-41 Dr.
W. Rosner Nakhla AM and Rosner W (1994) Steroids 59, 586-589
AR.sup.- human prostate cancer; androgen growth insensitive DU145
ATCC Stone KR et al. (1978) Int J Cancer 21, 274-281 AR.sup.- human
prostate cancer; androgen growth insensitive PC3 ATCC Kaighn ME et
al. (1979) Invest Urol 17, 16-23 AR.sup.- human prostate cancer;
androgen growth insensitive HT-29 ATCC Chen TR et al. (1987) Cancer
Genet Cytogenet 27, 125-134 Thyroid hormone responsive human colon
cancer .sup.1The use of two strains of MCF-7 cells has been
described (Sirbasku DA and Moreno-Cuevas (2000) In Vitro Cell Dev
Biol 36, 428-446). Clonal variations of this line are known
(Seibert K et al. (1983) Cancer Res 43, 2223-2239). Demonstration
of estrogen responsive MCF-7 tumor formation in vivo (Huseby RA et
al. (1984) Cancer Res 44, 2654-2659; Soule HD and McGrath CM (1980)
Cancer Lett 10, 177-189; Welsch CW et al. (1981) Cancer Lett 14,
309-316). .sup.2Estrogen responsive T47D tumors in vivo (Leung CKH
and Shiu RPC (1981) Cancer Res 41, 546-551). .sup.3Estrogen
responsive ZR-75-1 tumors in vivo (Osborne CK et al. (1985) Cancer
Res 45, 584-589). .sup.4Estrogen responsive GH.sub.4C.sub.1 tumors
in vivo (Riss TL and Sirbasku DA (1989) In Vitro Cell Dev Biol 25,
136-142). .sup.5Estrogen responsive GH.sub.3 tumors in vivo
(Sorrentino JM et al. (1976) J Natl Cancer Inst 56, 1149-1154).
.sup.6Estrogen responsive MTW9/PL2 tumors in vivo (Sirbasku DA
(1978) Cancer Res 38, 1154-1165; Danielpour D and Sirbasku DA
(1984) In Vitro 20, 975-980). .sup.7Estrogen responsive H301 tumors
in vivo (Sirbasku DA and Kirkland WL (1976) Endocrinology 98,
1260-1272; Liehr JG et al. (1986) J Steroid Biochem 24, 353-356).
.sup.8Androgen responsive LNCaP tumors in vivo (Sato N et al.
(1997) Cancer Res 57, 1584-1589; Gleave M et al (1991) Cancer Res
51, 3753-3761; Horoszewicz JS et al. (1983) Cancer Res 43,
1809-1818; Pretlow TG et al. (1991) Cancer Res 51, 3814-3817;
Passaniti A et al. (1992) Int J Cancer 51, 318-324).
[0245] General Cell Culture--Cell Passage Method. All stock
cultures were grown in medium containing phenol red. Stocks of the
cells were maintained at 37.degree. C. in a humid atmosphere of 5%
(v/v) CO.sub.2 and 95% (v/v) air in 17 to 20 mL of standard
D-MEM/F-12 with 2.2 g per liter sodium bicarbonate, 15 mM HEPES (pH
7.4), and serum. With all cell lines except the rat pituitary
cells, the serum used for stock culture was 10% (v/v) fetal bovine
serum (FBS). For the three rat pituitary tumor cell lines
GH.sub.4C.sub.1, GH.sub.1 and GH.sub.3, the medium contained 12.5%
(v/v) horse serum and 2.5% (v/v) FBS. To passage the cells, the
medium was removed and the dishes washed with 10 mL of saline.
Next, the cells were dissociated by incubation at room temperature
or at 37.degree. C. for 3 to 10 minutes with 1.5 mL of
trypsin/EDTA. The action of the trypsin was stopped by addition of
8 mL of D-MEM/F-12 containing 10% (v/v) FBS or 8 mL of the horse
serum/FBS combination. The cells were collected by centrifugation
at 1000.times.g for 5 minutes and suspended in 10 mL of fresh serum
containing medium. Aliquots were diluted into Isoton II (Coulter
Diagnostics) and cell numbers determined with a Model ZBI or Z1
Coulter Particle Counter. The new dishes (100-mm diameter with 15
to 20 mL of fresh medium) were seeded with 2.0.times.10.sup.5 to
1.0.times.10.sup.6 cells on an alternating three-four day schedule
or weekly as dictated by cell line growth rate. Cultures were used
for growth assays between three and six days after passage. Acidic
(yellow medium indicator color) cultures are not used for growth
assays.
[0246] General Cell Culture--Media Types Used. The assays done in
the presence of serum were initially in "low-Fe" D-MEM/F-12
containing phenol red (Moreno-Cuevas J E and Sirbasku D A (2000) In
Vitro Cell Dev Biol 36, 410-427). The issue of the significance of
the presence or absence of phenol red, a potential estrogen
(Berthois Y et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500),
has been dealt with in considerable detail (Moreno-Cuevas J E and
Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464). The Fe
(III) content of this medium was .ltoreq.0.2 .mu.M (Eby J E et al.
(1992) Anal Biochem 203, 317-325). Fe (III) levels of .gtoreq.1.0
.mu.M interfere with thyroid hormone and estrogen responsive rat
pituitary tumor cell growth in culture (Eby J E et al. (1992) Anal
Biochem 203, 317-325; Eby J E et al. (1993) J Cell Physiol 156,
588-600; Sato H et al. (1991) In Vitro Cell Dev Biol 27A, 599-602;
Sato H et al (1992) Mol Cell Endocrinol 83, 239-251). Although Fe
(III) might prevent estrogen responsiveness from being identified
in culture with MTW9/PL2 cells, as shown herein and reported
(Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol
36, 428-446; Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro
Cell Dev Biol 36, 447-464), this is not the case when serum is
present. Standard Fe (III)/Fe (II) containing D-MEM/F-12 was as
effective as the low-Fe medium. It is clear that the apotransferrin
in the serum effectively reduced the free Fe (III) in the medium to
less than cytotoxic levels. As stated above, apotransferrin binds
Fe (III) with very high affinity at pH 7.4 in plasma. The total
concentration of transferrin in serum is about 3 mg/mL. Usually,
two-thirds of the total is apotransferrin. This amount is more than
adequate to chelate Fe (III) in culture medium (Eby Y E et al.
(1992) Anal Biochem 203, 317-325). However, in assays in serum-free
defined medium, as described below, a Fe (III) chelator (e.g.
apotransferrin or DFX) is present in the serum-free defined medium
at sufficient levels to neutralize the toxic iron.
[0247] General Cell Culture--Growth Assay Methods. Cell growth
assays were initiated with stock cultures that were harvested by
trypsin/EDTA treatment as described above with one exception. It
was highly preferred to stop the action of trypsin with 3 mL of
soybean trypsin inhibitor (0.5% w/v in saline) instead of medium
containing serum. The use of trypsin inhibitor reduced the
possibility of contamination of the subsequent assay media by
serum-derived steroid hormones. The dissociated cells were
collected by centrifugation as described above and washed three
times with 10 mL volumes of serum-free standard D-MEM/F-12. After
each wash, care was taken to aspirate all medium from the cell
pellet and the walls of the centrifuge tubes. This minimized the
carryover of steroid hormones into the experimental test dishes. By
taking steps to avoid carryover of serum-containing medium, steroid
hormones are prevented from being retained by the cells in culture.
It is highly preferred to wash the cells in this way before
assaying to measure various steroid hormone effects in culture. It
has been reported that steroid hormones are retained long term by
breast cancer cells in culture (Strobl J S and Lippman M E (1979)
Cancer Res 39, 3319-3327). The above-described wash procedure
negates this problem. After the final wash, the cells were
suspended in 10 mL of serum-free D-MEM/F-12 and cell numbers
determined. When cells were to be assayed in medium without phenol
red discussed elsewhere herein and reported (Moreno-Cuevas J E and
Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464), the cells
were washed and resuspended in phenol red free D-MEM/F-12 purchased
from Gibco-BRL. The growth assays were initiated in 35-mm dishes
containing a total of 2.0 mL of medium and the final concentration
of all components except steroid hormones. The steroid hormone
stocks were diluted to appropriate concentrations in serum-free
D-MEM/F-12 and 20 .mu.L aliquots added to each dish. For all growth
assays, the medium was not changed after the initial inoculation.
Because several of the cell lines described in TABLE 2 grow in
serum containing medium and serum-free defined medium as mixtures
of suspension and attached cells, removal or changing of the medium
during the course of the assays causes substantial cell losses. For
all cell growth assays, the initial seed densities ranged from
5,000 to 12,000 cells per 35-mm diameter dish.
[0248] General Cell Culture--Steroid Hormone Preparations. A number
of hormone preparations are used to supplement the cell cultures.
Unlabeled steroid hormones were obtained from Sigma or Steraloids.
Stock solutions were prepared in sterile glass containers. The
powder (non-sterile) steroid is added to the bottle along with 200
ml of 70% aqueous ethanol (ready as sterile). The steroids dissolve
within an hour at room temperature, or when required were dissolved
by gentle heating on a hot plate (hand temperature test--no
boiling--no open flames). The stock solutions were stored at
4.degree. C. and renewed at six-month intervals. It is not
necessary or desirable to filter sterilize these solutions because
of steroid hormone loss on filter membranes. Stocks of 1.0 mM
steroid hormones were prepared. To prepare diluted stocks for
direct use in culture, 10 .mu.L of 1.0 mM steroid hormone is
diluted into 10 mL of D-MEM/F-12. This gives a stock of 1.0 .mu.M.
It is used in the assay dishes or diluted further in D-MEM/F-12 as
needed. The diluted steroids are discarded after each use because
they bind to the plastic with storage. The formula weight (FW) of
each of the common natural and synthetic hormones used is listed
below in TABLE 2 along the abbreviation used for each and the
amounts required to prepare 200 mL of stock.
TABLE-US-00003 TABLE 2 Preparation of Steroid Hormone Stocks for
Cell Culture and Hormone Binding Assays FORMULA MILLIGRAMS/ STEROID
HORMONES WEIGHT (FW) 200 mL 17.beta.-estradiol (E.sub.2) 272.4 54.4
Estrone (E.sub.1) 270.4 54.1 Estriol (E.sub.3) 288.4 57.7
Diethylstilbestrol (DES) 268.4 53.7 Tamoxifen Citrate (TAM) 563.6
112.7 Progesterone (PROG) 314.5 62.9 Hydrocortisone/Cortisol (C)
362.5 72.5 Dexamethasone (DEX) 392.5 78.5 Testosterone (T) 288.4
57.7 Dihydrotestosterone (DHT) 290.4 58.1
[0249] General Cell Culture--Harvest and Counting Cells. At the
termination of the experiments, each plate received 0.4 mL of crude
pancreatic trypsin dissolved in phosphate buffered saline was added
along with 0.3 mL of 0.29 M EDTA. After 4 to 40 minutes incubation
at room temperature or at 37.degree. C., the action of the trypsin
was stopped by addition of 0.6 mL of horse serum. The cell clumps
were dissociated further by one passage through a 201/2 or 23-gauge
needle and syringe. This suspension was then diluted to 10 mL with
Isoton II and cell numbers determined with a Coulter Counter. The
results are presented as the average of triplicate dishes for each
test medium. To determine day zero cell numbers, at least
triplicate 1.0 mL aliquots of the inoculum were collected for
counting during the seeding of the test dishes. Coulter Counter
standardization and monitoring were performed by the
manufacturer.
[0250] General Cell Culture--Quantification of Growth. The cell
number results are converted to cell population doublings (CPD) by
the following calculation:
CPD = Log 10 Average Cell Number on Collection Day Log 10 Average
Cell Number on Day Zero Log 10 2 ##EQU00001##
For the purposes of this Disclosure, the mitogenic response to sex
steroid hormones is designated the "steroidogenic effect." For
example, the "estrogenic effect" is calculated as the difference
between CPD measured in the presence of an estrogen minus CPD in
the absence of the steroid. These values equal cell number
increases of 2.sup.CPD. The term "androgenic effect" has the same
meaning except that it describes growth caused by androgens such as
DHT and T. CPD is used herein as a measure of growth because it is
a direct calculation of the number of times a cell population
undergoes cell division. Furthermore, CPD use permits a direct
measure of ED.sub.50 and ED.sub.100 Concentrations in different and
in replicate assays. The significance of differences between test
dishes and controls was evaluated by the student's t test. Values
of p<0.05 were accepted as significant. Standard deviations
(.+-.SD) are included when appropriate.
[0251] ATCC Cell Line Deposition. The rat breast cancer cell line
designated as MTW9/PL2 (ATCC accession number ______) was deposited
with the American Type Culture Collection, Rockville, Md. on
______, 2000. The hamster kidney tumor cell line designated as H301
(ATCC accession number ______) was deposited with the American Type
Culture Collection, Rockville, Md. on ______, 2000. The subject
cultures have been deposited under conditions that assure that
access to the cultures will be available during the pendency of
this patent application to one determined by the Commissioner of
Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and
35 U.S.C. 122. The deposits will be available as required by
foreign patent laws in countries wherein counterparts of the
subject application, or its progeny, are filed. However, it should
be understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
[0252] Cell Lines--Budapest Treaty Compliance. Further, the subject
culture deposits will be stored and made available to the public in
accord with the provisions of the Budapest Treaty for the Deposit
of Microorganisms, i.e., they will be stored with all the care
necessary to keep them viable and uncontaminated for a period of at
least five years after the most recent request for the furnishing
of a sample of each deposit, and in any case, for a period of at
least thirty (30) years after the date of deposit or for the
enforceable life of any patent which may issue disclosing the
cultures. The depositor acknowledges the duty to replace the
deposits should the depositor be unable to furnish a sample when
requested, due to the condition of the deposit. All restrictions on
the availability to the public of the subject culture deposits will
be irrevocably removed upon the granting of a patent disclosing
it.
[0253] Growth of Cells for Steroid Hormone Receptor Assays. Whole
cells were assayed for the presence of steroid hormone receptors by
a modification of described methods (Baxter J D et al. (1975)
Methods Enzymol 36, 240-248). The cells (9 to 10.times.10.sup.6)
were seeded in 850 cm.sup.2 roller bottles (Corning) containing 200
mL of standard phenol red containing D-MEM/F-12 supplemented with
2.0% (v/v) charcoal-dextran extracted (CDE) horse serum and grown
at 37.degree. C. for five to seven days. The cells were collected
from the plastic surface and the medium and concentrated by
centrifugation at 1000.times.g for 15 minutes. The cells were
washed with saline, resuspended in 20 mL of saline and treated with
2.0 mL, of trypsin/EDTA at room temperature for one minute. The
trypsin action was stopped with 6.0 mL of 0.5% (w/v) soybean
trypsin inhibitor in saline. The dispersed cells were collected and
suspended in phenol red-free D-MEM/F-12 to a density of 0.5 to
1.0.times.10.sup.6 per mL. The rationale for measuring steroid
hormone binding with whole cells rests with the intent to replicate
cell culture conditions. To derive complete information, the use of
whole cells avoids the possible loss of a new receptor that might
not withstand the cell extraction process or otherwise not be
recovered.
[0254] Whole Cell Steroid Binding Assays. Total estrogen binding
was measured with .sup.3H-E.sub.2
(2,4,6,7-.sup.3H-17.beta.-estradiol) at specific activity 96 Ci per
mmole (Amersham). Non-specific binding was assessed with
.sup.3H-E.sub.2 plus a 100-fold molar excess of unlabeled DES.
Specific binding was total binding minus non-specific binding. To
assay specific progesterone binding, the medium contained either
.sup.3H-progesterone [1,2-.sup.3H (n) progesterone] at specific
activity 92 Ci per mmole (ICN) or .sup.3H-progesterone plus a
100-fold molar excess of the unlabeled synthetic progestin R5020
(DuPont NEN). Specific androgen binding was measured using [1,2
.sup.3H(N)] DHT at specific activity 45 Ci per mmole (DuPont NEN)
and the combination of .sup.3H-DHT plus a 100-fold excess of
unlabeled DHT. Glucocorticoid specific binding was assayed with
[1,2 .sup.3H(N)] hydrocortisone at specific activity 53 Ci per
mmole (DuPont NEN) and .sup.3H-hydrocortisone plus a 100-fold
excess of unlabeled DEX. The steroid hormone binding incubations
were done in phenol red free D-MEM/F-12 in a total volume of 1160
.mu.L containing 1000 .mu.L of cells, 100 .mu.L of labeled steroid
and 60 .mu.L of unlabeled steroid hormone or medium. The
incubations were done in glass tubes for two hours at 37.degree. C.
with gentle agitation in an orbital shaker water bath followed by
cooling to 0.degree. C. for 15 to 30 minutes. The cells were
collected by centrifugation at 7.degree. C. as described above and
washed three times with 2 mL portions of ice-cold phenol red-free
D-MEM/F-12. The final collected cells were dissolved in 0.5 mL of
0.5N sodium hydroxide and the radioactivity quantified by liquid
scintillation counting. All samples were duplicates or triplicates.
To obtain dissociation (K.sub.d) and association (K.sub.a)
constants, the data were analyzed by the method of Scatchard
(Scatchard G (1949) Ann NY Acad Sci 51, 660-672).
[0255] Steroid Hormone Receptor Analysis by Western Immunoblotting.
The following antibodies were obtained from Affinity Bioreagents: a
rabbit polyclonal antibody against a bacterial fusion protein
containing the N-terminal domain of the human androgen receptor and
a mouse monoclonal antibody against the amino acid sequence 533
through 547 of the DNA binding domain of the progesterone receptor.
An affinity-purified rabbit polyclonal antibody corresponding to
the amino acid sequence 580 through 599 of the mouse estrogen
receptor was obtained from Santa Cruz Biotechnology. To analyze
steroid hormone receptor content, both cytosolic and nuclear
extracts were prepared. To obtain the cytosol, 20.times.10.sup.6
cells were washed with serum-free D-MEM/F-12 and resuspended in 5
mL of 0.01 M Tris-HCl, pH 7.4, containing 0.15 M NaCl and 1 mM EDTA
(Tris/EDTA). After cooling to ice-bath temperature, the cells were
disrupted by three treatments for ten seconds with a Tekmar
Polytron homogenizer. The homogenates were centrifuged at
800.times.g for 10 minutes followed by centrifugation at
150,000.times.g for one hour to obtain the cytosolic supernatants.
To prepare nuclear extracts, the pellets from the 800.times.g
centrifugation were homogenized again three times with 1.5 mL
Tris/EDTA as described above. The centrifugation supernatants from
the three homogenizations were combined to give the nuclear extract
from each cell line. Protein concentrations were determined using
the BCA kit from Pierce Chemical.RTM. kit with bovine serum albumin
as standard. When required, the samples were concentrated by
precipitation with 20% (w/v) trichloroacetic acid. The precipitates
were washed once with 500 .mu.L of 70% (v/v) ethanol and twice with
500 .mu.L of water. They were dissolved in 200 .mu.L of 0.01 M
Tris-HCl, pH 7.4, containing 1% (w/v) sodium dodecyl sulfate (SDS)
by warming to 65.degree. C. SDS-PAGE (Laemmli UK (1970) Nature
(Lond) 227, 680-685) was done using 8 to 15% (w/v) acrylamide
gradient gels with 3% (w/v) acrylamide stacking gels. Each sample
was diluted with four volumes of buffer containing 0.3125 M
Tris-HCl, pH 6.8, 10% (w/v) SDS, 50% (v/v) glycerol, 25% (v/v)
mercaptoethanol and 0.0025% (w/v) bromophenol blue. After heating
to 95.degree. C. for five minutes, the samples were applied to the
gels and electrophoresis carried out at 7.degree. C. The separated
proteins were transferred to nitrocellulose membranes using a
Milliblot Graphite Electroblotter I with a transfer buffer
containing 1.0 mM (3-[cyclohexylamino]-1-propanesulfonic acid), pH
11, with 10% (v/v) methanol. Transfer was done for 45 minutes at
390 milliamps at room temperature. The receptors were detected by
chemiluminescence using a kit from Tropix.RTM.. The protocol used
was that recommended by the manufacturer. For the detection of
androgen receptors, the membranes were incubated at room
temperature with a 1:100 dilution of the primary antiserum for one
hour and a 1:10000 dilution of second antibody for one hour. To
detect estrogen receptors, the primary antiserum was used at a
1:5000 dilution with incubation at room temperature for one hour
followed by one hour with a 1:10000 dilution of second antibody.
For estrogen and androgen receptors, the second antibody was an
affinity purified anti-rabbit immunoglobulin conjugated to alkaline
phosphatase. To detect progesterone receptors, the incubations were
done with 5 .mu.g/mL primary antibody for 24 hours at 7.degree. C.,
followed by incubation with a 1:1000 dilution of second antibody
for eight hours at room temperature. The second antibody was an
affinity purified anti-mouse immunoglobulin conjugated to alkaline
phosphatase.
[0256] Western Immunoblotting with other Primary Antibodies. The
SDS-PAGE and Western Immunoblotting method described above was used
throughout the Examples with the only significant modifications
being changes in primary antibodies, and if required, changes in
the secondary antibody. The changes are noted when introduced.
[0257] Labeled Steroid Hormone Binding to Whole Serum and the
Purified Inhibitor Including Scatchard Analysis. The binding
affinities of tritium labeled steroid hormones (purchased from
DuPont NEN) to serum and the purified inhibitor were analyzed by
the ion exchange filter method (Mickelson K E and Petra P H (1974)
FEBS Lett 44, 34-38). Total DHT binding was measured with
[1,2-.sup.3H(N)] DHT at specific activity 45 Ci/mmole. Nonspecific
binding was assessed with .sup.3H-DHT plus a 100-fold molar excess
of unlabeled DHT. Specific binding was total binding minus
nonspecific binding. For B.sub.2 specific binding, the incubations
contained either .sup.3H-E.sub.2
[2,4,6,7-.sup.3H-17.beta.-estradiol] at specific activity 96
Ci/mmole or .sup.3H-E.sub.2 plus a 100-fold molar excess of DES.
Glucocorticoid specific binding was assayed with [1,2.sup.-3H(N)]
hydrocortisone at specific activity 53 Ci/mmole and
.sup.3H-hydrocortisone plus a 100-fold molar excess of unlabeled
DEX. Progesterone specific binding was assayed with
.sup.3H-progesterone [1,2-.sup.3H (n) progesterone] alone at
specific activity 92 Ci/mmole or .sup.3H-progesterone plus a
100-fold molar excess of unlabeled synthetic progestin R5020. The
use of 100-fold unlabeled steroids to determine nonspecific binding
has been discussed (Chamness G C and McGuire W L (1975) Steroids
26, 538-542). Each assay contained 0.01 M Tris-HCl, pH 7.4, with 10
mM CaCl.sub.2. The binding conditions were optimized for time and
temperature. The incubations were done in glass tubes at 34 C in a
total volume of 660 .mu.L that included 50 .mu.g/mL of the phenyl
Sepharose pools and labeled and unlabeled steroid competitor. After
two hours, the incubations were cooled to ice bath temperature and
50 to 200 .mu.L aliquots applied to each DEAE-cellulose (DE-81) ion
exchange filter (2.3-cm, Fisher) positioned in a Millipore Vacuum
Filter Manifold.RTM.. Thereafter, the filters were washed with ten
one mL portions of ice-cold Tris/CaCl.sub.2 buffer. With control
incubations minus protein, <3% of the label was retained.
Radioactivity was quantified by liquid scintillation methods
(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol
36, 410-427). To obtain K.sub.d (dissociation constant) and K.sub.a
(association constant) values by Scatchard analysis (Scatchard G
(1949) Ann NY Acad Sci 51, 660-672), the incubations began with 50
nM .sup.3H-labeled steroid hormone and included five or six serial
two-fold dilutions done both minus and plus 100-fold excesses of
unlabeled steroid for each labeled hormone concentration. Data
points were the averages of triplicate incubations. K.sub.d/K.sub.a
were obtained from plots of (bound/free) versus (bound) hormone.
Best-fit slopes were estimated with either Apple MAC computer
software or with the PC based Graph Pad program.
[0258] Protein Assay and Quantification Methods. For cell growth
assays, the protein sample volumes added to the culture medium were
.ltoreq.20%. As required, chromatography samples were concentrated
using Amicon Ultrafiltration with YM-10 (molecular weight cut-off
10,000) low protein binding membranes and nitrogen gas pressure
(Sirbasku D A et al. (1991) Biochemistry 30, 295-304). Before
assay, all fractions were dialyzed against 0.05 M Tris-HCl, pH 7.4,
with 0.15 M NaCl using Spectropor 1 membranes (molecular weight
cutoff 6,000 to 8,000). They were sterilized with 0.2 .mu.M
membrane filtration units. The protein concentrations of serum, the
ammonium sulfate precipitation and all of the conventional
chromatography fractions were estimated as one A.sub.280nM equal to
one mg/mL. For the cortisol affinity isolated proteins,
concentrations were estimated either by the Pierce BCA.RTM. method
according to the instructions supplied or the dye binding method of
Bradford (Bradford MM (1976) Anal Biochem 72, 248-254).
Trichloroacetic acid (TCA) precipitation was used with BCA to
eliminate the interfering cortisol and DHT, With TCA, deoxycholate
was used to co-precipitate the protein and ethanol/water washes to
eliminate the steroid. Human IgG was used as standard for the
colorimetric protein determinations. The protein concentration of
several lots of horse serum averaged of 30.+-.5 mg/ml. Ammonium
sulfate precipitation was carried out as described (Sirbasku D A et
al. (1991) Biochemistry 30, 295-304). Before further use, the
protein was dissolved in 0.05 M Tris-HCl, pH 7.5, containing either
10 mM CaCl.sub.2 or 0.15 M NaCl and dialyzed with Spectropor
membranes against several four-liter volumes of the same buffer or
a buffer appropriate to the next chromatography step.
III. EXAMPLES
Example 1
Identification of Steroid Hormone Receptors in MTW9/PL2 Cells
[0259] In the course of searching for what regulates the growth of
estrogen responsive breast cancer and of androgen responsive
prostate cancer, an in vitro cell culture system was developed that
would serve as an accurate model for predicting in vivo
physiological effects. An estrogen responsive rat mammary tumor
cell line, the MTW9/PL2 cell line had already been developed
(Sirbasku D A (1978) Cancer Res 38, 1154-1165). The MTW9/PL2
population is the first highly steroid hormone-responsive rat
mammary tumor cell line to be established in culture from a
carcinogen-induced tumor. These cells have been shown previously to
form estrogen responsive tumors in W/Fu rats (Sirbasku D A (1978)
Cancer Res 38, 1154-1165; Danielpour D and Sirbasku D A (1984) In
Vitro 20, 975-980; Riss T L et al. (1986) J Tissue Culture Methods
10, 133-150). Nonetheless, they were not estrogen responsive in
culture (Sirbasku D A (1978) Proc Natl Acad Sci USA 75, 3786-3790).
It was thought possible that the cells had lost the estrogen
receptors (e.g. dedifferentiation). This Example presents evidence
confirming that the cells are estrogen receptor positive and are
suitable for use in in vitro and in vivo studies.
[0260] Identification of MTW9/PL2 Cell Estrogen Receptors by
.sup.3H-E.sub.2 Binding Methods. Examining the MTW9/PL2 cell line
anew, the MTW9/PL2 population was examined for .sup.3H-E.sub.2
binding to whole cells, to determine if estrogen receptors were
present. First, the effect of temperature on .sup.3H-E.sub.2
specific binding was examined (FIG. 1). At 37.degree. C., specific
binding reached a maximum in two hours and thereafter decreased
rapidly. At 23.degree. C., specific binding reached the same
maximum but at three hours. The decay in binding at 23.degree. C.
was not as pronounced as at 37.degree. C. At 7.degree. C., the rate
of specific binding reached a stable maximum at six hours. Similar
temperature effects have been observed for the kinetics of
.sup.3H-E.sub.2 binding to MCF-7 breast cancer cells (Horwitz K B
and McGuire W L (1978) J Biol Chem 253, 8185-8191; MacIndoe et al.
(1982) Steroids 39, 245-258).
[0261] Effect of .sup.3H-E.sub.2Concentration on Binding. Next, the
effect of the concentration of .sup.3H-E.sub.2 on binding at
37.degree. C. was characterized. Specific binding was saturated by
.gtoreq.5 nM .sup.3H-E.sub.2 (insert FIG. 2). One-half saturation
occurred at 2 to 3 nM .sup.3H-E.sub.2. A Scatchard analysis
(Scatchard G (1949) Ann NY Acad Sci 51, 660-672) of .sup.3H-E.sub.2
binding also was done at 37.degree. C. (FIG. 2) (N=2). It indicated
a single class of E.sub.2 binding sites with a dissociation
constant (K.sub.d) of 2.78.times.10.sup.-9 M. This analysis
indicated 38,400 estrogen receptors per cell. These values compared
closely to a K.sub.d of 1.89.times.10.sup.-9 M and the estimated
34,000 sites per cell determined for .sup.3H-E.sub.2 binding to the
original MTW9/PL cell population in 1982 (Leland F E et al. (1982)
In: Cold Spring Harbor Conferences on Cell Proliferation, Volume 9,
Growth of Cells in Hormonally Defined Media, Sato G, Pardee A B and
Sirbasku D A, eds, Cold Spring Harbor, N.Y., pp 741-750). Plainly,
the estrogen receptor content of this permanent cell population has
remained stable over several years.
[0262] Specificity of .sup.3H-E.sub.2 Binding. The specificity of
.sup.3H-E.sub.2 binding to MTW9/PL2 cell receptors was examined.
The effects of unlabeled DES, DHT or testosterone (T) on binding of
5 nM .sup.3H-E.sub.2 at 37.degree. C. were examined. The results
from one of these experiments (N=3) are shown in FIG. 3A. At
100-fold molar excess, unlabeled DES reduced .sup.3H-E.sub.2 total
binding by 85%. Conversely, 100-fold molar excesses of either DHT
or T did not displace .sup.3H-E.sub.2 total binding significantly.
Even at 1000-fold excess, T or DHT only reduced .sup.3H-E.sub.2
total binding by 15%. Next, the effects of unlabeled progesterone
and cortisol on .sup.3H-E.sub.2 binding to MTW9/PL2 cells were
investigated under conditions similar to those used in FIG. 3B, A
100-fold excess of either progesterone or cortisol reduced
.sup.3H-E.sub.2 binding by 30 to 50%. The results of the
.sup.3H-E.sub.2 binding competition studies presented here are
nearly identical to those done with cell extracts of the original
MTW9/PL population in 1982.
[0263] Comparison of the Labeled E.sub.2 Binding Dissociation
Constants (K.sub.d) of Several Estrogen Sensitive Cell Types.
Clearly, the assays with extracts measured the same affinity
binding sites as analyses with whole cells. This offers reasonable
evidence that the standard binding technology employed in these
studies is measuring the most common form of receptor present in
cells, no matter whether whole cells are assayed or cell extracts.
The affinity of the MTW9/PL2 estrogen receptor is that which is
characteristic of the ER.alpha.. The K.sub.d of the receptor
measures the concentration of ligand that one-half saturates the
sites. In TABLE 3, the K.sub.d values for labeled E.sub.2 are
presented as reported and presumably represent the ER.alpha.. Only
when the measurements are specific for the .beta. form is the
designation (ER.beta.) included.
TABLE-US-00004 TABLE 3 Comparison of E.sub.2 Binding Affinities
Expressed as Dissociation Constants (K.sub.d) WHOLE CELLS CELL
EXTRACTS CELL LINES K.sub.d for E.sub.2 K.sub.d for E.sub.2
REFERENCES MTW9/PL2 2.78 .times. 10.sup.-9 M 1.89 .times. 10.sup.-9
M Moreno-Cuevas JE and Sirbasku DA (2000) In Vitro Cell Dev Biol
36, 410-427 MCF-7 0.58 .times. 10.sup.-9 M 1.77 .times. 10.sup.-9 M
MacIndoe JH et al. (1982) Steroids 39, 247-258 MCF-7-Mason 4.0
.times. 10.sup.-9 M Horwitz KB et al. (1978) Cancer Res 38,
2434-2437 Unfilled nuclear MCF-7-Mason 0.4 .times. 10.sup.-9 M
Horwitz KB et al. (1978) Cancer Res 38, 2434-2437 Filled nuclear
MCF-7 0.1 .times. 10.sup.-9 M Reddel RR et al. (1985) Cancer Res
45, 1525-1531 MCF-7-L 0.08 .times. 10.sup.-9 M MCF-7-M 0.07 .times.
10.sup.-9 M T47D 1.0 .times. 10.sup.-9 M Horwitz KB et al. (1978)
Cancer Res 38, 2434-2437 Unfilled nuclear T47D 4.0 .times.
10.sup.-9 M Horwitz KB et al. (1978) Cancer Res 38, 2434-2437
Filled nuclear T47D 0.11 .times. 10.sup.-9 M Reddel RR et al.
(1985) Cancer Res 45, 1525-1531 ZR-75-1 0.09 .times. 10.sup.-9 M
Reddel RR et al. (1985) Cancer Res 45, 1525-1531 ZR-75-1 1.3
.times. 10.sup.-9 M Engel LW et al. (1978) Cancer Res 38, 3352-3364
H301 1.0 .times. 10.sup.-9 M Liehr JG and Sirbasku DA (1985) In:
Tissue Culture of Epithelial Cells, Taub M, ed, Plenum, New York,
pp 205-234 H301 0.87 .times. 10.sup.-9 M Soto AM et al. (1988)
Cancer Res 48, 3676-3680 GH.sub.3 0.25 .times. 10.sup.-9 M Moo JB
et al (1982) In: Growth of Cells in Hormonally Defined Media, Vol.
9, Cold Spring Harbor, New York, pp 429-444 GH.sub.3 0.31 .times.
10.sup.-9 M Haug E et al. (1978) Mol Cell Endocrinol 12, 81-95
Prostate and 0.2 .times. 10.sup.-9 M (ER.alpha.) Tremblay GB et al.
(1997) Mol Endocrinol 11, 353-365 Ovary 0.5 .times. 10.sup.-9 M
(ER.beta.) Transfection 0.05 to 0.1 .times. 10.sup.-9 M Kuiper GC
et al. (1998) Endocrinology 139, 4252-42-63 Studies (ER.beta.
only)
[0264] TABLE 3 presents only a fraction of the estrogen binding
data available in the literature. However, the K.sub.d values
presented are representative and do show a discernable pattern. The
lowest K.sub.d from a literature search was in the range
5.times.10.sup.-11 M to 1.0.times.10.sup.-10 M for the ER.beta. and
7.times.10.sup.-11 M to 1.1.times.10.sup.-19 M for the ER.alpha..
In general, the binding affinities as estimated by K.sub.d are
lower for receptors from human cells than those from rodent lines.
It is important to note that the results presented in TABLE 3
indicate that the lower limit of measuring estrogen receptor
affinities most likely has been reached. The use of the highest
specific activity tritium labeled steroids has been optimized and
simply cannot be used to measure 10 to 100-fold lower K.sub.d
concentrations. This opens the possibility of an as yet
unrecognized ER that mediates growth effects at lower
concentrations of estrogen than either the ER.alpha. or the
ER.beta..
[0265] Effect of Temperature on .sup.3H-Progesterone Binding.
Progesterone receptors in MTW9/PL2 cells were sought using the same
series of experiments done to identify estrogen receptors. The
effect of temperature on progesterone specific binding with
MTW9/PL2 cells is shown in FIG. 4. Maximum .sup.3H-progesterone
binding at 37.degree., 23.degree. C. and 7.degree. C. occurred at
2, 4 and 6 hours, respectively. After reaching an optimum, the
binding decayed at 37.degree. C. and 23.degree. C. but not at
7.degree. C.
[0266] Effect of .sup.3H-Progesterone Concentration on Binding The
saturability of .sup.3H-progesterone binding was examined at
37.degree. C. Labeled progesterone specific binding was saturated
at .gtoreq.5 nM (insert FIG. 5). One-half saturation occurred at
0.5 to 1 nM .sup.3H-progesterone. Scatchard analysis (N=2)
identified a single class of binding sites with a K.sub.d of
1.02.times.10.sup.-9 M and yielded an estimated 26,800 sites per
cell, as shown in FIG. 5. Previous studies in 1982 with extracts of
the original MTW9/PL cell population had given a K.sub.d of
3.29.times.10.sup.-9 M for .sup.3H-progesterone binding and an
estimated 180,000 sites per cell (data not shown). Comparison of
the number of progesterone sites then and now indicates a decrease.
However, a sufficient number remain to expect progesterone specific
gene expression or growth regulation (Alexander I E et al. (1989)
Mol Endocrinol 3, 1377-1386; Keydar I et al. (1979) Eur J Cancer
15, 659-670).
[0267] Effect of Other Steroid Hormones on .sup.3H-Progesterone
Binding. The effect of non-progestins on .sup.3H-progesterone
binding was investigated (FIG. 6). As control, the binding was
studied in the presence of increasing concentrations of the
synthetic progestin R5020. A 100-fold excess of the unlabeled R5020
reduced .sup.3H-progesterone binding by 82%. A 100-fold excess of
unlabeled DHT or T reduced binding by .ltoreq.20%. A 100-fold
excess of unlabeled E.sub.2 reduced progesterone binding by a
maximum of 20% (data not shown).
[0268] Assays for Androgen and Cortisol Receptors. Experiments
(N=3) were carried out to seek specific saturable binding sites for
androgens using .sup.3H-DHT. In experiments not shown, incubation
of MTW9/PL2 cells at 37.degree. C. for two hours with .ltoreq.20 nM
labeled DHT did not reveal saturable specific binding sites.
Studies using .ltoreq.20 nM .sup.3H-cortisol did not identify
specific saturable receptors for this corticosteroid.
[0269] Estrogen effects on Progesterone Receptor Expression in
MTW9/PL2 Cells. Estrogens induction of progesterone receptors in
target cells is generally taken as strong evidence of sex steroid
responsiveness by the criteria of regulation of gene expression
(Leavitt W W et al. (1977) Ann NY Acad Sci 286, 210-255; Toft D O
and O'Malley B W (1972) Endocrinology 90, 1041-1045; Horwitz K B
and McGuire (1978) J Biol Chem 253, 2223-2228; Haslam S Z and
Shyamala G (1979) Biochem J 182, 127-131; Haslam S Z and Shyamala G
(1979) Endocrinology 105, 786-795). In the next study, it was asked
whether this was the case with MTW9/PL2 cells. The cells were grown
for five to seven days in the absence of estrogens in standard
phenol red containing D-MEM/F-12 plus 2% (v/v) charcoal-dextran
extracted (CDE) horse serum. Thereafter, they were harvested and
inoculated into phenol red free medium in 100-mm diameter dishes
containing 1.0.times.10.sup.-8 M E.sub.2. Beginning at day 0
(inoculation day) and for each of the next five days, the cells
were assayed for progesterone receptors as described (Moreno-Cuevas
J E and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427).
All results were normalized to "receptors per cell" to correct for
growth over the duration of the experiment. The number of
progesterone receptors increased 1.8-fold within two days after
exposure to E.sub.2 (FIG. 7). In a replicate experiment, the
induction was 2.0-fold in two days. The effect of estrogen
concentration on progesterone receptor induction also was evaluated
(insert FIG. 7). Maximum induction occurred at 1.0 nM E.sub.2.
These results confirm that MTW9/PL2 cells are E.sub.2 responsive by
a criterion separate from growth.
[0270] Western Analysis for Androgen, Estrogen and Progesterone
Receptors. Western immunoblotting was used to analyze the MTW9/PL2
cells for the presence of steroid hormone receptors. Nuclear and
cytosolic extracts were compared to those from negative control rat
and human diploid fibroblasts and positive control T47D and LNCaP
cells. The T47D cells have androgen (Keydar I et al. (1979) Eur J
Cancer 15, 659-670; Horwitz K B et al. (1978) Cancer Res 38,
2434-2437), progesterone (Horwitz K B et al. (1978) Cancer Res 38,
2434-2437; Horwitz K B and Alexander P S (1983) Endocrinology 113,
2195-2201; Lessey B A et al. (1983) Endocrinology 112, 1267-1274)
and estrogen (Horwitz K B et al. (1978) Cancer Res 38, 2434-2437;
Keydar I et al (1979) Eur J Cancer 15, 659-670; Soto A M et al.
(1986) Cancer Res 46, 2271-2275) receptors. The androgen receptors
of LNCaP cells previously have been characterized by labeled
hormone binding analysis (Veldscholte J et al. (1990) Biochem
Biophys Res Commun 173, 534-540; Veldscholte J et al. (1990)
Biochim Biophys Acta 1052, 187-194) and Western immunoblotting
(Prins G S et al. (1991) Endocrinology 129, 3187-3199). Although
the LNCaP cells were initially reported to not have progesterone or
estrogen receptors (Schuurmans A L et al. (1988) Int J Cancer 42,
917-922; Brolin J et al. (1992) The Prostate 20, 281-295), more
recent evidence indicates that they express significant levels of
both (Castagnetta L et al. (1995) Endocrinology 136,
2309-2319).
[0271] Western Analysis--Androgen Receptors. As shown in (FIG. 8,
top panel), nuclear and cytosolic extracts of LNCaP cells gave an
intense band at 101 kDa that was the expected mass of the androgen
receptor as determined previously by immunoblotting (Prins G S et
al. (1991) Endocrinology 129, 3187-3199; Berrevoets C A et al.
(1993) J Steroid Biochem Mol Biol 46, 731-736). It was also nearly
the same mass as the 98.5 kDa predicated by molecular cloning
(Trapman J et al. (1988) Biochem Biophys Res Commun 153, 241-248;
Faber P W et al. (1989) Mol Cell Endocrinol 61, 257-262). The bands
migrating at 79 kDa and 21 kDa may be degradation products,
non-specific reactions between the antibody and unrelated proteins,
or may represent other forms of the androgen receptor (Prins G S et
al. (1991) Endocrinology 129, 3187-3199). The T47D cells showed the
same androgen receptors as LNCaP cells. In contrast, there was
little androgen receptor in the nuclear or cytosolic extracts of
MTW9/PL2 cells. The faint band identified was the same intensity as
an equivalent component seen in the extracts of fibroblasts (FIG.
8, top panel). Fibroblasts have been reported to have low levels of
androgen receptors (Eil C et al. (1983) Clin Endocrinol 19,
223-230; Eil C et al. (1980) Steroids 35, 389-404).
[0272] Western Analysis--Progesterone Receptors. Similar
experiments were done to identify progesterone receptors with
MTW9/PL2 cells. Receptors of 79 kDa (A form) and 107 kDa (B form)
were immunostained with both the cytosolic and nuclear extracts
(FIG. 8, middle panel). Another possible form was identified at 44
kDa. The progesterone receptors of MTW9/PL2 cells were the same
molecular mass as the A and B forms from chick oviduct (Dure L S et
al. (1980) Nature (Lond) 238, 784-786; Birnbaumer M et al. (1983) J
Biol Chem 258, 1637-1644). The forms in MTW9/PL2 cells also
compared closely to the 85.6 kDa and 109.6 kDa masses reported for
progesterone receptors of rat uterus (Ilenchuk T T et al. (1987)
Endocrinology 120, 1449-1456). Furthermore, the 79 kDa component (A
form) was the more abundant of the two receptors in MTW9/PL2 cells.
This was also the case for rat uterus (Ilenchuk T T et al. (1987)
Endocrinology 120, 1449-1456), The T47D positive controls showed
the same molecular masses of progesterone receptors although there
was greater immunostaining with the cytosolic extracts than with
the nuclear preparations. This receptor distribution between
cytosol and nucleus was similar to that found when T47D cells were
examined by labeled hormone binding (Horwitz K B et al. (1978)
Cancer Res 38, 2434-2437). The T47D progesterone receptor masses
observed in the present study were similar to those reported by
others studying human breast cancer cells (Horwitz K B and
Alexander PS (1983) Endocrinology 113, 2195-2201; Lessey B A et al.
(1983) Endocrinology 112, 1267-1274; Horwitz K B et al. (1985)
Recent Prog Hormone Res 41, 249-316). Additionally, the LNCaP cells
showed intense nuclear extract staining for the same forms of
progesterone receptors seen in extracts of MTW9/PL2 and T47D cells.
No progesterone receptors were identified in the negative control
rat or human fibroblasts.
[0273] Western Analysis--Estrogen Receptors. In the final study,
the estrogen receptors in MTW9/PL2 were sought (FIG. 8, bottom
panel). The major form of estrogen receptor in MTW9/PL2 cells was
molecular mass 67 kDa. Two presumed degradation products of 50 kDa
and 17 kDa were also observed. The results with MTW9/PL2 cells were
in agreement with the expected mass of the rat estrogen receptor
estimated at 67 kDa by molecular cloning (Koike S et al (1987)
Nucleic Acid Res 15, 2499-2513). The extracts from T47D and LNCaP
cells showed a similar estrogen receptor pattern. Control
fibroblasts showed no estrogen receptors. Initially, the band
identified at 50 kDa was thought to be a degradation product of the
67 kDa intact form of ER.alpha. (Moreno-Cuevas J E and Sirbasku D A
(2000) In Vitro Cell Dev Biol 36, 410-427); however, it may be an
ER.beta. because that new receptor has a mass of 54 kDa (Enmark E
et al. (1997) J Clin Endocrinol Metab 82, 4258-4265). The 50 kDa
band may also represent a variant form of ER.beta. (Gustafsson
J-.ANG. and Warner M (2000) J Steroid Biochem Mol Biol 74,
245-248). Alternately, this band may represent a new estrogen
receptor that regulates growth, hereby designated as estrogen
receptor gamma (ER.gamma.).
[0274] Thus, it is concluded that another positive acting ER exists
in the MCF-7 and T47D cells and its function is dominant and
sustains growth related gene expression even with the inhibitory
ER.alpha. present. The existence of two ER receptors is also
indicated in an older study of the growth of the GH.sub.4C.sub.1
rat pituitary tumor cells in culture (Amara J F and Dannies P S
(1983) Endocrinology 112, 1141-1143). These investigators
demonstrated a biphasic effect of E.sub.2 on these cells. At
picomolar concentrations, E.sub.2 caused growth. At higher
concentrations, E.sub.2 induced prolactin production secretion and
inhibited growth. If two receptors are operating, the growth
receptor is more sensitive to E.sub.2 whereas the ER regulating
gene expression (e.g. prolactin mRNA production) is activated by
higher concentrations of estrogen. This same biphasic action of
estrogen on the growth of T47D human breast cancers cells has also
been noted (Chalbos D et al. (1982) J Clin Endocrinol Metab 55,
276-283). Low concentrations promoted growth, whereas higher levels
were inhibitory. Indeed, a biphasic effect also was noted with the
MCF-7 cell line (Soto A M and Sonnenschein C (1985) J Steroid
Biochem 23, 87-94). When this observation is coupled with the clear
statements of Soto et al (Soto A M et al. (1986) Cancer Res 46,
2271-2275) that "the free estradiol levels needed for maximum
response are significantly lower than estrophilin (i.e. ER.alpha.)
K.sub.ds", there is further support for the view that an ER exists
that regulates growth and is more estrogen sensitive (i.e. lower
K.sub.d) than the classical ER.alpha.. While those investigators
conclude that the results exclusively supported their estrocolyone
hypothesis, and excluded ER.alpha. as the positive growth
regulator, there was no recognition of the possibility of a much
higher affinity receptor different than ER.alpha.. Finally, there
is one other issue that has perplexed endocrinologists and cancer
biologists for several years. Breast cancer is sometimes treatable
with high doses of estrogen (Segaloff A (1981) Banbury Report 8,
229-239). If the ER.alpha. is the only growth mediator, one is
forced into many other postulates to explain this observation
(Reese C C et al. (1988) Ann NY Acad Sci 538, 112-121). Indeed,
this may actually represent evidence that full occupation of
ER.alpha. is inhibitory and that another receptor is the positive
signal.
[0275] Variant estrogen receptors have been identified previously
by others. For example from the estrogen growth responsive T47D
human breast cancer cell line, there have been three isoforms of
the ER.alpha. identified in one study (Wang Y and Miksicek R J
(1991) Mol Endocrinol 5, 1707-1715) and another three in a
different study (Graham M L et al (1990) Cancer Res 50, 6208-6217).
With another two estrogen growth responsive human breast cancer
cell lines, the MCF-7 and ZR-75-1, another ER.alpha. variant was
identified that lacked the entire exon 4 of the receptor (Pfeffer U
et al. (1993) Cancer Res 53, 741-743). Variant receptors have also
been identified from human breast cancer biopsy specimens (Murphy L
C and Dotzlaw H (1989) Mol Endocrinol 3, 687-693). Another
truncated variant of ER.alpha. acts as a natural inhibitor of the
action of the wild-type ER.alpha. (i.e. unchanged receptor) (Fuqua
S A et al. (1992) Cancer Res 52, 483-486). Another type of variant
has received wide attention because it has constitutive
transcriptional activity without the steroid hormone ligand bound
(Fuqua S A et al. (1991) Cancer Res 51, 105-109). Even normal human
breast epithelial cells show several natural variants of ER.alpha.
(Yang J et al. (2000) Endocrine 12, 243-247). When all of these
results are considered as a group, it is clear that different forms
of the ER.alpha. are possible in cells. It is reasonable to
conclude that an alternate form of ER.alpha., possibly formed by
alternate splicing, or possibly arising from an as yet unrecognized
gene, may regulate estrogen dependent/responsive tumor cell growth.
Upon further investigation ER.gamma. may prove to be such a
variant.
[0276] Whatever mechanism is proposed for the action of the steroid
hormone (i.e. on growth), it can be seen from the data presented
herein, and subsequently reported elsewhere (Sirbasku D A and
Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446), it
takes a significant period to reverse the effects of the inhibitor.
This process cannot be simply due to a rapid effect on
transcription caused by steroid hormones (e.g. via a known estrogen
receptor). Cellular metabolic events, including the transformation
of E.sub.2 to an active steroid metabolite, may provide the growth
regulating ligand for one of the "orphan" estrogen receptors. An
alternative possibility is that the receptor may be activated by
metabolites formed from cholesterol metabolism (Gustafsson J-.ANG.
(1999) Science (Wash D.C.) 284, 1285-1286).
[0277] Discussion of Example 1. The evidence presented verifies
that the MTW9/PL2 cells are estrogen receptor positive (ER.sup.+)
with a significant number of progesterone receptors and possibly
low levels of androgen receptors. The estrogen receptor content and
affinity characteristics of the MTW9/PL2 cells indicate appropriate
stability for use as a testing standard and for commercial
applications. The long-term stability of this cell line in culture,
without alteration of its cell properties, is further discussed in
Example 3. These results and the information provided above, show
that this cell line is a unique asset for combination in vitro and
in vivo modalities that can be applied to evaluate a multitude of
compounds or preparations having, or potentially having, hormone or
antihormone activities.
Example 2
Preparations of Steroid Hormone Depleted Serum
[0278] Three Methods. In this example, evaluations of three methods
for preparing steroid hormone depleted serum are described. The
primary purpose was to prepare serum that supported large magnitude
sex steroid growth effects in culture and to identify the
dose-response concentrations that cause the effects, as
demonstrated in Examples that follow. This meant preparing serum
with .ltoreq.5 pg/mL estrogen (and other steroid hormones). This
concentration corresponds approximately to the lower limit of
detection of steroids by radio immunoassay. The methods tested
included (A) a two-step charcoal/dextran extraction of serum at
34.degree. C., (B) a one-step charcoal extraction at 56.degree. C.,
and (C) a one step treatment with Amberlite.TM. XAD.TM.-4 resin at
37.degree. C. One advantageous result established by this Example
is that sera prepared according to the preferred methods contain
significant amounts of active immunoglobulin inhibitors, in
contrast to previously known steroid depleted sera.
[0279] (A) Charcoal-Dextran Extraction at 34.degree. C.
[0280] 1. Preparation of the charcoal/dextran mixture. Activated
charcoal, untreated powder (100 to 400 mesh), was obtained from
Sigma (Catalog No. C5260). This preparation was done at room
temperature. The powder (30 g) was suspended in 600 mL of water and
stirred for 20-30 minutes at room temperature. The water used to
wash and suspend the charcoal was a purified source made by reverse
osmosis/ion exchange treatment/charcoal filtration/0.2 .mu.m pore
diameter filtration/distillation into glass (only) containers.
Next, 3.0 g of Dextran T70 (Pharmacia) was dissolved in 300 mL of
water, added to the charcoal suspension with stirring, and the
mixture stirred for 30-60 min at room temperature, preferably 60
min. The mixture was then washed with about 6-8 liters of distilled
water in a sintered glass funnel (2000 mL, ASTM 40-60, C#36060).
This wash removes impurities as well as fine particles of charcoal
that cannot be separated from serum by centrifugation. The
charcoal-dextran retentate was suspended in a final volume of 300
mL of distilled water to yield a suspension of 100 mg/mL charcoal
and 10 mg/mL dextran. Preferably the mixture is stirred vigorously
for about an hour, and then stored at room temperature for no more
than about 2-3 weeks prior to use.
[0281] 2. Charcoal-dextran extraction at 34.degree. C. of horse
serum (CDE-horse serum). This serum in 500 mL sterile bottles was
removed from the freezer (-17.degree. C.) and thawed at 4.degree.
C. for 24 to 48 hours. Alternatively, fresh serum could be used.
The thawed serum (still in the 500 mL sterile bottles) was placed
in an orbital shaker water bath (Lab-Line Orbit Shaker Water Bath)
equilibrated at 34.degree. C. The serum was incubated at 140 RPM
for 45-60 minutes to reach 34.degree. C. Approximately 250 mL
portions of the 34.degree. C. serum (total volume about 1 liter)
were transferred to one-liter Erlenmeyer flasks and tightly capped
with aluminum foil. These were incubated for 20-30 minutes
(preferably 30 minutes) in the 34.degree. C. orbital shaker water
bath at a medium-high rotation speed. Thereafter, 25 mL of the
charcoal/dextran suspension was added to each flask. The
charcoal-dextran suspension was stirred at room temperature while
removing the 25 mL aliquot. The final charcoal concentration in
each flask was 10 mg/mL, and the final concentration of dextran was
1 mg/mL. After addition of the charcoal-dextran mixture to each
flask, the extraction mixtures were shaken at 140-160 RPM at
34.degree. C. for two hours. After this, the mixture was cooled on
ice and the charcoal removed by centrifugation at 10,000.times.g
for about 60 minutes at room temperature. In some preparations the
temperature of the mixture gradually warmed to about 40.degree. C.
during centrifugation. The supernatants were pooled in a two-liter
beaker and 275 mL portions of the supernatant (serum) transferred
to fresh one-liter Erlenmeyer flasks. These were then incubated in
the orbital shaker water bath at 34.degree. C. for 20-30 minutes
(preferably 30 min) to re-equilibrate the temperature. A second
extraction was done by addition of a fresh aliquot (about 14 mL) of
the charcoal-dextran suspension. This re-extraction mixture was
incubated with shaking for another 2 hours at 34.degree. C. The
final charcoal concentration during this extraction was about 5
mg/mL. Afterward the bulk of the charcoal was removed by
centrifugation, as before. In some preparations the temperature of
the mixture reached about 41.degree. C., without harming the
quality of the serum. The supernatants were collected into a
two-liter beaker and filtered through 5 .mu.m pore diameter filters
to remove residual charcoal. If it was considered necessary for
particular preparations (for example, due to charcoal darkening
serum) the serum was also filtered with 0.45 .mu.m Millipore
filters. These filtrations were done with plastic reusable filter
holders and light vacuum. The steroid hormone depleted serum was
then sterilized using 0.2 .mu.m pore diameter filters. After
sterilization, aliquots of about 26 mL were dispensed into sterile
glass (50 mL) bottles or sterile 50 mL polypropylene tubes and
stored frozen at -17.degree. C. Although 34.degree. C. is preferred
in the above-described regime, and provides the best results,
satisfactory depletion of steroid hormones can be obtained over the
temperature range of about 30 to 37.degree. C. The 2 hour
incubation times for the extraction and re-extraction mixture (at
34.degree. C.) are preferred, but a time range of 30 minute to 3
hours could also be used with success, employing longer incubation
times for the lower temperatures within the 30-37.degree. C. range.
A .+-.25% variation in the charcoal concentration used for each
extraction had no detrimental effects on the final product.
[0282] (B) Charcoal-dextran Extraction at 56.degree. C. The
preparation of 56.degree. C. charcoal-dextran extracted serum was
done as described (Sirbasku D A and Kirkland W L (1976)
Endocrinology 98, 1260-1272; Riss T L and Sirbasku D A (1989) In
Vitro Cell Dev Biol 25, 136-142; Kirkland W L et al. (1976) J Natl
Cancer Inst 56, 1159-1164). Frozen serum was thawed for four hours
at 37.degree. C. followed by incubation in an orbital shaker water
bath at medium-high speed at 56.degree. C. for 15 minutes. The same
charcoal-dextran mixture described above was used in this
extraction. One-tenth volume (warmed to 56.degree. C.) was added to
the serum. Incubation was continued with shaking at 56.degree. C.
for 15 minutes. Thereafter, the flasks were cooled in an ice bath
and the charcoal removed by centrifugation at 8,000.times.g and
filtration with 5.0 or 0.45 .mu.m pore membrane filters. The serum
was 0.2 .mu.m pore filter sterilized and stored at -17.degree.
C.
[0283] (C) Amberlite.TM. XAD.TM.-4 Resin Treatment. In a different
procedure carried out to free CBG of storage cortisol, XAD resin
has been used to remove the steroid by incubation for 5 hrs at room
temperature (A. M. Nakhla, et al. (1988) Biochem. Biophys. Res.
Commun. 153, 1012-1018). Described as such, this method removed
only about 80% of cortisol from the purified protein. Careful
application of that method failed to yield serum suitable for the
purpose of this study. As an alternative to preparing steroid
hormone depleted serum by charcoal-dextran extraction, horse serum
was treated by incubation with Amberlite.TM. XAD.TM.-4 nonionic
polymeric absorbent (Aldrich, Catalog. No. 21, 648-8; or Sigma
Catalog No. XAD-4 37380-42-0). Specifically, a 500 mL bottle of
horse serum was thawed at 37.degree. C. and divided into 250 mL
portions that were each in a one-liter Ehlenmeyer flask. To each
flask was added 25 grams of moist XAD-4 resin. The mixtures of
serum and resin were then incubated with shaking in a rotary
Labline Orbital Shaker water bath at 34.degree. C. at about
two-thirds of the maximum rate for 24 hours (speed adjusted to
control foaming). This extraction can be done at temperatures from
30.degree. C. to 37.degree. C. At 30.degree. C., the extraction
requires 24 to 36 hours. At 37.degree. C., it requires 18-24 hours
to be complete. The 34.degree. C. and 37.degree. C. procedures are
preferred. Each flask was tightly capped with aluminum foil and
taped. After 24 hrs, the resin is allowed to settle by gravity, the
supernatant decanted, and then vacuum filtered using a glass fiber
filter in a Buchner funnel. The resulting serum was filter
sterilized using 0.2 .mu.m pore filter units. Aliquots of about 26
mL were frozen at -17.degree. C. in 50 mL sterile bottles or
tubes.
[0284] The charcoal method described above is readily applicable to
one to five liter volumes of serum per preparation. With use of
.ltoreq.50 mL per test substance, this is an adequate supply. To
prepare larger volumes of serum (i.e. .gtoreq.20 liters) for
extensive testing programs or commercial applications, the
charcoal-dextran methods will preferably employ industrial
filtration or other separation equipment to remove the carbon after
each extraction. The XAD-4 resin method as presented is adaptable
to one to five liters for testing purposes. For industrial
applications, where .gtoreq.20 to 100 liter batches are customarily
required, the resin method is preferred because of the need for
only one separation after extraction. However, where "foaming" of
the serum protein is to be avoided completely, charcoal extraction
is superior. The materials cost for charcoal-dextran has an
advantage when economy is a major consideration. It is less
expensive than XAD-4 resin on a per liter basis, although the resin
is commercially available at low cost when purchased in large
amounts (i.e. .gtoreq.50-100 kilograms).
[0285] Discussion of Example 2. Each of the methods presented have
advantages, depending on the particular needs and desires of the
user. The scale procedures described are useful to prepared
sufficient serum for testing of hormone activities or antihormone
activities or evaluation of toxicity of compounds in cell culture
assays. This 34.degree. C. method has been used to prepare CDE
human serum, porcine serum, rat serum, hamster serum, ovine serum,
fetal bovine serum, new born bovine serum (0 to 10 days old), young
donor bovine serum (10 days to 6 moths old) young adult bovine
serum (300 to 900 lbs), fetal horse serum, chicken serum, turkey
serum, dog serum, goat serum, rabbit serum and monkey serum.
Subsequent Examples demonstrate how these stripped sera are
preferably employed. The results demonstrate the broad utility of
the method of preparing charcoal-dextran extracted serum for
testing of cell lines from many species using homologous serum
assays. From these results it can also be readily appreciated that
these methods are applicable to testing of veterinary medicine
samples or compounds of significance to domestic animals, as well
as any application where a steroid hormone stripped serum is
used.
Example 3
Cancer Cell Line MTW9/PL2 Exhibits Estrogen Responsive Growth in
34.degree. C. Charcoal-Dextran Extracted Serum
[0286] Estrogenic Effects in Cultures Supplemented with CDE-horse
Serum. Unless otherwise stated, references in this and the
following Examples to "CDE-horse serum" refer to the 34.degree. C.
charcoal-dextran extraction process described in above. The
MTW9/PL2 cells were assayed for E.sub.2 responsiveness in cultures
supplemented with increasing concentrations of CDE-horse serum
(FIG. 9A). Concentrations .gtoreq.5% (v/v) promoted growth.
Typically within seven days cell numbers increased from 6,000 per
dish to more than 200,000 in 2 to 5% serum. This most likely
resulted from stimulation by serum-borne growth factors as well as
the mitogenic effect of transferrin (Danielpour D et al (1988) In
Vitro Cell Dev Biol 24, 42-52; Riss T L and Sirbasku D A (1987) In
Vitro Cell Dev Biol 23, 841-849; Riss T L et al. (1986) J Tissue
Culture Methods 10, 133-150). As serum concentrations exceeded 5%
(v/v), the effects of the growth promoters were counteracted by a
serum-borne inhibitor(s). At serum concentrations of 30 to 50%
(v/v), growth was completely inhibited. Usually only seed density
cell numbers were found after seven days in cultures containing 50%
(v/v) CDE-horse serum. In contrast, the presence of
1.0.times.10.sup.-8 M E.sub.2 completely reversed the serum
dependent inhibition. In cultures supplemented with 20 to 50% (v/v)
CDE-serum plus 1.0.times.10.sup.-8 M E.sub.2, cell numbers were
.gtoreq.400,000 per dish. Logarithmic quantifying of cell growth
was done by converting the cell number data in FIG. 9A into CPD. A
plot of these values is shown in FIG. 9B. The estrogenic effect is
also presented. In FIG. 9B, the difference was maximum at 30% (v/v)
CDE-horse serum. It was a 6.14 CPD or a 70-fold (i.e. 2.sup.CPD or
2.sup.6.14) increase in cell numbers in response to E.sub.2. In
randomly selected replicate experiments (N=9) done over a two-year
period with different preparations of CDE-horse serum, the average
maximum estrogen effect.+-.SEM was 6.43 CPD.+-.0.49 (range 5.63 to
7.22). This was an 86-fold (2.sup.6.43) estrogenic effect. The
modal concentration of serum that promoted maximum E.sub.2 effects
was 40% (range 20 to 50%).
[0287] Morphology of MTW9/PL2 Cells Growing in CDE-horse
Serum.+-.E.sub.2. The morphology of the cells growing under the
conditions was examined. The photomicrographs (Moreno-Cuevas J E
and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427) show
cells growing under optimum conditions in medium with 2.5% (v/v)
CDE-horse serum with and without E.sub.2, respectively. The
presence of the hormone had no effect on the appearance of the
cultures. The cells grew in clusters in suspension and had the same
morphology reported earlier for the parent MTW9/PL line grown in
medium containing 10% (v/v) fetal bovine serum (Sirbasku D A (1978)
Cancer Res 38, 1154-1165). When the concentration of CDE-horse
serum was increased to 50% (v/v) without steroid, many fewer cells
were present. Despite the near complete inhibition of growth, the
morphology of the cells was the same as in rapidly growing
cultures. Estrogen addition to this same medium caused substantial
growth. The morphology of the estrogen-stimulated cultures in 50%
serum was equivalent to that seen with or without estrogen in 2.5%
serum. The inhibitor had no effect on the microscopic morphology of
MTW9/PL2 cultures nor did it affect cell-cell adhesion or
cell-surface adhesion.
[0288] Estrogen Reversibility of the Growth Inhibition Caused by
CDE-horse Serum. It was examined whether inhibition caused by
CDE-horse serum was reversible even after several days in culture
(FIG. 10). The MTW9/PL2 cells were seeded into medium containing
50% (v/v) CDE-horse without E.sub.2 and cell numbers monitored
daily. Growth ceased within 48 hours; thereafter cell numbers
remained static. In parallel cultures, addition of E.sub.2 on days
two, four, and six after seeding caused resumption of growth (after
a lag period) at nearly the same rate as cultures that received
hormone at the time of inoculation. These results show that the
cells survived in the presence of the inhibitor without E.sub.2 for
at least six days.
[0289] Storage Stability of CDE-horse Serum. TABLE 4 shows the
effect of storage temperature on the estrogen mediating activity of
CDE-horse serum. The assays were done with MTW9/PL2 cells as shown
in FIGS. 9A and 9B. Stability was assessed by four criteria: (i)
the concentration of serum needed to give an estrogenic effect of
2.5 CPD (i.e. ED.sub.2.5), (ii) the percent serum needed for the
maximum estrogenic effect, and the magnitude of the estrogenic
effects (CPD) at (iii) 20% and (iv) 30% serum. CDE-horse serum was
stable at 23.degree. C. for three weeks without loss of activity as
assessed by all four criteria. Storage at 4.degree. C. was
detrimental within 24 days as measured by the CPD at 20% and 30%
(v/v) serum concentrations. Longer storage at 4.degree. C. was not
advisable. Storage at -17.degree. C. was most effective; the
activity was unchanged even after 90 days. In experiments not
shown, repeated freeze-thaw cycles caused an appreciable loss of
inhibitor activity.
TABLE-US-00005 TABLE 4 Summary of the Effects of Serum Storage
Temperature on Activity. % Serum Maximum needed for E.sub.2 Induced
2.5 CPD CPDs (ED.sub.2.5) (% serum, CPD CPD of E.sub.2 v/v, at 20%
at 30% Days of Induced for the (v/v) (v/v) Storage growth maximum)
serum serum Storage at 23.degree. C. 1 2.1 4.9 (10%) 5.0 3.2 3 5.2
5.4 (20%) 6.2 5.2 6 5.0 4.2 (10%) 3.5 0.9 14 2.9 6.0 (10%) 4.3 2.6
23 4.0 6.3 (10%) 3.9 2.5 Storage at 4.degree. C. 1 1.8 5.9 (10%)
4.9 4.0 7 6.8 5.7 (20%) 6.4 5.4 15 3.8 4.1 (30%) 5.5 4.2 24 5.3 5.3
(10%) 1.0 2.8 44 3.0 4.8 (5%) 0.04 0.26 55 2.2 5.0 (5%) 0.00 0.24
90 >50 2.1 (5%) 0.30 0.40 Storage at -17.degree. C. 1 2.6 5.2
(10%) 5.0 3.1 7 4.0 5.8 (30%) 6.8 5.8 44 3.3 5.8 (20%) 6.0 5.4 90
6.1 5.2 (30%) 6.2 5.9
[0290] Dose-Response Effects of Steroid Hormones in CDE-horse
Serum. The dose effects of a number of steroid hormones were
evaluated with MTW9/PL2 cells in medium containing 50% (v/v)
CDE-horse serum. The results of one of these studies (N=3) are
presented in FIG. 11. Estrogens were the most effective mitogens.
Their order of potency was E.sub.2>E.sub.1>E.sub.3. This
relative potency was expected based on the affinities of these
steroids for the estrogen receptors of other target tissues (Clark
J H and Markaverich B M (1983) Pharmacol Ther 21, 429-453). The
cell numbers in dishes containing 1.0.times.10.sup.-13 M E.sub.2
were 32-fold (p<0.01) higher than in dishes without the hormone.
Concentrations of 1.0.times.10.sup.-12 to 1.0.times.10.sup.-11 M
E.sub.2 promoted 6.73 CPD that was a 110-fold estrogenic effect in
seven days. The ED.sub.50 of E.sub.2 was about 0.5 to
1.0.times.10.sup.-12 M. Using E.sub.1 and E.sub.3, optimum growth
was achieved at 1.0.times.10.sup.-9 and 1.0.times.10.sup.-8 M,
respectively. In experiments not shown, the mitogenic potency of
the synthetic estrogen DES was assessed. At 1.0.times.10.sup.-8 M,
it caused the same growth as saturating concentrations of the
natural estrogens. The DES effect was 6.98 CPD in seven days that
was a 126-fold (2.sup.6.98) increase in cell number. The next most
potent hormone was DHT. It caused significant (p<0.05) growth at
super physiologic concentrations .gtoreq.1.0.times.10.sup.-8 M.
Progesterone also caused significant growth, but only at
supraphysiological concentrations .gtoreq.1.0.times.10.sup.-7 M.
Cortisol was ineffective at concentrations up to
1.0.times.10.sup.-5 M.
[0291] Estrogen Mitogenic Effects with MTW9/PL2 cells in CDE-serum
from Several Species. Serum from species other than horse were
examined to determine they also possessed estrogen reversible
inhibitory activity with rat MTW9/PL2 cells. These experiments are
shown in FIG. 12. All of the sera tested were charcoal dextran
extracted at 34.degree. C. CDE-porcine (FIG. 12A), and CDE-human
serum (FIG. 12B) showed patterns nearly identical to that of
CDE-horse serum. The maximum estrogenic effects with both sera were
six to seven CPD (N=3). CDE-rat serum also showed the same pattern
of estrogen reversible growth inhibition (FIG. 12C). CDE-ovine
serum showed estrogen reversible inhibition equivalent to CDE rat
serum (data not shown). With serum from rats, the maximum
estrogenic effect was four to five CPD (N=4). CDE-bovine serum
(adult donor herd) displayed the same pattern of activity as other
sera (FIG. 12D). CDE-fetal bovine serum showed a different pattern
(FIG. 12E). Even at 40% (v/v), there was no inhibition. With some
batches of this serum, there was no inhibition even at 50% (N=2).
With others (N=2), inhibition was found. In these experiments, the
estrogenic effects reached three to four CPD in 50% (v/v)
CDE-serum. Even with this variability, fetal bovine serum has less
activity than the serum from the adults of this species. The assays
with CDE-fetal horse serum (N=3) showed inhibition at 50% (v/v)
that was not reversible by 10 nM E.sub.2 (FIG. 12F). The present
study shows very clearly that estrogen growth effects were not
found in medium with 5% (v/v) fetal bovine serum, as also reported
(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol
36, 410-427). In fact, charcoal-dextran treated fetal bovine serum
at concentrations of .gtoreq.40% (v/v) does not cause inhibition of
estrogen target cell growth in culture.
[0292] Technical Conditions for Demonstrating Estrogen
Responsiveness in Culture and Evidence for a Serum-borne Inhibitor.
Conditions that permit the observation of very large magnitude
estrogen mitogenic effects with the permanent MTW9/PL2 cell line in
culture are defined herein. As mentioned in the Background of the
Invention, most existing rat mammary tumor cell lines are not
suitable for use in evaluating hormone responsiveness in vivo
because they are derived from outbred animals. This problem was
overcome by developing the MTW9/PL2 rat mammary tumor cell line
from a carcinogen-induced hormone responsive tumor (i.e. the MT-W9A
tumor), first induced and transplanted in an inbred W/Fu rat as
described (MacLeod R M et al. (1964) Cancer Res 75, 249-258). The
MTW9/PL2 cells form hormone responsive tumors when implanted in
these rats (Sirbasku D A (1978) Cancer Res 38, 1154-1165;
Danielpour D and Sirbasku D A (1984) In Vitro 20, 975-980; Riss T L
et al. (1986) J Tissue Culture Methods 10, 133-150). In culture,
the MTW9/PL2 cells showed the same hormone responsiveness expected
of rat and human breast epithelial cells, as shown herein and
subsequently reported (Moreno-Cuevas J E and Sirbasku D A (2000) In
Vitro Cell Dev Biol 36, 410-427; Sirbasku D A and Moreno-Cuevas J E
(2000) In Vitro Cell Dev Biol 36, 428-446; Moreno-Cuevas J E and
Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464).
[0293] The effects of the steroid hormones in culture were the same
as described for the growth of the original MT-W9A tumor in W/Fu
rats (MacLeod et al. (1964) Endocrinology 75, 249-258) and tumor
formation by the parental MTW9/PL cell line in this same strain of
rats (Sirbasku D A (1978) Cancer Res 38, 1154-1165). The present
embodiment is the first established cell line derived from a
carcinogen induced rat mammary tumor that continues to show large
magnitude growth responses to estrogens, progesterone and androgens
even after extended periods in culture, preferably when cultured
under the conditions disclosed herein. Thyroid hormone
responsiveness has also been demonstrated for MTW9/PL cells (Leland
F E et al. (1981) In: Functionally Differentiated Cell Lines, Sato
G, ed, Alan Liss, New York, pp 1-46). Of the other important
hormones known to influence the growth of the original MT-W9A
tumor, only prolactin remains to be investigated. Prolactin is not
mitogenic for the parental MTW9/PL cells under serum-free defined
conditions (Danielpour D et al. (1988) In Vitro Cell Dev Biol 24,
42-52). Continuing investigations are directed toward evaluating
the possibility that prolactin also reverses the effects of the
serum-borne inhibitor or otherwise acts as a cytokine to influence
the production of immunoglobulins in breast and other mucosal
tissues. The development of this cell line now permits not only
sensitive steroid hormone growth analysis in culture, but also
direct comparisons to the effectiveness of the same test substances
in animals. No other such rat mammary system is currently
available.
[0294] MTW9/PL2 Receptor Not Lost in Culture. The present results
showing an average 86-fold MTW9/PL2 cell number increase in seven
days in response to physiological concentrations of E.sub.2 have
several important technical implications. Most notably, they
contradict many earlier explanations for why estrogen stimulated
cell growth has been difficult to demonstrate in culture.
Originally, the lack of estrogenic effects in culture was thought
to be due to a dedifferentiation of cells that resulted in a loss
of functional receptors or some other aberration that disrupted the
growth response. In light of the present Disclosure, this
explanation now seems very unlikely. The present results show the
presence of similar levels of estrogen receptors in both the
original MTW9/PL cell line reported in 1982 and the current
MTW9/PL2 cells. Analyses made by others showing estrogen receptors
in established cell lines in culture (Horwitz K B et al. (1978)
Cancer Res 38, 2434-2437; Haug E (1976) Endocrinology 104, 429-437;
Soto A M et al. (1988) Cancer Res 48, 3676-3680; Keydar I et al.
(1979) Eur J Cancer 15, 659-670; Engel L W et al. (1978) Cancer Res
38, 3352-3364) also mitigate against this explanation. Furthermore,
the estrogen receptors of the MCF-7 cells were functional based on
the demonstration of estrogen inducibility of the progesterone
receptor (Horwitz K B and McGuire W L (1978) J Biol Chem 253,
2223-2228). As with the human breast cancer cells, the MTW9/PL2
line was also significantly estrogen responsive by this criterion.
When all of the available data is considered in light of the
presently disclosed observations, the notion that long-term culture
necessarily leads to loss of functional estrogen receptors is laid
to rest. A major advantage of the MTW9/PL2 line is its long-term
stability permitting series analyses over long periods of time
without concern for cell property changes.
[0295] Prolonged Steroid Hormone Retention by Culture Cells. It has
been suggested that prolonged retention of estrogens might be the
reason for a lack of responsiveness of target cells in culture
(Strobl J S and Lippman M E (1979) Cancer Res 39, 3319-3327).
Investigators have reported that the half-life of loss of
specifically bound .sup.3H-E.sub.2 from MCF-7 cells was about 24
hours at 37.degree. C. (Strobl J S and Lippman M E (1979) Cancer
Res 39, 3319-3327). Cells from stock cultures grown in
untreated/steroid hormone containing serum were proposed to retain
stimulating levels of estrogens. Even several washes over 78 hours
did not attenuate the problem (Strobl J S and Lippman M E (1979)
Cancer Res 39, 3319-3327). Conversely, the studies herein did not
identify this problem. All the assays reported here were done with
cells taken directly from cultures grown in steroid hormone
containing serum (e.g. fetal bovine serum). After trypsinization of
the MTW9/PL2 cells from stock culture, only three careful washes
with serum-free D-MEM/F-12 were performed before initiating the
growth assays. The results in FIG. 11 show clearly that
1.0.times.10.sup.-12 M E.sub.2 caused significant MTW9/PL2 cell
growth. Also, the results in FIG. 10 show that MTW9/PL2 cells cease
proliferation within 48 hours of starting a growth assay. These
observations either support the conclusion that prolonged steroid
hormone retention by cells is not as serious an issue as first
proposed or are evidence that the technical processes described
herein to prepare cells for assays have eliminated this problem.
With regard to the present investigation, all cell lines studied
showed this same property when prepared by the same technical
process for growth assays.
[0296] Merits of Charcoal Extraction. Other investigators have
challenged the use of charcoal extraction to deplete serum of
steroid hormones. It has been stated that this procedure absorbs or
otherwise alters serum to make it ineffective (Amara J F and
Dannies P S (1983) Endocrinology 112, 1141-1143; Wiese T E et al.
(1992) In Vitro Cell Dev Biol 28A, 595-602). To counter this
problem, either individual lots of untreated serum were used to
seek estrogenic effects (Wiese T E et al. (1992) In Vitro Cell Dev
Biol 28A, 595-602), or serum was prepared from animals after
endocrine ablation surgery (Amara J F and Dannies P S (1983)
Endocrinology 112, 1141-1143). One of the best examples of use of
surgically depleted serum came from the study of the
GH.sub.4C.sub.1 rat pituitary cells (Amara J F and Dannies P S
(1983) Endocrinology 112, 1141-1143). They were highly B.sub.2
responsive in medium supplemented with the serum from a gelded
horse (Amara J F and Dannies P S (1983) Endocrinology 112,
1141-1143). However, experience with serum derived by these methods
has not been as positive. For example, this issue was inventigated
in 1976 with the related GH.sub.3C.sub.14 rat pituitary tumor cell
line (Kirkland W L et al. (1976) J Natl Cancer Inst 56, 1159-1164),
and found that serum from ovariectomized sheep or adrenalectomized
and ovariectomized sheep did not support estrogen effects.
Furthermore, unextracted serum from different species can at times
support limited estrogenic effects. However, the estrogenic effects
are of lower magnitude than those in the CDE-serum described
herein. Based on the observation that CDE-serum from a number of
species was very effective, it seems highly unlikely that the
now-disclosed preferred 34.degree. C. procedure is deleterious.
However, it is clear that the 56.degree. C. charcoal method caused
a temperature dependent loss of the inhibitor (FIG. 26). The
presently described CDE-serum provides greater consistency and
reproducibility than the other proposed approaches (Amara I F and
Dannies P S (1983) Endocrinology 112, 1141-1143; Wiese T E et al.
(1992) In Vitro Cell Dev Biol 28A, 595-602). Another advantage is
that these results do not dependent significantly on the lot of
serum purchased. Furthermore, CDE-serum consistently provides
larger magnitude estrogenic effects than serum obtained by either
of the other approaches discussed above.
[0297] Steroid Hormone Conjugates are Non-problematic. While
charcoal treatment can be expected to remove the major classes of
steroid hormones from serum, there is a question about its effect
on the more soluble and potentially active conjugates. It has been
reported that hydrolysis of estrogen sulfates provided free
estrogens in human breast cancer cell cultures (Vignon F et al.
(1980) Endocrinology 106, 1079-1086). This abrogated the effects of
exogenous E.sub.2. Although the previous investigations did not
address estradiol sulfate, it was shown that more than 95% of
estrone sulfate and estradiol glucuronide were removed from serum
by a single 56.degree. C. charcoal extraction (Sirbasku D A and
Kirkland W L (1976) Endocrinology 98, 1260-1272). Additionally, in
previous studies MTW9/PL cells were incubated with tritium labeled
estradiol glucuronide for up to 24 hours under cell culture
conditions and found no organic solvent extractable free steroid.
Both past and current results indicate that the impact of the
estrogen conjugates has been overestimated. In the present study,
no precautions were taken to remove the conjugated forms of
estrogens from any of the sera tested. Despite this, it was found
that many different types of serum were effective after charcoal
extraction at 34.degree. C. Thus, it is concluded that removal of
steroid conjugates by digestion or any procedure beyond charcoal
treatment is unnecessary. This is a further advantage of the new
CDE method because the additional treatment to remove the steroid
conjugates could be prohibitive for larger scale production than a
few liters.
[0298] Plastic Product Use for Cell Culture. The present studies
were done with plastic ware made of polystyrene. Plastic is
manufactured using alkylphenols (Platt AE (1978) In: Encyclopedia
of Chemical Technology, Kirk R E, Othmer D F, eds, 3.sup.rd
Edition, Volume 26, Wiley, New York, pp 801-847). One of these
compounds, p-nonyl-phenol, has been reported to be estrogenic for
MCF-7 cells in culture (Soto A M et al. (1991) Environ Health
Perspect 92, 167-173). This xenobiotic most likely is present in
the cultures used in these studies. No precautions were taken to
exclude compounds leaching from plastic. In fact, the bioassay
procedures herein are done with polystyrene plastic ware and
culture dishes almost exclusively. If there had been a significant
contamination of the medium by such compounds, the estrogenic
effects reported in this study should not have been seen or should
have been markedly attenuated. An advantage of the assay systems
described herein is that they have no need for expensive and or
exotic substitutes for the common plastic ware used in cell culture
laboratories to conduct bioassays. Also, the CDE-serum can be
stored and shipped for commercial use in plastic containers without
concern for creation of plastic-induced artifacts.
[0299] Discussion of Example 3. Using the present 34.degree. C. CDE
and XAD-4 serum, estrogen responsiveness can be demonstrated in rat
tumor cells, where no such responsiveness had previously been
demonstrated. Further, because estrogen responsiveness and binding
affinity can now be compared, data indicating the existence of an
heretofore unknown estrogen receptor have been generated.
Example 4
Estrogen Responsive Growth of Additional Rodent and Human Cell
Lines in Charcoal-Dextran Extracted Horse and Human Serum
[0300] In addition to the above-described studies using the
MTW9/PL2 rat mammary tumor cell line, several other cell lines were
employed to define the conditions for demonstrating estrogen and
androgen responsive cell growth. Established cell lines from a
number of different steroid hormone target tissues were selected
for growth regulation analysis under those defined conditions.
[0301] Estrogen Mitogenic Effects with Established ER.sup.+ Rodent
Tumor and Human Carcinoma Cells in CDE-horse Serum. In the first
study of this series, the three GH rat pituitary tumor cell lines
were examined for estrogenic effects in CDE-horse serum. This was
considered important in light of their clear responsiveness to many
hormones (Tashjian A H Jr (1979) Methods Enzymol 58, 527-535).
Furthermore, these cells are from a tissue that grows coordinately
with mammary tissue in castrated rats administered exogenous
estrogens. As described above, this suggested a common regulation
mechanism. FIG. 13 shows an estrogenic effect .gtoreq.5 CPD with
GH.sub.4C.sub.1 cells in 10 days. The results with GH.sub.3 and
GH.sub.1 cells ranged between 4.0 and 5.2 CPD in 10 to 14 day
assays (data not shown). The same progressive estrogen reversible
CDE-serum inhibition was demonstrable with both rat mammary and rat
pituitary tumor cells in CDE-horse serum. To confirm the
effectiveness CDE-horse serum with human cells, the ZR-75-1 breast
cancer line was selected because of previous attempts to
demonstrate its estrogen responsiveness in culture (Allegra J C and
Lippman M E (1978) Cancer Res 38, 3823-3829; Darbre P D et al.
(1984) Cancer Res 44, 2790-2793; Darbre P et al. (1983) Cancer Res
43, 349-355). The ZR-75-1 cells showed the same CDE-serum caused
estrogen reversible inhibition as seen with rodent cell lines in
this serum. In 14 days, there was a 3.65 CPD (i.e. 12.5-fold)
estrogenic effect (FIG. 14). This was a greater response than
recorded in the ZR-75-1 cell studies cited above. Of all of the
cell lines studied, the MCF-7A was the least estrogen responsive
even in 50% CDE-horse (FIG. 15). The estrogenic effect was 2.65 CPD
in 10-12 days. This was still significant (p<0.01) as a
2.sup.2.65 or 6.3-fold increase in cell number caused by estrogen.
The present serum-derived inhibitor exhibited biological activity
exactly opposite the estrogen reversible inhibitors described by M
Tanji et al. (Tanji M et al. (2000) Anticancer Res. 20, 2779-2783;
Tanji Metal. (2000) Anticancer Res. 20, 2785-2789).
[0302] Additional Cell Lines Evaluated. Evidence is presented
herein that the MCF-7K, T47D, LNCaP, and H301 cells are highly sex
steroid hormone responsive in CDE-horse serum.
[0303] Kinetics of Estrogen Responsive Growth in CDE Serum
Containing Medium. In the experiments presented in FIGS. 16A and
16B, ER.sup.+ cell growth was measured daily for 15 days to
determine cell growth kinetics.+-.E.sub.2. The results with the
T47D line are presented as characteristic of human cells. When
evaluated in medium with partially inhibitory 20% (v/v) CDE horse
serum, the effect of E.sub.2 on cell number increase was not
apparent until after 4 days (FIG. 16A). Increasing the
concentration of CDE serum to 50% (v/v) further delayed the effect
of E.sub.2 (FIG. 16B). Clearly, whatever mechanism is proposed for
the action of the steroid hormone, it takes a significant period to
reverse the effects of the inhibitor. This process cannot be simply
due to a rapid effect on transcription caused by steroid hormones.
The interaction of .sup.3H-E.sub.2 with intracellular estrogen
receptors saturates in .ltoreq.1 hour at 37.degree. C. (Horwitz K B
and McGuire W L (1978) J Biol Chem 253, 8185-8191; MacIndoe J H et
al. (1982) Steroids 39, 245-258; Moreno-Cuevas J E and Sirbasku D A
(2000) In Vitro Cell Dev Biol 36, 410-427), while de novo hormone
induced protein synthesis requires only a few hours (Beato M (1989)
Cell 56, 335-344). Based on a growth lag of .gtoreq.4 days, it is
likely that steroid hormones initiate a cascade of signaling events
that are more complex than recognized today. To demonstrate that
the lag period was related to the inhibitor, T47D growth was
monitored daily in D-MEM/F-12 supplemented with 10% (v/v) fetal
bovine serum (FIG. 16A). This concentration of fetal bovine serum
shows no inhibitor (Moreno-Cuevas J E and Sirbasku D A (2000) In
Vitro Cell Dev Biol 36, 410-427). Cell growth in medium with fetal
bovine serum showed at most a one or two day lag period.
[0304] Effect of CDE-human Serum on Estrogen Responsive Cell
Growth. The next study examined whether human serum was a source of
inhibitor for steroid hormone sensitive cell lines from different
species and tissues. The results confirm that CDE-human serum
contains approximately the same level of inhibitor as CDE-horse
serum. Results are shown with T47D human breast cancer cells (FIG.
17A), LNCaP human prostatic carcinoma cells (FIG. 17B), MTW9/PL2
rat mammary tumor cells (FIG. 17C), two GH rat pituitary tumor cell
lines (FIGS. 17D and 17E), and the Syrian hamster H301 kidney tumor
cells (FIG. 17F). All lines showed the same biphasic response to
CDE-human serum. Low concentrations (i.e. S10%) promoted growth
whereas higher concentrations (i.e. .gtoreq.20%) progressively
inhibited growth. Only the absolute magnitudes of the estrogenic
effects varied. Replicate assays with MCF-7A, MCF-7K and ZR-75-1
cells gave the same outcomes (data not shown). The experiments
reported thus far herein support the conclusion that the inhibitor
is ubiquitous in mammals and is not species specific, also
subsequently reported (Sirbasku D A and Moreno-Cuevas (2000) In
Vitro Cell Dev Biol 36, 428-446).
[0305] Dose-response Effects of Steroid Hormones with Human Breast
Cancer Cells in CDE Serum. The studies presented thus far have
assessed estrogen effects using 10 nM E.sub.2. Although 10 nM
saturates growth, it is decidedly at the high boundary of
physiological. It is important to note that circulating estrogens
in non-pregnant females are generally thought to be in the range of
10.sup.-8 to 10.sup.-10 M (Clark J H et al. In: Williams Textbook
of Endocrinology (1992), Saunders, Philadelphia, pp 35-90). Tissue
concentrations are generally conceded to be lower due to SHBG that
reduces the "free" or "active" form of sex steroid hormones (Rosner
W (1990) Endocr Rev 11, 80-91). The next studies with T47D cells
determined the effective concentration ranges for the three most
common estrogens and compared these to non-estrogen steroid
hormones. FIG. 18 shows an analysis with T47D cells in D-MEM/F-12
containing 50% (v/v) CDE horse serum for 14 days. Estrogens were
the only physiologically relevant activators of T47D growth. As
expected from previous studies with breast cancer cells (Lippman M
E et al. (1977) Cancer Res 37, 1901-1907; Jozan S et al. (1979) J
Steroid Biochem 10, 341-342; Katenellenbogen B S (1980) Annu Rev
Physiol 42, 17-35) and other estrogen target tissues (Clark J H and
Markaverich B M (1983) Pharmacol Ther 21, 429-453), their order of
effectiveness was E.sub.2>E.sub.1>E.sub.3. E.sub.2 caused
significant (p<0.05) growth when present at 1.0.times.10.sup.-14
M and optimum growth at 1.0.times.10.sup.-10 M. Higher
concentrations were not inhibitory. The ED.sub.50 concentration of
E.sub.2 was .ltoreq.1.0.times.10.sup.-13 M. It is noteworthy that
even E.sub.3 was remarkably potent. Others also had commented that
E.sub.3 was more potent than expected (Lippman M E et al. (1977)
Cancer Res 37, 1901-1907). This observation may have special
significance because breast cancers that appear during pregnancy
can be particularly life threatening. Human maternal plasma has
greatly elevated levels of E.sub.3 during the last trimester of
pregnancy. Testosterone and DHT promoted growth but only at
supraphysiological concentrations (FIG. 18). Other investigators
have suggested that supraphysiological concentrations of androgens
act through the ER of human breast cancer cells (Zava D T and
McGuire W L (1978) Science (Wash D.C.) 199, 787-788). However,
another group has reported no effect of androgens on human breast
cancer cell proliferation (Soto A M and Sonnenschein C (1985) J
Steroid Biochem 23, 87-94). In the present study, progesterone and
cortisol were completely ineffective with T47D cells (FIG. 18).
Others have also reported negative results with these hormones and
human breast cancer cells (Schatz R W (1985) J Cell Physiol 124,
386-390; Soto A M and Sonnenschein C (1985) J Steroid Biochem 23,
87-94). The data presented in this Disclosure support the
conclusion that the new CDE serum culture conditions yield
physiologically relevant information.
[0306] Dose-response Effects of Steroid Hormones with Rat Pituitary
Tumor Cells in CDE Serum. The GH family of related cell lines
responds to a number of different classes of hormones (Amara J F
and Dannies P S (1983) Endocrinology 112, 1141-1143; Tashjian A H
Jr et al. (1970) J Cell Biol 47, 61-70; Tashjian A H Jr (1979)
Methods Enzymol 58, 527-535; Haug E (1979) Endocrinology 104,
429437; Schonbrunn A et al. (1980) J Cell Biol 85, 786-797;
Sorrentino J M et al. (1976) J Natl Cancer Inst 56, 1159-1164;
Ramsdell J S (1991) Endocrinology 128, 1981-1990; Hayashi I et al.
(1978) In Vitro 14, 23-30; Faivre-Bauman A et al. (1975) Biochem
Biophys Res Commun 67, 50-57). These cells also form steroid
hormone responsive tumors in W/Fu rats (Sorrentino J M et al.
(1976) J Natl Cancer Inst 56, 1149-1154). The GH.sub.4C.sub.1
strain was selected as an example for this next study because of
its marked E.sub.2 responsiveness in culture (Amara J F and Dannies
P S (1983) Endocrinology 112, 1141-1143; Sirbasku D A and
Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446; Sato H
et al. (1991) In Vitro Cell Dev Biol 27A, 599-602) and estrogen
requirement for tumor formation in rats (Riss T L and Sirbasku D A
(1989) In Vitro Cell Dev Biol 25, 136-142). The dose-response
effect of steroid hormones with GH.sub.4C.sub.1 rat pituitary tumor
cells in 50% CDE-horse serum was analyzed next. FIG. 19 shows the
results of these experiments. All three major estrogens promoted
growth. The potencies of E.sub.2 and E.sub.1 were equivalent
whereas E.sub.3 was substantially less effective. Even at
supraphysiologic concentrations, E.sub.3 did not promote the
saturation densities seen with E.sub.2 and E.sub.1. The lowest
concentration of E.sub.2 and E.sub.1 that gave significant
(p<0.05) growth was 1.0.times.10.sup.-12M. The ED.sub.50 of
E.sub.2 was .ltoreq.1.0.times.10.sup.-11 M. Optimum growth required
supraphysiological concentrations (i.e. 1.0.times.10.sup.-8 M) of
E.sub.2 and E.sub.1. In the present studies, the biphasic effect of
E.sub.2 reported by Amara and Dannies (Amara J F and Dannies P S
(1983) Endocrinology 112, 1141-1143) was not found. This may be
explained by the different conditions used to conduct the assays.
The matter of assay culture conditions with ER.sup.+ cells has been
discussed (Zugmaier G et al. (1991) J Steroid Biochem Mol Biol 39,
681-685). Certainly however, the low E.sub.2 concentration for
ED.sub.50 still speaks to a problem with ER.alpha. as the mediating
receptor. Furthermore, the pattern reported in this Example is
consistent with physiological facts. Tumor formation by GH cells
was greater in W/Fu rats treated with 25 mg estrogen pellets than
in untreated intact sexually mature females (Sorrentino J M et al.
(1976) J Natl Cancer Inst 56, 1149-1154). Without a doubt,
supraphysiological levels of estrogens were most effective in vivo.
In contrast to estrogens, progesterone and cortisol had no effect
on GH.sub.4C.sub.1 growth in culture (FIG. 19). These steroids also
did not promote GH cell tumor growth in vivo (Sorrentino J M et al.
(1976) J Natl Cancer Inst 56, 1149-1154). The findings with
androgens and GH.sub.4C.sub.1 cell growth shown in FIG. 19 revealed
another important contribution made by the work in CDE serum
supplemented cultures described herein. It has been shown before
that T promoted GH tumor growth in vivo (Sorrentino J M et al.
(1976) J Natl Cancer Inst 56, 1149-1154). It was proposed at that
time that T was effective because it was metabolized to estrogens
in the rat. Therefore, it was expected that T would be ineffective
in culture. The results in FIG. 19 confirm this expectation. In
this case, the new culture methods permitted resolution of an issue
arising from previous in vivo observations. The dose-response
results in FIG. 19 fortify a conclusion arrived at earlier that
cell culture can be used to uncover physiologically important new
information not accessible by in vitro methods (McKeehan W L et al.
(1990) In Vitro Cell Dev Biol 26, 9-23).
[0307] Dose-response Effects of Steroid Hormones with Hamster
Kidney Tumor Cells in CDE Serum. To explore the utility of the new
culture conditions further, steroid hormone effects on the H-301
Syrian hamster kidney tumor cells in D-MEM/F-12 containing 50%
(v/v) CDE-horse serum were investigated. This cell line has two
unique characteristics. First, tumors form from H301 cells in
Syrian hamsters only in response to exogenous estrogens (Sirbasku D
A and Kirkland W L (1976) Endocrinology 98, 1260-1272). It is very
important to note that normal physiologic levels in intact adult
female hamsters do not support tumor formation (Sirbasku D A and
Kirkland W L (1976) Endocrinology 98, 1260-1272). It is thought
that progesterone from the normal estrus cycle suppresses growth in
response to physiological levels of estrogen (Kirkman H and Robbins
M (1959) In: National Cancer Institute Monograph No. 1, National
Institutes of Health, Bethesda, Md.). Second, these cells only form
tumors in response to estrogens. The other major classes of steroid
hormones are ineffective in vivo. The relative effectiveness of the
three estrogens with H301 cells was investigated (FIG. 20). Their
potency was E.sub.2>E.sub.1>E.sub.3. As with rat tumor cells,
E.sub.3 was markedly less effective than E.sub.2 or E.sub.1.
E.sub.2 and E.sub.1 required 1.0.times.10.sup.-11 M and
1.0.times.10.sup.-10 M, respectively, to achieve significant
(p<0.05) growth. The ED.sub.50 concentration of E.sub.2 is about
5 to 9.times.10.sup.-11 M. As expected from in vivo results
(Sirbasku D A and Kirkland W L (1976) Endocrinology 98, 1260-1272),
this concentration was higher than for the rat pituitary tumor
cells (FIG. 19) or rat mammary tumor cells (FIG. 11). In fact, they
were as much as 100 to 1000-fold higher than for human breast
cancer cells (FIG. 18). In other tests shown in FIG. 20,
progesterone, cortisol, T and DHT were all inactive. The higher
estrogen concentrations required for significant growth of the
H-301 cells in culture, coupled with the marked estrogen
specificity, indicate that the medium conditions used in this study
yielded physiologically germane results.
[0308] Dose-response Effects of Steroid Hormones with Human
Prostatic Carcinoma Cells in CDE Serum. In the final dose-response
study, the potency of several classes of steroid hormones with the
LNCaP cells was analyzed. This was done in D-MEM/F-12 containing
50% (v/v) CDE horse serum. Due to a point mutation which permits
binding of both androgen and non-androgen hormones to the AR of
LNCaP cells (Veldscholte J et al. (1990) Biochem Biophys Res Commun
173, 534-540; Veldscholte J et al. (1990) Biochim Biophys Acta
1052, 187-194), the Inventor expected several classes of steroids
to promote growth, albeit at concentrations compatible with their
known affinities for the mutated receptor. This proved to be the
case, as shown in FIG. 21. DHT and E.sub.2 were the most potent
steroids. In fact, they were equipotent. Both caused significant
(p<0.05) growth at 1.0.times.10.sup.12 M. Contrary to other
reports (Schuurmans A L et al. (1988) The Prostate 12, 55-64;
Sonnenschein C et al. (1989) Cancer Res 49, 3474-3481; de Launoit Y
et al. (1991) Cancer Res 51, 5165-5170; Lee C et al. (1995)
Endocrinology 136, 796-803; Kim I et al. (1996) Endocrinology 137,
991-999), the present study did not find that high concentrations
of DHT inhibited LNCaP growth. The potency of the steroid hormones
tested was
DHT=E.sub.2>T>E.sub.1>progesterone>E.sub.3>cortisol.
As potencies declined, saturation densities also decreased. The
observed relative steroid potencies agreed with those of others
(Belanger C et al. (1990) Ann NY Acad Sci 595, 399-402), and
correlated with the expected binding of the various classes of
steroids to the mutated AR of the LNCaP line. Additionally, the
presently disclosed methods offered the advantage of greater growth
responses. The results in FIG. 21 not only lend support to the view
that cultures containing a high concentration of CDE serum yield
physiologically relevant information, but they also demonstrate
that the new charcoal extraction method disclosed herein
effectively depletes several classes of steroid hormones.
[0309] Comparisons of ED.sub.50 and K.sub.d as Evidence Supporting
a New ER Designated ER.gamma.. As mentioned in the Background of
the Invention, it is important to recognize that if a given
estrogen receptor is in fact a mediator of estrogen-induced growth,
then the steroid hormone concentrations required for one-half
maximum growth (i.e. ED.sub.50), or for optimum growth (i.e.
ED.sub.100), should be about the same. According to the theory of
hormone binding, the K.sub.d value represents the steroid
concentration that one-half saturates the existing receptors. The
following TABLE 5 summarizes the ED.sub.50 concentrations required
for a one-half maximum growth and the corresponding lowest K.sub.d
measured for the same or closely related cell lines:
TABLE-US-00006 TABLE 5 Comparisons of ED.sub.50 and K.sub.d as
Evidence Supporting a New ER Designated ER.gamma. Fold-higher
K.sub.d ED.sub.50 for E.sub.2 Concentration Induced Compared to
Cell Line Growth K.sub.d for E.sub.2 ED.sub.50 for Growth MTW9/PL2
1 .times. 10.sup.-12 M 1.8 .times. 10.sup.-9 M 1.8 .times. 10.sup.3
T47D 1 .times. 10.sup.-12 M 0.11 .times. 10.sup.-9 M 1.1 .times.
10.sup.3 GH.sub.4C.sub.1 1 .times. 10.sup.-11 M 0.25 .times.
10.sup.-9 M 25 H301 9 .times. 10.sup.-11 M 0.87 .times. 10.sup.-9 M
10
Clearly, to seek the new ER.gamma., the rat mammary or human breast
cells will be the best sources based on the differences between the
ED.sub.50 growth concentrations and the K.sub.d values for
ER.alpha. or ER.beta.. Because the ER.beta. was first obtained from
rat tissues, the MTW9/PL2 cells will be the preferred source of
ER.gamma..
[0310] One preferred application supported by the data in TABLE 5
is the use of the ER.gamma. for diagnosing and/or screening for
breast cancer. Measurement of the ER.gamma. specifically will
provide a more accurate determination of estrogen receptor status
and therefore permit more precise modeling of the therapy for each
patient. ER.gamma. will be identified by immunohistochemical
methods, labeled ligand binding with very high specific activity
isotopes, and by PCR and other molecular biology analyzes. Other
methods will also be applied. Similar analyses are expected to be
applicable to other estrogen receptor related or estrogen receptor
containing mucosal cancers including ovarian, uterine, vaginal,
cervical, colon, lung, stomach, pituitary, liver, pancreas, skin
and kidney, as described in co-owned, concurrently filed U.S.
patent application Ser. No. ______ (Atty. Dkt. No.
1944-00800)/PCT/US2001/______ (Atty. Dkt. No. 1944-00801) entitled
"Compositions and Methods for the Diagnosis, Treatment and
Prevention of Steroid Hormone Responsive Cancers," which is hereby
incorporated herein by reference.
[0311] The dose-response results presented in FIGS. 11, and 18
through 21 demonstrate the usefulness of the extracted sera, assays
and cell lines with regard to assessment of estrogenic activity or
androgenic activity in industrial, commercial, environmental,
medicinal, or other medical samples where activity measurement is
required at concentrations below the usual levels detectable by
radioimmunoassay. The sensitivity of this bioassay is unique.
[0312] Benign prostatic hypertrophy (BPH) is among the most common
afflictions of older men. About 50% of 60-year old men have BPH. At
85 years about 90% of men have BPH (Berry S J et al. (1984) J Urol
132, 474-479). There is a general view that estrogens may be
important in BPH (Henderson D et al. (1987) Steroids 50, 219-229;
Nakhla A M et al. (1994) Proc Natl Acad Sci USA 91, 5402-5405). The
paradox involved is that as men age androgen levels fall and SHBG
rises. These work in concert to further limit available androgen
(Davidson J M et al. (1983) J Clin Endocrinol Metab 57, 71-77;
Tenover J S et al. (1987) J Clin Endocrinol Metab 65, 1118-1126).
Furthermore, as part of the weight gain with age, estrogens become
more prominent in older men. Although it has been suggested that
estrogens cause LNCaP cell growth via an estrogen receptor, it
remains to be proven conclusively. Nonetheless, the ER.gamma. may
be expressed in BPH and prostatic cancer and therefore its use as a
diagnostic tool and a site for development of new antihormone
treatments of these diseases has great potential.
[0313] Discussion of Example 4. The results presented in this
Example have special significance with regard to support for the
conclusion that a new ER.gamma. regulates growth and is activated
by more than 10-fold at lower concentrations of E.sub.2 than
expected of the classical ED.alpha.. Example 4 also demonstrates
the utility of assays using 34.degree. C. CDE serum for
demonstrating estrogen responsive cell growth in a variety of
tissues.
Example 5
Thyroid Hormone Growth Effects in CDE-Horse Serum Prepared at
34.degree. C.
[0314] This Example demonstrates that not only steroid hormone but
also thyroid hormone growth effects can be demonstrated in cell
growth assays using the present 34.degree. C. CDE serum.
[0315] Thyroid Hormone Responsive Pituitary Tumor Cell Growth in
CDE-Serum Prepared at 34.degree. C. GH rat pituitary tumor cells
are highly thyroid hormone responsive in serum-free defined medium
(Eby J E et al. (1992) Anal Biochem 203, 317-325; Eby J E et al.
(1992) J Cell Physiol 156, 588-600; Sato H et al. (1991) In Vitro
Cell Dev Biol 27A, 599-602). An example of this responsiveness with
the GH.sub.3 line is shown in FIG. 22. However, in serum-free
defined medium, these cells are not E.sub.2 responsive when T.sub.3
is omitted from the medium (FIG. 23). During evaluation of the role
the GH cell lines in CDE-serum, in D-MEM/F-12 with 2.5% (v/v)
CDE-horse serum, T.sub.3 caused substantial growth of the
GH.sub.4C.sub.1, GH.sub.1 and GH.sub.3 rat pituitary tumor cell
lines (FIG. 24). However, at 50% (v/v) CDE-horse serum, only
supraphysiologic concentrations of thyroid hormone showed growth
effects (FIG. 25). Nonetheless, the 34.degree. C. CDE method
described in the preceding Examples is clearly functional to
demonstrate both steroid hormone and thyroid hormone growth effects
in culture. It is known that the thyroid hormone receptor is a
member of a superfamily of receptors that also includes the steroid
hormone receptors (Evans R M (1988) Science (Wash D.C.)
240:889-895). Testing of substances expected to have thyroid
hormone like activity can be performed with the GH cell lines in
the presence of low concentrations of CDE-serum.
[0316] Discussion of Example 5. The removal of thyroid hormones
from serum has been described before using the Bio-Rad.TM.
AG-1.times.8 ion exchange resin (Samuels H H et al. (1979)
Endocrinology 105, 80-85). Removal of T.sub.3/T.sub.4 by the
AG-1.times.8 method relies on their negative carboxylic acid charge
at neutral pH. However, ion exchange does not remove the
uncharged/hydrophobic steroid hormones. This Example demonstrates
that the 34.degree. C. CDE method described herein is more
effective than the AG-1 X8 method previously known.
Example 6
Effect of 56.degree. C. Versus 34.degree. C. CDE-Horse Serum on
MTW9/PL2 Cell Growth
[0317] Previously, unsuccessful attempts were made to identify
estrogen responsive tumor cell growth in cultures supplemented with
serum depleted of steroid hormones by a 56.degree. C. charcoal
extraction procedure (Kirkland W L et al. (1976) J Natl Cancer Inst
56, 1159-1164; Sirbasku D A and Kirkland W L (1976) Endocrinology
98, 1260-1272; Sirbasku D A (1978) Proc Natl Acad Sci USA 75,
3786-3790; Leland F E et al. (1982) In: Cold Spring Harbor
Conferences on Cell Proliferation, Volume 9, Growth of Cells in
Hormonally Defined Media, Cold Spring Harbor, N.Y., pp 741-750;
Liehr J G and Sirbasku D A (1985) In: Tissue Culture of Epithelial
Cells, Taub M, ed, Plenum, New York, pp 205-234; Riss T L and
Sirbasku D A (1989) In Vitro Cell Dev Biol 25, 136-142). In light
of the data presented in the foregoing Examples, it appears that
the 56.degree. C. method was the major problem. The high
temperature may have inactivated the inhibitor. Alternatively,
because the 56.degree. C. method was done for only a brief period,
it may not have sufficiently removed the steroid hormones. Clearly,
from the results presented above, even modest levels of residual
estrogens can promote growth. This latter possibility seemed likely
because the 56.degree. C. method removed only somewhat more than
90% of the serum steroid hormones (Kirkland W L et al. (1976) J
Natl Cancer Inst 56, 1159-1164; Sirbasku D A and Kirkland W L
(1976) Endocrinology 98, 1260-1272). To reevaluate this problem,
the E.sub.2 effects on MTW9/PL2 cell growth were compared in medium
supplemented with either 34.degree. C. or 56.degree. C. CDE-horse
serum. As expected, the assay with control 34.degree. C. treated
serum, prepared as described in Example 2, showed maximum
estrogenic effects of 6.01 CPD (FIG. 26). By comparison, the same
lot of serum that had been charcoal extracted at 56.degree. C.
showed a maximum estrogenic effect of only 2.96 CPD (FIG. 26). When
34.degree. C. CDE-serum was either charcoal extracted again at
56.degree. C., or heated at this temperature for 20 minutes without
charcoal, B.sub.2 induced growth was reduced to only 1.47 and 2.01
CPD, respectively (FIG. 26). A typical assay from which these
results were calculated is shown in (FIG. 26, insert). This
experiment demonstrates that 56.degree. C. treatment results in the
loss of the inhibitory activity in serum. It should be noted that
many investigators routinely "heat inactivate" serum at 56.degree.
C. for 20 to 30 minutes to destroy complement. The results indicate
that this heating should be avoided when the serum is to be used in
cell culture experiments testing steroid hormone growth
responsiveness.
[0318] Discussion of Example 6. This example makes clear some major
differences between the serum-borne inhibitor presently disclosed
and those previously described. Specifically, exposure to heat can
inactivate or alter the effect of inhibitors. For example, U.S.
Pat. Nos. 4,859,585 (Sonnenschein) and 5,135,849 (Soto) describe an
inhibitor that was derived from heat inactivated (i.e. 56.degree.
C. treated) serum and thereafter depleted of its endogenous
estrogens and androgens by a 37.5.degree. C. single step
charcoal-dextran procedure. The facts of that method are also
stated in a publication (Soto A M and Sonnenschein C (1984) Biochem
Biophys Res Commun 122, 1097-1103). In light of the results
presented in FIG. 26, it is likely that the inhibitor described by
Sonnenschein and Soto is a different molecular entity, as further
illustrated in Examples which follow. Among other differences, the
serum used to isolate the present active inhibitor has not been
inactivated by exposure to heat.
Example 7
Demonstration of Estrogenic Effects in XAD-4 Resin Treated Horse
Serum
[0319] Horse serum depleted of steroid hormones by XAD-4.TM.,
prepared as described in Example 2.C, was assayed to determine if
it demonstrated estrogen reversible inhibition of ER.sup.+ cancer
cell growth in culture. FIG. 27 shows the effects of XAD-4 treated
horse serum.+-.10 nM E.sub.2 with the MTW9/PL2 cell line.
Unmistakably, the pattern of cell response was the same as seen
with CDE-horse serum prepared as described in Example 2. A. At 50%
XAD-4 serum (v/v), an estrogenic effect of 5.2 CPD was observed in
7 days. FIG. 28 shows a similar experiment with T47D cells after 14
days. At 50% (v/v) XAD-4 treated serum, an estrogenic effect with
T47D cells of 5.3 CPD was observed. The magnitudes of the
estrogenic effects with both cell lines were the same as observed
with CDE-horse serum. Because both MTW9/PL2 and T47D cells are
sensitive to picomolar concentrations of estrogen, it was evident
that the XAD-4.TM. resin treatment effectively removed the
endogenous sex steroids present in serum.
[0320] Discussion of Example 7. There is no previous report of the
preparation of steroid depleted serum by this resin treatment
method. As indicated in Example 2, the XAD-4.TM. treatment method
has particular applicability for the industrial preparation of
large volumes of steroid hormone depleted serum, and will allow the
commercial supply of steroid depleted serum at reasonable cost. A
preferred application for this steroid hormone stripped serum is in
the biotechnology industry, in which cell culture is used to
produce medically and otherwise commercially significant proteins
and cellular products. Steroid hormone depleted serum has
applicability beyond the ER.sup.+ and AR.sup.+ cells described in
this report. For example, hybridoma cells are the sources of many
important monoclonal antibodies. Depletion of steroids from the
serum used to grow these cells will increase cell viability
(cortisol is a potent cytotoxic agent) and therefore increase
product yield. These and other applications of the XAD-4.TM.
treated serum for both commercial and diagnostic testing as well as
for industrial production of valuable cellular products are
foreseen.
Example 8
Testing of Substances for Estrogenic Activity
[0321] The purported estrogenic effects of phenol red were tested
and proven to be unfounded. Further, the methods described in this
Example exemplify methods that are generally effective for
assessing the steroidogenic activity of any substance.
[0322] Phenol Red as an Estrogen. It is widely believed that the
phenol red indicator in tissue culture medium acts is a weak
estrogen (Berthois Y et al. (1986) Proc Natl Acad Sci USA 83,
2496-2500). In the first report describing the phenol red problem,
the indicator itself was thought to act as an estrogen (Berthois Y
et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500). At the
concentration in standard culture media (e.g. D-MEM/F-12 is 8.1
mg/mL or 22.9 .mu.M), it was believed to stimulate ER.sup.+ cell
growth nearly as well as natural estrogens. Simply stated, this
meant that exogenous estrogens would have no effect because the
cells were already nearly completely stimulated. Further work by
the original investigators later demonstrated that it was the
lipophilic impurities in phenol red that were the true culprits
(Bindal R D et al. (1988) J Steroid Biochem 31, 287-293). The
chemical structure of one was determined to be
bis(4-hydroxyphenyl)[2-(phenoxysulfonyl)phenyl]methane (Bindal R D
and Katzenellenbogen J A (1988) J Med Chem 31, 1978-1983).
Interfering amounts of the impurities were identified in many
different commercially available preparations of phenol red (Bindal
R D et al. (1988) J Steroid Biochem 31, 287-293; Bindal R D and
Katzenellenbogen J A (1988) J Med Chem 31, 1978-1983). These
investigators concluded that many, if not most, phenol red
containing culture media had sufficient contaminants to at least
partially mask estrogenic effects. Despite the wide acceptance of
phenol red as an estrogen, experience has shown differently.
Instead, large estrogen mitogenic effects have been observed in
phenol red containing culture medium with ER.sup.+ MCF-7 human
breast cancer cells, T47D human breast cancer cells, MTW9/PL2 rat
mammary tumor cells, GH rat pituitary tumor cells, H301 Syrian
hamster kidney tumor cells and the androgen receptor positive
(AR.sup.+) and ER.sup.+ LNCaP human prostatic carcinoma cells, as
shown herein and subsequently reported (Moreno-Cuevas J E and
Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D
A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36, 428-446,
incorporated by reference). In these studies, even when phenol red
was present, estrogen-inducible cell number increases of 8 to
80-fold were observed. These responses were as large or larger than
any previously reported. They exceeded any reported in phenol red
free medium. Also, growth without the natural hormone was very
limited even with phenol red present (Moreno-Cuevas J E and
Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 410-427; Sirbasku D
A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol 36,
428-446).
[0323] Phenol Red Indicator is a "Red Herring". Phenol red was
further evaluated. Head-on comparisons of the growth of nine
different ER.sup.+ cell lines representing four target tissues and
three species in medium with and without phenol red were performed
(Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev Biol
36, 447-464). These studies were designed to specifically test
various aspects of published reports that phenol red is estrogenic.
Considering the results of these head-on comparisons, new
conclusions have been reached about the effects of phenol red in
culture, especially as they are relevant to experimental conditions
available today to most investigators. Even more important, the
test assays show the methods that can be used to determine if any
commercial preparation or other source material possesses
estrogenic activity. To do this, nine cell lines were employed in
the tests. Five different experimental protocols were used to
investigate phenol red. First, E.sub.2 responsive growth of all
nine ER.sup.+ cells lines was compared in medium with and without
the indicator. Second, using representative lines it was asked if
phenol red was mitogenic in indicator free medium. The
dose-response effects of phenol red were compared directly to those
of E.sub.2. Third, it was asked if tamoxifen inhibited growth
equally in phenol red containing an indicator-free medium, which
would also confirm or refute a report indicating that antiestrogen
effects should be seen only in phenol red containing medium.
Fourth, it was asked if phenol red displaced the binding of
.sup.3H-E.sub.2 using ER.sup.+ intact human breast cancer cells.
Fifth, E.sub.2 and phenol red were compared as inducers of the
progesterone receptor using a human breast cancer cell line. All of
the experiments reported (Moreno-Cuevas J E and Sirbasku D A (2000)
In Vitro Cell Dev Biol 36, 447-464) support the conclusion that the
concentration of phenol red contaminants in a standard culture
medium available today is not sufficient to cause estrogenic
effects. The real issue of how to demonstrate estrogenic effects in
culture resides elsewhere than phenol red. Demonstration of sex
steroid hormone mitogenic effects in culture depends upon
conditions that maximize the effects of a serum-borne inhibitor, as
described in foregoing Examples. When the effects of the inhibitor
are optimized, the presence or absence of phenol red makes no
everyday difference to the demonstration of estrogen mitogenic
effects with several target cell types from diverse species.
[0324] Phenol Red Testing for Estrogenic Activity with MCF-7A
Cells. The original reports of the effect of phenol red or its
impurities had used the MCF-7 human breast cancer cells to assess
estrogenic activity (Berthois Y et al. (1986) Proc Natl Acad Sci
USA 83, 2496-2500; Bindal R D et al. (1988) J Steroid Biochem 31,
287-293; Bindal R D and Katzenellenbogen J A (1988) J Med Chem 31,
1978-1983). The initial study began with the MCF-7A strain of this
population. As shown in FIG. 29A, growth was measured in the
presence of increasing concentrations of CDE-horse serum with and
without phenol red in the medium and .+-.E.sub.2. Concentrations of
.ltoreq.10% (v/v) CDE-horse serum supported more than 5 CPD. Higher
concentrations progressively inhibited in both indicator containing
and indicator free medium. In both types of medium, E.sub.2 was
required to reverse the serum inhibition. To confirm that E.sub.2
was equally effective in phenol red free and phenol red containing
medium, the estrogenic effects shown in FIG. 29A were compared in
both types of medium and at each serum concentration. The results
of this analysis are presented in FIG. 29B. The maximum estrogenic
effect at 50% (v/v) serum was 2.38 CPD (i.e. 2.sup.2.38 or
5.2-fold) in medium without indicator and 2.56 CPD (i.e. 2.sup.2.56
or 5.9-fold) with phenol red. This difference was not significant.
Only at 5% (v/v) serum was there a significantly (p<0.05)
greater estrogenic effect in phenol red free medium. However, in
replicate experiments this <1.0 CPD effect was inconsistent. At
all other serum concentrations, the growth differences between plus
and minus phenol red were not significant.
[0325] Test of Phenol Red Effects with MCF-7K Cells. The MCF-7K
strain was routinely more estrogen responsive than the MCF-7A line
(Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro Cell Dev Biol
36, 428-446). The MCF-7K cells also showed a serum concentration
dependent growth inhibition (FIG. 29C). The final degree of
inhibition at 50% (v/v) serum was independent of phenol red. Only
in the presence of 2.5, 5, 10 and 20% (v/v) CDE-horse serum were
the estrogenic effects significantly greater in phenol red free
(FIG. 29D). It is important to note that while these differences
were identified more often with the MCF-7K strain than the MCF-7A
line, they were invariably small. Plainly, no serum concentration
supported .gtoreq.1.0 CPD estrogenic effects in phenol red free
medium compared to indicator free medium (FIG. 29D). In fact,
deletion of phenol red improved estrogen responsiveness by an
average of only 0.6 CPD with the MCF-7K line. When judged by the
maximum estrogenic effects achievable with MCF-7K cells in 50%
(v/v) CDE-horse serum, plus and minus phenol red gave
indistinguishable results of CPD 3.01 (8.0-fold) and CPD 2.99
(7.9-fold), respectively (FIG. 29D).
[0326] Phenol Red Testing for Estrogenic Activity with T47D and
ZR-75-1 Cells. The same experiments just described above with the
MCF-7 cell strains were repeated with T47D and ZR-75-1 cells. These
lines were substantially more estrogen stimulated in CDE-serum than
MCF-7 cells (Sirbasku D A and Moreno-Cuevas J E (2000) In Vitro
Cell Dev Biol 36, 428-446) and hence were expected to be more
sensitive to phenol red/contaminants.
[0327] Phenol Red and T47D Cells. T47D cells were grown in medium
with CDE-horse serum both with and without phenol red (FIG. 30A).
Low concentrations of serum (i.e. .ltoreq.2%) promoted growth.
Higher concentrations progressively inhibited growth irrespective
of indicator content. In both media, E.sub.2 was required to
reverse the inhibition (FIG. 30A). In 50% (v/v) CDE-horse serum,
the maximum E.sub.2 responses were 2.sup.5.35 (41-fold) and
2.sup.5.29 (39-fold) in phenol red containing and indicator free
medium, respectively (FIG. 30B). Only at low serum concentrations
were phenol red effects observed in any experiment. In some
replicates, the phenol red effect was opposite to that expected.
For example, in the experiment shown in FIG. 30B, 0.5 to 2.5% serum
showed significantly (p<0.05) greater estrogenic effects in the
presence of phenol red. These results graphically illustrate the
hazards of interpreting 1.0 CPD responses either in favor of phenol
red/contaminants as estrogens or in opposition to this
proposal.
[0328] Phenol Red and ZR-75-1 Cells. ZR-75-1 cells showed similar
results as the T47D line. Serum caused an inhibition of growth that
was undoubtedly unrelated to phenol red (FIG. 30C). In both types
of medium, and at every serum concentration tested, F.sub.2 was
required to reverse the inhibition (FIG. 30C). In 50% (v/v) serum,
ZR-75-1 cells showed maximum estrogenic effects of 2.sup.3.39
(10.5-fold) and 2.sup.3.49 (11.2-fold) in medium with and without
indicator, respectively (FIG. 30D). As seen with T47D cells, the
ZR-75-1 line showed greater estrogenic effects in medium with
phenol red than in medium without indicator when the serum was 0.5,
5 or 10% (v/v) (FIG. 30D).
[0329] Phenol Red Testing for Estrogenic Activity with MTW9/PL2
Cells The next experiments were done with MTW9/PL2 rat mammary
tumor cells (FIG. 31A). They were inhibited by high concentrations
of CDE-horse serum with and without indicator. B.sub.2 was required
to reverse the inhibition in both types of medium (FIG. 31A). The
maximum estrogenic effects in 50% serum were 2.sup.5.82 (56-fold)
and 2.sup.5.69 (52-fold) with and without phenol red, respectively
(FIG. 31B). In the experiment shown in FIG. 31B, estrogenic effects
were unpredictably greater in phenol red free medium than in medium
with indicator. This was observed at low serum concentrations (i.e.
0.5 and 1.0%) and again at higher levels (i.e. 20 and 30%).
Although suggesting a phenol red effect, these results in fact only
serve to emphasize the pitfalls of accepting small changes as
meaningful even though they are significant at p<0.05. When
estrogenic effects were found with MTW9/PL2 cells in phenol red
free conditions, they invariably were .ltoreq.1.0 CPD. The sum of
the studies with MTW9/PL2 cells did not yield a predictable
correlation between estrogenic effects in the absence of the
indicator and serum concentrations.
[0330] Other Cell Lines Tested for Growth.+-.Phenol Red and
.+-.E.sub.2. The results presented above were replicated with the
GH.sub.1 and GH.sub.4C.sub.1 rat pituitary tumor cell lines as well
as with the F1301 cells and the LNCaP cell line (Moreno-Cuevas J E
and Sirbasku D A (2000) In Vitro Cell Dev Biol 36, 447-464). Again,
the presence or absence of the indicator in the medium containing
CDE-horse serum had no effect whatever on the demonstration of the
usual high estrogenic effects with these cells.
[0331] Direct Test of Phenol Red Estrogenic Activity. Three cell
lines were selected for a direct test of phenol red as a mitogen.
The MCF-7A line was used because it most closely approximated the
origin and passage age of the cells used to conduct the original
study of phenol red as a weak estrogen (Berthois Y et al. (1986)
Proc Natl Acad Sci USA 83, 2496-2500). The T47D cells were chosen
because they are the most estrogen responsive human breast cancer
cell line available today (Sirbasku D A and Moreno-Cuevas J E
(2000) In Vitro Cell Dev Biol 36, 428-446). The MTW9/PL2 cells were
chosen as an example of a highly estrogen responsive rodent origin
line (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev
Biol 36, 410-427; Sirbasku D A and Moreno-Cuevas J E (2000) In
Vitro Cell Dev Biol 36, 428-446). The assays were done in phenol
red free D-MEM/F-12 supplemented with 30% CDE-HS. This
concentration was chosen even though it is not as inhibitory as 50%
(v/v) serum. This selection was made to reduce possible
interactions of the phenol red/contaminant with serum proteins
while still retaining a significant inhibitory effect. Phenol red
concentrations of up to 16 mg/L were added to this medium. This
highest level was twice that in standard commercially formulated
Gibco-BRL D-MEM/F-12. Several different manufacturing lots of
aqueous phenol red gave equivalent results. The preparations used
in this study ranged in age from newly obtained to more than ten
year old laboratory stocks. These experiments gave unmistakable
results. There was no increase in the growth of any of the cell
lines in response to phenol red (FIG. 32A). By comparison, parallel
cultures receiving E.sub.2 showed sizable 2 to 5 CPD responses to
the natural hormone (FIG. 32B). E.sub.2 at 1.0.times.10.sup.-10 M
optimized growth of all three cell lines. The ED.sub.50
concentrations of E.sub.2 were 3.0.times.10.sup.-12 M. Significant
(p<0.05) estrogenic effects were observed at
1.0.times.10.sup.-12 M. The results presented in FIG. 32 indicate
that the culture conditions used in this study could reasonably be
expected to detect responses due to contaminants present at the
concentrations indicated in the original reports (Berthois Y et al.
(1986) Proc Natl Acad Sci USA 83, 2496-2500; Bindal R D et al.
(1988) J Steroid Biochem 31, 287-293; Bindal R D and
Katzenellenbogen J A (1988) J Med Chem 31, 1978-1983).
[0332] Comparison of E.sub.2 Potency in Medium with and without
Phenol Red. As described above in TABLE 5, the T47D and MTW9/PL2
cells grow significantly in response to 1.0.times.10.sup.-12 M
E.sub.2. The D-MEM/F-12 used in those studies also contained about
23 .mu.M phenol red. When the results of those studies were
compared to the experiments in FIG. 32B, done in D-MEM/F-12 without
indicator, the estrogen dose response curves were very similar. The
conclusion is straightforward. E.sub.2 dose-responses were not
affected by phenol red. If phenol red lipophilic contaminants were
present at the concentrations originally suggested (Berthois Y et
al. (1986) Proc Natl Acad Sci USA 83, 2496-2500; Bindal R D et al.
(1988) J Steroid Biochem 31, 287-293; Bindal R D and
Katzenellenbogen J A (1988) J Med Chem 31, 1978-1983) they should
have masked the observation of picomolar effects of exogenous
estrogens.
[0333] Effect of Phenol Red on Binding of .sup.3H-E.sub.2 to Intact
Cells. For the next study, intact T47D cells were used to measure
the effects of phenol red on estrogen receptor binding. The cells
were incubated with 5 nM .sup.3H-E.sub.2 and the effects of
addition of increasing concentrations of unlabeled E.sub.2 assessed
(TABLE 6). A 100-fold excess of unlabeled E.sub.2 displaced 75% of
the binding of .sup.3H-E.sub.2. By this criterion, 75% of the
binding of .sup.3H-E.sub.2 was specific to estrogen receptors
(Chamness G C and McGuire W L (1975) Steroids 26, 538-542). The
same analysis was conducted with aqueous preparations of phenol
red. Even at 16 mg/L, the indicator did not reduce the binding of
.sup.3H-E.sub.2 (TABLE 6). This was true no matter which batch of
indicator was analyzed (results not shown). The phenol red used for
the experiment shown in TABLE 6 was approximately the same age
(purchased in 1986) as the date of the original report (Berthois Y
et al. (1986) Proc Natl Acad Sci USA 83, 2496-2500). These results
raise the question how often preparations of phenol red purchased
at that time as an aqueous membrane filtered product contained a
sufficient level of contaminants to elicit an estrogenic
effect.
TABLE-US-00007 TABLE 6 Displacement of .sup.3H-E.sub.2 Binding to
Intact T47D Cells by Unlabeled E.sub.2 or Unlabeled Phenol Red
Indicator Free and Serum-free D-MEM/F-12 for Two Hours at
37.degree. C. Additions Counts per Minute Percent of Control
Control - No Additions 12,458 .+-. 1615 100% (5 nM .sup.3H-E.sub.2
only) 2.5 nM Unlabeled E.sub.2 12,177 .+-. 872 98% 5.0 nM Unlabeled
E.sub.2 8,756 .+-. 588 70% 50 nM Unlabeled E.sub.2 7,898 .+-. 744
63% 250 nM Unlabeled E.sub.2 4,892 .+-. 194 39% 500 nM Unlabeled
E.sub.2 3,494 .+-. 127 28% 1000 nM Unlabeled E.sub.2 2,543 .+-. 304
20% 1 mg/L Phenol Red 12,670 .+-. 727 102% 2 mg/L Phenol Red 13,874
.+-. 906 111% 4 mg/L Phenol Red 11,730 .+-. 566 94% 8 mg/L Phenol
Red 12,357 .+-. 664 99% 16 mg/L Phenol Red 13,748 .+-. 998 110%
[0334] Comparison of the E.sub.2 and Phenol Red Induction of
Progesterone Receptors. Another putative function of phenol red was
to induce progesterone receptors in estrogen sensitive cells. An
investigation was made as to whether the indicator induced an
increase in the progesterone receptors of T47D cells which contain
these sites (Horwitz K B et al. (1978) Cancer Res 38, 2434-2437).
In a first study, the kinetics of progesterone receptor induction
versus estrogen concentration in phenol red free medium were
investigated (FIG. 33A). E.sub.2 levels as low as
1.0.times.10.sup.-12 M caused a significant two-fold increase in
receptor content in four days. At 1.0.times.10.sup.-8 M, E.sub.2
induced a four-fold increase in progesterone receptors in four
days. Clearly, E.sub.2 induced a time and concentration dependent
increase in the progesterone receptors with T47D cells. Next, this
same analysis was done with phenol red over a concentration range
of 1 to 16 mg/L (FIG. 33B). Phenol red induced a small increase in
progesterone receptors at 8 and 16 mg/L after four days. This
induction was about the same as caused by 1.0.times.10.sup.-14 M
E.sub.2 (FIG. 33A). These results indicate that if estrogenic
contaminants are present in phenol red, they are most likely in the
10.sup.-14 M range even assuming equal receptor binding capacity to
E.sub.2. This point is important because the active agent is
thought to be only a trace impurity in many batches of phenol red
(Bindal R D et al. (1988) J Med Chem 31, 1978-1983). The impurities
bind to the estrogen receptor with only 50% of the affinity of
E.sub.2. The impurity was expected to be 0.002% of the phenol red
concentration. Based on test results that employed many different
batches of Gibco-BRL D-MEM/F-12, this concentration of the impurity
seems highly unlikely in the medium commercially available
today.
[0335] Discussion of Example 8. The studies of the effects of
phenol red or its lipophilic impurities demonstrate the usefulness
of the presently disclosed methods for the assessment of estrogenic
and androgenic activity of commercially prepared materials,
substances present or extracted from environmental or food sources
or other sources that are thought to contain such activities. The
testing can be approached by three separate methods as shown by
examples with phenol red. (1) Compounds or other preparations and
substances can be tested for growth activity with human or rodent
cell lines depending upon the information sought. Potency can be
established as UNITS based on E.sub.2 or any other estrogen or
androgen required. This permits direct expression of the estrogen
like activity or androgen like activity per volume or mass of the
substance under evaluation. Levels can be measured without regard
for expensive development of a radio immunoassay that in the end
still does not yield evidence of biological activity as a sex
steroid hormone analog (agonist or antagonist). The use of rodent
cell lines opens the possibility of direct comparison to in vivo
activity if required. (2) Another form of analysis is direct
measure of potency by .sup.3H-E.sub.2 or .sup.3H-DHT binding
displacement analysis from whole cells or extracted estrogen
receptors. An example with .sup.3H-E.sub.2 and whole cells is shown
in TABLE 6. The two different binding assays offer different
information. Whole cells have a predominance of hydrophobic sites
(i.e. membranes) that absorb lipophilic substances and therefore
may attenuate their activity. Use of cell extracted sex steroid
hormone receptors permits direct measure of the potential of a
substance to act as a hormone independent of its biological
effects. (3) Finally, use of the progesterone receptor analysis
permits evaluation of substances and preparations by a method
entirely independent of growth. This is a gene expression based
analysis that permits evaluation that can be used to supplement
growth data or be used in place of growth analysis. The MTW9/PL2
cells have been shown above to be suitable for this purpose.
Example 9
Testing of Substances for Inhibitor-Like Activity
[0336] In studies described in this Example, TGF.alpha.,
TGF.beta.1, EGF, IGF-I, IGF-II and insulin were tested for
inhibitor-like activity, using the cell growth assay described in
the General Materials and Methods section, and in the foregoing
Examples, substituting those proteins for the serum-borne inhibitor
contained in the preferred CDE serum.
[0337] TGF.beta.1 as a Substitute for the Serum-borne Estrogen
Reversible Inhibitor. Normal mouse mammary (Silberstein G B and
Daniel C W (1987) Science (Wash D.C.) 237, 291-293; Silberstein G B
et al. (1992) Dev Biol 152, 354-362) and normal human breast
epithelial cell growth is inhibited by TGF.beta. (Bronzert D A et
al. (1990) Mol Endocrinol 4, 981-989). Additionally, human breast
cancer cells are inhibited by TGF.beta. (Knabbe C et al. (1987)
Cell 48, 417-428; Arteaga C L et al. (1988) Cancer Res 48,
3898-3904; Arteaga C L et al. (1990) Cell Growth Diff 1, 367-374).
TGF also inhibits the GH.sub.4C.sub.1 rat pituitary tumor cells
(Ramsdell JS (1991) Endocrinology 128, 1981-1990) and the LNCaP
human prostatic carcinoma cells (Schuurmans A L et al. (1988) The
Prostate 12, 55-64; Wilding G et al. (1989) Mol Cell Endocrinol 62,
79-87; Carruba G et al (1994) Steroids 59, 412-420; Castagnetta L A
and Carruba G (1995) Ciba Found Symp 191, 269-286; Kim I Y et al.
(1996) Endocrinology 137, 991-999). In studies presented next,
replacement of the serum-borne inhibitor with TGF.beta. was
attempted. A number of related forms of this inhibitor are known
(Clark D A and Coker R (1998) Int J Biochem Cell Biol 30, 293-298;
Massague J (1998) Annu Rev Biochem 67, 753-791). TGF.beta.1 and
TGF.beta.2 are most often studied and commonly have similar
potencies. For example, they are equipotent with human breast
cancers cells (Zugmaier G et al. (1989) J Cell Physol 141,
353-361). TGF.beta.1 was chosen for the instant study. Without a
doubt, a number of the key cell lines used throughout the Examples
were inhibited by TGF.beta.. It was therefore considered essential
to ask if TGF was the estrogen reversible inhibitor.
[0338] TGF.beta.1 and MCF-7 Cells. Because MCF-7 cells are probably
the most studied human breast cancer line today, this next work
began with those cells. TGF.beta. has been described as a hormone
regulated autocrine inhibitor of the ER.sup.+ MCF-7 human breast
cancer cell growth (Knabbe C et al. (1987) Cell 48, 417-428). In
the present study, to test if TGF.beta.1 substituted for the
serum-borne inhibitor with these cells, they were grown in
D-MEM/F-12 containing 2.5% (v/v) CDE-horse serum plus increasing
concentrations of transforming growth factor and .+-.E.sub.2. The
results in FIG. 34A show that even 50 ng/mL of TGF.beta.1 caused
only a modest inhibition of MCF-7K cell growth. Cell numbers were
reduced from 350,000 to 200,000 per dish. This difference was
significant (p<0.05). Nevertheless, the estrogen reversal of the
inhibition was no larger than the E.sub.2 effect observed in
D-MEM/F-12 containing 2.5% (v/v) horse serum without TGF.beta.1
FIG. 34A. Furthermore, when the cell number data were expressed as
CPD (insert FIG. 34A), it was definite that TGF.beta.1 was at best
a very modest inhibitor and that there was no TGF.beta.1 related
estrogenic effect.
[0339] TGF.beta.1 and MTW9/PL2 Cells. The next study was performed
because the MTW9/PL2 cells are the only known estrogen growth
responsive rat cell line derived from a hormone responsive
carcinogen induced tumor. A similar analysis was done with the
MTW9/PL2 rat mammary tumor cells (FIG. 34B). TGF.beta.1 reduced
cell numbers from 350,000 to 100,000 per dish. This was significant
(p<0.05). However, the presentation of cell number results only
tends to exaggerate the effects of TGF.beta.1. When the results
were converted to CPD (FIG. 34B, insert), the actual inhibition was
1.5 CPD. This was at most a 25% decrease in growth rate. As shown,
there was no estrogen reversal of the TGF.beta.1 inhibition with
MTW9/PL2 cells.
[0340] TGF.beta.1 and other ER.sup.+ Cell Lines. The effects of
TGF.beta.1 at 50 ng/mL.+-.E.sub.2 were also investigated with the
other cell lines used in this study. The MCF-7A, T47D and ZR-75-1
human breast cancer cells were inhibited by TGF.beta.1 (FIG. 35A).
From these results, and those in FIG. 34A, it was clear that the
MCF-7 cells were the most sensitive of the ER.sup.+ human breast
cancer lines tested. Irrespective of the line, E.sub.2 had no
influence on the TGF.beta.1 mediated inhibitions (FIG. 35A). The
same experiments were done with the LNCaP cells and the
GH.sub.4C.sub.1 pituitary line (FIG. 35A). They were more sensitive
to TGF.beta.1 than breast cancer cells. Nonetheless, the TGF.beta.1
effects were not reversed by E.sub.2. When the cell number
decreases presented in FIG. 35A were converted to CPD, it was clear
that the TGF.beta.1 effects were negligible and that E.sub.2 was of
no significant consequence (FIG. 35B). Thus, TGF.beta.1 did not
substitute for the estrogen reversible inhibitor(s) in CDE serum
with any of the sex steroid sensitive ER.sup.+ cell lines
tested.
[0341] TGF.alpha. and EGF as Substitutes for the Estrogen
Reversible Inhibitor in CDE Serum. The EGF family of mitogens and
receptors has been linked to breast cancer proliferation, invasion
and progression (Dickson R B and Lippman M E (1987) Endocr Rev 8,
29-43; Normanno N et al. (1994) Breast Cancer Res Treat 29, 11-27;
Ether SP (1995) J Natl Cancer Inst 87, 964-973; de Jung J S et al.
(1998) J Pathol 184, 44-52 and 53-57). Most prominent among these
polypeptide mitogens has been the EGF analogue, TGF.alpha. (Dickson
R B and Lippman M E (1987) Endocr Rev 8, 29-43; de Jung J S et al.
(1998) J Pathol 184, 44-52 and 53-57). Estrogen induced secretion
of TGF.alpha. is thought to create an autocrine loop that promotes
breast cancer cell growth (Dickson R B et al. (1985) Endocrinology
118, 138-142; Dickson R B et al. (1986) Cancer Res 46, 1707-1713;
Dickson R B et al. (1986) Science (Wash D.C.) 232, 1542-1543;
Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; Derrick R
(1988) Cell 54, 593-595; Arrack B A et al. (1990) Cancer Res 50,
299-303; Kenney N J et al. (1993) J Cell Physiol 156, 497-514;
Normanno N et al. (1994) Breast Cancer Res Treat 29, 11-27; Dickson
R B et al. (1987) Proc Natl Acad Sci USA 84, 837-841; Salomon D S
et al. (1984) Cancer Res 44, 4069-4077; Liu S C et al. (1987) Mol
Endocrinol 1, 683-692). TGF.alpha. is also thought to potentiate
estrogen action in uterus (Nelson K G et al. (1992) Endocrinology
131, 1657-1664) as well as to regulate the EGF receptor in this
tissue (DiAugustine R P et al. (1988) Endocrinology 122, 2355-2363;
Huet-Hudson Y M et al. (1990) Mol Endocrinol 4, 510-523; Mukku V R
and Stancel G M (1985) J Biol Chem 260, 9820-9824). The culture
conditions described herein offer a new opportunity to test the
autocrine growth model under conditions not previously available.
Application of the new cell growth assays allowed a direct test to
determine if an autocrine/intacrine growth factor loop explains the
estrogen reversal of the serum inhibition.
[0342] EGF and TGF.alpha. as Substitutes for E.sub.2. Growth of the
MCF-7A, MCF-7K, T47D and ZR-75-1 cells was measured in D-MEM/F-12
containing increasing concentrations of CDE horse serum with and
without exogenous EGF or TGF.alpha.. The results with the four cell
lines are shown in FIGS. 36 A, 36B, 36C, and 36D, respectively. As
expected, CDE horse serum was progressively inhibitory at
concentrations >5% (v/v). The addition of growth saturating
concentrations (Karey K P and Sirbasku D A (1988) Cancer Res 48.
4083-4092) of EGF or TGF.alpha. did not reverse the effects of the
serum-borne inhibitor. In control cultures without added
polypeptide mitogens, E.sub.2 completely reversed the serum
inhibition. These results again confirm the same conclusion arrived
at earlier using an entirely different approach (Karey K P and
Sirbasku D A (1988) Cancer Res 48. 4083-4092). Direct evidence for
obligatory EGF/TGF.alpha. autocrine loops in estrogen responsive
cell growth simply has not yet been established. In fact, there is
solid in vivo evidence to challenge EGF/TGF.alpha. autocrine loop
participation in the action of estrogens (Arteaga C L et al. (1988)
Mol Endocrinol 2, 1064-1069).
[0343] IGF-I, IGF-II and Insulin as Substitutes for Estrogen
Action. Insulin-like growth factors I and II (IGF-I and IGF-II)
promote breast cancer cell growth (Furlanetto R W and DiCarlo J N
(1984) Cancer Res 44, 2122-2128; Myal Y et al. (1984) Cancer Res
44, 5486-5490; Dickson R B and Lippman M E (1987) Endocr Rev 8,
29-43; Karey K P and Sirbasku D A (1988) Cancer Res 48, 4083-4092;
Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,
911-920; Stewart A J et al. (1990) J Biol Chem 265, 2172-2178).
IGF-I related proteins (Huff K K et al. (1986) Cancer Res 46,
4613-4619; Huff K K et al. (1988) Mol Endocrinol 2, 200-208;
Dickson R B and Lippman M E (1987) Endocr Rev 8, 29-43; Minute F et
al. (1987) Mol Cell Endocrinol 54, 17-184, as well IGF-II (Yee D et
al. (1988) Cancer Res 48, 6691-6696; Osborne C K et al. (1989) Mol
Endocrinol 3, 1701-1709), are thought of as possible
autocrine/paracrine mitogens. Their secretion in response to
hormones has been proposed (Dickson R B and Lippman M E (1987)
Endocr Rev 8, 29-43; Huff K K et al. (1988) Mol Endocrinol 2,
200-208; Osborne C K et al. (1989) Mol Endocrinol 3, 1701-1709).
Insulin itself is likely an endocrine mediator. In the instant
study, it was investigated whether exogenous IGF-I addition to
cultures containing CDE-horse serum substituted for the inhibition
reversing effects of estrogens with human breast cancer cells.
FIGS. 37A and 37B show the results with the MCF-7K and MCF-7A
cells, respectively. Clearly, 1.0 .mu.g/mL IGF-I did not reverse
the serum inhibition. This was true despite the fact that this
concentration of added IGF-I was much more than growth saturating
(Karey K P and Sirbasku D A (1988) Cancer Res 48, 4083-4092).
Duplicate studies with the T47D cells gave the same results (FIG.
37C). It should be noted that IGF-I is active with breast cancer
cells even in the presence of serum (Furlanetto R W and DiCarlo J N
(1984) Cancer Res 44, 2122-2128; Myal Y et al. (1984) Cancer Res
44, 5486-5490; Osborne C K et al. (1989) Mol Endocrinol 3,
1701-1709; Stewart A J et al. (1990) J Biol Chem 265, 2172-2178;
Cullen K J et al. (1990) Cancer Res 53, 48-53) that contains
specific growth factor binding proteins (Rechler M et al. (1980)
Endocrinology 107, 1451-1459). Human breast cancer cells also
secrete binding proteins for the insulin-like growth factors (Yee D
et al. (1991) Breast Cancer Treat Res 18, 3-10). Binding of the
insulin-like factors to carrier proteins may attenuate activity
(Zapf J et al. (1978) J Clin Invest 63, 1077-1084), have both
inhibiting and activating effects (De Mellow J S et al. (1988)
Biochem Biophys Res Commun 156, 199-204), or enhance biological
action (Elgin R et al. (1987) Proc Natl Acad Sci USA 84, 3254-3258;
Blum W F et al. (1989) Endocrinology 125, 766-772). In parallel
studies (data not shown), the effects of IGF-II were assayed with
the same breast cancer lines under the conditions used with IGF-I.
Even at 500 ng/mL, IGF-II did not reverse the inhibitory effects of
10 to 50% (v/v) CDE serum. In another related test, insulin at 10
ng/mL to 10 .mu.g/mL did not reverse the inhibition caused by 50%
(v/v) CDE serum. The results with insulin, IGF-I and IGF-II were
mutually supportive because these mitogens promote growth via a
common receptor (Rechler M et al. (1980) Endocrinology 107,
1451-1459; Karey K P and Sirbasku D A (1988) Cancer Res 48,
4083-4092; Osborne C K et al. (1989) Mol Endocrinol 3, 1701-1709;
Stewart A J et al. (1990) J Biol Chem 265, 2172-2178). The insulin
results were also important in another way. This hormone does not
interact with binding proteins and hence their presence in medium
will not influence insulin action. These results again confirm the
same conclusion arrived at earlier using an entirely different
approach (Karey K P and Sirbasku D A (1988) Cancer Res 48.
4083-4092). Direct evidence for obligatory IGF-1/IGF-II autocrine
loops in estrogen responsive cell growth simply has not been
confirmed yet. In fact, there is solid in vivo evidence to the
challenge IGF-1/IGF-II autocrine loop participation in the action
of estrogens (Arteaga C L et al. (1989) J Clin Invest 84,
1418-1423).
[0344] Conceptual Derivations from this Study. These results also
have a direct bearing on a number of hypotheses advanced to explain
how estrogens cause target tissue cell growth. The development of
the new methods herein provided a unique opportunity to reevaluate
the most widely cited proposals under consideration. It was
concluded that serum contains an inhibitor that effectively blocks
ER.sup.+ and AR.sup.+ cell growth. Furthermore, physiologic
concentrations of sex steroid hormones reverse this inhibition. The
results were uniformly the same no matter from which species the
cell lines were derived or which species was the source of the
serum. In every case, the effects of the various classes of steroid
hormones on the different cell lines were consistent with their
known tumor forming/growth properties in vivo or published
responses in vitro. These results provide new insights into the
following proposed mechanisms.
[0345] Serum Factor Regulation--Demonstration of Estrogen
Responsiveness. The literature describing positive sex steroid
hormone growth effects is notably weighted in favor of the use of
serum-supplemented cultures. In fact, a review made of the
literature (Briand P and Lykkesfeldt A E (1986) Anticancer Res 6,
85-90; Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A, 595-602)
indicates that most past studies have used medium containing
.ltoreq.20% (v/v) steroid hormone depleted serum. Although other
investigators have reported estrogenic effects in "serum-free
defined culture", these studies actually used conditions that
included a prolonged preincubation in the presence of serum
(Allegra J C and Lippman M E (1978) Cancer Res 38, 3823-3829;
Briand P and Lykkesfeldt A E (1986) Anticancer Res 6, 85-90; Darbre
P D et al. (1984) Cancer Res 44, 2790-2793). The results presented
in preceding Examples demonstrate clearly that large magnitude
effects are readily demonstrable in medium with CDE-serum and that
as the CDE-serum concentrations increase to a maximum useable level
of 50%, cell growth is inhibited and estrogens invariably reverse
these effects. In light of those results, it was clear that the
presence of serum, or a factor(s) contained in serum, made possible
the demonstration of sex hormone dependent growth in culture.
[0346] The Endocrine Estromedin Hypothesis--Positive Indirect
Control. In 1978 it was proposed (Sirbasku D A (1978) Proc Natl
Acad Sci USA 75, 3786-3790) that growth of estrogen target tissues
was not mediated directly by these hormones, but was instead
controlled indirectly by steroid inducible circulating growth
factors (i.e. endocrine estromedins). Estromedins were proposed to
be secreted by target tissues such as uterus, kidney and pituitary,
and to act in concert to simultaneously promote the growth of all
ER.sup.+ target tissues (Sirbasku D A (1978) Proc Natl Acad Sci USA
75, 3786-3790; Sirbasku D A (1981) Banbury Report 8, 425-443; Ikeda
T et al. (1982) In Vitro 18, 961-979). The estromedin hypothesis
arose from the observation that reproducible in vitro direct
estrogen mitogenic effects were not identifiable (Sirbasku D A
(1978) Proc Natl Acad Sci USA 75, 3786-3790; Sirbasku D A (1981)
Banbury Report 8, 425-443; Ikeda T et al. (1982) In Vitro 18,
961-979). It must be emphasized that the original estromedin
hypothesis rested entirely upon the failure to demonstrate large
magnitude estrogen mitogenic effects in culture with cell lines
confirmed to form steroid hormone responsive tumors in host
animals. When estrogen effects were clearly observed with the
MTW9/PL2 rat mammary tumor cells in culture, as described herein
and reported (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro
Cell Dev Biol 36, 410-427; Sirbasku D A and Moreno-Cuevas J E
(2000) In Vitro Cell Dev Biol 36, 428-446), it was apparent that
the endocrine estromedin model required further evaluation. It was
reasoned that extension of these results to additional ER.sup.+
cell lines, including those from other species and diverse target
tissues, would either provide important support for the earlier
hypothesis or disprove it. In the work disclosed herein, this
reassessment has been accomplished. All of the ER.sup.+ cells
tested, as well as one androgen sensitive AR.sup.+ human cancer
line, manifested substantial growth in response to the appropriate
steroid hormones in cultures containing inhibiting concentrations
of CDE serum. There can be no doubt that steroid hormones act
positively to promote target tumor cell growth. The results
presented in this report plainly nullify the previous endocrine
estromedin model of steroid hormone responsive cell growth. The
disproval of the earlier endocrine estromedin model reopened the
question of how estrogens and other factors regulate sex steroid
responsive growth.
[0347] The Autocrine and Paracrine Models--Positive Indirect
Control. In the studies described in this Example, it was asked if
exogenous growth factors mimic the inhibitor reversing effects of
estrogens. The EGF/TGF.alpha. and insulin-like families were
focused on because of their high biological potencies and
physiologic relevance. These growth factors were expected to
substitute for steroid hormones based on the autocrine loop
mechanisms proposed earlier. Despite this expectation, polypeptide
growth factors did not substitute for the estrogens. They were
inactive in the presence of the serum-borne inhibitor. In point of
fact, deduction indicates that it makes no practical difference
whether the growth factors were autocrine or paracrine in origin.
The presence of the serum inhibitor in effect blocks all mitogenic
action except that exerted by the steroid hormones. This is a
preferred feature of the serum-borne inhibitor(s) disclosed herein,
and is further described in Examples which follow, when the use of
serum-free defined culture is described. These results also
indicate that the search for the regulatory mechanism controlling
estrogen dependent growth must seek new directions. Since the
estrogenic effects seen in CDE-serum are the largest yet recorded,
CDE is the preferred source of the regulator in the cell growth
assays.
[0348] Culture Parallels in vivo Growth Regulation. The results
shown in this Example have another important implication. Usually
normal in vivo tissues are bathed in growth factor containing
fluids. Mitogens within tissues may be of local origin or may be
derived from the circulation (Gospodarowitz D and Moran J S (1976)
Annu Rev Biochem 45, 531-558; Goustin A S et al. (1986) Cancer Res
46, 1015-1029). If growth factors have unrestricted freedom to
stimulate cell proliferation, normal formation and architecture of
the tissues would not develop nor could they be maintained.
Manifestly, tissue architecture would be disrupted. In fact, this i
s one definition of cancer (Sonnenschein C and Soto A M (2000) Mol
Carcinog 29, 205-211). The properties of a serum-borne inhibitor
that counterbalances unrestricted growth merit serious further
consideration with regard to how cancers develop in steroid hormone
sensitive tissues. Others researchers have also arrived at this
conclusion (Soto A M and Sonnenschein C (1985) J Steroid Biochem
23, 87-94).
[0349] The Estrocolyone Hypothesis--Negative Indirect Regulation.
The estrocolyone model (Soto A M and Sonnenschein C (1987) Endocr
Rev 8, 44-52) is an indirect negative mechanism based on regulation
of sex steroid hormone dependent cells via a serum-borne inhibitor.
The inhibitor blocks growth promoted by non-steroidal mitogens such
as growth factors and diferric transferrin. Sonnenschein and Soto
first proposed that estrocolyone acted at the cell surface via
specific receptors. The effects of sex steroid hormones were to
bind estrocolyone and prevent it from associating with the cells.
Only low physiologic concentrations of sex steroid hormones were
needed for this function. The special emphasis of this model was
that sex steroid hormones did not act through intracellular located
DNA binding receptors (i.e. cytosolic or nuclear sites). These
intracellular sites had no growth function. Hence, this was an
indirect negative mechanism (Soto A M and Sonnenschein C (1987)
Endocr Rev 8, 44-52). The results presented in this disclosure are
in agreement with the serum-borne mediator aspect of the
estrocolyone hypothesis. There is no doubt that serum from several
species contains a steroid hormone reversible inhibitor and that
its isolation and molecular characterization will be a major
advance with both practical and conceptual applications. With
regard to the action site of the steroid hormones, these results
differ from the estrocolyone hypothesis as described (Soto A M and
Sonnenschein C (1987) Endocr Rev 8, 44-52). As discussed in the
Background of the Invention, the tentative identification of
several estrocolyone candidates have been described, and in U.S.
Pat. Nos. 4,859,585 (Sonnenschein) and 5,135,849 (Soto), the issue
of properties was raised again, but with different conclusions than
published earlier.
[0350] The Positive Direct Model--Steroid Hormone Receptor
Mediation. The one mechanism most widely accepted regarding steroid
hormones and growth involves the nuclear located DNA binding
ER.alpha. receptor (Gorski J and Hansen J C (1987) Steroids 49,
461-475). Growth is thought to be mediation by specific cytosolic
and/or nuclear located receptors that ultimately alter DNA
transcription to regulate gene activity. Results from many
laboratories support this mechanism (Jensen E V and Jacobson H I
(1962) Recent Prog Horm Res 18, 387-414; Gorski J et al. (1968)
Recent Prog Horm Res 24, 45-80; Jensen E V et al. (1968) Proc Natl
Acad Sci USA 59, 632-638; Jensen E V and DeSombre E R (1973)
Science (Wash D.C.) 182, 126-134; Anderson J N et al. (1974)
Endocrinology 95, 174-178; O'Malley B W and Means A R (1974)
Science (Wash D.C.) 183, 610-620; Lippman M E (1977) Cancer Res 37,
1901-1907; Harris J and Gorski J (1978) Endocrinology 103, 240-245;
Markaverich B M and Clark J H (1979) Endocrinology 105, 1458-1462;
Katzenellenbogen B S (1980) Annu Rev Physiol 42, 17-35;
Katzenellenbogen B S (1984) J Steroid Biochem 20, 1033-1037; Clark
J H and Markaverich B M (1983) Pharm Ther 21, 429-453; Darbre P et
al. (1983) Cancer Res 43, 349-355; Darbre P D et al. (1984) Cancer
Res 44, 2790-2793; Huseby R A et al. (1984) Cancer Res 44,
2654-2659; Gorski J and Hansen J C (1987) Steroids 49, 461-475;
Katzenellenbogen B S et al. (1987) Cancer Res 47, 4355-4360;
O'Malley B W (1990) Mol Endocrinol 4, 363-369). As also discussed
in Example 1, the preferred positive action of estrogens is
activation of a new ER.gamma. that saturates/activates at lower
steroid concentrations than the ER.alpha. or the ER.beta..
[0351] Serum Proteins with Estrocolyone Steroid Binding
Characteristics. If the estrocolyone mechanism is in fact correct,
one must be able to identify at least one serum protein with very
high affinity binding (i.e. K.sub.d picomolar) for sex steroids.
There is, however, a major unresolved problem with that hypothesis.
Other than sex hormone binding globulin (SHBG), additional high
affinity estrogen binding in CDE human serum has not been found.
SHBG has K.sub.d of 1.7.times.10.sup.-9M for E.sub.2 at 37.degree.
C. (Rosner W and Smith R N (1975) Biochemistry 14, 4813-4820). This
affinity does not qualify as the high binding expected of
estrocolyone. Also, a search for estrocolyone in human serum only
resulted in identification of SHBG (Reny J-C and Soto A M (1992) J
Clin Endocrinol Metab 68, 938-945). No higher affinity binding
site/protein was found. The binding of labeled steroid hormones
with CDE-horse and CDE-rat serum was studied (results presented in
an Example which follows), and .sup.3H-E.sub.2 specific binding at
K.sub.d of 20 to 50 nM was found. This is a significant matter
because estrogenic effects are demonstrated in this disclosure at 1
to 10 picomolar. As further support for this point, the
estrocolyone authors found estrogenic effects at 10 to 30 picomolar
B.sub.2 (Soto A M and Sonnenschein C (1985) J Steroid Biochem 23,
87-94; Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52). The
lack of correlation between the concentration of steroid that
promotes growth and affinity of sex steroids for serum components
raises serious questions about this aspect of the estrocolyone
hypothesis. These observations also suggest that a very high
affinity intracellular ER.gamma. regulates growth.
[0352] A New Model of Steroid Hormone Responsive Cell Growth. A new
model best fits the available data. It brings together aspects of
both the direct positive mechanism and indirect negative control.
According to this model, regulation of steroid hormone target tumor
cell growth is a balance between positive and negative control
signals. This balance dictates either growth (i.e. cell division)
or quiescence (i.e. cell metabolism and tissue specific function
but without cell division). The positive mediators are the steroid
hormones acting mediated by a high sensitivity intracellular DNA
binding sex steroid receptor that ultimately activates gene
expression via intracellular located receptors; whereas negative
regulation is exerted by a serum-borne inhibitor that acts at the
cell surface. The results disclosed herein support the view that
growth is controlled directly by both negative and positive
mediators. In a subsequent Example, this model of negative and
positive response control mechanisms is further described and the
mediators are shown to be the secretory immunoglobulins acting on
cell surface (membrane) receptors.
[0353] TGF.beta. and Relevant Inhibition. The results presented
further define the molecular properties of the serum-borne
inhibitor by eliminating TGF.beta.1 as a candidate. This is an
important issue because of the well-known effects of TGF.beta. on
normal breast epithelial cells (Hosobuchi M and Stampfer M R (1989)
In Vitro Cell Dev Biol 25, 705-713) and ER.sup.- estrogen
insensitive breast cancer cells (Arteaga C L et al. (1988) Cancer
Res 48, 3898-3904). The results herein continue to confirm a
previously unrecognized entity that serves as the estrogen
reversible inhibitor in serum. Inhibitors that lack estrogen
reversibility can be eliminated from consideration.
[0354] Discussion of Example 9. From this series of experiments, it
can be readily appreciated that any other natural or synthetic
protein or other substance can be similarly tested for cancer cell
growth inhibiting activity akin to the serum-derived inhibitor in
the CDE horse serum. Also, the same XAD.TM.-4 and CDE extraction
protocols may also be applied to body fluids and secretions other
than serum, and the extracted fluids may be assayed as described
for inhibitor activity. Such fluids or secretions include plasma,
urine, seminal fluid, milk, colostrum, mucus and stool. An
XAD.TM.-4 column is especially suited for preparing a steroid
hormone depleted specimen from a small sample of body fluid.
Example 10
Serum-Free Defined Culture Medium Formulations
[0355] In this Example, formulations of various serum-free defined
culture media are discussed. Among other features, the preferred
embodiments of the present media provide useful tools for detecting
estrogenic effects.
[0356] During the course investigations leading to the present
invention, serum-free defined medium was used to identify IgA, IgM
and IgGs as estrogen and/or androgen reversible inhibitors of
target tumor cell growth in culture, as demonstrated in subsequent
Examples. However, before the full effects of the immunoglobulins
could be measured in serum-free defined medium, another issue had
to be resolved. The growth of hormone responsive cancer cell types
in serum-free medium based on standard preparations of D-MEM/F-12
was not as vigorous as expected. Despite the extensive purification
of the water used for cell culture procedures (Sirbasku D A et al.
(1991) Biochemistry 30, 295-304; Sirbasku D A et al. (1991)
Biochemistry 30, 7466-7477) and careful management of technical
issues (Moreno-Cuevas J E and Sirbasku D A (2000) In Vitro Cell Dev
Biol 36, 410-427), it was still apparent that the problem
persisted. It was found that thyroid hormone dependent cell growth
in culture was being inhibited by a normal component in standard
D-MEM/F-12 medium and that a serum-borne factor corrected the
problem. This work was done with established rat pituitary tumor
cell lines (Tashjian A H Jr (1979) Methods Enzymol 58, 527-535) in
serum-free defined medium (Sirbasku D A et al. (1991) 77, C47-055;
Sirbasku D A et al. (1991) Biochemistry 30, 295-304; Sirbasku D A
et al. (1991) Biochemistry 30, 7466-7477). As this work developed,
it was recognized that the serum factor was apotransferrin and that
its addition to serum-free defined medium permitted observation of
thyroid hormone dependent pituitary tumor cell growth (Sirbasku D A
et al. (1992) In Vitro Cell Dev Biol 28A, 67-71; Sato H et al.
(1992) Mol Cell Endocrinol 83, 239-251). Apotransferrin is a
M.sub.r 80,000 bilobular serum protein that binds one Fe (III) in
each lobe, albeit with slightly different affinities (Aisen P and
Liebman A (1972) Biochim Biophys Acta 257, 314-323; Chasteen N D
(1983) Trends Biochem Sci 8, 272-275; Evans R W and Williams J
(1978) Biochem J 173, 543-552). When Fe (III) saturated, the
protein is called diferric transferrin. This form of transferrin is
the major iron delivery system for the body (Young S P and Aisen P
(1981) Hepatology 1, 114-119; Ciechanover A et al. (1983) J Biol
Chem 258, 9681-9689). Because apotransferrin possesses very high
affinity for Fe (III) (i.e. .about.10.sup.20 at pH 7.4), there is
no significant free iron in blood. Considering the extraordinary
specificity of apotransferrin for Fe (III), it was concluded that
the presence of the ferric (FeIII) form of iron in culture medium
was deleterious to hormone responsive rat pituitary tumor cell
growth (Sato H et al. (1991) In Vitro Cell Dev Biol 27A, 599-602).
Additional work with apotransferrin and other Fe (III) chelators,
along with direct addition of Fe (III) to culture medium, confirmed
that this toxic metal was inhibiting thyroid hormone dependent
growth of rat pituitary tumor cells in culture (Eby J E et al.
(1992) Anal Biochem 203, 317-325; Eby J E et al. (1992) J Cell
Physiol 156, 588-600). In neutralizing studies, the very specific
Fe (III) chelator deferoxamine mesylate (a.k.a. deferoxamine or
desferrioxamine) stood out because of its very high affinity for
the iron (i.e. .about.10.sup.30.6) (Eby J E et al. (1993) J Cell
Physiol 156, 588-600) and its lack of toxicity to cells in culture.
Its addition to cell culture, at concentrations in small excess of
the few .mu.M levels of Fe (III) in medium, essentially neutralized
the toxic metal (Eby J E et al. (1992) Anal Biochem 203, 317-325).
Because deferoxamine is a low molecular weight bacterial product,
it is relatively inexpensive compared to serum-derived
apotransferrin. But without doubt, it is equally effective (Eby J E
et al. (1992) Anal Biochem 203, 317-325). Before these studies,
deferoxamine had never been used in serum-free defined medium to
protect hormone responsive cell growth from the toxic effects of Fe
(III). Prior to the present invention, the broad applicability of
that information had not yet been discovered nor had it been
discovered that some of the tools developed (e.g. a
deferoxamine-Sepharose.RTM. affinity matrix) were applicable to
estimation of the concentrations of biologically active Fe (III) in
chemical, industrial, environmental and biological samples.
Deferoxamine mesylate is a U.S. FDA approved drug used to treat
iron overload and iron toxicity in humans. It is marketed by
Novartis Pharmaceuticals, East Hanover, N.J., under the trade name
DESFERAL.RTM.. Deferoxamine mesylate is sold to researchers by
Sigma Chemicals (St. Louis, Mo.).
[0357] Serum-free Defined Mammalian Cell Culture--Development
Background. The use of serum-free defined medium to grow diverse
cell types in culture gained national and international recognition
with the publication by Hayashi and Sato (Hayashi I and Sato G H
(1976) Nature (Lond) 259, 132-134). They demonstrated a
breakthrough. The serum supplement commonly used in cell culture
medium could be replaceable entirely by mixtures of nutrients and
hormones in serum-free medium. This observation was expanded to
include cell types from many mammalian tissues (Barnes D and Sato G
(1980) Anal Biochem 102, 255-270; Barnes D and Sato G (1980) Cell
22, 649-655; Bottenstein J et al. (1979) Methods Enzymol 58,
94-109; Rizzino A et al. (1979) Nutr Rev 37, 369-378). Further
development and application of this technology has been reported
(Barnes D W, Sirbasku D A and Sato G H (Volume Editors) (1984) Cell
Culture Methods for Molecular Biology and Cell Biology, Volume 1:
Methods for Preparation of Media, Supplements, and Substrata for
Serum-free Animal Cell Culture; Volume 2: Methods for Serum-free
Culture of Cells of the Endocrine System; Volume 3: Methods for
Serum-free Culture of Epithelial and Fibroblastic Cells; Volume 4:
Methods for Serum-free Culture of Neuronal and Lymphoid Cells,
Allan R. Liss/John Wiley, New York). A national symposium organized
and directed by Drs. Gordon Sato, Authur Pardee and David Sirbasku
was held at the Cold Spring Harbor Laboratory to address the
unfolding technology required for serum-free defined medium growth
of cells in culture and to discuss its applications (Sato G H,
Pardee A B and Sirbasku D A (1982) Volume Editors, Cold Spring
Harbor Conferences on Cell Proliferation, Volume 9, Books A and B,
Growth of Cells in Hormonally Defined Media, Cold Spring Harbor,
N.Y.).
[0358] Serum-free Defined Culture--Nutrient Additions. A number of
nutrient additions to D-MEM/F-12 are needed to grow the cells used
in the presently described studies. The formulations of serum-free
defined medium employed are specific optimizations, modifications,
or necessary changes of earlier media that have been described
(Riss T L and Sirbasku D A (1987) Cancer Res 47, 3776-3782;
Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52;
Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,
911-920; Karey K P and Sirbasku D A (1988) Cancer Res 48,
4083-4092; Riss T L et al. (1988) In Vitro Cell Dev Biol 24,
1099-1106; Riss T L et al. 25, In Vitro Cell Dev Biol 25, 127-135;
Riss T L and Sirbasku D A (1989) In Vitro Cell Dev Biol 25,
136-142; Riss T L et al. (1986) J Tissue Culture Methods 10,
133-150; Sirbasku D A et al. (1991) Mol Cell Endocrinol 77,
C47-055; Sirbasku D A et al. (1991) Biochemistry 30, 295-304;
Sirbasku D A et al. (1991) Biochemistry 30, 7466-7477; Sato H et
al. (1991) In Vitro Cell Dev Biol 27A, 599-602; Sirbasku D A et al.
(1992) In Vitro Cell Dev Biol 28A, 67-71; Sato H et al. (1992) Mol
Cell Endocrinol 83, 239-251; Eby J E et al. (1992) Anal Biochem
203, 317-325; Eby J E et al. (1993) J Cell Physiol 156, 588-600;
Sirbasku D A and Moreno-Cuevas J E (2000) In vitro Cell Dev Biol
36, 428-446).
[0359] Serum-free Defined Medium Nutrient Supplements--Bovine Serum
Albumin. Bovine serum albumin (BSA) (Sigma Catalog No. A3912) was
made by "initial fractionation by heat shock and Fraction V",
minimum purity 98% (electrophoresis), according to the supplier. A
50 mg/mL stock solution of BSA was prepared in normal saline and
was sterilized using 0.2 .mu.m pore membrane filters. Aliquots are
stored at -20.degree. C. in plastic tubes. As will be discussed
below, the "heat shock" step that was used in most albumin
preparation methods inactivates the estrogen reversible inhibitor
disclosed herein.
[0360] Serum-free Defined Medium Nutrient Supplements--Linoleic
Acid--Albumin (Lin-Alb). This preparation was purchased from Sigma
as Linoleic Acid Albumin Conjugate (Catalog No. L8384). The
conjugate is supplied as a powder sterilized by irradiation. The
fatty acid content is 1% linoleic acid by weight. A stock solution
was typically prepared by dissolving the contents of a 500 mg
bottle in 10 mL of sterile normal saline to give a final
concentration of 50 mg/mL. Aliquots are stored at 4.degree. C. in
polystyrene tubes. This solution is never frozen. Mammalian cells
cannot produce polyunsaturated fatty acids. They must be supplied
in a soluble form. Fatty acids are carried physiologically bound to
albumin.
[0361] Serum-free Defined Medium Nutrient Supplements--Ethanolamine
(ETN). ETN was purchased from Sigma (Catalog No. A5629) (FW 61).
This liquid has a density of 1.0117 grams/mL. Using 0.610 mL in 100
mL of water, a 100 mM stock solution was prepared which was
sterilized using the 0.2 um pore membrane filters. The ETN was
stored at -20.degree. C. in polystyrene tubes. This nutrient is
required to sustain phospholipid metabolism required for all
membrane biosynthesis.
[0362] Serum-free Defined Medium Nutrient
Supplements--Phosphoethanolamine (PETN). This solid material was
purchased as o-phosphoryl-ethanolamine (FW 141) (Sigma Catalog No.
P0503). A 10 mM stock of PETN was prepared by dissolving 141 mg in
100 mL of water and sterilizing with 0.2 .mu.m pore membrane
filters. Aliquots were stored at -20.degree. C. in polystyrene
tubes. This component is an adjunct to ETN.
[0363] Serum-free Defined Medium Nutrient Supplements--Glutamine
(GLUT). This essential amino acid was purchased from Sigma (Catalog
No. G5763). It is "cell culture tested" according to the
manufacturer. Addition of glutamine (FW 146.1) to the culture media
is necessary because of its relatively short half-life (i.e. about
80% is lost in 20 days at 35.degree. C.). See the Sigma product
information for the decay curves at different temperatures and pH.
Purchased D-MEM/F-12 stored in the refrigerator for about three
weeks lost most of the original glutamine present. For serum-free
applications, additional supplementation is required to sustain
growth. For a preparation, 11.7 g was dissolved in 400 mL of water
to give 200 mM glutamine. This solution was sterilized using 0.2
.mu.m pore filter membranes. Aliquots are stored at -20.degree. C.
polystyrene tubes. The final glutamine concentration added to
serum-free defined medium is 2 mM. Glutamine is a major metabolite
and energy source for cells growing in culture.
[0364] Serum-free Defined Medium Nutrient Supplements--Reduced
Glutathione (GSH). Crystalline reduced glutathione (FW 307.3) was
purchased from Sigma (Catalog No. G4251). A stock of 40 mg/mL was
prepared by dissolving 400 mg in 10 mL of water. This stock was
very quickly sterilized with a 0.2 .mu.m pore filter unit. Aliquots
were quickly stored at -20.degree. C. in polystyrene tubes.
According to Sigma technical service, this sulfhydryl (--SH)
compound is unstable in aqueous solutions, including tissue culture
medium, and is rapidly converted to the oxidized GS-SG form by
exposure to air. Addition every two to four days to the culture
medium may be required for reducing agent requiring cells. Another
reducing agent that also is effective is mercaptoethanol. It is
more stable and often effective at lower concentrations than GSH.
Reducing agents act as "scavengers" of free radicals generated by
the oxygen atmosphere of cell culture.
[0365] Serum-free Defined Medium Nutrient Supplements--Selenium
(Se). A powder of sodium selenite (100 mg/vial) is obtained from
Collaborative Research or Sigma (Catalog No. S5261). It has been
sterilized by irradiation. The contents of a single vial are
dissolved in 100 mL of sterile water to give final stock of 1.0
mg/mL. This preparation should not be filter sterilized because Se
binds to filters. The final volume was diluted to 100 mL with
sterile saline. Aliquots are stored at -20.degree. C. in
polystyrene tubes. Selenium is an important cofactor for enzyme
systems that protect the cells from oxidation effects.
[0366] Serum-free Defined Medium Nutrient Supplements--Diferric
Transferrin (2FeTf). Iron Fe (III) saturated (98%) human
transferrin (diferric transferrin) was purchased from Collaborative
Research (Catalog No. 40304) or Sigma (Catalog No. T3309) as
bottles containing 1 gram of red colored powder. The contents of
one bottle are dissolved in 100 mL of normal saline. This red
colored solution is sterilized using 0.2 .mu.M pore membrane
filters. This stock is 10 mg/mL. Aliquots are stored at -20.degree.
C. in polystyrene tubes. All growing cells require diferric
transferrin as a source of iron for a great many metabolic
processes.
[0367] Serum-free Defined Medium Growth Factor
Supplements--Epidermal Growth Factor (EGF). EGF prepared from mouse
submaxillary gland (tissue culture grade) was purchased from
Collaborative Research (Catalog No. 40001) as 100 .mu.g in a
sterile vial or from Sigma (Catalog No. E4127). The original vials
are stored at 4.degree. C. according to the manufacturer's
instructions. To prepare a stock solution, 5.0 mL of sterile saline
was added to a vial to yield a 20 .mu.g/mL EGF solution. Aliquots
are stored frozen at -20.degree. C. polystyrene tubes. Repeated
freeze-thaw must be avoided. This growth factor is useful because
of its very broad cell specificity range.
[0368] Serum-free Defined Medium Growth Factor Supplements--Acidic
Fibroblast Growth Factor (aFGF). Acidic FGF is purchased from Sigma
(Catalog No. F5542). It is the human recombinant product from E.
coli. This product has very specific handling requirements. It is
provided sterilized in 25 .mu.g vials lyophilized from PBS
containing 1.25 mg of BSA. The contents of each vial are
reconstituted in 25 mL of sterile PBS containing 1.0 mg/mL of BSA
and 10 .mu.g/mL of heparin. Filtration of this product at this
concentration must absolutely be avoided. This solution is stored
at -20.degree. C. in polystyrene tubes. The solutions of aFGF
definitely cannot be freeze-thawed more than twice. This growth
factor is highly labile. Careless handling will result in problems.
Keratinocyte growth factor (KGF) can substitute for aFGF. The
fibroblast growth factor family is very important in growth of
urogenitial tissues including prostate.
[0369] Serum-free Defined Medium Growth Factor
Supplements--Heparin. Heparin is used to stabilize FGF in cell
culture (Gospodarowitz D and Cheng J (1986) J Cell Physiol 128,
475-484). Heparin is obtained from Sigma (Catalog No. H3149) as the
sodium salt, Grade 1-A, from porcine intestinal mucosa. A solution
of 1.0 mg/mL is made in saline and sterilized with 0.2 .mu.m pore
membrane filters. An aliquot of 250 .mu.L is added to the 25 mL of
aFGF reconstitution solution used above. Sterile heparin is stored
at 4.degree. C.
[0370] Serum-free Defined Medium Adhesion Protein
Supplement--Fibronectin (Fbn). Human plasma derived Fbn can be
purchased from many commercial sources. Bovine Fbn is also
available and is effective. Fbn is prepared from units of fresh
human plasma (unfrozen) or fresh bovine (unfrozen) plasma by two
methods (Retta S F et al. (1999) Methods in Molecular Biology 96,
119-124; Smith R L and Griffin C A (1985) Thrombosis Res 37,
91-101). Purity is evaluated by SDS-PAGE with Coomassie Brilliant
Blue staining or silver staining (Pierce Chemicals.RTM. kits).
Adhesion activity is confirmed with cells in serum-free defined
medium. Vitronectin can substitute for fibronectin.
[0371] Serum-free Defined Medium Iron (Fe (III) Chelator
Supplements--Deferoxamine mesylate (DFX). DFX (FW 656.8) is
purchased from Sigma (Catalog No. D9533). The stock solution is
made at 10 mM by adding 131 mg to 20 mL of highly purified water as
described above. The solution is sterilized by filtration with 0.2
.mu.M pore membranes. Aliquots are stored at -20.degree. C. in
polystyrene tubes.
[0372] Serum-free Defined Medium Iron (Fe (III) Chelator
Supplements--Apotransferrin (apoTf). Human serum ApoTf can be
purchased from Sigma (Catalog No. T4382). It is minimum 98%
iron-free. ApoTf is also prepared as described previously (Sirbasku
D A et al. (1991) Biochemistry 30, 295-304; Sirbasku D A et al.
(1991) Biochemistry 30, 7466-7477). ApoTf is prepared by dialysis
against citrate buffer pH 5.0-5.5 with 1 .mu.g/mL DFX present to
chelate >98% of the iron. Handling and storage were as described
for diferric transferrin but with great care to avoid contact with
iron sources.
[0373] Serum-free Defined Medium Nutrient Supplements--Bovine
Insulin (INS). This hormone was purchased from either of two
sources. From Gibco-BRL it is Insulin, Bovine Zinc Crystals for
Cell Culture Applications (Catalog No. 18125-039). It was also
obtained from Collaborative Research (Catalog No. 40305) and stored
at 4.degree. C., according to that manufacturer's recommendation.
Gibco-BRL recommends solid insulin storage at -5.degree. C. to
20.degree. C. A stock of 10 mg/mL in 0.01 N HCl was prepared by
adding 250 mg of insulin to 25 mL of the acid. The HCl was made by
adding 172 .mu.L of concentrated (11.6 N) HCl to 100 mL of water.
The final stock solution of 10 mg/mL of insulin is filter
sterilized using 0.2 .mu.m pore diameter membranes. Aliquots are
stored at 4.degree. C. in polystyrene tubes. Care was taken not to
freeze-thaw the aliquots of stock solution. Insulin is a very broad
range cell growth-stimulating factor as well as a regulator of
specific metabolic processes.
[0374] Serum-free Defined Medium Nutrient Supplements--Thyroid
Hormones. The preferred thyroid hormone is T.sub.3
(3',5-Triiodothyronine, FW 673, purchased from Sigma as Catalog No.
T2752). It is stored desiccated at -20.degree. C. To prepare
stocks, 0.5 N NaOH was made by addition of 20 grams of pellets to
one liter of water. Then, 67.3 mg of T.sub.3 was added. After
dissolving the T.sub.3 with stirring for a few minutes, 25 mL of
this stock was diluted up to 250 mL with water, for a final
concentration of 0.05 N NaOH. This dilution was sterilized using
the 0.2 .mu.m pore diameter filter. At this point, the final stock
for storage was 10 .mu.M T.sub.3. Aliquots of this final stock are
stored in polystyrene tubes at -20.degree. C. The second thyroid
hormone, thyroxin (T.sub.4, sodium salt, pentahydrate FW 888.9), is
prepared by the same procedure. For this stock solution, 88.9 mg of
T.sub.4 are used. T.sub.4 is purchased from Sigma (Catalog No.
T2501). T.sub.4 is used at 10 to 20 times higher concentrations
than T.sub.3. Care is taken not to freeze-thaw these preparations.
Thyroid hormones have a very broad range of biological effects on
metabolism and growth. Many cells in culture require these for
growth.
[0375] Compositions of Serum-free Defined Media. TABLE 7 presents
the formulations of the preferred serum-free defined media
developed for use in detecting high-level steroid hormone
reversible inhibition by steroid hormone-stripped serum fractions
and by purified inhibitors in serum-free cell growth assays. As
indicated in the footnotes to the table, when a particular
component is included in one of the formulations, the concentration
that provides a suitable cell growth medium can fall within the
indicated range.
TABLE-US-00008 TABLE 7 Composition of Serum-free Defined Media
Based on Standard Gibco-BRL D-MEM/F-12 Human Human Rat Rat Hamster
CELL TYPE Breast Prostate Mammary Pituitary Kidney MEDIUM NAME
DDM-2MF CAPM DDM-2A PCM-9 CAPM COMPONENT FINAL CONCENTRATIONS IN
THE DEFINED MEDIA Insulin.sup.1 500 ng/mL 10 .mu.g/mL 10 .mu.g/mL
10 .mu.g/mL 10 .mu.g/mL EGF.sup.2 20 ng/mL 20 ng/mL 20 ng/mL None
20 ng/mL AFGF.sup.3 None 10 ng/mL None None 10 ng/mL
Triiodothyronine.sup.4 0.3 nM 1.0 nM 0.3 nM 1.0 nM 1.0 nM Diferric
transferrin.sup.5 10 .mu.g/mL 10 .mu.g/mL 10 .mu.g/mL 10 .mu.g/mL
10 .mu.g/mL Ethanolamine.sup.6 50 .mu.M 50 .mu.M 50 .mu.M 10 .mu.M
50 .mu.M Phosphoethanolamine.sup.7 5 .mu.M None 5 .mu.M None None
Bovine Serum Albumin.sup.8 500 .mu.g/mL 1.0 mg/mL 500 .mu.g/mL 500
.mu.g/mL 1.0 mg/mL Linoleic acid-BSA.sup.9 150 .mu.g/mL None 150
.mu.g/mL None None Selenium.sup.10 20 ng/mL 10 ng/mL 20 ng/mL 10
ng/mL 10 ng/mL Reduced glutathione.sup.11 20 .mu.g/mL None 20
.mu.g/mL None None Glutamine.sup.12 2.0 mM None 2.0 mM None None
Heparin.sup.13 None 7.5 .mu.g/mL None None 7.5 .mu.g/mL
Deferoxamine.sup.14 5 .mu.M 10 .mu.M 5 .mu.M 10 .mu.M 10 .mu.M
Human Fibronectin.sup.15 25 .mu.g 20 .mu.g None None 20 .mu.g When
a component is added, the following are the effective concentration
ranges used: .sup.1INS range 100 ng/mL to 10 .mu.g/mL .sup.2EGF
range 1 ng/mL to 50 ng/mL .sup.3aFGF range 0.2 ng/mL to 20 ng/mL
.sup.4T.sub.3range 0.3 nM to 10 nM .sup.52FeTf range 2 .mu.g/mL to
50 .mu.g/mL .sup.6ETN range 5 .mu.M to 100 .mu.M .sup.7PETN range 5
.mu.M to 50 .mu.M .sup.8BSA range 0.2 mg/mL to 5.0 mg/mL
.sup.9Lin-Alb range 50 .mu.g/mL to 500 .mu.g/mL .sup.10Se range 5
ng/mL to 20 ng/mL .sup.11GSH range 1 .mu.g/mL to 50 .mu.g/mL
.sup.12Glut range 0.5 mM to 2.0 mM .sup.13Heparin range 1 .mu.g/mL
to 10 .mu.g/mL .sup.14DFX range 2 .mu.M to 20 .mu.M .sup.15Fbn
range 15 .mu.g to 50 .mu.g per 35-mm diameter dish
[0376] Serum-free Media Variations. Standard phenol red-containing
Gibco-BRL D-MEM/F-12 is a preferred basal medium to which the
defined media components are added. It contains 0.6 mM to 1.0 M
CaCl.sub.2. D-MEM/F-12 can be purchased from Gibco-BRL in the
liquid form or can be prepared from the powder formulation using
only highly purified water. Alternatively, another suitable basal
medium could be used as long as it provides at least the required
minimum amounts of necessary nutrients, vitamins and minerals to
maintain cell viability of the desired cell line. The calcium
concentration range preferred is 0.6 to 10 mM. Calcium stabilizes
the inhibitor in cell culture without impairing cell growth. The
human breast cancer cell medium, DDM-2MF, was a modification of the
original DDM-2 medium (Danielpour D et al. (1988) In Vitro Cell Dev
Biol 24, 42-52) and MOM-1 (Ogasawara M and Sirbasku D A (1988) In
Vitro Cell Dev Biol 24, 911-920) and contained modified hormone
concentrations, deferoxamine (DFX) and fibronectin. Aqueous salt
solutions such as tissue culture medium contain hydrolytic
polymeric forms of Fe (III) (Spiro T G et al. (1966) J Am Chem Soc
88, 2721-2726). DFX binds this form of Fe (III) with very high
affinity (Schubert J (1964) In; Iron Metabolism The Chemical Basis
of Chelation, Springer, Berlin, pp 466-498). If not removed, Fe
(III) inhibits hormone-responsive growth in serum-free defined
medium (Sirbasku D A et al. (1991) Mol Cell Endocrinol 77, C47-055;
Sato H et al. (1992) Mol Cell Endocrinol 83, 239-251; Eby J E et
al. (1993) J Cell Physiol 156, 588-600; Eby J E et al. (1992) Anal
Biochem 203, 317-325). Fibronectin was used with DDM-2MF to promote
cell attachment. The 35-mm diameter assay dishes were pre-coated by
incubation with the designated amount of fibronectin (TABLE 7) for
16 to 48 hours at 37.degree. C. in 2.0 mL of D-MEM/F-12. CAPM human
prostatic cancer cell medium was developed to support the growth of
tumor cells from this tissue. The composition of CAPM is described
in TABLE 7. CAPM also supports the growth of the H301 Syrian
hamster kidney tumor cells. DDM-2A, which is a modified form of
DDM-2 (Danielpour D et al. (1988) In Vitro Cell Dev Biol 24,
42-52), was preferred for growing MTW9/PL2 cells. PCM-9 defined
medium was developed for growing the rat pituitary cell lines. This
medium differs from previous PCM formulations (Sirbasku D A et al.
(1991) Mol Cell Endocrinol 77, C47-055; Sato H et al. (1992) Mol
Cell Endocrinol 83, 239-251; Eby J E et al. (1993) J Cell Physiol
156, 588-600; Eby J E et al. (1992) Anal Biochem 203, 317-325) in
that DFX was substituted for apotransferrin and the
triiodothyronine concentration was increased to 1.0 nM. Although
DFX and apotransferrin (2 to 50 .mu.g/mL) are the preferred
chelators based on their very high specificity and affinities for
Fe (III), EDTA at 1 to 10 .mu.M or sodium citrate at 10 to 1000
.mu.M also effectively neutralize the cytotoxic effects of Fe (III)
(Eby J E et al. (1993) J Cell Physiol 156, 588-600). Ascorbic acid
(vitamin C) also chelates Fe (III), but is used less often because
it is unstable in cell culture medium at 37.degree. C. in an oxygen
environment in the presence of salts and metals in the medium.
Also, at concentrations of 50 to 100 .mu.g/mL, apo-ovotransferrin
and apo-lactoferrin also were effective Fe (III) chelators in
serum-free defined medium (Eby J E et al. (1993) J Cell Physiol
156, 588-600). Although EGF, aFGF and insulin are the preferred
growth factors, several other human recombinant proteins are
effective. They have either been purchased or obtained as gifts
from Gibco-BRL, Sigma or IMCERA Bioproducts. Insulin-like growth
factors I and II (IGF-I and IGF-II) can be used to replace insulin,
transforming growth factor .alpha. (TGF.alpha.) replaces EGF,
TGF.beta. as an inhibitory supplement, and basic fibroblast growth
factor (bFGF) partially replaces aFGF. Insulin can be used to
replaced IGF-I and IGF-II. All of these protein growth factors are
dissolved under sterile conditions according to manufacturers'
instructions and stored as indicated.
[0377] Discussion of Example 10. The preferred serum-free media
described above provide an ideal scenario for the study of growth
responses of hormone responsive cancers without the myriad of
potential interactions accompanying the presence of serum with its
5000+ proteins and other compounds. The formulations presented
permit dissection of growth into its individual parts caused by
different stimulators. When of interest, a combination of a few
factors can be investigated to achieve an understanding of growth
promoter/inhibitor interactions (i.e. cross-talk). This is
exceptionally difficult to achieve in the presence of full serum.
The serum-free medium described herein provided a tool for the
assessment of growth inhibitor(s) isolated from CDE-horse serum,
whose actions are reversed by sex-steroid hormones, as mentioned at
the beginning of this Example and described in more detail in
subsequent Examples. The preferred serum-free media of the present
invention raise hope for the provision of new insight that could
help to clarify the mechanisms involved in the control of breast,
prostatic and other mucosal cancers under conditions not previously
available.
[0378] Moreover, because of widespread concern today about possible
contamination of commercial animal sera by disease causing agents
such as bovine spongiform encephalopathy ("mad cow disease"), there
is a great need for serum-free cell culture media that can support
a variety of cell types. The new media compositions fill that need.
The new serum-free media can be used not only for assays but also
for large scale testing purposes and industrial uses such as cell
culture production of a desirable protein. For example, an antigen
for vaccine production, or a monoclonal antibody can be prepared
without fear of contamination a by serum-derived infectious agent.
They are also useful for producing a quantity of virus for vaccine
manufacture or for producing recombinant viruses for gene therapy.
Basic cell culture methods for producing quantities of proteins or
viruses are well known in the art and have been described in the
literature.
Example 11
Serum-Free Defined Medium that Supports Hormone Sensitive and
Autonomous Cancer Cell Growth
[0379] In this Example, it is shown that media derived according to
the present methods are effective for supporting hormone sensitive
and autonomous cancer cell growth.
[0380] Selection of Models to Study Hormone Dependence and Autonomy
in Serum-free Defined Culture Media. One goal was to develop
serum-free defined media that can be used to directly compare
negative serum factor regulation with steroid hormone responsive
and steroid hormone autonomous cancers of the same tissue. That
meant establishing a medium that supported the growth of both cell
types. As models, human prostatic carcinoma and human breast
carcinoma cells were chosen because responsive and autonomous
(unresponsive) cell lines are currently available for both types of
cancers. Furthermore, as discussed above, these cancers have many
common characteristics including their tendency to pass from
steroid hormone receptor positive to steroid hormone receptor
negative in a process called tumor progression. During the course
of development of such defined media, one observation was made
consistently: breast cancer cells that were ER.sup.+ (i.e. estrogen
sensitive) and prostate cancer cells that were AR.sup.+ (i.e.
androgen sensitive) grew less well in defined medium based on
standard D-MEM/F12 than in defined medium based on "low-Fe"
D-MEM/F12. The results of an example with T47D cells in DDM-2MF are
shown in FIG. 38. The example with LNCaP cells in CAPM is shown in
FIG. 39. Another example is the thyroid hormone responsive MDCK
kidney tubule epithelial cells in CAPM as shown in FIG. 40.
Standard D-MEM/F-12 contains both ferric nitrate and ferrous
sulfate as nutrient additions. When purchased without these salts,
the medium was designated "low-Fe" D-MEM/F-12. The iron
concentrations in standard and "low-Fe" D-MEM/F-12 were 1.0 .mu.M
and 0.15 .mu.M, respectively (Eby J E et al (1992) Anal Biochem
203, 317-325). Even in "low-Fe" medium, iron is present as a
contaminant in the chemicals used to make the formulation, the 2.2
g/L NaHCO.sub.3 added as a metabolic requirement and buffer, and
the 15 mM HEPES buffer necessary for stabilizing the pH under
serum-free conditions (Eby J E et al (1992) Anal Biochem 203,
317-325). It is noteworthy that as low as 1.0 .mu.M Fe (III)
inhibits epithelial cell growth completely within five to seven
days. In another test the thyroid hormone responsive human HT-29
colonic carcinoma cells in CAPM also grew better in "low-Fe" than
standard D-MEM/F-12 (data not shown). This indicates that
restriction of Fe (III) in culture medium will have implications
even beyond sex steroid hormone dependent cells.
[0381] Modifications of the Usual Growth Assays for Experiments in
"low-Fe" Medium versus "Standard" Medium. Specific modifications of
the customary cell growth assays were required for assays done
under iron-restricted conditions. For example, the 35-mm assay
dishes were incubated for 16 to 24 hours prior with 20 to 25 .mu.g
of fibronectin in 2 mL of "low-Fe" D-MEM-F12 medium, Serum-free
components were added to "low-Fe" D-MEM/F-12 at double the
concentrations needed (2.times.) or to "standard" D-MEM/F-12 at
(2.times.) as the experiments dictated. Each assay dish received
1.0 mL of this solution. Next, the cells to be used in the assays
were washed three times in either "low-Fe" medium or "standard"
medium depending upon the experimental protocol. These washes were
done with the same care as described above in General Materials and
Methods. Each dish received 1.0 mL of cells in the appropriate
medium. At this point, the components final concentrations were
(1.times.) as summarized in TABLE 7. Also, TABLE 7 describes medium
containing deferoxamine as the Fe (III) chelator. Although less
preferred, due in part to cost considerations, specificity, and
affinity for Fe (III), as noted above, apotransferrin is also
effective, especially at the preferred apotransferrin concentration
of 50 .mu.g/mL. When apotransferrin binds Fe (III), it is converted
to one of three forms of ferric transferrin (Eby J E et al (1992)
Anal Biochem 203, 317-325). These become additional support for
cell growth in defined medium, thereby converting a toxic substance
to a useable nutrient.
[0382] Growth in Serum-free Defined Medium versus D-MEM/F-12 with
10% (v/v) Fetal Bovine Serum. To demonstrate the usefulness of the
formulations in TABLE 7, cell growth was compared in serum-free
defined medium.+-.steroid hormone versus growth supported by fetal
bovine serum. It is generally accepted that fetal bovine serum
represents one of the most effective sera for tissue culture. As an
example, growth of the LNCaP cells was compared in CAPM.+-.DHT
versus growth in 10% (v/v) fetal bovine serum (FIG. 41). CAPM plus
10 nM DHT supported growth at about 80-90% of the rate of fetal
bovine serum. Growth promoted by 10% fetal bovine serum obtained
from conventional commercial sources reached 6.57 (.+-.0.48) CPD
or, a 96-fold increase on cell number in 12 days. By day 12, cell
densities in CAPM nearly equaled those in serum. Growth promoted by
the serum-free medium reached 6.22 (.+-.0.35) CPD or 84-fold
increase. CAPM was able to support LNCaP growth even in the absence
of sex-steroid hormones. Maximum growth obtained without
sex-steroid hormones was of 5.35 (.+-.0.12) CPD or a 49-fold
increase. The androgenic effect is therefore marginal, with
differences of less than one CPD between the presence and absence
of DHT. Also shown, the cells did not grow in D-MEM/F-12 without
any additions (FIG. 41). Similar studies were done with other cell
lines to determine growth rates versus serum and to establish the
periods for single time assays (e.g. 7, 10, 12 or 14 days). FIG. 42
shows the same analysis with DU145 and PC3 cells in CAPM and in
D-MEM/F-12 with 10% fetal bovine serum. As the cell number data
show, growth was logarithmic. After 12 days, growth in the
serum-free medium was identical to that in 10% fetal bovine serum
for both cell lines. Growth of PC3 in 10% serum reached 6.98
(.+-.0.71) CPD or a 112-fold increase in cell number versus 6.97
(.+-.0.44) CPD or the same fold increase for cell numbers in
serum-free medium. Growth of DU145 in 10% fetal bovine serum was
6.71(.+-.0.58) CPD versus 6.73 (.+-.0.18) CPD in serum-free
conditions. The results in FIGS. 41 and 42 demonstrate by example
that the serum-free defined media in TABLE 7 are effective with
both hormone sensitive and hormone autonomous cells.
[0383] Determination of Component Concentrations and the
Requirement for a Fe (III) Chelator. The optimum concentration of
each single component was determined by dose-response analysis in
the presence of other components. The technology used to establish
early forms of serum-free defined media has been described
(Danielpour D et al. (1988) In Vitro Cell Dev Biol 24, 42-52;
Ogasawara M and Sirbasku D A (1988) In Vitro Cell Dev Biol 24,
911-920). An example of this process is shown in FIG. 43 with LNCaP
cells. Dose-response effects of bovine serum albumin,
apotransferrin, T.sub.3, ethanolamine, selenium, and EGF are shown.
The results show clearly that the addition of the iron chelator
apotransferrin was required for cell growth. After determining
optimum concentrations for each component, the contribution of each
to the total was assessed by another assay. Individual components
were deleted one at a time. As an example, the three most widely
used prostatic carcinoma cell lines were compared (i.e. LNCaP, PC3
and DU145) in CAPM that contained deferoxamine in place of
apotransferrin (FIG. 44). The deletions were done.+-.DHT. The first
and most striking result was the major differences between the
growth requirements of the DHT sensitive LNCaP cells and those of
the autonomous DU145 and PC3. Only the deletion of diferric
transferrin substantially prevented the growth of autonomous cells.
Also, it was clear that deletion of deferoxamine had only a small
(i.e. <20%) effect on growth of the DU145 and PC3 cells. The
DU145 and PC3 cell lines also were T.sub.3, insulin, EGF,
fibronectin and deferoxamine independent. As expected.+-.DHT had no
significant effect on DU145 or PC3. By contrast, LNCaP growth was
significantly (p<0.01) reduced or arrested completely by
deletion of fibronectin, T3, diferric transferrin or deferoxamine.
LNCaP growth also was inhibited by deletion of EGF or insulin, but
these effects were pronounced only in the absence of DHT.
[0384] Discussion of Example 11. The media described in TABLE 7
were optimized for the specific cell types designated.
Additionally, they were optimized to permit direct comparison of
the growth properties of ER.sup.+ and AR.sup.+ steroid hormone
sensitive tumor cell lines to their ER.sup.- and AR.sup.- steroid
hormone insensitive (also called autonomous) counterparts. This
careful optimization was done originally to study rat mammary tumor
cells of both types in DDM-2A defined media. The appropriate cell
lines for this approach have been developed from the MTW9/PL2
population and described (Danielpour D and Sirbasku D A (1984) In
Vitro 20, 975-980). The medium DDM-2MF has been developed for the
same purpose only for comparisons of ER.sup.+ and ER.sup.- forms of
these cancers. TABLE 1 lists the most important ER.sup.+ human
breast cancer cell lines in use today. In addition a number of
other ER.sup.- human breast cancer cells lines have been evaluated.
They are the MDA-MB-231 (Cailleau R et al. (1974) J Natl Cancer
Inst 53, 661-674), BT-20 (Lasfargues E Y and Ozzello L (1958) J
Natl Cancer Inst 21, 1131-1147), Hs0578T (Hackett A J et al. (1977)
J Natl Cancer Inst 58, 1795-1806), MDA-MD-330 (Cailleau R et al.
(1978) In Vitro 14, 911-915), and the myoepithelial HBL-100
(Gaffney E V (1982) Cell Tissue Res 227, 563-568). The
demonstration of ER.sup.- status of these lines has been described
(Reddel R R et al. (1985) Cancer Res 45, 1525-1531). With regard to
human prostatic cancer, the only reliable androgen responsive cell
line available today is the LNCaP (TABLE 1). Another, the ALVA-41,
has been described as androgen growth responsive (Nakhla A M and
Rosner W (1994) Steroids 59, 586-589). However, as shown in
subsequent Examples, this line is autonomous by the criterion of
lack of DHT effects in CDE-horse serum. Two other human prostate
cancer cell lines are commonly used as autonomous examples. These
lines are the DU145 (Stone K R et al. (1978) Int J Cancer 21,
274-281) and the PC3 (Kaighn M E et al. (1979) Invest Urol 17,
16-23). Previously, there was a defined medium established for PC3
cells (Kaighn M E et al. (1981) Proc Natl Acad Sci USA 78,
5673-5676). This medium was evaluated and did not support LNCaP
cell growth. However, others have reported "serum-free" media that
was stated to be effective with LNCaP, DU145, PC3 and ALVA-31 cells
(Hedlund T E and Miller G J (1994) The Prostate 24, 221-228). The
problem was this medium was not serum-free nor was it defined. The
experiments began with cells plated into 5% serum and then preceded
to use a serum fraction called fetuin to support growth. Fetuin is
a complex undefined mixture of .gtoreq.4% of the proteins in serum.
Under those conditions, an accurate analysis of hormonal and growth
factor effects cannot be done satisfactorily. The completely
serum-free CAPM in TABLE 7 supports the growth of all of these
prostate cell lines. In addition, CAPM has been applied to the
ER.sup.+ estrogen growth stimulated H301 Syrian hamster kidney
cells (Sirbasku D A and Moreno J E (2000) In Vitro Cell Dev Biol
36, 428-446) and its autonomous derivative cell line A195. As has
been reviewed (Evans R M (1988) Science (Wash D.C.) 240, 889-895),
steroid hormones and thyroid hormones belong to the same
superfamily of receptors. Both are important in growth. Therefore,
it was expected that some tissues might be thyroid hormone positive
regulated, while others might be positive regulated by steroid
hormones. CAPM has also been applied to the study of thyroid
hormone reversal of purified inhibitors with the human colonic
carcinoma cell line HT-29. Similar use has been made of CAPM with
the MDCK dog kidney tubule cell line (Leighton J et al. Science
(Wash D.C.) 158, 472-473). CAPM replaces a different defined medium
prepared for MDCK cells (Taub M et al. (1979) Proc Natl Acad Sci
USA 76, 3338-3342). It is likely that the prostaglandins in that
earlier medium interfere with the action of the thyroid hormones.
In any case, that medium was not useful for demonstration of
thyroid hormone reversal of purified MDCK cell growth inhibitors.
All of these observations support the view that a series of
uniquely optimized media have been formulated to define the growth
requirements of epithelial cells from several of the very prominent
cancers of humans. Furthermore, the technology developed promises
application to the optimization of growth of other types of
epithelial cells from a variety of target tissues.
Example 12
Differential Effects of Fe (III) on the Growth of Hormone
Responsive and Autonomous Human Breast and Human Prostate Cancer
Cells
[0385] This Example demonstrates that iron has an inhibiting effect
on steroid responsive cell growth, independent of the
above-described immunoglobulin effects, and which is
distinguishable from its effect on autonomous cells.
[0386] Approaches to Demonstration of Iron Toxicity. The fact that
standard D-MEM/F-12 contains sufficient Fe (III) to inhibit cell
growth, led to the next series of studies. Other approaches were
used to further demonstrate the deleterious effects of Fe (III) on
hormone responsive tumor cell growth. To add Fe (III) to culture
medium, it must be in a soluble form. Ferric ammonium citrate was
selected for use. However, ferric ammonium sulfate is also
effective. Ferric ammonium citrate is a mixture that contains 16.6%
of ferric iron by weight. The amount of mixture added to each dish
was adjusted to achieve the desired Fe (III) concentrations. Due to
the light sensitivity of the mixture, the solutions were prepared
fresh daily and the experiments carried out under restricted light
conditions. Also, the mixture was prepared in water. Buffers
without phosphate may be used, but they are generally less
effective. The ferric mixtures and the iron chelators EDTA,
deferoxamine mesylate and sodium citrate were purchased from
Sigma.
[0387] Iron Toxicity with Human ER.sup.+ Breast Cancer Cells. In
the first experiments, two ER.sup.+ cell lines were evaluated for
Fe (III) sensitivity in DDM-2MF defined medium prepared with 10
.mu.g/mL apotransferrin in place of the deferoxamine shown in TABLE
7. The effect of addition of ferric ammonium citrate on MCF-7A
growth.+-.B.sub.2 at 10 days is shown in FIG. 45. Either with or
without the steroid, Fe (III) was completely inhibitory at 10
.mu.M. There were no viable cells in the dishes at .gtoreq.10
.mu.M. The EI.sub.50 of Fe (III) with MCF-7A cells was 5 to 7
.mu.M. A similar analysis with T47D cells in DDM-2MF with 10
.mu.g/mL apotransferrin instead of deferoxamine showed complete
inhibition at 10 days with 2 .mu.M Fe (III) (FIG. 46). At .gtoreq.2
.mu.M there were no viable cells in the dishes either with or
without E.sub.2. The EI.sub.50 of FE (III) with T47D cells was 1
.mu.M.
[0388] Iron Toxicity with AR.sup.+ and AR.sup.- Human Prostate
Cancer Cell Lines. The effect of Fe (III) on AR.sup.+ LNCaP cell
growth was assessed in CAPM defined medium in which apotransferrin
(500 nM) was substituted for deferoxamine, and the results are
shown in FIG. 47. Clearly, 10 .mu.M Fe (III) arrested growth to
seed density levels (i.e. 12,000 cells per dish) in a 12-day assay.
The EI.sub.50 for LNCaP cells was 5 .mu.M. In another experiment in
CAPM, the effects of ferric ammonium citrate were evaluated with
AR.sup.+ LNCaP cells and AR.sup.- PC3 and DU145 cells (FIG. 48).
Again, Fe (III) inhibited LNCaP cells to seed densities levels by 8
to 10 .mu.M. However, effects on the androgen autonomous PC3 and
DU145 cells were markedly less (FIG. 48). Reductions of 10 to 30%
in cell number for PC3 and DU145, respectively, were observed in 10
.mu.M Fe (III). The inhibitory effects of Fe (III) on the androgen
independent PC3, DU145 and ALVA-41 cells were variable, and never
as marked as with the steroid hormone responsive LNCaP cells. The
insert in FIG. 48 shows a correlation between hormone
responsiveness and Fe (III) effects. The results show a correlation
between iron effects and thyroid hormone responsiveness. LNCaP
cells are T.sub.3 responsive whereas PC3 and DU145 are not.
[0389] Reversal of Fe (III) Inhibition by Iron Chelators. The
inhibitory/cytotoxic effects of Fe (III) were reversible by the
addition of iron chelators. Those studied were selected based on
data showing their relative affinities and specificities for Fe
(III) (Schubert J (1963) In: Iron Metabolism, Gross F, ed,
Springer-Verlag, Berlin, pp 466-496). Deferoxamine is most specific
and has the highest affinity for Fe (III). Citrate is next most
effective. EDTA is not as effective nor is it as specific as the
first two chelators. In experiments with T47D cells, the
deferoxamine usually present in the DDM-2MF medium was removed and
an additional 1.5 .mu.M Fe (III) added to ensure complete
inhibition of the cells. FIG. 49 shows the relative effects of
addition of these three chelators to T47D serum-free defined medium
cultures. The order of effectiveness was as expected from the
affinities and specificities of these chelators. Clearly, addition
of Fe (III) chelators restored growth. FIG. 50 shows a similar
study with LNCaP cells in CAPM defined medium from which the
deferoxamine also was removed and 1.5 .mu.M Fe (III) added. It was
clear that chelation of the Fe (III) restored growth. It should be
noted that this conclusion is reasonable based on the fact that
deferoxamine has near absolute specificity for Fe (III).
Concentrations as low as 0.5 .mu.M of deferoxamine were sufficient
to induce 3.5 CPD with LNCaP cells. Maximum growth with this
chelator (5.81 CPD) was obtained at 10 .mu.M. Citrate and EDTA were
also effective growth stimulators of LNCaP cells incubated at high
iron concentrations. Maximum growth was obtained with the addition
of 500 .mu.M and 10 .mu.M respectively. The growth induction
achieved with EDTA is lower than with citrate or deferoxamine. This
probably could be explained by the fact that EDTA is a less
discriminatory chelator, and essential metals other than iron were
affected. Concentrations of the chelators higher than the ones
showed in the FIGS. 49 and 50 were associated with cell damage and
death. In particular, chelation of calcium by citrate and EDTA will
cause cell death in culture. As controls, stimulation by chelators
was prevented by resupply of Fe (III) (data not shown).
[0390] Correlation Between Hormone Autonomy and Lack of Iron
Effects. In the next series of studies, data was sought supporting
the concept that loss of steroid hormone dependence correlates
positively with loss of Fe (III) effects. As shown in FIG. 39,
LNCaP cells grew better in `low-Fe" serum-free defined medium than
in defined medium based on "standard" D-MEM/F-12. This difference
was also evaluated with the androgen insensitive DU145 (FIG. 51)
and PC3 (FIG. 52) cells. The results were clear. The autonomous
lines grew equally well in CAPM based on both types of D-MEM/F-12.
The presence of the higher Fe (III) level in CAPM based on standard
D-MEM/F-12 had no effect. To confirm that these cell lines were
androgen autonomous as defined by the loss of steroid and inhibitor
growth regulation in CDE-serum, the next studies were done. DU145
cells showed no inhibition of growth in 50% CDE-serum (FIG. 53).
There was no androgenic effect whatsoever. A similar assay with PC3
cells showed essentially the same results (FIG. 54). There was no
inhibition even in 50% CDE-horse serum, and no androgenic effect.
Additionally, ALVA-41 cells are not iron sensitive (results not
shown), and also are not sensitive to the serum-borne inhibitor
(FIG. 55).
[0391] Discussion of Example 12. Together with the studies
presented above, it appears that AR.sup.+ cells are sensitive to
the serum-borne inhibitor, sensitive to the positive effects of
steroid hormone and sensitive to Fe (III) inhibition. In contrast,
the DU145 and PC3 cells are insensitive to the serum-borne
inhibitor, insensitive to the positive effects of androgen, and
insensitive to Fe (III). The results presented in this example
continue to demonstrate the requirement for the action of a
serum-borne mediator to demonstrate steroid hormone responsive cell
growth in culture. The use of CDE-serum was essential for the
demonstration of androgen and other steroid hormone responsiveness
in culture, but its use limits the understanding of stimulatory or
inhibitory roles of hormones or factors on prostate and other
cancer cells because of the inclusion of an undetermined amount of
undefined components. A serum-free medium circumvents this problem,
as shown in subsequent Examples.
[0392] In addition, autonomy may be the loss of the receptor for
the serum factor and/or the loss of the intracellular steroid
hormone receptor. If this hypothesis is correct it should be
possible to identify cells that possess steroid receptors but still
have lost "sensitivity" to the hormone by virtue of the lack of the
effect of the inhibitor. Most notably, this is the case with DU145
and ALVA-41 cells. As defined by immunohistochemistry, the DU145
cells are definitely AR.sup.+ (Brolin J et al. (1992) The Prostate
20, 281-295). As defined by a number of criteria, the ALVA-41 cells
are AR.sup.+ (Nakhla A M and Rosner W (1994) Steroids 59, 586-589).
A new concept explaining the progression of normal tissue cells to
hormone autonomous cancers is discussed in more detail in an
Example below.
[0393] Exposure of androgen responsive prostate cancer cells to Fe
(III) results in cell death. Compounds containing available Fe
(III) offer the possibility of new therapies for prostate cancer
localized to the tissue. It is proposed that deprivation of iron
will be a highly effective means of eliminating the most dangerous
hormone autonomous forms of prostate cancer. The measurement of
thyroid hormone receptors in prostate cancer should be initiated as
a diagnostic tool to determine iron sensitivity. Moveover, new
therapy mode for tumors containing mixtures of both hormone
responsive and autonomous cells is suggested, based on the
observation that deprivation of iron can equally kill both types of
cancer. This suggests that systemic Fe (III) therapy for
disseminated prostate cancer may be efficacious.
[0394] It is definitely possible that iron in the Fe (III) form and
compounds containing it will be effective anti-prostate cancer
treatments, and that direct injection (or painting) of localized
prostate tumors or metastasis at other sites (e.g. bone) might
effectively kill these cancers without concomitant systemic
effects. This therapy potentially could replace such protocols as
systemic chemotherapy (physically damaging), radiotherapy (damage
to collateral tissues) or the use of locally acting radioactive
gold chips that are complex to handle in the surgical environment
and must be implanted and removed surgically. Furthermore, iron
therapies can be repeated frequently by application via transrectal
or transurethral access, using conventional techniques. This
approach is unique and has not been discussed or suggested anywhere
else in the literature. Such iron treatments may be a useful
therapy for benign prostatic hypertrophy (BPH). As discussed above,
this condition is very common in older men and is treated usually
by surgery. Application of iron compounds is a new approach to
treatment of BPH, Similarly, a Fe (III) solution could be applied
to breast cancer lumpectomy or mastectomy sites at the time of
surgery, and/or applied by injection to the sites subsequent to
surgery.
Example 13
Growth in Serum-Free Defined Medium Versus Growth in
CDE-Serum.+-.E.sub.2
[0395] The defined media described in Example 10 were used to
verify the presence of a serum-borne inhibitor. The growth of six
different ER.sup.+ cell lines was compared in serum-free defined
media (TABLE 7) to the effects seen in cultures supplemented with
CDE-horse serum. These studies are shown in FIGS. 56 and 57.
Estrogenic effects are recorded for each set of conditions with
each cell line.
[0396] MCF-7K Cells in Serum-free and Serum Containing
Medium.+-.E.sub.2. The first studies were done with steroid hormone
responsive human cancer cell lines. FIG. 56A shows MCF-7K cell
growth in serum-free DDM-2MF.+-.10 nM E.sub.2. The population
replicated logarithmically for 12 days. E.sub.2 had no effect on
growth rate or saturation density. These results were in contrast
to assays done in D-MEM/F-12 supplemented with CDE horse serum
(FIG. 56B). Above 10% (v/v) serum, growth was progressively
inhibited. The inhibition caused by any serum concentration was
reversed by E.sub.2. Measured on assay day 10, a 3 CPD estrogenic
effect was observed which was a 2.sup.3 or 8-fold cell number
increase. The experiments were also done with MCF-7A cells with
similar results (data not shown). This effect in CDE-serum was as
great as that reported for a special response clone of the MCF-7
cell line (Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A,
595-602).
[0397] T47D Cells in Serum-free and Serum Containing
Medium.+-.E.sub.2. FIG. 56C shows the growth of T47D cells in
serum-free defined DDM-2MF.+-.10 nM E.sub.2. Although a small
effect of estrogen was observed on growth rate, the most
significant effect was an increase in stationary densities by 0.5
to 1.0 CPD. In contrast, the effect of E.sub.2 was much greater in
medium containing CDE horse serum (FIG. 56D). At 50% (v/v)
CDE-serum, growth was completely inhibited. The estrogenic effect
under these conditions was >5 CPD. This was more than a 2.sup.5
or 32-fold hormone effect on cell number. Comparison of these
results with those of others (Chalbos D et al (1982) J Clin
Endocrinol Metab 55, 276-283; Schatz R W et al. (1985) J Cell
Physiol 124, 386-390); Soto A M et al. (1986) Cancer Res 46,
2271-2275; Soto A M and Sonnenschein C (1987) Endocr Rev 8, 44-52;
Reese C C et al. (1988) Ann NY Acad Sci 538, 112-121) confirmed
that the conditions in FIG. 56D were substantially more effective.
Comparable experiments with the ZR-75-1 line gave results
intermediate between MCF-7 and T47D cells (data not shown). ZR-75-1
cells showed no effect of E.sub.2 in serum-free defined DDM-2MF.
This line grows more slowly than MCF-7 or T47D cells in defined
medium and in serum-supplemented cultures (Ogasawara M and Sirbasku
D A (1988) In Vitro Cell Dev Biol 24, 911-920). The maximum
estrogenic effects of the preferred embodiment recorded with
ZR-75-1 cells in D-MEM/F-12 with 50% (v/v) CDE-horse serum ranged
between 3 and 4 CPD after 14 days. This was greater than reported
by others in serum containing (Darbre P et al. (1983) Cancer Res
43, 349-355; Kenney N J et al. (1993) J Cell Physiol 156, 497-514)
or "serum-free" medium (Allegra J C and Lippman M E (1978) Cancer
Res 38, 3823-3829; Darbre P D et al. (1984) Cancer Res 44,
2790-2793).
[0398] LNCaP Cells in Serum-free and Serum Containing
Medium.+-.E.sub.2. In another study, the effects of E.sub.2 on the
growth of the LNCaP human prostatic carcinoma cell lines in defined
medium and in serum-supplemented culture were compared. This cell
line bears a point mutation in the AR that permits high affinity
binding of estrogens to the altered receptor (Veldscholte J et al.
(1990) Biochem Biophys Res Commun 173, 534-540; Veldscholte J et
al. (1990) Biochim Biophys Acta 1052, 187-194). In addition, it is
possible that estrogens cause LNCaP growth via a separate
functional ER (Castagnetta L A and Carruba G (1995) Ciba Found Symp
191, 269-286). Irrespective of mechanism, estrogens are known to
promote LNCaP growth (Belanger C et al. (1990) Ann NY Acad Sci 595,
399-402; Veldscholte J et al. (1990) Biochem Biophys Res Commun
173, 534-540; Veldscholte J et al. (1990) Biochim Biophys Acta
1052, 187-194; Castagnetta L A and Carruba G (1995) Ciba Found Symp
191, 269-286). As presented herein (FIG. 56E), this cell line in
serum-free defined CAPM showed essentially no E.sub.2 effect on
growth rate and .ltoreq.1.0 CPD on saturation density. When LNCaP
growth assays were done in medium with CDE-horse serum, the
mitogenic effect of E.sub.2 was >5 CPD (FIG. 56F). Estrogenic
effects herein were larger than reported by others with LNCaP cells
in serum containing culture (Belanger C et al. (1990) Ann NY Acad
Sci 595, 399-402; Castagnetta L A and Carruba G (1995) Ciba Found
Symp 191, 269-286).
[0399] LNCaP Cell Growth in CAPM Defined Medium with CDE-Horse
Serum and .+-.DHT or E.sub.2. To confirm that the serum-borne
inhibitor can be assessed even in the presence of all of the
components of serum-free defined medium, an example experiment is
shown in FIG. 58. The LNCaP cells were grown in serum-free CAPM
supplemented with increasing concentrations of CDE-horse serum
without steroids and in assay dishes with the CDE-serum plus 10 nM
E.sub.2 or 10 nM DHT. Without steroid, the CDE-horse serum showed
the expected progressive inhibition. Both the estrogen and androgen
reversed this inhibition completely at every serum concentration.
Clearly, the inhibitor in serum possesses a very special quality
that blocks the action of the many mitogenic agents present in
defined media.
[0400] GH.sub.4C.sub.1Cells in Serum-free and Serum Containing
Medium.+-.E.sub.2. In the next studies, shown in FIG. 57A, the
growth of rodent ER.sup.+ cell lines in defined medium and CDE
serum-containing medium with and without E.sub.2 were compared. The
study was with the GH.sub.4C.sub.1 rat pituitary tumor cell line.
In serum-free PCM-9, E.sub.2 had no effect on growth rate or
saturation density (FIG. 57A). In contrast, the cells were highly
estrogen responsive in CDE-horse serum (FIG. 57B). In .gtoreq.30%
(v/v) CDE-serum, the estrogenic effect was >4.5 CPD (i.e.
>22-fold cell number increase). The GH.sub.4C.sub.1 response
obtained was substantially greater than that previously reported in
cultures containing serum from a gelded horse (Amara J F and
Dannies P S (1983) Endocrinology 112, 1141-1143). Replicate studies
with the GH.sub.1 and GH.sub.3 rat pituitary tumor cells gave
results equivalent to those shown in FIGS. 57A and 57B (results not
shown).
[0401] MTW9/PL2 Cells in Serum-free and Serum Containing
Medium.+-.E.sub.2. FIG. 57C shows the effect of E.sub.2 on growth
of the MTW9/PL2 rat mammary tumor cells in serum-free DDM-2A. There
was a small effect on growth rate and a 1.0 CPD effect on
saturation density. When the same cells were assayed in D-MEM/F-12
containing CDE horse serum, the effect of E.sub.2 was remarkable
(FIG. 57D). Cell number differences of 2.sup.6 (i.e. 64-fold) were
recorded in 50% (v/v) serum in a seven-day assay. This result
agrees with those presented above in this disclosure. Furthermore,
comparison of MTW9/PL2 responses (FIG. 57D) to those of the human
breast cancer cells (FIGS. 56B and 56D) confirms that the ER.sup.+
rat cells are the most estrogen responsive mammary origin line yet
developed.
[0402] H301Cells in Serum-free and Serum Containing
Medium.+-.E.sub.2. In the final studies, the effect of E.sub.2 on
the growth of the H301 hamster kidney tumor cells in serum-free
medium was compared to that in CDE horse serum containing medium.
Estrogen had no effect on H301 cell growth in serum-free defined
CAPM (FIG. 57E). In contrast, E.sub.2 induced H-301 cell number
increases of >2.sup.4 (i.e. >16-fold) were recorded in
D-MEM/F-12 containing .gtoreq.30% (v/v) CDE serum (FIG. 57F). The
H301 response was similar to the MCF-7 cells in that 50% (v/v)
CDE-serum did not fully inhibit. The magnitude of the estrogenic
effect with H301 cells was equal to that reported by others
studying this line in cultures supplemented with CDE serum prepared
by different methods (Soto A M et al. (1988) Cancer Res 48,
3676-3680).
[0403] Discussion of Example 13. The new serum-free defined medium
serves as part of a model system for identifying physiologically
relevant new molecules. When completely serum-free defined
conditions were employed in the past, the effects of estrogens were
either marginal or insignificant as has been discussed above. The
earlier observations in completely serum-free defined culture
medium have been extended in the present investigation. Direct
comparisons were made between estrogenic effects in serum-free
defined culture and estrogenic effects in medium containing CDE
serum. The results were unequivocal. With every cell line tested,
CDE serum was required to demonstrate significant estrogenic
effects on logarithmic cell growth rates. A major advance provided
was the clear demonstration that high concentrations of serum are
required to observe large magnitude estrogenic effects.
Furthermore, the inhibitory effects of serum are dose dependent
even in the presence of the components used to formulate serum-free
medium. This indicates that growth is progressively negatively
regulated. This observation has physiological implications. Changes
in the serum concentration of the inhibitor, or changes in
availability to target tissues, will have direct effects on the
rate of cell replication. The results in FIGS. 56-58 point to serum
as the best source yet identified to obtain the component that
regulates sex steroid responsive growth. The tissue origin of the
serum regulator remains to be investigated.
Example 14
Action of DES on Human AR.sup.+ LNCaP Prostate Cancer Cells
[0404] In this Example, it is demonstrated that DES des not inhibit
steroidogenic cell growth and may be suitable for use in cancer
therapies, including but not limited to other therapies disclosed
herein.
[0405] LNCaP Cells and DES Action. Diethylstilbestrol (DES) is now
used as one of the primary treatments for prostatic cancer
(Seidenfeld J et al. (2000) Ann Intern Med 132, 566-577). Its
action is likely mediated through the hypothalamus-pituitary axis
(Seidenfeld J et al. (2000) Ann Intern Med 132, 566-577). DES
causes suppression of anterior pituitary gonadotrophins and
therefore suppresses testicular output of androgens. Although it is
thought that DES has no direct effects on prostate cancer cells,
the development of the assay methodology set out herein permitted a
direct assessment of this issue. The AR.sup.+ LNCaP cells were used
as a model for these tests (FIG. 59). As shown in FIG. 59A, 10 nM
DHT effectively reversed the inhibition caused by higher
concentrations of CDE-horse serum in D-MEM/F-12. Likewise, 10 nM
E.sub.2 also reversed the CDE-serum caused inhibition completely
(FIG. 59B). However, the same concentration of DES was entirely
ineffective (FIG. 59C). DES did not reverse the serum caused
inhibition. The synthetic estrogen had no direct positive effect on
LNCaP cell growth. In the final study of this series, DES addition
to medium containing DHT or E.sub.2 did not affect the reversal
caused by these two natural steroids (FIG. 59D). Therefore, DES is
not a direct inhibitor of androgen or estrogen promoted LNCaP cell
growth. The view that DES acts indirectly to cause chemical
castration is consistent with the present results. These results
are supported by other studies indicating that DES does not bind to
the AR of LNCaP cells (Montgomery B T et al. (1992) The Prostate
21, 63-73).
[0406] Discussion of Example 14. The fact that DES is a major
treatment for prostate cancer but does not act directly on the
tissue has therapeutic implications. For prostate cancer localized
to the organ, or specific metastases in other locations (e.g. bone,
liver or lung), direct application of Fe (III) offers a therapy
with a different mode of action. It is also possible that local Fe
(III) therapy (as described in Example 12) can be used in
conjunction with conventional systemic DES treatment to increase
effectiveness above that with either treatment alone. There is
another potential advantage of local Fe (III) treatment over
systemic DES treatment. DES has many side-effects in males. Some
present considerable discomfort or medical problems. Locally
applied Fe (III) is absorbed by the body to form non-toxic mono
ferric and diferric transferrin by chelation with the large pool of
available apotransferrin. The iron containing proteins formed are
no problem for the body because they are the natural physiological
forms of iron delivered to all tissues.
Example 15
Preparation of Inhibitor Depleted Serum for Control Studies and
Stability Properties of the Inhibitor
[0407] This Example, lists several acceptable techniques for useful
inactivated immunoglobulin inhibitors, and distinguishes the
inhibitors from the classical "estrocolyone."
[0408] Effect of Dialysis on Estrogenic Effects. CDE-horse serum
was dialyzed at 4.degree. C. against 0.05M Tris-HCl, pH 7.4, for up
to 72 hours with buffer changes every 24 hours using a Spectropor
dialysis membrane. The resulting serum was tested for estrogenic
effects with MWT9/PL2 cells as shown in FIG. 60. There was near a
total loss of estrogen reversible inhibitory activity accompanying
this treatment. It was found consistently (N=14) that this
treatment resulted in the appearance of an estrogen irreversible
inhibitor at serum concentrations above 10% (v/v). It was possible
that the estrogen reversible inhibitor was low molecular weight and
had passed through the dialysis membrane.
[0409] Ultrafiltration of CDE-Serum and Estrogenic Effects.
CDE-horse serum was submitted to nitrogen gas pressure
ultrafiltration using an Amicon unit and an YM-30 membrane (i.e. a
30,000 molecular weight cut-off). The filtrate was assayed with
MTW9/PL2 cells directly whereas the retentate was diluted to the
original volume with normal saline before assay. The filtrate (FIG.
61B) supported growth but without any estrogenic effect. The
retentate (FIG. 61A) demonstrated the usual high estrogenic effect
(i.e. 6 CPD) seen in the other MTW9/PL2 cell assays presented in
this Example. It is unlikely the Tris dialysis results described
above came from passage of the inhibitor through the membrane. The
ultrafiltration results confirm a molecular weight >30,000
daltons. The combined results of dialysis and ultrafiltration
suggest a lower molecular weight cofactor that might help stabilize
the estrogen reversible inhibitor.
[0410] Heat Treatment and Estrogenic Effects/Inhibitor Content of
CDE-serum. The heat stability of the estrogen reversible inhibitor
of CDE-horse serum was investigated at 50.degree. C. and 60.degree.
C. with the MTW9/PL cells. Heating at 50.degree. C. for 30 minutes
reduced the estrogen effect to 4.6 CPD (FIG. 62) instead of the
usual 5 to 6 CPD. The effect of heating at 50.degree. C. for up to
30 hours is shown in FIG. 63. By 20 hours, the estrogenic effect
with MTW9/PL2 cells was reduced to .ltoreq.1.0 CPD. Nonetheless,
this serum still supported full growth of the MTW9/PL2 cells (FIG.
64). Another effective method requiring less time is shown in (FIG.
65). Heating at 60.degree. C. for 90 minutes yielded serum that
supported high levels of growth (i.e. .gtoreq.6 CPD) but had lost
all inhibitor activity. This easy treatment, which is especially
fast and inexpensive to perform, provides a control serum that has
applications in assay of test substances.
[0411] Affi-Gel Blue Extraction of CDE-Serum. An aliquot of
CDE-horse serum was passed through a 5 mL Affi-Gel Blue.TM.
affinity chromatography column (Bio-Rad, Inc.), prepared according
to the manufacturer's instructions. The flow through fraction was
tested in the assay for estrogen mitogenic activity at 0 to 50%
(v/v). The results are shown in FIG. 66. The 5 mL Affi-Gel Blue.TM.
column removed more than 80% of the inhibitory activity in the
serum. Increasing the column bed to 10 mL resulted in removal of
more than 90% of the inhibitory activity in the serum.
[0412] Acid Treatment of CDE-serum. CDE-horse serum was adjusted to
pH 4.5 with HCl and incubated for 16 hrs at 4.degree. C. The
resulting serum readjusted to pH 7.4 and tested as previously done
with MTW9/PL2 cells. Acid treated CDE-serum promoted only limited
MTW9/PL2 cell growth and an estrogenic effect of <0.5 CPD. Not
only was the inhibitor acid labile, but the serum components that
support growth at <10% (v/v) were also adversely affected (data
not shown).
[0413] Urea Treatment of CDE-serum. CDE-horse serum was dialyzed
against 0.05 M Tris-HCl, pH 7.4, with 50 mM calcium chloride and 6
M urea for 16 hours at 4.degree. C. The urea was removed by
dialysis against the buffer without urea. The addition of
CaCl.sub.2 to the Tris buffer protects the activity (see results
below). The resulting serum was tested as previously described. As
shown in FIG. 67, the inhibitory activity was inactivated. Also,
the growth promoting activity of <10% (v/v) was also adversely
affected.
[0414] Discussion of Example 15. The preparation of
inhibitor-depleted serum has applications with regard to testing
compounds that might possess cytotoxic activity independent of any
steroid hormone-like cell growth stimulating ("steroidogenic")
effects or other hormone-like properties. The methods outlined will
permit assays of commercial, environmental, industrial and medical
compounds, substances and mixtures for inhibitor-like activity
and/or cytotoxic activity in the same preparations.
[0415] There is another very important application of this
technology. Development of compounds with estrogen reversible and
estrogen irreversible inhibitor-like activity, including peptides,
recombinant DNA products, or synthetic organic or inorganic
compounds can be sought using inhibitor-depleted serum as the assay
base. The new agents can be compared directly to the purified serum
inhibitor to determine their efficacy and potency. It is
anticipated that this technology will yield compounds that mimic
the serum inhibitor and can be used to treat various forms of
mucosal cancers including breast and prostate and colon. This
method is expected to allow rapid examination of many compounds.
The preferred preparation method for control serum is heating at
50.degree. C. for about 20 to 30 hours or 60.degree. C. for about
90 minutes. Affi-Gel Blue treatment is effective, but only with
small volumes of serum (e.g. 1 to 2 liters). Affi-Gel Blue is more
expensive and time consuming than the heating methods. Tris
dialysis, acid pH treatment and urea treatment are not as
satisfactory but can be applied as required under special
circumstances.
[0416] The results presented herein distinguish the estrogen
reversible inhibitor sought here from estrocolyone 1 (Soto A M et
al. (1992) J Steroid Biochem Mol Biol 43, 703-712). Estrocolyone 1
is stable to treatment with 6 M urea, stable at 60.degree. C. for 2
hours, and stable in 2 M acetic acid. Furthermore, estrocolyone
does not bind to Affi-Gel Blue. The serum-borne inhibitor described
herein does not share any of these properties.
Example 16
Effects of Conventional Purification Methods on the Properties of
the Estrogen Reversible Serum-Borne Inhibitor
[0417] This Example demonstrates the mainly adverse effects of
conventional purification techniques on the desired properties of
the present inhibitors. This Example also illustrates that the
conventional purification techniques can be used to produce certain
desired effects on the inhibitors.
[0418] CDE-horse Serum Effects Used to Calculate the ED.sub.50
Required for Purification Quantification. Conduct of purifications
requires measurement of specific activity (i.e. ED.sub.50) and
definition of units of activity. The results in FIG. 68 present
examples of how estimates of the ED.sub.50 concentrations protein
required for half-maximum estrogenic effects were determined. Two
representative commonly studied estrogen sensitive human cell lines
and two established rodent lines were selected for presentation.
FIGS. 68A, 68B, 68C and 68D show assay results with the MCF-7K and
ZR-75-1 human breast cancer cells, the MTW9/PL2 rat mammary tumor
cells, and the GH.sub.4C.sub.1 rat pituitary tumor cells,
respectively. With all four lines, the maximum estrogen reversible
inhibition was observed at 50% (v/v) CDE-serum. Under these
conditions, estrogen reversed cell number increases (i.e.
estrogenic effects) ranged from 2.sup.3.1 to 2.sup.5.5 (i.e.
2.sup.CPD) or 8-fold with MCF-7K cells to 45-fold with MTW9/PL2
cells. Serum concentrations of 8 to 16% (v/v) supported ED.sub.50
effects. This corresponded to protein concentrations of 2.4 to 4.8
mg/mL (TABLE 8). One unit of activity is the amount that achieves
ED.sub.50. To achieve maximum inhibition, 15.+-.2.5 mg/mL of
protein were required (i.e. 50% serum). The experiments presented
here support the previous conclusion that serum contains an
estrogen reversible inhibitor.
TABLE-US-00009 TABLE 8 Inhibitor Purification by General Methods
Chromatography/ Pool Elution ED.sub.50 of % Activity Activity
Separation Method Conditions Pools Recovered Half-life 1. CDE-horse
serum -- 2.4 to 4.8 mg/ml (100%) .ltoreq.24 days 2. Ammonium
Sulfate 40 to 75% saturation 6.7 mg/ml 80% .ltoreq.14 days 3.
DEAE-Sepharose; 0.05M Tris-HCl, pH 8.6, with NaCl elution steps
Pool I 1.33 .mu.g/ml 278% .ltoreq.14 days II 30 .mu.g/ml 111%
.ltoreq.14 days III 2.2 mg/ml 23% ND IV 390 .mu.g/ml 0% ND V 2.9
mg/ml 3% ND VI 223 .mu.g/ml 1042% .ltoreq.14 days VII 1.7 mg/ml 19%
ND 4. Phenyl Sepharose 0.05M Tris-HCl, pH 7.4, with a 3.0M NaCl to
buffer gradient Pooled fractions 100-130 94 .mu.g/ml 33% .ltoreq.14
days 5. Bio-Gel HTP 0.01M sodium phosphate, pH 7.2, with a linear
gradient of the buffer Pool I 224 .mu.g/ml 18% ND II 70 .mu.g/ml
0.7% .ltoreq.14 days III 421 .mu.g/ml 20% ND IV 260 .mu.g/ml 10.2%
ND V 36 .mu.g/ml 2.7% .ltoreq.14 days
[0419] Ammonium Sulfate Precipitation. Ammonium sulfate
precipitation was studied both to increase specific activity and to
decease the volume of the serum. The activity precipitated over a
broad concentration range. The precipitate from 0 to 40% saturated
ammonium sulfate contained <30% of the protein and activity of
whole CDE-serum. Replicate 40 to 75% saturation precipitates
contained approximately 80% of the total activity units and 60% of
the serum protein. The ED.sub.50 of this fraction ranged between
3.0 to 6.7 mg/ml (FIG. 69A). The ammonium sulfate results are
summarized in TABLE 8. The ammonium sulfate fractions were not
stable to freezing and thawing nor were they stable during storage
at 4 C or 23 C. To best preserve activity, the precipitated
material had to be used immediately in the next isolation step. Our
results differ from those in another report (Soto A M et al. (1992)
J Steroid Biochem Mol Biol 43, 703-712) indicating that the
estrogen reversible inhibitory activity was stable in ammonium
sulfate.
[0420] Proteinase Inhibitors. In replicate studies, the addition of
the proteinase inhibitors 4-amidinophenylmethanesulfonyl fluoride
(0.01 mg/ml), phenylmethylsulfonyl flouride (0.10 mg/ml),
N-tosyl-L-phenylalanine chloromethyl ketone (0.1 mg/ml), and
leupeptin (0.01 mg/ml) to the serum before precipitation, or to the
40 to 75% precipitated fraction, was not beneficial. Although
effective as a metaloproteinase inhibitor, EDTA was not used
because it was expected to remove stabilizing calcium.
[0421] Affi-Gel Blue Chromatography--General Considerations.
Affi-Gel Blue fractionation of CDE-horse serum was done as
described by the manufacturer for the isolation of albumin (Bio-Rad
Affi-Gel.RTM. BlueGel, 50 to 100 mesh, Instruction Manual, Catalog
Numbers 153-7301 and 153-7302). This study addressed two issues.
First, it was important to establish that the inhibitor localized
as a single protein, or at most only a few proteins. This issue has
not been addressed directly before. Affi-Gel BlueGel is a mild
method that effectively separates functionally active serum
proteins (Sirbasku D A et al. (1991) Biochemistry 30, 295-304;
Travis J et al. (1976) Biochem J 157, 301-306; Gianazza E and
Arnaud P (1982) Biochem J 201, 129-136; Iqbal M J and Johnson M W
et al. (1977) J Steroid Biochem 8, 977-983). Second, Affi-Gel
BlueGel offers a reliable and convenient means of isolating
relatively pure native serum albumin (Travis J et al. (1976)
Biochem J 157, 301-306; Gianazza E and Arnaud P (1982) Biochem J
201, 129-136). This is especially significant because reports from
other laboratories have cited albumin as "the" serum inhibitor
(Laursen I et al. (1990) Anticancer Res 10, 703-712; Sonnenschein C
et al. (1996) J Steroid Biochem Mol Biol 59, 147-154).
[0422] Affi-Gel Blue Chromatography--Technical Applications. A
one-liter column (5 cm.times.51 cm) was equilibrated with 0.05 M
Tris-HCl, pH 7.4. The 40 to 75% ammonium sulfate precipitated
material (438 mL, with 23.5 grams of protein) was dialyzed against
this buffer and applied to the column. After washing with
equilibration buffer, elution was done with increasing step
concentrations of 0.15, 1.0 and 3.0 M NaCl in the buffer. The four
pools contained 3.7, 3.2, 27.3 and 26.8% of the protein applied,
respectively. The flow-through and the 0.15 M NaCl pools did not
contain albumin, as expected (Travis J et al. (1976) Biochem J 157,
301-306; Gianazza E and Arnaud P (1982) Biochem J 201, 129-136).
The 1.0 and 3.0 M NaCl pools contained 70% albumin (Travis J et al.
(1976) Biochem J 157, 301-306; Gianazza E and Arnaud P (1982)
Biochem J 201, 129-136). SDS-PAGE and Coomassie Blue staining
confirmed albumin in these pools (results not shown). Assay of the
four pools showed no inhibitory activity (TABLE 8). Affi-Gel
BlueGel either retained the activity even with a 3.0 M NaCl wash,
or it caused inactivation. FIG. 69B shows an example assay of an
albumin rich pool. The same results were obtained with whole
CDE-serum applied to the same column (results not presented). The
same four pools shown in TABLE 8 also showed no recovery of the
estrogen reversible inhibitory activity. The Affi-Gel BlueGel
results in TABLE 8 suggested another use for this matrix. Passage
of CDE-horse serum through Affi-Gel Blue removed the majority of
the estrogen reversible inhibitory activity for MTW9/PL2 cells
(FIG. 70). This was effective even though the volume of serum
applied was more than five times the volume of the resin. The
maximum estrogenic effect seen with Affi-Gel BlueGel treated serum
as 1.5 CPD (FIG. 70) whereas the maximum effect in control
CDE-serum was 5.5 CPD (FIG. 68C) with MTW9/PL2 cells. The residual
activity in the by-pass fraction is likely due to IgA/IgM. Small
amounts of these immunoglobulins are usually in the by-pass of this
column (Gianazza E and Arnaud P (1982) Biochem J 201, 129-136).
[0423] Human Serum Albumin as Inhibitor. In studies not presented,
three preparations of human serum albumin were assayed for estrogen
reversible inhibitory activity with ER.sup.+ human and rodent cell
lines. Globulin containing (96 to 99% albumin), crystalline, and
Cohn's fraction V human serum albumin (all from Sigma) were not
inhibitory at concentrations up to 12 mg/mL. The assays showed the
same pattern as in FIG. 69B. These results further support our
earlier conclusion that albumin was not the estrogen reversible
inhibitor.
[0424] DEAE Sepharose Chromatography--General Considerations. We
next applied DEAE Sepharose chromatography. Because ammonium
sulfate precipitation provided no benefit beyond sample
concentration (TABLE 8), we instead used whole CDE-serum. The DEAE
Sepharose column was eluted with both step increases in NaCl
concentration and with linear gradients of NaCl. Eight permutations
of pH, NaCl elution concentrations, and gradient protocols were
analyzed. The results presented in FIG. 71 were the optimum
conditions identified.
[0425] DEAE Sepharose Chromatography--Technical Considerations.
DEAE Sepharose used at pH 8.6 provided the best separations. Seven
pools were obtained (FIG. 71). Activity assays of each pool are
summarized in TABLE 8. The flow-through fraction (pool I) and the
wash with equilibration buffer (pool II) together contained 389% of
the applied activity and 5.6% of the protein applied. Pool II was
particularly active, with the lowest ED.sub.50 concentration of any
from general chromatography (i.e. 30 .mu.g/mL). Pools III, IV and V
were less active although they still contained 126% of the units
and 37.7% of the protein. Pool VI contained 1042% of the activity
applied and 32% of the protein. Pool VI alone contained 10 times
more activity than applied to the column (TABLE 8). Pool VII was
markedly less active with only 19% of the activity and 4.5% of the
protein. Although 79.8% of the applied protein was recovered, more
than 15 times the expected units were recovered. Although the
reason(s) for the apparent increase is not clear, other
investigators (Dell'Aquila M L and Gaffney E V (1984) J Natl Cancer
Inst 73, 397-403) have reported identification of estrogen
irreversible inhibitors of the ER.sup.+ MCF-7, T47D and ZR-75-1
human breast cancer cell lines in fractions from DEAE Sepharose. It
is possible that the higher pH conditions have inactivated these
inhibitors and thereby allow greater effect of the estrogen
reversible form(s). It is also possible that exposure to high pH
alters the inhibitor to yield a more active form.
[0426] DEAE Sepharose Chromatography--Stability Considerations and
Evidence of more than one Activity. Activity was found in pools I,
II and VI. This suggested more than one estrogen reversible
inhibitor. In any case, there was a stability problem. An example
of this is shown in replicate assays of pool VI over a period of 21
days. The first assay of activity immediately after isolation is
shown in FIG. 69C. The effects on ER.sup.+ cell growth were
biphasic. At lower protein concentrations, estrogen reversible
inhibition was observed. At higher concentrations, the pool
material became irreversibly inhibitory. With all active DEAE
pools, this biphasic pattern was consistent. Additional sequential
assays initiated after 14 days storage at 4.degree. C. or
23.degree. C. showed another consistent finding. The maximum
estrogenic effect caused by E.sub.2 decreased >90% (FIG. 69D).
As estrogen reversible inhibition decayed, only the estrogen
irreversible inhibition remained (FIG. 69D). From these results,
decay most likely resulted in formation of an altered inhibitor
that was estrogen irreversible.
[0427] Phenyl Sepharose Chromatography. Phenyl Sepharose
chromatography has been previously reported to effectively enrich
human serum-derived estrocolyone 1 (Soto A M et al. (1992) J
Steroid Biochem Mol Biol 43, 703-712). When samples of that
estrogen reversible activity were applied under high salt
conditions, and the elution done with decreasing salt
concentrations, a single inhibitory fraction was separated from the
bulk of the proteins (Soto A M et al. (1992) J Steroid Biochem Mol
Biol 43, 703-712). In the present studies, further purification of
DEAR Sepharose pool VI was investigated using phenyl Sepharose.
FIG. 72 presents the results of the optimum of four elution
protocols investigated. Activity was located in fractions 100
through 130 that contained 12% of the applied protein. The specific
activity increased 2.3-fold compared to DEAE Sepharose pool VI
(TABLE 8). The initial assay results (data not shown) were similar
to those in FIG. 69C. Sequential assays again confirmed a rapid
inactivation ending with estrogen irreversible inhibition similar
to that shown in FIG. 69D. As seen before, an estrogen irreversible
inhibitor was generated upon standing in buffer.
[0428] HTP Bio-Gel Chromatography and other Methods. Further
purification of DEAE Sepharose pool VI was also attempted using HTP
Bio-Gel (hydroxylapatite). FIG. 73 shows the results of the most
effective of three HTP Bio-Gel elution protocols attempted. The
protein and total units of activity recovered from this column were
69.6% and 51.6% respectively. As summarized in TABLE 8, the
specific activities (i.e. ED.sub.50) of pools I, III and V were not
improved compared to DEAE pool VI. The specific activities of pools
II and V were significantly improvement. Sequential assays of these
showed responses similar to those in FIGS. 69C and 69D. The initial
estrogen reversible activity decayed within 14 days to irreversible
inhibition. In studies not presented, Concanavalin A Sepharose and
metal (Zn.sup.2+) chelate affinity chromatography were also
attempted with whole serum, the active fractions from DEAE
Sepharose and with the 40 to 75% ammonium sulfate precipitate.
These methods were not effective. When activity was obtained, it
decayed within two to three weeks in the same pattern as shown in
FIGS. 69C and 69D. Analysis of the report (Soto A M et al. (1992) J
Steroid Biochem Mol Biol 43, 703-712) attempting estrocolyone
isolation confirmed substantially the same instability and low
yield problems. In the studies described in this Example, it is
very clear that purification of the inhibitor had not yet been
achieved using conventional purification methods, although useful
ways of producing irreversible inhibitor compositions were
revealed.
[0429] Discussion of Example 16. All of the methods described in
this Example are conventional protein purification methods in
general use today. They commonly yield high specific activity or
high purity protein preparations. Because they were carried out
under what are considered non-denaturating conditions and
non-reducing conditions, the expected outcome was isolation of an
active estrogen reversible inhibitor. It is clear that a spectrum
of the usual methods will not yield the estrogen reversible
inhibitor in an active, stable form.
[0430] It should be noted that the ammonium sulfate experiments
alone clearly differentiate the present serum described inhibitor
from that described in U.S. Pat. Nos. 4,859,585 (Sonnenschein) and
5,135,849 (Soto). Those patents teach the use of a stable inhibitor
obtained by ammonium sulfate fractionation. In the present case,
however, an ammonium sulfate fraction yields unstable activity.
[0431] Affi-Gel BlueGel has usefulness as a method of preparation
of inhibitor depleted serum. It may be preferred under
circumstances where heating (the other very effective method) might
destroy some component in serum needed for a specialized mucosal or
other origin cancer cells. The Affi-Gel Blue results, and others
presented herein, support the view that serum albumin is not the
serum-borne estrogen reversible inhibitor activity sought
herein.
[0432] The results of the DEAE chromatography indicate that there
is more than one inhibitor. The number cannot be established by
that method, but elution localization suggests at least two and
possibly more. Further, in light of the results shown in Example
20, the DEAE elution pattern shown in FIG. 69D is consistent with
the two or more inactivated inhibitors being denatured forms of
immunoglobulins IgA/IgM/IgG1/IgG2 may be very potent antitumor
agents and worthy of consideration as new treatment modalities. It
should be noted that these fractions were very freeze-thaw
sensitive, especially in the absence of calcium. In every case
where activity was localized to a chromatographic pool, the
activity decayed within 21 to 28 days to an estrogen irreversible
agent. This suggests that a denaturation process is ongoing in
buffers mostly without calcium, and that a potentially important
product is formed that may have cancer therapeutic value. This may
be a means of generating very high potency irreversible inhibitors
of mammary cancers and other types of mucosal cancers.
Example 17
Calcium Stabilization and Correlation with .sup.3H-DHT Binding and
Immunoprecipitation by Antibodies Raised to Human SHBG
[0433] General Protein Isolation Principals. Before continuing the
isolation attempts described above, principals common to all
protein isolation attempts were applied. Information was sought
about the conditions that would best stabilize the activity, and in
doing so an understanding of new/appropriate methods of
purification was gained.
[0434] Effect of Calcium on Tris-HCl Dialysis Retention of
Estrogenic Activity. CDE-horse serum was dialyzed against 0.05M
Tris-HCl, pH 7.4, with 50 mM CaCl.sub.2 for 72 hrs, buffer changes
every 24 hrs at 4.degree. C. using a 6000-8000 molecular weight
cut-off dialysis membrane. The resulting serum was assayed with
MTW9/PL2 cells.+-.10 nM B.sub.2. As shown in FIG. 74, the usual
large magnitude estrogenic effects were identified. The presence of
calcium in the buffer completely prevented the inactivation found
when dialysis was done in buffer without calcium (compare to FIG.
60).
[0435] Chelex.TM. Treatment and Protective Effects of Calcium Ions
Against Heat Inactivation. CDE-horse serum was treated with
Chelex.TM. resin beads to remove free metal ions including calcium.
This was done to continue the evaluation of calcium as a
stabilizer. The serum was incubated with 10% (w/v) prewashed
Chelex.TM. 100 resin (100-200 mesh, sodium form) (Bio-Rad) for 2
hrs at room temperature. At the end of the incubation, the serum
was separated from the Chelex beads by 0.2 .mu.m pore filtration.
Calcium concentrations were determined with a calcium-detecting
probe. They were <10 nM. Next the Chelex treated serum was
incubated at 50.degree. C. either without added calcium or in the
presence of 1.0, 10 and 50 mM CaCl.sub.2. The serum was assayed
with MTW9/PL2 cells at the designated times shown in FIG. 75 (30%
Chelex treated serum.+-.10 nM E.sub.2) to determine estrogenic
effects. Without calcium, total activity was lost within 3 hours.
In the presence of increasing calcium, the activity was
progressively stabilized. At 50 mM CaCl.sub.2, <15% of the
activity was lost even after 30 hours at 50.degree. C. A control
with CDE-horse serum is shown. CDE-serum alone lost complete
activity by 20 hours at 50.degree. C. as expected from the results
in FIG. 64. Clearly, the addition of calcium stabilized the
activity.
[0436] Chelex.TM. Treatment and Protective Effects of Metal Ions
Against Heat Inactivation. CDE-horse serum was treated with
Chelex.TM. resin beads to remove free metal ions including calcium.
This was a continuation of the study above but with other metal
ions to determine if they substituted for calcium. The experiment
was conducted as described in the paragraph above but with the
change that the incubation temperature was lowered to 37.degree. C.
to permit more accurate estimation of the early kinetics of
inactivation. Chelex treated serum lost 80% activity in 30 hours at
this temperature (FIG. 76). With 10 mM CaCl.sub.2, protection was
nearly complete at this temperature for 30 hours. However, zinc,
magnesium and manganese ions offered no protection. Because these
are expected to be common substitutes for calcium, it is likely
that stabilization by calcium is quite specific.
[0437] Labeled Steroid Hormone Binding in CDE-Serum and Scatchard
Analysis. One of the basic tenets of the estrocolyone hypothesis is
that there are serum proteins that bind sex steroid hormones at
affinities (i.e. K.sub.d) in the picomolar range (Soto A M et al.
(1986) Cancer Res 46, 2271-2275). However, what was found instead
with CDE-horse serum is specific binding of .sup.3H-DHT in the of
K.sub.d range 1 to 5 nM as determined by Scatchard analysis
(Scatchard G (1949) Ann NY Acad Sci 51, 660-672) (FIG. 77). The
binding methods are presented in the General Materials and Methods
section. With CDE-rat serum the binding affinities were even higher
(results not shown). Routinely, the affinities for specific binding
of .sup.3H-DHT with CDE-rat serum were in the K.sub.d range 15 to
40 nM. With both CDE-horse and CDE-rat serum, binding K.sub.d for
.sup.3H-E.sub.2 was about two to five times higher concentrations
(results not presented). CDE-serum shows specific binding of sex
steroid hormones, but the affinity was not sufficiently high to
support the conclusions of others (Soto A M et al. (1986) Cancer
Res 46, 2271-2275) concerning picomolar affinities and the
estrocolyone hypothesis.
[0438] Correlation between Calcium Stabilization of Inhibitor
Activity and Calcium Stabilization of .sup.3H-DHT Binding to
CDE-horse Serum. As shown in FIG. 75 with CDE-serum that had been
Chelex treated, calcium protected the inhibitor activity from heat
inactivation. This study was repeated with another batch of Chelex
treated CDE-horse serum (FIG. 78). This study showed essentially
the same results as presented in FIG. 75. However, in parallel, the
Chelex treated serum was also assayed for .sup.3H-DHT binding (FIG.
78B). Clearly, as the calcium concentration was increased in the
serum, there was protection of .sup.3H-DHT binding that paralleled
the protection of the estrogenic effect shown in FIG. 78A. These
results implied a relationship between the estrogen reversible
inhibitor activity being sought and a sex steroid hormone binding
protein.
[0439] Immunoprecipitation of the .sup.3H-DHT Binding Activity and
Estrogenic Activity in CDE-horse Serum. In the final studies of
this series, rabbit antibodies against human SHBG (Accurate
Chemicals) were assayed for immunoprecipitation of the .sup.3H-DHT
binding activity of CDE-horse serum. After incubation of the serum
with the designated dilutions antiserum, Protein A/G-Sepharose
(Pierce kit) was added to absorb the immune complexes, and the
resulting serum assayed for binding under conditions described in
General Materials and Methods. The results of this study are shown
in FIG. 79A. Increasing antibody decreased the steroid binding
activity. In parallel, the same samples were used to assess
estrogenic effects with MTW9/PL2 cells (FIG. 79B). Increasing
anti-SHBG decreased the estrogenic effect by decreasing the
concentration of the inhibitor in the serum. There appeared to be
some type of cross-reaction, but it was still not clear that this
proved SHBG-like properties for the estrogen reversible inhibitor.
In both FIGS. 79A and 79B, addition of control rabbit serum had no
effect.
[0440] Discussion of Example 17. The effect of calcium on both the
estrogenic activity and the binding of .sup.3H-DHT to CDE-serum was
remarkably similar to data presented by others concerning the
stability of human SHBG (Rosner W et al. (1974) Biochim Biophys
Acta 351, 92-98). Other investigators have raised the issue of
classical SHBG as the sex hormone reversible inhibitor of target
cell growth. This seems highly unlikely, however, in light of the
results presented above. Both CDE horse serum and CDE rat serum
contain concentrations of inhibitor about equal to any of the other
serum types investigated. Furthermore, it is accepted knowledge
that horse and adult rat serum do not contain SHBG (Corvol P and
Bardin C W (1973) Biol Reprod 8, 277-282; Renior J-M et al. (1980)
Proc Natl Acad Sci USA 77, 4578-4582; Wenn R V et al. (1977)
Endokrinologie 69, 151-156). Nevertheless, anti-human SHBG
purchased from Accurate Chemicals not only immunoprecipitated the
activity in serum, but also the .sup.3H-DHT binding activity. This
data initially suggested that the inhibitor was a SHBG like
activity (Sirbasku D A et al. "Serum factor regulation of estrogen
responsive mammary tumor cell growth." Proceedings of the 1997
Meeting of the "Department of Defense Breast Cancer Research
Program: An Era of Hope", (Abstract) pp. 739-740, Washington, D.C.,
Oct. 31-Nov. 4, 1997). However, there were enough physical
differences to indicate that the activity was not actually SHBG and
that the cross-reaction with anti-SHBG was possibly misleading.
[0441] Despite the ambiguity in its identity at that point, it was
clear that the estrogen reversible inhibitor sought by herein had
different properties than that described in U.S. Pat. Nos.
4,859,585 (Sonnenschein) and 5,135,849 (Soto). In those patents,
the activity was not shown to cross-react with anti-human SHBG nor
was it stated to share SHBG-like properties. The putative kinship
to SHBG provided impetus to use a method that had already been
applied to the purification of SHBG in order to identify the
inhibitor. In Example 18 the purification of the estrogen
reversible inhibitor activity is described, performing the first,
and third through sixth isolation.
Example 18
Cortisol Affinity and Phenyl Sepharose Isolation of the "SHBG-like"
Estrogen Reversible Inhibitor from CDE-Horse Serum
[0442] Outcome of the Search for the Estrogen Reversible
Inhibitors. As cited above, neither horse or rat serum contains
SHBG. Therefore, these were the preferred sera to begin isolation.
Partial purification of the inhibitor from serum has been achieved
initially by a two-step procedure. The partially purified inhibitor
fractions are different than the serum derived inhibitor described
in U.S. Pat. No. 4,859,585 (issued to Sonnenschein and Soto), which
has been more recently identified as a subtype domain of albumin.
By contrast, it has been discovered that IgA and IgM, preferably in
dimeric/polymeric form, are steroid hormone reversible inhibitors
of cell growth.
[0443] Two-step Cortisol-agarose and phenyl Sepharose Isolation
Method. Based on the perceived SHBG-like properties described
above, a new approach to the purification was taken. This method
used a two-step cortisol-agarose affinity and phenyl-Sepharose
chromatography protocol. It had been employed by others to
simultaneously yield purified human cord serum CBG and SHBG
(Fernlund P and Laurell C-B (1981) J Steroid Biochem 14, 545-552).
The method first required the synthesis of the cortisol affinity
matrix. The cortisol-agarose affinity matrix was synthesized and
the initial purifications done as described (Fernlund P and Larell
C-B (1981) J Steroid Biochem 14, 545-552). An 80 mL bed volume
cortisol-agarose column (2.5 cm.times.17.8 cm) was equilibrated
with a buffer containing 0.05 M piperazine, pH 5.5, with 0.2 M
NaCl. Two liters of horse serum were charcoal-dextran extracted at
34.degree. C. as described above. For two of the six preparations
used in these studies, the serum was depleted of steroid hormones
by the Amberlite.TM. XAD-4 resin method. There was no resulting
difference in the purifications. After removing a 30 mL sample for
pre-column activity assay, the remaining volume was adjusted to pH
5.5 with 1.0 N HCl. This was applied to the column at a flow rate
of 30 to 40 mL per hour. Throughout the purification, the flow
rates were maintained with a peristaltic pump. The effluent was
collected and a sample and adjusted to pH 7.2 for post-column
assessment of estrogen reversible inhibitory activity. After all of
the serum had been applied, the column was washed for 7 days at the
same flow rate with the equilibration buffer until the A.sub.280nm
of the effluent was <0.06 versus water.
[0444] To recover the activity, the cortisol-agarose column was
eluted with a 500 mL linear gradient formed with 250 mL of the
piperazine/NaCl buffer and 250 mL of the buffer with 1.0 mg/mL
cortisol and 10% (v/v) methanol. After completion of the gradient,
the column was washed with one volume of the cortisol/methanol
buffer. A total volume of 600 mL was collected as 10 mL fractions.
As reported by Fernlund & Laurell (Fernlund P and Laurell C-B
(1981) J Steroid Biochem 14, 545-552), two separate A.sub.280nm or
protein concentration ranges could be recognized, but their
separation and individual chromatography on phenyl-Sepharose was no
more effective than pooling the entire 600 mL gradient elution and
using it for the next step. The total volume from the cortisol
gradient was reduced 5 to 8-fold by nitrogen gas pressure Amicon
ultrafiltration (YM-10 membrane) and applied directly to the next
column without dialysis or pH adjustment.
[0445] A 28 mL bed volume phenyl-Sepharose (1.5 cm.times.16 cm) was
equilibrated with 0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl.
The concentrated cortisol gradient volume was applied at a flow
rate of 60 mL/hour (10 mL fractions). The first A.sub.280nm peak
observed was a mixture of cortisol and CBG (Fernlund P and Laurell
C-B (1981) J Steroid Biochem 14, 545-552). These fractions were
combined as cortisol affinity-phenyl Sepharose pool I (CA-PS-pool
I). The column was then washed with equilibration buffer until the
A.sub.280nm was reduced to 0.002 versus water. The next buffer
applied was 0.05 M Tris-HCl, pH 7.5 (60%, v/v) containing 40% (v/v)
ethylene glycol. The A.sub.280nm peak observed with this wash was
combined to form CA-PS-pool II that corresponded to SHBG from human
serum (Fernlund P and Laurell C-B (1981) J Steroid Biochem 14,
545-552). The two pools were separately concentrated to
approximately 40 mL each and dialyzed separately against storage
buffer which was 0.05 M Tris-HCl, pH 7.5, containing 0.15 NaCl,
0.05 M CaCl.sub.2 and 60% (v/v) glycerol. The dialysis further
concentrated each sample. As last additions, 0.1 mM cortisol was
added to CA-PS-pool I and 0.1 mM DHT to CS-PS-pool II. The pools
were stored unfrozen at -20 C. Six replicate isolations were done.
The protein yields ranged from 22.8 to 37.7 for CA-PS-pool I and
5.82 to 12.2 mg for Ca-PS-pool II. Based on an average of 60 grams
of protein per two liters of CDE-horse serum (i.e. 30 mg/mL),
CA-PS-pool II represented about 0.013% of the total protein in
serum.
[0446] Cortisol affinity and phenyl Sepharose Isolation Results and
SDS-PAGE Molecular Weight Estimation. The chromatography profiles
from the two-step cortisol affinity and phenyl Sepharose isolation
of the inhibitor(s) activity from CDE-horse serum are shown in FIG.
80. The elution from phenyl Sepharose gave the CA-PS-pools I and
II. CA-PS-pool I contained predominantly 58 kDa CBG (Rosner W and
Bradlow H L (1971) J Clin Endocrinol Metab 33, 193-198) as
confirmed by SDS-PAGE and Western immunoblotting with rabbit
anti-horse CBG as well as by partial N.sup..alpha. amino acid
sequencing of the first 10 to 20 residues (results not presented).
SDS-PAGE analyses of three example preparations of CA-PS-pool II
are shown in FIG. 81A. Components of 67, 58, 54, and 29 kDa were
identified. These were compared to the 48 and 46 kDa units
identified for purified human SHBG (Khan M S et al. (1985) Steroids
45, 463-472) (FIG. 81A).
[0447] Native Molecular Weight Estimation. Analyzes done under
non-reducing and non-denaturing conditions using Superdex molecular
sieve FPLC at neutral pH in buffers identified components
CA-PS-pool I in the exclusion volume at .gtoreq.900 kDa, and
components approximately 350 and 168 kDa (Sirbasku D A et al.
"Serum factor regulation of estrogen responsive mammary tumor cell
growth." Proceedings of the 1997 Meeting of the "Department of
Defense Breast Cancer Research Program: An Era of Hope", (Abstract)
pp. 739-740, Washington, D.C., Oct. 31-Nov. 4, 1997). Comparison of
the results from denaturing and non-denaturing conditions confirmed
that the CA-PS-pool II was still heterogeneous and that the
activity was most likely a subunit containing high molecular weight
protein(s).
[0448] Removal of Storage Solution Components before Bioassay.
Before conducting bioassays of the inhibitory activity in the
phenyl-Sepharose pools, the glycerol and steroid hormones in the
storage buffers were removed. If DHT is not removed completely from
CA-PS-pool II, the inhibitory activity was substantially diminished
or eliminated entirely. Samples (0.5 to 15 mL) were introduced into
Slide-A-Lyzer.RTM. (Pierce) cassettes of molecular weight cutoff
10,000. The cassettes were incubate twice with stirring in two
liters of Tris-HCl, pH 7.4, containing 10 mM CaCl.sub.2 for four
hours at 34.degree. C. to remove excess free steroids and glycerol.
Next, the cassettes were transferred to the same buffer containing
20% (v/v) of a charcoal-dextran mixture prepared as described
above. After 18 hours at 37.degree. C., the cassettes were
transferred to another two-liter volume of the same buffer
containing 10% (v/v) of the charcoal-dextran mixture and dialysis
continued with stirring for another 6 to 8 hours. Finally, the
cassettes were rinsed lightly with water and the dialyzed material
recovered according to manufacturers instructions. The contents
were sterilized by 0.2-.mu.m-pore membrane filtration and stored at
4.degree. C. These preparations were usually used within a few
weeks.
[0449] Assay of CA-PS-pool I Estrogen Reversible Inhibitory
Activity with MTW9/PL2 Cells. When assayed with MTW9/PL2 cells,
CA-PS-pool I contained 20 to 25% of the units of estrogen
reversible inhibitory activity recovered from the phenyl Sepharose
column (data not shown). With two preparations not presented, the
cortisol gradient pool shown in FIG. 80 was made 1.5 M NaCl before
application to the phenyl Sepharose column equilibrated at the same
higher salt concentration. Under these conditions, the CA-PS-pool I
contained >90% CBG, as estimated by SDS-PAGE, but showed either
no estrogen reversible activity or only traces (results not
presented). Irrespective of the ionic strength or pH of the
cortisol affinity pool applied to phenyl Sepharose, ethylene glycol
was required to elute the majority of the activity.
[0450] Assay of CA-PS-pool II Estrogen Reversible Inhibitory
Activity with Several ER.sup.+ Cell Lines. Despite method
variations with phenyl Sepharose, CA-PS-pool II always contained
.gtoreq.75% of the activity recovered. In a crucial test of
significance, CA-PS-pool II was assayed to determine if it replaced
the effects of CDE-serum with eight different ER.sup.+ cell lines.
The results are shown in FIG. 82. The estrogen reversible
inhibitory effects of CA-PS-pool H were investigated with five
rodent tumor cell lines derived from three different estrogen
target tissue tumors, and three separate estrogen sensitive human
breast cancer cell lines. The cells were added to medium with 2.5%
(v/v) CDE-horse serum plus increasing concentrations of CA-PS-pool
II.+-.10 nM E.sub.2. The first lines evaluated were the GH.sub.1,
GH.sub.3, and GH.sub.4C.sub.1 rat pituitary tumor cells (FIGS. 82A,
82B and 82C, respectively). They were chosen first because these
lines are well known for hormone regulation of differentiated
tissue specific functions in culture and exceptional sensitivity to
a variety of hormones including estrogens (Tashjian A H Jr (1979)
Methods Enzymol 58, 527-535; Haug E and Gautvik K M (1976)
Endocrinology 99, 1482-1489; Haug E (1979) Endocrinology 104,
429-437; Amara J F and Dannies P S (1983) Endocrinology 112,
1141-1143). At 10 .mu.g/mL, CA-PS-pool H was fully inhibitory with
all three GH lines. Growth was reduced to near seed density levels
(i.e. <0.5 CPD). By this measure, >1,700-fold increase in
potency had been achieved versus full CDE-serum. The ED.sub.50 with
the GH cells was 6 to 8 .mu.g/mL which was a 300 to 800-fold
specific activity increase compared to full serum. E.sub.2 reversed
the effects of the CA-PS-pool II at every inhibitory concentration.
CA-PS-pool II replaced the effects of full CDE-serum with these
cells. FIGS. 82D and 82E show similar experiments with the estrogen
sensitive H301 hamster kidney tumor cells and the MTW9/PL2 rat
mammary cells, respectively. CA-PS-pool II was most inhibitory at
15 .mu.g/mL with both lines. The ED.sub.50 were in the range of 5
to 10 .mu.g/mL. As with the GH lines, E.sub.2 completely reversed
the effects of the inhibitor. Again, CA-PS-pool II replaced the
effects of full CDE-serum with these cells. With human breast
cancer cell lines MCF-7K, ZR-75-1 and T47D, the results were
similar (FIGS. 82F, 82G, and 82H, respectively). Addition of 10 to
15 .mu.g/mL of CA-PS-pool II caused maximum inhibition. The
ED.sub.50 concentrations were 6 to 9 .mu.g/mL. As with ER.sup.+
rodent cell lines, E.sub.2 completely reversed the inhibition
caused by CA-PS-pool II. Again, CA-PS-pool II replaced the effects
of full CDE-serum with these cells.
[0451] Cortisol-agarose Affinity Removal of the Inhibitor from
CDE-serum. Next it was determined if the cortisol affinity
chromatography had not removed the majority of the activity from
serum. To test this, three cell lines were analyzed with pre- and
post cortisol column samples. FIGS. 83A and 83B show the effect of
a single column passage on the inhibitory activity for T47D human
breast cells. The ED.sub.50 of the pre-column CDE-serum was 7%
(v/v). Post column, even 50% (v/v) serum did not achieve ED.sub.50.
FIGS. 83C and 83D show the same studies with the GH.sub.3 rat
pituitary cells. In this case, a single column passage completely
depleted the activity. Complete depletion was also observed with
the H-301 hamster kidney cell line (FIGS. 83E and 83F).
[0452] Storage Conditions and SHBG Related Properties. At
completion of the two-step isolation, the pools were stored in the
presence of sufficient glycerol to prevent freezing at -20.degree.
C. In experiments not shown, the estrogen reversible inhibitor was
progressively less stable without addition of glycerol, calcium
and/or steroid hormone. Dialysis against buffers without calcium is
most definitely to be avoided. Freeze/thaw is very harmful, even
with calcium and DHT present. Assays of -20.degree. C. glycerol
stored CA-PS-pool H over a two year period indicated no decay in
activity. Clearly, the storage conditions known to stabilize
functional SHBG (Fernlund P and Laurell C-B (1981) J Steroid
Biochem 14, 545-552; Rosner W et al. (1974) Biochim Biophys Acta
351, 92-98) also favored retention of estrogen reversible inhibitor
activity in CA-PS-pool II.
[0453] Labeled Steroid Hormone Binding to CA-PS-pool I. CA-PS-pool
I was determined to contain CBG by criteria cited above.
Additionally, this pool was examined by Scatchard analysis for
binding of tritium labeled steroid hormones. The results are
summarized in TABLE 9. The association constants (K.sub.a) of the
labeled hormones showed the order
cortisol>progesterone>>>sex steroid hormones. The
K.sub.a of cortisol binding at 34.degree. C. was
1.41.times.10.sup.9 M.sup.-1 that was equal to that of native rat
CBG when analyzed at 4.degree. C. (Rosner W (1990) Endocr Rev 11,
80-91). However, it was higher than the K.sub.a of
5.2.times.10.sup.7 M.sup.-1 for human CBG measured at 23.degree. C.
(Rosner W and Bradlow HL (1971) J Clin Endocrinol Metab 33,
193-198). The binding characteristics of steroids to CBG from
several species have been studied (Rosner W (1972) J Steroid
Biochem 3, 531-542). The similarity of the results further supports
the conclusion that CA-PS-pool I contains predominantly CBG.
[0454] Labeled Steroid Hormone Binding to CA-PS-pool H. The
estrogen reversible inhibitor activity in CDE-serum correlated with
the binding of tritium labeled sex steroid hormones. This suggested
a relationship between the estrogen reversible inhibitor and SHBG.
However, the K.sub.a for .sup.3H-DHT binding to CDE-serum at
34.degree. C. was 3.90.times.10.sup.7 M.sup.-1. However, it is
important to note that this was at least 20 times lower than that
of purified human SHBG at 0.99.times.10.sup.9 M.sup.-1 for DHT or
2.2.times.10.sup.8 M.sup.-1 for E.sub.2 at 37.degree. C. (Rosner W
and Smith R N (1975) Biochemistry 14, 4813-4820). To determine if
CA-PS-pool II possessed the same sex hormone binding properties as
whole CDE-serum, and/or human SHBG, the next study was conducted.
Scatchard analysis of .sup.3H-DHT binding to CA-PS-pool II was done
at 34.degree. C. The estimated K.sub.a was 5.88.times.10.sup.7
M.sup.-1. Replicates (N=3) gave a K.sub.a range
4.5-10.times.10.sup.7 M.sup.-1. Computer analysis indicated a
single class of binding sites although correlation coefficients
were approximately 0.7. Similar analyses were done with
.sup.3H-E.sub.2, .sup.3H-progesterone and .sup.3H-cortisol. The
results with all four labeled steroids are summarized in TABLE 9.
The K.sub.a order was
DHT>E.sub.2>>>cortisol>progesterone. The K.sub.a for
sex steroid hormone binding to the CA-PS-pool II was similar to
whole CDE-serum but 20 to 50-fold lower than human SHBG.
TABLE-US-00010 TABLE 9 Summary of the Scatchard Analysis of
phenyl-Sepharose pools I and II with four labeled steroid hormones
Steroid Hormone CA-PS-Pool I CA-PS-Pool II (3H-labeled) K.sub.d (M)
K.sub.a (M.sup.-1) K.sub.d (M) K.sub.a (M.sup.-1) Cortisol .sup.
7.10 .times. 10.sup.-10 1.41 .times. 10.sup.9 1.89 .times.
10.sup.-6 5.30 .times. 10.sup.5 Progesterone 1.70 .times. 10.sup.-9
5.90 .times. 10.sup.8 7.89 .times. 10.sup.-6 1.17 .times. 10.sup.5
17.beta.-estradiol 1.05 .times. 10.sup.-5 9.51 .times. 10.sup.4
2.83 .times. 10.sup.-8 3.55 .times. 10.sup.7 Dihydro- 6.05 .times.
10.sup.-6 1.64 .times. 10.sup.5 1.43 .times. 10.sup.-8 6.99 .times.
10.sup.7 testosterone
[0455] Western immunoblotting with anti-human SHBG. The above shows
that the estrogen reversible inhibitor shared immunological
properties with human SHBG. To investigate further, Western
immunoblotting of CA-PS-pool II was done with anti-human SHBG. The
results are presented in FIG. 81B. Western analysis with the
anti-SHBG recognized the same four components seen with Coomassie
Blue staining in FIG. 81A. These same four components have also
been identified with whole CDE-serum using Western analysis with
anti-human SHBG (data not shown). In Western immunoblotting studies
not presented, anti-human SHBG did not identify horse serum
albumin. This confirmed that the 67 kDa Coomassie Blue stained
component present in the CA-PS-pool II was not 68 kDa horse serum
albumin. These results provided additional support for the
conclusion that albumin is not the estrogen reversible inhibitor
activity of serum. These results also very clearly demonstrated
that the SHBG used to raise antibodies in rabbit had not been
purified to homogeneity, but rather had been used at a more crude
state. (It was also confirmed by the manufacturer of the anti-SHBG
antibody that the SHBG fraction used for antibody production was
not highly purified and had not been size fractionated.)
[0456] Discussion of Example 18. There has been one very critical
problem with the estrocolyone hypothesis. Estrocolyone has never
been purified and shown to act as described (Soto A M and
Sonnenschein C (1987) Endocr Rev 8, 44-52). The active pool
isolated from the two-step procedure (i.e. CA-PS-pool II) certainly
does not bind steroid hormones with sufficient affinity to act as
estrocolyones (TABLE 9). Growth is activated at picomolar
concentrations while the affinity (Kd) of E.sub.2 with CA-Pool II
is about 10.sup.-8 M. This discrepancy is simply far too large to
accept the role of estrogens in growth as binding the inhibitor and
thereby preventing its action on target cells (Soto A M and
Sonnenschein C (1987) Endocr Rev 8, 44-52). The fact that proteins
in CA-PS-pool II bind steroids is not germane to the mechanism of
action of these hormones in growth regulation under physiological
conditions.
[0457] The results of steroid hormone binding may however be
germane to the use of high dose treatments of breast cancer. Care
must be taken when considering that high doses of estrogen,
androgen, progesterone and cortisol all have the potential for
binding the active agent in CA-PS-pool II and therefore may reduce
the effective concentration of inhibitor. The assays described in
this Example can be applied to biological fluids and plasma to
determine if steroid concentrations are excessive and to evaluate
proper levels with changes in treatment regimes.
[0458] The results presented herein indicate that the proposed new
model of cell growth is a favored mechanism. Steroid hormones
appear to act as positive agents via internal high affinity
receptors (e.g. ER.gamma.) whereas serum-borne inhibitors act at
the surface to block growth. The combination of the two signals
dictates cell proliferation rates. This data further supports the
assertion that the ER.gamma. can be used for diagnostic purposes in
ER.sup.+ cancers in the same way that conventional ER receptor
screening is now performed.
[0459] A highly enriched fraction of serum protein was prepared
whose estrogen reversible inhibitory activity is stable and whose
effects replicate those seen with full serum with a variety of sex
steroid hormone target tumor cell types in culture. Because early
studies mistakenly indicated that the inhibitor shared various
properties with SHBG, a two-step cortisol-agarose affinity and
phenyl-Sepharose chromatography protocol was applied. A highly
enriched "SHBG-like" preparation was obtained. At 10 to 15
.mu.g/mL, it replicated the E.sub.2 reversible inhibition caused by
30 to 50% (v/v) serum with steroid responsive human breast cancer
cells, and responsive rat mammary, rat pituitary and Syrian hamster
kidney tumor cells in culture. The inhibitor retained full activity
for more than one year when stored unfrozen at -20.degree. C. in
the presence of calcium, dihydrotestosterone and glycerol. This
study demonstrated that the longstanding problem of inhibitor
stability has been overcome and that a high specific activity
preparation was now available to further probe molecular identity.
These results clearly differentiate this inhibitor preparation from
any previously described type of estrogen reversible inhibitor
(i.e. estrocolyone). Moreover, no previous inhibitor composition,
at a concentration .ltoreq.15 .mu.g/mL, can supplant the effects of
full serum to give estrogenic effects .gtoreq.3 CPD with several
ER.sup.+ cell lines from different tissues and different
species.
[0460] The most active inhibitor preparation obtained in this study
appeared to have multiple components present. The separation and
identification of these components, as discussed in Example 20,
would yield additional assays and preferred reagents and
methodologies for testing new hormone-like and anti-hormone like
substances. The active serum-derived inhibitor fraction can be used
directly in tests of new compounds, substances, mixtures and
preparations from natural and synthetic sources to estimate both
estrogenic and androgenic activity in culture. Large-scale
preparation of this purified serum fraction is possible by using
larger affinity columns and proportionately increased serum
volumes, similar to existing technology employed for purifying
other biological products. It is advantageous that only small
quantities of the purified serum fraction are needed for cell
growth assays.
Example 19
Serum-Free Assay Systems for Measuring Large Magnitude Steroid
Hormone Mitogenic Responses with the Two-Step Purified
Inhibitor
[0461] The above-described studies with several different sex
steroid sensitive cell lines demonstrated that the effects of a
partially purified estrogen reversible inhibitor could readily be
assayed in the presence of a low concentration (i.e. 2.5%) of
CDE-serum. The next step was to eliminate the serum completely and
to show estrogen responsiveness under far more defined
conditions.
[0462] Second Analysis of Serum-free Growth.+-.E2. Experiments were
conducted using completely serum-free medium, and the magnitude of
the estrogenic effects observed in defined medium was again
compared to those seen in medium containing CDE-serum. ER.sup.+
tumor cell growth was measured first in serum-free defined
culture.+-.10 nM B.sub.2. Similar experiments have been reported in
FIGS. 56 and 57. The reassays are included because the first
experiments were done two years earlier. The results show the
stability of the cell lines used and the fact that serum-free
defined medium is highly reproducible. More recent results are
shown with the MCF-7K human breast cancer cells (FIG. 84A), the
T47D human breast cancer cells (FIG. 84B), the GH.sub.4C.sub.1 rat
pituitary tumor cells (FIG. 84C), and the H301 Syrian hamster
kidney tumor cells (FIG. 84D). All four-cell lines grew
logarithmically for several days in defined and reached densities
of 0.5 to 1.0.times.10.sup.6 cells per 35-mm dish. The media
formulations were based on standard D-MEM/F-12 as described in
TABLE 7. Growth rates were optimized to 70% or more of D-MEM/F-12
containing 10% (v/v) fetal bovine serum. The results presented in
FIG. 84 show little or no E.sub.2 effect on growth in defined
medium. Barnes and Sato (Barnes D and Sato G (1980) Nature (Lond)
281, 388-389) have reported similar negative results with another
strain of MCF-7 cells in a different formulation of defined medium.
Considering the variety of cell types assayed herein, the present
results and the results of others, the lack of estrogenic effects
in serum-free defined medium is not related to chemical composition
of any one medium nor is there a major problem with time dependent
variation of cell line properties.
[0463] Effects of CDE-Serum on ER.sup.+ Cells in Different
Formulations of Serum-free Defined Medium. The experiments in FIG.
85 were done to show that serum could be added to different
formulations of defined medium and still cause estrogen reversible
inhibition. Effects are shown with CDE-horse serum.+-.10 nM E.sub.2
and T47D cells DDM-2MF (FIG. 85A), MTW9/PL2 cells in DDM-2A (FIG.
85B) and GH.sub.4C.sub.1 cells in PCM-9 (FIG. 85C). Definitely, the
serum-borne inhibitor(s) was fully effective in three different
formulations of defined medium and with three different estrogen
target tissue cell types.
[0464] Effects of CA-PS-pool II on ER.sup.+ Cell Growth in
Serum-free Defined Medium. The estrogen reversible inhibitory
effects of CA-PS-pool H were examined with eight ER.sup.+ cell
lines growing in different serum-free defined media (FIG. 86). The
cell lines were the MCF-7K cells (FIG. 86A), the T47D cells (FIG.
86B), the ZR-75-1 human breast cancer cells (ATCC) (FIG. 86C), the
GH.sub.1 (ATCC) (FIG. 86D), GH.sub.3 (ATCC) (FIG. 86E), and
GH.sub.4C.sub.1 (FIG. 86F) rat pituitary tumor cells, the MTW9/PL2
rat mammary tumor cells (FIG. 86G), and the H301 Syrian hamster
kidney tumor cells (FIG. 86H). At 20 to 30 .mu.g/mL, this fraction
completely inhibited growth. The inhibition was totally reversed by
10 nM E.sub.2. The E.sub.2 effects on cell number were in the range
from 33 to 72-fold (i.e. CPD=2.sup.5.04 to 2.sup.6.18). The
activity was not replaced by serum albumin at 5 mg/mL (data not
shown). The estrogen mitogenic effects seen in defined medium
containing only a few .mu.g/mL of protein were equal to or greater
than those seen in medium containing 30 to 50% (v/v) CDE-horse
serum with every ER.sup.+ cell line tested (TABLE 10). Plainly, the
serum-free conditions established herein are the most defined model
assay systems yet established to demonstrate estrogen
responsiveness in vitro.
TABLE-US-00011 TABLE 10 Summary of the Maximum Estrogenic Effects
in D-MEM/F-12 plus CDE-horse Serum .+-. 10 nM E2 versus those in
Serum-free Defined Medium Supplemented with CA-PS-pool II MAXIMUM
ESTROGENIC MAXIMUM EFFECTS IN ESTROGENIC SERUM-FREE EFFECTS IN
MEDIUM PLUS CELL LINES CDE-SERUM CA-PS-POOL II MCF-7K 3.40 CPD 5.84
CPD (23.sup.3.40 = 10.5-fold) (2.sup.5.84 = 57.3-fold) T47D 5.38
CPD 5.88 CPD (2.sup.5.38 = 41.6-fold) (2.sup.5.88 = 58.9-fold)
ZR-75-1 3.84 CPD 5.21 CPD (2.sup.3.84 = 14.3-fold) (2.sup.5.21 =
37.0-fold) GH.sub.1 4.71 CPD 5.04 CPD (2.sup.4.71 = 26.2-fold)
(2.sup.5.04 = 32.9-fold) GH.sub.3 4.78 CPD 5.04 CPD (2.sup.4.78 =
27.4-fold) (2.sup.5.04 = 32.9-fold) GH.sub.4C.sub.1 4.82 CPD 5.11
CPD (2.sup.4.82 = 28.2-fold) (2.sup.5.11 = 34.5-fold) MTW9/PL2 6.22
CPD 6.18 CPD (2.sup.6.22 = 74.5-fold) (2.sup.6.18 = 72.5-fold)
H-301 4.33 CPD 6.01 CPD (2.sup.4.33 = 20.1-fold) (26.sup.6.01 =
64.4-fold) CPD (2.sup.CPD = Fold Cell Number Increases Above
Controls Without Estrogen)
[0465] Discussion of Example 19. The studies presented in FIG. 86
and TABLE 10 summarized unequivocally, and for the very first time,
that large magnitude estrogen mitogenic responses can be observed
in completely serum-free defined media containing 2 mg/mL total
protein. Furthermore, the responses shown in FIG. 86 either equal
or exceed others previously observed in partially serum-free media
with ZR-75-1 human breast cancer cells (Allegra J C and Lippman M E
(1978) Cancer Res 38, 3823-3829; Darbe P D et al. (1984) Cancer Res
44, 2790-2793) or with a variety of other estrogen sensitive
(ER.sup.+) human and rodent cell lines in medium with hormone
depleted or deficient serum (Amara J F and Dannies P S (1983)
Endocrinology 112, 1141-1143; Natoli C et al. (1983) Breast Cancer
Res Treat 3, 23-32; Soto A M et al. (1986) Cancer Res 46,
2271-2275; Wiese T E et al. (1992) In Vitro Cell Dev Biol 28A,
595-602).
[0466] These results have a number of important implications.
First, they support the aspect of the estrocolyone hypothesis (Soto
A M and Sonnenschein C (1987) Endocr Rev 8, 44-52) that relates to
the presence in serum of a meaningful inhibitor(s). Also, there is
no doubt that the inhibitor(s) is completely estrogen reversible.
However, the present experiments do not confirm that the steroid
hormones interact with sufficient affinity with the inhibitor to
support that aspect of the estrocolyone hypothesis. The results in
TABLE 9 indicate that this aspect of the estrocolyone hypothesis is
highly unlikely.
[0467] The estrogen reversibility of the inhibitor with every
target cell type studied under the rigorous conditions of
serum-free defined culture suggests physiologic relevance. The
large magnitude of the effects is a strong statement in favor of
significance. This is especially clear when considering the fact
that the first experiments with 30 to 50% (v/v) serum contained 15
to 25 mg/mL of protein, whereas the later tests using serum-free
medium required only 20 .mu.g/mL of isolated protein.
[0468] The active fraction isolated from horse serum represented
only 0.01 to 0.04% (w/w) of the total protein. Nonetheless, it
effectively regulated eight ER.sup.+ cell lines derived from three
species and three different target tissues. These observations are
evidence that a broadly applicable serum fraction has been
identified. Furthermore, the serum-free medium results suggest that
a common agent(s) may coordinately regulate estrogen responsive
tissue growth in vivo and that the concept of estrogen reversible
negative control may be far-reaching. The results support the
conclusion that in vitro studies can be used to identify important
new aspects of in vivo endocrine physiology.
[0469] The results in defined medium have practical applications.
Cells in serum-free medium grow in response to nutrients, growth
factors, metal delivery proteins, adhesion proteins, and various
classes of hormones. All of these components are mitogenic in the
sense that they contribute to cell replication. Nonetheless, the
addition of only 20 .mu.g/mL of inhibitor to block growth
completely bears directly on the question of the progression of
normal steroid target cells to fully hormone autonomous
cancers.
[0470] The inhibitor preparation used herein has the properties of
a family of tissue regulators first named "chalones". These
proposed cell regulators are water-soluble and tissue specific (but
not species specific) proliferation inhibitors that are reversible
by physiologic stimuli including hormones (Bullough W S (1975) Life
Sci 16, 323-330; Finkler N and Acker P (1978) Mt Sinai J Med 45,
258-264). The studies presented here support this classic concept
as it applies to sex steroid hormone target tissues. As further
demonstrated in subsequent Examples, the molecular identification
of the serum inhibitor(s) promises not only to further support the
role of estrogens as "necessary", but also to establish that
"chalone-like" entities likely are the missing "sufficient"
components that account for estrogen regulation of tissue growth.
The application of serum-free defined medium conditions along with
the use of a high specific activity fraction to demonstrate
estrogen responsiveness in culture is unique.
Example 20
Chemical and Immunological Properties of the Partially Purified
CA-PS-Pool II Inhibitors and Identification as IgA and IgM
[0471] This Example describes chemical and physical confirmation
that the sought-after serum-borne cancer cell growth inhibitor(s)
include at least IgA and IgM.
[0472] Proteinase and Chemical Fragmentation followed by HPLC and
Amino Acid Sequencing. Although it was clear from SDS-PAGE (FIG.
81A) that the CA-PS-pool II preparation was not homogeneous,
chemical fragmentation with cyanogen bromide and proteolytic
enzymes was used for protein/peptide sequencing in an attempt to
identify at least some of the proteins present, employing standard
techniques (Work T S and Burton R H, eds (1981) Laboratory
Techniques in Biochemistry and Molecular Biology, Volume 9, G.
Allen, Sequencing of Proteins and Peptides, Elsevier/North-Holland,
Amsterdam, pp 43-71). This set of protocols yielded many peptides.
These were applied to reverse phase HPLC columns in trifluoroacetic
acid and eluted with organic solvents as described (Ogasawara M et
al. (1989) Biochemistry 28, 2710-2721). The separated peptides were
sequenced. The analyses of several peptides are shown in FIG. 87.
Attempts were made to align these sequences with human SHBG (hm
SHBG) (Walsh K A et al. (1986) Biochemistry 25, 7584-7590), rabbit
SHBG (rb SHBG) (Griffin P R et al. (1989) J Biol Chem 264,
19066-19075), rat androgen binding protein (rt ABP) (GENBANK
registration) and a partial sequence of hamster androgen binding
protein (hs ABP) (GENBANK registration). A BLAST SEARCH match
showed .ltoreq.30% homology for the peptides sequenced. If the
material in CA-PS-pool II had significant shared primary structure
with SHBG, it could not be confirmed.
[0473] Antibodies Against the CA-PS-Pool II Components. Preparative
SDS-PAGE was done on the Ca-PS-pool II fraction, and after
localization of the 54 kDa band, the 54 kDa band was eluted and
prepared for rabbit antibody production by HTI (Ramona, Calif.).
The antibodies raised were very potent and reacted with CA-PS-pool
II (FIG. 88). They did not cross react with CBG (CA-PS-pool I).
However, despite great care, it was evident that the anti-54 kDa
was raised against a mixture of 67, 58 and 54 kDa subunits (FIG.
88). The reaction was definitely strongest with the 54 kDa
component, but clearly identifiable with the 67 kDa and 58 kDa
bands as well. This apparent problem turned out to be an advantage,
and allowed positive identification of the active agents in
CA-PS-pool II. It was investigated whether the activity in
CA-PS-pool II might have been isolated because of affinity for the
agarose matrix rather than as a consequence of the steroid hormone
ligand attached to agarose, noting from interpretation of unrelated
studies, that agarose alone can bind immunoglobulins and give
SDS-PAGE bands at 67, 58 and 54 kDa. Therefore, it was thought
possible that IgG was the estrogen reversible inhibitor.
[0474] Antibodies Against the 54 kDa Component of CA-PS-Pool II and
Blocking of the Estrogen Reversible Inhibitor Activity. Based on
the results in FIG. 88, it was apparent that the 54 kDa antiserum
might be used to determine if the biological activity resided in
any of the 67, 58 or 54 kDa bands. The next study was done to
resolve this important issue. The results were pivotal. FIG. 89
shows that the purified material in CA-PS-pool II was completely
inhibitory at 20 to 40 .mu.g/mL. Addition of even a 1:5000 dilution
of anti-54 kDa blocked the effect of the inhibitor. In control
studies, rabbit pre-immune serum had no effect even at 1:100 a
dilution (data not shown). It was evident that anti-54 kDa serum
contained the antibody to the activity.
[0475] Anti-54 kDa Serum Recognizes Authentic Horse IgA, IgM and
IgG. Next, authentic horse IgA was obtained from Accurate
Chemicals, and horse IgM was obtained from Accurate Chemicals and
Custom Monoclonal International. The material from Custom
Monoclonals was custom purified by an affinity method with a
monoclonal antibody against horse IgM Fc and further purified by
molecular sieve chromatography to be sure of elimination of other
immunoglobulins (a common problem). IgGs were obtained from Zymed
(San Francisco, Calif.), Sigma (St. Louis, Mo.) or The Binding Site
(San Diego, Calif.). The Western analysis shown in FIG. 90
demonstrates these results. The results show clear cross-reaction
with 67 kDa IgM heavy chain, 58 kDa IgA heavy chain and 54 kDa IgG
heavy chain but no reaction with horse albumin.
[0476] Assay of Estrogenic Effects Controlled by Commercially
Purchased Horse IgG, IgA and IgM in 2.5% CDE-horse Serum with
MTW9/PL2 Cells. FIG. 91 demonstrates that at concentrations up to
59 .mu.g/mL, horse IgG did not cause inhibition of MTW9/PL2 cell
growth in 2.5% CDE-horse serum. There was no significant estrogenic
effect caused by IgG. FIG. 92 shows very clearly that commercially
prepared horse serum derived IgM (Custom Monoclonals), was very
active. At concentrations of 20 to 50 .mu.g/mL, IgM completely
inhibited the growth of the MTW9/PL2 cells (i.e. <1.0 CPD).
Addition of 10 nM B.sub.2 reversed the inhibition nearly
completely. Estrogenic effects of 4 to 5 CPD were seen (FIG. 92).
FIG. 93 shows the same general results with commercially prepared
horse serum derived IgA (Accurate). The only apparent difference
was that IgA was slightly more effective than IgM. These results
clearly proved that the active components in CA-PS-pool II were IgA
and IgM. That these Igs would prove to be the inhibitor was
completely unexpected. Although these two active classes of
immunoglobulins (IgA and IgM) are well-established secretory
products of normal breast cells, there was no previous suggestion
in the prior art that they play a role in the negative regulation
of estrogen-dependent cell growth. These immunoglobulins are major
proteins in milk whose hormone-related local production in breast
tissue is well documented, and their function in the body's
secretory immune system is well known.
[0477] Alternate Methods of Obtaining Horse Serum IgG, IgM and IgA.
IgG can be purified using a Hytrap matrix, which is a mixture of
immobilized Protein A and Protein G, employing a technique
described by others (Lindmark R et al. (1983) J. Immunol Meth 62,
1-13; Kronvall G et al. (1969) J Immunol 103, 828-833; Akerstrom B
et al. (1986) J Biol Chem 261, 10240-10247). IgM can be obtained
using a mannan binding protein isolation method normally applied
with human serum (Nevens J R et al. (1992) J Chrom 597, 247-256).
However, yields are low. Another method based on anti-IgM
immunoglobulins linked covalently to Sepharose is far more
effective. This same procedure with immobilized anti-IgA
immunoglobulins can be used to isolate IgA (Tharakan J In: Antibody
Techniques, Malik V S & Lillehoj E P, Eds, 1994, Academic
Press, San Diego, Calif., Chapter 15). Horse IgA can also be
purified using an immobilized Jacalin lectin method usually
reserved for human samples (Roque-Barreira M C et al. (1986) Braz J
Med Biol Res 19, 149-157). However, it can be modified for
non-human species. The buffers are modified to contain 10 to 50 mM
CaCl.sub.2 to bind IgA from other species. Even then, yields are
not high. The preferred methods for horse IgA and IgM use
immobilized antibodies.
[0478] Purification of Rat Serum Immunoglobulins. Three isolations
of the estrogen reversible inhibitor from separate one-liter
batches of adult rat serum were conducted. This was done for two
important reasons. First, the estrogen reversible activity in all
types of adult serum, including rat, were assayed with a highly
estrogen sensitive MTW9/PL2 rat mammary tumor cell line. It was
useful to confirm the horse serum purification results with a
homologous experimental system. Second, the confirmation that rat
IgA and IgM regulated rat mammary tumor cell growth would open the
possibility of combined testing of new therapeutic substances both
in vitro and in vivo. To summarize, the same "CBG" and "SHBG"
fractions were obtained from rat serum by the methods of Fernlund
& Laurell as had been obtained from horse serum. The
chromatography profiles of the rat separations (not presented) were
very similar to those presented in FIG. 80. The only major
difference was that no rat CBG was obtained. At pH 5.5, rat CBG did
not significantly bind to the affinity matrix. Rat serum CA-PS-pool
I and CA-PS-pool II both contained only two Coomassie Blue stained
bands when analyzed by SDS-PAGE (FIG. 94A). These were
approximately 55 kDa and 54 kDa. They were somewhat lower molecular
weights than found with horse, and there were fewer bands. To test
if either rat band was IgG, a Western analysis was performed with
rabbit anti-rat IgG (FIG. 94B). The antibody did not recognize the
Coomassie stained bands but did react with control IgG. However,
when examined with very specific heavy chain monoclonal antibodies
raised to rat IgG1, IgA, and IgM (purchased from Zymed), the
Western analysis was clear (FIG. 95). Both the commercially
purified rat immunoglobulins (purchased from Zymed) and the
two-step purified pools showed cross-reaction with anti-IgA
(weakly), anti-IgG1 subtype (strong reaction) and anti-IgM
(moderate reaction) (FIGS. 95A, 95B, and 95C, respectively).
[0479] Rat and Horse Serum Active Pools Isolated by the Two-Step
Procedure of Fernlund and Laurell have the same Classes of
Immunoglobulins. The same classes of immunoglobulins obtained by
the two-step procedure of Fernlund and Laurell (Fernlund P and
Laurell C-B (1981) J Steroid Biochem 14, 545-552) with horse serum
were found when rat serum was the starting material. This was
considered to be further confirmation that binding to the agarose
matrix was more important than to the immobilized cortisol. It
should be noted that in the original Fernlund and Laurell report
using human cord serum does not address possible immunoglobulin
contamination, however (Fernlund P and Laurell C-B (1981) J Steroid
Biochem 14, 545-552). This is particularly curious because human
immunoglobulins bind to agarose (Smith R L and Griffin C A (1985)
Thombosis Res 37, 91-101).
[0480] Amino Acid Sequencing Of Rat "SHBG-like" Proteins. Protein
fragmentation and amino acid sequencing of rat "SHBG-like" proteins
were done as described above for horse CA-PS-pool II. The analyses
of several peptides are shown in FIG. 96. Attempts were made to
align these sequences with human SHBG (hm SHBG) (Walsh K A et al.
(1986) Biochemistry 25, 7584-7590), rabbit SHBG (rb SHBG) (Griffin
P R et at (1989) J Biol Chem 264, 19066-19075), rat androgen
binding protein (rt ABP) (GENBANK registration) and a partial
sequence of hamster androgen binding protein (hs ABP) (GENBANK
registration). A BLAST SEARCH match showed .ltoreq.30% homology for
the peptides sequenced. If the rat "SHBG-like" pools have
significant shared primary structure with SHBG or rat androgen
binding protein, it could not be confirmed by these studies.
[0481] Labeled Steroid Hormone Binding to The "SHBG-like" Pools
from Rat Serum. As described in TABLE 9, CA-PS-pool H from horse
serum binds sex steroids with an affinity of about 10.sup.-8 M.
This same Scatchard analysis was done with an active fraction from
rat serum. TABLE 11 shows the results of these studies with four
labeled steroid hormones. It is clear that sex steroid hormones
bind with a higher affinity than progesterone or cortisol. The
binding affinities of rat and horse preparations were very similar.
In both cases, the affinities tend to rule out the estrocolyone
hypothesis because it requires E.sub.2 binding in the picomolar
range.
TABLE-US-00012 TABLE 11 Summary of the Scatchard Analysis of the
"SHBG-like" Pools from Rat Serum with Labeled Steroid Hormones
Steroid Hormone CA-PS-Pool II (3H-labeled) K.sub.d (M) K.sub.a
(M.sup.-1) Cortisol 5.7 .times. 10.sup.-6 1.8 .times. 10.sup.5
Progesterone 6.9 .times. 10.sup.-6 1.4 .times. 10.sup.5
17.beta.-estradiol 4.1 .times. 10.sup.-8 2.4 .times. 10.sup.7
Dihydrotestosterone 2.4 .times. 10.sup.-8 4.1 .times. 10.sup.7
[0482] Evaluation of the Rabbit Anti-SHBG Cross-Reaction with the
Active Pools from the Two-Step Isolation of Fernlund and Laurell.
As shown above in FIG. 81B, Western analysis with the anti-SHBG
detected horse IgA, IgM and IgG. Additionally, anti-SHBG
immunoprecipitated the estrogenic activity of horse serum (FIG.
79B). To extend these results, it was established that rabbit
anti-human SHBG recognized a number of the major classes and
subclasses of rat immunoglobulins. SDS-PAGE with Coomassie blue
staining (FIG. 97A) was compared to identification of the same
proteins by Western analysis with anti-SHBG (FIG. 97B). These
results leave very little doubt that the SHBG used to raise
antibodies in rabbits was not homogeneous but in fact was a "crude"
preparation contaminated with several immunoglobulins.
[0483] Test of Rat IgG, IgA and IgM for Estrogen Reversible
Inhibitory Activity with MTW9/PL2 Rat Mammary Tumor Cells. All of
the rat immunoglobulins described in this section were purchased
from Zymed as the highest quality available. Their activity was
assessed with MTW9/PL2 cells in 2.5% (v/v) CDE-rat serum, as
described above. The activity of rat IgG (all subclasses combined)
was assessed (FIG. 98). There was no inhibitory effect at up to 50
.mu.g/mL. Rat IgA was a potent estrogen reversible inhibitor (FIG.
99). At 20 to 50 .mu.g/mL, it completely inhibited growth. Addition
of 10 nM E.sub.2 completely reversed the inhibition. The estrogenic
effects recorded were >5 CPD. The results with rat IgM were very
similar (FIG. 100). At 20 to 50 .mu.g/mL, it completely inhibited
growth. Addition of 10 nM E.sub.2 reversed the inhibition. The
estrogenic effects recorded were >5 CPD. It is essential to note
that IgA or IgM replaced the effect of full CDE-rat serum with
MTW9/PL2 cells. With a completely homologous system (i.e. cell
line, basal 2.5% CDE-serum, and immunoglobulins), the results were
clear. IgA and IgM were the sought after serum-borne inhibitors
from rat.
[0484] Discussion of Example 20. The identification of IgA and IgM
as serum-borne inhibitors fully separates these inhibitors from the
teachings of U.S. Pat. Nos. 4,859,585 (Sonnenschein) and 5,135,849
(Soto), which arrived at no molecular identification of the
inhibitor. The series of investigations presented above demonstrate
that a very longstanding problem has been solved. While the
solution is significant, an even more an important consequence of
this knowledge is the fact that for the very first time, mucosal
cell hormone dependent growth has been linked to a natural immune
regulation. Moreover, this information has direct application to
the diagnosis, genetic screening, prevention and therapy of breast
and prostate cancer and a high likelihood of applications to other
mucosal cancers, as described in more detail in U.S. patent
application Ser. No. ______ (Atty. Dkt. No.
1944-00800)/PCT/US2001/______ (Atty. Dkt. No. 1944-00801) entitled
"Compositions and Methods for the Diagnosis, Treatment and
Prevention of Steroid Hormone Responsive Cancers," which is hereby
incorporated herein by reference.
[0485] During the purification of both the horse serum and the rat
serum estrogen reversible activity, SUPERDEX.TM. (Pharmacia)
molecular sieve chromatography of the final mixtures indicated the
presence of <20% 160 kDa monomeric immunoglobulins. The majority
of the material was of much larger mass. Because IgA exists
naturally as monomer, dimer and polymers, there was a question
concerning which of these is/are inhibitory form(s). The
SUPERDEX.TM. results strongly favor the dimer/polymer form. This
was confirmed also with commercially prepared IgA that was obtained
from hybridoma and myeloma cell lines. The IgA from these was
>80% dimer/polymer. It was very active as an inhibitor. In light
of these results, it is suggested that these forms are the "good"
type of IgA in the body, and that direct measurement of their
concentration in plasma and body fluids has diagnostic and
prognostic applications.
[0486] The introduction of test methods done with minimum serum
plus purified immunoglobulin inhibitor ("spiked serum") provides a
new approach to substances, mixtures and compounds that might be
influenced by serum components. For example, a serum composition
might contain steroid hormone free serum, such as a standard,
commercially available fetal bovine serum preparation, and a
predetermined amount of an immunoglobulin inhibitor, i.e., one or
more of IgA, IgM or IgG. Testing under these conditions, with a
known amount of inhibitor in the serum, may be desirable or
required when the substance has potential for
inactivation/activation by a serum component or when it has
lipophilic properties that require a minimum protein concentration
in the medium to prevent loss.
[0487] Another valuable application of the immunoglobulin
inhibitors will be in identifying substances that may have direct
effects on the action of the immunoglobulins to cause inactivation.
An assay of this nature is unique in the sense that incubation of
substances with the immunoglobulin can be done before the assay to
determine effects on natural immune responses. Changes in
environmental/chemical factors that affect the body's immune system
are of major medical concern. They also are of great concern to
veterinary medicine. Chemicals/nutritional supplements may affect
immune function of domestic animals and thereby affect human food
supplies.
[0488] This series of investigations demonstrate at least two
immunoglobulin inhibitors in serum. More than one inhibitor was
suggested by the conventional purification data in a preceding
Example, and was proved true in succeeding examples. There may
still be other useful estrogen reversible immunoglobulin inhibitors
in serum that are yet to be identified from serum or tissue
sources. The methods described in this Example have direct
application to the search for new compounds that mimic the effect
of the immunoglobulins as estrogen reversible inhibitors. Such
application opens a new avenue of search for anticancer drugs.
Example 21
Regulation of Steroid Hormone-Responsive and Thyroid
Hormone-Responsive Cancer Cell Growth in Serum-Free Defined Medium
by Secretory and Plasma Forms of IgA and Plasma and Cell Culture
Derived IgM
[0489] This Example demonstrates that the determination of whether
purified IgA and IgM from several species mimic the sex steroid
hormone reversible inhibitors isolated from horse in serum was
sought. These studies included ER.sup.+ tumor cells derived from
rodents as well ER.sup.+ and AR.sup.+ cells from human cancers.
Completely serum-free defined culture conditions were used to
perform cell growth assays using the purified inhibitors. The total
protein concentration in the media was <2 mg/mL. The estrogenic
and androgenic effects observed in these assays are unique, as like
effects have not been achieved previously in completely serum-free
defined medium.
[0490] Sources of Purified IgA and IgM. Human IgM was purified from
human plasma as described using immobilized mannan-binding protein
(Nevens J R et al. (1992) J Chromatography 597, 247-256). As an
example of the effectiveness of this isolation, FIG. 101 shows
SDS-PAGE and Coomassie Blue Staining with two preparations of human
plasma IgM prepared, Human IgA1 and IgA2 were purified using
immobilized Jacalin (Roque-Barreira M C and Campos-Neto A (1985) J
Immunol 134, 1740-1743; Kondoh H et al. (1986) J Immunol Methods
88, 171-173; Pack TD (1999) American Biotechnology Laboratory 17,
16-19; Loomes L M et al. (1991) J Immunol Methods 141, 209-218).
Rat IgA and IgM were purchased from Zymed. The effectiveness of the
Jacalin method with human plasma is shown in FIG. 102. Horse IgA
and IgM were purchased from Accurate, Sigma and Custom Monoclonals.
IgA and IgM from other species or as products from cell culture are
purchased from Sigma or Accurate. Human IgA and IgM were bought
also from Sigma and Accurate. Human secretory (milk) IgA (sIgA) was
purchased from Sigma or Accurate.
[0491] A. MTW9/PL2 rat mammary tumor cells. For this series of
experiments the serum-free defined medium was the preferred
formulation of DDM-2A described in TABLE 7. The cell growth assays
with this cell line in DDM-2A testing increasing concentrations of
human plasma IgM is shown in FIG. 103. Human plasma IgM completely
inhibited growth by 20 to 60 .mu.g/mL. The ED.sub.50 was about 12
.mu.g/mL. Based on an IgM M.sub.r of 950,000, the ED.sub.50
concentration was 1.3.times.10.sup.-8 M. Complete inhibition was at
2.2.times.10.sup.-8 M. These concentrations are certainly within
the physiological range of IgM in the plasma and body fluids such
as breast milk. Based on these studies, a comparison was done in
completely serum-free defined DDM-2A medium of the effects of 40
.mu.g/mL of rat plasma IgA.+-.E.sub.2, rat plasma IgM.+-.E.sub.2,
and horse plasma IgM.+-.E.sub.2 (FIG. 104, expressed as (A) cell
numbers and (B) CPD). From the CPD calculations it was clear that
no matter the species source, IgA and IgM were very potent estrogen
reversible inhibitors of MTW9/PL2 cell growth.
[0492] One problem occurred with the MTW9/PL2 cell assays that
initially caused concern. Human IgA was purchased from Sigma as the
milk derived immunoglobulin. It was far less expensive than plasma
IgA. For reasons that at first were not clear, this material was at
best only partially inhibitory and often not inhibitory. As will be
discussed below with GH.sub.1 cells, this turned out to be a
significant clue to the mechanism of action of the immunoglobulins.
Nonetheless, it is known that the heavy chains of IgM and IgA from
different species share primary structure homology. This is not
true of the variable regions of the light chains. The results
presented support the possibility of Fc-like receptor mediation of
the IgA and IgM effects on MTW9/PL2 cells.
[0493] B. GH.sub.1, GH.sub.3 and GH.sub.4C.sub.1 rat pituitary
tumor cells. For this series of experiments the serum-free defined
medium was the preferred formulation of PCM-9 described in TABLE 7.
The next serum-free defined medium studies were done with GH.sub.1
cells. Example assays are shown. This cell line was highly estrogen
responsive in the presence of homologous rat myeloma derived IgA
(FIG. 105). Maximum estrogenic effect was >5 CPD or more than a
32-fold estrogen-induced increase in cell number in 10 days. A
similar assay with human plasma derived IgA showed nearly the same
results (FIG. 106). Indeed, human IgA showed greater inhibition at
10 .mu.g/mL. Another study with human IgM demonstrated that it was
also an estrogen reversible inhibitor of GH.sub.1 cell growth (FIG.
107). It was not as inhibitory as IgA with this cell line, but
certainly still effective. As discussed above, in the Background of
the Invention, during the secretion process a fragment of about 80%
of the poly-Ig receptor (including the five extracellular domains)
becomes attached to the dimeric/polymeric form of IgA to form
secretory IgA or sIgA. The receptor fragment is called the
"secretory component". After secretion, sIgA can be readily
isolated from human milk. The effect of milk derived secretory IgA
(sIgA) was evaluated with the GH.sub.1 cells in PCM-9, and the
results of a representative study are shown in FIG. 108. These
results were strikingly different than those obtained with plasma
derived IgA (pIgA) (FIG. 106). SIgA was not inhibitory even at 20
.mu.g/mL. Considering why the two different forms of IgA behaved so
differently, the poly-Ig receptor was recognized as a potential
candidate for the mediator of the action of IgA/IgM. As discussed
in the Introduction of this Detailed Description, this receptor has
not been previously associated with any growth related function.
The poly-Ig receptor is concerned with process of transcytosis of
IgA/IgM, as conceptually illustrated in FIG. 109. SIgA already has
the receptor bound in the sense of the secretory piece in
association with the Fc domains of the dimer. FIG. 110 illustrates
schematically the structures of inactive monomeric IgA, the
connecting or joining "J" chain, the structure of the active dimer
with "J" chain, the secretory piece or secretory component, and the
dimeric IgA structure plus secretory component attached, as
generally understood. The illustration shows that the Fc domains of
dimeric IgA are blocked by the secretory piece/component. Access to
the Fc domains is required for binding to the poly-Ig receptor.
[0494] The present series of cell growth assays above were
continued with the related GH.sub.3 cells, again in serum-free
defined the preferred formulation of PCM-9 medium. Rat myeloma
derived IgA was an effective estrogen reversible inhibitor of these
cells in a 9 day growth assay (FIG. 111). The maximum estrogenic
effect exceeded 5 CPD. A similar assay with rat IgM was conducted
(FIG. 112). It showed even greater inhibition at 10 .mu.g/mL than
with IgA. The estrogenic effect recorded in 10 days was nearly 6
CPD. These same assays were next repeated with the human
immunoglobulins. Human pIgA was an estrogen reversible inhibitor of
GH.sub.3 cell growth (FIG. 113). It was not as effective as its rat
counterpart, but the estrogenic effect with the human
immunoglobulin was still 4 CPD. Also, human IgM was effective with
GH.sub.3 cells (FIG. 114). Again the estrogenic effect was about 4
CPD. In the final study with GH.sub.3 cells, it was again apparent
that human milk derived sIgA was not inhibitory (FIG. 115).
[0495] The studies above with GH.sub.1 and GH.sub.3 cells were
continued with the related GH.sub.4C.sub.1 line, again in
serum-free defined PCM-9 medium. Rat myeloma derived IgA was an
effective estrogen reversible inhibitor of these cells in a 9 day
growth assay (FIG. 116). The maximum estrogenic effect approached 5
CPD. A similar assay with rat plasma IgM was conducted (FIG. 117).
It showed slightly less inhibition than IgA. The estrogenic effect
recorded in 10 days was nearly 4 CPD. These same assays were next
repeated with the human immunoglobulins. Human pIgA was an estrogen
reversible inhibitor of GH.sub.4C.sub.1 cell growth (FIG. 118). It
was not as effective as its rat counterpart, but the estrogenic
effect with the human immunoglobulin was still almost 4 CPD. Also,
human pIgM was effective with GH.sub.4C.sub.1 cells (FIG. 119). The
estrogenic effect was about 5 CPD. In the final study with
GH.sub.4C.sub.1 cells it was again apparent that human milk derived
sIgA was not inhibitory (FIG. 120).
[0496] C. H301 Syrian hamster kidney tumor cells. The studies with
this cell line were done in the preferred formulation of CAPM
defined medium described in TABLE 7. Because hamster IgA and IgM
were not available, these experiments began with plasma IgA from
mouse (FIG. 121). Mouse IgA was very effective with hamster H301
cells. The estrogenic effect was >5 CPD. Human plasma IgA was
also effective (FIG. 122A). The maximum estrogenic effect reached 4
CPD. Secretory IgA was inactive (FIG. 122B). With this cell line,
human IgM also was an estrogen reversible inhibitor. As shown in
FIG. 123, a dose-response study demonstrated that in serum-free
defined medium with 40 .mu.g/mL of human plasma IgM, concentrations
of 0.1 to 1.0 picomolar E.sub.2 caused significant growth
(p<0.01). This data demonstrate the extraordinary sensitivity of
the serum-free defined cell growth assays in the presence of
immunoglobulin. The data in FIG. 123 provide strong support for the
view that the H301 cells can be used to characterize the new
ER.gamma. and characterized in preceding Examples.
[0497] D. MCF-7A and MCF-7K human breast cancer cells. For this
series of experiments the serum-free defined medium was the
preferred formulation of DDM-2MF described in TABLE 7. Two highly
applied MCF-7 human breast cancer cell strains were applicable to
this series of investigations. As shown with MCF-7A cells in
DDM-2MF serum-free defined medium, plasma IgA was highly effective
as an estrogen reversible inhibitor. The estrogenic effect exceeded
4 CPD in 10 days (FIG. 124A). In contrast, sIgA was inactive (FIG.
124B). With the MCF-7K strain, the results were nearly identical.
Plasma IgA was effective (FIG. 125A) and sIgA was inactive (FIG.
125B). The estrogenic effects caused by pIgA were replicated by
substitution of plasma IgM. With MCF-7A and MCF-7K, pIgM was an
effective estrogen reversible sustaining estrogenic effects of
>4 CPD (FIGS. 126 and 127, respectively). In a final study of
this series, an E.sub.2 dose-response experiment was conducted with
MCF-7K cells in DDM-2MF plus 40 .mu.g/mL of plasma IgM. The results
were remarkable. Estrogen at as low as 0.1 picomolar caused more
than one-half maximum growth response (FIG. 128). The extraordinary
sensitivity of this assay methodology is clearly established. These
results add more evidence that a very high affinity estrogen
receptor (i.e. ER.gamma.) regulates growth and is yet to be defined
in human breast cancer cells.
[0498] E. T47D human breast cancer cells. The T47D cell line was
assayed for immunoglobulin effects in the preferred formulation of
serum-free defined medium DDM-2MF described in TABLE 7. As shown in
FIG. 129A, human plasma IgA was a very effective estrogen
reversible inhibitor with T47D cells. The maximum estrogenic effect
was 6 CPD or a 72-fold cell number increase in 12 days. In
contrast, sIgA was inactive at up to 20 .mu.g/mL (FIG. 129B).
Likewise, human plasma IgM is effective (FIG. 130), demonstrating
complete inhibition of cell growth by 20 .mu.g/mL IgM. The
estrogenic effect was 5 CPD in 12 days. In experiments not shown,
the effects of plasma derived IgM were compared to myeloma derived
IgM. This study yielded the same estrogenic effects with both
sources of IgM. Again, the antigenic determinant appears to be
unimportant. The results support the view that the heavy chains
dictate the activity. In other studies with T47D cells in defined
medium containing 40 .mu.g/mL, the dose-response effects with
E.sub.2 showed more than one-half maximum growth at 0.1 picomolar
(FIG. 131). These results continue to fortify the theme that the
methods described in this Example allow investigation of potential
estrogenic compounds and substances that might be present in
samples of industrial or biological materials at very low
concentrations. It is also apparent that the data supports the view
that a high affinity ER.gamma. regulates growth.
[0499] F. ZR-75-1 human breast cancer cells. For these experiments
the serum-free medium was the preferred formulation of DDM-2MF
described in TABLE 7. Plasma IgA was an estrogen reversible
inhibitor with ZR-75-1 cells (FIG. 132A). The estrogenic effect was
recorded at 5 CPD in 14 days. As seen before with the other
ER.sup.+ cell lines above, sIgA was not an inhibitor with ZR-75-1
cells (FIG. 132B). Plasma IgM was also assayed with the ZR-75-1
cells (FIG. 133). It was a potent estrogen reversible inhibitor
under these completely serum-free defined conditions. As discussed
above, this line had been thought to be estrogen responsive in
serum-free culture. However, the former methods were not
serum-free. As disclosed herein, it has now been established in
entirely different culture conditions and shown that this line is
truly estrogen growth responsive in culture.
[0500] G. HT-29 human colon cancer cells. For this series of
experiments the serum-free defined medium was the preferred
formulation of CAPM described in TABLE 7. As expected from
endocrine physiology, colon is not a sex steroid hormone growth
regulated tissue as are others such as breast, uterus, ovary and
pituitary. However, it was discovered that this tissue is thyroid
hormone growth responsive. As shown in FIG. 134, HT-29 human
colonic carcinoma cells grow in CAPM independently of the presence
of thyroid hormone. This growth is promoted by the other factors
present in CAPM minus T.sub.3. However addition of plasma IgM at 40
.mu.g/mL had a dramatic effect. In the absence of T.sub.3 HT-29
cell growth was inhibited to .ltoreq.1.0 CPD in 10 days. Addition
of increasing concentrations of T.sub.3 restored growth (FIG. 134).
This demonstrates that colonic cancer cells respond to thyroid
hormones in the same manner that ER.sup.+ cells respond to E.sub.2.
Estrogens and thyroid hormones belong to the same superfamily of
receptors and both are required for normal physiologic growth and
development (Williams G R and Franklyn J A (1994) Baillieres Clin
Endocinol Metab 8, 241-266; Tsai M J and O'Malley B W (1994) Annu
Rev Biochem 63, 451-486). This is the first demonstration of a
secretory immunoglobulin acting directly as a thyroid hormone
reversible growth inhibitor of a human origin colon cancer cell
line.
[0501] H. LNCaP human prostatic carcinoma cells. For this series of
experiments the serum-free defined medium was the preferred
formulation of CAPM described in TABLE 7. LNCaP cells were
negatively regulated by plasma IgA (FIG. 135A). The immunoglobulin
was a DHT reversible inhibitor that was completely effective at 10
.mu.g/mL. The androgenic effect was >5 CPD in 12 days. As seen
with the ER.sup.+ cell lines above, sIgA was not inhibitory with
LNCaP cells (FIG. 135B). Two different types of human IgM were also
compared with LNCaP cells (FIG. 136). They were plasma derived and
myeloma derived IgM. Despite the differences in antigen binding
domains, both forms were equally inhibitory and both forms were
reversed by 10 nM DHT. These results indicate that the Fc/heavy
chain of IgM is the functional activator of the inhibition.
[0502] Summary of the estrogenic effects of IgM on ER.sup.+ cell
growth. FIG. 137 presents a summary of the effects of IgM derived
from different species with a variety of ER.sup.+ cell lines. This
summary presents the maximum estrogenic effects recorded under
conditions described above in serum-free defined medium with each
cell line.+-.10 nM E.sub.2. Estrogenic effects ranged from 4 to
>7 CPD. Comparison of the results in FIG. 137 with those in
TABLE 10 show in general that the results achieved in completely
defined medium are equal to or greater than those seen in CDE-serum
cultures.
[0503] Discussion of Example 21. These methods will permit
evaluation of industrial, environmental, biological, medical,
veterinary medicine and other potential sources of estrogenic or
androgenic activity under the most sensitive conditions yet
developed. Estrogenic activity is measurable at .ltoreq.1.0
picomolar concentrations. Two cell lines, MTW9/PL2 and H301, are
preferred potential sources of identification of the new growth
regulatory ER.gamma.. The evidence presented with MCF-7 and T47D
human breast cancer cells support the presence of a new growth
regulatory ER.gamma.. Because the purified IgA and IgM described
herein are as effective as serum-borne inhibitors, the serum-free
methods described herein provide unique tools to search for
ER.gamma.. Assays conducted under these conditions permit
estimation of estrogen sensitivities in ranges not approachable by
other technology. These methods can also be adapted to measurement
of the inhibitor in biological fluids available in only small
supply. For example, coupled with use of XAD-4 resin extraction to
remove steroids, bodily fluids and other source materials can be
assayed on small scale to determine the concentration of effective
inhibitor. This is of particular interest because IgA in plasma is
>90% inactive monomer and <10% active dimer/polymer.
Measurement of IgA by conventional methods gives total
concentrations, and does not determine the concentration/presence
of active inhibitor. The present biological activity method has
distinct features and advantages, and can serve as an adjunct
measurement.
[0504] Serum-free defined medium assays can be used to search for
new compounds that mimic the action of immunoglobulins to block
cancer cell growth in its early stages. This screening can be done
under conditions in which serum proteins might interfere. Compounds
so-identified can next be evaluated by addition of CDE-serum or
XAD-4 treated serum to determine if serum proteins interfere and to
determine drug efficacy in vitro under both serum-free defined
medium conditions and serum supplemented conditions. Serum-free
defined medium method can be used for screening of compounds that
may either enhance or inhibit immune function at the epithelial
cell level. Compounds with these activities may have utility as
immune enhancers to help reduce the risk of cancer development.
These assay methods offer a screening tool for such compounds that
has not been available before. Larger magnitude effects permit
greater accuracy with the new assay methods when estimating effects
of substances that are less potent than natural estrogens.
Example 22
Effect of Tamoxifen Antiestrogen in Serum-Free Defined Medium
[0505] This Example illustrates the use of one of the present
assays to detect the estrogenic or anti-estrogenic effect of a
substance. In particular, the classical antiestrogen was assayed as
a demonstration of the usefulness of the present assay system.
[0506] Background of Tamoxifen Effects and Clinical Applications.
The antiestrogenic effects of tamoxifen are well documented. Most
evidence suggests this compound and its active metabolite
4-hydroxyl-tamoxifen prevent growth of ER.alpha. positive cells via
interaction with the receptor (Coezy E et al. (1982) Cancer Res 42,
317-323; Bardon S et al. (1984) Mol Cell Endocrinol 35, 89-96;
Reddel R R et al. (1985) Cancer Res 45, 1525-1531). However, it has
also been suggested that tamoxifen blocks growth factor promoted
MCF-7 breast cancer cell growth (Vignon F et al. (1987) Biochem
Biophys Res Commun 146, 1502-1508). Also, tamoxifen has high
affinity binding sites and actions distinct from the estrogen
receptor (Sutherland R L et al. (1980) Nature (Lond) 288, 273-275;
Phaneuf S et al. (1995) J Reprod Fertil 103, 121-126). Despite its
complex actions, tamoxifen has widespread use as a treatment for
breast cancer (Fisher B et al. (1998) J Natl Cancer Inst 90,
1371-1388; Jaiyesimi I A et al (1995) J Clin Oncol 13, 513-529;
Clinical Trial Report (1997) J Clin Oncol 15, 1385-1394; Clinical
Trial Report (1987) Lancet 2(8552), 171-175; Forrest A P et al.
(1996) Lancet 348(9029), 708-713; Tormey D C et al. (1996) J Natl
Cancer Inst 88, 1828-1833; Gundersen S et al. (1995) Breast Cancer
Res Treat 36, 49-53; Gelber R D et al. (1996) Lancet 347(9008),
1066-1071; Raabe N K et al. (1997) Acta Oncol 36, 2550260).
[0507] Serum-free Medium Effects of Tamoxifen. The effects of
tamoxifen (TAM) were reexamined under completely serum-free defined
conditions. It is very important to note that throughout the
Examples herein, data is presented showing that estrogens have
either had no effect on growth in defined medium or at most a 1.0
CPD effect that was related to saturation density. This was true no
matter if phenol red was present or absent from the medium, as
shown in Example 8 and reported (Moreno-Cuevas J E and Sirbasku D A
(2000) In Vitro Cell Dev Biol 36, 447-464). In similar assays,
1.0.times.10.sup.-7 M tamoxifen was completely inhibitory with T47D
cells in culture, as shown in FIG. 138. The study shown in FIG. 138
examined the concentrations of tamoxifen needed to fully inhibit
T47D cell growth in the preferred formulation of DDM-2MF serum-free
defined medium without any source of estrogens. The expected
outcome was no tamoxifen inhibition. As shown, estrogen alone had
only a 1.0 CPD effect in serum-free defined medium. However,
tamoxifen had unexpected effects revealed by the use of serum-free
defined medium. Tamoxifen effectively arrested growth at
1.0.times.10.sup.-7M. Higher concentrations were cytotoxic. It
should be noted that tamoxifen had the same effect as
immunoglobulins IgA and IgM. To demonstrate this fact another way,
the experiment in FIG. 139 shows that estrogens completely reverse
the effect of 1.0.times.10.sup.-7 M tamoxifen. This sequence of
experiments shows the same results as that shown above with plasma
IgA and IgM and ER.sup.+ cell lines.
[0508] Discussion of Example 22. The observation of inhibition of
cell growth by a classical antiestrogen demonstrates the usefulness
of this technology to search for other antiestrogenic compounds.
Furthermore, because of the current intense focus on the search for
SERMs (i.e. Selective Estrogen Receptor Modulators) the serum-free
technology disclosed herein has particularly useful applications.
Specific types of SERMS can be sought for different cell types.
Those SERMs that do not cause breast cancer cell growth can be
readily identified by this technology. Those SERMs with multiple
activities can be identified before conducting expensive animal
testing.
[0509] The technology presented permits a clear definition of
antiestrogens with "mixed" functions (e.g. tamoxifen-like, that act
at several sites) versus those with a "pure" function mediated only
by the estrogen receptor. To date, no similar easily applied in
vitro method based on serum-free defined medium and secretory
immunoglobulins is available that produces growth as an endpoint of
the assay. An entirely new function for the drug tamoxifen is
proposed, in which the tamoxifen mimics the immune system effects
on ER.sup.+ cancers thereby inhibiting growth. Estrogen reverses
these effects, not as a consequence of interaction with the
classical ER.alpha., but as a consequence of the ER.gamma..
Tamoxifen may also be an antagonist of ER.gamma., and this possible
use for tamoxifen is now proposed.
[0510] The serum-free defined medium technology presented herein
has direct application to the assay of a great variety of drugs now
in use by women either before the onset of breast cancer or after
the onset. Drugs or preparations such as antidepressants, herbal
extracts, soy products, other food, plant or microorganism
extracts, estrogenic creams and cosmetic preparations can be
assessed for anti-estrogenic or estrogenic activity.
[0511] Serum-free assay methods are also applicable to exploration
of additional antiandrogenic compounds. Furthermore, in view of the
possible role of estrogens as well as androgens in prostate growth,
this technology can be used to search for compounds with both
activities.
Example 23
IgG1 and IgG2 as Immunoglobulin Regulators of Estrogen and Androgen
Responsive Cancer Cell Growth
[0512] This Example investigates and discusses the relative
effectiveness of certain IgGs as inhibitors of steroid hormone
responsive cancer cell growth.
[0513] The IgG Subclasses and the Importance of Assessing Each for
Activity. As reviewed above, in the Background of the Invention,
three classes of immunoglobulins are secreted by mucosal tissues.
The IgG class is lowest in concentration in secretions, but still
physiologically important because of its capacity to neutralize
pathogens by different mechanisms. Additionally, the studies above
showed that bulk purified mixtures of all subclasses of horse and
rat IgG were not estrogen reversible inhibitors for MTW9/PL2 rat
mammary tumor cells. The human clinical importance of understanding
and measuring IgG subclasses has been growing steadily. From a few
clinical reports per year in 1970, the literature now exceeds four
hundred reports a year. These assays are significant for several
reasons. (i) They provide a clearer picture of an individual's
susceptibility to disease. (ii) An awareness that treatment for
subclass deficiencies is important. (iii) The subclasses can be
used to assess the state of a number of diseases. (iv) The
importance of IgG subclass difference between ethnic groups and
different races is a potential area for expanded control of
disease.
[0514] Test of Rat IgG Subclasses as Estrogen Reversible Inhibitors
of MTW9/PL2 Rat Mammary Tumor Cell Growth. The IgG subclasses of
rat are IgG1, IgG2A, IgG2B and IgG2C. These IgGs, obtained from
commercial sources previously identified herein, were tested at 15
.mu.g/mL with MTW9/PL2 cells in DDM-2A serum-free defined medium
(FIG. 140). All four IgG subclasses were compared to rat pIgA and
rat pIgM. The latter two were estrogen reversible inhibitors, as
expected (FIG. 140). However, the four IgG subclasses were not
inhibitors at a concentration that was effective with IgA or IgM.
The estrogenic effects recorded in cultures with them were no
larger than seen in serum-free defined medium alone (FIG. 140).
Clearly, IgG are not effective steroid hormone modulators in
rat.
[0515] Test of Human IgG Subclasses as Estrogen Reversible
Inhibitors of Breast and Prostate Cancer Cell Growth. The
subclasses of human IgG are IgG1, IgG2, IgG 3 and IgG4. They are
formed with both .lamda. and .kappa. light chains. A series of
studies was performed, and it was found that with human breast
cancer cells, only IgG1.kappa. was a significant estrogen
reversible inhibitor. FIG. 141 shows a comparison of its activity
to human pIgA and pIgM. At 40 .mu.g/mL, it was 37% as effective as
pIgM. A similar study with LNCaP cells showed that only IgG1.kappa.
had activity greater than the estrogenic effect seen in CAPM
serum-free defined medium only (FIG. 142). In some experiments with
prostate cells, IgG2.kappa. also showed androgen reversible
inhibitory activity (data not shown).
[0516] Discussion of Example 23. The effect of IgG1.kappa. raises
an issue not encountered with IgA or IgM. The preference for the
.kappa. light chain implies that a different receptor mediates the
effects of this immunoglobulin. This immunoglobulin may have
greater effect on normal breast or prostate cells as an inhibitor.
It is also believed that part of the transformation/progression
process leading to hormone responsive cancers is an attenuation of
the effectiveness of IgG1.kappa. as an inhibitor. The present IgG1
observations have other applications, as well, including the
measurement of the IgG1.kappa. subclass in different populations
such as black American, Asian, white, Native American and Hispanic
with contrasting susceptibilities to breast and prostate cancer, or
individuals within any one ethnic group, may provide additional
information and confirmation of the usefulness of such
measurements. These measurements can be made in bodily fluids or
plasma. Measurement in milk and breast fluid may provide an
indication of susceptibility to the development of breast
cancer.
[0517] Irrespective of the receptor that mediates the growth
response of IgG1.kappa., this receptor will be a candidate for the
missing transcytosis receptor for IgG. Its molecular identification
has utility in diagnostic specimens of breast, prostate and other
cancers and can be used to determine new uses of the immune system
for therapeutic applications. Once it is completely identified, the
receptor that mediates the IgG1 growth inhibition effect will
provide another target for development of compounds that mimic the
immune system inhibition of cancer cell growth.
Example 24
Mediation of IgA/IgM Effects by the Poly-Ig Receptor
[0518] In this Example, it was determined that a poly-Ig receptor
or a poly-Ig like receptor mediates the inhibition of cell growth
by IgA and IgM. The negative response to IgA and IgM is mediated by
the mucosal poly-Ig receptor or a very similar structure with the
same immunoglobulins specificity as well as the same immunological
and M.sub.r properties. The known poly-Ig receptor is a M.sub.r
100,000 transmembrane protein with several properties that place it
in the Ig superfamily of receptors (Kraj{hacek over (c)}i P et al.
(1992) Eur J Immunol 22, 2309-2315; Williams A F and Barclay A N
(1988) Annu Rev Immunol 6, 381-405).
[0519] Genetic Properties of the Poly-Ig Receptor. The complete
genomic and cDNA sequences of the poly-Ig receptor have been
determined (Kraj{hacek over (c)}i P et al. (1991) Hum Genet 87,
642-648; Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22,
2309-2315). Poly-Ig receptor gene has been localized to chromosome
1 at 1q31-q42 locus Kraj{hacek over (c)}i P et al. (1991) Hum Genet
87, 642-648; Kraj{hacek over (c)}l P et al. (1992) Eur J Immunol
22, 2309-2315; Kraj{hacek over (c)}i P et al. (1995) Adv ExpMed
Biol 371A, 617-623). The long arm of chromosome 1 had initially
been described as the location of the most frequent ctyogenetic
abnormalities found in human breast carcinoma (Bieche I et al.
(1995), Clin Cancer Res 1, 123-127). More recently this conclusion
was modified to state that distal alterations of the short arm of
chromosome 1 are the most frequent cytogenetic abnormalities in
human breast carcinoma (Bieche I et al. (1999) Genes Chromosomes
Cancer 24, 255-263). The gene encoding the poly-Ig receptor is
linked to D1S58 on the long arm of chromosome 1 (Kraj{hacek over
(c)}i P et al. (1992) Hum Genet. 90, 215-219). This locus (i.e.
D1S58) is a known site for "allelic imbalances" in a remarkable 75%
of all breast cancers (Loupart M-L et al. (1995) Genes Chromosomes
Cancer 12, 16-23). Allelic imbalances include "Allelic Loss,
Allelic Gain, and Imbalances". Loss of heterozygosity (LOH) is
consistently high along the length of the long arm of chromosome 1
at D1S58 (i.e. 46%) in breast cancers (Loupart M-L et al. (1995)
Genes, Chromosomes & Cancer 12, 16-23). LOH is strongly
associated with development of cancer. Viewed in light of the
present invention, these published published reports gain new
meaning and significance. The report describing changes in D1S58
did not specify what gene or type of gene or function might be
impaired by damage to this locus (Loupart M-L et al. (1995) Genes,
Chromosomes & Cancer 12, 16-23). The present results indicate
that this "hot spot" is either the authentic poly-Ig receptor
acting in its new capacity as a growth regulator, or a very closely
related receptor with similar molecular weight, ligand binding and
immunological properties. However, it must be recognized that the
functional form of the growth regulatory receptor may arise from
alternate splicing of the poly-Ig receptor gene. Alternate splicing
of the poly-Ig receptor gene is known in rabbit (Deitcher D L and
Mostov K E (1986) Mol Cell Biol 6, 2712-2715; Frutiger S (1987) J
Biol Chem 262, 1712-1715) and bovine tissue (Kulseth M A et al.
(1995) DNA Cell Biol 14, 251-256). It has yet to be proven (or
disproved) in humans. Certainly this possibility is still open with
hormone responsive cancer cells. Alternately the 1q31-q41 region of
chromosome 1 contains several other genes of immunological interest
(Kraj{hacek over (c)}i P et al. (1991) Hum Genet 87, 642-648;
Kraj{hacek over (c)}i P et al. (1992) Eur J Immunol 22, 2309-2315;
Bruns GAP and Sherman SL (1989) Cytogenet Cell Genet 51, 67-77).
There can be little doubt that the discovery of immune negative
regulation of growth mediated by the poly-Ig receptor, or one very
related, is an advance. It was arrived at not by the genetic
approach described above which screens genes without regard for
function, but instead by a functional approach based on the
biochemical, endocrine and cell biology studies described
above.
[0520] Structural Properties of the Poly-Ig Receptor. A very
detailed structural analysis of the human poly-Ig receptor has been
presented by others (Kraj{hacek over (c)}i P et al. (1992) Eur J
Immunol 22, 2309-2315). Altogether, eleven exons cover the entire
coding sequence. The five extracellular domains designated D1, D2,
D3, D4 and D5 were coded for by exons E3, E4, E5 (D3 & D4), E5
and E6, respectively. The five extracellular domains are repeating
disulfide stabilized Ig-like domains with homology to the Ig
superfamily of receptors. The functions of D2, D3 and D4 are not
well defined. The functions of D1 and D5 are well studied. D1 is
the binding site for the Fc domains of IgA and IgM (Frutiger S et
al. (1986) J Biol Chem 262, 1712-1715; Bakos M-A et al. (1993) J
Immunol 151, 1346-1352; Roe M et al. (1999) J Immunol 162,
6046-6052). The presence of a "J" chain in the immunoglobulins
appears essential for receptor binding and secretion (Vaerman J-P
et al. (1998) Eur J Immunol 28, 171-182). D1 is highly conserved
among species. The amino acid sequence of the D1 loop responsible
for IgA/IgM binding has been established as residues 15.fwdarw.37
(Bakos M-A et al. (1991) J Immunol 147, 3419-3426; Bakos M-A et al.
(1993) J Immunol 151, 1346-1352; Bakos M-A et al. (1994) Mol
Immunol 31, 165-168). A monoclonal antibody recognizing this
sequence blocks the binding of the immunoglobulins. Also, anti-J
chain blocks binding (Vaerman J-P et al. (1998) Eur J Immunol 28,
171-182). The D5 domain forms a covalent disulfide bond with IgA to
form secretory sIgA. Exon 8 codes for the membrane spanning
sequence/domain. Exons 9, 10 and 11 code for the three cytoplasmic
domains that regulate various aspects of transcytosis (Breitfeld P
P et al. (1990) J Biol Chem 265, 13750-13757; Reich V et al. (1996)
J Cell Sci 109, 2133-2139; Kraj{hacek over (c)}i P et al. (1992)
Eur J Immunol 22, 2309-2315). These domains are highly conserved
(Banting G et al. (1989) FEBS Lett 254, 177-183). One serine
residue is particularly important for transcytosis (Hirt R P et al.
(1993) Cell 74, 245-255).
[0521] Clinical Studies of Secretory Component (Poly-Ig Receptor)
Expression in Breast and Colon Cancer. Others have performed a
study of the protein and mRNA expression of the poly-Ig receptor
with a sample of human colon cancers (Kraj{hacek over (c)}i P et
al. (1996) Br J Cancer 73, 1503-1510). Expression of secretory
component was found in 33 colorectal adenomas (31 patients) and in
19 colorectal carcinomas from 19 patients. Although that study
provides evidence that colon adenomas (i.e. a predisposition to
colon cancer) and confirmed cancers express poly-Ig receptor, the
investigators did not attempt to translate the observations further
than to propose a role in "cellular dysplasia".
[0522] Likewise, the levels of secretory component have been
measured by others in breast tumors from 95 patients with primary
or metastatic disease (Stern J E et al. (1985) Cancer Immunol
Immunother 19, 226-230). These authors proposed that low levels of
secretory component (SC) were found in metastatic lesions and that
this "could indicate a potential for SC involvement in immune
regulation of tumor growth," referring to conventional
antigen-antibody recognition immune effects. However, it was not
undertaken to identify growth effects related to either the
immunoglobulins IgA/IgM or to identify a role of the poly-Ig
receptor directly. Furthermore, this study was incomplete. There
was no attempt made to determine the estrogen receptor status of
the primary or metastatic disease. Therefore, there was no
correlation to growth state based on the most accepted criterion of
steroid hormone receptor status. This line of study appears to have
stopped with 1985 observation. The present invention has directly
addressed the problem by demonstrating growth regulation by the
secretory immune system using several different ER.sup.+
cancers.
[0523] Lines of Evidence Supporting Poly-Ig Receptor or a
Poly-Ig-like Receptor in Negative Growth Regulation. The present
series of studies and observations indicate that the IgA/IgM
mediating receptor has the properties of the poly-Ig receptor.
Supporting facts include the following: (1) The source of the
active IgA is not the deciding factor. Plasma or myeloma derived
IgA are equally effective. Also, species makes little or no
apparent difference in activity. IgA isolated from various species
has major sequence homology in the .alpha. heavy chains but
differences in the variable chains. This is consistent with
mediation by an Fc superfamily receptor. (2) IgA purchased
commercially from myeloma cell sources contains predominantly
dimeric and polymeric immunoglobulin. It is highly active as an
inhibitor. This is consistent with mediation by the poly-Ig
receptor. (3) Cultures containing the active CA-PS-pool II material
(see Examples 18 and 20) are predominantly dimeric/polymeric forms
of immunoglobulins. These preparations are active in
serum-supplemented and serum-free defined medium. This is
consistent with the expected binding to the poly-Ig receptor. (4)
IgM is at least as active, or more active than IgA on a molar
basis. The source of the IgM can be from plasma or myeloma cells.
They are equally effective. This is also expected of the poly-Ig
receptor. (5) Anti-secretory component antibodies completely
blocked the inhibitory effects of IgA and IgM. This not only
indicates poly-Ig receptor mediation, it supports the view that IgA
and IgM act via the same receptor. The poly-Ig receptor is known to
conduct transcytosis of both of these immunoglobulins.
[0524] Secretory IgA is invariably inactive as an inhibitor. It has
the five extracellular domains of poly-Ig receptor attached. By
contrast, plasma derived IgA is fully active. To prove that pIgA
does not have the secretory component whereas sIgA contains the 80
kDa receptor fragment, the Western analysis in FIG. 143 was done.
Secretory IgA shows an 80 kDa cross-reaction band with
anti-secretory component whereas pIgA shows no reaction. This was
the expected result and provides additional support for the view
that the poly-Ig receptor is the mediator. Because secretory
component is isolated from milk sIgA, these results show that the
secretory component used for immunization of the rabbits was free
of the other subunits in IgA. This was a good control for the next
experiments.
[0525] In the next experiments, anti-human secretory component
antiserum was used to block the inhibiting effects of IgA and IgM.
FIG. 144 shows the results with the T47D cells in serum-free
defined medium DDM-2MF with human plasma IgM alone and with a
series of dilutions of the antiserum. As shown, 10 nM B.sub.2
completely reversed the IgM inhibition. Dilutions of 1:500 to
1:5000 also blocked the inhibition. In the insert in FIG. 144, a
control study with pre-immune rabbit serum demonstrated it had no
inhibitor blocking activity. A similar study was done with LNCaP
cells in serum-free defined CAPM with human pIgA (FIG. 145). As
shown, 10 nM E.sub.2 completely reversed the pIgA inhibition.
Anti-serum dilutions of 1:00 and 1:1000 also reversed the
inhibition. Differences between the effective dilutions with T47D
and LNCaP cells is due to changes in lots of commercially prepared
antiserum.
[0526] To determine if IgA/IgM responsive cells expressed 100 kDa
poly-Ig receptor, the Western analysis shown in FIG. 146 was done.
Amounts of extracts of the designated cell types were analyzed with
a 1:1000 dilution of rabbit anti-human secretory component. As
expected MDCK cells were positive. This cell line has been studied
for several years as a model of poly-Ig receptor sorting and
function. LNCaP cells showed the same receptor (FIG. 146). Cell
lines that were negative were ALVA-41, DU145, human fibroblasts,
and PC3 cells (FIG. 146). As shown in multiple experiments in
preceding Examples, LNCaP cells are IgA/IgM inhibited. The results
of the Western analyses show that they express the poly-Ig
receptor.
[0527] In the final experiments of this series, pIgA was tested
with two of the cell lines that were poly-Ig receptor negative by
the Western analysis shown in FIG. 146. The results with DU145
cells are shown in FIG. 147. Plasma IgA was not an inhibitor. A
similar study with PC3 cells is shown in FIG. 148. Again, pIgA was
not an inhibitor even at 50 .mu.g/mL. These results demonstrate
cells that lack the poly-Ig receptor are also insensitive to pIgA.
The HT-29 colon cancer cells are known to express only the
authentic form of the poly-Ig receptor. They are also negatively
growth regulated by IgM.
[0528] Discussion of Example 24. For the first time a relationship
between immunoglobulin growth regulation and the poly-Ig receptor
is demonstrated. This receptor has in the past been studied only
from the perspective of a transcytosis receptor. In view of all
these results, the poly-Ig receptor very likely has more function
than transcytosis only. Ongoing investigations are directed to
identifying gene changes in the authentic poly-Ig receptor gene,
which may include point mutations, deletions, insertions, and
premature termination. The receptor mediating the effects of
IgA/IgM may be an alternate splicing form of the original
transcytosis receptor. Alternatively, changes in the regulation of
expression may influence the presence or absence of this receptor.
The positive correlation between the presence of ER and AR, and
expression of the growth regulating poly-Ig receptor indicates
regulation or positive influence by steroid hormones.
[0529] One of the primary themes of cancer research has been that
loss of "tumor suppressor genes" causes the release of cells from
negative regulation and thereby contributes to the progression to
cancer. The present invention indicates that the poly-Ig receptor
has a "tumor suppressor" function. It is present in cells that are
regulated by IgA/IgM and absent in cells that are insensitive to
immune inhibitors. This is a new aspect of cancer immunology not
recognized before the present invention. For the first time, the
poly-Ig receptor is connected to the D1S58 linked locus that is a
"hot spot" for genetic changes in breast cancer. It is now proposed
that this locus or near neighbors contain the growth regulating
form of authentic transcytosis poly-Ig receptor or a very similar
immunoglobulin superfamily receptor. Alternately the 1q31-q41
region of chromosome 1 contains several other genes of
immunological interest that include the receptor for IgA/IgM. Those
genes can be employed as screens for breast and other mucosal cell
cancers. They are expected to indicate susceptibility and may be
useful in prognosis and other diagnostic applications with human
tissue and cancer samples. Analyses of allelic imbalances in the
receptor gene are also foreseen as a new tool to determine
susceptibility and prognosis for development of breast and other
mucosal cancers, as will be the detection of mutations in the
growth regulating intracellular domains of the receptor. The known
amino acid sequence of the poly-Ig receptor does not contain the
immunoreceptor tyrosine-based inhibitory motif (ITIM) common to a
new family of inhibitory motif receptors (Cambier J C (1997) Proc
Natl Acad Sci USA 94, 5993-5995). Other amino acid sequences may
serve this same function.
Example 25
Mediation of IgG1.kappa. Effects by a Fc-Like Receptor
[0530] In this Example, it is shown that the inhibiting effects of
IgG1.kappa. were mediated by an Fc receptor or Fc.gamma.-type
receptor. It is highly unlikely that IgG1 acts via the poly-Ig
receptor. The poly-Ig receptor has a requirement for "J" chain for
binding (hence its specificity for dimeric/polymeric IgA or
pentameric IgM each of which have one J chain). Also, as shown in
TABLE 12, Fc.gamma. receptors are localized in leukocyte series or
bone marrow origin cells. There is no convincing evidence of their
presence in epithelial cells or in secretory cells of the mucosa.
It is now proposed that the receptor being sought is one analogous
to the Fc.gamma. in at least two significant properties. First, it
binds monomeric IgG1 via the Fc domain of the immunoglobulin with
some participation of the .kappa. light chain. Second, that the
receptor has inhibitory activity akin to a new family of Fc
receptors. The amino acid sequence of the new IgG1.kappa. receptor
is expected to have an immunoreceptor tyrosine-based inhibitory
motif (ITIM) (VxYxxL) common to the new family of inhibitory motif
receptors (Cambier J C (1997) Proc Natl Acad Sci USA 94,
5993-5995). Other amino acid sequences may serve this same
function.
[0531] It is proposed that the Fc.gamma. family of receptors
contains members that possess the very special property of
mediating cell growth inhibition. The methods of identification are
outlined below.
TABLE-US-00013 TABLE 12 Properties of the Fc.gamma. Family of
Receptors Fc.gamma. R1 Fc.gamma. RII Fc.gamma. RIII (CD 64) (CD 32)
(CD 16) IgG1 Binding K.sub.a = 10.sup.8 M.sup.-1 K.sub.a = 2
.times. 10.sup.6 M.sup.-1 K.sub.a = 5 .times. 10.sup.5 M.sup.-1
Binding Order IgG 1 > IgG1 > IgG1 = IgG3 = IgG3 = IgG3 IgG4
> IgG4 > IgG2 IgG2 Found in these Macrophages Macrophages
Natural Killer Cells Cell Types Neutrophils Neutrophils Macrophages
Eosinophils Eosinophils Neutrophils Platelets Eosinophils B
Cells
[0532] Discussion of Example 25. The amino acid sequence of a new
Fc family receptor may include immunoreceptor tyrosine-based
inhibitory motif (ITIM) common to a new family of inhibitory motif
receptors (Cambier J C (1997) Proc Natl Acad Sci USA 94,
5993-5995). The results obtained in the present studies support the
involvement of Fc receptors of mucosal cells that include one of
the known members of the family of ITIMs or other amino acid
sequences that serve this same function. Ongoing work includes
genetic mapping to a specific chromosome number and locus. The
genomic DNA sequence of the new receptor (or existing receptor if
already known), introns and exons, will be obtained. This receptor
may be used for diagnostic and clinical proposes, and as a screen
for genetic susceptibility to breast and prostate and other mucosal
cancers, as described in more detail in U.S. patent application
Ser. No. ______ (Atty. Dkt. No. 1944-00800)/PCT/US2001/______
(Atty. Dkt. No. 1944-00801) entitled "Compositions and Methods for
the Diagnosis, Treatment and Prevention of Steroid Hormone
Responsive Cancers," which is hereby incorporated herein by
reference. Identification of mutations and changes associated with
progression from normal cells to autonomous cancer cells is also
expected, and, along with detection of changes in regulation of
expression and allelic imbalances in the receptor gene, will have
very useful research, diagnostic and clinical applications.
Example 26
Immunoglobulin Inhibitors as Tools for Identifying the Receptors
that Mediate the IgA/IgM/IgG Cell Growth Regulating Effects
[0533] As shown by Examples 24 and 25, the present Immunoglobulin
Inhibitors can be used as reagents for identifying their mediating
receptors.
[0534] The Mediating Receptors--Inhibitory Function. As discussed
above, serum contains a great variety of mitogenic agents. On this
point the present results in 50% (v/v) serum were especially
relevant. This concentration of serum is a rich source of mitogens
including insulin and the insulin-like growth factors. Nutrients
and other serum components also have growth-promoting effects.
Examples include diferric transferrin, unsaturated fatty acids
bound to albumin, complex lipids and ethanolamine. Clearly, the
inhibitor(s) also blocks their growth effects, which lends support
to the conclusion that the mediating receptor for the serum-borne
agent must have special properties. For instance, the
immunoreceptor tyrosine-based inhibitory motif (ITIM) class of Fc
receptors is of particular interest with respect to identifying the
mediator(s) of immunoglobulin inhibition of cancer cell growth,
because the hallmark of the ITIM receptors is that they have an
intracellular amino acid sequence motif I/VxYxxL that signals cell
growth shutdown after ligand binding, and therefore shuts off
growth factor dependent growth. In the preceding Examples it is
demonstrated that steroid hormones are selectively capable of
reversing the effects of the serum inhibitor(s). Plainly, as
predicted by the estrocolyone hypothesis, serum contains an
inhibitor(s) that has a dominant role in the regulation of
proliferation of steroid hormone target cells. The isolated IgA and
IgM blocked growth factor dependent growth in serum-free defined
medium. Because of its "master switch" character, the newly
identified immunoglobulin inhibitors have many important and useful
applications. Moreover, the results of the present investigations
support the view that the inhibitor(s) will have biological
implications extending well beyond estrogen and androgen target
tissues.
[0535] The Receptor Mediating IgA/IgM/IgG Inhibitory Effects. The
results shown in the foregoing Examples strongly indicate that the
IgA/IgM growth inhibition is mediated either by the poly-Ig
receptor or a very closely related receptor. Establishing a growth
regulating function for this "transcytosis" receptor will open new
directions in medical diagnosis, treatment and prevention of
cancers of mucosal epithelial tissues. It will be determined
whether the poly-Ig receptor, or a poly-Ig like receptor mediates
the growth regulating effects of IgA on human breast and prostate
cancer cells in culture. For this study, the poly-Ig receptor in
these cancer cells will be identified using well-known PCR cloning
technology, .sup.125I-labeled IgA chemical cross-linking and
Western and immunohistochemistry methods described in the
literature.
[0536] Next, blocking polyclonal antibodies or blocking monoclonal
antibodies will be employed to show that the poly-Ig receptor
mediates the growth response. The antibodies will be raised against
the poly-Ig receptor using known techniques. Reversal of the
inhibitory effect of IgA and IgM by blocking the poly-Ig receptor
will suggest that the poly-Ig receptor is not just a simple
transport receptor, but that it has a central role in breast and
prostate cancer cell growth regulation. There is no existing
paradigm for breast or prostate cell growth regulation that
involves the poly-Ig receptor or for that matter any receptor
specific for the IgA class of immunoglobulins including Fc.alpha.
receptors (Fridman W H (1991) FASEB J 5, 2684-2690).
[0537] The different forms and domains of IgG, IgA and IgM that act
as inhibitors of normal prostate and breast and other mucosal
epithelial cell growth and the hormone responsive and hormone
autonomous forms of these cancers in serum-free defined culture
medium will be determined and used as tools to evidence or confirm
the identity of the receptor(s) responsible for mediating the
growth regulatory effect. The properties of the ligand that elicits
a response will be evidence supporting the identity of the
receptor. Poly-Ig receptor is activated by Fc-domains as are
Fc.gamma. receptors. Normal cells are likely to be most inhibited
by IgG, IgA and IgM, whereas the ER.sup.+ and AR.sup.+ cells will
likely be inhibited primarily by IgA/IgM, and ER.sup.- and AR.sup.-
cells will likely not be inhibited by any of the three classes of
immunoglobulins, as predicted by the conceptual model described
below. The methods employed will include direct tests of the
activity of IgG, IgA and IgM on cell growth as well as assessment
of the activity of specific size forms and Fc versus Fab fragments.
Antibodies such as anti-J chain and anti-Fc will be used to extend
these studies to demonstrate that the Fc is the active domain and
that Fc binding receptors are involved.
[0538] More specifically, AR.sup.+ LNCaP cells, the AR.sup.- PC3
and DU145 cells, and the AR.sup.+ ALVA-41 cells will be studied.
Normal human prostate and breast epithelial cells will be obtained
from Clonetics. Growth assays will be done in completely serum-free
CAPM (prostate) and DDM-2MF (breast), as described above. IgA1 and
IgA2 will be purified from human serum and colostrum, using
techniques that are well known and have been described in the
literature. Initial small samples will be obtained from a
commercial supplier such as The Binding Site (San Diego, Calif.).
The monomeric, dimeric and polymeric forms of IgA will be separated
using techniques that are well known and have been described in the
literature. If only IgA2 has activity, it will be further separated
into the A2(m)1 and A2(m)2 allotypes, using well-known techniques
that have been described in the literature. Because the initial
IgA/IgM inhibitor preparations evaluated were mostly dimeric and
monomeric, those forms are expected to be the most active in these
series of tests. Confirmation that the most active forms are
dimeric/polymeric IgA/IgM will be strong evidence for poly-Ig
receptor mediation. Should the monomers be revealed as the only
active inhibitor forms, however, it would favor Fc or Fc
superfamily receptors, in which case the Fc.alpha. will be
investigated as a possible mediator.
[0539] IgA will be fragmented with a specific protease to yield Fc
and Fab fragments from IgA, using techniques that are well known
and have been described in the literature. The Fab and Fc fragments
of IgM will be obtained using a Pierce Chemicals kit based on
immobilized trypsin. Fab and Fc fragments of IgG1 will be obtained
using another Pierce kit. If only Fc fragments of IgA and IgM are
active, mediation by the poly-Ig receptor is likely. If the Fc of
IgG1 is active, it will indicate an Fc receptor as the
mediator.
[0540] The immunoglobulin inhibitors will also be used as tools or
biological reagents to confirm whether IgG acts via a receptor
different than IgA/IgM. Based on the results reported above,
identification of Fc.gamma. like receptors and the poly-Ig receptor
(or related receptor) with normal cells, ER.sup.+ cells and
AR.sup.+ cells is expected, and no functional receptors are
expected in ER.sup.- cells or AR.sup.- cells. .sup.125I-labeled
IgG1, IgA and IgM will be prepared using chloramine T or Iodogen
beads or coated tube (Pierce Chemicals kits). Binding parameters,
binding constants, analyses of the effects of reciprocal additions
of labeled and unlabeled immunoglobulins to identify separate or
similar binding sites, and determination of the effects of addition
of purified secretory component on IgA and IgM binding will be
performed as previously described or using well known published
techniques. Specific binding will be as total binding minus binding
in a 100-fold excess of unlabeled protein. For each form with
activity, time, concentration and temperature dependence of binding
will be assessed. Scatchard analysis will be used to estimate the
number of sites per cell and the association constants (K.sub.a).
Reciprocal competitions with unlabeled and labeled immunoglobulins
will be used to define interaction with the same or different
receptors. This latter point is important because binding of both
IgA and IgM to the same site strongly favors the poly-Ig receptor
and plainly contra-indicates Fc.alpha. (IgA) or Fc.mu. (IgM)
receptors, which are members of a superfamily in which each member
is specific for a (monomer) class of immunoglobulins. In addition,
the effects of blocking antibodies such as anti-secretory
component, anti J chain and anti Fc will be assessed with all three
cell types. Where indicated, chemical cross-linking with
.sup.125I-labeled Ig will be performed to define the mass of the
receptors. Optionally, metabolic labeling and/or
immunoprecipitation techniques will be used instead, employing
well-known techniques.
[0541] Western immunoblotting with normal, steroid hormone receptor
positive and steroid hormone receptor negative cell types will be
performed to identify the receptors present. Immunohistochemistry
will be applied to identify the poly-Ig receptor and Fc.gamma.
receptors on all three types of cells using the blocking
antibodies. Using a full-length human poly-Ig receptor cDNA clone,
S1 nuclease protection assays will be conducted with RNA from
normal prostate and breast cells, ER.sup.+ and ER.sup.- breast
cancer cells, and AR.sup.+ and AR.sup.- prostate cancer cells to
identify mRNA. In the cases of ER.sup.+ and AR.sup.+ or ER.sup.- or
AR.sup.- cells, this method will help to identify truncated or
otherwise altered receptors or non-functional receptors. As
described in certain of the preceding examples, Western blots have
already been conducted, as well as cell growth assays with receptor
blocking antibodies. The remaining analyses will be done with
normal cells as well as all other ER.sup.- or AR.sup.- lines. All
blocking antibodies are dialyzed against buffer containing charcoal
to remove interfering steroid hormones. Rabbit polyclonal anti
secretory component will be raised (e.g., by HTI BioProducts,
Ramona, Calif.) and rabbit polyclonal anti-human J chain and
specific antibodies against the Fc receptors for IgG and IgA are
commercially available (Accurate). The specificity of all antiserum
will be checked by Western analysis.
[0542] To identify the receptors mediating the androgen reversible
inhibition of normal and/or AR.sup.+ cells, PCR cloning methods
will additionally be used to determine the cDNA sequences of the
poly-Ig receptor and Fc.gamma. receptors from normal, AR.sup.+ and,
if indicated, from AR.sup.+ cells. This method will provide clear
answers to the question of the relationship of the human poly-Ig
receptor and Fc.gamma. receptors to immune system negative
regulation. It is expected that the receptors will be found to be
either identical to known sequences or altered in sequence to
convert them to "inhibitory motif" receptors. Based on the known
cDNA sequence of the poly-Ig receptor from HT-29 cells, PCR cloning
technology will be applied to obtain a full-length clone from the
LNCaP and T47D cells. Ongoing investigations are directed to
comparing receptor sequences from normal prostate and breast cells
to identify any changes. Based on the known sequence of the
Fc.gamma.RIIB1 receptor, these same studies will be repeated. The
receptors identified by cloning will be examined for the
immunoreceptor tyrosine-based inhibitory motif (ITIM) amino acid
sequence I/VxYxxL or related sequences. Concomitantly, the cells
will be examined by Western analysis for SHP-1 and SHP-2
phosphatase mediators of the inhibition of growth factor activity.
These markers are not only associated with the inhibitory motif but
also other inhibitory receptors. More specifically, an LNCaP and
T47D full-length poly-Ig receptor clone will be prepared and
compared to the reported sequence of the poly-Ig receptor. The same
technology will be applied to the poly-Ig receptor from normal
prostate cells, and, if indicated, from the AR.sup.+ lines. Because
these cell lines are expected to express the known poly-Ig
receptor, or a related form, the PCR approach is applicable. The
same approach will be used with the Fc.gamma. like receptor.
However, in this case, because these receptors are predominantly
lymphoid origin, the form in epithelial cells may be substantially
different. Standard cloning methods will be employed to obtain the
complete cDNA sequence of the Fc.gamma. like receptor from normal
and LNCaP cells. Total RNA will be extracted and mRNA purified by
oligo dT cellulose chromatography (also for Northern analysis).
cDNA synthesis will be done with oligo dT primers and AMV reverse
transcriptase followed by Rnase H to remove RNA. Second strand
synthesis will be done with hexameric random primers and DNA pol.
I. Treatment with T4 DNA pol, Rnase H and Rnase A creates blunt
ends. EcoRI methylation is followed by EcoRI linkers and ligation
into a cloning vector. (Stragene) vectors based on .lamda.gt10
(hybridization screening) and .lamda.gt11 (secretory component
antibody screening). Both vectors will accept inserts larger than
the receptor. The cDNA sequence of human poly-Ig receptor known is
the genomic sequence. These will be used to prepare sequence
specific primers for PCR. The primers will encompass the 5' and 3'
non-coding sequences to ensure a complete cDNA. The PCR products
will be subcloned using the TA kit from Invitrogen. The sequencing
of PCR clones will be done by the dideoxy chain termination method
(Lone Star Labs, Houston, Tex.). From these, determination of
whether there have been significant alterations in the receptor
during the transition from normal to ER.sup.- and AR.sup.- cancer
cells is expected. From sequence data, the ITIM amino acid
sequences indicating an inhibitory motif receptor will be sought.
It is important to note, however, that the absence of these
sequences does not necessarily rule out an inhibitory function. The
Western analyses for SHP-1 and SHP-2 will be valuable for
indicating an inhibitory function even in the absence of ITIM or
when the ITIM is in a modified form.
[0543] Discussion of Example 26. Without wishing to be bound by a
particular theory, it is proposed that the inhibitory effect of
IgG1 is more marked with normal cells than with ER.sup.+ or
AR.sup.+ cancer cell lines and an early step in the pathway to
malignancy involves loss by the cell of IgG1 regulation. From
preliminary investigations, it is suggested that the IgA and IgM
receptors are likely to be a common poly-Ig receptor or poly-Ig
like receptor, which in normal cells is expected to be the same as
in steroid hormone receptor positive cell lines. In contrast, the
IgG1 receptor, likely an Fc gamma type receptor, is expected to
either be altered or its expression greatly reduced in ER.sup.+ and
AR.sup.+ cell lines. The demonstration that IgG1 has a major growth
inhibiting effect on normal cells may lead to immunization against
breast cancer by administering or enhancing IgG1 in at-risk
tissues. Characterization of an inhibitory role for IgG1 via an
Fc.gamma.-like receptor is expected to lead to important
innovations in medical diagnosis, treatment and prevention of
cancers of mucus epithelial tissues.
Example 27
[0544] Conceptual Model for Cascading Loss of Cell Growth
Inhibition in Cancer Cells Concept. The isolated inhibitors, now
identified as IgA, IgM and IgG1, controlled breast and prostate
cell growth by acting as a steroid hormone reversible inhibitor
even when tested under the very rigorous conditions of serum-free
defined culture. These active natural inhibitors are present in
blood, bodily secretions and mucosal epithelial tissues. The
isolated inhibitors readily prevented the growth of these types of
cancer cells when they were still in the early (i.e., hormone
responsive) stage, but not in the late, non-hormone responsive
stage. These results have many implications with regard to the
diagnosis, genetic screening, treatment and prevention of breast,
prostate, colon and other mucosal cancers. Without wishing to be
bound by a particular theory, considering the present experimental
results and discoveries, a new conceptual model for understanding
how estrogens cause ER.sup.+ breast cancer cell growth and for
understanding how the natural progression of breast cancers occurs
to give rise to highly malignant (and dangerous) hormone autonomous
forms is proposed. This same model is applicable to other mucosal
tissues that respond to the steroid hormone family of hormones,
including androgens and thyroid hormones.
[0545] Progression Concept based on the Breast Cancer
Model--Generally Applicable to Mucosal Tissue Cancers. It is well
established that breast cancers pass through a characteristic
natural history that involves a gradual evolution from near normal
growth patterns into cancers that are completely steroid hormone
autonomous (i.e. they are no longer stimulated by steroid
hormones). These are usually designated estrogen receptor negative
(ER.sup.-). As disclosed herein, it has been found that autonomous
(ER.sup.-) breast cancer is accompanied by a loss in sensitivity to
IgA or IgM. Fully autonomous breast cancers are not inhibited by
these secretory immunoglobulins. In light of the outcome of the
present investigations, it appears that autonomous breast cancers
lack the poly-Ig receptor that mediates the growth inhibiting
effects of IgA and IgM. These results are of extraordinary
significance because for the first time they pinpoint a specific
genetic change (i.e. in the poly-Ig receptor) that might account
for the majority (i.e. approximately 75%) of breast cancers termed
"sporadic" and for which there is as yet no clear genetic change
identified. Indeed, these results also provide an excellent
opportunity to implement gene therapy based on reintroduction of
the poly-Ig or poly-Ig like receptor into completely autonomous
cancers to regain immunological regulation.
[0546] It is well established in the literature that IgG1 is
present in serum during childhood, when breast tissue growth is
precisely regulated to body size (isometric growth). The other
inhibitors, IgA and IgM, are very low at this time, but increase in
serum at puberty. Because adult women have increased positive
stimuli for breast cell proliferation due to estrogen production,
the presence of IgA and IgM may provide additional protection.
[0547] It is now proposed that alterations in immune regulation
lead to the progression of breast and prostate cells from normal
control to ER.sup.+ and AR.sup.+ cancer cells and that additional
alternations in immune control contribute to the development of
fully autonomous cancers, according to the following model
presented in TABLE 13:
TABLE-US-00014 TABLE 13 Model for Progression of Steroid Hormone
Dependent Cancers from Normal Growth Regulation by the Immune
System to Steroid Responsive Cancers and on to Fully Hormone
Autonomous Cancers ##STR00001##
[0548] Inhibitory Motif Receptors. The receptors mediating the
immune response regulation must be at or very near the beginning of
the onset of breast cancer. Using the tools developed in the
present series of investigations, it is expected that inhibitory
motif receptors for these immunoglobulins will be identified. It is
now proposed that the mediating receptors are members of the Ig
superfamily, which includes Fc receptors and a new class of Ig
inhibitory motif receptors. This new class of receptors has
emerging importance because of the increasing recognition of the
role of negative regulation of cell growth. These receptors have
both common and unique properties. They bind immunoglobulins via
the Fc domains and hence can be classified as Fc receptors. One of
these is, in fact, Fc.gamma.RIIB that binds IgG1 (TABLE 12) and
causes inhibition of antigen activation of B cells. There are many
other examples (Cambier J C (1997) Proc Natl Acad Sci USA 94,
5993-5995). Among these are more than 15 receptors now designated
Signal-Regulatory Proteins (SIRPs). These all express a special
inhibitor motif of six amino acids (I/VxYxxL) that is now referred
to as the "immunoreceptor tyrosine-based inhibitory motif" or ITIM.
One of the most marked characteristics of the ITIM containing SIRPs
is that this motif recruits two phosphatases (SHP-1 and SHP-2) to
result in the inhibition of all growth factor dependent
proliferation. This is similar to what was observed with IgG1, IgA
and IgM and ER.sup.+ breast cancer cells and AR.sup.+ prostate
cancer cells serum-free defined medium. This work is expected to
aid in the identification of the missing genes for sporadic breast
cancers and a more complete understanding of the cascade of gene
changes that lead to complete loss of immune control of breast cell
growth.
[0549] Similarly, it is suggested that alterations in immune
regulation also lead to the progression of prostate cells from
normal control to AR.sup.+ cancer cells and that additional
alterations in immune control contribute to the development of
AR.sup.- fully autonomous cancers. Further studies are directed at
identifying a cascade of gene changes leading to complete loss of
immune control of cell proliferation.
[0550] Similarly, it is also proposed that alterations in immune
regulation also lead to the progression of colon cancer cells from
thyroid hormone receptor (THR) normal control to THR cancer cells
and that additional alterations in immune control contribute to the
development of THR.sup.+ fully autonomous cancers. Further studies
are directed at identifying a cascade of gene changes leading to
complete loss of immune control of cell proliferation
[0551] In continuing investigations, tests to determine whether
steroid hormone independent breast and prostate cancer cell growth
results from either the loss of the poly-Ig receptor or an
inactivation of its function are being carried out. A series of
steroid hormone dependent and steroid hormone independent breast
and prostate cancer cell lines will be compared for their
inhibitory growth responses to IgA, the presence of poly-Ig
receptor m-RNA, the expression of the receptor by .sup.125I-IgA
binding analysis and immunohistochemistry localization of receptor.
Detection of an absence of the receptor or an inability to bind IgA
will suggest that cancer cell autonomy arises due to a loss of
secretory immune system regulation. Such a result would be entirely
new in the field of hormone dependent cancers and would provide a
new immune mechanism responsible for conversion from hormone
dependence to autonomy. New immunotherapies can be developed based
on activating the receptor in hormone responsive cancers and new
gene therapies based on reestablishing the function of this
receptor in autonomous breast cancers.
[0552] Ongoing investigation is directed at resolving whether
hormone autonomous breast cancer cell lines have functional poly-Ig
receptors. The ER.sup.- cell lines to be studied are the
MDA-MB-231, BT-20, MDA-MB-330 the non-tumorus HBL-100, and the
Hs578t and Hs578Bst. Each will be evaluated for growth in
serum-free medium.+-.IgA and .+-.E.sub.2. This study will determine
if autonomous cells have lost immune system negative regulation. To
determine if the receptor is lost, the S1 nuclease protection
assays will be used to seek its mRNA. A kit from AMBION will be
used. In addition, .sup.125I-I labeled IgA will be used to
determine specific binding characteristics as described above.
Immunohistochemistry will be employed to confirm and/or extend the
binding data. If the receptor mRNA and protein are absent, these
methods should confirm that fact. If they are present but
nonfunctional, these methods should confirm that fact as well.
[0553] Discussion of Example 27. The proposed model for progression
of mucosal cancers from normal cells to fully autonomous cancers is
based on the experimental results presented and is unique. No
previous recognition has been published of the roles of IgA, IgM
and IgG1 in breast, prostate, or other mucosal cancers. Application
of this model has diagnostic implications. Breast, prostate and
other cancers can be examined for content of the receptors for IgA,
IgM and IgG1 to determine stage of the cancer. This information can
be compared to the determination of estrogen receptor and
progesterone receptor status to aid in decisions regarding
immunotherapy with immune modulators or the immunoglobulins or the
use of combined anti-hormone and immune therapy modalities. Tumors
that are negative for all of the immunoglobulin receptors are prime
candidates for gene therapy to replace the receptors and thereby
reestablish immune surveillance, as further described in U.S.
patent application Ser. No. ______ (Atty. Dkt. No.
1944-00800)/PCT/US2001/______ (Atty. Dkt. No. 1944-00801) entitled
"Compositions and Methods for the Diagnosis, Treatment and
Prevention of Steroid Hormone Responsive Cancers," which is
incorporated herein by reference.
Example 28
IgA/IgM Based Test to Detect Lowered Levels of Steroid Hormone
Reversible Cell Growth Inhibitors in Plasma or Body Secretions
[0554] Toward identifying individuals with high susceptibility to
breast cancer or prostate cancer, the level of the inhibitory form
of IgA (i.e., IgA dimer) will be measured in an individual's
plasma, or the secretory IgA and polymeric IgM will be measured in
a bodily secretion. Decreases in plasma levels of IgA or decreased
secretory capacity into milk or structural alterations in IgA may
confer greater susceptibility to breast cancer. Levels are expected
to be low in susceptible individuals and to fall with increasing
age in normal individuals, substantially mirroring the age
distribution pattern associated with breast and prostate cancer
incidence. An antibody raised against the D5 domain disulfide
regions, with IgA attached, is an example of an assay for the
dimeric/polymeric IgA. In secretory fluids, direct measure of sIgA
can be done along with a measure of secretory component by
radioimmunoassay or other methods using ELISA or biotin-avidin
technology. The levels of IgM can be measured directly although
their levels are more subject to wide variations. Also, "J" chain
can be measured, but only in samples treated to remove the free
(unbound) form known to be in plasma.
[0555] Another useful test process is use of rectal or nasal
passage antigen challenge and measurement of the appearance of the
specific antibody against the antigen in plasma and secretory
fluids by standard high capacity clinical test methods. This will
directly measure the immune status of the individual. Those with
optimum capacity can be separated from individuals with impaired
secretory immune system function. Impaired function of the
secretory immune system may indicate susceptibility to cancer.
[0556] The testing is carried out by first treating a plasma
specimen to deplete or substantially remove the steroid hormone
content without inactivating or removing the endogenous poly IgA
dimer and poly IgM molecules. The hormone depleted specimen is then
tested for cell growth inhibitory activity in the presence of added
steroid hormone in an in vitro assay employing cultured tumor cells
incubated in a defined serum-free medium. Procedures for preparing
the steroid hormone depleted plasma or serum and for conducting the
assay are described in the preceding examples, XAD.TM.-4 is
particularly suited for treating small biological specimens. These
extraction methods yield steroid hormone depleted serum that allows
identification of 30 to 100-fold estrogen and androgen growth
effects (cell number measurement) in culture in 7 to 14 days with
human breast and human prostate cancer cells as well at rat
mammary, rat pituitary and Syrian hamster kidney tumor cells.
[0557] The results are compared to similar tests using positive and
negative control plasmas or serums, which have defined levels of
IgA dimer and poly IgM. In this way the tumor cell growth
inhibitory activity of the individual's plasma is measured. Because
the in vitro assay system employs a cell line that forms breast or
prostate tumors when implanted in vivo, the in vitro assay results
are believed to be suggestive of the in vivo condition of the
individual.
[0558] Alternatively, or additionally, plasma and bodily fluids may
be monitored for autoimmune antibodies that block the inhibitory
action of IgA and IgM. An expected increase in autoimmune
antibodies with increasing age is expected to coincide with
increased cancer incidence, or the incidence of cancer may be high
in individuals with early onset disease.
[0559] Each and every claim is incorporated into the specification
as an embodiment of the present invention. Thus the claims are a
further description and are an addition to the preferred
embodiments of the present invention. While the preferred
embodiments of the invention have been shown and described,
modifications thereof can be made by one skilled in the art without
departing from the spirit and teachings of the invention. The
embodiments described herein are exemplary only, and are not
intended to be limiting. Many variations and modifications of the
invention disclosed herein are possible and are within the scope of
the invention. Accordingly, the scope of protection is not limited
by the description set out above, but is only limited by the claims
which follow, that scope including all equivalents of the subject
matter of the claims. The disclosures U.S. Provisional Patent
Application Nos. 60/203,314; 60/208,348; 60/208,111; 60/229,071 and
60/231,273, and all patents, patent applications and publications
cited herein are hereby incorporated herein by reference.
* * * * *