U.S. patent application number 10/277603 was filed with the patent office on 2003-11-27 for progression elevated gene-3 and uses thereof.
Invention is credited to Fisher, Paul B..
Application Number | 20030219376 10/277603 |
Document ID | / |
Family ID | 21979687 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219376 |
Kind Code |
A1 |
Fisher, Paul B. |
November 27, 2003 |
Progression elevated Gene-3 and uses thereof
Abstract
This invention provides a vector suitable for introduction into
a cell, comprising: a) an inducible PEG-3 regulatory region; and b)
a gene encoding a product that causes or may be induced to cause
the death or inhibition of cancer cell growth. In addition, this
invention further provides the above-described vectors, wherein the
inducible PEG-3 regulatory region is a promoter. This invention
further provides the above-described vectors, wherein the gene
encodes an inducer of apoptosis. In addition, this invention
provides the above-described vectors, wherein the gene is a tumor
suppressor gene. In addition, this invention provides the
above-described vectors, wherein the gene encodes a viral
replication protein. This invention also provides the
above-described vectors, wherein the gene encodes a product toxic
to cells or an intermediate to a product toxic to cells. In
addition, this invention provides the above-described vectors,
wherein the gene encodes a product causing enhanced immune
recognition of the cell. This invention further provides the
above-described vectors, wherein the gene encodes a product causing
the cell to express a specific antigen.
Inventors: |
Fisher, Paul B.; (Scarsdale,
NY) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Family ID: |
21979687 |
Appl. No.: |
10/277603 |
Filed: |
October 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10277603 |
Oct 22, 2002 |
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09052753 |
Mar 31, 1998 |
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6472520 |
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09052753 |
Mar 31, 1998 |
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PCT/US98/05793 |
Mar 20, 1998 |
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PCT/US98/05793 |
Mar 20, 1998 |
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08821818 |
Mar 21, 1997 |
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6146877 |
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Current U.S.
Class: |
424/1.49 ;
424/93.2; 435/456; 536/23.2 |
Current CPC
Class: |
A61K 39/00 20130101;
G01N 33/57496 20130101; A61K 38/00 20130101; A61K 2035/126
20130101; C12N 2830/85 20130101; C12N 15/85 20130101; C12N 2830/008
20130101; A61K 2035/128 20130101; C12N 2799/022 20130101; C07K
14/47 20130101; C12N 2830/002 20130101; A61K 48/0058 20130101; A01K
2217/05 20130101; A61K 48/00 20130101; G01N 33/68 20130101; C40B
30/04 20130101 |
Class at
Publication: |
424/1.49 ;
424/93.2; 435/456; 536/23.2 |
International
Class: |
A61K 051/00; A61K
048/00; C07H 021/04; C12N 015/861 |
Goverment Interests
[0001] The invention disclosed disclosed herein was made with
United States Government support under National Institute of Health
Grant CA 35675. Accordingly, the United States Government has
certain rights in this invention.
Claims
25. A method of treating cancer in a subject, comprising: a.
administering the vector of claim 14 to the subject; and b.
administering gancyclovir or acyclovir to the subject.
26. A method of treating cancer in a subject, comprising: a.
administering the vector of claim 24 to the subject; and b.
administering an antibody or a fragment of an antibody to the
antigen of claim 24 to the subject.
27. The method of claim 26, wherein the antibody is toxic or linked
to a toxic substance.
28. The method of claim 26, wherein the antibody is labeled and
used for tumor imaging.
29. The method of claim 27, wherein the antibody is
radioactive.
30. A pharmaceutical composition comprising the vector of claim 2
and a carrier.
Description
[0002] Throughout this application, various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citations for these
references may be found at the end of this application, preceding
the claims.
BACKGROUND OF THE INVENTION
[0003] The carcinogenic process involves a series of sequential
changes in the phenotype of a cell resulting in the acquisition of
new properties or a further elaboration of
transformation-associated traits by the evolving tumor cell (1-4).
Although extensively studied, the precise genetic mechanisms
underlying tumor cell progression during the development of most
human cancers remain enigmas. Possible factors contributing to
transformation progression, include: activation of cellular genes
that promote the cancer cell phenotype, i.e., oncogenes; activation
or modification of genes that regulate genomic stability, i.e., DNA
repair genes; loss or inactivation of cellular genes that function
as inhibitors of the cancer cell phenotype, i.e. tumor suppressor
genes; and/or combinations of these genetic changes in the same
tumor cell (1-6). A useful model system for defining the genetic
and biochemical changes mediating tumor progression is the type 5
adenovirus (Ads)/early passage rat embryo (RE) cell culture system
(1,7-14). Transformation of secondary RE cells by Ads is often a
sequential process resulting in the acquisition of and further
elaboration of specific phenotypes by the transformed cell (7-10).
Progression in the Ad5-transformation model is characterized by the
development of enhanced anchorage-independence and tumorigenic
potential (as indicated by a reduced latency time for tumor
formation in nude mice) by progressed cells (1,10). The progression
phenotype in AdS-transformed RE cells can be induced by selection
for growth in agar or tumor formation in nude mice (7-10), referred
to as spontaneous-progression, by transfection with oncogenes (13),
such as Ha-ras, v-src, v-raf or the E6/E7 region of human
papillomavirus type (HPV)-18, referred to as oncogene-mediated
progression, or by transfection with specific signal transducing
genes (14), such as protein kinase C, referred to as growth
factor-related, gene-induced progression.
[0004] Progression, induced spontaneously or after gene transfer,
is a stable cellular trait that remains undiminished in
Ad5-transformed RE cells even after extensive passage (>100) in
monolayer culture (13). However, a single-treatment with the
demethylating agent 5-azacytidine (AZA) results in a stable
reversion in transformation progression in >95% of cellular
clones (10,13,14). The progression phenotype is also suppressed in
somatic cell hybrids formed between normal or unprogressed
transformed cells and progressed cells (11-13). These findings
suggest that progression may result from the activation of specific
progression-promoting genes or the selective inhibition of
progression-suppressing genes, or possibly a combination of both
processes.
[0005] The final stage in tumor progression is acquisition by
transformed cells of the ability to invade local tissue, survive in
the circulation and recolonize in a new area of the body, i.e.,
metastasis (15-17). Transfection of a Ha-ras oncogene into cloned
rat embryo fibroblast (CREF) cells (18) results in morphological
transformation, anchorage-independence and acquisition of
tumorigenic and metastatic potential (19-21). Ha-ras-transformed
CREF cells exhibit major changes in the transcription and
steady-state levels of genes involved in suppression and induction
of oncogenesis (21,22). Simultaneous overexpression of the Ha-ras
suppressor gene Krev-1 in Ha-ras-transformed CREF cells results in
morphological reversion, suppression of agar growth capacity and a
delay in in vivo oncogenesis (21). Reversion of transformation in
Ha-ras+Krev-1 transformed CREF cells correlates with a return in
the transcriptional and steady-state mRNA profile to that of
untransformed CREF cells (21,22). Following long latency times,
Ha-ras+Krev-1 transformed CREF cells form both tumors and
metastases in athymic nude mice (21). The patterns of gene
expression changes observed during progression, progression
suppression and escape from progression suppression supports the
concept of "transcriptional switching" as a major component of
Ha-ras-induced transformation (21,22).
[0006] To identify potential progression inducing genes with
elevated expression in progressed versus unprogressed
AdS-transformed cells we used subtraction hybridization (13,23).
This approach resulted in the cloning of PEG-3 that is expressed at
elevated levels in progressed cells (spontaneous, oncogene-induced
and growth factor-related, gene-induced) than in unprogressed cells
(parental Ad5-transformed, AZA-suppressed, and suppressed hybrids).
Transfection of PEG-3 into unprogressed parental Ad5-transformed
cells induces the progression phenotype, without significantly
altering colony formation in monolayer culture or affecting cell
growth. PEG-3 expression is also elevated following DNA damage and
oncogenic transformation of CREF cells by various oncogenes.
Sequence analysis indicates that PEG-3 has 73 and 68% nucleotide
(nt) and 59 and 72% amino acid (aa) similarities, respectively,
with the gadd34 and MyD116 gene. However, unlike gadd34 and MyD116
that encode proteins of .about.65 and .about.72 kDa, respectively,
PEG-3 encodes a protein of .about.50 kDa with only .about.28 and
.about.40% aa similarities to gadd34 and Myd116, respectively, in
its carboxyl terminus. These results indicate that PEG-3 represents
a new member of the gadd34/MyD116 gene family with both similar and
distinct properties. Unlike gadd34 and MyD116, which dramatically
suppress colony formation (24), PEG-3 only modestly alters colony
formation following transfection, i.e., .ltoreq.20% reduction in
colony formation in comparison with vector transfected cells.
Moreover, a direct correlation only exists between expression of
PEG-3, and not gadd34 or Myd116, and the progression phenotype in
transformed rodent cells. These findings provide evidence for a
potential link between constitutive induction of a stress response,
characteristic of DNA damage, and induction of cancer
progression.
SUMMARY OF THE INVENTION
[0007] This invention further provides an inducible PEG-3
regulatory region functionally linked to a gene encoding a product
that causes or may be induced to cause the death or inhibition of
cancer cell growth.
[0008] In addition, this invention further provides the
above-described vectors, wherein the inducible PEG-3 regulatory
region is a promoter.
[0009] This invention further provides the above-described vectors,
wherein the gene encodes an inducer of apoptosis.
[0010] In addition, this invention provides the above-described
vectors, wherein the gene is a tumor suppressor gene.
[0011] In addition, this invention provides the above-described
vectors, wherein the gene encodes a viral replication protein.
[0012] This invention also further provides the above-described
vectors, wherein the gene encodes a product toxic to cells or an
intermediate to a product toxic to cells.
[0013] In addition, this invention provides the above-described
vectors, wherein the gene encodes a product causing enhanced immune
recognition of the cell.
[0014] This invention further provides the above-described vectors,
wherein the gene encodes a product causing the cell to express a
specific antigen.
[0015] In addition, this invention provides a method of treating
cancer in a subject, comprising: a) administering one of the
above-described vectors to the subject; and b) administering an
antibody or a fragment of an antibody to the the above-described
antigen to the subject.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1: Northern blot illustrating PEG-3 expression in
Ad5-transformed RE cells displaying different stages of
transformation progression. Fifteen .mu.g of cellular RNA isolated
from the indicated cell types, were electrophoresed, transferred to
nylon membranes and hybridized with an .about.700 bp 3' region of
the PEG-3 gene (top) and then stripped and probed with GAPDH
(bottom).
[0017] FIG. 2: Northern blot illustrating PEG-3 expression in gamma
irradiated and oncogene transformed CREF cells. The experimental
procedure was as described in the legend to FIG. 1. CREF cells were
gamma irradiated with 10 Gy and RNA was isolated 4 and 24 hr later.
Fifteen .mu.g of cellular RNA isolated from the indicated cell
types, were electrophoresed, transferred to nylon membranes and
hybridized with an .about.700 bp 3' region of the PEG-3 gene (top)
and then stripped and probed with GAPDH (bottom).
[0018] FIGS. 3A and 3B: Predicted amino acid sequences of the rat
PEG-3, gadd34 and MyD116 proteins. Sequences shared by the three
genes are shaded. PEG-3 encodes a putative protein of 457 aa (MW of
.about.50 kDa), the gadd34 gene encodes a putative protein of 589
aa (MW .about.65 kDa) and the MyD116 gene encodes a putative
protein of 657 aa (MW of .about.72 kDa).
[0019] FIG. 4: Results of in vitro translation of the rat PEG-3
gene. Lane Luciferase is the in vitro translation of the luciferase
gene (.about.61 kDa), positive control. The blank lane contains the
same reaction mixture without mRNA, negative control. Lane PEG-3
contains the translated products of this cDNA. Rainbow protein
standards (Amersham) were used to determine the sizes of the in
vitro translated products.
[0020] FIG. 5: An autoradiogram illustrating the transcription of
the rat PEG-3, gadd34 and MyD116 genes as a function of DNA damage
and transformation progression. Nuclear run-on assays were
performed to determine comparative rates of transcription. Nuclei
were isolated from CREF cells treated with MMS (100 .mu.g/ml for 2
hr followed by growth for 4 hr in complete medium) or gamma
irradiation (10 Gy followed by 2 hr growth in complete medium). DNA
probes include, PEG-3 (1), MyD116 (2), gadd34 (3), GAPDH (4) and
pBR322 (5).
[0021] FIG. 6: Histogram illustrating the effect of transfection
with PEG-3, mda-7 and p21 (mda-6) on colony formation of E11 and
E11-NMT cells in monolayer culture. Target cells were transfected
with 10 .mu.g of a Zeocin vector (pZeoSV), the PEG-3 gene cloned in
pZeoSV (PEG-3), the pREP4 vector, the mda-7 gene cloned in pREP4
(mda-7) and the mda-6 (p21) gene cloned in pREP4 (p21 (mda-6)), as
indicated. Data represents the average number of Zeocin or
hygromycin (pREP4 transfection) resistant colonies .+-.S.D. for 4
plates seeded at 1.times.10.sup.5 cells/6-cm plate.
[0022] FIG. 7: Histogram illustrating the effect of stable PEG-3
expression on anchorage independent growth of E11 cells. Agar
cloning efficiency of E11, Zeocin resistant pZeoV (vector)
transfected E11 and Zeocin resistant pZeoPEG transfected E11 cells.
Average number of colonies developing in 4 replicate plates
.+-.S.D.
[0023] FIG. 8: Autoradiogram illustrating the expression of PEG-3,
Ad.sup.5 E1A and GAPDH RNA in pZeoPEG transfected E11 cells. The
experimental procedure was as described in the legend to FIG. 1.
Blots were probed sequentially with PEG-3 (top), Ad.sup.5 E1A
(middle) and GAPDH (bottom). The E11-ZeoPEG clones are the same
clones analyzed for anchorage-independence in FIG. 7.
[0024] FIG. 9: Autoradiogram showing PEG-3 expression in normal
human melanocyte and melanoma cell lines. Fifteen .mu.g of cellular
RNA isolated from the indicated cell types, were electrophoresed,
transferred to nylon membranes and hybridized with an .about.700 bp
3' region of the PEG-3 gene (top) and then stripped and probed with
GAPDH (bottom). Cell types include: FM516-SV, normal human
melanocyte immortalized with the SV40 T-antigen; MeWo, WM239,
C8161, F0-1 and Ho-1, metastatic human melanoma; WM35, early radial
growth phase (RGP) primary human melanoma; and WM278, early
vertical growth phase (VGP) primary human melanoma.
[0025] FIG. 10: Autoradiogram showing the effect of treatment with
DNA damaging agents on PEG-3 expression in human melanoma cells.
The indicated cell type was exposed to methyl methanesulfonate
(MMS) (100 .mu.g/ml for 2 hr and then grown in medium) lacking MMS
for 2 hr) or gamma irradiation (JR) (10 gy and cells were grown for
4 or 24 hr in medium) prior to RNA isolation. Fifteen .mu.g of
cellular RNA isolated from the indicated cell types and conditions,
were electrophoresed, transferred to nylon membranes and hybridized
with an .about.700 bp 3' region of the PEG-3 gene (top) and then
stripped and probed with GAPDH (bottom). HO-1 and F0-1 cells
express wild-type p53 protein (p53 wt) and SK MEL 110 expresses a
mutant p53 (p53 mut).
[0026] FIG. 11: Nucleotide sequence of rat Progression Elevated
Gene-3 (PEG-3). The initiation and termination codons are
underlined.
[0027] FIG. 12: Amino acid sequence of Progression Elevated Gene-3
(PEG-3). PEG-3 protein contains 457 amino acids and with M.W. of
approximately 50 kDa.
[0028] FIGS. 13A-13C: Nucleotide and amino acid sequence of a human
PEG-3 cDNA.
[0029] FIG. 14: Sequence of the rat PEG-3 promoter. This region of
DNA consists of 2,616 nucleotides. This DNA sequence contains the
putative initiation site of transcription of the rat PEG-3 gene.
For luciferase assays a .about.2,200 nucleotide region of the PEG-3
promoter was cloned into a luciferase reporter vector.
[0030] FIG. 15: Relative PEG-3 promoter luciferase activity in
unprogressed (E11 and E11-NMT-AZA) and progressed (E11-NMT and
E11-ras) transformed rodent cells.
[0031] FIG. 16: Relative PEG-3 promoter luciferase activity in
normal and transformed CREF cells. These include unprogressed and
non-tumorigenic (CREF, Rat 1, CREF-ras+Krev-1 B1, and CREF-wt3A
(Ad5) and progressed and oncogenic (CREF-Trans 6:4 NMT,
CFEF-Ha-ras, CREF-ras+Krev-1 BIT, CREF-ras+Krev-1 B1M, CREF-A2
(H5hr1), CREF-src, CREF-HPV-18, CREF-raf and CREF-PKC B1) cells. *
Indicates non-tumorigenic cell type.
[0032] FIG. 17: Induction of PEG-3 promoter luciferase activity in
CREF and CREF-PEG-Luc cells after MMS (100 .mu.g/ml) treatment.
CREF (O) and CREF-PEG-Luc (X).
[0033] FIG. 18: The Rapid Promoter Screening (RPS) assay system for
identifying compounds and experimental conditions regulating
important physiological processes. The systems outlined above
represent sensitive biosensor monitoring approaches for defining
conditions and compounds that can regulate cellular phenotype,
thereby altering the functionality of the PEG-3 promoter. Briefly,
the PEG-3 promoter is linked to a reporter gene, such as
luciferase, and stable cell clones are generated that contain
either an inducible PEG-Luc gene (CREF) or a suppressible PEG-Luc
gene (E11-NMT or various transformed CREF cells, including 4NMT,
CREF-ras, CREF-src, CREF-HPV etc.). The PEG-3-Luc containing clones
can then be used as sensitive indicators of alterations in cellular
physiology resulting from DNA damage, induction of cancer
progression, induction of oncogenic transformation, treatment with
chemoprevention agents, inhibitors of cancer progression,
inhibitors of angiogenesis and agents specifically involved in
regulating defined oncogenic pathways. The RPS approach can be
adapted for manual or rapid automated screening.
[0034] FIG. 19: Northern blot analysis of human PEG-3. A 500 bp
probe from the 3' end of the human PEG-3 gene was used to probe a
Northern blot containing the following tumor and normal cell
lines.
[0035] MCF-7, T47D: human breast carcinoma
[0036] LNCaP, PC-3: human prostate carcinoma
[0037] T98G, GBM-18: human glioblastoma
[0038] HO-1, LO-1, SH-1, C8161, FO-1: human melanoma
[0039] NhPEC: normal human prostate epithelial cells
(Clonetics)
[0040] HBL-100: immortalized normal human breast cells
[0041] HeLa: human ovarian carcinoma
[0042] REF(RAD): irradiated CREF-Trans 6 cells
[0043] E11, E11NMT: Sprague Dawley rat embryo cells transformed
with mutant adenovirus H5ts125
[0044] 4NMT: CREF-Trans 6 cells transformed with LNCaP high
molecular weight DNA
[0045] HONE-1: human nasopharyngeal carcinoma
[0046] FIG. 20: Northern blot analysis of human PEG-3. A 500 bp
probe from the 3' end of the human PEG-3 gene was used to probe a
normal human tissue blot (Clonetics)
[0047] FIG. 21: Titration of CREF-Trans 6 4NMT cells containing the
rat PEG-3 promoter/luciferase reporter gene. Cells were grown in 96
well plates, lysed as described and the luciferase activity was
read in a luminometer.
[0048] FIG. 22: Effects of an antisense oligonucleotide to the
PTI-1 oncogene on expression of the rat PEG-3 promoter/luciferase
reporter gene in CREF-Trans 6 4NMT cells. 4NMT cells were treated
with an antisense oligonucleotide to the bridge region of the PTI-1
gene for 24, 48, and 72 hours. Cells were lysed and luciferase
activity was determined using a luminometer.
[0049] FIG. 23: Luciferase activation in the presence of various
oncogenes expressed in CREF-Trans 6 cells transfected with the rat
PEG-3 promoter/luciferase reporter. CREF-Trans 6 cells were stably
transfected with the following oncogenes: ras, src, and PTI-1. 4NMT
cells were transfected with high molecular weight DNA from the
human prostate carcinoma cell line LNCaP and expresses PTI-1. Cells
grown in 96 well microtiter plates, lysed, and assayed for
luciferase activity.
[0050] FIG. 24: Schematic diagram of specific deletion mutants in
the rat PEG-3 promoter. The deletion mutants are labeled as 2 to
11, with 1 being the unmodified promoter. Numbers in brackets
indicate the beginning nucleotide remaining after deletion from the
5 region of the rat PEG-3 promoter. The TATA box is located at
nucleotide 1751 from the 5' region of the promoter.
[0051] FIG. 25: Relative PEG-3 promoter luciferase activity in E11
cells after transfection with an intact rat PEG-3 promoter (1) and
various deletion mutants of the rat PEG-3 promoter (2 through 11).
Further information in FIG. 24.
[0052] FIG. 26: Relative PEG-3 promoter luciferase activity in
E11-NMT cells after transfection with an intact rat PEG-3 promoter
(1) or various deletion mutants of the rat PEG-3 promoter (2
through 11). Further information in FIG. 24.
[0053] FIG. 27: Comparison of the relative PEG-3 promoter
luciferase activity in E11 and E11-NMT cells after transfection
with an intact rat PEG-3 promoter (1) or various deletion mutants
of the rat PEG-3 promoter (2 through 11). Further information in
FIG. 24.
[0054] FIG. 28: Relative PEG-3 promoter luciferase activity in E11
cells transformed by the PKC .beta..sub.l gene (E11-PKC) after
transfection with an intact rat PEG-3 promoter (1) or various
deletion mutants of the rat PEG-3 promoter (2 through 11). Further
information in FIG. 24.
[0055] FIG. 29: Relative PEG-3 promoter luciferase activity in CREF
cells transformed by human papilloma virus type 18 (CREF-HPV) after
transfection with an intact rat PEG-3 promoter (1) or various
deletion mutants of the rat PEG-3 promoter (2 through 11). Further
information in FIG. 24.
[0056] FIG. 30: Relative PEG-3 promoter luciferase activity in CREF
cells transformed by the Ha-ras oncogene (CREF-ras) after
transfection with an intact rat PEG-3 promoter (1) or various
deletion mutants of the rat PEG-3 promoter (2 through 11). Further
information in FIG. 24.
[0057] FIG. 31: Relative PEG-3 promoter luciferase activity in
CREF-Trans 6 cells transformed by human prostatic carcinoma (LNCaP)
DNA (CREF-Trans 6:4 NMT) after transfection with an intact rat
PEG-3 promoter (1) or various deletion mutants of the rat PEG-3
promoter (2 through 11). Further information in FIG. 24.
[0058] FIG. 32: Schematic outline of CURE (Cancer Utilized Reporter
Execution) strategy and construction of CIRAs (Cancer Inhibitory
Recombinant Adenoviruses). The PEG-3 promoter is linked to various
genes, including (1) wt-p53, (2) mda-7, (3) p21, (4) Ad-E1A, (5)
HSV-TK, (6) ImStim (immunostimulatory gene, or (7) Antigen
(molecule encoding an immunogenic molecule increasing tumor
reactivity with immune cells). The various constructs display
slective gene expression as a function of PEG-3 promoter
activation, which is restricted to cancer cells. The genes are
incorporated into a replication defective (1, 2, 3, 5, 6, and 7) or
replication competent (1, 2, 3, 4, 5, 6, and 7) adenovirus (Ad).
These adenoviruses can then be used to infect human cancer cells
resulting in PEG-3 promoter activation of gene expression resulting
in transcription of the linked gene and production of the encoded
gene product. When normal cells are infected with any of the
adenovirus constructs (CIRAs), the promoter is inactive or
marginally active resulting in either no or small quantities of the
encoded gene product. In these contexts, no physiological change
should occur in normal cells. In contrast, when expressed in tumor
cells the PEG-3 promoter is active resulting in transcription of
the linked gene and production of the encoded gene product. In the
case of Ad 1, 2, and 3, infection of cancer cells results in
wt-p53, mda-7 or p21 protein. These proteins will result in
inhibition of cancer growth and in specific contexts will induce
programmed cell death (apoptosis). In the case of Ad 4, which is a
replication competent Ad, induction of Ad E1A will result in viral
replication and lysis of the cancer cell. In the case of Ad 5,
induction of the HSV-TK gene renders the cell sensitive to growth
inhibition and toxicity following administration of gangcyclovir or
acyclovir. In the case of Ad 6, induction of an ImStim
(Immunostimulatory gene), such as GM-CSF, IL-2, a cytokine (immune
interferon, interleukin 6, etc.) or an immunomodulating protein,
renders the cancer cell susceptible to immunological attack and
cell lysis. In the case of Ad 7, induction of antigenic expression
(with and without expression of co-stimulatory molecules), renders
the cancer cell susceptible to antibody mediated toxicity. This can
result from an interaction with an antibody with direct antitumor
activity, an antibody linked to an immunotoxin, an antibody linked
to a high energy radionuclide, etc. Ad 7 CIRAs expressing proteins
with T-cell epitopes or T-cell epitopes themselves can be used to
sensitize cancer cells to killing by CD8 cytotoxic killer T-cells.
In principle, CIRAs can be produced that will result in the
targeted destruction of only cancer cells (the basis of the CURE
technology). In addition to using CIRAs, CURE can also be used with
alternative transfer systems, including a retrovirus, an
adeno-associated virus, a herpes virus, a vaccinia virus, a
liposome preparation, physical delivery technology, naked DNA
technology, etc.
DETAILED DESCRIPTION OF THE INVENTION
[0059] This invention provides an isolated nucleic acid molecule
encoding a Progression Elevated Gene-3 protein. The nucleic acid
may be DNA, cDNA, genomic DNA or RNA.
[0060] This invention also encompasses DNAs and cDNAs which encode
amino acid sequences which differ from those of Progression
Elevated Gene-3 protein, but which should not produce phenotypic
changes. Alternatively, this invention also encompasses DNAs and
cDNAs which hybridize to the DNA and cDNA of the subject invention.
Hybridization methods are well-known to those of skill in the
art.
[0061] The DNA molecules of the subject invention also include DNA
molecules coding for polypeptide analogs, fragments or derivatives
of antigenic polypeptides which differ from naturally-occurring
forms in terms of the identity or location of one or more amino
acid residues (deletion analogs containing less than all of the
residues specified for the protein, substitution analogs wherein
one or more residues specified are replaced by other residues and
additional analogs where in one or more amino acid residues is
added to a terminal or medial portion of the polypeptides) and
which share some or all properties of naturally-occurring forms.
These molecules include: the incorporation of codons "preferred"
for expression by selected non-mammalian hosts; the provision of
sites for cleavage by restriction endonuclease enzymes; and the
provision of additional initial, terminal or intermediate DNA
sequences that facilitate construction of readily expressed
vectors.
[0062] The DNA molecules described and claimed herein are useful
for the information which they provide concerning the amino acid
sequence of the polypeptide and as products for the large scale
synthesis of the polypeptide by a variety of recombinant
techniques. The molecule is useful for generating new cloning and
expression vectors, transformed and transfected prokaryotic and
eukaryotic host cells, and new and useful methods for cultured
growth of such host cells capable of expression of the polypeptide
and related products.
[0063] Moreover, the isolated nucleic acid molecules encoding a
Progression Elevated Gene-3 are useful for the development of
probes to study the progression of cancer. This invention also
provides isolated nucleic acid molecule encoding a human
Progression Elevated Gene-3 protein.
[0064] This invention provides a nucleic acid molecule of at least
12 nucleotides capable of specifically recognizing a nucleic acid
molecule encoding a Progression Elevated Gene-3 protein. In a
preferred embodiment, this nucleic acid molecule has a unique
sequence of the Progression Elevated Gene-3. The unique sequence of
the Progression Elevated Gene-3 may easily be determined by
comparing its sequence with known sequences which are available in
different databases. The nucleic acid molecule may be DNA or
RNA.
[0065] This nucleic acid molecule of at least 15 nucleotides
capable of specifically hybridizing with a sequence of a nucleic
acid molecule encoding a Progression Elevated Gene-3 protein can be
used as a probe. Nucleic acid probe technology is well-known to
those skilled in the art who will readily appreciate that such
probes may vary greatly in length and may be labeled with a
detectable label, such as a radioisotope or fluorescent dye, to
facilitate detection of the probe. DNA probe molecules may be
produced by insertion of a DNA molecule which encodes Progression
Elevated Gene-3 protein into suitable vectors, such as plasmids or
bacteriophages, followed by transforming into suitable bacterial
host cells, replication in the transformed bacterial host cells and
harvesting of the DNA probes, using methods well-known in the art.
Alternatively, probes may be generated chemically from DNA
synthesizers.
[0066] RNA probes may be generated by inserting the Progression
Elevated Gene-3 molecule downstream of a bacteriophage promoter
such as T3, T7 or SP6. Large amounts of RNA probe may be produced
by incubating the labeled nucleotides with the linearized
Progression Elevated Gene-3 fragment where it contains an upstream
promoter in the presence of the appropriate RNA polymerase.
[0067] This invention provides a method of detecting expression of
the Progression Elevated Gene-3 in a sample which contains cells
comprising steps of: (a) obtaining RNA from the cells; (b)
contacting the RNA so obtained with a labelled probe of the
Progression Elevated Gene-3 under hybridizing conditions permitting
specific hybridization of the probe and the RNA; and (c)
determining the presence of RNA hybridized to the molecule, thereby
detecting the expression of the Progression Elevated Gene-3 in the
sample. mRNA from the cell may be isolated by many procedures
well-known to a person of ordinary skill in the art. The
hybridizing conditions of the labelled nucleic acid molecules may
be determined by routine experimentation well-known in the art. The
presence of mRNA hybridized to the probe may be determined by gel
electrophoresis or other methods known in the art. By measuring the
amount of the hybrid made, the expression of the Progression
Elevated Gene-3 protein by the cell can be determined. The
labelling may be radioactive. For an example, one or more
radioactive nucleotides can be incorporated in the nucleic acid
when it is made.
[0068] The RNA obtained in step (a) may be amplified by polymerase
chain reaction (PCR) with appropriate primers. The appropriate
primers may be selected from the known Progression Elevated Gene-3
sequences. Instead of detection by specific PEG-3 probe as
described in the preceding paragraph, the specific amplified DNA by
PCR is an indication that there is expression of Progression
Elevated Gene-3.
[0069] This invention provides an isolated nucleic acid molecule
encoding a Progression Elevated Gene-3 protein operatively linked
to a regulatory element. In an embodiment, the vector is a
plasmid.
[0070] This invention provides a host vector system for the
production of a polypeptide having the biological activity of a
Progression Elevated Gene-3 protein which comprises the vector
having the sequence of Progression Elevated Gene-3 and a suitable
host. The suitable host includes but is not limited to a bacterial
cell, yeast cell, insect cell, or animal cell.
[0071] The isolated Progression Elevated Gene-3 sequence can be
linked to different vector systems. Various vectors including
plasmid vectors, cosmid vectors, bacteriophage vectors and other
viruses are well-known to ordinary skilled practitioners. This
invention further provides a vector which comprises the isolated
nucleic acid molecule encoding for the Progression Elevated Gene-3
protein.
[0072] As an example to obtain these vectors, insert and vector DNA
can both be exposed to a restriction enzyme to create complementary
ends on both molecules which base pair with each other and are then
ligated together with DNA ligase. Alternatively, linkers can be
ligated to the insert DNA which correspond to a restriction site in
the vector DNA, which is then digested with the restriction enzyme
which cuts at that site. Other means are also available and known
to an ordinary skilled practitioner.
[0073] In an embodiment, the rat PEG-3 sequence is cloned in the
EcoRI site of pZeoSV vector. This plasmid, pPEG-3, was deposited on
Mar. 6, 1997 with the American Type Culture Collection (ATCC),
12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the
provisions of the Budapest Treaty for the International Recognition
of the Deposit of Microorganism for the Purposes of Patent
Procedure. Plasmid, pPEG-3, was accorded ATCC Accession Number
97911.
[0074] This invention further provides a host vector system for the
production of a polypeptide having the biological activity of the
Progression Elevated Gene-3 protein. These vectors may be
transformed into a suitable host cell to form a host cell vector
system for the production of a polypeptide having the biological
activity of the Progression Elevated Gene-3 protein.
[0075] Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation
sequences for ribosome binding. For example, a bacterial expression
vector includes a promoter such as the lac promoter and for
transcription initiation the Shine-Dalgarno sequence and the start
codon AUG. Similarly, a eukaryotic expression vector includes a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors may
be obtained commercially or assembled from the sequences described
by methods well-known in the art, for example the methods described
above for constructing vectors in general. Expression vectors are
useful to produce cells that express the Progression Elevated
Gene-3 protein.
[0076] This invention further provides an isolated DNA, cDNA or
genomic DNA molecule described hereinabove wherein the host cell is
selected from the group consisting of bacterial cells (such as E.
coli), yeast cells, fungal cells, insect cells and animal cells.
Suitable animal cells include, but are not limited to Vero cells,
HeLa cells, Cos cells, CV1 cells and various primary mammalian
cells.
[0077] This invention further provides a method of producing a
polypeptide having the biological activity of the Progression
Elevated Gene-3 protein which comprising growing host cells of a
vector system containing Progression Elevated Gene-3 sequence under
suitable conditions permitting production of the polypeptide and
recovering the polypeptide so produced.
[0078] This invention provides a mammalian cell comprising a DNA
molecule encoding a Progression Elevated Gene-3 protein, such as a
mammalian cell comprising a plasmid adapted for expression in a
mammalian cell, which comprises a DNA molecule encoding a
Progression Elevated Gene-3 protein and the regulatory elements
necessary for expression of the DNA in the mammalian cell so
located relative to the DNA encoding the Progression Elevated
Gene-3 protein as to permit expression thereof.
[0079] Various mammalian cells may be used as hosts, including, but
not limited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa
cells, Ltk.sup.- cells, Cos cells, etc. Expression plasmids such as
that described supra may be used to transfect mammalian cells by
methods well-known in the art such as calcium phosphate
precipitation, electroporation or DNA encoding the Progression
Elevated Gene-3 protein may be otherwise introduced into mammalian
cells, e.g., by microinjection, to obtain mammalian cells which
comprise DNA, e.g., cDNA or a plasmid, encoding a Progression
Elevated Gene-3 protein.
[0080] This invention also provides a purified Progression Elevated
Gene-3 protein and a fragment thereof. As used herein, the term
"purified Progression Elevated Gene-3 protein" shall mean isolated
naturally-occurring Progression Elevated Gene-3 protein or protein
manufactured such that the primary, secondary and tertiary
conformation, and posttranslational modifications are identical to
naturally-occurring material as well as non-naturally occurring
polypeptides having a primary structural conformation (i.e.
continuous sequence of amino acid residues). Such polypeptides
include derivatives and analogs. The fragment should bear
biological activity similar to the full-length Progression Elevated
Gene-3 protein.
[0081] This invention also provides a polypeptide encoded by the
isolated vertebrate nucleic acid molecule having a sequence of a
Progression Elevated Gene-3.
[0082] This invention provides an antibody capable of specifically
binding to a Progression Elevated Gene-3 protein. The antibody may
be polyclonal or monoclonal. This invention provides a method to
select specific regions on the Progression Elevated Gene-3 to
generate antibodies. The protein sequence may be determined from
the DNA sequence. The hydrophobic or hydrophilic regions in the
protein will be identified. Usually, the hydrophilic regions will
be more immunogenic than the hydrophobic regions. Therefore the
hydrophilic amino acid sequences may be selected and used to
generate antibodies specific to the Progression Elevated Gene-3
protein.
[0083] Polyclonal antibodies against these peptides may be produced
by immunizing animals using the selected peptides. Monoclonal
antibodies are prepared using hybridoma technology by fusing
antibody producing B cells from immunized animals with myeloma
cells and selecting the resulting hybridoma cell line producing the
desired antibody. Alternatively, monoclonal antibodies may be
produced by in vitro techniques known to a person of ordinary skill
in the art. Specific antibody which only recognizes the Progression
Elevated Gene-3 protein will then be selected. The selected
antibody is useful to detect the expression of the Progression
Elevated Gene-3 in living animals, in humans, or in biological
tissues or fluids isolated from animals or humans.
[0084] This invention provides a method of transforming cells which
comprises transfecting a host cell with a suitable vector having
the sequence of a Progression Elevated Gene-3. This invention also
provides the transformed cells produced by this method.
[0085] This invention provides a method for determining whether
cells are in progression comprising steps of: a) measuring the
expression of the Progression Elevated Gene-3; and b) comparing the
expression measured in step a) with the expression of Progression
Elevated Gene-3 in cells which are known not to be in progression,
wherein an increase of the expression indicates that the cells are
in progression. In an embodiment, the expression of Progression
Elevated Gene-3 is measured by the amount of Progression Elevated
Gene-3 mRNA expressed in the cells. In another embodiment, the
expression of Progression Elevated Gene-3 is measured by the amount
of the Progression Elevated Gene-3 protein expressed in the
cells.
[0086] This invention provides a method for determining whether a
cancer cell is in a progression stage comprising measuring the
expression of Progression Elevated Gene-3 in the cancer cell,
wherein an increase in the amount indicates that the cancer cell is
in progression.
[0087] This invention provides a method for diagnosing the
aggressiveness of cancer cells comprising measuring the expression
of Progression Elevated Gene-3 in the cancer cell, wherein an
increase in the amount of the expression indicates that the cancer
cell is more aggressive.
[0088] This invention provides a pharmaceutical composition for
reversing the progression state of cells comprising an amount of
the nucleic acid molecule capable of specifically hybridizing the
Progression Elevated Gene-3 protein effective to inhibit the
expression of Progression Elevated Gene-3 and a pharmaceutically
acceptable carrier.
[0089] Pharmaceutically acceptable carriers are well-known to those
skilled in the art. Such pharmaceutically acceptable carriers may
be aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, saline and
buffered media. Parenteral vehicles include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's or fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Preservatives and other
additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases and the
like.
[0090] This invention provides a pharmaceutical composition for
reversing the progression state of cells comprising an amount of
the antibody or a functional fragment thereof which is capable of
specifically recognizing the Progression Elevated Gene-3 protein
effective to neutralize the action of the Progression Elevated
Gene-3 protein and a pharmaceutically acceptable carrier.
[0091] This invention provides a method for producing cells which
are resistant to progression comprising inhibiting or eliminating
the expression of Progression Elevated Gene-3 in the cells. This
invention also provides cells resulting from the method.
[0092] This invention provides a method for protecting cells from
therapeutic damage comprising inhibiting or eliminating the
expression of Progression Elevated Gene-3 in the cells. In an
embodiment, the damage is resulted from chemotherapy. In another
embodiment, the damage is resulted from physical agent. Such
physical agent includes but is not limited to
gamma-irradiation.
[0093] One method to inhibit the expression of Progression Elevated
Gene-3 is by expression of effective amount antisense RNA in the
cell thereby inhibiting the expression of Progression Elevated
Gene-3. The expression of Progression Elevated Gene-3 may be
eliminated by deletion of the gene or introduction of mutation(s)
to the gene.
[0094] This invention provides a transgenic nonhuman living
organism expressing Progression Elevated Gene-3 protein. In an
embodiment, the living organism is animal.
[0095] One means available for producing a transgenic animal, with
a mouse as an example, is as follows: Female mice are mated, and
the resulting fertilized eggs are dissected out of their oviducts.
The eggs are stored in an appropriate medium. DNA or cDNA encoding
a Progression Elevated Gene-3 is purified from a vector by methods
well-known in the art. Inducible promoters may be fused with the
coding region of the DNA to provide an experimental means to
regulate expression of the trans-gene. Alternatively or in
addition, tissue specific regulatory elements may be fused with the
coding region to permit tissue-specific expression of the
trans-gene. The DNA, in an appropriately buffered solution, is put
into a microinjection needle (which may be made from capillary
tubing using a pipet puller) and the egg to be injected is put in a
depression slide. The needle is inserted into the pronucleus of the
egg, and the DNA solution is injected. The injected egg is then
transferred into the oviduct of a pseudopregnant mouse (a mouse
stimulated by the appropriate hormones to maintain pregnancy but
which is not actually pregnant), where it proceeds to the uterus,
implants, and develops to term. As noted above, microinjection is
not the only method for inserting DNA into the egg cell, and is
used here only for exemplary purposes.
[0096] This invention provides a cell having an exogenous indicator
gene under the control of the regulatory element of a Progression
Elevated Gene-3. In an embodiment, the cell is at progression. This
cell may be produced by introducing an indicator gene to an
E11-NMT, CREF-ras or CREF-src cell.
[0097] In a separate embodiment, the cell having an exogenous
indicator gene under the control of the regulatory element of a
Progression Elevated Gene-3 is not at progression. This cell may be
produced by introducing an indicator gene to the E11 or the CREF
cell.
[0098] The indicator gene codes for beta-galactosidase, luciferase,
chloramphenicol transferase or secreted alkaline phosphatase. Other
indicator gene such as the Green Fluorescent Protein gene may be
similar used in this invention. The indicator provides an easily
detectable signal when the PEG-3 is expressed.
[0099] This invention provides a method for determining whether an
agent is capable of inhibiting DNA damage and repair pathways,
cancer progression or oncogene mediated transformation comprising
contacting an amount of the agent with the cell having an exogenous
indicator gene under the control of the regulatory element of a
Progression Elevated Gene-3, wherein a decrease of expression of
the indicator gene indicates that the agent is capable of
inhibiting DNA damage and repair pathways, cancer progression or
oncogene mediated transformation. This invention provides a method
for determining whether an agent is capable of inducing DNA damage
and repair pathways, cancer progression or oncogene mediated
transformation comprising contacting an amount of the agent with
the cell having an exogenous indicator gene under the control of
the regulatory element of a Progression Elevated Gene-3 is not at
progression, wherein an increase of expression of the indicator
gene after the contact indicates that the agent is capable of
inducing DNA damage and repair pathways, cancer progression or
oncogene mediated transformation.
[0100] Large scale of agents may be screened by the above two
methods through automation. Indicator gene which produces color
reaction may be selected.
[0101] This invention provides a cell having an exogenous suicidal
gene or genes under the control of the regulatory element of a
Progression Elevated Gene-3. Such "suicidal gene" will disrupt the
normal progress of the cell. Preferably, the switching on of the
suicidal gene will lead to cell death or halt in cell growth.
Example of such genes are genes which lead to apotosis.
[0102] This invention provides a nucleic acid molecule comprising a
sequence of the promoter of a Progression Elevated Gene-3
protein.
[0103] This invention also provides a nucleic acid molecule
comprising Cis-Acting Regulatory Elements of the promoter of a
Progression Elevated Gene-3 protein.
[0104] This invention also provides a Trans-Acting Regulatory
Element that activates the expression of Progression Elevated
Gene-3.
[0105] This invention further provides Trans-Acting Regulatory
Element that suppresses the expression of Progression Elevated
Gene-3.
[0106] This invention also provide an isolated nucleic acid
molecule comprising sequence encoding the Trans-Acting Regulatory
Element.
[0107] This invention provides an isolated nucleic acid molecule
encoding a Progression Elevated Gene-3 protein. This invention also
provides the above-described nucleic acid, wherein the nucleic acid
encodes a human Progression Elevated Gene-3 protein.
[0108] In addition, this invention provides the above-described
nucleic acid, wherein the nucleic acid encodes a rodent Progression
Elevated Gene-3 protein.
[0109] This invention further provides an isolated nucleic acid
comprising substantially the same sequence as the sequence set
forth in FIG. 11 or 13 or the complement of the sequence set forth
in FIG. 11 or 13.
[0110] This invention also provides an isolated nucleic acid
sequence comprising a nucleic acid sequence that specifically
hybridizes to the sequence set forth in FIG. 11 or 13 or the
complement of the sequence set forth in FIG. 11 or 13.
[0111] This invention provides an isolated nucleic acid comprising
substantially the same sequence as the nucleic acid sequence
encoding the 80 C-terminal-most amino acids, wherein the nucleic
acid sequence is set forth in FIG. 11 or 13 or the complement of
the sequence set forth in FIG. 11 or 13.
[0112] This invention also provides an isolated nucleic acid
comprising substantially the same sequence as the nucleic acid
sequence encoding the 80 C-terminal-most amino acids, wherein the
nucleic acid sequence is set forth in FIG. 11 or 13 or the
complement of the sequence set forth in FIG. 11 or 13.
[0113] This invention also provides an isolated nucleic acid
sequence comprising a nucleic acid sequence that specifically
hybridizes to the nucleic acid sequence encoding the 80
C-terminal-most amino acids, wherein the nucleic acid sequence is
set forth in FIG. 11 or 13 or the complement of the sequence set
forth in FIG. 11 or 13.
[0114] This invention further provides the above-described nucleic
acid, wherein the nucleic acid is DNA, cDNA, genomic DNA, or
RNA.
[0115] This invention provides a nucleic acid molecule comprising a
promoter of Progression Elevated Gene-3.
[0116] This invention also provides a nucleic acid molecule
comprising cis-acting regulatory element of Progression Elevated
Gene-3 protein.
[0117] In addition, this invention provides a trans-acting
regulatory element that activates the expression of Progression
Elevated Gene-3.
[0118] This invention also provides a trans-acting regulatory
element that suppresses the expression of Progression Elevated
Gene-3.
[0119] This invention further provides an isolated nucleic acid
molecule comprising sequence encoding the trans-acting regulatory
element of claim 12 or 13.
[0120] This invention provides a purified Progression Elevated
Gene-3 protein.
[0121] This invention provides the polypeptide encoded by the
above-described nucleic acids.
[0122] This invention also provides an isolated polypeptide
comprising substantially the same sequence as the sequence set
forth in FIG. 12 or 13.
[0123] This invention provides the above-described polypeptide,
wherein the polypeptide has tumor progression activity or the
presence of the polypeptide positively correlates with the
progression phenotype.
[0124] In addition, this invention also provides an isolated
polypeptide comprising substantially the same sequence as the
sequence of the 80 C-terminal-most amino acids set forth in FIG. 12
or 13 or the complement of the sequence set forth in FIG. 12 or
13.
[0125] This invention provides a nucleic acid molecule comprising
12 or more nucleotides that specifically hybridize with the
above-described nucleic acids.
[0126] This invention also provides an antisense polynucleotide
comprising a sequence complementary to the above-described nucleic
acids.
[0127] This invention further provides an upstream nucleic acid
sequence comprising nucleotides 1-500 as set forth in FIG. 14.
[0128] In addition, this invention provides an upstream nucleic
acid sequence comprising nucleotides 1-1000 as set forth in FIG.
14.
[0129] This invention provides an upstream nucleic acid sequence
comprising the nucleic acid sequence set forth in FIG. 14.
[0130] This invention also provides an antisense nucleic acid
comprising 15 or more nucleotides capable of specifically
hybridizing to the above-described upstream nucleic acid
sequences.
[0131] This invention further provides an antisense nucleic acid
comprising 15 or more nucleotides that specifically hybridizes to
the nucleotides 1-500 as set forth in FIG. 14.
[0132] This invention provides an isolated polypeptide comprising
at least a portion of a progression-associated protein, or a
variant thereof, wherein: a) the progression-associated protein
comprises a sequence encoded by a nucleotide sequence set forth in
FIG. 11 or 13; and b) the portion retains at least one
immunological or biological activity characteristic of the
progression-associated protein.
[0133] This invention provides the above-described polypeptides,
wherein the portion is immunologically active.
[0134] This invention also provides an isolated nucleic encoding
the above-described polypeptides.
[0135] This invention provides a vector which comprises the
above-described isolated nucleic acids.
[0136] This invention also provides the above-described isolated
nucleic acids operatively linked to a regulatory element.
[0137] This invention further provides the above-described vector,
wherein the vector is a plasmid.
[0138] This invention also provides the above-described plasmid,
designated PGEN-3 (ATCC Accession No. 97911).
[0139] This invention provides a host vector system for the
production of a polypeptide having the biological activity of a
Progression Elevated Gene-3 protein which comprises the
above-described vectors and a suitable host.
[0140] This invention further provides the above-described vectors,
wherein the suitable host is a bacterial cell, yeast cell, insect
cell, or animal cell.
[0141] In addition, this invention also provides an expression
vector comprising the above-described nucleic acids.
[0142] This invention provides a host cell transformed or
transfected with the above-described expression vectors.
[0143] This invention further provides an antibody or
antigen-binding fragment thereof that specifically binds to the
above-described polypeptides.
[0144] This invention provides a kit for inhibiting tumor
progression, comprising an antisense nucleic acid capable of
specifically hybridizing to the above-described nucleic acids.
[0145] This invention provides the above-described kit, wherein the
antisense nucleic acid is linked to a promoter.
[0146] This invention further provides the above-described kits,
wherein the antisense nucleic acid linked to a promoter is part of
an expression vector.
[0147] In addition, this invention provides the above-described
kits, wherein the expression vector is adapted for expression in a
mammalian cell.
[0148] This invention further provides a method of detecting
expression of the Progression Elevated Gene-3 in a sample which
contains cells comprising steps of: a) obtaining RNA from the
cells; b) contacting the nucleic acid so obtained with a labeled
form of the above-described nucleic acids under hybridizing
conditions; and c) detecting the presence of RNA hybridized to the
molecule, thereby detecting the expression of the Progression
Elevated Gene-3 in the sample.
[0149] This invention also provides the above-described method,
further comprising amplification of the RNA obtained in step a
prior to the contacting of step b.
[0150] In addition, this invention provides the above-described
methods, wherein the expression of Progression Elevated Gene-3 is
measured by the amount of Progression Elevated Gene-3 mRNA
expressed in the cells.
[0151] This invention provides the above-described methods, wherein
the expression of Progression Elevated Gene-3 is measured by the
amount of the Progression Elevated Gene-3 protein expressed in the
cells.
[0152] This invention provides a method for determining whether a
cancer cell is in a progression stage comprising measuring the
expression of Progression Elevated Gene-3 in the cancer cell,
wherein an increase in the amount indicates that the cancer cell is
in progression.
[0153] This invention provides a method for diagnosing the
aggressiveness of cancer cells comprising measuring the expression
of Progression Elevated Gene-3 in the cancer cell, wherein an
increase in the amount of the expression indicates that the cancer
cell is more aggressive.
[0154] This invention further provides a method of monitoring tumor
progression in a subject, comprising: a) obtaining at least one
nucleic acid sample from the subject; and b) determining the
quantity of the above-described nucleic acids in the nucleic acid
sample.
[0155] This invention provides a method of monitoring DNA damage in
a subject, comprising: a) obtaining at least one nucleic acid
sample from the subject; and b) determining the quantity of the
above-described nucleic acids in the nucleic acid sample.
[0156] In addition, this invention provides the above-described
methods, wherein the quantity of nucleic acid positively correlates
with transformation progression.
[0157] This invention also provides the above-described methods,
wherein two or more nucleic acid samples are taken from the subject
at different times.
[0158] This invention also provides the above-described methods,
further comprising using the nucleic acid samples in determining
differences in the expression of the above-described nucleic acids
over time.
[0159] This invention further provides a kit for diagnosing tumor
progression, comprising a nucleic acid consisting of a sequence of
15 or more nucleotides that specifically hybridizes to the
above-described nucleic acids.
[0160] In addition, this invention also provides a kit for
diagnosing tumor progression, comprising an antibody to the
above-described polypeptides.
[0161] This invention provides a method for determining whether
cells are in progression, comprising the steps of: a) measuring
expression of PEG-3 in a sample of cells; and b) comparing the
expression measured in step a with the expression of PEG-3 in cells
that are not in progression, thereby determining whether the cells
are in progression.
[0162] This invention further provides a method for determining
whether a cancer in a patient is in progression, comprising
detecting in a biological sample obtained from the patient the
above-described polypeptides, thereby determining whether a cancer
in the patient is in progression.
[0163] A method for determining whether a cancer in a patient is in
progression, comprising detecting, in a biological sample obtained
from the patient, a nucleic acid encoding the above-described
polypeptides or a portion thereof, thereby determining whether a
cancer in the patient is in progression.
[0164] This invention provides the above-described methods, wherein
the detecting comprises: preparing cDNA from RNA molecules in the
biological sample; and specifically amplifying cDNA molecules
encoding at least a portion of the above-described
polypeptides.
[0165] This invention also provides a method for monitoring the
progression of a cancer in a patient, comprising: a) detecting, in
a biological sample obtained from a patient, the above-described
polypeptides at a first point in time; b) repeating step (a) at a
subsequent point in time; and c) comparing the amounts of
polypeptide detected in steps a and b, thereby monitoring the
progression of a cancer in the patient.
[0166] This invention further provides the above-described methods,
wherein the step of detecting comprises contacting a portion of the
biological sample with a monoclonal antibody that specifically
recognizes the above-described polypeptides.
[0167] This invention provides the above-described methods, wherein
the biological sample is a portion of a tumor.
[0168] This invention provides a method for monitoring the
progression of a cancer in a patient, comprising: a) detecting, in
a biological sample obtained from a patient, an amount of an RNA
molecule encoding the above-described polypeptides at a first point
in time; b) repeating step a at a subsequent point in time; and c)
comparing the amounts of RNA molecules detected in steps a and b,
thereby monitoring the progression of a cancer in the patient.
[0169] This invention also provides a diagnostic kit, comprising:
a) the above-described antibody or fragment thereof; and b) a
second antibody or fragment thereof that binds to (i) the
monoclonal antibody recited in step a; or (ii) the above-described
polypeptide; wherein the second monoclonal antibody is conjugated
to a reporter group.
[0170] This invention provides a cell comprising an exogenous
indicator gene under the control of the regulatory element of a
Progression Elevated Gene-3.
[0171] This invention further provides above-described cell,
wherein the cell is an E11 or CRF.
[0172] This invention further provides above-described cell,
wherein the cell is not at progression.
[0173] In addition, this invention provides above-described cell,
wherein the cell is an E11-NMT, CREF-ras, CREF-HPV, or CREF-src
cell.
[0174] This invention also provides above-described cell, wherein
the cell is at progression.
[0175] This invention provides above-described cell, wherein the
indicator gene encodes beta-galactosidase, luciferase,
chloramphenicol transferase or a secreted alkaline phosphatase.
[0176] This invention further provides a method for determining
whether an agent is capable of inhibiting DNA damage and repair
pathways, cancer progression or oncogene mediated transformation,
comprising contacting the agent with the above-described cells,
wherein a decrease of expression of the indicator gene indicates
that the agent is capable of inhibiting DNA damage and repair
pathways, cancer progression or oncogene mediated
transformation.
[0177] This invention also provides a method for determining
whether an agent is capable of inducing DNA damage and repair
pathways, cancer progression or oncogene mediated transformation,
comprising contacting the agent with the above-described cells,
wherein an increase of expression of the indicator gene after the
contact indicates that the agent is capable of inducing DNA damage
and repair pathways, cancer progression or oncogene mediated
transformation.
[0178] This invention provides a method for identifying an agent
that modulates the expression of PEG-3, comprising: a) contacting a
candidate agent with a cell transformed or transfected with a
reporter gene under the control of a PEG-3 promoter or a regulatory
element thereof under conditions and for a time sufficient to allow
the candidate agent to directly or indirectly alter expression of
the promoter or regulatory element thereof; and b) determining the
effect of the candidate agent on the level of reporter protein
produced by the cell, thereby identifying an agent that modulates
expression of PEG-3.
[0179] This invention also provides the above-described method,
wherein the agent activates the PEG-3 promoter indirectly by
interacting with an oncogene.
[0180] This invention further provides a method for identifying an
agent that modulates the ability of PEG-3 to induce progression,
comprising: a) contacting a candidate agent with the
above-described polypeptides, under conditions and for a time
sufficient to allow the candidate agent and polypeptide to
interact; and b) determining the effect of the candidate agent on
the ability of the polypeptide to induce progression, thereby
identifying an agent that modulates the ability of PEG-3 to induce
progression.
[0181] This invention also provides a cell comprising the
above-described nucleic acids encoding PEG-3 linked to a tissue
specific promoter.
[0182] This invention provides a cell comprising a reporter gene
linked to a PEG-3 promoter.
[0183] In addition, this invention provides the above-described
cells, wherein the reporter gene encodes luciferase or beta
galactosidase.
[0184] This invention further provides the above-described cell,
wherein the cell comprises 4NMT or tumorigenic CREF-Trans 6
cells.
[0185] This invention provides the above-described cell, wherein
the cell comprises CREF-ras, CREF-src, or CREF-HPV cells.
[0186] This invention further provides the above-described cell,
wherein the reporter gene encodes a cell surface protein.
[0187] This invention provides the above-described cell, wherein
the cell surface protein is in a position accessible for binding to
an antibody.
[0188] This invention provides a transgenic animal, comprising the
above-described cells.
[0189] This invention further provides the use of the
above-described cells to identify compounds that induce DNA
damage.
[0190] This invention also provides the use of the above-described
cells to identify compounds that induce cancer progression.
[0191] This invention provides the use of the above-described cells
to identify compounds that induce oncogenic transformation.
[0192] In addition, this invention provides the use of the
above-described cells to identify compounds that induce or inhibit
angiogenesis.
[0193] This invention further provides a method of identifying
compounds that induce oncogenic transformation, comprising:
exposing the above-described cells to the compound and identifying
compounds that activate the PEG-3 promoter.
[0194] This invention further provides a method of identifying
compounds that induce DNA damage, comprising: exposing the
above-described cells to the compound and identifying compounds
that activate the PEG-3 promoter.
[0195] This invention provides a method of identifying compounds
that regulate angiogenesis, comprising: exposing the
above-described cells to the compound and identifying compounds
that affect the activity of the PEG-3 promoter.
[0196] This invention provides above-described method, wherein the
activity of the PEG-3 promoter is monitor by assessing the level of
expression of the reporter gene.
[0197] This invention further provides above-described method,
wherein cells are plated in microtiter plates for rapid
screening.
[0198] This invention provides above-described method, wherein the
cell is obtained from a transgenic animal and exposed to the
compound in vitro.
[0199] This invention also provides above-described method, wherein
the cell is obtained by transfection or transformation and exposed
to the compound in vitro.
[0200] This invention further provides a method of identifying
compounds that induce oncogenic transformation, comprising:
exposing the above-described transgenic animal to the compound and
identifying compounds that activate the PEG-3 promoter.
[0201] In addition, this invention provides a method of identifying
compounds that induce DNA damage, comprising: exposing the
above-described transgenic animal to the compound and identifying
compounds that activate the PEG-3 promoter.
[0202] This invention further provides a method of identifying
compounds that regulate angiogenesis, comprising: exposing the
above-described transgenic animal to the compound and identifying
compounds that affect the activity of the PEG-3 promoter.
[0203] This invention provides the above-described methods, wherein
the activity of the PEG-3 promoter is monitor by assessing the
level of expression of the reporter gene.
[0204] This invention also provides a method of producing a
Progression Elevated Gene-3 protein which comprises growing the
above-described vector under conditions permitting production of
the protein and recovering the protein so produced.
[0205] This invention further provides a pharmaceutical composition
for reversing the progression state of cells comprising an amount
of the above-described nucleic acids effective to inhibit the
expression of Progression Elevated Gene-3 and a pharmaceutically
acceptable carrier.
[0206] This invention provides a pharmaceutical composition for
reversing the progression state of cells comprising an amount of
the above-described antibody or a functional fragment thereof
effective to neutralize the action of the Progression Elevated
Gene-3 protein and a pharmaceutically acceptable carrier.
[0207] This invention further provides a method for producing cells
which are resistant to progression comprising inhibiting or
eliminating the expression of Progression Elevated Gene-3 in the
cells.
[0208] In addition, this invention provides the cells resulting
from the above-described methods.
[0209] This invention provides a transgenic nonhuman living
organism expressing the above-described polypeptides.
[0210] In addition, this invention provides above-described
transgenic, wherein the organism is an animal.
[0211] This invention further provides a pharmaceutical
composition, comprising: a) the above-described polypeptides; and
b) a physiologically acceptable carrier.
[0212] This invention provides a vaccine, comprising: a) the
above-described polypeptides; and b) an immune response
enhancer.
[0213] This invention further provides a pharmaceutical
composition, comprising: a) the above-described nucleic acids; and
b) a physiologically acceptable carrier.
[0214] This invention provides a pharmaceutical composition,
comprising: a) the above-described antibody; and b) a
physiologically acceptable carrier.
[0215] This invention further provides a method for inhibiting the
progression of a cancer in a subject, comprising administering to
the subject an agent that inhibits expression of PGEN-3.
[0216] This invention provides the above-described methods, wherein
PGEN-3 is one of the above-described polypeptides.
[0217] This invention provides the above-described methods, wherein
agent is one of the above-described nucleic acids.
[0218] In addition, this invention provides a method for preparing
the above-described polypeptides, comprising the steps of: a)
culturing one of the above-described host cells under conditions
suitable for the expression of the polypeptide; and b) recovering
the polypeptide from the host cell culture.
[0219] This invention further provides a method for producing cells
that are resistant to progression, comprising inhibiting or
eliminating the expression of a PEG-3 gene in the cells.
[0220] This invention further provides a method for protecting
cells from chemotherapeutic damage, comprising inhibiting or
eliminating the expression of PEG-3 in the cells.
[0221] This invention provides a cell transformed or transfected
with a reporter gene under the control of a human PEG-3 promoter or
regulatory element thereof.
[0222] This invention provides the above-described polypeptides,
wherein the progression phenotype comprises anchorage-independent
growth, tumorigenesis, angiogenesis, or metastasis.
[0223] This invention further provides the above-described methods,
wherein the cancer is melanoma.
[0224] This invention provides the above-described methods, wherein
the cancer is brain cancer.
[0225] This invention further provides the above-described methods,
wherein the cancer is cervical cancer.
[0226] This invention provides the above-described methods, wherein
the cancer is prostate cancer.
[0227] In addition, this invention provides the above-described
methods, wherein the cancer is breast cancer.
[0228] This invention further provides the above-described methods,
wherein the cancer is nasal pharyngeal cancer.
[0229] In addition, this invention provides the above-described
methods, wherein the cancer is neoblastoma multiforme cancer.
[0230] Methods to introduce a nucleic acid molecule into cells have
been well known in the art. Naked nucleic acid molecule may be
introduced into the cell by direct transformation. Alternatively,
the nucleic acid molecule may be embedded in liposomes.
Accordingly, this invention provides the above methods wherein the
nucleic acid is introduced into the cells by naked DNA technology,
adenovirus vector, adeno-associated virus vector, Epstein-Barr
virus vector, Herpes virus vector, attenuated HIV vector,
retroviral vectors, vaccinia virus vector, liposomes,
antibody-coated liposomes, mechanical or electrical means. The
above recited methods are merely served as examples for feasible
means of introduction of the nucleic acid into cells. Other methods
known may be also be used in this invention.
[0231] This invention further provides the above-described methods,
wherein the cancer is melanoma.
[0232] This invention provides the above-described methods, wherein
the cancer is epithelial cancer.
[0233] This invention provides the above-described methods, wherein
the epithelial cancer is brain, breast, cervical, prostate, lung or
colorectal cancer.
[0234] In addition, this invention further provides the
above-described methods, wherein the cancer is derived from a
central nervous system tumor.
[0235] This invention provides the above-described methods, wherein
the central nervous system tumor comprises a neuroblastoma or
glioblastoma cancer.
[0236] This invention further provides the above-described methods,
wherein the cell comprises an endothelial cells.
[0237] In addition, this invention provides the above-described
methods, wherein endothelial cell growth or proliferation is
induced.
[0238] This invention further provides the above-described methods,
wherein endothelial cell growth or proliferation is inhibited.
[0239] This invention provides an inducible PEG-3 regulatory region
functionally linked to a gene encoding a product that causes or may
be induced to cause the death or inhibition of cancer cells.
[0240] This invention further provides an inducible PEG-3
regulatory region functionally linked to a gene encoding a product
that causes or may be induced to cause the death or inhibition of
cancer cell growth.
[0241] This invention also provides a vector suitable for
introduction into a cell, comprising: a) an inducible PEG-3
regulatory region; and b) a gene encoding a product that causes or
may be induced to cause the death or inhibition of cancer cell
growth.
[0242] Numerous vectors for expressing the inventive proteins may
be employed. Such vectors, including plasmid vectors, cosmid
vectors, bacteriophage vectors and other viruses, are well known in
the art. For example, one class of vectors utilizes DNA elements
which are derived from animal viruses such as bovine papilloma
virus, polyoma virus, adenovirus, vaccinia virus, baculovirus,
retroviruses (RSV, MMTV or MOMLV), Semliki Forest virus or SV40
virus. Additionally, cells which have stably integrated the DNA
into their chromosomes may be selected by introducing one or more
markers which allow for the selection of transfected host cells.
The markers may provide, for example, prototrophy to an auxotrophic
host, biocide resistance or resistance to heavy metals such as
copper. The selectable marker gene can be either directly linked to
the DNA sequences to be expressed, or introduced into the same cell
by cotransformation.
[0243] Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation
sequences for ribosome binding. Additional elements may also be
needed for optimal synthesis of mRNA. These additional elements may
include splice signals, as well as enhancers and termination
signals. For example, a bacterial expression vector includes a
promoter such as the lac promoter and for transcription initiation
the Shine-Dalgarno sequence and the start codon AUG. Similarly, a
eukaryotic expression vector includes a heterologous or homologous
promoter for RNA polymerase II, a downstream polyadenylation
signal, the start codon AUG, and a termination codon for detachment
of the ribosome. Such vectors may be obtained commercially or
assembled from the sequences described by methods well known in the
art, for example the methods described above for constructing
vectors in general.
[0244] These vectors may be introduced into a suitable host cell to
form a host vector system for producing the inventive proteins.
Methods of making host vector systems are well known to those
skilled in the art.
[0245] Suitable host cells include, but are not limited to,
bacterial cells (including gram positive cells), yeast cells,
fungal cells, insect cells and animal cells. Suitable animal cells
include, but are not limited to HeLa cells, Cos cells, CvI cells
and various primary mammalian cells. Numerous mammalian cells may
be used as hosts, including, but not limited to, the mouse
fibroblast cell N1H-3T3 cells, CHO cells, HeLa cells, Ltk.sup.-
cells and COS cells. Mammalian cells may be transfected by methods
well known in the art such as calcium phosphate precipitation,
electroporation and microinjection.
[0246] In an embodiment, inducible promoters may be fused with the
coding region of the DNA to provide an experimental means to
regulate expression. Alternatively or in addition, tissue specific
regulatory elements may be fused with the coding region to permit
tissue-specific expression.
[0247] In addition, this invention further provides the
above-described vectors, wherein the inducible PEG-3 regulatory
region is a promoter.
[0248] This invention also provides the above-described vectors,
wherein the inducible PEG-3 regulatory region is an enhancer.
[0249] This invention further provides the above-described vectors,
wherein the gene encodes an inducer of apoptosis.
[0250] In addition, this invention provides the above-described
vectors, wherein the gene is a tumor suppressor gene.
[0251] In an embodiment, tumor suppressors include agents that
inhibit tumor growth. In another embodiment, tumor suppressors
include agents that inhibit, reverse, or reduce the cancer
progression phenotype.
[0252] This invention also provides the above-described vectors,
wherein the tumor suppressor gene is p53.
[0253] In addition, this invention provides the above-described
vectors, wherein the tumor suppressor gene is mda-7.
[0254] This invention also provides the above-described vectors,
wherein the tumor suppressor gene is p21.
[0255] In addition, this invention provides the above-described
vectors wherein the gene encodes a viral replication protein.
[0256] This invention further provides the above-described vectors,
wherein the gene is E1A.
[0257] This invention also provides the above-described vectors,
wherein the gene is E1B.
[0258] This invention also further provides the above-described
vectors, wherein the gene encodes a product toxic to cells or an
intermediate to-a product toxic to cells.
[0259] In an embodiment, products toxic to cells include chemicals
that reduce a cells chances and/or duration of survival. In an
embodiment, the products toxic to cells are radioactive. In another
embodiment, the products toxic to cells induce DNA damage. In
another embodiment, the products toxic to cells inhibit a critical
enzyme or regulatory protein. In another embodiment, the products
toxic to cells contain or induce free radicals. One skilled in the
art would recognize a vast array of other products that are toxic
to cells.
[0260] Further, this invention provides the above-described
vectors, wherein the gene encodes thymidine kinase.
[0261] In addition, this invention provides the above-described
vectors, wherein the gene encodes a product causing enhanced immune
recognition of the cell.
[0262] Further, this invention provides the above-described
vectors, wherein the gene is GM-CSF.
[0263] This invention also provides the above-described vectors,
wherein the gene is IL-2.
[0264] This invention also provides the above-described vector,
wherein the gene encodes a cytokine.
[0265] Further, this invention provides the above-described vector,
wherein the cytokine is IF-gamma.
[0266] In addition, this invention provides the above-described
vector, wherein the cytokine is IL-6.
[0267] This invention also provides the above-described vector,
wherein the gene encodes an immunomodulator.
[0268] Further, this invention provides the above-described vector,
wherein the gene encodes a T-cell epitope.
[0269] This invention also provides the above-described vector,
wherein the gene encodes a T-cell reactive protein.
[0270] This invention further provides the above-described vectors,
wherein the gene encodes a product causing the cell to express a
specific antigen.
[0271] In an embodiment, the gene causes the cells to express an
antigen on their surface; thus, allowing the cells to be targeted
by antibodies specific to the antigen.
[0272] This invention also provides a method of treating cancer in
a subject, comprising: a) administering the one or more of the
above-described vectors to the subject; and b) administering
gancyclovir or acyclovir to the subject.
[0273] In addition, this invention provides a method of treating
cancer in a subject, comprising: a) administering one of the
above-described vectors to the subject; and b) administering an
antibody or a fragment of an antibody to the the above-described
antigen to the subject.
[0274] Further, this invention provides the above-described
methods, wherein the antibody is toxic or linked to a toxic
substance.
[0275] This invention also provides the above-described methods,
wherein the antibody is labeled and used for tumor imaging.
[0276] Methods of labeling include, but are not limited to,
radioactive labeling; enzymatic labeling, wherein the enzyme
directly or indirectly produces a detectable product; and
fluorescent labeling.
[0277] Further, this invention provides the above-described
methods, wherein the antibody is radioactive.
[0278] In addition, this invention provides a pharmaceutical
composition comprising one or more of the the above-described
vectors and a carrier.
[0279] Pharmaceutically acceptable carriers are well known to those
skilled in the art and include, but are not limited to, 0.01-0.1M
and preferably 0.05M phosphate buffer or 0.8% saline. Additionally,
such pharmaceutically acceptable carriers may be aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers such as those
based on Ringer's dextrose, and the like. Preservatives and other
additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases and the
like.
[0280] This invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative of the invention as described more fully in the
claims which follow thereafter.
[0281] Experimental Details
[0282] Cancer is a progressive multigenic disorder characterized by
defined changes in the transformed phenotype that culminates in
metastatic disease. Determining the molecular basis of progression
should lead to new opportunities for improved diagnostic and
therapeutic modalities. Through the use of subtraction
hybridization, a gene associated with transformation progression in
virus and oncogene transformed rat embryo cells, progression
elevated gene-3 (PEG-3), has been cloned. PEG-3 shares significant
nucleotide and amino acid sequence homology with the hamster growth
arrest and DNA damage inducible gene gadd34 and a homologous murine
gene, MyD116, that is induced during induction of terminal
differentiation by interleukin-6 in murine myeloid leukemia cells.
PEG-3 expression is elevated in rodent cells displaying a
progressed transformed phenotype and in rodent cells transformed by
various oncogenes, including Ha-ras, v-src, mutant type 5
adenovirus (Ad5) and human papilloma virus-18. The PEG-3 gene is
transcriptionally activated in rodent cells, as is gadd34 and
MyD116, after treatment with DNA damaging agents, including methyl
methanesulfonate and gamma irradiation. In contrast, only PEG-3 is
transcriptionally active in rodent cells displaying a progressed
phenotype. Although transfection of PEG-3 into normal and
AdS-transformed cells only marginally suppresses colony formation,
stable overexpression of PEG-3 in Ad5-transformed rat embryo cells
elicits the progression phenotype. These results indicate that
PEG-3 is a new member of the gadd and MyD gene family with similar
yet distinct properties and this gene may directly contribute to
the transformation progression phenotype. Moreover, these studies
support the hypothesis that constitutive expression of a DNA damage
response may mediate cancer progression.
[0283] First Series of Experiments
[0284] Materials and Methods
[0285] Cell Lines, Culture Conditions and Anchorage-Independent
Growth Assays. The isolation, properties and growth conditions of
the E11, E11-NMT, E11-NMT.times.CREF somatic cell hybrids,
E11.times.E11-NMT somatic cell hybrids and the E11-NMT AZA clones
have been described (1,7-13). E11-ras R12 and E11-HPV E6/E7 clones
were isolated by transfection with the Ha-ras or the HPV-18 E6/E7
genes, respectively. The isolation, properties and growth
conditions of CREF, CREF-H5hr1 A2, CREF-ras, the CREF-ras/Krev1 B1,
B1 T and B1 M and the CREF-ras/Krev1 B2, B2 T, and B2 M clones have
been described (21). CREF-src and CREF-HPV 18 clones were isolated
by transfection with the v-src and HPV-18 E6/E7 genes,
respectively. All cells were grown in Dulbecco's modified Eagle's
minimum essential medium supplemented with 5% fetal bovine serum at
37.degree. C. in a 5% CO.sub.2 plus 95% air humidified incubator.
Anchorage independence assays were performed by seeding various
cell densities in 0.4% Noble agar on a 0.8% agar base layer both of
which contain growth medium (7).
[0286] Cloning and Sequencing of the PEG-3 cDNA. The PEG-3 gene was
cloned from E11-NMT cells using subtraction hybridization as
described (23). A full-length PEG-3 cDNA was obtained using the
rapid amplification of cDNA end (RACE) procedure and direct
ligation (25,26). Sequencing was performed by the dideoxy-chain
termination (Sanger) method (27). The coding region of PEG-3 was
cloned into a pZeoSV vector (Invitrogen) as described (25,26).
[0287] RNA Analysis and In Vitro Transcription Assays. Total
cellular RNA was isolated by the guanidinium/phenol extraction
method and Northern blotting was performed as described (28).
Fifteen .mu.g of RNA were denatured with glyoxal/DMSO and
electrophoresed in 1% agarose gels, transferred to nylon membranes
and hybridized sequentially with .sup.32P-labeled PEG-3, Ad5 E1A
and GAPDH probes (28,29). Following hybridization, the filters were
washed and exposed for autoradiography. The transcription rates of
PEG-3, gadd34, MyD116, GAPDH and pBR322 was determined by nuclear
run-on assays (12,21).
[0288] In Vitro Translation of PEG-3. Plasmid, pZeoSV, containing
PEG-3 cDNA was linearized by digestion with Xho I and used as a
template to synthesize mRNA. In vitro translation of PEG-3 mRNA was
performed with a rabbit relticulocyte lysate translation kit as
described by Promega.
[0289] DNA Transfection Assays. To study the effect of PEG-3 on
monolayer colony formation the vector (pZeoSV) containing no insert
or a pZeoSV-PEG-3 construct containing the PEG-3 coding region were
transfected into the various cell types by the lipofectin method
(GIBCO/BRL) and Zeocin resistant clones were isolated or efficiency
of Zeocin colony formation was determined (29,30).
[0290] Results and Discussion
[0291] Expression of the PEG-3 Gene Correlates Directly with the
Progression Phenotype in Viral and Oncogene Transformed Rodent
Cells. A critical component of cancer development is progression, a
process by which a tumor cell develops either qualitatively new
properties or displays an increase in the expression of traits that
enhance the aggressiveness of a tumor (1-4). Insight into this
process offers the potential of providing important new targets for
intervening in the neoplastic process (1-4). In the Ad5 transformed
RE cell culture model system, enhanced anchorage-independent growth
and in vivo tumorigenic aggressiveness, i.e., markers of the
progression phenotype, are stable traits that can be induced
spontaneously or by gene transfer (oncogenes and growth
factor-related genes) (Table 1).
1TABLE 1 Expression of PEG-3 in Ad5-transformed RE cells directly
correlates with expression of the progression phenotype Agar
Cloning Efficiency Tumorigenicity Progression Cell Type.sup.a
(%).sup.b in Nude Mice.sup.c Phenotype.sup.d RE <0.001 0/10
Prog.sup.- CREF <0.001 0/18 Prog.sup.- E11 2.9 .+-. 0.3 8/8 (36)
Prog.sup.- E11-NMT 34.3 .+-. 4.1 6/6 (20) Prog.sup.+ CREF .times.
E11-NMT 2.0 .+-. 0.3 0/6 Prog.sup.- F1 CREF .times. E11-NMT 1.5
.+-. 0.1 0/6 Prog.sup.- F2 CREF .times. E11-NMT 72.5 .+-. 9.4 3/3
(17) Prog.sup.+ R1 CREF .times. E11-NMT 57.4 .+-. 6.9 3/3 (17)
Prog.sup.+ R2 E11 .times. E11-NMT 5.6 .+-. 0.7 3/3 (56) Prog.sup.-
IIId E11 .times. E11-NMT 41.0 .+-. 4.9 3/3 (19) Prog.sup.+ IIIdTD
E11 .times. E11-NMT 0.3 .+-. 0.0 3/3 (44) Prog.sup.- A6 E11 .times.
E11-NMT 29.3 .+-. 3.5 N.T. Prog.sup.+ A6TD E11 .times. E11-NMT 1.5
.+-. 0.2 3/3 (31) Prog.sup.- 3b E11 .times. E11-NMT 29.5 .+-. 2.8
3/3 (23) Prog.sup.+ IIA E11-NMT AZA C1 2.8 .+-. 0.5 N.T. Prog.sup.-
E11-NMT AZA B1 1.6 .+-. 0.3 3/3 (41) Prog.sup.- E11-NMT AZA C2 2.0
.+-. 0.1 3/3 (50) Prog.sup.- E11-ras R12 36.8 .+-. 4.6 3/3 (18)
Prog.sup.+ E11-HPV E6/E7 31.7 .+-. 3.1 3/3 (22) Prog.sup.+
.sup.aCell line descriptions can be found in Materials and Methods.
.sup.bAnchorage-independent growth was determined by seeding
variable numbers of cells in 0.4% agar on a 0.8% agar base layer.
Results are the average number of colonies from 4 replicate plates
.+-. S.D. .sup.cTumorigenicity was determined by injecting nude
mice with 2 .times. 10.sup.6 or 1 .times. 10.sup.7(RE, CREF and
CREF .times. E11-NMT hybrids). Results are the number of animals
with tumors per number of animals injected and the number in
parentheses indicate average latency time in days, i.e., first
appearance of a palpable tumor. #N.T. = not tested. .sup.dProg =
progression phenotype is not expressed; Prog* = progression
phenotype is expressed.
[0292] Upon treatment of progressed cells with AZA, the progression
phenotype can be stably reversed (1,10). A reversion of progression
also occurs following somatic cell hybridization of progressed
cells with unprogressed Ads-transformed cells or with normal CREF
cells. A further selection of these unprogressed Ad5-transformed
cells by injection into nude mice results in acquisition of the
progressed phenotype following tumor formation and establishment in
cell culture. These studies document that progression in this model
system is a reversible process that can be stably produced by
appropriate cellular manipulation. In this context, the
Ad5-transformed RE model represents an important experimental tool
for identifying genes that are associated with and that mediate
cancer progression.
[0293] To directly isolate genes elevated during progression we
employed an efficient subtraction hybridization approach previously
used to clone the p21 gene (melanoma differentiation associated
gene-6; mda-6) (23,25) and a novel cancer growth suppressing gene
mda-7 (26,29). For this approach, cDNA libraries from a progressed
mutant Ad5 (H5ts125)-transformed RE clone, E11-NMT (10), and its
parental unprogressed cells, E11 (10,31), were directionally cloned
into the .lambda. Uni-ZAP phage vector and subtraction
hybridization was performed between double-stranded tester
(E11-NMT) and single-stranded driver DNA (E11) by mass excision of
the libraries (23). With this strategy in combination with the RACE
procedure and DNA ligation techniques a full-length PEG-3 cDNA
displaying elevated expression in E11-NMT versus E11 cells was
cloned. Northern blotting analysis indicates that PEG-3 expression
is .gtoreq.10-fold higher in all progressed Ad5-transformed RE
cells, including E11-NMT, specific E11-NMT.times.CREF somatic cell
hybrid clones, R1 and R2, expressing an aggressive transformed
phenotype and specific E11.times.E11-NMT somatic cell hybrid
clones, such as IIa that display the progression phenotype (FIG. 1
and Table 1). PEG-3 mRNA levels also increase following induction
of progression by stable expression of the Ha-ras and HPV-18 E6/E7
oncogenes in E11 cells (FIG. 1). A further correlation between
expression of PEG-3 and the progression phenotype is provided by
E11.times.E11-NMT clones, such as IIId and A6, that initially
display a suppression of the progression phenotype and low PEG-3
expression, but regain the progression phenotype and PEG-3
expression following tumor formation in nude mice, i.e., IIIdTD and
A6TD (Table 1 and FIG. 1). In contrast, unprogressed
Ad5-transformed cells, including E11, E11-NMT.times.CREF clones F1
and F2, E11.times.E11-NMT clones IIId, A6 and 3b and AZA-treated
E11-NMT clones B1, C1 and C2, have low levels of PEG-3 RNA. These
results provide evidence for a direct relationship between the
progression phenotype and PEG-3 expression in this Ad5-transformed
RE cell culture system. They also demonstrate that the final
cellular phenotype, i.e., enhanced anchorage-independence and
aggressive tumorigenic properties, is a more important determinant
of PEG-3 expression than is the agent (oncogene) or circumstance
(selection for tumor formation in nude mice) inducing
progression.
[0294] A second rodent model used to study the process of cancer
progression employs CREF clones modified by transfection to express
dominant acting oncogenes (such as Ha-ras, v-src, HPV-18 and the
mutant adenovirus H5hr1) and tumor suppressor genes (such as
Krev-1, RB and wild-type p53) (19-22 and unpublished data). In this
model system, Ha-ras-transformed CREF cells are morphologically
transformed, anchorage-independent and induce both tumors and lung
metastases in syngeneic rats and athymic nude mice (19-22). The
Krev-1 (Ha-ras) suppressor gene reverses the in vitro and in vivo
properties in Ha-ras transformed cells (21). Although suppression
is stable in vitro, Ha-ras/Krev-1 CREF cells induce both tumors and
metastases after extended times in nude mice (21). Expression of
PEG-3 is not apparent in CREF cells, whereas tumorigenic CREF cells
transformed by v-src, HPV-18, H5hr1 and Ha-ras contain high levels
of PEG-3 RNA (FIG. 2). Suppression of Ha-ras induced transformation
by Krev-1 inhibits PEG-3 expression. However, when Ha-ras/Krev-1
cells escape tumor suppression and form tumors and metastases in
nude mice, PEG-3 expression reappears, with higher expression in
metastatic-derived than tumor-derived clones (FIG. 2). These
findings provide further documentation of a direct relationship
between induction of a progressed and oncogenic phenotype in rodent
cells and PEG-3 expression. As indicated above, it is the phenotype
rather than the inducing agent that appears to be the primary
determinant of PEG-3 expression in rodent cells.
[0295] The PEG-3 Gene Displays Sequence Homology with the Hamster
gadd34 and Mouse MyD116 Genes and is Inducible by DNA Damage. The
cDNA sizes of PEG-3, gadd34 and MyD116 are 2210, 2088 and 2275 nt,
respectively. The nt sequence of PEG-3 is .about.73% and the aa
sequence is .about.59% homologous to the gadd34 (32) gene (FIG. 3
and data not shown). PEG-3 also shares significant sequence
homology, .about.68% nt and .about.72% aa, with the murine
homologue of gadd34, MyD116 (33,34) (FIG. 3 and data not shown).
Differences are apparent in the structure of the 3' untranslated
regions of PEG-3 versus gadd34/MyD116. ATTT motifs have been
associated with mRNA destabilization. In this context, the presence
of 3 ATTT sequences in Gadd34 and 6 tandem ATTT motifs in MyD116
would predict short half-lives for these messages. In contrast,
PEG-3 contains only 1 ATTT motif suggesting that this mRNA may be
more stable. The sequence homologies between PEG-3 and
gadd34/MyD116 are highest in the amino terminal region of their
encoded proteins, i.e., .about.69 and .about.76% homology with
gadd34 and Myd116, respectively, in the first 279 aa. In contrast,
the sequence of the carboxyl terminus of PEG-3 significantly
diverges from gadd34/Myd116, i.e., only .about.28 and .about.40%
homology in the carboxyl terminal 88 aa. In gadd34 and MyD116 a
series of similar 39 aa are repeated in the protein, including 3.5
repeats in gadd34 and 4.5 repeats in MyD116. In contrast, PEG-3
contains only 1 of these 39 aa regions, with 64% and 85% homology
to gadd34 and MyD116, respectively. On the basis of sequence
analysis, the PEG-3 gene should encode a protein of 457 aa with a
predicted MW of .about.50 kDa. To confirm this prediction, in vitro
translation analyses of proteins encoded by the PEG-3 cDNA were
determined (FIG. 4). A predominant protein after in vitro
translation of PEG-3 has a molecular mass of .about.50 kDa (FIG.
4). In contrast, gadd34 encodes a predicted protein of 589 aa with
an M.sub.w of .about.65 kDa and MyD116 encodes a predicted protein
of 657 aa with an M.sub.w of .about.72 kDa. The profound similarity
in the structure of PEG-3 versus gadd34/MyD116 cDNA and their
encoded proteins suggest that PEG-3 is a new member of this gene
family. Moreover, the alterations in the carboxyl terminus of PEG-3
may provide a functional basis for the different properties of this
gene versus gadd34/MyD116.
[0296] The specific role of the gadd34/MyD116 gene in cellular
physiology is not known. Like hamster gadd34 and its murine
homologue MyD116, PEG-3 steady-state mRNA and RNA transcriptional
levels are increased following DNA damage by methyl
methanesulfonate (MMS) and gamma irradiation (.gamma.IR) (FIGS. 2
and 5 and data not shown). In contrast, nuclear run-on assays
indicate that only the PEG-3 gene is transcriptionally active
(transcribed) as a function of transformation progression (FIG. 5).
This is apparent in CREF cells transformed by Ha-ras and in E11-NMT
and various E11-NMT subclones either expressing or not expressing
the progression phenotype (FIG. 5). The gadd34/MyD116 gene, as well
as the gadd45, MyD118 and gadd153 genes, encode acidic proteins
with very similar and unusual charge characteristics (24). PEG-3
also encodes a putative protein with acidic properties similar to
the gadd and MyD genes (FIG. 3). The carboxyl-terminal domain of
the murine MyD116 protein is homologous to the corresponding domain
of the herpes simplex virus 1 .gamma..sub.134.5 protein, that
prevents the premature shutoff of total protein synthesis in
infected human cells (35,36). Replacement of the carboxyl-terminal
domain of .gamma..sub.134.5 with the homologous region from MyD116
results in a restoration of function to the herpes viral genome,
i.e., prevention of early host shutoff of protein synthesis (36).
Although further studies are required, preliminary results indicate
that expression of a carboxyl terminus region of MyD116 results in
nuclear localization (36). Similarly, gadd45, gadd153 and MyD118
gene products are nuclear proteins (24,37). Moreover, both gadd45
and MyD118 interact with the DNA replication and repair protein
proliferating cell nuclear antigen (PCNA) and the cyclin-dependent
kinase inhibitor p21 (37). MyD118 and gadd45 also modestly
stimulate DNA repair in vitro (37). The carboxyl terminus of PEG-3
is significantly different than that of MyD116 (FIG. 3). Moreover,
the carboxyl-terminal domain region of homology between MyD116 and
the .gamma..sub.134.5 protein is not present in PEG-3. In this
context, the localization, protein interactions and properties of
PEG-3 may be distinct from gadd and MyD genes. Once antibodies with
the appropriate specificity are produced it will be possible to
define PEG-3 location within cells and identify potentially
important protein interactions mediating biological activity. This
information will prove useful in elucidating the function of the
PEG-3 gene in DNA damage response and cancer progression.
[0297] PEG-3 Lacks Potent Growth Suppressing Properties
Characteristic of the gadd and Myd Genes. An attribute shared by
the gadd and MyD genes is their ability to markedly suppress growth
when expressed in human and murine cells (24,37). When transiently
expressed in various human tumor cell lines, gadd34/MyD116 is
growth inhibitory and this gene can synergize with gadd45 or
gadd153 in suppressing cell growth (24). These results and those
discussed above suggest that gadd34/MyD116, gadd45, gadd153 and
MyD118, represent a novel class of mammalian genes encoding acidic
proteins that are regulated during DNA damage and stress and
involved in controlling cell growth (24,37). In this context, PEG-3
would appear to represent a paradox, since its expression is
elevated in cells displaying an in vivo proliferative advantage and
a progressed transformed and tumorigenic phenotype.
[0298] To determine the effect of PEG-3 on growth, E11 and E11-NMT
cells were transfected with the protein coding region of the PEG-3
gene cloned into a Zeocin expression vector, pZeoSV (FIG. 6). This
construct permits an evaluation of growth in Zeocin in the presence
and absence of PEG-3 expression. E11 and E11-NMT cells were also
transfected with the p21 (mda-6) and mda-7 genes, previously shown
to display growth inhibitory properties (25,26,29). Colony
formation in both E11 and E11-NMT cells is suppressed 10 to 20%,
whereas the relative colony formation following p21 (mda-6) and
mda-7 transfection is decreased by 40 to 58% (FIG. 6 and data not
shown). Colony formation is also reduced by 10 to 20% when PEG-3 is
transfected into CREF, normal human breast (HBL-100) and human
breast carcinoma (MCF-7 and T47D) cell lines (data not shown).
Although the gadd and MyD genes were not tested for growth
inhibition in E11 or E11-NMT cells, previous studies indicate
colony formation reductions of >50 to 75% in several cell types
transfected with gadd34, gadd45, gadd153, MyD116 or MyD118 (24,37).
The lack of dramatic growth suppressing effects of PEG-3 and its
direct association with the progression state suggest that this
gene may represent a unique member of this acidic protein gene
family that directly functions in regulating progression. This may
occur by constitutively inducing signals that would normally only
be generated during genomic stress. In this context, PEG-3 might
function to alter genomic stability and facilitate tumor
progression. This hypothesis is amenable to experimental
confirmation.
[0299] PEG-3 Induces a Progression Phenotype in Ad5-Transformed RE
Cells. An important question is whether PEG-3 expression simply
correlates with transformation progression or whether it can
directly contribute to this process. To distinguish between these
two possibilities we have determined the effect of stable elevated
expression of PEG-3 on expression of the progression phenotype in
E11 cells. E11 cells were transfected with a Zeocin expression
vector either containing or lacking the PEG-3 gene and random
colonies were isolated and evaluated for anchorage independent
growth (FIG. 7). A number of clones were identified that display a
5- to 9-fold increase in agar cloning efficiency in comparison with
E11 and E11-Zeocin vector transformed clones. To confirm that this
effect was indeed the result of elevated PEG-3 expression,
independent Zeocin resistant E11 clones either expressing or not
expressing the progression phenotype were analyzed for PEG-3 mRNA
expression (FIG. 8). This analysis indicates that elevated
anchorage-independence in the E11 clones correlates directly with
increased PEG-3 expression. In contrast, no change in Ad5 E1A or
GAPDH mRNA expression is detected in the different clones. These
findings demonstrate that PEG-3 can directly induce a progression
phenotype without altering expression of the Ad5 E1A transforming
gene. Further studies are required to define the precise mechanism
by which PEG-3 elicits this effect.
[0300] Cancer is a progressive disease characterized by the
accumulation of genetic alterations in an evolving tumor (1-6).
Recent studies provide compelling evidence that mutations in genes
involved in maintaining genomic stability, including DNA repair,
mismatch repair, DNA replication, microsattelite stability and
chromosomal segregation, may mediate the development of a mutator
phenotype by cancer cells, predisposing them to further mutations
resulting in tumor progression (38). Identification and
characterization of genes that can directly modify genomic
stability and induce tumor progression will provide significant
insights into cancer development and evolution. This information
would be of particular benefit in defining potentially novel
targets for intervening in the cancer process. Although the role of
PEG-3 in promoting the cancer phenotype remains to be defined, the
current studies suggest a potential causal link between
constitutive induction of DNA damage response pathways, that may
facilitate genomic instability, and cancer progression. In this
context, constitutive expression of PEG-3 in progressing tumors may
directly induce genomic instability or it may induce or amplify the
expression of down-stream genes involved in this process. Further
studies are clearly warranted and will help delineate the role of
an important gene, PEG-3, in cancer.
[0301] Conclusion
[0302] Subtraction hybridization results in the identification and
cloning of a gene PEG-3 with sequence homology and DNA damage
inducible properties similar to gadd34 and MyD116. However, PEG-3
expression is uniquely elevated in all cases of rodent progression
analyzed to date, including spontaneous and oncogene-mediated, and
overexpression of PEG-3 can induce a progression phenotype in
Ad5-transformed cells. Our studies suggest that PEG-3 may represent
an important gene that is both associated with (diagnostic) and
causally related to cancer progression. They also provide a
potential link between constitutive expression of a DNA damage
response pathway and progression of the transformed phenotype.
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[0341] Second Series of Experiments
[0342] Development of Biosensor Systems to Efficiently and
Selectively Detect Agents Inducing and Inhibiting DNA Damage
Pathways, Oncogenic Transformation and Cancer Progression
[0343] The PEG-3 gene is induced in a p53-independent manner in
E11, CREF and human melanoma cells following treatment with DNA
damaging agents, such as gamma irradiation (1 and unpublished
data). Nuclear run-on assays, that measure rates of gene
transcription, indicate that induction of PEG-3 by DNA damage and
expression of PEG-3 in cells displaying the progression phenotype
(such as E11-NMT and CREF cells transformed by various oncogenes)
involves elevated transcription of this gene (1). This data
supports the hypothesis that the appropriate transcriptional
regulating factors are inducible following DNA damage in cells and
they are constitutively expressed in progressed cells. Since
transcription of genes involves elements located in the promoter
region of genes, current data supports the hypothesis that the
promoter region of the PEG-3 gene is directly regulated as a
function of genotoxic stress, oncogenic transformation and during
cancer progression. This finding will be exploited by isolating the
promoter of PEG-3 (as described below), linking this DNA sequence
to a .beta.-galactosidase (.beta.-gal) reporter gene and
constructing cells that either constitutively express this reporter
gene (E11-NMT-.beta.-gal, CREF-ras-.beta.-gal and
CREF-src-.beta.-gal) or cells that contain a DNA damage inducible
reporter gene (E11-.beta.-gal and CREF-.beta.-gal). The
E11-NMT-.beta.-gal, CREF-ras-.beta.-gal and CREF-src-.beta.-gal
constructs can be used as sensitive and selective monitors for
agents that can inhibit DNA damage and repair pathways, cancer
progression and oncogene mediated transformation. Conversely, the
E11-.beta.-gal and CREF-.beta.-gal cell constructs can be used as
sensitive and selective monitors for conditions and agents that
induce DNA damage and repair pathways and may also induce the
progression and oncogene-mediated transformed phenotypes. The
ability to modify .beta.-gal expression, as a function of
activation or suppression of the PEG-3 promoter region or factors
that interact with the promoter region, can easily be assessed
using the appropriate substrate
(5-bromo-4-chloro-3-indolyl-beta-D-galact- o-pyranoside (X-gal)
that is converted into a final product (5-bromo-4-chloro-3-indole)
that has a blue color. E11-NMT-.beta.-gal cells will normally stain
blue following addition of the appropriate substrate. However,
should expression from the PEG-3 promoter region be suppressed this
will extinguish .beta.-gal expression thereby resulting in a loss
of blue staining following addition of the substrate. These rapid,
efficient and selective biosensor systems can easily be formatted
for the screening of an infinite number of compounds with potential
cancer progression suppression, oncogene suppression and DNA damage
inhibiting functions. E11-.beta.-gal and CREF-.beta.-gal cells will
normally not stain blue following addition of the substrate.
However, should the promoter region be activated, following
induction of appropriate DNA damage and repair pathways or
expression of specific oncogenes, the .beta.-gal gene will be
activated resulting in a blue stain following addition of the
substrate. These rapid, efficient and selective biosensor systems
can easily be formatted for the screening of an infinite number of
compounds with potential cancer progression, oncogene
transformation and DNA damage inducing properties. These model
systems will prove valuable in identifying agents and elucidating
pathways involved in cancer progression, oncogenic transformation
and DNA damage induction and repair. This should lead to the
development of novel therapeutics to prevent genomic damage and
instability, thereby inhibiting cancer progression and oncogene
mediated-transformation, and the identification of new classes of
agents that can prevent DNA damage and enhance DNA damage
repair.
[0344] Identification and Characterization of the Promoter Region
of PEG-3, Cis-Acting Regulatory Elements of the PEG-3 Promoter and
Trans-Acting Regulatory Elements That Activate (or Repress) PEG-3
Expression.
[0345] Overview. Nuclear run-on studies indicate that the PEG-3
gene is constitutively transcribed in progressed E11-NMT, CREF
cells treated with methyl methanesulfonate (MMS) or gamma
irradiation and in CREF-cells transformed by various oncogenes,
such as Ha-ras and v-src. Studies will, therefore, be conducted to
(i) clone the 5'-flanking region of the PEG-3 gene and analyze its
activity in E11 and E11-NMT, CREF and DNA damaged CREF and CREF
cells transformed by various oncogenes; (ii) identify cis-acting
regulatory elements in the promoter region of the PEG-3 gene which
are responsible for the differential induction of expression in the
different cell types and under different experimental conditions;
and (iii) identify and characterize trans-acting regulatory
elements which activate (or repress) expression of the PEG-3
gene.
[0346] To elucidate the mechanism underlying the transcriptional
regulation of the PEG-3 gene the 5'-flanking region of this gene
will be analyzed. This will be important for studies determining
regulatory control of the PEG-3 gene including autoregulation,
developmental regulation, tissue and cell type specific expression,
DNA damage induction and differential expression in cells
displaying a progressed cancer phenotype. The isolation of the
promoter region will also be necessary for creating a biosensor
model for monitoring and analyzing factors involved in mediating
DNA damage and repair and oncogenic transformation and cancer
progression. Once the appropriate sequence of the PEG-3 gene
regulating the initiation of transcription has been identified,
studies can be conducted to determine relevant trans-acting
regulatory factors that bind to specific cis-acting regulatory
elements and activate or repress the expression of the PEG-3 gene.
These molecules may provide important clues for understanding the
pathways governing DNA damage and repair mechanisms underlying
cancer progression. Ultimately, such an understanding may uncover
important targets for directly modifying and intervening in these
phenotypes and processes.
[0347] Cloning the promoter region of the PEG-3 gene and testing
its function. To identify the promoter region of PEG-3 we have used
a human PromoterFinder.TM. DNA Walking Kit (Clontech) (2,3), This
PCR-based method facilitates the cloning of unknown genomic DNA
sequences adjacent to a known cDNA sequence. Using this approach an
.about.2 kb fragment of PEG-3 that may contain the promoter region
of this gene has been isolated. The putative 5' flanking-region of
PEG-3 has been subcloned into the pBluescript vector and sequenced
by the Sanger dideoxynucleotide procedure. To verify the
transcriptional start site deduced from the cDNA, primer extension
analysis will be performed (4). In case of the identification of
multiple putative ATG or start sites RNase protection assays will
be performed using oligonucleotides spanning the 5' end of the
PEG-3 cDNA sequence (4,5). To define the boundary of the PEG-3
promoter region, a heterologous expression system containing a
bacterial chloramphenicol acetyltransferase (CAT) or luciferase
gene without promoter or enhancer will be employed (4,5,6).
Putative promoter inserts of varying sizes will be subcloned into a
CAT expression vector (6,7). Internal deletion constructs will be
generated by taking advantage of either internal restriction sites
or by partial digestion of internal sites if these sites are not
unique. These constructs will be transfected into E11-NMT cells
that display high levels of PEG-3 expression. The CAT construct
with minimal 5'-flanking region showing the highest degree of
expression will be identified as the PEG-3 gene promoter.
[0348] The functionality of the PEG-3 promoter will be determined
in E11-NMT, CREF cells treated with MMS and gamma irradiation and
CREF cells transformed by the Ha-ras and v-src oncogenes. Various
CAT constructs will be transfected into these cell lines by the
lipofectin method or electroporation (Gene Pulser, Bio-Rad) as
previously described (4,8). To correct for DNA uptake and cell
number used for each transfection experiment, the CAT constructs
will be cotransfected with plasmids containing bacterial .beta.-gal
gene under the control of an Rous sarcoma virus (RSV) promoter. The
CAT and .beta.-galactosidase enzymatic activities will be
determined using standard protocols (4,6,7). Minimal 5'-flanking
region displaying the highest CAT activity will be identified as
the promoter region for that tissue cell type or experimental
condition. If no induction of CAT activity is apparent, further
subcloning and screening of cosmid or phage clones would be
performed until a PEG-3 promoter of sufficient length to mediate
CAT induction in E11-NMT, CREF cells treated with MMS and gamma
irradiation and CREF cells transformed by the Ha-ras and v-src
oncogenes is obtained.
[0349] Once the promoter of PEG-3 is identified it will be
subcloned into a vector adjacent to a bacterial .beta.-gal gene,
PEG-3-Prom-.beta.-gal fusion (4). This construct will allow
activation of the .beta.-gal gene as a function of transcription
from the PEG-3 promoter. The vector construct will also contain a
bacterial antibiotic resistance gene, such as the neomycin or
hygromycin gene, that will permit selection of cells containing the
PEG-3-Prom-.beta.-gal fusion. This vector will be transfected into
E11, E11-NMT, CREF and CREF cells transformed by Ha-ras and v-src
and antibiotic resistant colonies will be selected in G418
(neomycin gene) or hygromycin (hygromycin gene) as previously
described (1,8,9). Antibiotic resistant colonies will be isolated
and maintained as independent cell lines. Clones constitutively
expressing the PEG-3-Prom-.beta.-gal gene (E11-NMT and CREF cells
transformed by the Ha-ras and v-src oncogenes) will be identified
by growth in the appropriate substrate resulting in a blue color.
Similarly, clones containing an inducible PEG-3-Prom-.beta.-gal
gene (E11 and CREF cells) will be identified by treating cells with
MMS or gamma irradiation, incubation in the appropriate substrate
and identifying clones that develop a blue color. Clones displaying
the appropriate properties will be further characterized by
Southern blotting (DNA organization) and Northern blotting (RNA
expression). Clones with constitutive or inducible .beta.-gal
expression will then be tested for alteration in expression as a
function of culture conditions (low serum, high cell density,
etc.), exposure to various DNA damaging agents, incubation in
agents known to specifically inhibit or enhance oncogene and
progression phenotypes (such as caffeic acid phenethyl ester,
phorbol ester tumor promoters, farnesyl transferase inhibitors,
etc.), chemotherapeutic agents, viral infection, etc. These studies
will provide useful baseline information as to the potential use of
the specific constructs as biosensor monitors for agents capable of
modifying cancer progression, oncogenic transformation and DNA
damage and repair pathways.
[0350] Identifying cis-acting elements in the PEG-3 promoter
responsible for expression in progressed cancer cells, oncogene
transformed CREF cells and DNA damaged cells. Once a functional
PEG-3 promoter has been identified studies will be conducted to
locate cis-acting elements responsible for expression of PEG-3 in
E11-NMT, oncogene transformed CREF (Ha-ras and v-src) and MMS
treated CREF cells. To identify cis-acting DNA sequences, the DNA
fragment displaying maximal promoter function in a transient
transfection assay in E11-NMT, oncogene transformed CREF and MMS
treated CREF cells will be sequenced. Potential regulatory elements
will be defined by comparison to previously characterized
transcriptional motifs. The importance of these sequences in
regulating PEG-3 expression will be determined by introducing point
mutations in a specific cis element into the promoter region using
previously described site-specific mutagenesis techniques (4,9-12)
or with recently described PCR-based strategies, i.e., ExSite.TM.
PCR-based site-directed mutagenesis kit and the Chameleon.TM.
double-stranded site-directed mutagenesis kit (Stratagene, CA). The
mutated promoter constructs will be cloned into CAT expression
vectors and tested for their effects on the promoter function by
transfecting into different cell types displaying CAT activity. If
increased detection sensitivity is required, the various promoter
region mutants will be subcloned into a luciferase reporter
construct (7).
[0351] Identifying trans-acting nuclear proteins that mediate
transcriptional enhancing activity of the PEG-3 in progressed
cancer cells, oncogene transformed CREF cells and in DNA damaged
CREF cells. The current view on regulation of eukaryotic gene
expression suggests that trans-acting proteins bind to specific
sites within cis-elements of a promoter region resulting in
transcriptional activation (13,14). Experiments will be performed
to identify trans-acting factors (nuclear proteins) and determine
where these factors interact with cis-regulatory elements. To
achieve this goal, two types of studies will be performed, one
involving gel retardation (gel shift) assays (4,15-17) and the
second involving DNase-I footprinting (methylation interference)
assays (4).
[0352] Gel shift assays will be used to analyze the interactions
between cis-acting elements in the PEG-3 promoter and trans-acting
factors in mediating transcriptional control (4,15-17). To begin to
identify the trans-acting factors, different non-labeled DNAs
(including TATA, CAT, TRE, Sp-I binding site, NF.kappa.B, CREB,
TRE, TBP, etc.) can be used as competitors in the gel shift assay
to determine the relationship between the trans-acting factors and
other previously identified transcriptional regulators. It is
possible that the trans-acting factors regulating transcriptional
control of the PEG-3 gene may be novel. To identify these factors
extracts will be purified from E11-NMT cells by two cycles of
heparin-Sepharose column chromatography, two cycles of DNA affinity
chromatography and separation on SDS-polyacrylamide gels (18,19).
Proteins displaying appropriate activity using gel shift assays
will be digested in situ with trypsin, the peptides separated by
HPLC and the peptides sequenced (20). Peptide sequences will be
used to synthesize degenerate primers and RT-PCR will be used to
identify putative genes encoding the trans-acting factor. These
partial sequences will be used with cDNA library screening
approaches and the RACE procedure, if necessary, to identify
full-length cDNAs encoding the trans-acting factors (21-23). Once
identified, the role of the trans-acting factors in eliciting PEG-3
induction following DNA damage in CREF and constitutive expression
in E11-NMT, CREF-ras and CREF-src cells will be determined.
[0353] The functionality of positive and negative trans-acting
factors will be determined by transiently and stably expressing
these genes in E11 and E11-NMT cells to determine effects on the
progression phenotype, CREF and CREF-ras and CREF-src cells to
determine effects on oncogene transformation and in CREF and MMS
treated CREF cells to determine the effects of DNA damage on PEG-3
induction. Positive effects would be indicated if overexpressing a
positive trans-acting factor facilitates progression, expression of
the oncogenic phenotype and/or a DNA-damage inducible response,
whereas overexpressing a negative trans-acting factor inhibits
progression, oncogene transformation and/or a DNA-damage inducible
response.
[0354] Antisense approaches will be used to determine if blocking
the expression of positive or negative trans-acting factors can
directly modify progression, oncogenic transformation and/or DNA
damage repair pathways. A direct effect of positive trans-acting
factors in affecting cellular phenotype would be suggested if
antisense inhibition of the positive acting factor partially or
completely inhibits the progression and oncogene transformation
phenotypes and/or DNA-damage and repair pathways. Conversely, a
direct effect of negative trans-acting factors in inhibiting
expression of PEG-3 and progression, oncogene transformation and/or
DNA-damage and repair pathways would be suggested if antisense
inhibition of the negative factor facilitates PEG-3 expression and
the progression, oncogene transformation and/or DNA-damage
inducible response pathways. Depending on the results obtained,
cis-element knockouts could be used to further define the role of
these elements in regulating PEG-3 expression.
[0355] For DNase-I footprinting assays, nuclear extracts from E11,
E11-NMT, CREF, CREF-ras, CREF-src and MMS treated CREF cells will
be prepared and DNase-I footprinting assays will be performed as
described (4,6). The differential protection between nuclear
extracts from E11-NMT and E11 and MMS treated CREF, CREF-ras and
CREF-src cells will provide relevant information concerning the
involvement of trans-acting factors in activation and the location
of specific sequences in the cis-regulatory elements of the PEG-3
promoter mediating this activation. If differential protection is
not detected using this approach, the sensitivity of the procedure
can be improved by using different sized DNA fragments from the
PEG-3 promoter region or by using partially purified nuclear
extracts (4,6).
[0356] The studies briefly described above will result in the
identification and cloning of the PEG-3 promoter region, the
identification of cis-acting regulatory elements in the PEG-3
promoter and the identification of trans-acting regulatory elements
that activate (or repress) expression of the PEG-3 gene in
unprogressed and progressed cancer cells, untransformed and
oncogene transformed cells and undamaged and DNA damaged cells.
Experiments will also determine if cells containing a
PEG-3-Prom-.beta.-gal fusion gene can be used as a biosensor
monitoring system for the progression, oncogene transformation and
DNA damage and repair pathways. These reagents will be useful in
defining the mechanism underlying the differential expression of
PEG-3 in progressed and oncogene transformed cancer cells and
during induction of DNA damage and repair. This information should
prove valuable in designing approaches for selectively inhibiting
PEG-3 expression, and therefore potentially modifying cancer and
DNA damage resulting from treatment with physical and chemical
carcinogens.
[0357] Identifying a human homologue of the rat PEG-3 gene and
defining the genomic structure and the pattern of expression of the
PEG-3 gene. Probing Northern blots containing total cytoplasmic RNA
from human melanoma cells displaying different stages of cancer
progression, i.e., normal melanocytes, early radial growth phase
(RGP) primary human melanoma, early and late vertical growth phase
(VGP) primary human melanoma and metastatic human melanoma cells,
indicate that PEG-3 expression is highest in more advanced
metastatic human melanoma (FIG. 9). Treatment of human melanoma
cells, containing a wild-type p53 or a mutant p53 gene, with gamma
irradiation results in enhanced PEG-3 expression (FIG. 10). These
results suggest that a human homologue of rat PEG-3 is present in
human melanoma cells and induction of this gene correlates with
cancer progression and DNA damage. Human genomic clones of PEG-3
will be isolated by screening a human melanoma genomic lambda
library with sequences corresponding to the carboxyl terminus of
PEG-3 (that is significantly different from gadd34 and MyD116) and
by PCR based genomic DNA amplification procedures (4) The isolated
positive clones will be characterized by restriction mapping, and
suitable restriction fragments will be subcloned into the
pBluescript vector (Strategene) (24). Exons will be identified by
hybridization of the genomic fragments of a panel of PEG-3 clones
and subsequent comparison of the genomic DNA sequences to that of
the cDNA (25,26). A given intron/exon boundary will be indicated
when the sequence from the genomic clones diverges from that of the
cDNA. The size of each intron will be estimated by restriction
mapping (4,25,26). An alternative approach for identifying
intron/exon junctions will use a set of different restriction
endonucleases to digest the human genomic DNAs. Restriction
fragments resulting from this digestion will be ligated with
appropriate cDNA sequences and the other specific primer to the
linker sequences. By using a panel of PEG-3 cDNA oligonucleotides
as primers, PCR products will be generated, that contain most, if
not all, uncloned genomic DNA adjacent to PEG-3 exon sequence
(25,26). The PCR products obtained will be cloned and sequenced to
deduce the intron/exon boundaries of the PEG-3 gene.
[0358] Having a human genomic clone of PEG-3 will permit a direct
determination of possible structural alterations and mutations in
the PEG-3 gene (or its promoter) in human cancers. Tumor and normal
tissue samples will be collected in pairs from patients. Genomic
DNAs will be extracted from these samples (4) and analyzed by
Southern blotting with appropriate restriction enzymes for possible
heterozygous deletions, homozygous deletions, insertions and/or
rearrangements (27,28). To detect point mutations, pairs of
oligonucleotide primers for the exons will be designed for
single-strand conformation polymorphism (SSCP) analysis
(27,28).
[0359] The studies briefly described above will delineate the
structure of the human PEG-3 gene and identify structural changes
in the PEG-3 gene (or its promoter) in cancer versus normal tissue.
A high frequency of structural alterations and mutations,
especially those that can potentially alter the expression and
functionality of the PEG-3 protein, in normal versus cancer tissue
or in early versus late stage cancers, would suggest that these
alterations in the PEG-3 gene may be involved in initiation and/or
progression of this cancer. Additionally, experiments to determine
the state of methylation of the PEG-3 promoter region should prove
informative (29).
[0360] If specific mutations in PEG-3 (or its promoter) are found
to correlate with cancer development and/or evolution, the effect
of such mutations on the in vitro and in vivo biological properties
of cells can be determined. Mutations will be introduced that alter
the normal PEG-3 gene to generate a progression specific PEG-3 gene
product. To achieve these goals, the PEG-3 gene will be mutagenized
at specific sites, using the ExSite.TM. PCR-based site-directed
mutagenesis kit and the Chameleon.TM. double-stranded site-directed
mutagenesis kit (Stratagene, La Jolla, Calif.). We have documented
experience in introducing mutations in defined regions of the
adenovirus genome and characterizing these genetic changes (9-12).
Once identified and characterized, mutant constructs of the PEG-3
gene will be transfected into appropriate target cells to determine
the effects of specific mutations in PEG-3 on cellular
phenotype.
REFERENCES FOR THE SECOND SERIES OF EXPERIMENTS
[0361] 1. Su Z-z, Shi Y & Fisher P B (1994) Proc Natl Acad Sci
USA, in submission.
[0362] 2. Siebert P, Chen S & Kellogg D (1995) CLONTECHniques,
X (2)L: 1-3.
[0363] 3. Siebert P, Chenchik A, Kellogg D E, Lukyanov K A &
Lukyanov S A (1995) Nucleic Acids Res, 23: 1087-1088.
[0364] 4. Sambrook J, Fritsch E F & Maniatis T. In: Molecular
Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor
Laboratories Press, Cold Spring Harbor, N.Y., 1989.
[0365] 5. Duigou G J, Su Z-z, Babiss L E, Driscoll B, Fung Y-KT
& Fisher P B. (1991) Oncogene 6:1813-1824.
[0366] 6. Shen R, Goswami S K, Mascareno E, Kumar A & Siddiqui
MAQ. (1991) Mol Cell Biol 11: 1676-1685.
[0367] 7. Fisher A L, Ohsako S & Caudy M. (1996) Mol Cell Biol
16:2670-2677.
[0368] 8. Jiang H, Lin J J, Su Z-z, Goldstein N I & Fisher P B
(1995) Oncogene 11:2477-2486.
[0369] 9. Babiss L E, Fisher P B & Ginsberg H S. (1984) J Virol
49:731-740.
[0370] 10. Babiss L E, Fisher P B & Ginsberg H S. (1984) J
Virol 52: 389-395.
[0371] 11. Herbst R S, Hermo H Jr, Fisher P B & Babiss L E.
(1988) J Virol 62:4634-4643.
[0372] 12. Su Z-z, Shen R, Young C S H & Fisher P B. (1993) Mol
Carcinog 8:155-166.
[0373] 13. Maniatis T, Goodbourn S & Fischer A. (1987) Science
236:1237-1244.
[0374] 14. Ptashne M. (1988) Nature 335:683-689.
[0375] 15. Su Z-z, Yemul S, Stein C A & Fisher P B. (1995)
Oncogene 10:2037-2049.
[0376] 16. Jiang H, Lin J, Young S-m, Goldstein N I, Waxman S,
Davila V, Chellappan SP & Fisher P B. (1995) Oncogene
11:1179-1189.
[0377] 17. Su Z-z, Shen R, O'Brian C A & Fisher P B. (1994)
Oncogene 9:1123-1132.
[0378] 18. Kamat J P, Basu K, Satyamoorthy L, Showe L & Howe C
C (1995) Mol Rep Dev 41:8-15.
[0379] 19. Basu A, Dong B, Krainer A R & Howe C C (1997) Mol
Cell Biol 17:677-686.
[0380] 20. Aebersold R H, Leavitt R A, Saavedra R A, Hood L E &
Kent S B H (1987) Proc Natl Acad Sci USA 84:6970-6974.
[0381] 21. Jiang H, Lin J, Su Z-z, Kerbel R S, Herlyn M, Weissman R
B, Welch D R & Fisher P B. (1995) Oncogene 10: 1855-1864.
[0382] 22. Jiang H, Lin J J, Su Z-z, Goldstein N I & Fisher P B
(1995) Oncogene 11:2477-2486.
[0383] 23. Lin J J, Jiang H & Fisher P B (1996) Mol Cell
Different 4:317-333.
[0384] 24. Reddy P G, Su Z-z & Fisher P B Methods in Molecular
Genetics, vol. 1, KW Adolph, Ed, Academic Press, Inc, Orlando,
Fla., pp 68-102, 1993.
[0385] 25. Hong F D, Huang H-S, To H, Young L-J H S, Oro A,
Bookstein R, Lee EY-HP & Lee W H (1989) Proc Natl Acad Sci USA
86:5502-5506.
[0386] 26. Sun J, Rose J B & Bird P (1995) J Biol Chem
270:16089-16096.
[0387] 27. Puffenberger E G, Hosoda K, Washington S S, Nakao K,
dewit D, Yanagisawa M and Charkravarti A. (1994) Cell
79:1257-1266.
[0388] 28. Washimi O, Nagatake M, Osada H, Ueda R, Koshikawa T,
Seki T, Takahashi T and Takahashi T (1995) Cancer Res
55:514-517.
[0389] 29. Babiss L E, Zimmer S G & Fisher P B (1985) Science
228:1099-1101.
[0390] Third Series of Experiments
[0391] Expression of PEG-3 in human melanoma cells. Studies were
also performed to evaluate PEG-3 expression in human melanoma cells
and to determine whether induction or increased expression occurs
during DNA damage. PEG-3 is expressed de novo in advanced stage
tumorigenic and metastatic human melanoma cell lines (MeWo, WM239,
C8161, F0-1 and H0-1), whereas expression is reduced in
immortalized normal human melanocyte (FM516-SV) and RGP (WM35) and
early VGP (WM278) primary melanomas (FIG. 9). Moreover, PEG-3
expression is enhanced following exposure to gamma irradiation, but
is not elevated following a similar dose of MMS (100 mg/ml)
inducing PEG-3 expression in CREF cells (FIG. 10). Using a p53
mutant and p53 wild-type human melanoma cell lines, it is apparent
that PEG-3 induction by gamma irradiation in human melanoma can
occur by a wild-type p53 independent pathway (FIG. 10). These
results indicate that the PEG-3 response is not restricted to
rodent cells treated with specific DNA damaging agents, but instead
is a more general response in mammalian cells. Furthermore, there
appears to be a direct relationship between PEG-3 expression and
human melanoma progression.
[0392] Clarifying the role of PEG-3 in human cancer progression. To
define the role of the PEG-3 gene in human cancer progression it
will be essential to obtain a human homoloque of this gene. This
will be achieved by low stringency hybridization screening of a
human melanoma cDNA library (1) and by PCR-based approaches using
primers designed from the rat PEG-3 sequences that are highly
homologous with gadd34 and MyD116 (4,5). Once a full-length PEG-3
(Hu) cDNA is obtained it will be sequenced and in vitro translated
to insure production of the appropriate sized protein (3-5). This
gene can then be used to define patterns of expression, by Northern
blotting analysis, in normal, benign and metastatic human tumor
cell lines and primary patient-derived samples (2-5). This survey
will indicate the level of coordinate expression between PEG-3 and
human cancer progression. Clearly, if PEG-3 is shown to be a
regulator of the progression phenotype in human malignancies, a
large number of interesting and important experiments could be
conducted to amplify on this observation. However, these studies
would not be in the current scope of this grant because of limited
personnel and resources. The types of studies that could and should
be conducted include: (a) production of monoclonal antibodies
interacting with PEG-3 (Hu) and evaluation of these reagents for
cancer diagnostic purposes; (b) cellular localization studies with
PEG-3 (Hu) monoclonal antibodies to define potential targets for
activity; (c) mapping the chromosomal location of PEG-3 (Hu) in the
genome to determine any association between previously identified
regions associated with cancer; (d) identification and
characterization of the genomic structure of PEG-3 (Hu) and
determining if alterations in structure correlate with cancer
progression; (e) determine by nuclear run-on and mRNA degradation
assays if PEG-3 (Hu) expression is controlled at a transcriptional
or postranscriptional level; (f) identification and
characterization, if PEG-3 expression is regulated
transcriptionally, of the promoter region of PEG-3 (Hu) to define
the mechanism of regulation of this gene in progressed cancer
cells; (q) the identification and characterization of cis-acting
elements and trans-regulating factors (nuclear proteins) regulating
PEG-3 (Hu) expression; (h) defining the role of PEG-3 expression in
vivo by creating knockout mice and tissue specific knockout mice;
and (i) determining, using transgenic mice and the tyrosinase
promoter, the role of overexpression of PEG-3 in normal melanocyte
development. These studies would provide important information
about a potentially exciting and novel gene with direct relevance
to human cancer progression.
REFERENCES FOR THE THIRD SERIES OF EXPERIMENTS
[0393] 1. Jiang, H. and P. Fisher Use of a sensitive and efficient
subtraction hybridization protocol for the identification of genes
differentially regulated during the induction of differentiation in
human melanoma cells. Mol Cell Different. 1: 285-299, 1993.
[0394] 2. Jiang, H., et al. The melanoma differentiation associated
gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21
is differentially expressed during growth, differentiation and
progression in human melanoma cells. Oncogene 10: 1855-1864,
1995.
[0395] 3. Jiang, H., et al. Subtraction hybridization identifies a
novel melanoma differentiation associated gene, mda-7, modulated
during human melanoma differentiation, growth and progression.
Oncogene, 11: 2477-2486, 1995.
[0396] 4. Shen, R., et al. Identification of the human prostatic
carcinoma oncogene PTI-1 by rapid expression cloning and
differential RNA display. PNAS, USA 92: 6778-6782, 1995.
[0397] 5. Su, Z-Z, et al. Surface-epitope masking and expression
cloning identifies the human prostate carcinoma tumor antigen gene
PCTA-1 a member of the galectin gene family. PNAS, USA,
93:7252-7257, 1996.
[0398] Fourth Series of Experiments
[0399] The present invention is based, in part, on the
identification of certain cDNA molecules that correspond to
progression-associated mRNA molecules. As used herein, a
progression-associated mRNA is a mRNA whose expression correlates
with tumor cell progression (i.e., the level of RNA is at least
2-fold higher in progressing tumor cells). A progression-associated
cDNA molecule comprises the sequence of a progression-associated
mRNA (and/or a complementary sequence). Similarly, a
progression-associated protein or polypeptide comprises a sequence
encoded by a progression-associated mRNA, where the level of
protein or polypeptide correlates with tumor cell progression
(i.e., the level of protein is at least 2-fold higher in
progressing tumor cells). Progression-associated sequences
described herein are also called "progression elevated" genes
(PEG).
[0400] Progression-Associated Polynucleotides. Any polynucleotide
that encodes a progression-associated polypeptide, or a portion or
variant thereof as described herein, is encompassed by the present
invention. Such polynucleotides may be single-stranded (coding or
antisense) or double-stranded, and may be DNA (genomic, cDNA or
synthetic) or RNA molecules. Additional non-coding sequences may,
but need not, be present within a polynucleotide of the present
invention, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
[0401] Progression-associated polynucleotides may be prepared using
any of a variety of techniques. For example, such a polynucleotide
may be amplified from human genomic DNA, from tumor cDNA or from
cDNA prepared from any of a variety of tumor-derived cell lines
(typically cell lines characterized by a progression phenotype),
via polymerase chain reaction (PCR). For this approach,
sequence-specific primers may be designed based on the sequences
provided herein, and may be purchased or synthesized. An amplified
portion may then be used to isolate a full length gene from a human
genomic DNA library or from a tumor cDNA library, using well known
techniques, as described below. Alternatively, a full length gene
can be constructed from multiple PCR fragments.
[0402] cDNA molecules encoding a native progression-associated
protein, or a portion thereof, may also be prepared by screening a
cDNA library prepared from mRNA of a cell that is in progression,
such as E11-NMT or MCF-7 cells, as described herein. Such libraries
may be commercially available, or may be prepared using standard
techniques (see Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
1989, and references cited therein). A library may be a cDNA
expression library and may, but need not, be subtracted using well
known subtractive hybridization techniques.
[0403] There are many types of screens that may be employed,
including any of a variety of standard hybridization methods. For
initial screens, conventional subtractive hybridization techniques
may be used.
[0404] A progression-associated cDNA molecule may be sequenced
using well known techniques employing such enzymes as Klenow
fragment of DNA polymerase I, Sequenase.RTM. (US Biochemical Corp.,
Cleveland Ohio) Taq polymerase (Perkin Elmer, Foster City Calif.),
thermostable T7 polymerase (Amersham, Chicago, Ill.) or
combinations of recombinant polymerases and proofreading
exonucleases such as the ELONGASE Amplification System (Gibco BRL,
Gaithersburg, Md.). An automated sequencing system may be used,
using instruments available from commercial suppliers such as
Perkin Elmer and Pharmacia.
[0405] The sequence of a partial cDNA may be used to identify a
polynucleotide sequence that encodes a full length
progression-associated protein using any of a variety of standard
techniques. Within such techniques, a library (cDNA or genomic) is
screened using one or more polynucleotide probes or primers
suitable for amplification. Preferably, a library is size-selected
to include larger molecules. Random primed libraries may also be
preferred for identifying 5' and upstream regions of genes. Genomic
libraries are preferred for obtaining introns and extending 5'
sequence.
[0406] For hybridization techniques, a partial sequence may be
labeled (e.g., by nick-translation or end-labeling with .sup.32P)
using well known techniques. A bacterial or bacteriophage library
is then screened by hybridizing filters containing denatured
bacterial colonies (or lawns containing phage plaques) with the
labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
1989). Hybridizing colonies or plaques are selected and expanded,
and the DNA is isolated for further analysis. cDNA clones may be
analyzed to determine the amount of additional sequence by, for
example, PCR using a primer from the partial sequence and a primer
from the vector. Restriction maps and partial sequenced may be
generated to identify one or more overlapping clones. The complete
sequence may then be determined using standard techniques, which
may involve generating a series of deletion clones. The resulting
overlapping sequences are then assembled into a single contiguous
sequence. A full length cDNA molecule can be generated by ligating
suitable fragments, using well known techniques.
[0407] Alternatively, there are numerous amplification techniques
for obtaining a full length coding sequence from a partial cDNA
sequence. In an embodiment, amplification is performed via PCR. Any
of a variety of commercially available kits may be used to perform
the amplification step. Primers may be designed using, for example,
software well known in the art. Primers are preferably 22-30
nucleotides in length, have a GC content of at least 50% and anneal
to the target sequence at temperatures of about 68.degree. C. to
72.degree. C. The amplified region may be sequenced as described
above, and overlapping sequences assembled into a contiguous
sequence.
[0408] One such amplification technique is inverse PCR (see Triglia
et al., Nucl. Acids Res. 16:8186, 1988), which uses restriction
enzymes to generate a fragment in the known region of the gene. The
fragment is then circularized by intramolecular ligation and used
as a template for PCR with divergent primers derived from the known
region. Within an alternative approach, sequences adjacent to a
partial sequence may be retrieved by amplification with a primer to
a linker sequence and a primer specific to a known region. The
amplified sequences are typically subjected to a second round of
amplification with the same linker primer and a second primer
specific to the known region. A variation on this procedure, which
employs two primers that initiate extension in opposite directions
from the known sequence, is described in WO 96/38591. Additional
techniques include capture PCR (Lagerstrom et al., PCR Methods
Applic. 1:111-19, 1991), walking PCR (Parker et al., Nucl. Acids.
Res. 19:3055-60,1991) and rapid amplification of cDNA end (RACE)
procedures (see Jiang et al., Oncogene 10:1855-1864, 1995; Jiang et
al., Oncogene 11:2477-2486, 1995). Other methods employing
amplification may also be employed to obtain a full length cDNA
sequence.
[0409] In certain instances, it is possible to obtain a full length
cDNA sequence by analysis of sequences provided in an expressed
sequence tag (EST) database, such as that available from GenBank.
In an embodiment, searches for overlapping ESTs may be performed
using well known programs (e.g., NCBI BLAST searches), and such
ESTs may be used to generate a contiguous full length sequence
[0410] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present invention.
[0411] As noted above, antisense polynucleotides and portions of
any of the above sequences are also contemplated by the present
invention. In an embodiment, such polynucleotides may be prepared
by any method known in the art, including chemical synthesis by,
for example, solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro or in
vivo transcription of DNA sequences encoding a
progression-associated protein, or a portion thereof, provided that
the DNA is incorporated into a vector downstream of a suitable RNA
polymerase promoter (such as T3, T7 or SP6). Large amounts of RNA
probe may be produced by incubating labeled nucleotides with a
linearized Progression Elevated Gene-3 fragment downstream of such
a promoter in the presence of the appropriate RNA polymerase.
Certain portions of a PEG-3 polynucleotide may be used to prepare
an encoded polypeptide, as described herein. In addition, or
alternatively, a portion may function as a probe (e.g., for
diagnostic purposes, such as to monitor or study the progression of
cancer), and may be labeled by a variety of reporter groups, such
as radionuclides, fluorescent dyes and enzymes. Such portions are
preferably at least 10 nucleotides in length, more preferably at
least 12 nucleotides in length and still more preferably at least
15 nucleotides in length. Within certain preferred embodiments, a
portion for use as a probe comprises a sequence that is unique to a
PEG-3 gene. A portion of a sequence complementary to a coding
sequence (i.e., an antisense polynucleotide) may also be used as a
probe or to modulate gene expression. cDNA constructs that can be
transcribed into antisense RNA may also be introduced into cells of
tissues to facilitate the production of antisense RNA.
[0412] Any polynucleotide may be further modified to increase
stability in vivo. Possible modifications include, but are not
limited to, the addition of flanking sequences at the 5' and/or 3'
ends; the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages in the backbone; and/or the inclusion of
nontraditional bases such as inosine, queosine and wybutosine, as
well as acetyl-methyl-, thio- and other modified forms of adenine,
cytidine, guanine, thymine and uridine.
[0413] Nucleotide sequences as described herein may be joined to a
variety of other nucleotide sequences using established recombinant
DNA techniques. For example, a polynucleotide may be cloned into
any of a variety of cloning vectors, including plasmids, phagemids,
lambda phage derivatives and cosmids. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors and sequencing vectors. In an embodiment, a
vector will contain an origin of replication functional in at least
one organism, convenient restriction endonuclease sites and one or
more selectable markers. Additional initial, terminal and/or
intermediate DNA sequences that, for example, facilitate
construction of readily expressed vectors may also be present. For
example, regulatory elements required for expression include
promoter sequences to bind RNA polymerase and transcription
initiation sequences for ribosome binding. A bacterial expression
vector may include a promoter such as the lac promoter and for
transcription initiation the ShineDalgarno sequence and the start
codon AUG. Similarly, a eukaryotic expression vector may include a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors may
be obtained commercially or assembled from the sequences described
by methods well-known in the art, for example, the methods
described above for constructing vectors. Other elements that may
be present in a vector will depend upon the desired use, and will
be apparent to those of ordinary skill in the art.
[0414] For example, insert and vector DNA can both be exposed to a
restriction enzyme to create complementary ends on both molecules
which base pair with each other and are then ligated together with
DNA ligase. Alternatively, linkers can be ligated to the insert DNA
which correspond to a restriction site in the vector DNA, which is
then digested with the restriction enzyme which cuts at that site.
Other means are also available and known to an ordinary skilled
practitioner.
[0415] In one embodiment, a rat PEG-3 sequence is cloned in the
EcoRI site of a pZeoSV vector. The resulting plasmid, designated
pPEG-3, was deposited on Mar. 6, 1997 with the American Type
Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.
20852, U.S.A. under the provisions of the Budapest Treaty for the
International Recognition of the Deposit of Microorganism for the
Purposes of Patent Procedure. The plasmid, pPEG-3, was accorded
ATCC Accession Number 97911.
[0416] Vectors as described herein may be transfected into a
suitable host cell, such as a mammalian cell, by methods well-known
in the art. Such methods include calcium phosphate precipitation,
electroporation and microinjection.
[0417] Progression-Associated Polypeptides. Polypeptides within the
scope of the present invention comprise at least a portion of a
progression-associated protein or variant thereof, where the
portion is immunologically and/or biologically active. A
polypeptide may further comprise additional sequences, which may or
may not be derived from a native progression-associated protein.
Such sequences may (but need not) possess immunogenic or antigenic
properties and/or a biological activity.
[0418] As used herein, immunologically active polypeptides include,
but are not limited to, a polypeptide that is recognized (i.e.,
specifically bound) by a B-cell and/or T-cell surface antigen
receptor. In an embodiment, immunological activity may be assessed
using well known techniques, such as those summarized in Paul,
Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and
references cited therein. Such techniques include screening
polypeptides derived from the native polypeptide for the ability to
react with antigen-specific antisera and/or T-cell lines or clones,
which may be prepared using well known techniques. An
immunologically active portion of a progression-associated protein
reacts with such antisera and/or T-cells at a level that is not
substantially lower than the reactivity of the full length
polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). In
an embodiment, such screens may be performed using methods well
known to those of ordinary skill in the art, such as those
described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also
be predicted via computer analysis.
[0419] Biologically active polypeptides include, but are not
limited to, polypeptides that possesses one or more structural,
regulatory and/or biochemical functions of the native
progression-associated protein. For example, a polypeptide may
induce progression in cells at levels comparable to the level of
native protein. Appropriate assays designed to evaluate the
activity may then be designed based on existing assays known in the
art, and on the assays provided herein.
[0420] As noted above, polypeptides may comprise one or more
portions of a variant of an endogenous protein, where the portion
is immunologically and/or biologically active (i.e., the portion
exhibits one or more antigenic, immunogenic and/or biological
properties characteristic of the full length protein). Preferably,
such a portion is at least as active as the full length protein
within one or more assays to detect such properties. A polypeptide
variant as used herein includes, but is not limited to, a
polypeptide that differs from a native protein in substitutions,
insertions, deletions and/or amino acid modifications, such that
the antigenic, immunogenic and/or biological properties of the
native protein are not substantially diminished. In an emboidment,
a variant retains at least 80% sequence identity to a native
sequence. In another emboidment, a variant retains at least 90%
sequence identity to a native sequence. In another emboidment, a
variant retains at least 95% sequence identity to a native
sequence. Guidance in determining which and how many amino acid
residues may be substituted, inserted, deleted and/or modified
without diminishing immunological and/or biological activity may be
found using any of a variety of computer programs known in the art,
such as DNAStar software. In an embodiment, properties of a variant
may be evaluated by assaying the reactivity of the variant with
antisera and/or T-cells as described above and/or evaluating a
biological property characteristic of the native protein.
[0421] In an embodiment, a variant contains conservative
substitutions. A conservative substitution comprises a substitution
wherein an amino acid is substituted for another amino acid that
has similar properties, such that one skilled in the art of peptide
chemistry would expect the secondary structure and hydropathic
nature of the polypeptide to be substantially unchanged. In an
embodiment, amino acid substitutions may be made on the basis of
similarity on polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain nonconservative
changes.
[0422] Variants within the scope of this invention also include
polypeptides in which the primary amino acid structure of a native
protein is modified by forming covalent or aggregative conjugates
with other polypeptides or chemical moieties such as glycosyl
groups, lipids, phosphate, acetyl groups and the like. Covalent
derivatives may be prepared, for example, by linking particular
functional groups to amino acid side chains or at the N- or
C-termini.
[0423] The present invention also includes polypeptides with or
without associated native-pattern glycosylation. Polypeptides
expressed in yeast or mammalian expression systems may be similar
to or slightly different in molecular weight and glycosylation
pattern than the native molecules, depending upon the expression
system. Expression of DNA in bacteria such as E. coli provides
non-glycosylated molecules. In an embodiment, N-glycosylation sites
of eukaryotic proteins are characterized by the amino acid triplet
Asn-A.sub.l-Z, where A.sub.l is any amino acid except Pro, and Z is
Ser or Thr. Variants having inactivated N-glycosylation sites can
be produced by techniques known to those of ordinary skill in the
art, such as oligonucleotide synthesis and ligation or
site-specific mutagenesis techniques, and are within the scope of
this invention. Alternatively, N-linked glycosylation sites can be
added to a polypeptide.
[0424] As noted above, polypeptides may further comprise sequences
that are not related to an endogenous progression-associated
protein. For example, an N-terminal signal (or leader) sequence may
be present, which co-translationally or post-translationally
directs transfer of the polypeptide from its site of synthesis to a
site inside or outside of the cell membrane or wall (e.g., the
yeast a-factor leader). The polypeptide may also comprise a linker
or other sequence for ease of synthesis, purification or
identification of the polypeptide (e.g., poly-His or
hemagglutinin), or to enhance binding of the polypeptide to a solid
support. Fusion proteins capped with such peptides may also be
resistant to intracellular degradation in E. coli. Protein fusions
encompassed by this invention further include, for example,
polypeptides conjugated to an immunoglobulin Fc region or a leucine
zipper domain as described, for example, in published PCT
Application Wo 94/10308. Polypeptides comprising leucine zippers
may, for example, be oligomeric, dimeric or trimeric. All of the
above protein fusions may be prepared by chemical linkage or as
fusion proteins, as described below.
[0425] Also included within the present invention are alleles of a
progression-associated protein. Alleles are alternative forms of a
native protein resulting from one or more genetic mutations (which
may be amino acid deletions, additions and/or substitutions),
resulting in an altered mRNA. Allelic proteins may differ in
sequence, but overall structure and function are substantially
similar.
[0426] Progression-associated polypeptides, variants and portions
thereof may be prepared from nucleic acid encoding the desired
polypeptide using well known techniques. To prepare an endogenous
protein, an isolated cDNA may be used. To prepare a variant
polypeptide, standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis may be used, and
sections of the DNA sequence may be removed to permit preparation
of truncated polypeptides. Briefly, host cells of a vector system
containing a PEG-3 sequence under suitable conditions permitting
production of the polypeptide may be grown, and the polypeptide so
produced may then be recovered.
[0427] Any of a variety of expression vectors known to those of
ordinary skill in the art may be employed to express recombinant
polypeptides of this invention. Expression may be achieved in any
appropriate host cell that has been transformed or transfected with
an expression vector containing a DNA sequence that encodes a
recombinant polypeptide. Suitable host cells include prokaryotes,
yeast, insect cells and animal cells. In an embodiment, the host
cells employed are E. coli, yeast, primary mammalian cells or a
mammalian cell line such as COS, Vero, HeLa, fibroblast NIH3T3,
CHO, Ltk.sup.- or CV1. Following expression, supernatants from
host/vector systems which secrete recombinant protein or
polypeptide into culture media may be first concentrated using a
commercially available filter. Following concentration, the
concentrate may be applied to a suitable purification matrix such
as an affinity matrix or an ion exchange resin. One or more reverse
phase HPLC steps can be employed to further purify a recombinant
polypeptide.
[0428] Portions and other variants having fewer than about 100
amino acids, and generally fewer than about 50 amino acids, may
also be generated by synthetic means, using techniques well known
to those of ordinary skill in the art. For example, such
polypeptides may be synthesized using any of the commercially
available solid-phase techniques, such as the Merrifield
solid-phase synthesis method, where amino acids are sequentially
added to a growing amino acid chain. See Merrifield, J. Am. Chem.
Soc. 85:2149-2146, 1963. Various modified solid phase techniques
are also available (e.g., the method of Roberge et al., Science
269:202-204, 1995). Equipment for automated synthesis of
polypeptides is commercially available from suppliers such as
Applied BioSystems, Inc. (Foster City, Calif.), and may be operated
according to the manufacturer's instructions.
[0429] In an embodiment, an isolated polypeptide or polynucleotide
is one that is removed from its original environment. For example,
a naturally-occurring protein is isolated if it is separated from
some or all of the coexisting materials in the natural system. A
polynucleotide is considered to be isolated if, for example, it is
cloned into a vector that is not a part of the natural
environment.
[0430] Antibodies and Fragments Thereof. The present invention
further provides antibodies, and antigen-binding fragments thereof,
that specifically bind to a progression-associated protein. In an
embodiment, an antibody, or antigen-binding fragment specifically
binds to a progression-associated protein if it reacts at a
detectable level (within, for example, an ELISA) with a
progression-associated protein or a portion or variant thereof, and
does not react detectably with unrelated proteins. In certain
embodiments, antibodies that inhibit PEG-3 induced progression are
used.
[0431] Antibodies may be prepared by any of a variety of techniques
known to those of ordinary skill in the art. See, e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988. In an embodiment, antibodies can be produced by
cell culture techniques, including the generation of monoclonal
antibodies as described herein, or via transfection of antibody
genes into suitable bacterial or mammalian cell hosts, in order to
allow for the production of recombinant antibodies. In an
embodiment, monoclonal antibodies may be produced by in vitro
techniques known to a person of ordinary skill in the art.
[0432] Polypeptides comprising specific portions of a PEG-3 protein
may be selected for the generation of antibodies using methods well
known in the art. In general, hydrophilic regions are more
immunogenic than the hydrophobic regions. In an embodiment,
hydrophilic portions are used for the generation of antibodies.
[0433] In one such technique, an immunogen comprising the
polypeptide is initially injected into any of a wide variety of
mammals (e.g., mice, rats, rabbits, sheep or goats). In this step,
the polypeptides of this invention may serve as the immunogen
without modification. Alternatively, particularly for relatively
short polypeptides, a superior immune response may be elicited if
the polypeptide is joined to a carrier protein, such as bovine
serum albumin or keyhole limpet hemocyanin. The immunogen is
injected into the animal host, preferably according to a
predetermined schedule incorporating one or more booster
immunizations, and the animals are bled periodically. Polyclonal
antibodies specific for the polypeptide may then be purified from
such antisera by, for example, affinity chromatography using the
polypeptide coupled to a suitable solid support.
[0434] Monoclonal antibodies specific for the antigenic polypeptide
of interest may be prepared, for example, using the technique of
Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and
improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity (i.e., reactivity with the
polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and
myeloma cells may be combined with a nonionic detergent for a few
minutes and then plated at low density on a selective medium that
supports the growth of hybrid cells, but not myeloma cells. In an
embodiment, the selection technique uses HAT (hypoxanthine,
aminopterin, thymidine) selection. After a sufficient time, usually
about 1 to 2 weeks, colonies of hybrids are observed. Single
colonies are selected and their culture supernatants tested for
binding activity against the polypeptide. Hybridomas having high
reactivity and specificity are preferred.
[0435] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction. The
antibodies of this invention may be used in the purification
process in, for example, an affinity chromatography step.
Antibodies with a high degree of specificity for PEG-3 may then be
selected. Such antibodies may be used, for example, to detect the
expression of PEG-3 in living animals, in humans, or in biological
tissues or fluids isolated from animals or humans.
[0436] In certain embodiments, antigen-binding fragments of
antibodies are used. Such fragments include Fab fragments, which
may be prepared using standard techniques. In an embodiment,
immunoglobulins are purified from rabbit serum by affinity
chromatography on Protein A bead columns (Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988) and digested by papain to yield Fab and Fc fragments. The Fab
and Fc fragments may be separated by affinity chromatography on
protein A bead columns.
[0437] Methods for Identifying Binding Agents and Modulating
Agents. The present invention further provides methods for
identifying compounds that bind to and/or modulate the activity of
a progression-associated protein. Such agents may be identified by
contacting a polypeptide as provided herein with a candidate
compound or agent under conditions and for a time sufficient to
allow interaction with the polypeptide. Any of a variety of well
known binding assays may then be performed to assess the ability of
the candidate compound to bind to the polypeptide, and assays for a
biological activity of the polypeptide may be performed to identify
agents that modulate (i.e., enhance or inhibit) the biological
activity of the protein. Depending on the design of the assay, a
polypeptide may be free in solution, affixed to a solid support,
present on a cell surface or located intracellularly. Large scale
screens may be performed using automation.
[0438] Alternatively, compounds may be screened for the ability to
modulate expression (e.g., transcription) of PEG-3. For such assays
a promoter for PEG-3 may be isolated using standard techniques. The
present invention provides nucleic acid molecules comprising such a
promoter or a cis- or trans-acting regulatory element thereof. Such
regulatory elements may activate or suppress expression of
PEG-3.
[0439] One method for identifying a promoter region uses a
PCR-based method to clone unknown genomic DNA sequences adjacent to
a known cDNA sequence (e.g., a human PromoterFinder.TM.DNA Walking
Kit, available from Clontech). This approach may generate a 5'
flanking region, which may be subcloned and sequenced using
standard methods. Primer extension and/or RNase protection analyses
may be used to verify the transcriptional start site deduced from
the cDNA.
[0440] To define the boundary of the promoter region, putative
promoter inserts of varying sizes may be subcloned into a
heterologous expression system containing a suitable reporter gene
without a promoter or enhancer may be employed. Suitable reporter
genes may include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase
or the Green Fluorescent Protein gene, and may be generated using
well known techniques Internal deletion constructs may be generated
using unique internal restriction sites or by partial digestion of
non-unique restriction sites. Constructs may then be transfected
into cells that display high levels of PEG-3 expression (e.g.,
E11-NMT). In an embodiment, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the PEG-3 gene promoter.
[0441] Once a functional PEG-3 promoter is identified, cis- and
trans-acting elements may be located. In an embodiment, cis-acting
sequences may be identified based on homology to previously
characterized transcriptional motifs. Point mutations may then be
generated within the identified sequences to evaluate the
regulatory role of such sequences. Such mutations may be generated
using site-specific mutagenesis techniques or a PCR-based strategy.
The altered promoter is then cloned into a reporter gene expression
vector, as described above, and the effect of the mutation on
reporter gene expression is evaluated. Trans-acting factors that
bind to cis-acting sequences may be identified using assays such as
gel shift assays. Proteins displaying binding activity within such
assays may be partially digested, and the resulting peptides
separated and sequenced. Peptide sequences may be used to design
degenerate primers for use within RT-PCR to identify cDNAs encoding
the trans-acting factors.
[0442] To evaluate the effect of a candidate agent on PEG-3
expression, a promoter or regulatory element thereof may be
operatively linked to a reporter gene as described above. Such a
construct may be transfected into a suitable host cell, such as
E11-NMT or transfected forms of CREF Trans 6, including CREF-Trans
6:4NMT (expressing PTI-1), T24 (expressing ras), CREF-src
(expressing src) and CREF-HPV (expressing HPV). It has been found,
within the context of the present invention, that the PEG-3
promoter is constitutively expressed in tumor cell lines, but not
in normal cells. Clones that constitutively express high levels of
reporter protein may be selected and used within a variety of
screens. Such clones are encompassed by the present invention.
[0443] In an embodiment, cells may be used to screen a
combinatorial small molecule library. Briefly, cells are incubated
with the library (e.g., overnight). Cells are then lysed and the
supernatant is analyzed for reporter gene activity according to
standard protocols. Compounds that result in a decrease in reporter
gene activity are inhibitors of PEG-3 transcription, and may be
used to inhibit DNA damage and repair pathways, cancer progression
and/or oncogene mediated transformation.
[0444] This invention further provides methods for identifying
agents capable of inducing DNA damage and repair pathways, cancer
progression and/or oncogene mediated transformation. Briefly,
candidate compounds may be tested as described above, except that
the cells employed (which comprise a PEG-3 promoter or regulatory
element thereof operatively linked to a reporter gene) are not in
progression. For example, CREF-Trans 6 cells may be employed.
Within such assays, an increase in expression of the reporter gene
after the contact indicates that the compound is capable of
inducing DNA damage and repair pathways, cancer progression or
oncogene mediated transformation.
[0445] Within other embodiments, cells may comprise one or more
exogenous suicidal genes under the control of a promoter or
regulatory element of PEG-3. Such suicidal genes disrupt the normal
progress of the cell following transcription from the promoter.
Preferably, the switching on of the suicidal gene will lead to cell
death or halt in cell growth. Example of such genes are genes which
lead to apoptosis.
[0446] Pharmaceutical Compositions and Vaccines. Within certain
aspects, compounds such as polypeptides, antibodies, nucleic acid
molecules and/or other agents that modulate PEG-3 expression or
activity may be incorporated into pharmaceutical compositions or
vaccines. In an embodiment, pharmaceutical compositions comprise
one or more such compounds and a physiologically acceptable
carrier. In an embodiment, certain vaccines may comprise one or
more polypeptides and an immune response enhancer, such as an
adjuvant or a liposome (into which the compound is incorporated).
Pharmaceutical compositions and vaccines may additionally contain a
delivery system, such as biodegradable microspheres which are
disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Pharmaceutical compositions and vaccines within the scope of the
present invention may also contain other compounds, which may be
biologically active or inactive.
[0447] A pharmaceutical composition or vaccine may contain DNA
encoding an antisense polynucleotide or a polypeptides as described
above, such that the polynucleotide or polypeptide is generated in
situ. The DNA may be present within any of a variety of delivery
systems known to those of ordinary skill in the art, including
nucleic acid expression systems, bacteria and viral expression
systems. Appropriate nucleic acid expression systems contain the
necessary DNA sequences for expression in the patient (such as a
suitable promoter and terminating signal). Bacterial delivery
systems involve the administration of a bacterium (such as
Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of
the polypeptide on its cell surface. In a preferred embodiment, the
DNA may be introduced using a viral expression system (e.g.,
vaccinia or other pox virus, retrovirus, or adenovirus), which may
involve the use of a non-pathogenic (defective), replication
competent virus. Techniques for incorporating DNA into such
expression systems are well known to those of ordinary skill in the
art. The DNA may also be "naked," as described, for example, in
Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen,
Science 259:1691-1692, 1993. The uptake of naked DNA may be
increased by coating the DNA onto biodegradable beads, which are
efficiently transported into the cells.
[0448] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration. Such carriers include, but are not limited to,
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents include propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, saline and buffered media.
[0449] Compositions of the present invention may be formulated for
any appropriate manner of administration, including for example,
topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous or intramuscular administration. Intravenous vehicles
include fluid and nutrient replenishers, electrolyte replenishers
such as those based on Ringer's dextrose, and the like. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, a fixed
oil, a fat, a wax or a buffer. For oral administration, any of the
above carriers or a solid carrier, such as mannitol, lactose,
starch, magnesium stearate, sodium saccharine, talcum, cellulose,
glucose, sucrose, and magnesium carbonate, may be employed.
Biodegradable microspheres (e.g., polylactate polyglycolate) may
also be employed as carriers for the pharmaceutical compositions of
this invention. For certain topical applications, formulation as a
cream or lotion, using well known components, is preferred.
[0450] Such compositions may also comprise buffers (e.g., neutral
buffered saline or phosphate buffered saline), carbohydrates (e.g.,
glucose, mannose, sucrose or dextrans), mannitol, proteins,
polypeptides or amino acids such as glycine, antioxidants,
chelating agents such as EDTA or glutathione, adjuvants (e.g.,
aluminum hydroxide) and/or preservatives. Preservatives and other
additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases and the
like. Compositions of the present invention may also be formulated
as a lyophilizate. Compounds may also be encapsulated within
liposomes using well known technology.
[0451] Any of a variety of adjuvants may be employed in the
vaccines of this invention to nonspecifically enhance the immune
response. In an embodiment, the adjuvant contains a substance
designed to protect the antigen from rapid catabolism, such as
aluminum hydroxide or mineral oil, and a stimulator of immune
responses, such as lipid A, Bortadella pertussis or Mycobacterium
tuberculosis derived proteins. Suitable adjuvants are commercially
available as, for example, Freund's Incomplete Adjuvant and
Complete Adjuvant (Difco Laboratories, Detroit, Mich.), Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.), alum,
biodegradable microspheres, monophosphoryl lipid A and quil A.
Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be
used as adjuvants.
[0452] The compositions described herein may be administered as
part of a sustained release formulation (i.e., a formulation such
as a capsule or sponge that effects a slow release of compound
following administration). In an embodiment, such formulations may
be prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain a polypeptide, polynucleotide or antibody
dispersed in a carrier matrix and/or contained within a reservoir
surrounded by a rate controlling membrane. Carriers for use within
such formulations are biocompatible, and may also be biodegradable;
preferably the formulation provides a relatively constant level of
cyclic peptide release. The amount of active compound contained
within a sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0453] Cancer Therapy. In further aspects of the present invention,
the compounds described herein may be used for therapy of cancer.
Within such aspects, the compounds (which may be polypeptides,
antibodies, nucleic acid molecules or other modulating agents) are
preferably incorporated into pharmaceutical compositions or
vaccines, as described-above. Suitable patients for therapy may be
any warm-blooded animal, preferably a human. A patient may or may
not be afflicted with cancer, as determined by standard diagnostic
methods. Accordingly, the above pharmaceutical compositions and
vaccines may be used to prevent the development of cancer or to
treat a patient afflicted with cancer.
[0454] Within certain aspects, cells may be protected from
therapeutic damage (e.g., due to chemotherapy or a physical agent
such as gamma-irradiation) and/or rendered resistant to progression
by inhibiting or eliminating the expression and/or activity of
PEG-3 in the cells. One method for inhibiting the expression of
PEG-3 comprises providing an effective amount of antisense RNA in
the cell. In an embodiment, such antisense technology can be used
to control gene expression through triple-helix formation, which
compromises the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors or regulatory
molecules (see Gee et al., In Huber and Carr, Molecular and
Immunologic Approaches, Futura Publishing Co. (Mt. Kisco, N.Y.;
1994). Alternatively, an antisense molecule may be designed to
hybridize with a control region of a gene (e.g., promoter, enhancer
or transcription initiation site), and block transcription of the
gene; or to block translation by inhibiting binding of a transcript
to ribosomes. In an embodiment, the expression of PEG-3 may be
eliminated by deleting the gene or introducing mutation(s) into the
gene.
[0455] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). The route, duration and frequency of administration
will be determined by such factors as the condition of the patient,
the type and severity of the patient's disease and the method of
administration. Routes and frequency of administration may vary
from individual to individual, and may be readily established using
standard techniques. In an embodiment, the pharmaceutical
compositions and vaccines may be administered by injection (e.g.,
intracutaneous, intramuscular, intravenous or subcutaneous),
intranasally (e.g., by aspiration) or orally. Preferably, between 1
and 10 doses may be administered over a 52 week period. Preferably,
6 doses are administered, at intervals of 1 month, and booster
vaccinations may be given periodically thereafter. Alternate
protocols may be appropriate for individual patients.
[0456] In an embodiment, an appropriate dosage and treatment
regimen provides the active compound(s) in an amount sufficient to
provide therapeutic and/or prophylactic benefit. Such a benefit
should results in an improved clinical outcome (e.g., more frequent
remissions, complete or partial, or longer disease-free survival)
in treated patients as compared to non-treated patients.
[0457] Appropriate dosages of polypeptides, polynucleotides,
antibodies and modulating agents may be determined using
experimental models and/or clinical trials. In an embodiment, the
use of the minimum dosage that is sufficient to provide effective
therapy is used. In an embodiment, patients may be monitored for
therapeutic effectiveness using assays suitable for the condition
being treated or prevented, which will be familiar to those of
ordinary skill in the art. Suitable dose sizes will vary with the
size of the patient, but will typically range from about 0.1 mL to
about 5 mL.
[0458] Cancer Detection, Diagnosis and Monitoring. Polypeptides,
polynucleotides and antibodies, as described herein, may be used
within a variety of methods for detecting a cancer, determining
whether a cancer is in progression, and monitoring the progression
and/or treatment of a cancer in a patient. Within such methods, any
of a variety of methods may be used to detect PEG-3 activity or the
level of PEG-3 mRNA or protein in a sample. Suitable biological
samples include tumor or normal tissue biopsy, mastectomy, blood,
lymph node, serum or urine samples, or other tissue, homogenate or
extract thereof obtained from a patient.
[0459] Methods involving the use of an antibody may detect the
presence or absence of PEG-3 in any suitable biological sample.
There are a variety of assay formats known to those of ordinary
skill in the art for using an antibody to detect polypeptide
markers in a sample. See, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For
example, the assay may be performed in a Western blot format,
wherein a protein preparation from the biological sample is
submitted to gel electrophoresis, transferred to a suitable
membrane and allowed to react with the antibody. The presence of
the antibody on the membrane may then be detected using a suitable
detection reagent, as described below.
[0460] In another embodiment, the assay involves the use of
antibody immobilized on a solid support to bind to the polypeptide
and remove it from the remainder of the sample. The bound
polypeptide may then be detected using a second antibody or reagent
that contains a reporter group. Alternatively, a competitive assay
may be utilized, in which a polypeptide is labeled with a reporter
group and allowed to bind to the immobilized antibody after
incubation of the antibody with the sample. The extent to which
components of the sample inhibit the binding of the labeled
polypeptide to the antibody is indicative of the reactivity of the
sample with the immobilized antibody, and as a result, indicative
of the concentration of polypeptide in the sample.
[0461] The solid support may be any material known to those of
ordinary skill in the art to which the antibody may be attached.
For example, the solid support may be a test well in a microtiter
plate or a nitrocellulose filter or other suitable membrane.
Alternatively, the support may be a bead or disc, such as glass,
fiberglass, latex or a plastic material such as polystyrene or
polyvinylchloride. The support may also be a magnetic particle or a
fiber optic sensor, such as those disclosed, for example, in U.S.
Pat. No. 5,359,681.
[0462] The antibody may be immobilized on the solid support using a
variety of techniques known to those in the art, which are amply
described in the patent and scientific literature. In the context
of the present invention, immobilization includes both noncovalent
association, such as adsorption, and covalent attachment (which may
be a direct linkage between the antigen and functional groups on
the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or to
a membrane is preferred. In such cases, adsorption may be achieved
by contacting the antibody, in a suitable buffer, with the solid
support for a suitable amount of time. The contact time varies with
temperature, but is typically between about 1 hour and 1 day. In an
embodiment, contacting a well of a plastic microtiter plate (such
as polystyrene or polyvinylchloride) with an amount of antibody
ranging from about 10 ng to about 1 .mu.g, and preferably about
100-200 ng, is sufficient to immobilize an adequate amount of
polypeptide.
[0463] Covalent attachment of antibody to a solid support may also
be achieved by first reacting the support with a bifunctional
reagent that will react with both the support and a functional
group, such as a hydroxyl or amino group, on the antibody. For
example, the antibody may be covalently attached to supports having
an appropriate polymer coating using benzoquinone or by
condensation of an aldehyde group on the support with an amine and
an active hydrogen on the binding partner using well known
techniques.
[0464] In certain embodiments, the assay for detection of
polypeptide in a sample is a two-antibody sandwich assay. This
assay may be performed by first contacting an antibody that has
been immobilized on a solid support, commonly the well of a
microtiter plate, with the biological sample, such that the
polypeptide within the sample are allowed to bind to the
immobilized antibody. Unbound sample is then removed from the
immobilized polypeptide-antibody complexes and a second antibody
(containing a reporter group) capable of binding to a different
site on the polypeptide is added. The amount of second antibody
that remains bound to the solid support is then determined using a
method appropriate for the specific reporter group.
[0465] More specifically, once the antibody is immobilized on the
support as described above, the remaining protein binding sites on
the support are typically blocked. Any suitable blocking agent
known to those of ordinary skill in the art, such as bovine serum
albumin or Tween 20.TM. (Sigma Chemical Co., St. Louis, Mo.). The
immobilized antibody is then incubated with the sample, and
polypeptide is allowed to bind to the antibody. The sample may be
diluted with a suitable diluent, such as phosphate-buffered saline
(PBS) prior to incubation. In an embodiment, an appropriate contact
time (i.e., incubation time) is that period of time that is
sufficient to detect the presence of polypeptide within a sample
obtained from an individual with cancer. Preferably, the contact
time is sufficient to achieve a level of binding that is at least
95% of that achieved at equilibrium between bound and unbound
polypeptide. Those of ordinary skill in the art will recognize that
the time necessary to achieve equilibrium may be readily determined
by assaying the level of binding that occurs over a period of time.
At room temperature, an incubation time of about 30 minutes is
generally sufficient.
[0466] Unbound sample may then be removed by washing the solid
support with an appropriate buffer, such as PBS containing 0.1%
Tween 20.TM.. The second antibody, which contains a reporter group,
may then be added to the solid support. Preferred reporter groups
include enzymes (such as horseradish peroxidase), substrates,
cofactors, inhibitors, dyes, radionuclides, luminescent groups,
fluorescent groups and biotin. The conjugation of antibody to
reporter group may be achieved using standard methods known to
those of ordinary skill in the art.
[0467] The second antibody is then incubated with the immobilized
antibody-polypeptide complex for an amount of time sufficient to
detect the bound polypeptide. An appropriate amount of time may be
determined by assaying the level of binding that occurs over a
period of time. Unbound second antibody is then removed and bound
second antibody is detected using the reporter group. The method
employed for detecting the reporter group depends upon the nature
of the reporter group. For radioactive groups, scintillation
counting or autoradiographic methods are generally appropriate.
Spectroscopic methods may be used to detect dyes, luminescent
groups and fluorescent groups. Biotin may be detected using avidin,
coupled to a different reporter group (commonly a radioactive or
fluorescent group or an enzyme). Enzyme reporter groups may be
detected by the addition of substrate (i.e. for a specific period
of time), followed by spectroscopic or other analysis of the
reaction products.
[0468] To determine whether cells are in progression, expression of
PEG-3 in the cells is evaluated and compared with the level of
expression in cells that are not in progression. In an embodiment,
the signal detected from the reporter group that remains bound to
the solid support is compared to a signal that corresponds to a
predetermined cut-off value established from cells that are not in
progression. In an embodiment, the cut-off value is the average
mean signal obtained when the immobilized antibody is incubated
with samples from cells that are not in progression. In an
embodiment, a sample generating a signal that is three standard
deviations above the predetermined cut-off value may be considered
positive for progression. In an embodiment, the cut-off value is
determined using a Receiver Operator Curve, according to the method
of Sackett et al., Clinical Epidemiology: A Basic Science for
Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly,
in this embodiment, the cut-off value may be determined from a plot
of pairs of true positive rates (i.e., sensitivity) and false
positive rates (100%-specificity) that correspond to each possible
cut-off value for the diagnostic test result. The cut-off value on
the plot that is the closest to the upper left-hand corner (i.e.,
the value that encloses the largest area) is the most accurate
cut-off value, and a sample generating a signal that is higher than
the cut-off value determined by this method may be considered
positive. Alternatively, the cut-off value may be shifted to the
left along the plot, to minimize the false positive rate, or to the
right, to minimize the false negative rate. In an embodiment, a
sample generating a signal that is higher than the cut-off value
determined by this method is considered positive for
progression.
[0469] In a related embodiment, the assay is performed in a
flow-through or strip test format, wherein the antibody is
immobilized on a membrane, such as nitrocellulose. In the
flow-through test, the polypeptide within the sample bind to the
immobilized antibody as the sample passes through the membrane. A
second, labeled antibody then binds to the antibody-polypeptide
complex as a solution containing the second antibody flows through
the membrane. The detection of bound second antibody may then be
performed as described above. In the strip test format, one end of
the membrane to which antibody is bound is immersed in a solution
containing the sample. The sample migrates along the membrane
through a region containing second antibody and to the area of
immobilized antibody. Concentration of second antibody at the area
of immobilized antibody indicates the presence of cells in
progression. Typically, the concentration of second antibody at
that site generates a pattern, such as a line, that can be read
visually. The absence of such a pattern indicates a negative
result. In an embodiment, the amount of antibody immobilized on the
membrane is selected to generate a visually discernible pattern
when the biological sample contains a level of polypeptide that
would be sufficient to generate a positive signal in the
two-antibody sandwich assay, in the format discussed above.
Preferably, the amount of antibody immobilized on the membrane
ranges from about 25 ng to about 1 .mu.g, and more preferably from
about 50 ng to about 1 .mu.g. Such tests can typically be performed
with a very small amount of biological sample.
[0470] The presence or absence of cells in progression in a patient
may also be determined by evaluating the level of mRNA encoding
PEG-3 within the biological sample (e.g., a biopsy, mastectomy
and/or blood sample from a patient) relative to a predetermined
cut-off value. Such an evaluation may be achieved using any of a
variety of methods known to those of ordinary skill in the art such
as, for example, in situ hybridization and amplification by
polymerase chain reaction. In an embodiment, probes and primers for
use within such assays may be designed based on the sequences
provided herein, or on similar sequences identified in other
individuals. Probes may be used within well known hybridization
techniques, and may be labeled with a detection reagent to
facilitate detection of the probe. Such reagents include, but are
not limited to, radionuclides, fluorescent dyes and enzymes capable
of catalyzing the formation of a detectable product.
[0471] Primers may be used within detection methods involving
polymerase chain reaction (PCR), such as RT-PCR, in which PCR is
applied in conjunction with reverse transcription. Typically, RNA
is extracted from a sample tissue and is reverse transcribed to
produce cDNA molecules. PCR amplification using specific primers
generates a progression-associated cDNA molecule, which may be
separated and visualized using, for example, gel electrophoresis.
Amplification is typically performed on samples obtained from
matched pairs of tissue (tumor and non-tumor tissue from the same
individual) or from unmatched pairs of tissue (tumor and non-tumor
tissue from different individuals). The amplification reaction may
be performed on several dilutions of cDNA spanning two orders of
magnitude. A two-fold or greater increase in expression in several
dilutions of the tumor sample as compared to the same dilutions of
the non-tumor sample is typically considered positive.
[0472] Within certain specific embodiments, expression of PEG-3 may
be detected in a sample that contains cells by: (a) obtaining RNA
from the cells; (b) contacting the RNA so obtained with a labeled
(e.g., radioactively) probe of PEG-3 under hybridizing conditions
permitting specific hybridization of the probe and the RNA; and (c)
determining the presence of RNA hybridized to the molecule. As
noted above, mRNA may be isolated and hybridized using any of a
variety of procedures well-known to a person of ordinary skill in
the art. The presence of mRNA hybridized to the probe may be
determined by gel electrophoresis or other methods known in the
art. By measuring the amount of the hybrid formed, the expression
of the PEG-3 protein by the cell can be determined. Alternatively,
RNA obtained from the cells may be amplified by polymerase chain
reaction (PCR) with appropriate primers derived from a known PEG-3
sequence. The presence of specific amplified DNA following PCR is
an indicative of PEG-3 expression in the cells.
[0473] Certain in vivo diagnostic assays may be performed directly
on the tumor. One such assay involves contacting tumor cells with
an antibody or fragment thereof that binds to a
progression-associated protein. The bound antibody or fragment may
then be detected directly or indirectly via a reporter group. Such
antibodies may also be used in histological applications.
[0474] Within related aspects, the present invention provides
methods for diagnosing the aggressiveness of cancer cells. Such
methods are performed as described above, wherein an increase in
the amount of the expression indicates that a cancer cell is more
aggressive.
[0475] In other aspects of the present invention, the progression
and/or response to treatment of a cancer may be monitored by
performing any of the above assays over a period of time, and
evaluating the change in the level of the response (i.e., the
amount of polypeptide or mRNA detected). For example, the assays
may be performed every month to every other month for a period of 1
to 2 years.
[0476] In an embodiment, a cancer is progressing in those patients
in whom the level of the response increases over time. In contrast,
a cancer is not progressing when the signal detected either remains
constant or decreases with time.
[0477] The present invention further provides kits for use within
any of the above diagnostic methods. Such kits typically comprise
two or more components necessary for performing the assay. Such
components may be compounds, reagents and/or containers or
equipment. For example, one container within a kit may contain a
monoclonal antibody or fragment thereof that specifically binds to
a progression-associated polypeptide. Such antibodies or fragments
may be provided attached to a support material, as described above.
One or more additional containers may enclose elements, such as
reagents or buffers, to be used in the assay. Such kits may also
contain a detection reagent (e.g., an antibody) that contains a
reporter group suitable for direct or indirect detection of
antibody binding.
[0478] Transgenic Organisms. The present invention also provides
transgenic nonhuman living organism expressing PEG-3 protein. In an
embodiment, the living organism is animal.
[0479] One means available for producing a transgenic animal, with
a mouse as an example, is as follows: Female mice are mated, and
the resulting fertilized eggs are dissected out of their oviducts.
The eggs are stored in an appropriate medium. PEG-3 DNA or cDNA is
purified from a vector by methods well-known in the art. Inducible
promoters may be fused with the coding region of the DNA to provide
an experimental means to regulate expression of the trans-gene.
Alternatively or in addition, tissue specific regulatory elements
may be fused with the coding region to permit tissue-specific
expression of the trans-gene. The DNA, in an appropriately buffered
solution, is put into a microinjection needle (which may be made
from capillary tubing using a pipes puller) and the egg to be
injected is put in a depression slide. The needle is inserted into
the pronucleus of the egg, and the DNA solution is injected. The
injected egg is then transferred into the oviduct of a
pseudopregnant mouse (a mouse stimulated by the appropriate
hormones to maintain pregnancy but which is not actually pregnant),
where it proceeds to the uterus, implants, and develops to term. As
noted above, microinjection is not the only method for inserting
DNA into the egg cell, and is used here only for exemplary
purposes.
EXAMPLE
[0480] Identification of Human PEG-3. This Example illustrates the
identification of a human PEG-3 cDNA molecule.
[0481] Initially, PEG-3 gene expression was examined in various
human tumor cell lines using a rat PEG-3 cDNA 3'-end fragment as a
probe under low stringency conditions. Hybridization was performed
at 65.degree. C. overnight in the following solution: 800 .mu.l of
5M NaCl, 80 pl of 0.5M EDTA, 2 ml of 1M Na(PO.sub.4) (pH 6.4), 10
ml 10% SDS, 23.12 ml H.sub.2O, for a total of 40 ml. Following
hybridization, washing was performed in 1.times.SSC, 0.1%SDS at
room temperature for 15 minutes, and then twice at 65.degree. C.
for 30 minutes. Overnight exposures indicated that the MCF-7 cell
line highly expresses a human PEG-3 homolog and MCF-7 was used to
provide mRNA resources for the establishment of a cDNA library.
[0482] To establish an MCF-7 cDNA library, poly (A.sup.+) RNA was
extracted and purified of from MCF-7 cells, and cDNA was generated
using oligo (dT) as a primer through reverse transcription.
.lambda.Bk-MCV was used as a vector to generate cDNA library. The
original MCF-7 cDNA library was generated with 1.times.10.sup.6 pfu
and insert size was about 0.4 Kb-4 Kb.
[0483] The MCF-7 cDNA library was screened using a 600 bp rat PEG-3
cDNA 3'-end fragment as a probe at low stringency. Prehybridization
and hybridization were performed in the following solution:
2 100% Formamide 50 ml 20X SSC 25 ml 50X Denhardt's 10 ml 1 m
Na(PO.sub.4) (pH 5 ml 6.8) 100 mg/ml SSDNA 1 ml 10% SDS 1 ml
H.sub.2O 8 ml 100 ml
[0484] Hybridization was performed at 42.degree. C. overnight.
Washing was performed in 1.times.SSC, 0.1% SDS at room temperature
for 15 minutes, and then twice at 65.degree. C. for 30 minutes.
Exposures were performed overnight.
[0485] Twenty-five positive clones were isolated from the MCF-7
cDNA library using the above condition through primary screening,
secondary screening, and third screening. After restriction mapping
and sequencing, all 25 positive clones were confirmed to have an
insert of human PEG-3 cDNA 3'-end. The size for all these inserts
was about 400.about.500 bp.
[0486] Northern blots were performed to evaluate the human PEG-3
gene expression pattern in normal human tissues and human tumor
cell lines, using a 400 bp human PEG-3 cDNA 3'-end fragment as a
probe.
[0487] Hybridization was performed at 65.degree. C. overnight in
the following solution: 800 .mu.l of 5M NaCl, 80 .mu.l of 0.5M
EDTA, 2 ml of 1M Na(PO.sub.4) (pH 6.4), 10 ml 10% SDS, 23.12 ml
H.sub.2O, for a total of 40 ml. Following hybridization, washing
was performed in 1.times.SSC, 0.1%SDS at room temperature for 15
minutes, and then twice at 65.degree. C. for 30 minutes. Overnight
exposures indicated 2 mRNA species of human PEG-3 gene that express
in a high level in most human tumor cell lines. These two mRNA
species are about 1.5 and 2.8 Kb in size. No expression of PEG-3
gene was detected in all normal tissues except skeletal muscle
which expresses 1.5 Kb species of human PEG-3 mRNAs in a low
level.
[0488] 5' RACE was used to generate the full length human PEG-3
cDNA, using poly (A.sup.+) RNA extracted from MCF-7 cells as a
template. PEG gene specific primers were designed from the human
PEG-3 cDNA 400 bp fragment, including primer A
(CTAAGGCGTGTCCATGCTCTGGCC), primer B (CTCCTCTGCCTGGGCAATG) and
primer C (CGAGCAAAGCGGCTTCGATC). First strand cDNA synthesis was
carried out using human PEG-3 gene specific primer A or B through
reverse transcription. cDNA was purified by GlassMax DNA isolation
spin cartridge purification and TdT tailed. PCR of dc-tailed cDNA
was carried out using nested primer B or primer C. After PCR, the
PCR products were separated using 1% agarose at 100 voltages for 1
hour. Two dominant fragments (1.7 Kb and 0.9 Kb) were observed
after electrophoresis and cut for subcloning using AT cloning
vector. After sequencing some subclones, the 1.7 Kb fragment was
confirmed to cover all coding regions of the human PEG-3 cDNA, and
0.9 Kb fragment was a truncated product of the human PEG-3 cDNA
with a start at the first internal repeat of PEG-3 cDNA and also
had a 25 bp unique sequence at the 5'-end. The 5' and 3' sequences
of the 1.7 kb fragment are shown in FIG. 13.
[0489] The human PEG-3 gene also was found to express in human
primary tumor samples using the RT-PCR method. Total RNAs extracted
from primary human tumor sample were used as template, with an
oligo (dT) primer. Reverse transcription was carried out in
42.degree. C. for one hour. For PCR, First strand cDNA generated
from reverse transcription was used as the template, and the
primers were the human PEG-3 gene specific primers designed from
the human PEG-3 gene cDNA 3'-end. PCR conditions were as
follows:
3 Denaturation 94.degree. C.-5' 1 cycle Denaturation 94.degree.
C.-30" Annealing of 60.degree. C.-30' primers {close oversize
brace} 35 cycles Primer extension 72.degree. C.-2' Followed by
Final extension 72.degree. C.-7' Indefinite hold 4.degree. C.,
until samples are removed
[0490] Electrophoresis was used to separate PCR products of all
tested samples, in a 1.5% agarose gel, for 1 hour at 100 V.
[0491] Fifth Series of Experiments
[0492] Cancer is often a multistep process in which a tumor cell
either develops qualitatively new phenotypes or an enhanced
expression of transformation related properties. Defining the
molecular determinants of progression should lead to improved
cancer diagnosis and strategies for therapy. Subtraction
hybridization identified a novel gene associated with induction of
transformation progression in virus and oncogene transformed rat
embryo cells, progression elevated gene-3 (PEG-3). PEG-3 expression
correlates directly with the progression phenotype in rodent cells.
Ectopic expression of PEG-3 in transformed rodent cells elicits an
aggressive oncogenic phenotype, whereas antisense inhibition of
PEG-3 expression eliminates cancer aggressiveness. PEG-3 has
sequence homology to the growth arrest and DNA damage inducible
hamster gene gadd34, implicating DNA damage and repair processes in
progression. A working hypothesis is that PEG-3 expression is a
downstream event in oncogenic transformation and progression and
activation of PEG-3 may directly alter the expression of genes
involved in cancer progression, including genes associated with
tumorigenesis, metastasis and angiogenesis. Studies are evaluating
the effect of transient and stable expression of sense and
antisense PEG-3 constructs in transformed cells on transformation
progression in vitro and in vivo. Since induction of PEG-3 during
progression and as a consequence of DNA damage involves
transcriptional activation, the promoter region of the PEG-3 gene
has been isolated and will be investigated to identify and
characterize cis-acting and trans-acting regulatory elements which
control gene expression. Using the PEG-3 promoter, sensitive
indicator cell lines have been developed for identifying compounds
capable of inducing and inhibiting cancer progression. These
studies are providing important insights into a novel progression
gene with potential relevance to cancer development and evolution.
The PEG-3 gene may serve as a target for selectively intervening in
the progression process, thereby preventing cancer aggressiveness
and metastasis.
[0493] Cancer is a progressive process with defined temporal stages
culminating in metastatic potential by evolving tumor cells.
Although extensively scrutinized the molecular determinants of
cancer progression remain unclear. Well-characterized cell culture
systems are valuable experimental tools for defining the
biochemical and molecular basis of progression. Two rodent model
systems are providing insights into the genes and processes
regulating malignant progression of the transformed cell.
[0494] In adenovirus type 5 (Ad5) transformed rat embryo (RE)
cells, progression can occur spontaneously by tumor formation in
nude mice or by ectopic expression of oncogenes and signal
transducing growth-regulating genes. In all contexts of
progression, the demethylating agent 5-azacytidine (AZA) can
reverse this process resulting in an unprogressed phenotype in
>95% of treated clones. Inhibition of progression also occurs in
this system after forming somatic cell hybrids between progressed
and unprogressed cells. Using an immortal cloned rat embryo
fibroblast (CREF) cell culture system, progression to metastasis
and reversion of progression can be regulated by appropriate
genetic manipulation using the Ha-ras oncogene and the Krev-1
suppressor gene. These experimental findings support the hypothesis
that progression may involve the selective inactivation of genes
that suppress progression (progression suppressing genes) and/or
the induction of genes that promote progression (progression
enhancing genes). Identification and characterization of both types
of genetic elements would prove of immense value for defining this
important component of the cancer process and could provide useful
target molecules for intervening in the neoplastic process.
[0495] To elucidate the molecular basis of progression we are using
a subtraction hybridization approach. Subtraction hybridization
between progressed and unprogressed Ad5 transformed RE cells
resulted in the cloning of progression elevated gene-3, PEG-3, that
displays coordinate expression with the progression and
transformation phenotypes in Ad5 and oncogene transformed rat
embryo cultures. PEG-3 is a novel gene sharing nucleotide
(.about.73 and .about.68%) and amino acid (.about.59 and
.about.72%) sequence homology with the hamster growth arrest and
DNA damage inducible gene gadd34 and a homologous murine gene,
MyD116, that is induced during induction of differentiation by IL6
in murine myeloid leukemia cells. It is hypothesized that
overexpression of PEG-3 in transformed and progressed tumor cells
may facilitate progression by regulating the expression of genes
that control the cancer process, including genes directly promoting
tumorigenesis, metastasis and/or angiogenesis. Our research is
providing important information relative to the role of a novel DNA
damage-inducible gene, PEG-3, in cancer development and
progression. This information should be valuable in designing
refined and sensitive techniques for cancer detection and for
identifying cancer preventing compounds. It may also provide a
platform for developing new and improved cancer therapeutics.
[0496] The carcinogenic process involves a series of sequential
changes in the phenotype of a cell resulting in the acquisition of
new properties or a further elaboration of
transformation-associated traits by the evolving tumor cell (1-4).
Although extensively studied, the precise genetic mechanisms
underlying tumor cell progression during the development of most
human cancers remain unknown. Possible factors contributing to
transformation progression, include: activation of cellular genes
that promote the cancer cell phenotype, i.e., oncogenes; activation
of genes that regulate genomic stability, i.e., DNA repair genes;
activation of genes that mediate cancer aggressiveness and
angiogenesis, i.e., progression elevated genes; loss or
inactivation of cellular genes that function as inhibitors of the
cancer cell phenotype, i.e., tumor and progression suppressor
genes; and/or combinations of these genetic changes in the same
tumor cell (1-6). A useful model for defining the genetic and
biochemical changes mediating tumor progression is the Ad5/early
passage RE cell culture system (1,7-15). Transformation of
secondary RE cells by AdS is often a sequential process resulting
in the acquisition of and further elaboration of specific
phenotypes by the transformed cell (7-10). Progression in the
Ad5-transformation model is characterized by the development of
enhanced anchorage-independence and tumorigenic capacity (as
indicated by a reduced latency time for tumor formation in nude
mice) by progressed cells (1,10). The progression phenotype in
Ad5-transformed rat embryo cells can be induced by selection for
growth in agar or tumor formation in nude mice (7-10), referred to
as spontaneous-progression, by transfection with oncogenes (11,14),
such as Ha-ras, v-src, v-raf or E6/E7 region of human papilloma
virus type-18 (HPV-18), referred to as oncogene-mediated
progression, or by transfection with specific signal transducing
genes (15), such as protein kinase C (PKC), referred to as growth
factor-related, gene-induced progression.
[0497] Progression, induced spontaneously or after gene transfer,
is a stable cellular trait that remains undiminished in
Ad5-transformed RE cells even after extensive passage (>100) in
monolayer culture (1,10,14). However, a single-treatment with the
demethylating agent AZA results in a stable reversion in
transformation progression in >95% of cellular clones
(1,10,11,14,15). The progression phenotype is also suppressed in
somatic cell hybrids formed between normal or unprogressed
transformed cells and progressed cells (12-14). These findings
suggest that progression may result from the activation of specific
progression-promoting (progression elevated) genes or the selective
inhibition of progression-suppressing genes, or possibly a
combination of both processes. To identify potential progression
inducing genes with elevated expression in progressed versus
unprogressed AdS-transformed cells we are using subtraction
hybridization (14,16,17). The subtraction hybridization approach
resulted in cloning of PEG-3 displaying elevated expression in
progressed cells (spontaneous, oncogene-induced and growth
factor-related, gene-induced) than in unprogressed cells (parental
Ad5-transformed, AZA-suppressed, and suppressed somatic cell
hybrids) (17). These findings document a direct correlation between
expression of PEG-3 and the progression phenotype in this rat
embryo model system.
[0498] The nucleotide sequence of PEG-3 is .about.73 and .about.68%
and the amino acid sequence is .about.59 and 72% homologous to
gadd34 (18) and MyD116 (19,20), respectively (17). The sequence
homologies between PEG-3 and gadd34/MyD116 are highest in the amino
terminal region of their encoded proteins, i.e., .about.69 and
.about.76% homology with gadd34 and MyD116 respectively, in the
first 279 aa (17). In contrast, the sequence of the carboxyl
terminus of PEG-3 significantly diverges from gadd34/MyD116, i.e.,
only .about.28 and .about.49% homology in the carboxyl 88 aa (17).
The specific function of the gadd34/MyD116 gene is not known. Like
hamster gadd34 and its murine homologue MyD116, PEG-3 expression is
induced in CREF cells by MMS and gamma irradiation (17). The
gadd34/MyD116 gene, as well as the gadd45, MyD118 and gadd153
genes, encode acidic proteins with very similar and unusual charge
characteristics (21). PEG-3 also encodes a putative protein with
acidic properties similar to the gadd and MyD genes. The
carboxyl-terminal domain of the murine MyD116 protein is homologous
to the corresponding domain of the herpes simplex virus 1
.gamma..sub.134.5 protein, that prevents the premature shutoff of
total protein synthesis in infected human cells (22,23).
Replacement of the carboxyl-terminal domain of .gamma..sub.134.5
with the homologous region from MyD116 results in a restoration of
function to the herpes viral genome, i.e., prevention of early host
shutoff of protein synthesis (23). Although further studies are
necessary, preliminary results indicate that expression of a
carboxyl terminus region of MyD116 results in nuclear localization
(23). Similarly, both gadd153 and gadd45 gene products are nuclear
proteins (21). When transiently expressed in various human tumor
cell lines, gadd34/MyD116 is growth inhibitory and this gene can
synergize with gadd45 or gadd153 in suppressing cell growth (21).
In contrast, ectopic expression of PEG-3 in normal CREF (cloned rat
embryo fibroblast) and HBL-100 (normal breast epithelial) cells and
cancer (E11 and E11-NMT (Ad5-transformed rat embryo) and MCF-7 and
T47D (human breast carcinoma) cells does not significantly inhibit
cell growth or colony formation (17) (unpublished data). These
results suggest that gadd34/MyD116, gadd45, gadd153 and MyD118,
represent a novel class of mammalian genes encoding acidic proteins
that are regulated during DNA damage and stress and involved in
controlling cell growth. In this context, PEG-3 would appear to
represent an enigma, since it is not growth suppressive and its
expression is elevated in cells displaying an in vivo proliferative
advantage and a progressed transformed and tumorigenic phenotype
(17). PEG-3 may represent a unique member of this acidic protein
gene family that directly functions in regulating progression,
perhaps by constitutively inducing signals that would normally only
be induced during genomic stress. Additionally, PEG-3 may modify
the expression of down-stream genes involved in mediating cancer
aggressiveness, i.e., tumor- and metastasis-mediating genes and
genes involved in tumor angiogenesis. In these contexts, PEG-3
could function to modify specific programs of gene expression and
alter genomic stability, thereby facilitating tumor progression.
This hypothesis is amenable to experimental confirmation.
[0499] The final stage in tumor progression is the acquisition by
transformed cells of the ability to invade local tissue, survive in
the circulation and recolonize in a new area of the body, i.e.,
metastasis (24,25). Transfection of a Ha-ras oncogene into CREF
cells (26) results in morphological transformation,
anchorage-independence and acquisition of tumorigenic and
metastatic potential (27-29). Ha-ras-transformed CREF cells exhibit
profound changes in the transcription and steady-state levels of
genes involved in suppression and induction of oncogenesis (29,30).
Simultaneous overexpression of the Ha-ras suppressor gene Krev-1 in
Ha-ras-transformed CREF cells results in morphological reversion,
suppression of agar growth capacity and a delay in in vivo
oncogenesis (29). Reversion of transformation in Ha-ras+Krev-1
transformed CREF cells correlates with a return in the
transcriptional and steady-state mRNA profile to that of
nontransformed CREF cells (29,30). Following long latency times,
Ha-ras+Krev-1 transformed CREF cells form both tumors and
metastases in athymic nude mice (29). The patterns of gene
expression changes observed during progression, progression
suppression and escape from progression suppression supports the
concept of transcriptional switching as a major component of
Ha-ras-induced transformation (29,30).
[0500] Analysis of PEG-3 expression in CREF cells and various
oncogene-transformed and suppressor gene-reverted CREF cells
indicates a direct relationship between PEG-3 expression and
transformation and oncogenic progression (17). Northern blotting
indicates that CREF cells do not express PEG-3, whereas PEG-3
expression occurs in CREF cells transformed by several
diverse-acting oncogenes, including Ha-ras, v-src, HPV 18 and
mutant Ad5 (H5hr1) (17). Suppression of Ha-ras-induced
transformation by Krev-1 results in suppression of PEG-3
expression. However, both tumor-derived and metastasis-derived
Krev-1 Ha-ras-transformed CREF cells express PEG-3. The highest
relative levels of PEG-3 mRNA are consistently found in the
metastasis-derived Ha-ras+Krev-1 transformed CREF cells. These
results indicate a direct relationship between PEG-3 expression and
the transformed and oncogenic capacity of CREF cells. In addition,
PEG-3 expression directly correlates with human melanoma
progression, with the highest levels of expression found in
metastatic human melanoma and reduced levels observed in normal
human melanocytes, radial growth phase (RGP) primary melanomas and
early vertical growth phase (VGP) primary melanomas (unpublished
data). Although further studies with increased samples are
required, these intriguing results suggest that PEG-3 may be
relevant in human as well as rodent cancers.
[0501] A fundamentally important question is the role of PEG-3 in
cancer progression. PEG-3 could simply correlate with
transformation progression or alternatively it could directly
regulate this process. To distinguish between these possibilities,
E11 cells (not expressing PEG-3) were genetically engineered to
express PEG-3 (17). When assayed for growth in agar or
aggressiveness in vivo in nude mice, E11-PEG-3 cells display a
progression phenotype akin to that seen in E11-NMT cells (17.,31).
Moreover, antisense inhibition of PEG-3 in E11-NMT (normally
expressing PEG-3) results in suppression of the progression
phenotype in vitro and in vivo (31). Although the mechanism by
which PEG-3 affects cancer progression in vivo remains to be
determined, a potential role for induction of angiogenesis by PEG-3
is suggested (31). Tumors isolated from nude mice infected with
E11-NMT and E11-PEG-3 clones are highly vascularized and they
contain large numbers of blood vessels, whereas E11 and
E11-NMT-PEG-3 AS tumors grow slower and they remain compact without
extensive blood vessel involvement (31). Further studies are
necessary to determine the mechanism by which PEG-3 expression
modifies angiogenesis.
[0502] Experimental Studies
[0503] Model system for analyzing progression and suppression of
the transformed phenotype: oncogene-transformed and signal
transduction gene-transformed early passage rat embryo (RE) cell
cultures. Transformation of early passage RE cells with Ad5 or
mutants of Ad5 is a multistep process involving temporal
acquisition of enhanced transformation-related phenotypes by the
evolving transformed cells, i.e., progression. The progression
process can be accelerated by selecting cells for increased
anchorage-independence in vitro, injecting Ad5-transformed cells
into nude mice and isolating tumor-derived clonal cell lines or by
transfection with specific oncogenes (including Ha-ras, V-src,
V-raf and E6/E7 of HPV-18) or with signal transducing genes
(including the .beta..sub.1 isoform of PKC (7-15,16). In early
passage RE cells transformed by the mutant Ad5, H5ts125, the
progression phenotype, induced spontaneously by nude mouse tumor
formation or by transfection with the Ha-ras oncogene or the
.beta..sub.1 PKC gene, can be reversed by treating cells with AZA
(10,11,15). Suppression of the progression phenotype also occurs in
intraspecific somatic cell hybrids formed between normal CREF and
E11-NMT (spontaneously progressed nude-mouse tumor derived
H5ts125-transformed RE) cells or between E11 (non-progressed
H5ts125-transformed RE) and E11-NMT cells (12,14). These findings
indicate that progression in Ad5-transformed, Ad5+oncogene (Ha-ras)
transformed or Ad5+ signal transducing gene (.beta..sub.1 PKC)
transformed cells is a reversible process that behaves genetically
as a recessive phenotype. Progression may, therefore, involve the
selective inactivation of progression-suppression genes and/or the
activation of progression-inducing genes.
[0504] Identification and cloning genes associated with cancer
progression. To identify genes expressed at elevated levels in
progressed E11-NMT versus unprogressed E11 cells we are using a
subtraction hybridization approach developed in our laboratory
(16,17). For the subtraction hybridization approach, tester
(E11-NMT) and driver (E11) cDNA libraries were directionally cloned
into the commercially available .lambda. Uni-ZAP phage vector and
subtraction hybridization was then performed between
double-stranded tester DNA (E11-NMT) and single-stranded driver DNA
(E11) prepared by mass excision of the libraries. The subtracted
cDNAs were then cloned into the .lambda. Uni-ZAP phage vector and
used to probe Northern blots initially containing E11-NMT and E11
RNAs. cDNAs displaying elevated expression in E11-NMT versus E11
cells were identified, used to screen additional RNA samples and
appropriate clones were sequenced. One cDNA clone, PEG-3, displays
the predicted association with expression of the progression
phenotype (17). Expression of PEG-3 is apparent in a wide and
diverse spectrum of progressed transformed RE clones, including
spontaneously progressed (E11-NMT), progressed CREF.times.E11-NMT
somatic cell hybrids (R1 and R2), a progressed E11.times.E11-NMT
somatic cell hybrid (IIa), progressed tumor-derived
E11.times.E11-NMT somatic cell hybrids (A6-TD and IIId-TD), an HPV
18 progressed clone (E11-E6/E7), a Ha-ras progressed clone
(E11-Ras-12) and a .beta..sub.1 protein kinase C progressed clone
(E11-PKC B1) (16). In contrast, PEG-3 expression is not detected or
is apparent at reduced levels in the same series of cell lines that
do not express the progression phenotype, including unprogressed
E11, unprogressed CREF.times.E11-NMT somatic cell hybrid clones (F1
and F2), unprogressed E11.times.E11-NMT somatic cell hybrid clones
(IIId, A6 and 3b), and unprogressed E11-NMT subclones isolated
after AZA treatment (E11-NMT-AZA clone C1, B1 and C2) (FIG. 15).
These results document a direct correlation between expression of
progression and PEG-3 in RE cells displaying specific stages of
cancer progression (17).
[0505] Model system to analyze progression and suppression of the
transformed, tumorigenic and metastatic phenotype:
Ha-ras-transformed and Ha-ras+Krev-1-transformed CREF cells. A
second rodent model used to study the process of cancer progression
employs a specific clone of Fischer rat embryo fibroblast cells,
CREF, modified by transfection to express dominant acting oncogenes
(such as Ha-ras, v-src, v-raf, and HPV 18) and tumor suppressor
genes (such as Krev-1, RB and p53) (27-30,32-34). In this model
system, Ha-ras-transformed CREF cells are morphologically
transformed, anchorage-independent and induce both tumors and lung
metastases in syngeneic rats and athymic nude mice (27-30). The
Krev-1 (Ha-ras) suppressor gene reverses the in vitro and in vivo
properties in Ha-ras transformed cells (29,30). Although
suppression is stable in vitro, Ha-ras/Krev-1 CREF cells induce
both tumors and metastases after extended latency times in nude
mice (29). CREF cells, as well as Ha-ras/Krev-1 reverted cells,
contain RNA transcripts and steady-state mRNA for several cancer
suppressing genes, whereas these cells do not express transcripts
or mRNAs for several cancer promoting genes (29). During the
processes of transformation suppression and escape from
transformation suppression changes in the transcription and steady
state RNA levels of defined genes are observed (29,30).
[0506] Expression of PEG-3 occurs in tumorigenic CREF cells
transformed by v-src, HPV-18, H5hr1 (mutant of Ad5) and Ha-ras
(17). Suppression of Ha-ras induced transformation by
Krev-linhibits PEG-3 expression. However, when Ha-ras/Krev-1 cells
escape tumor suppression and form tumors and metastases in nude
mice, PEG-3 expression reappears. Treatment of CREF cells with
gamma irradiation and MMS results in PEG-3 expression by 4 hr and
continued expression at 24 hr (17 and data not shown). These
results indicate that PEG-3 expression is inducible by DNA damage
and suggests a direct association between PEG-3 expression and
oncogenic transformation and tumor progression.
[0507] Analysis of PEG-3 in Rodent Progression Models.
[0508] (1) PEG-3 is a DNA damage-inducible gene. To define the
level of regulation of PEG-3 in normal and transformed cells
nuclear run-on assays were performed (17). These studies document
that PEG-3 is transcriptionally induced in CREF cells as a function
of DNA damage, resulting from gamma irradiation or MMS treatment.
The same DNA-damage induction protocol also induces MyD116 and
gadd34 transcription in CREF cells. In contrast, analysis of
transformed CREF cells (Ha-ras), unprogressed rodent cells (E11,
E11-NMT AZA C1 and E11.times.E11-NMT 3b) and progressed rodent
cells (E11-NMT and E11.times.E11-NMT IIa) indicate that PEG-3, but
not MyD116 or gadd34, is transcribed as a consequence of
transformation progression (17). These results document that PEG-3
is a DNA damage inducible gene that is constitutively expressed in
transformed and progressed cells. They further demonstrate that a
primary level of regulation of PEG-3 occurs at a transcriptional
level.
[0509] (2) PEG-3 lacks growth inhibitory and oncogenic
transformation inducing properties. An attribute shared by the gadd
and MyD genes is their ability to markedly suppress growth when
expressed in human and murine cells (21,35). When transiently
expressed in various human and murine cell lines, gadd34/MyD116 is
growth inhibitory and this gene can synergize with gadd45 or
gadd153 in suppressing cell growth (21). To determine the effect of
PEG-3 on growth, E11 and E11-NMT cells were transfected with the
protein coding region of the PEG-3 gene cloned into a Zeocin
expression vector, pZeoSV (17). This construct permits an
evaluation of growth in Zeocin in the presence and absence of PEG-3
expression. E11 and E11-NMT cells were also transfected with the
p21 (mda-6) and mda-7 genes, previously shown to display growth
inhibitory properties (36-38). Colony formation in both E11 and
E11-NMT cells is suppressed 10-20% by PEG-3, whereas the relative
colony formation following p21 (mda-6) and mda-7 transfection is
decreased by 40-58% (17 and data not shown). Colony formation is
also reduced by 10-20% when PEG-3 is transfected into CREF, normal
human breast (HBL-100), and human breast carcinoma (MCF-7 and T47D)
cell lines (data not shown). These results document that PEG-3 is
distinct from the gadd and MyD genes since it does not
significantly alter growth when expressed in various human and
rodent cell lines. To determine if PEG-3 has transforming ability
or if it can elicit an oncogenic phenotype in rodent cells,
CREF-Trans 6 cells (39,40) were transfected with the PEG-3 gene in
a pZeoSV vector and analyzed for transformation in monolayer
culture, growth in agar and tumor formation in athymic nude mice.
PEG-3 did not induce morphological transformation or growth in agar
and pooled Zeocin resistant PEG-3 expressing CREF-Trans 6 cells did
not produce tumors in nude mice (data not shown). These results
indicate that PEG-3 does not have transforming or oncogenic
potential when expressed in normal rodent cells.
[0510] (3) PEG-3 controls the progression phenotype in
Ad5-transformed RE cells. A consequential question is whether PEG-3
expression simply correlates with transformation progression or
whether it can directly contribute or regulate this process. To
distinguish between these possibilities we have determined the
effect of stable elevated expression of PEG-3 on expression of the
progression phenotype in E11 cells. E11 cells were transfected with
a Zeocin expression vector either containing or lacking the PEG-3
gene, and random colonies were isolated and evaluated for anchorage
independent growth (17). A number of clones were identified that
displayed a 5- to 9-fold increase in agar cloning efficiency in
comparison with E11 and E11-Zeocin vector-transformed clones. Only
the three PEG-3-transfected E11 clones displaying elevated agar
growth, i.e., E11-ZeoPEG-A, E11-ZeoPEG-B and E11-ZeoPEG-C,
expressed PEG-3 mRNA (17). These findings demonstrate that PEG-3
can directly induce a progression phenotype, as monitored by
anchorage independence, in H5ts125-transformed E11 cells.
[0511] (4) PEG-3 expression correlates with cancer aggressiveness
and angiogenesis in Ad5-transformed RE cells. Studies were
conducted to determine the effect of forced PEG-3 expression in E11
cells and the consequence of antisense inhibition of expression of
PEG-3 in E11-NMT cells on tumorigenesis in nude mice. When injected
subcutaneously into nude mice, stable PEG-3 expressing E11 induced
tumors in 100% of animals (n=10) with a shorter latency time than
observed with E11 and even E11-NMT cells (data not shown). In
contrast, E11-NMT cells containing an antisense PEG-3 gene display
a reduction in agar colony formation (data not shown) and an
extension of tumor latency time in comparison with E11-NMT cells
(data not shown). Tumors that developed were analyzed and found to
be significantly larger and highly vascularized in E11-PEG-3 and
E11-NMT cells as compared to E11 and E11-NMT AS PEG-3 cells (data
not shown). Sectioning of tumors indicate extensive blood vessel
formation in E11-PEG-3 and E11-NMT cells but not in E11 parental
cells or AS PEG-3 expressing E11-NMT cells (data not shown). These
results indicate that modifying PEG-3 expression in E11 and E11-NMT
cells can directly effect tumorigenesis and blood vessel formation
(angiogenesis).
[0512] (5) Isolation and initial characterization of the PEG-3
promoter. To begin to define the mechanism by which DNA damage and
progression transcriptionally induce PEG-3 expression we have
identified a putative region of genomic DNA that contains the
promoter of this gene. This was achieved using the GenomeWalker.TM.
Kit from CLONTECH (Palo Alto, Calif.) that relies on an approach
described by Siebert et al. (41,42). Using this methodology a rat
genomic DNA fragment upstream of the 5' untranslated region of the
PEG-3 cDNA has been isolated and cloned. The size of this DNA
fragment is .about.2.1 kb and its sequence is shown in FIG. 14.
This promoter has been linked to a luciferase reporter construct
and evaluated for expression in different cell types. Additionally,
PEG-Luc reporter constructs have been stably integrated into
CREF-Trans 6, human prostate cancer DNA transformed CREF-Trans 6
(CREF-Trans 6:4 NMT, 4NMT), Ha-ras-transformed CREF (CREF-Ha-ras),
V-src-transformed CREF (CREF-src) and human papilloma virus
18-transformed CREF (CREF-HPV-18) cells. In these stable
transfectants, luciferase is inducible by DNA damage (CREF-PEG-Luc)
or constitutively expressed (4NMT-PEG-Luc, CREF-Ha-ras-Luc,
CREF-src-Luc and CREF-HPV-18-Luc). Using a PEG-Luc reporter
construct and transient transfection assays we demonstrate enhanced
expression in progressed E11-NMT and E11-Ha-ras cells versus
unprogressed E11 and E11-NMT-AZA cells (FIG. 15). In this system,
the PEG-3 promoter is constitutively active in unprogressed cells
and a relative increase of 5- to 10-fold is apparent in the
progressed cells. Studies were also performed to determine if a
relationship exists between oncogenic transformation induced by
diverse oncogenes in CREF and CREF-Trans 6 cells and expression of
the PEG-3 promoter (FIG. 16). In all cases of progression to an
oncogenic phenotype the PEG-3 promoter is more active than in CREF
or transformed CREF cells not displaying an oncogenic phenotype.
The relative fold-induction of luciferase is higher in the CREF
series than in the E11/E11-NMT series, whereas the absolute levels
of luciferase activity are lower in the CREF series. This reflects
a lower de novo (essentially null) expression of PEG-3 in CREF and
CREF-Trans 6 cells. The final test for promoter activity employed
CREF-Trans 6 cells and CREF-Trans 6 cells containing a stable
PEG-Luciferase gene (CREF-PEG-Luc cl 1). Using these cells we
demonstrate induction of luciferase activity in a temporal manner
as a function of DNA damage induced by treatment with MMS (100
.mu.g/ml) (FIG. 17). In the DNA damaged CREF cells the absolute
levels of luciferase that are induced are lower than found in
oncogenically transformed CREF cells, thereby accounting for the
lower relative fold increase apparent in luciferase activity. These
results indicate that we have identified rat genomic sequences
containing the promoter region of the PEG-3 gene that contains all
of the elements necessary for responsiveness to cellular
alterations occurring during cancer progression, oncogenic
transformation and DNA damage.
[0513] Experimental Assay Protocols for Monitoring Luciferase
Activity: Data presented in FIGS. 15 and 16. Cells were seeded at
2.times.10.sup.5/35-mm plate, .about.24 hr later cells were treated
with lipofectin containing 4 .mu.g of PEG-Luc plus a .beta.-Gal
control plasmid for 7 to 8 hr and the plates were washed and
incubated in complete medium for 48 hr. Cells were lysed in cell
lysate buffer E3971 (Promega), added to luciferase substrate E1500
(Promega) and luciferase activity was determined using a
luminometer. In FIG. 15, data reflects fold-change in luciferase
activity versus E11 cells. In FIG. 16, data reflects fold-change in
luciferase activity of transformed cells versus CREF cells. FIG.
17: CREF cells were treated as described above for 48 hr and then
exposed to 100 .mu.g/ml of MMS for 24, 18, 8, 4 and 2 hr prior to
lysate preparation and assaying for luciferase activity.
Untransfected CREF-PEG-Luc (containing an integrated PEG-Luc gene)
were treated with 100 .mu.g/ml of MMS for 24, 12, 8 and 4 hr prior
to lysate preparation and assaying for luciferase activity. Data
reflects fold-change in luciferase activity versus CREF or
CREF-PEG-Luc, respectively. All luciferase activities were
normalized to .beta.-Gal activities.
[0514] Defining the mechanism underlying the differential
expression of PEG-3 as a function of cancer progression, oncogenic
transformation and DNA damage. Nuclear run-on assays indicate that
PEG-3 expression directly correlates with an increase in the rate
of RNA transcription (17). This association is supported by the
isolation of a genomic fragment upstream of the 5' untranslated
region of the PEG-3 cDNA and demonstration that this sequence
linked to a luciferase reporter gene is activated as a function of
cancer progression, oncogenic transformation and DNA damage (FIGS.
15, 16 & 17). Additionally, changes in the stability of PEG-3
mRNA may also contribute to differential expression of this gene as
a function of cancer progression, oncogene expression and DNA
damage. To address this issue mRNA stability (RNA degradation)
assays will be performed as described in detail previously (43).
Our analysis focuses on the effect of cancer progression (E11-NMT,
R1 and R2 cells), oncogenic transformation (Ha-ras, V-src, H5hr1
and HPV-18 transformed CREF cells) and DNA damage (gamma
irradiation and MMS-treatment of CREF cells). Appropriate controls,
E11, untransformed CREF cells and CREF cells not treated with DNA
damaging agents, respectively, and experimental samples will be
incubated without additions or in the presence of 5 mg/ml of
actinomycin D (in the dark), and 30, 60 and 120 min later, total
cellular RNA will be isolated and analyzed for gene expression
using Northern hybridization. RNA blots will be quantitated by
densitometric analysis using a Molecular Dynamics densitometer
(Sunnyvale, Calif.). These straight forward experiments will
indicate if the stability of PEG-3 is altered in cells as a direct
consequence of spontaneous progression, expression of defined
oncogenes or as a consequence of DNA damage.
[0515] Most eukaryotic genes are regulated at the level of
initiation of gene transcription. Detailed characterization of many
different eukaryotic transcriptional units has led to the general
concept that specific interactions of short DNA sequences, usually
located at the 5'-flanking region of the corresponding genes
(cis-acting elements), with certain cellular proteins (trans-acting
elements) play a major role in determining the rate of initiation
of gene transcription. To elucidate the mechanism underlying the
transcriptional regulation of the PEG-3 gene the 5'-flanking region
of this gene will be analyzed. This will be important for
determining regulatory control of the PEG-3 gene including
autoregulation, developmental regulation, tissue and cell type
specific expression and differential expression in progressed
versus unprogressed cells, enhanced expression as a function of
oncogenic transformation and induction of expression as a
consequence of DNA damage. Once the appropriate regions of the
PEG-3 gene regulating the initiation of transcription has been
confirmed, studies will be conducted to determine the relevant
trans-acting regulatory factors that bind to specific cis-acting
regulatory elements and activate or repress expression of the PEG-3
gene. The experiments outlined below are designed to: [1] define
the 5'-flanking regions of the PEG-3 gene involved in mediating
differential activity of PEG-3 in progressed, oncogenically
transformed and DNA damaged cells; [2] identify cis-acting
regulatory elements in the promoter region of the PEG-3 gene which
are responsible for the differential induction of PEG-3 expression;
and [3] identify and characterize trans-acting regulatory elements
that activate (or repress) expression of the PEG-3 gene.
[0516] (1) Primary analysis of the functional regions of the PEG-3
promoter. Using a genomic walking strategy we have identified a
5'-flanking promoter region of the PEG-3 gene that appears to
encompass a functionally complete PEG-3 promoter (FIG. 14). To
define important transcriptional regulatory regions of the PEG-3
promoter, a heterologous expression system containing a luciferase
gene without promoter or enhancer has been developed using the
full-length promoter construct (44-46). Internal deletion mutations
will be generated either by taking advantage of internal
restriction sites or by a nested exonuclease III base deletion
strategy. These constructs will be transfected into E11 and
E11-NMT, untransformed and transformed CREF (H5hr1, Ha-ras, v-src
and HPV-18) and control CREF and gamma irradiation or MMS treated
CREF cells. On the basis of transfection analyses of various
deletion and point mutations it will be possible to define elements
responsible for induction of PEG-3 as a consequence of cancer
progression, specific transformation pathways or DNA damage
response.
[0517] Transcription of PEG-3 in E11-NMT cells, as determined by
nuclear run-on assays, is >20-fold higher than in E11 cells,
whereas transient transfection of the PEG-3 promoter-luciferase
gene into these two cell types indicates only an .about.5-fold
increase in activity in E11-NMT versus E11 cells. This could
indicate that the PEG-3 gene is repressed in non-expressing cells
(such as E11) through a cis-acting mechanism that is non-functional
on transiently transfected promoters. Various luciferase constructs
will be transfected into the different cell types by the
lipofectamine method or electroporation (Gene Pulser, Bio-Rad) as
previously described (44,47). To correct for DNA uptake and cell
number used for each transfection experiment, the luciferase
constructs will be transfected with plasmids containing bacterial
.beta.-galactosidase gene under the control of an Rous sarcoma
virus (RSV) promoter (44-46). Studies will be conducted using
multiple adult rat tissue Northern blots (CLONTECH) containing poly
A.sup.+ RNA and probing with PEG-3 (as well as gadd34 and MyD116)
to define which rat tissue normally express PEG-3. Previous studies
document that genes expressing in more than one tissue often
require different sequences flanking the 5'-end of the gene. It is
possible that PEG-3 expression in any normal tissue or under
different circumstances in rat cells, i.e., progression, oncogenic
transformation or DNA damage, may be regulated by different
5'-sequences. In that case, we will obtain variable luciferase
activities for different luciferase constructs in the various cell
lines. Transcription motifs contributing to PEG-3 regulation in a
tissue, cell type or specific progression, transformation or DNA
damage pathway will thus be identified.
[0518] (2) Identifying cis-acting elements in the PEG-3 promoter
responsible for expression during cancer progression, oncogenic
transformation and DNA damage. On the basis of the deletion studies
described above, the potential location of cis-acting elements
responsible for expression of PEG-3 during cancer progression,
oncogenic transformation and DNA damage will be identified. The
.about.2.1 kb PEG-3 promoter has been sequenced and potential
regulatory elements have been identified by comparison to
previously characterized transcriptional motifs. The PEG-3 promoter
contains a number of potentially important transcriptional motifs
including PEA3 (AGGAAA), E2A (GCAGGTG), GRE (TGTTCT), E2F
(TTTTGGCCG), TRE (GGTCA), acute phase reactive regulating element
(GTGGGA), SP1 (GGGCGG), AP1 (TGACTCA), AP2 (TCCCCAACCC) and NF1
(TGGATTTGAGCCA). The importance of these sequences in regulating
PEG-3 expression during cancer progression, oncogenic
transformation and DNA damage will be determined by introducing
point mutations in a specific cis element into the promoter region
using previously described site-specific mutagenesis techniques
(44,47-50) or with recently described PCR-based strategies, i.e.,
ExSite.TM. PCR-based site-directed mutagenesis kit and the
Chameleon.TM. double-stranded site-directed mutagenesis kit
(Stratagene, CA). The mutated promoter constructs will be cloned
into luciferase expression vectors and tested for their effects on
the promoter function by transfection into different cell types and
monitoring luciferase activity. Since the promoter region for the
PEG-3 gene is located in front of the luciferase reporter gene in
the various pPEG-Luciferase constructs, the change in luciferase
activity for each construct will permit a direct comparison of the
activity of the mutant promoter to that of the unmodified PEG-3
promoter.
[0519] After the regulatory regions of the PEG-3 promoter are
confirmed experiments will be conducted to address a number of
important questions relative to cancer progression, oncogenic
transformation and DNA damage induction of PEG-3 expression. (i)
Nuclear run-on and transient transfection assays with
pPEG-Luciferase constructs will be used to determine the effect of
changes in DNA methylation (AZA and phenyl butyrate treatment) on
PEG-3 expression in E11-NMT cells, treatment with different classes
of DNA damaging and cancer modulating agents (such as TPA,
retinoids, UV-C, gamma irradiation, methylating carcinogens,
topoisomerase inhibitors, okadaic acid, etc.) on PEG-3 expression
in CREF and CREF-PEG-Luc cl 1 cells (PEG-Luciferase stably
transformed CREF clone) and exposure to cancer modulating agents
(such as the Krev-1 gene, dominant negative inhibitors of specific
oncogenes, chemicals such as CAPE, retinoids, sodium butyrate,
interferon, TNF-.alpha. and additional progression modulating
agents) on PEG-3 expression in oncogenically transformed CREF cells
(1,8-10,18,21,28,29,32,33,51); (ii) The level of PEG-3
transcription in cells displaying different stages of cancer
progression and oncogenic transformation, including rodent model
systems of cancer progression (such as the Dunning rat prostate
model, metastatic murine melanoma variants, etc.) and additional
rodent cells transformed by various oncogenes. These studies will
indicate if expression of PEG-3 occurs in additional pathways of
progression and transformation. (iii) Transfection of varying
lengths of the 5' flanking region and internal deletion luciferase
constructs into rodent cells displaying different stages of
progression, transformed by different classes of oncogenes and
treated with various DNA damaging and cancer promoting and
inhibiting agents. These regulatory elements will be sequenced and
compared with previously characterized transcriptional motifs to
identify potential positive and negative regulatory elements; (iv)
In addition to mutagenesis studies (to define functional motifs
regulating transcriptional regulation of the PEG-3 promoter),
cotransfection studies will be conducted with cDNAs containing
putative positive acting regulatory elements and a minimal PEG-3
promoter-Luciferase construct into unprogressed and progressed
rodent cells, untransformed CREF and oncogenically transformed CREF
and untreated and DNA damage treated CREF cells. These studies will
indicate if the introduction of specific putative positive acting
regulatory elements can enhance PEG-3 expression in cells
cotransfected with a minimal PEG-3 promoter region. The potential
role of putative cis-acting negative regulatory elements will be
addressed by cotransfection with a complete PEG-3 promoter region
into the same target cells. These studies will provide relevant
information about the potential role of inhibitory elements in
regulating PEG-3 expression. (v) Experiments will also be performed
to evaluate the status of the endogenous PEG-3 gene during cancer
progression, oncogenic transformation and DNA damage. This will be
approached by using DNase hypersensitivity assays to look for
structural changes in this gene (44). Although not within the scope
of the present studies, future studies could involve the
identification of a human PEG-3 cDNA, elucidation of the human
PEG-3 promoter and analysis of the level of PEG-3 expression in
human progression model systems. These studies would be quite
informative in providing a potential link between PEG-3 expression
and cancer progression in human cells.
[0520] (3) Identifying trans-acting nuclear proteins that mediate
transcriptional enhancing activity of the PEG-3 gene during cancer
progression, oncogenic transformation and DNA damage. The current
view on regulation of eukaryotic gene expression suggests that
trans-acting proteins bind to specific sites within cis-elements of
a promoter region resulting in transcriptional activation (52,53).
Experiments will be performed to identify trans-acting factors
(nuclear proteins) and determine where these factors interact with
cis-regulatory elements. To achieve this goal, two types of studies
will be performed, one involving gel retardation (gel shift) assays
(15,44,54,55) and the second involving DNase-I footprinting
(methylation interference) assays (44,56,57).
[0521] Gel shift assays will be used to analyze the interactions
between cis-acting elements in the PEG-3 promoter and trans-acting
factors in mediating transcriptional control (15,54,55). For this
assay, .sup.32P-labeled cis-elements will be incubated with nuclear
extracts from E11 and E11-NMT, CREF and transformed CREF (Ha-ras,
v-src, H5hr1 and HPV-18) and untreated CREF and CREF treated with
MMS (100 .mu.g/ml for 8 hr) or gamma irradiation (10 Gy for 4 hr)
and reaction mixtures will be resolved on 5 or 8% polyacrylamide
gels. After autoradiography, the pattern of retarded DNAs on the
gel will provide information concerning the interaction between
trans-acting factors and specific regions of the cis-acting
elements in the PEG-3 promoter. Non-labeled cis-acting elements
(self-competition) will be added as a competitor to duplicate
samples to eliminate the possibility of non-specific binding and to
confirm that the interaction is really conferred by the
trans-acting factor. To begin to identify the transacting factors,
different non-labeled DNAs (including those corresponding to
sequences identified in the PEG-3 promoter, such as TATA, PEA3,
E2A, GRE, E2F, TRE, acute phase reactive regulating element, SP1,
API.sub.1, AP2 and NF1) can be used as competitors in the gel shift
assay to determine the relationship between the trans-acting
factors and previously identified transcriptional regulators. It is
possible that the trans-acting factors regulating transcriptional
control of the PEG-3 promoter may be novel. To identify these
factors extracts will be purified from E11 and E11-NMT, CREF and
transformed CREF and untreated and DNA damaged CREF cells by two
cycles of heparin-Sepharose column chromatography, two cycles of
DNA affinity chromatography and separation on SDS-polyacrylamide
gels (58,59). Proteins displaying appropriate activity using gel
shift assays will be digested in situ with trypsin, the peptides
separated by HPLC and the peptides sequenced (60). Peptide
sequences will be used to synthesize degenerate primers and RT-PCR
will be used to identify putative genes encoding the trans-acting
factor. These partial sequences will be used with cDNA library
screening approaches and the RACE procedure, if necessary, to
identify full-length cDNAs encoding the trans-acting factors
(17,47,61,62). Once identified, the role of the trans-acting
factors in eliciting cancer progression will be analyzed. (i) The
functionality of positive and negative trans-acting factors will be
determined by transiently and stably expressing these genes in E11
and E11-NMT cells to determine effects on anchorage independence
and tumorigenic potential in nude mice (stable expression).
Positive effects would be indicated if overexpressing a positive
trans-acting factor facilitates the progression phenotype, whereas
overexpressing a negative trans-acting factor inhibits the
progression phenotype. (ii) Antisense approaches will be used to
determine if blocking the expression of positive or negative
trans-acting factors can directly modify the progression state. A
direct effect of a positive trans-acting factor in affecting
progression would be suggested if antisense inhibition of the
positive factor partially or completely inhibits the progression
phenotype in E11-NMT, i.e., growth in agar is reduced and tumor
latency time is extended. Conversely, a direct effect of negative
trans-acting factors in inhibiting progression would be suggested
if antisense inhibition of the negative factor enhances the ability
of E11 to grow in agar and reduces tumor latency time. A potential
problem with these types of studies would be encountered if the
factors are involved in the regulation of many genes, e.g.,
Fos/Jun, and the antisense effects may, therefore, be non-specific.
Although not within the scope of the present proposal, depending on
the results obtained, cis-element knockouts could be used to
further define the role of these elements in regulating PEG-3
expression.
[0522] For DNase-I footprinting assays, nuclear extracts from E11
and E11-NMT, CREF and transformed CREF and untreated CREF and DNA
damaged (MMS and gamma irradiation) CREF cells will be prepared and
DNase-I footprinting assays will be performed as described
(44,63,64). The promoter necessary for PEG-3 expression, identified
from the experiments described above, will be terminally labeled
with .sup.32P and incubated with crude nuclear extracts from the
different cell types and experimental conditions described above
using established protocols (44,63,64). The reaction mixture that
has been digested with DNase-I enzyme will be terminated and the
digested products will be analyzed on an 8% sequencing gel. The
differential protection between nuclear extracts from progressed
versus unprogressed, untransformed and oncogenically transformed
and undamaged and DNA damaged cells will provide relevant
information concerning the involvement of trans-acting factors in
activation and the location of specific sequences in the
cis-regulatory elements of the PEG-3 promoter mediating this
activation. If differential protection is not detected using this
approach, the sensitivity of the procedure can be improved by using
different sized DNA fragments from the PEG-3 promoter region or by
using partially purified nuclear extracts (44,63,64).
[0523] The studies described above will result in the
characterization of the PEG-3 promoter region, the identification
of cis-acting regulatory elements in the PEG-3 promoter and the
identification of trans-acting regulatory elements that activate
(or repress) expression of the PEG-3 gene as a function of cancer
progression, oncogenic transformation and DNA damage. This
information could prove valuable in designing approaches for
selectively inhibiting PEG-3 expression, and therefore modifying
cellular phenotypes related to cancer progression and response to
DNA damage.
[0524] The PEG-3 promoter as a sensitive biosensor monitoring
system for identifying compounds with the capacity to modulate
cancer progression and oncogenic transformation. As documented in
this grant proposal, PEG-3 expression is elevated as a function of
cancer progression, oncogenic transformation and DNA damage (17).
Moreover, the PEG-3 promoter displays increased activity in cells
displaying these different phenotypes (FIGS. 15, 16 & 17).
These observations suggest that cell cultures stably expressing
PEG-3 may prove effective as biosensor monitoring systems for
identifying compounds and experimental conditions that can regulate
important physiological processes, including cancer progression,
oncogenic transformation, DNA damage and angiogenesis. This
approach is called the Rapid Promoter Screening (RPS) assay system
(FIG. 18). This strategy uses stable cell lines containing a
PEG-Luciferase transgene for evaluating the activation or
suppression of transcription from the PEG-3 promoter. Stable
expression systems are preferable to transfection of the
PEG-Luciferase gene into target cells since they are not dependent
on variable transfection efficiencies or affected by cellular
heterogeneity. Moreover, cells containing a stable PEG-Luciferase
transgene can be used to develop a high throughput RPS assay system
with the capacity to evaluate large numbers of compounds in a
simple manual or automated assay. Initial proof-of-principle for
the RPS assay system has now been obtained. CREF and 4NMT clones
have been established that stably express a PEG-Luciferase
transgene (FIG. 17). Treatment of CREF-PEG-Luc clones with the DNA
damaging agent MMS (100 .mu.g/ml) results in a temporal induction
of luciferase activity (FIG. 17). Similarly, MMS treatment of
parental CREF cells transiently transfected with a PEG-Luciferase
construct results in similar kinetics of luciferase induction.
Stable PEG-Luciferase expressing clones of 4NMT cells, tumorigenic
CREF-Trans 6 cells transformed with human LNCaP DNA and expressing
the PTI-1 oncogene (65), results in 4NMT-PEG-Luc clones
constitutively expressing luciferase activity (unpublished data).
When treated with an antisense phosphorothioate oligonucleotide
that modifies the transformed state by suppressing PTI-1
expression, the level of luciferase expression is inhibited
(unpublished data). These results support the suggestion that this
approach will prove amenable for developing an RPS biosensor
monitoring assay system to detect agents inducing DNA damage,
enhancing cancer progression, inducing oncogenic transformation
pathways and regulating angiogenesis (CREF-PEG-Luc) or agents
inhibiting these processes (4NMT-PEG-Luc).
[0525] Initial studies will focus on the CREF-PEG-Luc and
4NMT-PEG-Luc assay systems. These cells will be used to test the
utility of the RPS biosensor monitoring system with well
characterized reagents capable of modifying specific physiological
pathways. The two cell types will be treated with various agents,
including DNA damaging (a spectrum of compounds that elicit
different DNA repair pathways, used alone and in combination with
agents that directly modify DNA repair processes), oncogenic
transformation pathway inducing (such as phorbol ester tumor
promoters, tyrosine kinase pathway modifiers and compounds
affecting DNA methylation) and angiogenesis inducing and inhibiting
agents (such as TNF-.alpha., BFGF, alpha interferon, beta
interferon, thrombospondin and pleiotropin) and then monitored for
luciferase activity. If these studies are successful, i.e., the
assay systems can identify agents that have an impact on specific
biological pathways, they would provide the basis for future
expanded studies using this strategy. These studies would include:
(i) Screening small molecules, produced by recombinatorial
chemistry, to identify potentially important and clinically useful
modulators of DNA damage and repair, cancer progression, oncogenic
transformation and angiogenesis; (ii) Evaluating stable
CREF-ras-PEG-Luc, CREF-src-PEG-Luc, S CREF-HPV-PEG-Luc and
additional PEG-Luc transformed cell lines. These luciferase
expressing cells could be used to directly identify inhibitory
molecules suppressing specific oncogenic transformation pathways.
(iii) CREF-PEG-Luc or CREF-PEG-.beta.-Gal (beta galactosidase)
containing cells could be used to identify transforming cDNAs
capable of initiating cellular transformation and consequently
inducing PEG transcription. The human tumor cDNA containing
CREF-PEG-Luc or CREF-PEG-.beta.-Gal expressing clones could then be
used to identify the putative transforming human tumor cDNAs. (iv)
Transfection of normal cDNAs into transformed CREF-PEG or
CREF-.beta.-Gal expressing cells could be used to identify
potentially novel cancer suppressor genes, by their ability to
inhibit PEG transcription. Appropriately modified cells could then
be used to clone the putative human tumor suppressor cDNA. (v) A
PEG-.beta.-Gal transgene could be inserted into mouse ovum to
create transgenic mice harboring this gene. These animals could
then be used as a sensitive in vivo indicator for evaluating DNA
damage resulting from exposure to chemotherapeutic agents,
evaluating gene regulation leading to cancer formation and
identifying early stages in the conversion of a normal cell into a
cancer cell.
[0526] PEG-3 Promoter-Luciferase Biosensor Monitoring Assay System:
The basic protocol for this assay will involve incubating target
cells for varying times with compounds or growing cells under
experimental conditions that either induce (CREF-PEG-Luc) or
inhibit (4NMT-PEG-Luc) luciferase activity and assaying for such
activity. As part of the experimental procedures, appropriate
control reagents will be used. These will include MMS for the
CREF-PEG-Luc cell culture system and PTI-1 antisense
phosphorothioate oligonucleotide bridge primers for the
4NMT-PEG-LUC cell culture system. Two formats can be used for
assaying compounds, one employing cells plated in 35 mm-tissue
culture plates and the other employing cells plated in 96-well
microtiter plates. The former approach will be used to evaluate
small numbers of compounds or experimental conditions (maximum of
60 plates (20 agents tested in triplicate) in a single assay), and
the latter approach can be adapted for screening large numbers of
compounds in an automated fashion (96 agents tested per assay
block, using multiple assay blocks). After incubating cells for
different times with test reagents, growth medium will be removed,
the cells will be washed with serum-free growth medium and the
cells will be lysed using Reporter Lysis Buffer (Promega, Cat #
E4531). Samples (placed in microcentrifuge tubes) or plates
(96-well microtiter format) can be stored at -70.degree. C. (to be
assayed within 24-hr or stored for several weeks with samples
remaining stable through several freeze-thaw cycles). Samples or
plates are centrifuged to remove debris and a 10 .mu.l aliquot is
removed for monitoring luciferase assay using a luminometer.
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[0593] Sixth Series of Experiments
[0594] PEG-3 Promoter/PTI-1 System
[0595] Rationale: Cancer is a progressive, multigenic disorder
characterized by changes in the transformed phenotype that
culminates in metastatic disease. Through the use of subtraction
hybridization, a novel gene associated with transformation
progression in virus- and oncogene-transformed rat embryo cells was
cloned. The gene, designated PEG-3, shares significant nucleotide
and amino acid sequence homology with the hamster growth arrest and
DNA-damage inducible gene gadd34 and a homologous murine gene,
MyD116, that is induced during induction of terminal
differentiation by IL-6 in murine myeloid leukemia cells. PEG-3
expression is elevated in rodent cells displaying a
progressed-transformed phenotype and in rodent cells transformed by
various oncogenes including Ha-ras, v-src, mutant type 5
adenovirus, and human papilloma virus type 18 (HPV). PEG-3 is
transcriptionally activated in rodent cells, as is gadd34 and
MyD116, after treatment with DNA damaging agents. However, only
PEG-3 is active in rodent cells displaying a transformed progressed
phenotype. PEG-3 has been shown to be upregulated in ras, src, HPV,
and PTI-1 transformed CREF-Trans 6 cells but not in CREF-Trans 6
itself. In addition, the gene is also expressed in various human
tumor lines but not in normal cell lines.
[0596] Recently, the PEG-3 promoter has been isolated and linked to
a luciferase reporter gene. Transient transfection of the
promoter/reporter construct into cell lines of both tumor and
normal origin has demonstrated that the promoter is constitutively
expressed in the tumor cell lines but not in normal cells. We have
stably transfected the PEG-3/luciferase construct into various
transformed derivatives of CREF-Trans 6 containing the oncogenes
PTI-1, ras, and src, as well cells transformed by HPV. These cell
lines are used as targets for our small molecule combinatorial
libraries.
[0597] The PEG-3 Promoter Assay. Cell Lines: Stable transfectants
expressing a PEG-3 promoter/luciferase construct were prepared in
CREF-Trans 6:4NMT (PTI-1 positive), T24 (ras positive); CREF-src,
and CREF-HPV. Clones were isolated that constitutively expressed
high levels of luciferase. These clones are currently being used as
targets in GenQuest's small molecule screening assays.
[0598] The following protocol is being used to screen small
molecule libraries:
[0599] a. Cells (e.g., 4NMT; PTI-1 positive) are plated in 96 well
opaque microtiter plates at a cell concentration of
1-2.times.10.sup.4 cells/well in 100 ul of growth medium.
[0600] b. A stock solution of the combinatorial library is prepared
in DMSO (final concentration=1 mM). Further dilutions are made in
growth medium to final concentrations of 20 uM, 2 uM, and 0.2
uM.
[0601] c. 100 ul/well of each sample is added to the cells. The
final concentrations are 10, 1, and 0.1 uM.
[0602] d. Growth medium alone is added to the first two wells (Row
A, wells 1 and 2) of each plate; a positive control is added to the
next two wells (Row A, wells 3 and 4). In the case of PTI-1, an
antisense oligonucleotide to the bridge region has been shown to
down-regulate luciferase activity within 24 hours.
[0603] e. Plates are incubated overnight at 37C. Medium is removed
and cells are washed with PBS. Cells are lysed using Reporter
Lysis.Buffer supplied by the kit manufacturer (Luciferase Reporter
Gene Assay kit; Boehringer Mannheim, cat. #1 669 893).
[0604] f. Plates are placed .about.70C overnight. Alternatively,
the plates can be stored for several weeks (the manufacturer notes
that luciferase is stable in their Reporter Lysis Buffer) over
several freeze thaw cycles).
[0605] 9. Plates are thawed and 50 ul of the luciferase reaction
mixture is added. Plates are immediately read in a 96 well
luminometer (Tropix TR717 microplate luminometer).
[0606] h. A hit is defined as the decrease in luciferase activity
compared to control.
[0607] This assay can be completed in 48 hours:
[0608] i. Overnight incubation with library
[0609] ii. Luciferase assay: 4 hours
[0610] For viability assays.
[0611] a. Plates are set up as above except in transparent 96 well
microtiter plates.
[0612] b. After a 24 hour incubation, 10 ul of WST-1 (Cell
Proliferation Reagent; Boehringer Mannheim 1 544 807) is added to
each well.
[0613] c. Plates are read at 30, 60, 120, and 180 minutes in an
ELISA reader at 450 nM).
[0614] d. Viability is calculated as a percentage of the untreated
cells.
[0615] Deletion Analysis of the Rat PEG-3 Promoter
[0616] A series of deletion mutants have been produced by
mutagenesis (Erase-a-Base System, Promega), linked to a luciferase
reporter gene and transfected into different cell types to define
the regions of the PEG-3 promoter that mediate differential
expression in progressed versus unprogressed, oncogenically
transformed versus nontransformed and DNA damaged versus undamaged
cells. The mutagenesis procedure was performed as described by
Promega. The position of the various deletion mutants (determined
by sequence analysis) relative to the promoter are shown in FIG.
24. The various deletion clones are indicated by numbers ranging
from 2 to 11, with 1 being the complete PEG-3 promoter, and the
clone designations are indicated in brackets. These include: 1
(46): intact rat PEG-3 promoter; 2 (22): missing 223 nt from the 5'
region of the rat PEG-3 promoter; 3 (33): missing 386 nt from the
5' region of the rat PEG-3 promoter; 4 (42): missing 507 nt from
the 5' region of the rat PEG-3 promoter; 5 (14): missing 784 nt
from the 5' region of the rat PEG-3 promoter; 6 (8): missing 809 nt
from the 5' region of the rat PEG-3 promoter; 7 (65): missing 853
nt from the 5' region of the rat PEG-3 promoter; 8 (74): missing
1190 nt from the 5' region of the rat PEG-3 promoter; 9 (17):
missing 1215 nt from the 5' region of the rat PEG-3 promoter; 10
(114): missing 1511 nt from the 5' region of the rat PEG-3
promoter; and 11 (20): missing 1737 nt from the 5' region of the
rat PEG-3 promoter. The different deletion mutants are being used
to define the regions of the PEG-3 promoter that can enhance or
suppress expression of the PEG-3 gene. These constructs can now be
used to determine the regulatory control of the PEG-3 gene
including autoregulation, developmental regulation, tissue and cell
type specific expression and differential expression in progressed
versus unprogressed, enhanced expression as a function of oncogenic
transformation and induction of expression as a consequence of DNA
damage.
[0617] Expression of the intact PEG-3 promoter and various deletion
mutants of the PEG-3 promoter in the different cell types is shown
in FIGS. 25 to 31. The different cell types include: E11 (FIG. 12),
E11-NMT (FIG. 26), a comparison of E11 versus E11-NMT (FIG. 14),
E11-PKC (FIG. 28), CREF-HPV (FIG. 29), CREF-ras (FIG. 30) and
CREF-Trans 6:4 NMT (FIG. 31).
[0618] Seventh Series of Experiments
[0619] CURE (Cancer Utilized Reporter Execution): A Strategy for
Selectively Inhibiting the Growth and/or Killing of Cancer
Cells
[0620] Use of CIRAs (Cancer Inhibitory Recombinant Adenoviruses) in
CURE. Cancer is a progressive process with defined temporal stages
culminating in metastatic potential by evolving tumor cells.
Although extensively scrutinized the molecular determinants of
cancer progression remain unclear. Well-characterized cell culture
systems are valuable experimental tools for defining the
biochemical and molecular basis of progression. Two rodent model
systems are providing insights into the genes and processes
regulating malignant progression of the transformed cell.
[0621] In adenovirus type 5 (Ad5) transformed rat embryo (RE)
cells, progression can occur spontaneously by tumor formation in
nude mice or by ectopic expression of oncogenes and signal
transducing growth-regulating genes. In all contexts of
progression, the demethylating agent 5-azacytidine (AZA) can
reverse this process resulting in an unprogressed phenotype in
>95% of treated clones. Inhibition of progression also occurs in
this system after forming somatic cell hybrids between progressed
and unprogressed cells. Using an immortal cloned rat embryo
fibroblast (CREF) cell culture system, progression to metastasis
and reversion of progression can be regulated by appropriate
genetic manipulation using the Ha-ras oncogene and the Krev-1
suppressor gene. These experimental findings support the hypothesis
that progression may involve the selective inactivation of genes
that suppress progression (progression suppressing genes) and/or
the induction of genes that promote progression (progression
enhancing genes). Identification and characterization of these
genetic elements would prove of immense value for defining this
significant and defining component of the cancer process and could
provide useful target molecules for intervening in the neoplastic
process.
[0622] To elucidate the molecular basis of progression we are using
a subtraction hybridization approach. Subtraction hybridization
between progressed and unprogressed Ad5 transformed RE cells
resulted in the cloning of progression elevated gene-3, PEG-3, that
displays coordinate expression with the progression and
transformation phenotypes in AdS and oncogene transformed rat
embryo cultures. PEG-3 is a novel gene sharing nucleotide
(.about.73 and .about.68%) and amino acid (.about.59 and
.about.72%) sequence homology with the hamster growth arrest and
DNA damage inducible gene gadd34 and a homologous murine gene,
MyD116, that is induced during induction of differentiation by IL6
in murine myeloid leukemia cells. Like gadd34 and MyD116, PEG-3
expression is induced by DNA damage. Induction following DNA damage
results from increased RNA transcription and elevated steady-state
levels of the PEG-3 gene. PEG-3 expression is also elevated in
temperature sensitive mutant adenovirus transformed Sprgaue-Dawley
rat embryo cells as a consequence of increased expession of the
transformed phenotype, i.e., elevated cancer progression.
Additionally, PEG-3 expression increases in CREF cells as a
consequence of oncogenic transformation by diverse acting
oncogenes, including Ha-ras, v-src, human papilloma virus type 18,
v-raf, mutant type 5 adenovirus (H5hr1) and prostate tumor inducing
gene-1 (PTI-1). These experimental results support the hypothesis
that PEG-3 expression is regulated by cancer progression, oncogenic
transformation and DNA damage.
[0623] To define the mode of action of PEG-3 a 5' DNA sequence
containing the promoter region of this gene has been isolated and
analyzed. The PEG-3-Promoter (PEG-Prom) (.about.2.1 kb) has been
linked to a luciferase reporter gene (PEG-Prom-Luc) and evaluated
for expression in various cell types. Elevated levels of
PEG-Prom-Luc activity are apparent in transformed rodent cells
displaying a progressed transformed phenotype, DNA damaged rodent
and human cells, oncogenically transformed rodent cells and
histologically distinct human cancer cells (including metastatic
melanoma, glioblastoma multiforme and carcinomas of the breast,
cervix, colon, lung, nasopharyngx and prostate). On the basis of
the selective activity of the PEG-Prom for cancer cells, genetic
vectors are being constructed that display targeted expression of
growth arresting and apoptosis-inducing genes or genes encoding an
enzyme permitting activation of a toxic product in cancer cells.
Additionally, genetic vectors can be constructed using the CURE
protocol that target the expression of molecules on the surface of
only cancer cells permitting the directed therapy of cancer using
immunological reagents (monoclonal antibodies, cytotoxic T-cells,
TILs, etc.) or toxic chemicals. These novel vectors are the basis
for the CURE (Cancer Utilized Reporter Execution) protocol (FIG.
32). As one application of CURE, recombinant adenoviruses are being
constructed that permit the efficient delivery of CURE vectors into
cells. These vectors are designed as CIRAs (Cancer Inhibitory
Recombinant Adenoviruses) and they contain the PEG-Prom driving
expression of target genes, including wild-type p53 (wt p53),
melanoma differentiation associated gene-7 (mda-7), adenovirus E1A
and E1B (Ad E1A and E1B) or herpes simplex type 1 thymidine kinase
gene (HSV TK) (FIG. 32). When cancer cells are infected with the
CIRAs, the appropriate genes are activated resulting in a direct
growth inhibition or apoptosis (wt p53 or mda-7), cell death
following adenovirus replication (Ad E1A and E1B) or cell death
following administration of gangcyclovir (HSV TK).
[0624] The carcinogenic process involves a series of sequential
changes in the phenotype of a cell resulting in the acquisition of
new properties or a further elaboration of
transformation-associated traits by the evolving tumor cell (rev
1-4). Although extensively studied, the precise genetic mechanisms
underlying tumor cell progression during the development of most
human cancers remain unknown. Possible factors contributing to
transformation progression, include: activation of cellular genes
that promote the cancer cell phenotype, i.e., oncogenes; activation
of genes that regulate genomic stability, i.e., DNA repair genes;
activation of genes that mediate cancer aggressiveness and
angiogenesis, i.e., progression elevated genes; loss or
inactivation of cellular genes that function as inhibitors of the
cancer cell phenotype, i.e., tumor and progression suppressor
genes; and/or combinations of these genetic changes in the same
tumor cell (rev 1-6). A useful model for defining the genetic and
biochemical changes mediating tumor progression is the Ad5/early
passage RE cell culture system (1,7-15). Transformation of
secondary RE cells by Ad5 is often a sequential process resulting
in the acquisition of and further elaboration of specific
phenotypes by the transformed cell (7-10). Progression in the
Ad5-transformation model is characterized by the development of
enhanced anchorage-independence and tumorigenic capacity (as
indicated by a reduced latency time for tumor formation in nude
mice) by progressed cells (1,10). The progression phenotype in
Ad5-transformed rat embryo cells can be induced by selection for
growth in agar or tumor formation in nude mice (7-10), referred to
as spontaneous-progression, by transfection with oncogenes (11,14),
such as Ha-ras, v-src, v-raf or E6/E7 region of human papilloma
virus type-18 (HPV-18), referred to as oncogene-mediated
progression, or by transfection with specific signal transducing
genes (15), such as protein kinase C (PKC), referred to as growth
factor-related, gene-induced progression.
[0625] Progression, induced spontaneously or after gene transfer,
is a stable cellular trait that remains undiminished in
Ad5-transformed RE cells even after extensive passage (>100) in
monolayer culture (1,10,14). However, a single-treatment with the
demethylating agent AZA results in a stable reversion in
transformation progression in >95% of cellular clones
(1,10,11,14,15). The progression phenotype is also suppressed in
somatic cell hybrids formed between normal or unprogressed
transformed cells and progressed cells (12-14). These findings
suggest that progression may result from the activation of specific
progression-promoting (progression elevated) genes or the selective
inhibition of progression-suppressing genes, or possibly a
combination of both processes. To identify potential progression
inducing genes with elevated expression in progressed versus
unprogressed AdS-transformed cells we are using a subtraction
approach (14,16,17). The subtraction hybridization approach
resulted in cloning of PEG-3 displaying elevated expression in
progressed cells (spontaneous, oncogene-induced and growth
factor-related, gene-induced) than in unprogressed cells (parental
Ad5-transformed, AZA-suppressed, and suppressed somatic cell
hybrids) (17). These findings document a direct correlation between
expression of PEG-3 and the progression phenotype in this rat
embryo model system.
[0626] The nucleotide sequence of PEG-3 is .about.73 and .about.68%
and the amino acid sequence is .about.59 and 72% homologous to
gadd34 (18) and MyD116 (19,20), respectively (17). The sequence
homologies between PEG-3 and gadd34/MyD116 are highest in the amino
terminal region of their encoded proteins, i.e., .about.69 and
.about.76% homology with gadd34 and MyD116 respectively, in the
first 279 aa (17). In contrast, the sequence of the carboxyl
terminus of PEG-3 significantly diverges from gadd34/MyD116, i.e.,
only .about.28 and .about.49% homology in the carboxyl 88 aa (17).
The specific function of the gadd34/MyD116 gene is not known. Like
hamster gadd34 and its murine homologue MyD116, PEG-3 expression is
induced in CREF cells by MMS and gamma irradiation (17). The
gadd34/MyD116 gene, as well as the gadd45, MyD118 and gadd153
genes, encode acidic proteins with very similar and unusual charge
characteristics (21). PEG-3 also encodes a putative protein with
acidic properties similar to the gadd and MyD genes. The
carboxyl-terminal domain of the murine MyD116 protein is homologous
to the corresponding domain of the herpes simplex virus 1
.gamma..sub.134.5 protein, that prevents the premature shutoff of
total protein synthesis in infected human cells (22,23).
Replacement of the carboxyl-terminal domain of .gamma..sub.134.5
with the homologous region from MyD116 results in a restoration of
function to the herpes viral genome, i.e., prevention of early host
shutoff of protein synthesis (23). Although further studies are
necessary, preliminary results indicate that expression of a
carboxyl terminus region of MyD116 results in nuclear localization
(23). Similarly, both gadd153 and gadd45 gene products are nuclear
proteins (21). When transiently expressed in various human tumor
cell lines, gadd34/MyD116 is growth inhibitory and this gene can
synergize with gadd45 or gadd153 in suppressing cell growth (21).
In contrast, ectopic expression of PEG-3 in normal CREF (cloned rat
embryo fibroblast) and HBL-100 (normal breast epithelial) cells and
cancer (E11 and E11-NMT (Ad5-transformed rat embryo) and MCF-7 and
T47D (human breast carcinoma) cells does not significantly inhibit
cell growth or colony formation (17). These results suggest that
gadd34/MyD116, gadd45, gadd153 and MyD118, represent a novel class
of mammalian genes encoding acidic proteins that are regulated
during DNA damage and stress and involved in controlling cell
growth. In this context, PEG-3 would appear to represent an enigma,
since it is not growth suppressive and its expression is elevated
in cells displaying an in vivo proliferative advantage and a
progressed transformed and tumorigenic phenotype (17). PEG-3 may
represent a unique member of this acidic protein gene family that
directly functions in regulating progression, perhaps by
constitutively inducing signals that would normally only be induced
during genomic stress. Additionally, PEG-3 may modify the
expression of down-stream genes involved in mediating cancer
aggressiveness, i.e., tumor- and metastasis-mediating genes and
genes involved in tumor angiogenesis. In these contexts, PEG-3
could function to modify specific programs of gene expression and
alter genomic stability, thereby facilitating tumor progression.
This hypothesis is amenable to experimental confirmation.
[0627] The final stage in tumor progression is the acquisition by
transformed cells of the ability to invade local tissue, survive in
the circulation and recolonize in a new area of the body, i.e.,
metastasis (rev. 24,25). Transfection of a Ha-ras oncogene into
CREF cells (26) results in morphological transformation,
anchorage-independence and acquisition of tumorigenic and
metastatic potential (27-29). Ha-ras-transformed CREF cells exhibit
profound changes in the transcription and steady-state levels of
genes involved in suppression and induction of oncogenesis (30,31).
Simultaneous overexpression of the Ha-ras suppressor gene Krev-1 in
Ha-ras-transformed CREF cells results in morphological reversion,
suppression of agar growth capacity and a delay in in vivo
oncogenesis (30). Reversion of transformation in Ha-ras+Krev-1
transformed CREF cells correlates with a return in the
transcriptional and steady-state mRNA profile to that of
nontransformed CREF cells (30,31). Following long latency times,
Ha-ras+Krev-1 transformed CREF cells form both tumors and
metastases in athymic nude mice (30). The patterns of gene
expression changes observed during progression, progression
suppression and escape from progression suppression supports the
concept of transcriptional switching as a major component of
Ha-ras-induced transformation (30,31).
[0628] Analysis of PEG-3 expression in CREF cells and various
oncogene-transformed and suppressor gene-reverted CREF cells
indicates a direct relationship between PEG-3 expression and
transformation and oncogenic progression (17). Northern blotting
indicates that CREF cells do not express PEG-3, whereas PEG-3
expression occurs in CREF cells transformed by several
diverse-acting oncogenes, including Ha-ras, v-src, HPV 18 and
mutant Ad5 (H5hr1) (17). Suppression of Ha-ras-induced
transformation by Krev-1 results in suppression of PEG-3
expression. However, both tumor-derived and metastasis-derived
Krev-1 Ha-ras-transformed CREF cells express PEG-3. The highest
relative levels of PEG-3 mRNA are consistently found in the
metastasis-derived Ha-ras+Krev-1 transformed CREF cells. These
results indicate a direct relationship between PEG-3 expression and
the transformed and oncogenic capacity of CREF cells. In addition,
PEG-3 expression directly correlates with human melanoma
progression, with the highest levels of expression found in
metastatic human melanoma and reduced levels observed in normal
human melanocytes, radial growth phase (RGP) primary melanomas and
early vertical growth phase (VGP) primary melanomas.
[0629] An important question is the role of PEG-3 in cancer
progression. PEG-3 could simply correlate with transformation
progression or alternatively it could directly regulate this
process. To distinguish between these possibilities, E11 cells (not
expressing PEG-3) were genetically engineered to express PEG-3
(17). When assayed for growth in agar or aggressiveness in vivo in
nude mice, E11-PEG-3 cells display a progression phenotype akin to
that seen in E11-NMT cells (17,31). Moreover, antisense inhibition
of PEG-3 in E11-NMT (normally expressing PEG-3) results in
suppression of the progression phenotype in vitro and in vivo (31).
Although the mechanism by which PEG-3 affects cancer progression in
vivo remains to be determined, a potential role for induction of
angiogenesis by PEG-3 is suggested (31). Tumors isolated from nude
mice infected with E11-NMT and E11-PEG-3 clones are highly
vascularized and they contain large numbers of blood vessels,
whereas E11 and E11-NMT-PEG-3 AS tumors grow slower and they remain
compact without extensive blood vessel involvement (31). Further
studies are necessary to determine the potentially important
relationship between PEG-3 expression and angiogenesis.
[0630] Defining the mechanism underlying the differential
expression of PEG-3 as a function of cancer progression, oncogenic
transformation and DNA damage. Nuclear run-on assays indicate that
PEG-3 expression directly correlates with an increase in the rate
of RNA transcription (17). This association is supported by the
isolation of a genomic fragment upstream of the 5' untranslated
region of the PEG-3 cDNA and demonstration that this sequence
linked to a luciferase reporter gene is activated as a function of
cancer progression, oncogenic transformation and DNA damage.
Additionally, changes in the stability of PEG-3 mRNA may also
contribute to differential expression of this gene as a function of
cancer progression, oncogene expression and DNA damage. To address
this issue mRNA stability (RNA degradation) assays will be
performed as described in detail previously (32). Our analysis will
focus on the effect of cancer progression (E11-NMT, R1 and R2
cells), oncogenic transformation (Ha-ras, V-src, H5hr1 and HPV-18
transformed CREF cells) and DNA damage (gamma irradiation and
MMS-treatment of CREF cells). Appropriate controls, E11,
untransformed CREF cells and CREF cells not treated with DNA
damaging agents, respectively, and experimental samples will be
incubated without additions or in the presence of 5 .mu.g/ml of
actinomycin D (in the dark), and 30, 60 and 120 min later, total
cellular RNA will be isolated and analyzed for gene expression
using Northern hybridization. RNA blots will be quantitated by
densitometric analysis using a Molecular Dynamics densitometer
(Sunnyvale, Calif.) (32). These straight forward experiments will
indicate if the stability of PEG-3 is altered in cells as a direct
consequence of spontaneous progression, expression of defined
oncogenes or as a consequence of DNA damage.
[0631] Most eukaryotic genes are regulated at the level of
initiation of gene transcription. Detailed characterization of many
different eukaryotic transcriptional units has led to the general
concept that specific interactions of short DNA sequences, usually
located at the 5'-flanking region of the corresponding genes
(cis-acting elements), with certain cellular proteins (trans-acting
elements) play a major role in determining the rate of initiation
of gene transcription. To elucidate the mechanism underlying the
transcriptional regulation of the PEG-3 gene the 5'-flanking region
of this gene is being analyzed. These experiments are important and
they will determine regulatory control of the PEG-3 gene including
autoregulation, developmental regulation, tissue and cell type
specific expression and differential expression in progressed
versus unprogressed cells, enhanced expression as a function of
oncogenic transformation and induction of expression as a
consequence of DNA damage. Once the appropriate regions of the
PEG-3 gene regulating the initiation of transcription has been
confirmed, studies will be conducted to determine the relevant
trans-acting regulatory factors that bind to specific cis-acting
regulatory elements and activate or repress expression of the PEG-3
gene. The experiments outlined below are designed to: [1] define
the 5'-flanking regions of the PEG-3 gene involved in mediating
differential activity of PEG-3 in progressed, oncogenically
transformed and DNA damaged cells; [2] identify cis-acting
regulatory elements in the promoter region of the PEG-3 gene which
are responsible for the differential induction of PEG-3 expression;
and [3] identify and characterize trans-acting regulatory elements
that activate (or repress) expression of the PEG-3 gene.
[0632] Primary analysis of the functional regions of the PEG-3
promoter. Using a genomic walking strategy we have identified a
5'-flanking promoter region of the PEG-3 gene that appears to
encompass a functionally complete PEG-3 promoter. To define
important transcriptional regulatory regions of the PEG-3 promoter,
a heterologous expression system containing a luciferase gene
without promoter or enhancer has been developed using the
full-length promoter construct (33-35). Internal deletion mutations
will be generated either by taking advantage of internal
restriction sites or by a nested exonuclease III base deletion
strategy. These constructs will be transfected into E11 and
E11-NMT, untransformed and transformed CREF (HShr1, Ha-ras, v-src
and HPV-18) and control CREF and gamma irradiation or MMS treated
CREF cells. On the basis of transfection analyses of various
deletion and point mutations it will be possible to define elements
responsible for induction of PEG-3 as a consequence of cancer
progression, specific transformation pathways or DNA damage
response.
[0633] Transcription of PEG-3 in E11-NMT cells, as determined by
nuclear run-on assays, is >20-fold higher than in E11 cells,
whereas transient transfection of the PEG-3 promoter-luciferase
gene into these two cell types indicates only an .about.5-fold
increase in activity in E11-NMT versus E11 cells (FIG. 15). This
could indicate that the PEG-3 gene is repressed in non-expressing
cells (such as E11) through a cis-acting mechanism that is
non-functional on transiently transfected promoters. Various
luciferase constructs will be transfected into the different cell
types by the lipofectamine method or electroporation (Gene Pulser,
Bio-Rad) as previously described (33,36). To correct for DNA uptake
and cell number used for each transfection experiment, the
luciferase constructs will be transfected with plasmids containing
bacterial .beta.-galactosidase gene under the control of an Rous
sarcoma virus (RSV) promoter (33-35). Studies will be conducted
using multiple adult rat tissue Northern blots (CLONTECH)
containing poly A.sup.+ RNA and probing with PEG-3 (as well as
gadd34 and MyD116) to define which rat tissue normally express
PEG-3. Previous studies document that genes expressing in more than
one tissue often require different sequences flanking the 5'-end of
the gene. It is possible that PEG-3 expression in any normal tissue
or under different circumstances in rat cells, i.e., progression,
oncogenic transformation or DNA damage, may be regulated by
different 5'-sequences. In that case, we will obtain variable
luciferase activities for different luciferase constructs in the
various cell lines. Transcription motifs contributing to PEG-3
regulation in a tissue, cell type or specific progression,
transformation or DNA damage pathway will thus be identified.
[0634] Identifying cis-acting elements in the PEG-3 promoter
responsible for expression during cancer progression, oncogenic
transformation and DNA damage. On the basis of the deletion studies
described above, the potential location of cis-acting elements
responsible for expression of PEG-3 during cancer progression,
oncogenic transformation and DNA damage will be identified. The
.about.2.1 kb PEG-3 promoter has been sequenced and potential
regulatory elements have been identified by comparison to
previously characterized transcriptional motifs. The PEG-3 promoter
contains a number of potentially important transcriptional motifs
including PEA3 (AGGAAA), E2A (GCAGGTG), GRE (TGTTCT), E2F
(TTTTGGCCG), TRE (GGTCA), acute phase reactive regulating element
(GTGGGA), SP1 (GGGCGG), AP1 (TGACTCA), AP2 (TCCCCAACCC) and NF1
(TGGATTTGAGCCA). The importance of these sequences in regulating
PEG-3 expression during cancer progression, oncogenic
transformation and DNA damage will be determined by introducing
point mutations in a specific cis element into the promoter region
using previously described site-specific mutagenesis techniques
(33,37-40) or with recently described PCR-based strategies, i.e.,
ExSite.TM. PCR-based site-directed mutagenesis kit and the
Chameleon.TM. double-stranded site-directed mutagenesis kit
(Stratagene, CA). The mutated promoter constructs will be cloned
into luciferase expression vectors and tested for their effects on
the promoter function by transfection into different cell types and
monitoring luciferase activity. Since the promoter region for the
PEG-3 gene is located in front of the luciferase reporter gene in
the various pPEG-Luciferase constructs, the change in luciferase
activity for each construct will permit a direct comparison of the
activity of the mutant promoter to that of the unmodified PEG-3
promoter.
[0635] After the regulatory regions of the PEG-3 promoter are
confirmed experiments will be conducted to address a number of
important questions relative to cancer progression, oncogenic
transformation and DNA damage induction of PEG-3 expression. (1)
Nuclear run-on and transient transfection assays with
pPEG-Luciferase constructs will be used to determine the effect of
changes in DNA methylation (AZA and phenyl butyrate treatment) on
PEG-3 expression in E11-NMT cells, treatment with different classes
of DNA damaging and cancer modulating agents (such as TPA,
retinoids, UV-C, gamma irradiation, methylating carcinogens,
topoisomerase inhibitors, okadaic acid, etc.) on PEG-3 expression
in CREF and CREF-PEG-Luc cl 1 cells (PEG-Luciferase stably
transformed CREF clone) and exposure to cancer modulating agents
(such as the Krev-1 gene, dominant negative inhibitors of specific
oncogenes, chemicals such as CAPE, retinoids, sodium butyrate,
interferon, TNF-.alpha. and additional progression modulating
agents) on PEG-3 expression in oncogenically transformed CREF cells
(1,8-10,18,21,28,29,41-43); (2) The level of PEG-3 transcription in
cells displaying different stages of cancer progression and
oncogenic transformation, including rodent model systems of cancer
progression (such as the Dunning rat prostate model, metastatic
murine melanoma variants, etc.) and additional rodent cells
transformed by various oncogenes. These studies will indicate if
expression of PEG-3 occurs in additional pathways of progression
and transformation. (3) Transfection of varying lengths of the 5'
flanking region and internal deletion luciferase constructs into
rodent cells displaying different stages of progression,
transformed by different classes of oncogenes and treated with
various DNA damaging and cancer promoting and inhibiting agents.
These regulatory elements will be sequenced and compared with
previously characterized transcriptional motifs to identify
potential positive and negative regulatory elements; (4) In
addition to mutagenesis studies (to define functional motifs
regulating transcriptional regulation of the PEG-3 promoter),
cotransfection studies will be conducted with cDNAs containing
putative positive acting regulatory elements and a minimal PEG-3
promoter-Luciferase construct into unprogressed and progressed
rodent cells, untransformed CREF and oncogenically transformed CREF
and untreated and DNA damage treated CREF cells. These studies will
indicate if the introduction of specific putative positive acting
regulatory elements can enhance PEG-3 expression in cells
cotransfected with a minimal PEG-3 promoter region. The potential
role of putative cis-acting negative regulatory elements will be
addressed by cotransfection with a complete PEG-3 promoter region
into the same target cells. These studies will provide relevant
information about the potential role of inhibitory elements in
regulating PEG-3 expression. (5) Experiments will also be performed
to evaluate the status of the endogenous PEG-3 gene during cancer
progression, oncogenic transformation and DNA damage. This will be
approached by using DNase hypersensitivity assays to look for
structural changes in this gene (33). Although not within the scope
of the present studies, future studies could involve the
identification of a human PEG-3 cDNA, elucidation of the human
PEG-3 promoter and analysis of the level of PEG-3 expression in
human progression model systems. These studies would be quite
informative in providing a potential link between PEG-3 expression
and cancer progression in human cells.
[0636] Identifying trans-acting nuclear proteins that mediate
transcriptional enhancing activity of the PEG-3 gene during cancer
progression, oncogenic transformation and DNA damage. The current
view on regulation of eukaryotic gene expression suggests that
trans-acting proteins bind to specific sites within cis-elements of
a promoter region resulting in transcriptional activation (44,45).
Experiments will be performed to identify trans-acting factors
(nuclear proteins) and determine where these factors interact with
cis-regulatory elements. To achieve this goal, two types of studies
will be performed, one involving gel retardation (gel shift) assays
(15,33,46,47) and the second involving DNase-I footprinting
(methylation interference) assays (33,48,49).
[0637] Gel shift assays will be used to analyze the interactions
between cis-acting elements in the PEG-3 promoter and trans-acting
factors in mediating transcriptional control (15,46,47). For this
assay, .sup.32P-labeled cis-elements will be incubated with nuclear
extracts from E11 and E11-NMT, CREF and transformed CREF (Ha-ras,
v-src, H5hr1 and HPV-18) and untreated CREF and CREF treated with
MMS (100 .mu.g/ml for 8 hr) or gamma irradiation (10 Gy for 4 hr)
and reaction mixtures will be resolved on 5 or 8% polyacrylamide
gels. After autoradiography, the pattern of retarded DNAs on the
gel will provide information concerning the interaction between
trans-acting factors and specific regions of the cis-acting
elements in the PEG-3 promoter. Non-labeled cis-acting elements
(self-competition) will be added as a competitor to duplicate
samples to eliminate the possibility of non-specific binding and to
confirm that the interaction is really conferred by the
trans-acting factor. To begin to identify the transacting factors,
different non-labeled DNAs (including those corresponding to
sequences identified in the PEG-3 promoter, such as TATA, PEA3,
E2A, GRE, E2F, TRE, acute phase reactive regulating element, SP1,
AP1, AP2 and NF1) can be used as competitors in the gel shift assay
to determine the relationship between the trans-acting factors and
previously identified transcriptional regulators. It is possible
that the trans-acting factors regulating transcriptional control of
the PEG-3 promoter may be novel. To identify these factors extracts
will be purified from E11 and E11-NMT, CREF and transformed CREF
and untreated and DNA damaged CREF cells by two cycles of
heparin-sepharose column chromatography, two cycles of DNA affinity
chromatography and separation on SDS-polyacrylamide gels (50,51).
Proteins displaying appropriate activity using gel shift assays
will be digested in situ with trypsin, the peptides separated by
HPLC and the peptides sequenced (52). Peptide sequences will be
used to synthesize degenerate primers and RT-PCR will be used to
identify putative genes encoding the trans-acting factor. These
partial sequences will be used with cDNA library screening
approaches and the RACE procedure, if necessary, to identify
full-length cDNAs encoding the trans-acting factors (17,36,51,52).
Once identified, the role of the trans-acting factors in eliciting
cancer progression will be analyzed. (1) The functionality of
positive and negative trans-acting factors will be determined by
transiently and stably expressing these genes in E11 and E11-NMT
cells to determine effects on anchorage independence and
tumorigenic potential in nude mice (stable expression). Positive
effects would be indicated if overexpressing a positive
trans-acting factor facilitates the progression phenotype, whereas
overexpressing a negative trans-acting factor inhibits the
progression phenotype. (2) Antisense approaches will be used to
determine if blocking the expression of positive or negative
trans-acting factors can directly modify the progression state. A
direct effect of a positive trans-acting factor in affecting
progression would be suggested if antisense inhibition of the
positive factor partially or completely inhibits the progression
phenotype in E11-NMT, i.e., growth in agar is reduced and tumor
latency time is extended. Conversely, a direct effect of negative
trans-acting factors in inhibiting progression would be suggested
if antisense inhibition of the negative factor enhances the ability
of E11 to grow in agar and reduces tumor latency time. A potential
problem with these types of studies would be encountered if the
factors are involved in the regulation of many genes, e.g.,
Fos/Jun, and the antisense effects may, therefore, be non-specific.
Although not within the scope of the present proposal, depending on
the results obtained, cis-element knockouts could be used to
further define the role of these elements in regulating PEG-3
expression.
[0638] For DNase-I footprinting assays, nuclear extracts from E11
and E11-NMT, CREF and transformed CREF and untreated CREF and DNA
damaged (MMS and gamma irradiation) CREF cells will be prepared and
DNase-I footprinting assays will be performed as described
(33,53,54). The promoter necessary for PEG-3 expression, identified
from the experiments described above, will be terminally labeled
with .sup.32P and incubated with crude nuclear extracts from the
different cell types and experimental conditions described above
using established protocols (33,53,54). The reaction mixture that
has been digested with DNase-I enzyme will be terminated and the
digested products will be analyzed on an 8% sequencing gel. The
differential protection between nuclear extracts from progressed
versus unprogressed, untransformed and oncogenically transformed
and undamaged and DNA damaged cells will provide relevant
information concerning the involvement of trans-acting factors in
activation and the location of specific sequences in the
cis-regulatory elements of the PEG-3 promoter mediating this
activation. If differential protection is not detected using this
approach, the sensitivity of the procedure can be improved by using
different sized DNA fragments from the PEG-3 promoter region or by
using partially purified nuclear extracts (33,53,54).
[0639] The studies described above will result in the
characterization of the PEG-3 promoter region, the identification
of cis-acting regulatory elements in the PEG-3 promoter and the
identification of trans-acting regulatory elements that activate
(or repress) expression of the PEG-3 gene as a function of cancer
progression, oncogenic transformation and DNA damage. This
information could prove valuable in designing approaches for
selectively inhibiting PEG-3 expression, and therefore modifying
cellular phenotypes related to cancer progression and response to
DNA damage.
[0640] Isolation and initial characterization of the PEG-3
promoter.
[0641] Targeted adenovirus gene delivery system for selectively
inhibiting proliferation or inducing toxicity in PEG-3 expressing
cancer cells: CURE (Cancer Utilized Reporter Execution) and CIRAs
(Cancer Inhibitory Recombinant Adenoviruses). Gene based therapies
that exploit differences between cancer cells and normal cells
represent potentially significant technologies for improved cancer
therapy. This approach has been used to selectively target the
replication of an E1B, 55-kDa gene-attenuated adenovirus
(ONYX-015), to cancer cells containing a mutant p53 gene (55,56).
Moreover, a minimal promoter/enhancer construct derived from the 5'
flanking region of the human prostate specific antigen (PSA)
promoter has been used to drive the expression of the Ad5 E1A gene
in a replication competent Ad, thereby selectively inducing viral
replication and toxicity in PSA-expressing prostate cancer cells
(57). Similarly, viruses expressing the herpes simplex thymidine
kinase (TK) gene have been used in combination with gancyclovir or
acyclovir to target toxicity in cancer cells expressing herpes
simplex viral thymidine kinase (58-61). In this context, a virus
containing a gene promoter displaying restrictive or selective
expression of a linked gene (with the capacity to inhibit growth or
induce toxicity either directly or indirectly) would represent an
extremely valuable therapeutic reagent.
[0642] As documented experimentally, the PEG-3 promoter displays
elevated expression in progressed cancer cells, oncogenically
transformed cancer cells and DNA damaged cells. The absolute level
of induction of luciferase activity in progressed cancer cells and
oncogenically transformed cells is .gtoreq.10-fold higher than in
DNA damaged CREF cells. In this respect, it is anticipated that the
activity of the PEG-3 promoter will be reduced and less effective
in driving a linked gene in a recombinant replication competent or
incompetent Ad when expressed in DNA damaged versus progressed or
oncogenically transformed cells. However, it is likely that
treatment with DNA damaging agents will even further augment the
activity of the PEG-3 promoter in cancer cells. Moreover,
preliminary experiments using a large panel of human cancer cell
lines, including metastatic melanoma, glioblastoma multiforme and
carcinomas of the breast, cervix, colon, lung, nasopharyngx and
prostate, indicate that the PEG-3 promoter is active, whereas no
activity is apparent in their normal cellular counterparts
(unpublished data). On the basis of these considerations, the PEG-3
promoter would appear to be an ideal genetic tool for the
construction of "cancer inhibitory recombinant adenoviruses"
(CIRAs). These CIRAs could be used as part of a protocol called
"cancer utilized reporter execution" (CURE) to selectively induce
growth suppression, apoptosis or toxicity uniquely in cancer
cells.
[0643] We propose to construct evaluate CURE using CIRAs that
permit expression of a gene inhibiting growth and or inducing
toxicity specifically in cancer cells using the PEG-3 promoter to
control gene expression. Additionally, the PEG-3 promoter can be
used to drive expression of a gene encoding an antigenic epitope
that will increase the immunogenicity and killing of modified cells
by the immune system (activated T-cells and/or antibodies). Several
types of CIRAs will be generated and tested for biological efficacy
using the CURE protocol.
[0644] 1. Recombinant Ad expressing a wild-type p53 gene controlled
by the PEG-3 promoter, Ad.PEG-wtp53. These viruses, using
conventional cytomegalovirus promoters to drive wild-type p53
expression, are proving efficacious in treating a number of tumor
types in humans (62). It is predicted that Ad.PEG-wtp53 would
inhibit the growth or induce apoptosis in cancer cells containing
defects in p53, which occur in a very high percentage of cancer
cells (63). Moreover, even if low level PEG-3 promoter activity
occurs in normal cells, the subsequent low level of p53 expression
should not significantly affect cell growth or viability in normal
cells.
[0645] 2. Recombinant Ad expressing the novel mda-7 cancer growth
suppressor gene controlled by the PEG-3 promoter, Ad.PEG-mda-7. The
mda-7 gene is selectively growth suppressive in cancer versus
normal cells (64). Moreover, the inhibitory effect of mda-7 in
tumor cells is independent of p53 status and occurs in cancer cells
with diverse genetic defects (64). This novel tumor suppressor gene
would appear ideally suited for therapeutic applications (CURE) and
the generation of CIRAs, since expression in normal cells (even
following infection with 100 pfu/cell of Ad.mda-7 S virus) does not
elicit a biological phenotype (65).
[0646] 3. Recombinant Ad expressing the cyclin-dependent kinase
inhibitor p21 (66) controlled by the PEG-3 promoter, Ad.PEG-p21.
The p21 gene can induce growth arrest and apoptosis in specific
cancer cells (67). In addition, recent studies indicate that an Ad
expressing p21 can be used to inhibit tumor growth in animals (68).
Since p21 can also modify growth in normal cells, the use of the
PEG-3 promoter to selectively drive p21 in cancer cells would
appear to be preferable vehicle for gene therapy than a virus
constitutively expressing p21.
[0647] 4. Recombinant replication competent Ad expressing the viral
E1A gene controlled by the PEG-3 promoter, Ad.PEG-E1A. In
principle, this virus should only replicate in cancer cells that
allow activation of the PEG-3 promoter. The successful application
of this type of virus for cancer therapy will be contingent upon
sufficient E1A expression to permit switch-on of additional Ad
genes permitting virus replication and cytotoxicity only in the
cancer cells. A potential problem that might be encountered using
this type of CIRA is minimal activity of the PEG-3 promoter
resulting in low levels of Ad5 E1A expression in normal cells. This
could prove problematic since previous studies have documented that
even very low levels of Ad E1A can result in virus production (69).
Moreover, since CREF cells are nonpermissive for Ad replication
(1,27), this type of CIRA requires testing in a human cell line.
This could be accomplished by using 293 cells, a human embryonic
kidney cell line transformed by sheared Ad5 and constitutively
expressing the Ad5 E1A and E1B genes. Preliminary studies indicate
that the PEG-3 promoter is very active in 293 cells, whereas it
displays no activity in normal human fibroblast or epithelial
cells.
[0648] 5. Recombinant virus expressing a HSV 1 TK gene controlled
by the PEG-3 promoter, Ad.PEG-TK. This virus should produce viral
TK in cancer cells as a result of activation of the PEG-3 promoter.
By applying gancyclovir or acyclovir it would be possible to
selectively kill cancer cells expressing elevated levels of viral
TK. Previous studies provide precedents for the use of the HSV 1 TK
gene and gancyclovir or acyclovir to selectively kill cancer cells
(58-61).
[0649] 6. Recombinant virus expressing an antigenic
immunostimulating gene, such as GM-CSF and/or IL-2, controlled by
the PEG-3 promoter, Ad.PEG-ImStim. This virus can be used to infect
cells resulting in targeted expression, because of PEG-3 promoter
utilization, specifically in cancer cells. The altered
immunoreactivity in the tumor cells will elicit enhanced immune
recognition and elimination of the tumor.
[0650] 7. Recombinant virus expressing a defined antigen (with and
without a co-stimulatory molecule) controlled by the PEG-3
promoter, Ad.PEG-Antigen. This virus can be used to infect cells
resulting in targeted expression, because of PEG-3 promoter
utilization, of the antigen (with or without the co-stimulatory
molecule) specifically in cancer cells. The expression of this
antigen on the surface of the cancer cells can then be used to
target an antibody for immaging of tumor cells in a patient and/or
inducing toxicity (by using an antibody with cytotoxic properties,
an antibody conjugated with a toxin or an antibody carrying a high
energy emitting radionuclide). By using appropriate vectors, all of
the approaches briefly described above can also be used to treat
patients with systemic tumors and metastases.
[0651] The methodologies for construction of the different CIPAs
are routine and we have extensive experience in this approach
(37-40,64). We therefore do not expect any problems in producing
the appropriate viruses. Once the various recombinant viruses have
been constructed they will be evaluated for biological efficacy
using in vitro assays (64) and if active using in vivo tumor
xenograft models (27-29). As initial tests for proof of practice of
CURE and the CIRA approach, CREF, CREF-ras, CREF-src and
CREF-HPV-18 cells will be infected with the specific recombinant
virus (Ad.PEG-wtp53, Ad.PEG-mda-7, Ad.PEG-p21 or Ad.PEG-TK) or a
recombinant virus not containing any gene insert (Ad.vec) and
colony forming ability in monolayer culture will be determined
(64). As indicated above, to test the Ad.PEG-E1A construct we will
use 293 and normal human kidney cells. In the case of the Ad.PEG-TK
virus system, infected cells will be cultured in the presence or
absence of gancyclovir or acyclovir. A biologically relevant
endpoint would be a statistically significant reduction of colony
formation in transformed cells, but not in normal cells, for the
CIRA versus the Ad.vec. This could be verified by monitoring
expression of the transduced gene (RNA and protein levels)
following viral infection. The levels of expression of the
transduced gene should be significantly higher in transformed cells
versus parental CREF cells (or in 293 versus normal human kidney
cells). If these results occur with any or all of the CIRAs,
further studies would be performed to assay for effects on tumor
growth and metastasis using previously described procedures
(27-29). This would include injecting tumor cells infected with
recombinant virus prior to injection into animals and establishing
tumors in animals followed by repeated administration (2.times. per
week) of recombinant virus. If successful in reducing or
eliminating cancer cells in vivo, the CIRA concept could ultimately
prove of immense value for the targeted therapy of human cancers.
Obvious extensions of this approach would be to isolate a human
PEG-3 promoter and construct recombinant viruses in which this
promoter drives the gene of choice. It is realized that the use of
CIRAs for the therapy of human cancer depends on a number of
important considerations. The rat or human PEG-3 promoter must
display differential expression in human cancer versus normal human
cells and the level of expression of the PEG-3 promoter must be
sufficiently high in the cancer cells to allow for expression of
adequate amounts of the linked gene to induce a biological effect
in these cells. In preliminary studies, the rat PEG-3 promoter
displays differential expression in human cancer versus normal
cells suggesting that it my provide the appropriate reagent for the
CURE approach using CIRAs. Moreover, the level of expression in
normal tissue must be negligible, although this should not be a
problem when using Ad.PEG-wtp53, Ad.PEG-mda-7 or Ad.PEG-p21.
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[0723] product causing the cell to express a specific antigen.
Sequence CWU 1
1
8 1 457 PRT Rat 1 Met Ala Pro Ser Pro Arg Pro Gln His Val Leu His
Trp Lys Glu Ala 1 5 10 15 His Ser Phe Tyr Leu Leu Ser Pro Leu Met
Gly Phe Leu Ser Arg Ala 20 25 30 Trp Ser Arg Leu Arg Gly Pro Glu
Val Ser Glu Ala Trp Leu Ala Glu 35 40 45 Thr Val Ala Gly Ala Asn
Gln Ile Glu Ala Asp Ala Leu Leu Thr Pro 50 55 60 Pro Pro Val Ser
Glu Asn His Leu Pro Leu Arg Glu Thr Glu Gly Asn 65 70 75 80 Gly Thr
Pro Glu Trp Ser Lys Ala Ala Gln Arg Leu Cys Leu Asp Val 85 90 95
Glu Ala Gln Ser Ser Pro Pro Lys Thr Trp Gly Leu Ser Asp Ile Asp 100
105 110 Glu His Asn Gly Lys Pro Gly Gln Asp Gly Leu Arg Glu Gln Glu
Val 115 120 125 Glu His Thr Ala Gly Leu Pro Thr Leu Gln Pro Leu His
Leu Gln Gly 130 135 140 Ala Asp Lys Lys Val Gly Glu Val Val Ala Arg
Glu Glu Gly Val Ser 145 150 155 160 Glu Leu Ala Tyr Pro Thr Ser His
Trp Glu Gly Gly Pro Ala Glu Asp 165 170 175 Glu Glu Asp Thr Glu Thr
Val Lys Lys Ala His Gln Ala Ser Ala Ala 180 185 190 Ser Ile Ala Pro
Gly Tyr Lys Pro Ser Thr Ser Val Tyr Cys Pro Gly 195 200 205 Glu Ala
Glu His Arg Ala Thr Glu Glu Lys Gly Thr Asp Asn Lys Ala 210 215 220
Glu Pro Ser Gly Ser His Ser Arg Val Trp Glu Tyr His Thr Arg Glu 225
230 235 240 Arg Pro Lys Gln Glu Gly Glu Thr Lys Pro Glu Gln His Arg
Ala Gly 245 250 255 Gln Ser His Pro Cys Gln Asn Ala Glu Ala Glu Glu
Gly Gly Pro Glu 260 265 270 Thr Ser Val Cys Ser Gly Ser Ala Phe Leu
Lys Ala Trp Val Tyr Arg 275 280 285 Pro Gly Glu Asp Thr Glu Glu Glu
Glu Asp Ser Asp Leu Asp Ser Ala 290 295 300 Glu Glu Asp Thr Ala His
Thr Cys Thr Thr Pro His Thr Ser Ala Phe 305 310 315 320 Leu Lys Ala
Trp Val Tyr Arg Pro Gly Glu Asp Thr Glu Glu Glu Asp 325 330 335 Asp
Gly Asp Trp Asp Ser Ala Glu Glu Asp Ala Ser Gln Ser Cys Thr 340 345
350 Thr Pro His Thr Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly
355 360 365 Glu Asp Thr Glu Glu Glu Asp Asp Ser Glu Asn Val Ala Pro
Val Asp 370 375 380 Ser Glu Thr Val Asp Ser Cys Gln Ser Thr Gln His
Cys Leu Pro Val 385 390 395 400 Glu Lys Thr Lys Gly Cys Gly Glu Ala
Glu Pro Pro Pro Phe Gln Trp 405 410 415 Pro Ser Ile Tyr Leu Asp Arg
Ser Gln His His Leu Gly Leu Pro Leu 420 425 430 Ser Cys Pro Phe Asp
Cys Arg Ser Gly Ser Asp Leu Ser Lys Pro Pro 435 440 445 Pro Gly Ile
Arg Ala Leu Arg Phe Leu 450 455 2 590 PRT Rat 2 Met Ala Pro Ser Pro
Arg Pro Gln His Ile Leu Leu Trp Arg Asp Ala 1 5 10 15 His Ser Phe
His Leu Leu Ser Pro Leu Met Gly Phe Leu Ser Arg Ala 20 25 30 Trp
Ser Arg Leu Arg Val Pro Glu Ala Pro Glu Pro Trp Pro Ala Glu 35 40
45 Thr Val Thr Gly Ala Asp Gln Ile Glu Ala Asp Ala His Pro Ala Pro
50 55 60 Pro Leu Val Pro Glu Asn His Pro Pro Gln Gly Glu Ala Glu
Glu Ser 65 70 75 80 Gly Thr Pro Glu Glu Gly Lys Ala Ala Gln Gly Pro
Cys Leu Asp Val 85 90 95 Gln Ala Asn Ser Ser Pro Pro Glu Thr Leu
Gly Leu Ser Asp Asp Asp 100 105 110 Lys Gln Gly Gln Asp Gly Pro Arg
Glu Gln Gly Arg Ala His Thr Ala 115 120 125 Gly Leu Pro Ile Leu Leu
Ser Pro Gly Leu Gln Ser Ala Asp Lys Ser 130 135 140 Leu Gly Glu Val
Val Ala Gly Glu Glu Gly Val Thr Glu Leu Ala Tyr 145 150 155 160 Pro
Thr Ser His Trp Glu Gly Cys Pro Ser Glu Glu Glu Glu Asp Gly 165 170
175 Glu Thr Val Lys Lys Ala Phe Arg Ala Ser Ala Asp Ser Pro Gly His
180 185 190 Lys Ser Ser Thr Ser Val Tyr Cys Pro Gly Glu Ala Glu His
Gln Ala 195 200 205 Thr Glu Glu Lys Gln Thr Glu Asn Lys Ala Asp Pro
Pro Ser Ser Pro 210 215 220 Ser Gly Ser His Ser Arg Ala Trp Glu Tyr
Cys Ser Lys Gln Glu Gly 225 230 235 240 Glu Ala Asp Pro Glu Pro His
Arg Ala Gly Lys Tyr Gln Leu Cys Gln 245 250 255 Asn Ala Glu Ala Glu
Glu Glu Glu Glu Ala Lys Val Ser Ser Leu Ser 260 265 270 Val Ser Ser
Gly Asn Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly 275 280 285 Glu
Asp Thr Glu Asp Asp Asp Asp Ser Asp Trp Gly Ser Ala Glu Glu 290 295
300 Glu Gly Lys Ala Leu Ser Ser Pro Thr Ser Pro Glu His Asp Phe Leu
305 310 315 320 Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp Thr Glu Asp
Asp Asp Asp 325 330 335 Ser Asp Trp Gly Ser Ala Glu Glu Glu Gly Lys
Ala Leu Ser Ser Pro 340 345 350 Thr Ser Pro Glu His Asp Phe Leu Lys
Ala Trp Val Tyr Arg Pro Gly 355 360 365 Glu Asp Thr Glu Asp Asp Gln
Asp Ser Asp Trp Gly Ser Ala Glu Lys 370 375 380 Asp Gly Leu Ala Gln
Thr Phe Ala Thr Pro His Thr Ser Ala Phe Leu 385 390 395 400 Lys Thr
Trp Val Cys Cys Pro Gly Glu Asp Thr Glu Asp Asp Asp Cys 405 410 415
Glu Val Val Val Pro Glu Asp Ser Glu Ala Ala Asp Pro Asp Lys Ser 420
425 430 Pro Ser His Glu Ala Gln Gly Cys Leu Pro Gly Glu Gln Thr Glu
Gly 435 440 445 Leu Val Glu Ala Glu His Ser Leu Phe Gln Val Ala Phe
Tyr Leu Pro 450 455 460 Gly Glu Lys Pro Ala Pro Pro Trp Thr Ala Pro
Lys Leu Pro Leu Arg 465 470 475 480 Leu Gln Arg Arg Leu Thr Leu Leu
Arg Thr Pro Thr Gln Asp Gln Asp 485 490 495 Pro Glu Thr Pro Leu Arg
Ala Arg Lys Val His Phe Ser Glu Asn Val 500 505 510 Thr Val His Phe
Leu Ala Val Trp Ala Gly Pro Ala Gln Ala Ala Arg 515 520 525 Arg Gly
Pro Trp Glu Gln Leu Ala Arg Asp Arg Ser Arg Phe Ala Arg 530 535 540
Arg Ile Ala Gln Ala Glu Glu Lys Leu Gly Pro Tyr Leu Thr Pro Ala 545
550 555 560 Phe Arg Ala Arg Ala Trp Ala Arg Leu Gly Asn Pro Ser Leu
Pro Leu 565 570 575 Ala Leu Glu Pro Ile Cys Asp His Thr Phe Phe Pro
Ser Gln 580 585 590 3 657 PRT Rat 3 Met Ala Pro Ser Pro Arg Phe Gln
His Val Leu His Trp Arg Asp Ala 1 5 10 15 His Asn Phe Tyr Leu Leu
Ser Pro Leu Met Gly Leu Leu Ser Arg Ala 20 25 30 Trp Ser Arg Leu
Arg Gly Pro Glu Val Pro Glu Ala Trp Leu Ala Lys 35 40 45 Thr Val
Thr Gly Ala Asp Gln Ile Glu Ala Ala Ala Leu Leu Thr Pro 50 55 60
Thr Pro Val Ser Gly Asn Leu Leu Pro His Gly Glu Thr Glu Glu Ser 65
70 75 80 Gly Ser Pro Glu Gln Ser Gln Ala Ala Gln Arg Leu Cys Leu
Val Glu 85 90 95 Ala Glu Ser Ser Pro Pro Glu Thr Trp Gly Leu Ser
Asn Val Asp Glu 100 105 110 Tyr Asn Ala Lys Pro Gly Gln Asp Asp Leu
Arg Glu Lys Glu Met Glu 115 120 125 Arg Thr Ala Gly Lys Ala Thr Leu
Gln Pro Ala Gly Leu Gln Gly Ala 130 135 140 Asp Lys Arg Leu Gly Glu
Val Val Ala Arg Glu Glu Gly Val Ala Glu 145 150 155 160 Pro Ala Tyr
Pro Thr Ser Gln Leu Glu Gly Gly Pro Ala Glu Asn Glu 165 170 175 Glu
Asp Gly Glu Thr Val Lys Thr Tyr Gln Ala Ser Ala Ala Ser Ile 180 185
190 Ala Pro Gly Tyr Lys Pro Ser Thr Pro Val Pro Phe Leu Gly Glu Ala
195 200 205 Glu His Gln Ala Thr Glu Glu Lys Gly Thr Glu Asn Lys Ala
Asp Pro 210 215 220 Ser Asn Ser Pro Ser Ser Gly Ser His Ser Arg Ala
Trp Glu Tyr Tyr 225 230 235 240 Ser Arg Glu Lys Pro Lys Gln Glu Gly
Glu Ala Lys Val Glu Ala His 245 250 255 Arg Ala Gly Gln Gly His Pro
Cys Arg Asn Ala Glu Ala Glu Glu Gly 260 265 270 Gly Pro Glu Thr Thr
Phe Val Cys Thr Gly Asn Ala Phe Leu Lys Ala 275 280 285 Trp Val Tyr
Arg Pro Gly Glu Asp Thr Glu Glu Glu Asp Asn Ser Asp 290 295 300 Ser
Asp Ser Ala Glu Glu Asp Thr Ala Gln Thr Gly Ala Thr Pro His 305 310
315 320 Thr Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp
Thr 325 330 335 Glu Glu Glu Asp Ser Asp Ser Asp Ser Ala Glu Glu Asp
Thr Ala Gln 340 345 350 Thr Gly Ala Thr Pro His Thr Ser Ala Phe Leu
Lys Ala Trp Val Tyr 355 360 365 Arg Pro Gly Glu Asp Thr Glu Glu Glu
Asn Ser Asp Leu Asp Ser Ala 370 375 380 Glu Glu Asp Thr Ala Gln Thr
Gly Ala Thr Pro His Thr Ser Ala Phe 385 390 395 400 Leu Lys Ala Trp
Val Tyr Arg Pro Gly Glu Asp Thr Glu Glu Glu Asn 405 410 415 Ser Asp
Leu Asp Ser Ala Glu Glu Asp Thr Ala Gln Thr Gly Ala Thr 420 425 430
Pro His Thr Ser Pro Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu 435
440 445 Asp Thr Glu Asp Asp Thr Glu Glu Glu Glu Asp Ser Glu Asn Val
Ala 450 455 460 Pro Gly Asp Ser Glu Thr Ala Asp Ser Ser Gln Ser Pro
Cys Leu Gln 465 470 475 480 Pro Gln Arg Cys Leu Pro Gly Glu Lys Thr
Lys Gly Arg Gly Glu Glu 485 490 495 Pro Pro Leu Phe Gln Val Ala Phe
Tyr Leu Pro Gly Glu Lys Pro Glu 500 505 510 Ser Pro Trp Ala Ala Pro
Lys Leu Pro Leu Arg Leu Gln Arg Arg Leu 515 520 525 Arg Leu Phe Lys
Ala Pro Thr Arg Asp Gln Asp Pro Glu Ile Pro Leu 530 535 540 Lys Ala
Arg Lys Val His Phe Ala Glu Lys Val Thr Val His Phe Leu 545 550 555
560 Ala Val Trp Ala Gly Pro Ala Gln Ala Ala Arg Arg Gly Pro Trp Glu
565 570 575 Gln Phe Ala Arg Asp Arg Ser Arg Phe Ala Arg Arg Ile Ala
Gln Ala 580 585 590 Glu Glu Lys Leu Gly Pro Tyr Leu Thr Pro Asp Ser
Arg Ala Arg Ala 595 600 605 Trp Ala Arg Leu Arg Asn Pro Ser Leu Pro
Gln Ser Glu Pro Arg Ser 610 615 620 Ser Ser Glu Ala Thr Pro Leu Thr
Gln Asp Val Thr Thr Pro Ser Pro 625 630 635 640 Leu Pro Ser Glu Thr
Pro Ser Pro Ser Leu Tyr Leu Gly Gly Arg Arg 645 650 655 Gly 4 2137
DNA Rat 4 ctgcagtact tgtacattgc taaataaaga gagggactcc aggaggagca
gcctgggtct 60 aagaggtagg cagaaggagg ttttaggggc ctgagcacaa
gcttgaggag agaaaggtta 120 ttaaaaagcc agacgcttac aggtctcaga
agggctagcc agaaactgtg gctggggtta 180 aggaaagggt ttaagagtgt
gggcttttgg ttctgaggat gtagaacgtg aatgttgaga 240 gaagaaccaa
gtggcggagt tgggtgtgag caatgctatt aggaatttga ggcagggatt 300
cacgcgctgc tgtgactatt ttttaacaat gactcagtgc tgtgacctga tactgtttcc
360 agagcgactt ctaaacaaat tccccctttc taggccagac acatggcccc
aagcccaaga 420 ccccagcatg tcctgcactg gaaggaagcc cactctttct
acctcctgtc tccactgatg 480 ggcttcctca gccgggcctg gagccgcctg
agggggcccg aggtctcaga ggcctggttg 540 gcagaaacag tagcaggagc
aaaccagata gaggctgatg ctctgttgac gcctcccccg 600 gtctctgaaa
atcacctacc tctccgagag actgaaggaa atggaactcc tgaatggagt 660
aaagcagccc agaggctctg ccttgatgtg gaagcccaaa gttcccctcc taaaacttgg
720 ggactttcag agtattgatg aacataatgg gaagccagga caagatggcc
ttagagagca 780 agaagtggag cacacagctg gcctgcctac actacagccc
cttcacctgc aaggggcaga 840 taagaaagtt ggggaggtgg tggctagaga
agagggtgtg tccgagctgg cttaccccac 900 atcacactgg gagggtggtc
cagctgagga tgaagaggat acagaaaccg tgaagaaggc 960 tcaccaggcc
tctgctgctt ccatagctcc aggatataaa cccagcactt ctgtgtattg 1020
cccaggggag gcagaacatc gagccacgga ggaaaaagga acagacaata aggctgaacc
1080 ctcaggctcc cactccagag tctgggagta ccacactaga gagaggccta
agcaggaggg 1140 agaaactaag ccagagcaac acagggcagg gcagagtcac
ccttgtcaga atgcagaggc 1200 tgaggaagga ggacctgaga cttctgtctg
ttctggcagt gccttcctga aggcctgggt 1260 gtatcgccca ggagaggaca
cagaggagga agaagacagt gatttggatt cagctgagga 1320 agacacagct
catacctgta ccacccccca tacaagtgcc ttcctgaagg cctgggtcta 1380
tcgcccagga gaggacacag aagaggaaga tgacggtgat tgggattcag ctgaggaaga
1440 cgcgtctcag agctgtacca ccccccatac aagtgccttc ctgaggcctg
ggtctatcgc 1500 ccaggagagg acacagaaga ggaagacgac agtgagaatg
tggccccagt tgactcagaa 1560 acagttgact cttgccagag tacccagcat
tgtctaccag tagagaagac caagggatgt 1620 ggagaagcag agccccctcc
cttccagtgg ccttctattt acctggacag aagccagcac 1680 caccttgggc
tgcccctaag ctgccccttc gactgcagaa gcggctcaga tctttcaaag 1740
cccccgcccg gaatcagggc cctgagattc ctctgaaggg tagaaaggtg cacttctctg
1800 agaaagttac agtccatttc cttgctgtct gggcaggacc agcccaggct
gctcgtcgag 1860 gcccctggga gcagtttgca cgagatcgaa gccgctttgc
tcgacgcatt gccgtcctcg 1920 tctcttccac tgcctgagcc ttgctcttcc
actgaggcca cacccctcag ccaagatgtg 1980 accactccct ctccccttcc
cagtgaaatc cctcctccca gcctggactt gggaggaagg 2040 cgggctaagc
ctgagtagtt ttttgtgtat tctatgagtg ttagtctctt aatacgaata 2100
tgtaacgcct tttgcatttg taaaaaaaaa aaaaaaa 2137 5 457 PRT Rat 5 Met
Ala Pro Ser Pro Arg Pro Gln His Val Leu His Trp Lys Glu Ala 1 5 10
15 His Ser Phe Tyr Leu Leu Ser Pro Leu Met Gly Phe Leu Ser Arg Ala
20 25 30 Trp Ser Arg Leu Arg Gly Pro Glu Val Ser Glu Ala Trp Leu
Ala Glu 35 40 45 Thr Val Ala Gly Ala Asn Gln Ile Glu Ala Asp Ala
Leu Leu Thr Pro 50 55 60 Pro Pro Val Ser Glu Asn His Leu Pro Leu
Arg Glu Thr Glu Gly Asn 65 70 75 80 Gly Thr Pro Glu Trp Ser Lys Ala
Ala Gln Arg Leu Cys Leu Asp Val 85 90 95 Glu Ala Gln Ser Ser Pro
Pro Lys Thr Trp Gly Leu Ser Asp Ile Asp 100 105 110 Glu His Asn Gly
Lys Pro Gly Gln Asp Gly Leu Arg Glu Gln Glu Val 115 120 125 Glu His
Thr Ala Gly Leu Pro Thr Leu Gln Pro Leu His Leu Gln Gly 130 135 140
Ala Asp Lys Lys Val Gly Glu Val Val Ala Arg Glu Glu Gly Val Ser 145
150 155 160 Glu Leu Ala Tyr Pro Thr Ser His Trp Glu Gly Gly Pro Ala
Glu Asp 165 170 175 Glu Glu Asp Thr Glu Thr Val Lys Lys Ala His Gln
Ala Ser Ala Ala 180 185 190 Ser Ile Ala Pro Gly Tyr Lys Pro Ser Thr
Ser Val Tyr Cys Pro Gly 195 200 205 Glu Ala Glu His Arg Ala Thr Glu
Glu Lys Gly Thr Asp Asn Lys Ala 210 215 220 Glu Pro Ser Gly Ser His
Ser Arg Val Trp Glu Tyr His Thr Arg Glu 225 230 235 240 Arg Pro Lys
Gln Glu Gly Glu Thr Lys Pro Glu Gln His Arg Ala Gly 245 250 255 Gln
Ser His Pro Cys Gln Asn Ala Glu Ala Glu Glu Gly Gly Pro Glu 260 265
270 Thr Ser Val Cys Ser Gly Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg
275 280 285 Pro Gly Glu Asp Thr Glu Glu Glu Glu Asp Ser Asp Leu Asp
Ser Ala 290 295 300 Glu Glu Asp Thr Ala His Thr Cys Thr Thr Pro His
Thr Ser Ala Phe 305 310 315 320 Leu Lys Ala Trp Val Tyr Arg Pro Gly
Glu Asp Thr Glu Glu Glu Asp 325 330 335 Asp Gly Asp Trp Asp Ser Ala
Glu Glu Asp Ala Ser Gln Ser Cys Thr 340 345 350 Thr Pro His Thr Ser
Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly 355 360 365 Glu Asp Thr
Glu Glu Glu Asp Asp Ser Glu Asn Val Ala Pro Val Asp 370 375 380
Ser
Glu Thr Val Asp Ser Cys Gln Ser Thr Gln His Cys Leu Pro Val 385 390
395 400 Glu Lys Thr Lys Gly Cys Gly Glu Ala Glu Pro Pro Pro Phe Gln
Trp 405 410 415 Pro Ser Ile Tyr Leu Asp Arg Ser Gln His His Leu Gly
Leu Pro Leu 420 425 430 Ser Cys Pro Phe Asp Cys Arg Ser Gly Ser Asp
Leu Ser Lys Pro Pro 435 440 445 Pro Gly Ile Arg Ala Leu Arg Phe Leu
450 455 6 2111 DNA Human CDS (294)...(2027) PEG3 6 tgagattgac
tcagttcgca gcttgtggaa gattacatgc gagaaaaagc gcgactccgc 60
atccctttgc cgggacagcc cttgcgacag cccgtgagac atcacgtccc cgagccccac
120 ctttgccggg acagcctttg cgacagcccg tgagacatca cgtccccgag
ccccacgcct 180 gagggcgaca tgaacgcgct ggccttgaga gcaatccgga
cccacgaccg cttttggcaa 240 accgaaccgg acctccagcc cccggggtga
cgcgcagccc gccggccaga cac atg 296 Met 1 gcc cca agc cca aga ccc gag
cat gtc ctg cac tgg aag gaa gcc cac 344 Ala Pro Ser Pro Arg Pro Glu
His Val Leu His Trp Lys Glu Ala His 5 10 15 tct ttc tac ctc ctg tct
cca ctg atg ggc ttc ctc agc cgg gcc tgg 392 Ser Phe Tyr Leu Leu Ser
Pro Leu Met Gly Phe Leu Ser Arg Ala Trp 20 25 30 agc cgc ctg agg
ggg ccc gag gtc tca gag gcc tgg ttg gca gaa aca 440 Ser Arg Leu Arg
Gly Pro Glu Val Ser Glu Ala Trp Leu Ala Glu Thr 35 40 45 gta gca
gga gca aac cag ata cag gct gat gct ctg ttg acg cct ccc 488 Val Ala
Gly Ala Asn Gln Ile Gln Ala Asp Ala Leu Leu Thr Pro Pro 50 55 60 65
ccg gtc tct gaa aat cac cta cct ctc cga gag act gaa gga aat gga 536
Pro Val Ser Glu Asn His Leu Pro Leu Arg Glu Thr Glu Gly Asn Gly 70
75 80 act cct gaa tgg agt aaa gca gcc cag agg ctc tgc ctt gat gtg
gaa 584 Thr Pro Glu Trp Ser Lys Ala Ala Gln Arg Leu Cys Leu Asp Val
Glu 85 90 95 gcc caa agt tcc cct cct aaa act tgg gga ctt tca gat
att gat gaa 632 Ala Gln Ser Ser Pro Pro Lys Thr Trp Gly Leu Ser Asp
Ile Asp Glu 100 105 110 cat aat ggg aag cca gga caa gat ggc ctt aga
gag caa gaa gtg gag 680 His Asn Gly Lys Pro Gly Gln Asp Gly Leu Arg
Glu Gln Glu Val Glu 115 120 125 cac aca gct ggc ctg cct aca cta cag
ccc ctt cac ctg caa ggg gca 728 His Thr Ala Gly Leu Pro Thr Leu Gln
Pro Leu His Leu Gln Gly Ala 130 135 140 145 gat aag aaa gtt ggg gag
gtg gtg gct aga gaa gag ggt gtg tcc gag 776 Asp Lys Lys Val Gly Glu
Val Val Ala Arg Glu Glu Gly Val Ser Glu 150 155 160 ctg gct tac ccc
aca tca cac tgg gag ggt ggt cca gct gag gat gaa 824 Leu Ala Tyr Pro
Thr Ser His Trp Glu Gly Gly Pro Ala Glu Asp Glu 165 170 175 gag gat
aca gaa acc gtg aag aag gct cac cag gcc tct gct gct tcc 872 Glu Asp
Thr Glu Thr Val Lys Lys Ala His Gln Ala Ser Ala Ala Ser 180 185 190
ata gct cca gga tat aaa ccc agc act tct gtg tat tgc cca ggg gag 920
Ile Ala Pro Gly Tyr Lys Pro Ser Thr Ser Val Tyr Cys Pro Gly Glu 195
200 205 gca gaa cat cga gcc acg gag gaa aaa gga aca gac aat aag gct
gaa 968 Ala Glu His Arg Ala Thr Glu Glu Lys Gly Thr Asp Asn Lys Ala
Glu 210 215 220 225 ccc tca ggc tcc cac tcc aga ttc tgg gag tac cac
act aga gag agg 1016 Pro Ser Gly Ser His Ser Arg Phe Trp Glu Tyr
His Thr Arg Glu Arg 230 235 240 cct aag cag gag gga gaa act aag cca
gag caa cac agg gca ggg cag 1064 Pro Lys Gln Glu Gly Glu Thr Lys
Pro Glu Gln His Arg Ala Gly Gln 245 250 255 agt cac cct tgt cag aat
gca gag tct gag gaa gga gga cct gag act 1112 Ser His Pro Cys Gln
Asn Ala Glu Ser Glu Glu Gly Gly Pro Glu Thr 260 265 270 tct gtc tgt
tct ggc agt gcc ttc ctg aag gcc tgg gtg tat cgc cca 1160 Ser Val
Cys Ser Gly Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro 275 280 285
gga gag gac aca gag gag gaa gaa gac cct gat ttg gat tca gct gag
1208 Gly Glu Asp Thr Glu Glu Glu Glu Asp Pro Asp Leu Asp Ser Ala
Glu 290 295 300 305 gaa gac aca gct cat acc tgt acc acc ccc cat aca
agt gcc ttc ctg 1256 Glu Asp Thr Ala His Thr Cys Thr Thr Pro His
Thr Ser Ala Phe Leu 310 315 320 aag gcc tgg gtc tat cgc cca gga gag
gac aca gaa gag gaa gat gac 1304 Lys Ala Trp Val Tyr Arg Pro Gly
Glu Asp Thr Glu Glu Glu Asp Asp 325 330 335 ggt gat tgg gat tca gct
gag gaa gac gca gct cag agc tgt acc acc 1352 Gly Asp Trp Asp Ser
Ala Glu Glu Asp Ala Ala Gln Ser Cys Thr Thr 340 345 350 ccc cat aca
agt gcc ttc ctg aag gcc tgg gtc tat cgc cca gga gag 1400 Pro His
Thr Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu 355 360 365
gac aca gaa gag gaa gac gac agt gag aat gtg gcc cca gtt gac tca
1448 Asp Thr Glu Glu Glu Asp Asp Ser Glu Asn Val Ala Pro Val Asp
Ser 370 375 380 385 gaa aca gtt gac tct tgc cag agt acc cag cat tgt
cta cca gta gag 1496 Glu Thr Val Asp Ser Cys Gln Ser Thr Gln His
Cys Leu Pro Val Glu 390 395 400 aag acc aag gga tgt gga gaa gca gag
ccc cct ccc ttc cag gtg gcc 1544 Lys Thr Lys Gly Cys Gly Glu Ala
Glu Pro Pro Pro Phe Gln Val Ala 405 410 415 ttc tat tta cct gga cag
aag cca gca cca cct tgg gca gcc cct aag 1592 Phe Tyr Leu Pro Gly
Gln Lys Pro Ala Pro Pro Trp Ala Ala Pro Lys 420 425 430 ctg ccc ctt
cga ctg cag aag cgg ctc aga tct ttc aaa gcc ccc gcc 1640 Leu Pro
Leu Arg Leu Gln Lys Arg Leu Arg Ser Phe Lys Ala Pro Ala 435 440 445
cgg aat cag ggc cct gag att cct ctg aag ggt aga aag gtg cac ttc
1688 Arg Asn Gln Gly Pro Glu Ile Pro Leu Lys Gly Arg Lys Val His
Phe 450 455 460 465 tct gag aaa gtt aca gtc cat ttc ctt gct gtc tgg
gca gga cca gcc 1736 Ser Glu Lys Val Thr Val His Phe Leu Ala Val
Trp Ala Gly Pro Ala 470 475 480 cag gct gct cgt cga ggc ccc tgg gag
cag ttt gca cga gat cga agc 1784 Gln Ala Ala Arg Arg Gly Pro Trp
Glu Gln Phe Ala Arg Asp Arg Ser 485 490 495 cgc ttt gct cga cgc att
gcc cag gca gag gag cag ctg ggt cct tac 1832 Arg Phe Ala Arg Arg
Ile Ala Gln Ala Glu Glu Gln Leu Gly Pro Tyr 500 505 510 ctt acc cct
gct ttc agg gcc aga gca tgg aca cgc ctt aga aac cta 1880 Leu Thr
Pro Ala Phe Arg Ala Arg Ala Trp Thr Arg Leu Arg Asn Leu 515 520 525
ccc ctt cct ctg tcg tcc tcg tct ctt cca ctg cct gag cct tgc tct
1928 Pro Leu Pro Leu Ser Ser Ser Ser Leu Pro Leu Pro Glu Pro Cys
Ser 530 535 540 545 tcc act gag gcc aca ccc ctc agc caa gat gtg acc
act ccc tct ccc 1976 Ser Thr Glu Ala Thr Pro Leu Ser Gln Asp Val
Thr Thr Pro Ser Pro 550 555 560 ctt ccc agt gaa atc cct cct ccc agc
ctg gac ttg gga gga agg cgg 2024 Leu Pro Ser Glu Ile Pro Pro Pro
Ser Leu Asp Leu Gly Gly Arg Arg 565 570 575 ggc taagcctgag
tagttttttg ttatttattt attttaatac gaaataaagc 2077 Gly cttttgattt
gtagtgaaaa aaaaaaaaaa aaaa 2111 7 578 PRT Human 7 Met Ala Pro Ser
Pro Arg Pro Glu His Val Leu His Trp Lys Glu Ala 1 5 10 15 His Ser
Phe Tyr Leu Leu Ser Pro Leu Met Gly Phe Leu Ser Arg Ala 20 25 30
Trp Ser Arg Leu Arg Gly Pro Glu Val Ser Glu Ala Trp Leu Ala Glu 35
40 45 Thr Val Ala Gly Ala Asn Gln Ile Gln Ala Asp Ala Leu Leu Thr
Pro 50 55 60 Pro Pro Val Ser Glu Asn His Leu Pro Leu Arg Glu Thr
Glu Gly Asn 65 70 75 80 Gly Thr Pro Glu Trp Ser Lys Ala Ala Gln Arg
Leu Cys Leu Asp Val 85 90 95 Glu Ala Gln Ser Ser Pro Pro Lys Thr
Trp Gly Leu Ser Asp Ile Asp 100 105 110 Glu His Asn Gly Lys Pro Gly
Gln Asp Gly Leu Arg Glu Gln Glu Val 115 120 125 Glu His Thr Ala Gly
Leu Pro Thr Leu Gln Pro Leu His Leu Gln Gly 130 135 140 Ala Asp Lys
Lys Val Gly Glu Val Val Ala Arg Glu Glu Gly Val Ser 145 150 155 160
Glu Leu Ala Tyr Pro Thr Ser His Trp Glu Gly Gly Pro Ala Glu Asp 165
170 175 Glu Glu Asp Thr Glu Thr Val Lys Lys Ala His Gln Ala Ser Ala
Ala 180 185 190 Ser Ile Ala Pro Gly Tyr Lys Pro Ser Thr Ser Val Tyr
Cys Pro Gly 195 200 205 Glu Ala Glu His Arg Ala Thr Glu Glu Lys Gly
Thr Asp Asn Lys Ala 210 215 220 Glu Pro Ser Gly Ser His Ser Arg Phe
Trp Glu Tyr His Thr Arg Glu 225 230 235 240 Arg Pro Lys Gln Glu Gly
Glu Thr Lys Pro Glu Gln His Arg Ala Gly 245 250 255 Gln Ser His Pro
Cys Gln Asn Ala Glu Ser Glu Glu Gly Gly Pro Glu 260 265 270 Thr Ser
Val Cys Ser Gly Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg 275 280 285
Pro Gly Glu Asp Thr Glu Glu Glu Glu Asp Pro Asp Leu Asp Ser Ala 290
295 300 Glu Glu Asp Thr Ala His Thr Cys Thr Thr Pro His Thr Ser Ala
Phe 305 310 315 320 Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp Thr
Glu Glu Glu Asp 325 330 335 Asp Gly Asp Trp Asp Ser Ala Glu Glu Asp
Ala Ala Gln Ser Cys Thr 340 345 350 Thr Pro His Thr Ser Ala Phe Leu
Lys Ala Trp Val Tyr Arg Pro Gly 355 360 365 Glu Asp Thr Glu Glu Glu
Asp Asp Ser Glu Asn Val Ala Pro Val Asp 370 375 380 Ser Glu Thr Val
Asp Ser Cys Gln Ser Thr Gln His Cys Leu Pro Val 385 390 395 400 Glu
Lys Thr Lys Gly Cys Gly Glu Ala Glu Pro Pro Pro Phe Gln Val 405 410
415 Ala Phe Tyr Leu Pro Gly Gln Lys Pro Ala Pro Pro Trp Ala Ala Pro
420 425 430 Lys Leu Pro Leu Arg Leu Gln Lys Arg Leu Arg Ser Phe Lys
Ala Pro 435 440 445 Ala Arg Asn Gln Gly Pro Glu Ile Pro Leu Lys Gly
Arg Lys Val His 450 455 460 Phe Ser Glu Lys Val Thr Val His Phe Leu
Ala Val Trp Ala Gly Pro 465 470 475 480 Ala Gln Ala Ala Arg Arg Gly
Pro Trp Glu Gln Phe Ala Arg Asp Arg 485 490 495 Ser Arg Phe Ala Arg
Arg Ile Ala Gln Ala Glu Glu Gln Leu Gly Pro 500 505 510 Tyr Leu Thr
Pro Ala Phe Arg Ala Arg Ala Trp Thr Arg Leu Arg Asn 515 520 525 Leu
Pro Leu Pro Leu Ser Ser Ser Ser Leu Pro Leu Pro Glu Pro Cys 530 535
540 Ser Ser Thr Glu Ala Thr Pro Leu Ser Gln Asp Val Thr Thr Pro Ser
545 550 555 560 Pro Leu Pro Ser Glu Ile Pro Pro Pro Ser Leu Asp Leu
Gly Gly Arg 565 570 575 Arg Gly 8 2614 DNA Rat 8 acatgggcac
gcgtggtcga cggcccgggc tggctgggca acacgggttc agcccaggtt 60
tcatagtaag ttccagacac tcctggaaaa acaatacagg tccctgacaa aagaaaaaac
120 aaaacaaagg aaacagaaac atgcgttttt aaaaaagaag gaggagactc
catgaaggca 180 ggccttgggt ggggtcactg cttctctgta cacaggagga
gaattgccaa gatcttccgg 240 acagtgtgga ctatactgta agaccctctc
aatacagaca gactggacag gcatagtgac 300 acatgccttt aatgcctgca
gtactcagga ggaggtggca ggtggaacgg ctgttctttg 360 aggttcaaga
ccagcgtgga ctacagagtg agttccagga caggcagggc tacacagaaa 420
aatcctgtct gaaaacaaaa caaaacccag acagacacac caaaaacagc caagggacca
480 gagagatggg tcagggccta atcacttgct actctttgca gaggacccaa
atttagttcc 540 tataaccctc catgagaagc ttcacaattg tctctaactc
aattccaccc gtgttccgac 600 ctccatatgc accagacatg atatactcac
acatacgcac aaacacacac acacacacac 660 acacacacac acacacacac
acacacacac ggaaaacata taaaataaag atttaaaaaa 720 tctttttctt
ttggccgggg tgtgtgggag agcatctgag ccatctcacc agcccagggt 780
gcagctcttt ttcttttttt cggagctggg gaccgaaccc agagccttgt gcttgctagg
840 caagtgctct accactgagc taaatcccca accccggagc acgtctttaa
tcccagaatc 900 aggaggtaga ggtaatgaga tcccagtgag cccaaggtca
gccgagtcta caaagtgagt 960 tccaggacag ccagaactaa tcttggaaaa
acaaacaagg gctggtgagg tggttcagta 1020 gttaagaaca ctggctgctc
ttccagaggt cctgagttca ttctcagtaa ccacatggtg 1080 gggatctgat
gcctgttctg gcatgcagat atacatgcag atagtgcact cctacattta 1140
aaaaaaaaag acataaataa tattttaaaa cattgggcgt tttgtcttct aataaaactt
1200 cactgctatc ttctaataaa aattcactgc tagccgcggg gtgtggtggc
cccatacctt 1260 taatcccaac aacttgagag gcagaggcag gcggaccttt
gagtttgaag ctagcctggt 1320 ctacagagtg agttcaagat agccacggat
agtcagaaag tcctgtttcg aacctctccc 1380 caaccaaatc actcctgtaa
tcccagcact ctggaggcag tagcaggtta gtccctgctt 1440 ctcagagaga
ggagagagag agagagagag agagagagga gacacacaca cacagagaca 1500
gagaggagag agaaagagaa agagaatggg acagcatgtg actgcctgat gaagttggcg
1560 tgcttgctca aaagttctgc gagattgacg gctctctgga tttgagccaa
ggacacgcct 1620 gggaagccac ggtgacctca caaggcccgg aatctccgcg
agaatttcag tgttgttttc 1680 ctctctccac ctttctcagg gacttccgaa
actccgcctc tccggtgacg tcagatagcg 1740 ctcgtcagac tataaactcc
cgggtgatcg tgttggcgca gattgactca gttcgcagct 1800 tgtggaagat
tacatgcgag accccgcgcg actccgcatc cctttgccgg gacagccttt 1860
gcgacagccc gtgagacatc acgtccccga gccccagcct gagggcgaca tgaacgcgct
1920 ggccttgaga gcaatccgga cccacgatcg cttttggcaa accgaaccgg
accgaaccgg 1980 acctccagcc cccggggtga cgcgcagtcg ccggtgagtg
ggggatgggg cggcctttgg 2040 gggagtgctg gggaggactt tctttggcga
tggaggctag gagagtgttg tgggatctag 2100 gggagactgg ggaggaaccc
agatttgagg aaacggcact gaaagccgga tgctttattt 2160 ggtccgagag
aggagagccc aggtctagtc tctacattga agggcagggg tcctgaacta 2220
gaactgcagt acttgtacat tgctaaataa agagagggac tccaggagga gcagcctggg
2280 tctaagaggt aggcaggaga aggttttagg ggcctgagca caagcttgag
gagagaaagg 2340 ttattaaaaa gccagacgtt acaggtctca gaagggctag
ccagaaactg tggcttgggg 2400 ttaaggaaag ggtttaagag tgtgggcttt
tggttctgag gatgtaggaa cgtgaatgtt 2460 gagagaagaa ccaagtggcg
gagttgggtg tgagcaatgc tattaggaat ttgaggcagg 2520 gattcacgct
gctgtgacta ttttttaaca atgactcagt gctgtgacct gatactgttt 2580
ccagagcgac ttctaaacaa attcccccct ttct 2614
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