U.S. patent application number 10/408969 was filed with the patent office on 2003-09-11 for uses of antileukoprotease in carcinoma.
This patent application is currently assigned to Board of Trustees of University of Arkansas. Invention is credited to O' Brien, Timothy J., Shigemasa, Kazushi, Tanimoto, Hirotoshi, Underwood, Lowell J..
Application Number | 20030170759 10/408969 |
Document ID | / |
Family ID | 22574904 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170759 |
Kind Code |
A1 |
O' Brien, Timothy J. ; et
al. |
September 11, 2003 |
Uses of antileukoprotease in carcinoma
Abstract
The present invention provides a method of detecting/monitoring
tumor growth and progression in a tissue by measuring the level of
antileukoprotease. Also provided is a method of treating an
individual having a tumor by administering antileukoprotease to
inhibit the activity of stratum corneum chymotryptic enzyme.
Specifically, the tumor is an ovarian carcinoma.
Inventors: |
O' Brien, Timothy J.;
(Little Rock, AR) ; Underwood, Lowell J.; (Little
Rock, AR) ; Tanimoto, Hirotoshi; (Kagawa, JP)
; Shigemasa, Kazushi; (Hiroshima, JP) |
Correspondence
Address: |
Benjamin Aaron Adler, Ph.D., J.D.
Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
Board of Trustees of University of
Arkansas
|
Family ID: |
22574904 |
Appl. No.: |
10/408969 |
Filed: |
April 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10408969 |
Apr 8, 2003 |
|
|
|
09692820 |
Oct 18, 2000 |
|
|
|
60159972 |
Oct 18, 1999 |
|
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
C12Q 1/37 20130101; A61K
38/57 20130101; G01N 33/57449 20130101 |
Class at
Publication: |
435/7.23 |
International
Class: |
G01N 033/574 |
Claims
What is claimed is:
1. A method of detecting growth of an ovarian tumor and
ovarian-derived metastatic tumor in a sample from an individual,
comprising the step of: measuring the level of antileukoprotease in
said individual, wherein if said level of antileukoprotease exceeds
the mean basal level of antileukoprotease in nondiseased
individuals by 2 standard deviation or greater, said individual has
growth in an ovarian or ovarian-derived tumor.
2. The method of claim 1, wherein said tumor is selected from the
group consisting of a low malignant potential tumor and an ovarian
carcinoma.
3. The method of claim 2, wherein said ovarian carcinoma is
selected from the group consisting of a serous carcinoma, a
mucinous carcinoma, an endometrioid carcinoma and a clear cell
carcinoma.
4. The method of claim 1, wherein said antileukoprotease is
detected in a sample selected from the group consisting of an
ovarian secretion, an ovarian biopsy, a metastatic tumor biopsy and
blood.
5. The method of claim 1, wherein said antileukoprotease is
detected by a method selected from the group consisting of western
blot analysis, immunohistochemical staining, and immunochemical
methods.
6. A method of treating an individual having a tumor selected from
the group consisting of an ovarian tumor and ovarian-derived
metastatic tumors, comprising the step of: administering
antileukoprotease to said individual.
7. The method of claim 5, wherein said tumor is selected from the
group consisting of a low malignant potential tumor and an ovarian
carcinoma.
8. The method of claim 7, wherein said ovarian carcinoma is
selected from the group consisting of a serous carcinoma, a
mucinous carcinoma, an endometrioid carcinoma and a clear cell
carcinomas.
9. The method of claim 6, wherein said antileukoprotease is
administered systemically.
10. The method of claim 6, wherein said antileukoprotease is
administered locally
11. A method of preventing metastasis of a tumor selected from the
group consisting of an ovarian tumor and ovarian derived metastatic
tumors, comprising the step of: administering antileukoprotease to
an individual having said tumor.
12. The method of claim 11, wherein said tumor is selected from the
group consisting of a low malignant potential tumor and an ovarian
carcinoma.
13. The method of claim 12, wherein said ovarian carcinoma is
selected from the group consisting of a serous carcinoma, a
mucinous carcinoma, an endometrioid carcinoma and a clear cell
carcinoma.
14. The method of claim 11, wherein said antileukoprotease is
administered systemically.
15. The method of claim 11, wherein said antileukoprotease is
administered locally
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This non-provisional patent application claims benefit of
provisional patent application U.S. Serial No. 60/159,972, filed
Oct. 18, 1999, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
protein chemistry and cancer therapy. More specifically, the
present invention relates to antileukoprotease, a peptide inhibitor
of stratum corneum chymotryptic enzyme, and its uses in carcinoma
diagnosis and treatment.
[0004] 2. Description of the Related Art
[0005] Proteases mediate specific proteolysis involved in
processing of precursors of protein hormones, activation of
regulatory enzymes in blood coagulation and complement activation,
and the tissue rearrangement involved in tumor progression (1). In
the process of tumor invasion and metastasis, proteases mediate the
digestion of neighboring extracellular matrix components during
initial tumor growth. This allows shedding of minor cells into the
surrounding environment, providing the basis for invasion of
basement membranes in target metastatic organs. Proteolytic
digestion is also required for release and activation of many
growth and angiogenic factors (2-4).
[0006] A large number of reports have demonstrated increased
production of several classes of proteases, including matrix
metalloproteases (MMP's), cysteine proteases, aspartic pro teases
and serine proteases in tumor cells (5-9). The proteolysis of the
extracellular matrix is a highly complicated process, which
probably involves a cascade of events requiring a variety of
proteases (10). In this cascade, the integrated capacity for
extracellular matrix digestion, tumor cell invasion, and metastatic
growth may be mediated by proteases with unique specificities. This
hypothesis is supported by findings that some agents specifically
inhibit one of these proteases to reduce tumor cell invasion
(11,12).
[0007] Stratum corneum chymotryptic enzyme (SCCE) was originally
isolated from a keratinocyte derived library and was identified as
a serine protease (13,14). Analysis of mRNA showed that two
transcripts of 1.2 kb and 2.0 kb were present, and abundant
expression of the stratum corneum chymotryptic enzyme gene was
restricted to human skin. Immunohistochemical studies confirmed
that stratum corneum chymotryptic enzyme was a tissue-specific
enzyme only expressed by the stratum corneum (15). The nucleotide
sequence includes an open reading frame for a stratum corneum
chymotryptic enzyme precursor protein consisting of 253 amino
acids. This inactive precursor becomes proteolytically active after
tryptic removal of a 7 amino acid peptide from the amino terminal
end of the propeptide. Recent studies have revealed that stratum
corneum chymotryptic enzyme appears to catalyze the degradation of
intercellular cohesive structures between corneocytes in the
outermost cornified layer of the skin and contributes to the cell
shedding process at the skin surface (14, 16, 17). This process
occurs possibly through the degradation of matrix components
including the desmosomal protein desmoglein I.
[0008] Protease inhibitor antileukoprotease (ALP), also known as
secretory leukocyte proteinase inhibitor (SLPI), has been
identified as a potent inhibitor of leukocyte elastase, cathepsin
G, chymotrypsin and trypsin (18). Antileukoprotease has been cloned
from skin tissue and shown to be a specific inhibitor of the
stratum corneum chymotryptic enzyme (SCCE) (17). This serine
protease is produced and released into mucus by secretory cells in
the parotid, bronchus, cervix and testicular glands (18). There, it
is thought to play a physiological role in preventing the
proteolytic degradation of these tissues. However, little has been
known about the expression of antileukoprotease in human cancer
tissues, including ovarian cancer.
[0009] The prior art is deficient in the lack of effective means of
using antileukoprotease as a diagnostic or monitoring tool of
carcinomas. The present invention fulfills this long-standing need
and desire in the art.
SUMMARY OF THE INVENTION
[0010] The present invention demonstrates that antileukoprotease
(ALP) is overexpressed in low malignant potential tumors and
carcinomas in ovary, while little or no transcript is present in
normal adult and fetal tissues. This indicates that
antileukoprotease may be used as a diagnostic or monitoring tool of
ovarian tumors.
[0011] In one embodiment of the present invention, there is
provided a method of detecting growth of an ovarian or
ovarian-derived metastatic tumor in an individual suspected to have
such a tumor, comprising the step of detecting the level of
antileukoprotease in a test tissue, a secretion from a test tissue
or the blood. If the level exceeds the mean basal level of
antileukoprotease in nondiseased individuals by 2 standard
deviation or more, the individual has ovarian and ovarian derived
metastatic tumor growth.
[0012] In still another embodiment of the present invention, there
is provided a method of treating an individual having a ovarian
tumor by administering antileukoprotease to the individual.
[0013] In yet another embodiment of the instant invention, a method
of preventing metastasis of an ovarian tumor is provided wherein
antileukoprotease is administered to an individual having such a
tumor.
[0014] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention given for
the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings form a
part of the specification. It is to be noted, however, that the
appended drawings illustrate preferred embodiments of the invention
and therefore are not to be considered limiting in their scope.
[0016] FIGS. 1A-1D show the results of northern blot analysis of
antileukoprotease expression in various tissues. FIG. 1A confirms
the presence of antileukoprotease transcript in tumor tissues as
opposed to normal tissues. ALP mRNA was detected as a 1.4 kb
transcript in ovarian cancers but was not detected in normal ovary.
FIG. 1B shows that the antileukoprotease transcript was not
detected in fetal tissues. FIG. 1C and FIG. 1D show that
antileukoprotease transcript was not detected in many normal adult
tissues but was abundantly expressed in others including lung.
[0017] FIG. 2 shows the overexpression of antileukoprotease in
ovarian carcinoma specimens as detected by quantitative PCR of
antileukoprotease transcript. Expression levels of
antileukoprotease relative to .beta.-tubulin are significantly
elevated in many cancer samples relative to levels observed in
normal ovary.
[0018] FIG. 3 shows a graphical representation of
antileukoprotease/.beta.- -tubulin ratios in normal ovaries, LMP
tumors, and carcinomas. ALP mRNA expression levels were
significantly elevated in LMP tumors and carcinomas as compared
with levels in normal ovaries.
[0019] FIGS. 4A-4E show immunohistochemical staining results for
antileukoprotease in various ovarian tumors. FIG. 4A shows
immunohistochemical staining or normal uterine endocervix at
50.times. magnification. The endocervix section acts as a positive
control as normal endocervical glandular cells and the mucin in the
endocervical glands is positive for ALP. FIG. 4B, showing a
100.times. magnification of normal ovarian surface epithelium, is
negative for antileukoprotease immunohistochemical staining.
Positive antileukoprotease immunohistochemical staining was
observed in both the cell membrane and cytoplasm of ovarian cancer
cells of FIG. 4C (endometrioid adenocarcinoma, X50). In FIG. 4D
(mucinous adenocarcinoma, X50), the mucin as well as tumor glands
of a mucinous carcinoma were positive for antileukoprotease
staining. Positive staining for antileukoprotease was also observed
in secretions from the clear cell carcinoma shown in FIG. 4E (clear
cell carcinoma, X50) and in the serous carcinomas shown in FIGS. 4F
and 4G.
DETAILED DESCRIPTION OF THE INVENTION
[0020] This invention encompasses a demonstration of overexpression
of antileukoprotease (ALP) in carcinoma tissues. It shows that many
low malignant potential tumors and most carcinomas tested have a
statistically significant overexpression of antileukoprotease.
Antileukoprotease is a secreted protein with a distribution of
cysteines known to provide extraordinary stability to protease
inhibitors. This molecule therefore fulfills many criteria
necessary for a valuable marker of tumor growth and progression.
Antileukoprotease is overexpressed in tumor cells secreted to the
extracellular space and is a relatively small protein
(approximately 100 amino acids) providing good opportunity for
uptake into the circulation. Moreover, antileukoprotease is a very
stable protein thus providing the potential for a relatively long
half life in the circulation.
[0021] In one embodiment of the present invention, there is
provided a method of detecting growth of an ovarian or ovarian
derived metastatic tumor in an individual suspected of having such
a tumor, comprising the step of detecting the level of
antileukoprotease in a test sample, wherein if the level exceeds
the mean basal level of antileukoprotease in nondiseased
individuals by 2 standard deviation and greater, the individual has
growth in an ovarian or ovarian-derived tumor. Preferably, the
tumor may be a low malignant potential tumor or a carcinoma.
Examples of carcinoma include serous carcinoma, mucinous carcinoma,
endometrioid carcinoma and clear cell carcinoma. The test samples
may be an ovarian secretion, an ovarian biopsy, a metastatic tumor
biopsy and blood. The antileukoprotease may be detected by various
means know to one having ordinary skill in this art including
western blot analysis, immunohistochemical staining, or other
immunochemical methods. In still another embodiment of the present
invention, there is provided a method for treating an individual
having an ovarian or ovarian-derived metastatic tumor, comprising
the step of administering antileukoprotease to the individual.
Preferably, the tumor may be a low malignant potential tumor or an
ovarian carcinoma. If it is an ovarian carcinoma, the tumor may be
a serous carcinoma, a mucinous carcinoma, an endometrioid carcinoma
and a clear cell carcinomas. The antileukoprotease may be
administered systemically or locally.
[0022] In yet another embodiment of the present invention, there is
provided a method for method of preventing metastasis of an ovarian
tumor or ovarian derived metastatic tumor comprising the step of
administering antileukoprotease to an individual having such a
tumor. Preferably, the tumor may be a low malignant potential tumor
or an ovarian carcinoma. If it is an ovarian carcinoma, the tumor
may be a serous carcinoma, a mucinous carcinoma, an endometrioid
carcinoma and a clear cell carcinomas. The antileukoprotease may be
administered systemically or locally.
[0023] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
EXAMPLE 1
[0024] Tissue Samples
[0025] Fresh surgical specimens of ovarian tumors were obtained
from low malignant potential tumors and carcinomas. Clinical
staging was determined according to the criteria of the
International Federation of Gynecology and Obstetrics (FIGO).
Normal ovaries were obtained from patients who underwent surgery
for benign gynecological disease. The materials were obtained
immediately after the surgical procedures and frozen in liquid
nitrogen and stored at -80.degree. C. prior to mRNA isolation. Two
ovarian cancer cell lines, SW626 and CaOv3, were also used.
EXAMPLE 2
[0026] mRNA Extraction and cDNA Synthesis
[0027] Extraction of mRNA from tissue specimens and cDNA synthesis
therefrom were carried out by the methods described previously
(19). mRNA was isolated by using a RiboSep.TM. mRNA isolation kit
(Becton Dickenson Labware). In this procedure, poly A+ mRNA was
isolated directly from the tissue lysate using the affinity
chromatography media oligo(dT) cellulose. The amount of mRNA
recovered was quantitated by UV spectrophotometry. The cDNA was
synthesized with either 2.0 .mu.g or 5.0 .mu.g of mRNA by random
hexamer priming using a 1st strand.TM. cDNA synthesis kit
(CLONTECH). The efficiency of the cDNA synthesis was estimated
using glucose 3-phosphate dehydrogenase (G3PDH) amplimers
(Clontech, Palo Alto, Calif., USA) as a positive control.
EXAMPLE 3
[0028] Quantitative PCR
[0029] mRNA overexpression of antileukoprotease was determined
using a quantitative PCR approach as previously reported (19-21).
The ALP target sequences were amplified in parallel with the
.beta.-tubulin gene. .beta.-tubulin has previously been established
to be a consistently expressed internal control for both normal and
tumor tissues (19,21). The following specific oligonucleotide
primers were used for antileukoprotease amplification by PCR:
Primer set 1- forward 5'-TGCATTGACAACGAGGC-3' (SEQ ID No. 1) and
reverse 5'-CTGTCTTGACATTGTTG -3' (SEQ ID No. 2); and, primer set
2-forward 5'-CCTTCAAAGCTGGAGTCTGT-3' (SEQ ID No. 3) and reverse
(CCAAAGGAGGATATCAGTGG-3' (SEQ ID No. 4). The primers for the
.beta.-tubulin internal control amplification were, forward
5'-CGCATCAACGTGTACTACAA-3' (SEQ ID No. 5) and reverse
5'-TACGAGCTGGTGGACTGAGA-3' (SEQ ID No. 6). The predicted sizes of
the amplified gene were 400 bp for ALP primer set 1, 469 bp for ALP
primer set 2 and 454 bp for .beta.-tubulin. The primer sequences
used in this study were designed according to the cDNA sequences
described for antileukoprotease and by Hall et al. for
.beta.-tubulin (22).
[0030] The PCR reaction mixture consisted of cDNA derived from 50
ng of mRNA, 5 pmol of sense and antisense primers for both the
stratum corneum chymotryptic enzyme gene and the .beta.-tubulin
gene, 200 .mu.mol of dNTPs, 5 .mu.Ci of [.alpha.-.sup.32P]dCTP and
0.25-0.625 units of Taq DNA polymerase with reaction buffer
(Promega) in a final volume of 25 .mu.l. The target sequences were
amplified in parallel with the .beta.-tubulin gene. Thirty cycles
of PCR were carried out in a Thermal Cycler (Perkin-Elmer Cetus,
Foster City, Calif., USA). Each cycle of PCR included 30 seconds of
denaturation at 95.degree. C., 30 seconds of primer annealing at
58-60.degree. C. and 30 seconds of extension at 72.degree. C. It
was previously established (19,21) and confirmed for the stratum
corneum chymotryptic enzyme that co-amplification with
.beta.-tubulin under these conditions for 30 cycles remains linear
for both products. Tubes containing all ingredients except
templates were included in all runs as negative control
reactions.
[0031] The PCR products were separated on 2% agarose gels and the
radioactivity of each PCR product was determined by using a Phospho
Imager (Molecular Dynamics). In the present study, the gene
expression of the stratum corneum chymotryptic enzyme was
calculated as the ratio of ALP to .beta.-tubulin as measured by the
phospho imager. The overexpression cut-off value was defined at two
standard deviation above the mean expression level for normal
ovarian expression.. For statistical analysis, the .chi..sup.2 test
and Fischer's exact probability were used for contingency analysis
and the unpaired student's T-test was used for the comparison of
the mean values of normal ovary and tumors. Significance was
defined as p<0.05.
EXAMPLE 4
[0032] Northern Blot Analysis
[0033] Ten .mu.g of mRNA were loaded onto a 1% formaldehyde-agarose
gel, resolved by electrophoresis and blotted on a Hybond-N+ nylon
membrane (Amersham, Amersham, UK). .sup.32P-labeled cDNA probes
were made by Prime-a-Gene Labeling System (Promega, Madison, Wis.).
The PCR products were amplified using the specific primers
described above as probes. The blots were prehybridized for 30 min
and hybridized for 60 min at 68.degree. C. with a .sup.32P-labeled
cDNA probe in ExpressHyb Hybridization Solution (CLONTECH, Palo
Alto, Calif.). Control hybridization to determine relative gel
loading was performed with the .beta.-tubulin probe.
[0034] Normal human tissues including spleen, thymus, prostate,
testis, ovary, small intestine, colon, peripheral blood leukocyte,
heart, brain, placenta, lung, liver, skeletal muscle, kidney,
pancreas and normal human fetal tissues; brain, lung, liver and
kidney (Human Multiple Tissue Northern Blot; CLONTECH, Palo Alto,
Calif.) were all examined using the same hybridization
procedure.
EXAMPLE 5
[0035] Immunohistochemistry
[0036] Polyclonal rabbit antibodies were generated by immunization
with a poly-lysine linked multiple antigen peptide (a 12 amino acid
sequence near the carboxy terminal end of ALP). Immunohistochemical
localization of antileukoprotease antigen was examined using normal
ovaries, mucinous LMP tumor and adenocarcinomas (including serous
adenocarcinomas, mucinous adenocarcinoma and clear cell carcinomas)
in the same series of the samples as were used for mRNA isolation.
Formalin fixed and paraffin-embedded sections, 4 .mu.m thick, were
cut and mounted on aminopropyltriethoxysilane treated slides.
Slides were routinely deparaffinized with xylene and rehydrated
with a series of ethanol washes. Nonenzymatic antigen retrieval was
performed by microwave heat treatment seven times for three minutes
in a 0.01 M sodium citrate buffer (pH 6.0). Immunohistochemical
staining was performed manually using the avidin-biotin peroxidase
complex technique (Vectastain Elite ABC kit, Vector Laboratories,
Burlingame, Calif., USA).
[0037] This indirect immunoperoxidase staining procedure was
performed at room temperature. Endogenous peroxidase and
nonspecific background staining were blocked by incubating the
slides with methanol containing 0.3% H.sub.2O.sub.2 for 30 minutes.
The slides were washed with phosphate-buffered saline (PBS) for 30
minutes, blocked with normal goat serum for thirty minutes, and
incubated with the above rabbit anti-antileukoprotease polyclonal
antibody for two hours. After washing with phosphate-buffered
saline (PBS) for 30 minutes, sections were incubated with
biotinylated anti-rabbit IgG for 30 minutes. After washing with PBS
for 30 minutes, slides were incubated with ABC reagent for 30
minutes. The final products were visualized using the
3-amino-9-ethylcarbazole (AEC) substrate system (DAKO Corporation,
Carpinteria, Calif.) and were counterstained with Mayer hematoxylin
for 20 seconds before mounting. Positive controls and negative
controls were used for each section. Normal endocervix was used as
a positive control. Negative controls were prepared by using normal
rabbit serum on sections instead of the primary antibody. All
experiments were duplicated. The stained slides were examined
microscopically by 3 observers. The presence of more than 10%
focally distributed positive tumor cells was the criterion for a 1+
positive staining and more than 50% of positive tumor cells was the
criterion for a 2+ positive staining. When less than 10% of the
cells showed positive nuclear staining, the staining was considered
negative
EXAMPLE 6
[0038] Western Blot
[0039] Approximately 20 ng of MDA-MBA-435S and HeLa cell lysates
were separated on a 15% SDS-PAGE gel and electroblotted to PVDF at
100 V for 40 minutes at 4.degree. C. The proteins were fixed to the
membrane by incubation in 50% MeOH for 10 minutes. The membrane was
blocked overnight in TBS, pH 7.8 containing 0.2% non-fat milk.
Primary antibody was added to the membrane at a dilution of 1:100
in 0.2% milk/TBS and incubated for 2 hours at room temperature. The
blot was washed and incubated with a 1:3000 dilution of
alkaline-phosphatase conjugated goat anti-rabbit IgG (BioRad) for
one hour at room temperature. The blot was washed and incubated
with a chemiluminescent substrate before a 10 second exposure to
X-ray film for visualization.
EXAMPLE 7
[0040] Northern blot analysis of antileukoprotease expression in
ovarian carcinomas
[0041] To evaluate the mRNA expression of antileukoprotease in
ovarian tumors and to examine the size of the mRNA transcript,
Northern blot analysis with a .sup.32P-labeled antileukoprotease
probe was performed in representative cases of each type of ovarian
carcinoma (FIG. 1A). Northern blot analysis revealed a 1.4 kb
transcript in all of the subtypes of ovarian carcinoma including
serous, mucinous, endometrioid, and clear cell carcinomas (FIG. 1A,
lanes 2-4). No transcript was observed in normal ovarian tissue
(FIG. 1A, lane 1). These results demonstrate that an appropriately
sized transcript of the antileukoprotease gene is expressed in the
ovarian carcinomas.
EXAMPLE 8
[0042] Northern blot analysis of antileukoprotease expression in
normal tissues
[0043] Northern blot analysis with a .sup.32P-labeled
antileukoprotease probe was also performed in normal fetal and
adult tissue samples. In the fetal tissues, little or no
antileukoprotease expression was detected in fetal brain, lung,
liver, kidney and pancrease (FIG. 1B). Antileukoprotease transcript
was also not detected in many normal adult tissues, including
heart, brain, placenta, liver, skeletal muscle, kidney, and
pancreas (FIGS. 1C and 1D). Only adult lung showed an abundance of
the antileukoprotease transcript (FIG. 1D). Lower levels of
expression were observed in adult prostate, ovaries, and the colon
(FIG. 1C).
[0044] Further data supporting tumor expression of
antileukoprotease was obtained using polyclonal antibodies
developed to multiple antigen peptide (MAP peptide), a 12 amino
acid sequence near the carboxyl terminal of antileukoprotease (data
not shown).
EXAMPLE 9
[0045] Semi-quantitative PCR analysis of Antileukoprotease (ALP)
Overexpression.
[0046] To confirm the results of the northern blot analysis,
semi-quantitative PCR was performed with primers for
antileukoprotease and the .beta.-tubulin internal control on 34
ovarian carcinomas and 10 normal ovarian tissue samples. FIG. 2
shows an example of a semi-quantitative PCR evaluation of
antileukoprotease expression using oligonucleotides specific for
antileukoprotease and the .beta.-tubulin internal control. It was
apparent that many ovarian carcinoma samples, when compared to
normal ovarian samples, exhibit elevated levels of
antileukoprotease transcript relative to the levels of the control
.beta.-tubulin transcript, providing additional evidence that
antileukoprotease is often overexpressed in ovarian carcinoma
specimens.
[0047] The relative expression antileukoprotease of ALP and
.beta.-tubulin in each sample were measured by phospho imager
analysis. The results for each tissue sample are presented in Table
1. It can be noted that many low malignant potential tumors and
most carcinomas have a statistically significant overexpression of
antileukoprotease. In many cases, this expression exceeds the mean
for normal by 4SD. It should also be noted that in at least one
ovarian tumor cell line, CAOV3, significant overexpression of
antileukoprotease was observed (Table 1).
1TABLE 1 A complete analysis of expression comparing normal ovarian
tissue to low malignant potential tumors and to overt carcinomas
Lab No. Hist. Type ALP alp 456 normal ovary 0.0033 0 856 normal
ovary 0.0228 0 858 normal ovary 0.0246 0 1235 normal ovary 0.04 0
1338 normal ovary 0.0997 0 1339 normal ovary 0.1058 0 1343 normal
ovary 0.0433 0 1344 normal ovary 0.1014 0 1345 normal ovary 0.069 0
2296 normal ovary 0.0146 0 481 s adenoma (LMP) 0.3214 4+.sup. 1448
s adenoma (LMP) 0.0251 0 1452 s adenoma (LMP) 0.2815 4+.sup. 1444 s
adenoma (LMP) 0.5005 4+.sup. 1447 s adenoma (LMP) 0.3741 4+.sup.
1450 s adenoma (LMP) 0.2195 4+.sup. 1036 m adenoma (LMP) 0.0775 0
1451 m adenoma (LMP) 0.0515 0 1456 m adenoma (LMP) 0.2771 4+.sup.
1242 s carcinoma 0.1502 2+.sup. 515 s carcinoma 1.5101 4+.sup. 1032
s carcinoma 0.28 4+.sup. 1240 s carcinoma 1.2282 4+.sup. 1245 s
carcinoma 0.9342 4+.sup. 465 s carcinoma 0.8166 4+.sup. 1026 s
carcinoma 0.4244 4+.sup. 464 s carcinoma 0.0827 0 468 s carcinoma
0.5117 4+.sup. 1033 s carcinoma 0.036 0 1039 s carcinoma 0.1749
2+.sup. 960 s carcinoma 0.3775 4+.sup. 962 s carcinoma 0.446
4+.sup. 1819 s carcinoma 0.5974 4+.sup. 1443 m carcinoma 0.1122 0
1219 m carcinoma 0.8489 4+.sup. 1990 m carcinoma 1.3723 4+.sup. 484
m carcinoma 0.0007 0 1244 m carcinoma 0.0376 0 1816 m carcinoma
0.4697 4+.sup. 2295 e carcinoma 0.2437 4+.sup. 2299 e carcinoma
0.4789 4+.sup. 2300 e carcinoma 0.4094 4+.sup. 947 c carcinoma
0.5468 4+.sup. 948 c carcinoma 0.5086 4+.sup. sw626 ovarian cancer
0.0633 0 cell line caov3 ovarian cancer 0.1862 2+.sup. cell line
Mean 0.05245 Confidence level (95.0%) 0.02769603 Standard Error
0.01224319 Median 0.04165 Mode .sup. #Num! Standard Deviation
0.03871635 Mean + 2SD .sup. 0.12989 = 2+ Mean + 4SD .sup. 0.20733 =
4+ Sample Variance 0.00149896 Kurtosis -1.60048588 Skewness
0.37295213 Range 0.1025 Minimum 0.0033 Maximum 0.1058 Sum 0.5245
Count 10
EXAMPLE 10
[0048] Statistical Analysis Semi-quantitative PCR Results
[0049] FIG. 3 summarizes the relative expression ratios of
antileukoprotease to .beta.-tubulin in normal ovaries, LMP tumors,
and ovarian carcinomas. The exact ratios (mean .+-.SD) are listed
in Table 2 for normal ovary (0.05.+-.0.04), LMP tumor
(0.24.+-.0.16) and carcinoma (0.50.+-.0.41). In many carcinoma
cases as well as LMP tumors, antileukoprotease mRNA expression was
significantly elevated compared to that in normal ovary (LMP tumor,
p<0.01; carcinoma, p<0.01; unpaired T-test). There was no
statistical difference between the antileukoprotease expression
levels and the clinical stage, histological grade or histological
type. It should be noted that in one ovarian cancer cell line,
CaOv3, significant overexpression of antileukoprotease was observed
(ratio=0.19), while another cell line, SW626, showed a relatively
low level of antileukoprotease expression (ratio=0.06) (Table
1).
2TABLE 2 Relative Expression Levels of ALP and ALP Overexpression
Rates in Ovarian Tumors Ratio ALP ALP/.beta.-tubulin overexpression
Tissue Type N Mean SD rates.sup.a Normal Ovary 10 0.05 0.04 0/10
(0%) LMP Tumor 9 .sup. 0.24.sup.b 0.16 6/9 (67%) Serous 6 0.29 0.16
.sup. 5/6 (83%).sup.d Mucinous 3 0.14 0.12 1/3 (33%) Ovarian Cancer
25 .sup. 0.50.sup.c 0.41 20/25 (80%) Clinical Stage Stage 1/2 7
0.54 0.45 6/7 (8.6%) Stage 3 18 0.49 0.41 14/18 (78%) Histological
Grade Grade 1/2 13 0.62 0.52 10/13 (77%) Grade 3 12 0.38 0.22 10/12
(83%) Histological Type Serous 14 0.54 0.44 12/14 (86%) Mucinous 6
0.47 0.55 3/6 (50%) Endometrioid 3 0.38 0.12 3/3 (100%) Clear Cell
2 0.53 0.03 2/2 (100%) .sup.aOverexpression was defined as
.gtoreq.2 SD over the mean normal value .sup.bSignificant, LMP
tumor vs. normal ovary; p < 0.01, unpaired T test
.sup.cSignificant, cancer vs. normal ovary; p < 0.01, unpaired T
test .sup.dSignificant, serous tumors (17/20) vs. mucinous tumors
(4/9), p < 0.05, .chi..sup.2 test
[0050] Table 2 also summarizes the mean antileukoprotease
overexpression rates by tissue subtypes in ovarian tumors. Overall,
antileukoprotease mRNA overexpression was found in 6 of 9 LMP
tumors (67%) and in 20 of 25 carcinoma cases (80%). All 10 normal
ovaries showed relatively low levels of antileukoprotease mRNA
expression. With regard to histological type, increased expression
of antileukoprotease was found in 5 of 6 LMP serous tumors (83%)
and in 12 of 14 serous carcinomas, whereas only 1 of 2 LMP mucinous
tumors (33%) and 3 of 6 mucinous carcinomas (50%) showed
overexpression of antileukoprotease. Thus, there was a statistical
difference of the antileukoprotease overexpression rates between
serous tumors (17/20) and mucinous tumors (4/9) including LMP
tumors and carcinomas (p<0.05, .chi..sup.2 test). The
antileukoprotease overexpression rates determined for clinical
stage and histological grad did not show any significant
difference.
[0051] Table 3 indicates the relationship between antileukoprotease
and SCCE overexpression status in ovarian tumor cases including LMP
tumors and carcinomas. SCCE overexpression status was analyzed in
the same series of the samples. Out of twenty-six antileukoprotease
overexpression cases, 23 cases showed SCCE overexpression, whereas
only two out of eight cases showed SCCE overexpression with normal
expression of antileukoprotease. Thus, there was a significant
positive correlation between antileukoprotease and SCCE
overexpression status in ovarian tumor cases (p<0.01,
.chi..sup.2 test).
3TABLE 3 ALP and SCCE Overexpression Status in Ovarian Tumors
Including LMP Tumors and Carcinomas SCCE mRNA Expression SCCE SCCE
Overexpression.sup.a Normal Range.sup.b ALP mRNA Expression Cases
Cases ALP Overexpression.sup.a Cases 23 3 ALP Normal Range.sup.b
Cases 3 6 (p < 0.01, .chi..sup.2 Test) .sup.aOverexpression was
defined as .gtoreq.2 SD over the mean normal value .sup.bNormal
range = mean .+-. 2SD for normal ovary value
EXAMPLE 11
[0052] Immunohistochemical Analysis
[0053] To further confirm the presence of the antileukoprotease
protein in ovarian tumor cells. Antileukoprotease expression in
both normal ovarian epithelia and ovarian tumor tissues was
analyzed by immunohistochemical staining with the polyclonal serum
describe supra. A comparison of immunohistochemical staining of
normal ovary and various sub-types of ovarian carcinoma is shown in
FIGS. 4A-4G. Different patterns of staining were noted in tumor
cell cytoplasm, vesicles and membranes. In a normal endocervical
specimen, which was used as a positive control, antileukoprotease
protein was detected in endocervical glandular cells as well as on
the mucin in the gland (FIG. 4A). Little or no staining was
observed with normal ovarian surface epithelium (FIG. 4B). However,
positive staining was observed both in the cytoplasm and on the
cell membrane of ovarian cancer cells (FIG. 4C; (endometrioid
adenocarcinoma).
[0054] Positive antileukoprotease staining was observed on the
mucin as well as in the tumor glands of mucinous carcinoma (FIG.
4D). The secretion of clear cell carcinomas also stained positive
for antileukoprotease (FIG. 4E). The extracellular location of
antileukoprotease was especially detectable in endometrioid and
clear cell tumors (FIGS. 4C and 4E respectively). Serous carcinomas
were also positive for ALP immunohistochemical staining (FIGS. 4F
and 4G). More than 10% positive tumor cell staining of ALP was
detected in one of two (50%) benign adenomas, two (100%) of two LMP
tumors, and 17 of 25 (68%) adenocarcinomas while little or no
staining was observed in all three normal ovary specimens. A more
complete analysis of the immunohistochemical findings is presented
in Table 4. 9 out of 12 borderline and low malignant potential
tumors stained positively for antileukoprotease while 12 out of 19
carcinomas showed positive staining.
4TABLE 4 A complete analysis of ALP immunohistochemical findings
Case Stage Histology Grade ALP Prognosis 1 normal ovary 0- 2 normal
ovary 0- 3 normal ovary 0- 4 mucinous B 0- Alive 5 mucinous B 2+
Alive 6 1a serous LMP G1 1+ Alive 7 1a mucinous LMP G1 1+ Alive 8
1a mucinous ca G1 1+ weak Alive 9 1a mucinous ca G2 0- Alive 10 1a
endometrioid ca G1 2+ Alive 11 1c serous ca G1 1+ Alive 12 1c
mucinous ca G1 2+ Alive 13 1c mucinous ca G1 2+ Alive 14 1c clear
cell ca G2 1+ Alive 15 1c clear cell ca G2 0- Alive 16 2c serous ca
G3 1+ Alive 17 3a mucinous ca G2 2+ Alive 18 3b serous ca G1 2+
Alive 19 3c serous ca G1 0- Dead 20 3c serous ca G3 0- Alive 21 3c
serous ca G2 2+ Alive 22 3c serous ca G1 2+ unknown 23 3c serous ca
G3 0- Alive 24 3c serous ca G2 0- Dead 25 3c mucinous ca G1 2+ Dead
26 3c mucinous ca G2 2+ unknown 27 3c endometrioid ca G2 1+ Alive
28 3c endometrioid ca G1 0- Dead 29 3c endometrioid ca G2 0- Alive
30 3c endometrioid ca G2 1+ Dead 31 3c endometrioid ca G3 2+ Alive
32 3c clear cell ca G3 2+ Dead B Borderline ca carcinoma LMP Low
malignant potential
EXAMPLE 12
[0055] Analysis and Implications of ALP Expression in Ovarian
Tumors
[0056] In recent years, aberrant expression of serine proteases
such as plasminogen activator has been shown to correlate
positively with the invasiveness and metastatic potential of tumor
cells (9). More significantly, the serine protease known as
prostate-specific antigen (PSA) has been used successfully as a
tumor marker for the early diagnosis of prostate cancer (23).
Serine proteases play important roles in the cascade of events
involved in the malignant processes, and at least for prostate
cancer, provide sufficient signal to allow detection of early
disease.
[0057] Specific inhibitors for most of the proteolytic enzymes have
been identified and it has been contemplated that these inhibitors
inhibit extracellular degradation, which in turn prevents tumor
cell invasion. For example, plasminogen-activator-inhibitor 1 is
suggested to protect the tumor stroma from ongoing
urokinase-plasminogen-activator mediated proteolysis in many human
tumors (24). The proteolytic activity associated with tumors is
probably a highly regulated cascade and the interplay between
proteases and their inhibitors may play a specific role in tumor
development and progression.
[0058] In the process of studying protease enzymes in ovarian
tumors, several candidate genes have been identified to be
overexpressed. In an effort to identify other genes which are
overexpressed early in the carcinogenic progression of ovarian
cancer, a screening strategy was developed using redundant primers
to evolutionary conserved domains of extracellular proteases such
as the conserved catalytic triad domain of the serine protease
family (viz. His--Asp--Ser). In the present study,
antileukoprotease, a specific inhibitor of SCCE, was found in be
expressed in abundance in carcinoma tissues, with little or no
expression in normal ovary.
[0059] Antileukoprotease (ALP) is a mucosal secretory protein that
has been identified as a potent protease inhibitor of leukocyte
serine proteases (18). Antileukoprotease is a secreted protein with
a distribution of cysteines known to provide extraordinary
stability to inhibitors. Immunocytochemical localization studies
have revealed its presence in respiratory tissues, salivary gland,
cervical gland, and lacrimal gland (25). Moreover,
antileukoprotease can be extracted from the human stratum corneum
and is constitutively produced and released from human keratinocyte
cell cultures (26). Therefore, Wiedow et al. (27) have suggested
that antileukoprotease might not only regulate serine protease
activities in mucus secretions but in skin as well.
[0060] Recently, Franzke et al. (17) reported that
antileukoprotease is the major inhibitor of SCCE in the epidermis
and that it seems to be involved in the regulation of desquamation
under physiological and pathological conditions. In vivo, SCCE mRNA
is expressed in the upper spinous and granular layers of the
epidermis (15, 16), and active SCCE can be isolated from human
horny layers (13). The fact that inhibition of SCCE causes the
concomitant complete inhibition of cell shedding from plantar
stratum corneum (desquamation) in vitro (28) led to the hypothesis
that SCCE may be involved in the process of physiological
desquamation (13-16,28). The fact that SCCE is overexpressed in
ovarian tumors supports the potential of SCCE as a target for
inhibition of down regulation in therapeutic interventions aimed at
preventing the spread or metastasis of ovarian cancer.
[0061] Herein, Northern blot hybridization has shown that the
antileukoprotease transcript is abundant in ovarian carcinomas but
is not detected in normal ovaries. Semi-quantitative PCR analysis
supports the observation that antileukoprotease mRNA levels are
significantly higher in ovarian tumors as compared to normal
ovaries. These results were confirmed by immunohistochemistry
experiments which confirmed that antileukoprotease is present in
ovarian tumor cells and the mucin secreted therefrom, whereas
little or no staining is observed in normal ovarian surface
epithelium.
[0062] Positive correlation has been demonstrated between SCCE and
antileukoprotease mRNA overexpression in ovarian tumor samples
including LMP tumors and carcinomas. This observation is, in one
sense, paradoxical, since antileukoprotease levels would be
expected to be low if SCCE plays an important role during ovarian
cancer development and progression. However, the present results
demonstrate that co-transcriptional activation SCCE and
antileukoprotease seems to occur during transformation and initial
tumor growth of ovarian cancer. These data are entirely consistent
with similar data observed for SCCE/ALP expression in
differentiated keratinocytes. In light of the fact that
desquamation of skin cells is SCCE dependent and can be inhibited
by ALP, it is suggested that some dis-synchrony in time or space
allows SCCE activation in the presence of ALP.
[0063] The present findings show that the presence of SCCE and
antileukoprotease together in tumor cells may similarly allow the
shedding or desquamation of malignant cells through a similar
dis-synchrony. The fact that inhibition of SCCE activity prevents
normal desquamation of skin cells points to the potential of SCCE
as a target for inhibition or down regulation of the spread or
metastasis of ovarian carcinoma. Because antileukoprotease is a
specific inhibitor of SCCE, it may also be useful in the abatement
of tumor growth, and progression in
low-antileukoprotease-expressing ovarian cancers, although there is
already high-level-ALP expression observed in many ovarian
cancers.
[0064] It has been demonstrated herein that the overexpression of
antileukoprotease is a common event in ovarian tumors. Because
antileukoprotease is a secreted protein and antileukoprotease
appears in abundance only in tumor tissues as demonstrated by
Northern blot analysis and semi-quantitative analyses, it has a
potential for being present in the circulation of tumor-bearing
patients. The overexpression of antileukoprotease in LMP tumors and
stage I carcinomas is of particular note as the antileukoprotease
is produced directly by tumor cells instead of underlying stromal
tissues. As a result of this, assays may be developed for the early
detection of ovarian cancer based on the detection the
antileukoprotease protein. This molecule fulfills many criteria
necessary for a valuable marker of tumor growth and progression. It
is overexpressed in tumor cells secreted to the extracellular space
and is a relatively small protein (approximately 100 amino acids)
providing good opportunity for uptake into the circulation.
Moreover, antileukoprotease is a very stable protein providing
potential for a relatively long half life in the circulation. In
addition, even though antileukoprotease has been shown to directly
inhibit SCCE activity, it co-existence with SCCE during
desquamation of keratinocytes suggests that a similar mechanism of
antileukoprotease sequestration of lack of ability to inhibit SCCE
may allow the desquamation or shedding of ovarian tumor cells.
[0065] The following references were cited herein.
[0066] 1. Neurath, H. The diversity of proteolytic enzymes. In:
Beynon et al., (eds.), Proteolytic enzymes, Oxford, IRL Press
1989:1-13.
[0067] 2. Liotta, et al., Cell 1991; 64:327-336.
[0068] 3. Duffy, M. J. Clin Exp Metastasis 1992; 10:145-155.
[0069] 4. Tryggvason, et al., Biochem Biophys Acta 1987;
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[0070] 5. Powell, W. C. et al., Cancer Res 1993, 53:417-422.
[0071] 6. MacDougall, J. R. et al., Cancer and Metastasis Reviews
1995; 14:351-362.
[0072] 7. Rempel, S. A. et al., Cancer Res 1994; 54:6027-6031.
[0073] 8. Nazeer, T. et al., Am J Clin Pathol 1992; 97:764-769.
[0074] 9. Duffy, M. J. et al., Cancer (Phila) 1988; 62:531-533.
[0075] 10. Liotta, L. A. et al., Cell 1991; 64:327-336.
[0076] 11. Wang, M. et al., Cancer Res 1988; 48:6262-6271.
[0077] 12. Reich, R. et al., Cancer Res 1988; 48:3307-3312.
[0078] 13. Egelrud, T. J Invest Dermatol 1993; 101:200-204.
[0079] 14. Hansson, et al., J Biol Chem 1994;269:19420-19426.
[0080] 15. Sondell, et al., J Histochem Cytochem
1994;42:459-465.
[0081] 16. Sondell, et al., J Invest Dermatol 1995;
104:819-823.
[0082] 17. Franzke, et al., J Biol Chem 1996;271:21886-21890.
[0083] 18. Thompson, R. C., et al., Proc Natl Acad Sci USA 1986;
83:6692-6696, 1986.
[0084] 19. Shigemasa, et al., J Soc Gynecol Invest
1997;4:95-102.
[0085] 20. Tanimoto, et al., Gynecol Oncol 1997;66:308-312.
[0086] 21. Shigemasa, K. et al., Int J. Gynecol Cancer 1997,
7(4):296-303.
[0087] 22. Hall, et al., Mol Cell Biol 1983; 3:854-862.
[0088] 23. McCormack, R. T. et al., Urology 1995; 45:729-744.
[0089] 24. Pappot et al., Biol Chem Hoppe Seyler 1995;
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[0090] 25. Franken C. et al., J Histochem Cytochem 1989;
37:493-498.
[0091] 26. Wiedow, O. (1995) Isolierung und Charakterisierung von
Serinprotease Inhibitoren der menschlichen Epidermis, Koster,
Berlin
[0092] 27. Wiedow, O. et al., J Invest Dermatol 1993;
101:305-309.
[0093] 28. Lundstrom, A. et al., J Invest Dermatol 1988;
91:340-343.
[0094] 29. Garver, R. I. et al., Gene Ther 1994; 1:46-50.
[0095] 30. Lunderstrom, et al.,. (1988) J. Invest. Dermatol. 101,
200-204.
[0096] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically incorporated by reference.
[0097] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods, procedures,
treatments, molecules, and specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the claims.
Sequence CWU 1
1
6 1 17 DNA Artificial sequence Forward oligonucleotide primer for
amplification of antileukoprotease 1 tgcattgaca acgaggc 17 2 17 DNA
Artificial sequence Reverse oligonucleotide primer for PCR
amplification of antileukoprotease 2 ctgtcttgac attgttg 17 3 20 DNA
Artificial sequence Forward oligonucleotide primer for PCR
amplification of antileukoprotease 3 ccttcaaagc tggagtctgt 20 4 20
DNA Artificial sequence Reverse oligonucleotide primer for PCR
amplification of antileukoprotease 4 ccaaaggagg atatcagtgg 20 5 20
DNA Artificial sequence Forward oligonucleotide primer for PCR
amplification of (-tubulin 5 cgcatcaacg tgtactacaa 20 6 20 DNA
Artificial sequence Forward oligonucleotide primer for PCR
amplification of (-tubulin 6 tacgagctgg tggactgaga 20
* * * * *