U.S. patent application number 10/390045 was filed with the patent office on 2003-09-11 for method of detecting androgen-regulated gene.
This patent application is currently assigned to Henry M. Jackson Foundation for the Advancement of Military Medicine. Invention is credited to Moul, Judd W., Segawa, Takehiko, Srivastava, Shiv, Xu, Linda L..
Application Number | 20030170713 10/390045 |
Document ID | / |
Family ID | 27391020 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170713 |
Kind Code |
A1 |
Srivastava, Shiv ; et
al. |
September 11, 2003 |
Method of detecting androgen-regulated gene
Abstract
This invention relates to androgen-regulated nucleic acids, a
polynucleotide array containing these androgen-regulated nucleic
acids, and methods of using the polynucleotide array in the
diagnosis and prognosis of prostate cancer.
Inventors: |
Srivastava, Shiv; (Potomac,
MD) ; Moul, Judd W.; (Bethesda, MD) ; Xu,
Linda L.; (Rockville, MD) ; Segawa, Takehiko;
(Rockville, MD) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Henry M. Jackson Foundation for the
Advancement of Military Medicine
|
Family ID: |
27391020 |
Appl. No.: |
10/390045 |
Filed: |
March 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10390045 |
Mar 18, 2003 |
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09769482 |
Jan 26, 2001 |
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6566130 |
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60178772 |
Jan 28, 2000 |
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60179045 |
Jan 31, 2000 |
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Current U.S.
Class: |
435/6.14 ;
435/287.2 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 14/4748 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Goverment Interests
[0002] The invention described herein may be manufactured,
licensed, and used for governmental purposes without payment of
royalties to us thereon.
Claims
We claim:
1. A polynucleotide array comprising: (a) a planar, non-porous
solid support having at least a first surface; and (b) a first set
of polynucleotide probes attached to the first surface of the solid
support, wherein the first set of polynuceotide probes comprises
polynucleotide sequences derived from genes that are up-regulated
or down-regulated in response to androgen.
2. The polynucleotide array of claim 1, wherein the polynucleotide
sequences are derived from the genes of LNCaP cells that are
up-regulated or down-regulated in response to androgen.
3. The polynucleotide array of claim 2, wherein the androgen is
R1881.
4. The polynucleotide array of claim 1, wherein the first set of
polynuceotide probes comprises polynucleotide sequences derived
from the polynucleotide sequences listed in Table 3.
5. The polynucleotide array according to claim 1, further
comprising a second set of polynucleotide probes, wherein the
second set of polynucleotide probes comprises polynucleotide
sequences derived from genes that are involved in testosterone
biosynthesis and metabolism.
6. The polynucleotide array according to claim 4, further
comprising a second set of polynucleotide probes, wherein the
second set of polynucleotide probes comprises polynucleotide
sequences derived from genes whose expression is altered in
prostate cancer or is specific to prostate tissue.
7. The polynucleotide array according to claim 6, wherein the
second set of polynucleotide probes comprises polynucleotide
sequences derived from the polynucleotide sequences listed in Table
6.
8. The polynucleotide array according to claim 1, further
comprising a second set of polynucleotide probes, wherein the
second set of polynucleotide probes comprises polynucleotide
sequences derived from the polynucleotide sequences listed in Table
8.
9. The polynucleotide array according to claim 7, further
comprising a third, fourth, fifth, and sixth set of polynucleotide
probes, wherein the third set of polynucleotide probes comprises
polynucleotide sequences derived from the polynucleotide sequences
listed in Table 4, the fourth set of polynucleotide probes
comprises polynucleotide sequences derived from the polynucleotide
sequences listed in Table 5, the fifth set of polynucleotide probes
comprises polynucleotide sequences derived from the polynucleotide
sequences listed in Table 7, and the sixth set of polynucleotide
probes comprises polynucleotide sequences derived from the
polynucleotide sequences listed in Table 8.
10. The polynucleotide array of claim 1, wherein at least one of
the genes that is up-regulated in response to androgen is the
polynucleotide sequence of SEQ ID NO:2.
11. The polynucleotide array of claim 1, wherein at least one of
the polynucleotide sequences comprises the polynuleotide sequence
of SEQ ID NO:2
12. A method of identifying an expression profile of
androgen-regulated genes in a target cell, comprising hybridizing
the nucleic acids of the target cell with the polynucleotide array
of claim 1 to obtain a hybridization pattern, wherein the
hybridization pattern represents the expression profile of
androgen-regulated genes in the target cell.
13. A method of identifying an expression profile of
androgen-regulated genes in a target cell, comprising hybridizing
the nucleic acids of the target cell with the polynucleotide array
of claim 10 to obtain a hybridization pattern, wherein the
hybridization pattern represents the expression profile of
androgen-regulated genes in the target cell.
14. The method acording to claim 12, wherein the target cell is a
prostate cell.
15. A method of diagnosing or prognosing prostate cancer,
comprising (a) hybridizing nucleic acids of a target cell of a
patient with the polynucleotide array of claim 1 to obtain a first
hybridization pattern, wherein the first hybridization pattern
represents an expression profile of androgen-regulated genes in the
target cell; (b) comparing the first hybridization pattern of the
target cell to a second hybridization pattern, wherein the second
hybridization pattern represents an expression profile of
androgen-regulated genes in prostate cancer, and (c) diagnosing or
prognosing prostate cancer in the patient.
16. A method of diagnosing or prognosing prostate cancer,
comprising (a) hybridizing nucleic acids of a target cell of a
patient with the polynucleotide array of claim 10 to obtain a first
hybridization pattern, wherein the first hybridization pattern
represents an expression profile of androgen-regulated genes in the
target cell; (b) comparing the first hybridization pattern of the
target cell to a second hybridization pattern, wherein the second
hybridization pattern represents an expression profile of
androgen-regulated genes in prostate cancer, and (c) diagnosing or
prognosing prostate caner in the patient.
17. The method acording to claim 15, wherein the target cell is a
prostate cell.
18. An isolated nucleic acid molecule selected from: (a) the
polynucleotide sequence of SEQ ID NO:2; and (b) an isolated nucleic
acid molecule that encodes a polypeptide having an amino acid
sequence of SEQ ID NO:3.
19. A recombinant vector that directs the expression of the nucleic
acid molecule of claim 18.
20. A host cell transfected or transduced with the vector of claim
19.
21. The host cell of claim 20 selected from bacterial cells, yeast
cells, and animal cells.
22. A method of detecting prostate cancer in a patient, comprising:
(a) detecting mRNA corresponding to the polynucleotide sequence of
SEQ ID NO:2 in a biological sample from the patient; and (b)
correlating the amount of mRNA corresponding to the polynucleotide
sequence of SEQ ID NO:2 in the sample with the presence of prostate
cancer in the patient.
23. An isolated polypeptide, comprising the amino acid sequence of
SEQ ID NO:3.
24. An isolated antibody that binds to the polypeptide of claim 23.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is based upon U.S. provisional
applications S. Nos. 60/178,772, and 60/179,045, filed Jan. 28,
2000, and Jan. 31, 2000, respectively, priority to which is claimed
under 35 U.S.C. .sctn.119(e). The entire disclosures of U.S.
provisional applications S. Nos. 60/178,772, and 60/179,045, are
expressly incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to the quantitative evaluation
of gene expression. More particularly, the present invention
relates to novel, androgen-regulated nucleic acids, polynucleotide
arrays containing these nucleic acids, and methods of using the
array in the evaluation of hormone-related cancers, such as
prostate cancer.
BACKGROUND
[0004] Prostate cancer (CaP) is the most common malignancy in
American men and second leading cause of cancer mortality (1).
Serum-prostate specific antigen (PSA) tests have revolutionized the
early detection of CaP (2). Although PSA has revolutionized early
detection of prostate cancer, there is still a very high false
positive rate. The increasing incidence of CaP has translated into
wider use of radical prostatectomy as well as other therapies for
localized disease (3-5). The wide spectrum of biologic behavior (6)
exhibited by prostatic neoplasms poses a difficult problem in
predicting the clinical course for the individual patient (3-5).
Traditional prognostic markers such as grade, clinical stage, and
pretreatment PSA have limited prognostic value for individual men
(3-5). A more reliable technique for the evaluation and prognosis
of CaP is desirable.
[0005] Molecular studies have shown a significant heterogeneity
between multiple cancer foci present in a cancerous prostate gland
(7,8). These studies have also documented that the metastatic
lesion can arise from cancer foci other than those present in
dominant tumors (7). Approximately 50-60% of patients treated with
radical prostatectomy for localized prostate carcinomas are found
to have microscopic disease that is not organ-confined, and a
significant portion of these patients relapse (9). Therefore,
identification and characterization of genetic Iterations defining
CaP onset and progression is crucial in understanding the biology
and clinical course of the disease.
[0006] Despite recent intensive research investigations, much
remains to be learned about specific molecular defects associated
with CaP onset and progression (6, 10-15). Alterations of the tumor
suppressor gene p53, bcl-2 and the androgen receptor (AR), are
frequently reported in advanced CaP (6, 10-15). However, the exact
role of these genetic defects in the genesis and progression of CaP
is poorly understood (6, 10-15). Recent studies have shown that the
"focal p53 immunostaining" or bcl-2 immunostaining in radical
prostatectomy specimens were independent prognostic markers for
cancer recurrence after surgery (16-19). Furthermore, the
combination of p53 and bcl-2 alterations was a stronger predictor
of cancer recurrence after radical prostatectomy (18).
[0007] The roles of several new chromosome loci harboring putative
proto-oncogenes or tumor suppressor genes are being currently
evaluated in CaP (7-13). High frequency of allelic losses on
8p21.sup.-22, 7q31.1, 10q23-25 and 16q24 loci have been shown in
CaP (6, 10-15). PTEN1/MMAC1, a recently discovered tumor suppressor
gene on chromosome 10q25, is frequently altered in advanced CaP
(20, 21). Gains of chromosome 8q24 harboring c-myc and prostate
stem-cell antigen (PSCA) genes have also been shown in prostate
cancer (22, 23). Studies utilizing comparative genomic
hybridization (CGH) have shown frequent losses of novel chromosomal
loci including 2q, 5q and 6q and gains of 11p, 12q, 3q, 4q and 2p
in CaP (24, 25). The inventors have recently mapped a 1.5 megabase
interval at 6q 16-21 which may contain the putative tumor
suppressor gene involved in a subset of prostate tumors. The risk
for 6q LOH to non-organ confined disease was five fold higher than
for organ confined disease (26). Chromosome regions, 1q24-25 and
Xq27-28 have been linked to familial CaP (27, 28).
[0008] It is evident that multiple molecular approaches need to be
explored to identify CaP-associated genetic alterations. Emerging
strategies for defining cancer specific genetic alterations and
characterizing androgen regulated genes in rat prostate and LNCaP
human prostate cancer cell models include, among others, the study
of global gene expression profiles in cancer cells and
corresponding normal cells by differential display (DD) (29) and
more recent techniques, such as serial amplification of gene
expression (SAGE) (30) and DNA micro-arrays (31; U.S. Pat. Nos.
5,744,305 and 5,837,832 which are herein incorporated by reference)
followed by targeted analyses of promising candidates. Our
laboratory has also employed DD, SAGE and DNA microarrays to study
CaP associated gene expression alterations (32-33). Each of these
techniques, however, is limited. The number of transcripts that can
be analyzed is the major limitation encountered in subtractive
hybridization and differential display approaches. Furthermore,
while cDNA microarray approaches can determine expression of a
large number of genes in a high throughput manner, the current
limitations of cDNA arrays include the presence of specific arrays
used for analyses and the inability to discover novel genes.
[0009] While alterations of critical tumor-suppressor genes and
oncogenes are important in prostate tumorogenesis, it is also
recognized that hormonal mechanisms play equally important roles in
prostate tumorogenesis. The cornerstone of therapy in patients with
metastatic disease is androgen ablation, commonly referred to as
"hormonal therapy (34)," which is dependent on the inhibition of
androgen signaling in prostate cancer cells. Androgen ablation can
be achieved, for example, by orchiectomy, by the administration of
estrogen, or more recently by one of the luteinizing
hormone-releasing hormone agonists. Recent clinical trials have
demonstrated the efficacy of combining an antiandrogen to
orchiectomy or a luteinizing hormone-releasing hormone to block the
remaining androgens produced by the adrenal glands. Although
approximately 80% of patients initially respond to hormonal
ablation, the vast majority of patients eventually relapse (35),
presumably due to neoplastic clones of cells which become
refractory to this therapy.
[0010] Alterations of the androgen receptor gene by mutations in
the hormone binding domain of the AR or by amplification of the AR
gene have been reported in advanced stages of CaP. Much remains to
be learned, however, about the molecular mechanisms of the
AR-mediated cell signaling in prostate growth and tumorogenesis
(36-43). Our earlier studies have also described mutations of the
AR in a subset of CaP (40). Mutations of the AR are reported to
modify the ligand (androgen) binding of the AR by making the
receptor promiscuous, so that it may bind to estrogen,
progesterone, and related molecules, in addition to the androgens
(36.38,42). Altered ligand binding specificity of the mutant AR may
provide one of the mechanisms for increased function in cancer
cells. Amplifications of the AR gene in hormone-refractory CaP
represent yet another scenario where increase in AR function is
associated with tumor progression (44,45).
[0011] Several growth factors commonly involved in cell
proliferation and tumorogenesis, e.g., IGF 1, EGF, and others, have
been shown to activate the transcription transactivation functions
of the AR (46). The co-activator of the AR transcription factor
functions may also play a role in prostate cancer (47). Recent
studies analyzing expression of the androgen-regulated genes (ARGs)
in hormone sensitive and refractory CWR22 nude mice xenograft
models (48) have also shown expression of several androgen
regulated genes in AR positive recurrent tumors following
castration, suggesting activation of AR in these tumors (49).
[0012] In addition to the alterations of the androgen signaling
pathway(s) in prostate tumor progression, androgen mechanisms are
suspected to play a role in the predisposition to CaP. Prolonged
administration of high levels of testosterone has been shown to
induce CaP in rats (50-52). Although recent evidence suggests an
association of androgen levels and risk of CaP, this specific
observation remains to be established. (53). An independent line of
investigations addressing the length of inherited polyglutamine
(CAG) repeat sequence in the AR gene and CaP risk have shown that
men with shorter repeats were at high risk of distant metastasis
and fatal CaP (54,55). Moreover, the size distribution of AR CAG
repeats in various ethnic groups has also suggested a possible
relationship of shorter CAG repeats and increased prostate cancer
risks in African-American men (56,57). Biochemical experiments
evaluating AR-CAG repeat length and in vitro transcription
transactivation functions of the AR revealed that AR with shorter
CAG repeats possessed a more potent transcription trans-activation
activity (58). Thus, molecular epidemiologic studies and
biochemical experimentation suggest that gain of AR function,
consequently resulting in transcriptional transactivation of
downstream targets of the AR gene, may play an important role in
CaP initiation. However, downstream targets of AR must be defined
in order to understand the biologic basis of these
observations.
[0013] The biologic effects of androgen on target cells, e.g.,
prostatic epithelial cell proliferation and differentiation as well
as the androgen ablation-induced cell death, are likely mediated by
transcriptional regulation of ARGs by the androgen receptor
(reviewed in 59). Abrogation of androgen signaling resulting from
structural changes in the androgen gene or functional alterations
of AR due to modulation of AR functions by other proteins would
have profound effects on transcriptional regulation of genes
regulated by AR and, thus, on the growth and development of the
prostate gland, including abnormal growth characterized by benign
prostatic hyperplasia and prostatic cancer. The nature of ARGs in
the context of CaP initiation and progression, however, remains
largely unknown. Since forced proliferation of the AR prostate
cancer cells lacking AR induces cell-death related phenotypes (60),
the studies utilizing AR expression via heterologous promoters in
cell cultures have failed to address the observations relating to
gain of AR functions and prostate cancer progression. Moreover,
suitable animal models to assess gain of AR functions do not exist.
Therefore, the expression profile of androgen responsive genes
(ARGs) has potential to serve as read-out of the AR signaling
status. Such a read-out may also define potential biomarkers for
onset and progression of those prostate cancers which may involve
abrogation of the androgen signaling pathway. Furthermore,
functional analysis of androgen regulated genes will help
understand the biochemical components of the androgen signaling
pathways.
SUMMARY OF THE INVENTION
[0014] The present invention relates to the identification and
characterization of a novel androgen-regulated gene that exhibits
abundant expression in prostate tissue. The novel gene has been
designated PMEPA1. The invention provides the isolated nucleotide
sequence of PMEPA1 or fragments thereof and nucleic acid sequences
that hybridize to PMEPA1. These sequences have utility, for
example, as markers of prostate cancer and other prostate-related
diseases, and as targets for therapeutic intervention in prostate
cancer and other prostate-related diseases. The invention further
provides a vector that directs the expression of PMEPA1, and a host
cell transfected or transduced with this vector.
[0015] In another embodiment, the invention provides a method of
detecting prostate cancer cells in a biological sample, for
example, by using nucleic acid amplification techniques with
primers and probes selected to bind specifically to the PMEPA1
sequence.
[0016] In another aspect, the invention relates to an isolated
polypeptide encoded by the PMEPA1 gene or a fragment thereof, and
antibodies generated against the PMEPA1 polypeptide, peptides, or
portions thereof, which can be used to detect, treat, and prevent
prostate cancer.
[0017] The present invention also relates to a polynucleotide array
comprising (a) a planar, non-porous solid support having at least a
first surface; and (b) a first set of polynucleotide probes
attached to the first surface of the solid support, where the first
set of polynuceotide probes comprises polynucleotide sequences
derived from genes that are up-regulated, such as PMEPA1, or
down-regulated in response to androgen, including genes downstream
of the androgen receptor gene and genes upstream of the androgen
receptor gene that modulate androgen receptor function. In another
embodiment of the invention the polynucleotides immobilized on the
solid support include genes that are known to be involved in
testosterone biosynthesis and metabolism. In another embodiment of
the invention the oligonucleotides immobilized on the solid support
include genes whose expression is altered in prostate cancer or is
specific to prostate tissue.
[0018] In another embodiment, the invention provides a method for
the diagnosis or prognosis of prostate cancer, comprising (a)
hybridizing nucleic acids of a target cell of a patient with a
polynucleotide array, as described above, to obtain a first
hybridization pattern, where the first hybridization pattern
represents an expression profile of androgen-regulated genes in the
target cell; (b) comparing the first hybridization pattern of the
target cell to a second hybridization pattern, where the second
hybridization pattern represents an expression profile of
androgen-regulated genes in prostate cancer, and (c) diagnosing or
prognosing prostate cancer in the patient.
[0019] Thus, a first aspect of the present invention is directed
towards a method for analysis of radical prostatectomy specimens
for the expression profile of those genes involved in androgen
receptor-mediated signaling. In a preferred embodiment, computer
models may be developed for the analysis of expression profiles.
Another aspect of the invention is directed towards a method of
correlating expression profiles with clinico-pathologic features.
In a preferred embodiment, computer models to identify gene
expression features associated with tumor phenotypes may be
developed. Another aspect of the invention is directed towards a
method of distinguishing indolent prostate cancers from those with
a more aggressive phenotype. In a preferred embodiment, computer
models to such cancers may be developed. Another aspect of the
invention is directed towards a method of analyzing tumor specimens
of patients treated by radical prostate surgery to help define
prognosis. Another aspect of the invention is directed towards a
method of screening candidate genes for the development of a blood
test for improved prostate cancer detection. Another aspect of the
invention is directed towards a method of identifying androgen
regulated genes that may serve as biomarkers for response to
treatment to screen drugs for the treatment of advanced prostate
cancer.
[0020] This invention is further directed to a method of
identifying an expression profile of androgen-regulated genes in a
target cell, comprising hybridizing the nucleic acids of the target
cell with a polynucleotide array, as described above, to obtain a
hybridization pattern, where the hybridization pattern represents
the expression profile of androgen-regulated genes in the target
cell.
[0021] Additional features and advantages of the invention will be
set forth in the description which follows, and in part will be
apparent from the description, or may be learned by practice of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a Northern blot showing that PMEPA1 is expressed
at high levels in prostate tissue. Multiple tissue northern blots
were hybridized with PMEPA1 and GAPDH probes. The arrows indicate
the two variants of the PMEPA1 transcript.
[0023] FIG. 2 shows the androgen-dependent expression of PMEPA1.
FIG. 2A is a Northern blot using PMEPA1 probe with mRNA derived
from LNCaP cells with or without R1881 treatment for various
durations. FIG. 2B is a Northern blot of PMEPA1 expression in
primary epithelial cell cultures of normal prostate and prostate
and breast cancer cell lines.
[0024] FIG. 3 shows PMEPA1 expression in CwR22 xenograft tumors.
Lane 1, sample from CWR22 tumor (androgen dependent). Lanes 2-5,
samples from 4 individual CW2R tumors (AR positive but androgen
independent).
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides a method useful in the
diagnosis and prognosis of prostate cancer. An aspect of the
invention provides a method to identify ARGs, such as PMEPA1, that
exhibit stable transcriptional induction/repression in response to
androgen and have potential as surrogate markers of the status of
the androgen signaling in normal and cancerous epithelial cells of
prostate.
[0026] A second aspect of the invention provides for use of the
expression profiles resulting from these methods in diagnostic
methods, including, but not limited to, characterizing the
treatment response to "hormonal therapy," correlating expression
profiles with clinico-pathologic features, distinguishing indolent
prostate cancers from those with a more aggressive phenotype,
analyzing tumor specimens of patients treated by radical prostate
surgery to help define prognosis, screening candidate genes for the
development of a polynucleotide array for use as a blood test for
improved prostate cancer detection, and identifying androgen
regulated genes that may serve as biomarkers for response to
treatment to screen drugs for the treatment of advanced prostate
cancer.
[0027] As will be readily appreciated by persons having skill in
the art, these gene sequences and ESTs described herein can easily
be synthesized directly on a support, or pre-synthesized
polynucleotide probes may be affixed to a support as described, for
example, in U.S. Pat. Nos. 5,744,305, 5,837,832, and 5,861,242,
each of which is incorporated herein by reference. Furthermore,
such arrays may be made in a wide number of variations, combining,
probes derived from sequences identified by the inventors as
up-regulated or down-regulated in response to androgen and listed
in Table 3 (genes and ESTs derived from the inventors' SAGE library
that are up-regulated and down-regulated by androgens) with any of
the sequences described in Table 4 (candidate genes and ESTs whose
expression are potentially prostate specific or restricted), Table
5 (previously described genes and ESTs, including those associated
with androgen signaling, prostate specificity, prostate cancer, and
nuclear receptors/regulators with potential interaction with
androgen receptors), Table 6 (genes and ESTs identified from the
NIH CGAP database that are differentially expressed in prostate
cancer), Table 7 (androgen regulated genes and ESTs derived from
the CPDR Genome Systems ARG Database) and Table 8 (other genes
associated with cancers). Tables 3-8 are located at the end of the
specification at the end of the "Detailed Description" section and
before the "References." In Table 3, genes in bold type are known
androgen-regulated genes based on Medline Search. In Table 4, genes
in bold type are known prostate-specific genes.
[0028] Such arrays may be used to detect specific nucleic acid
sequences contained in a target cell or sample, as described in
U.S. Pat. Nos. 5,744,305, 5,837,832, and 5,861,242, each of which
is incorporated herein by reference. More specifically, in the
present invention, these arrays may be used in methods for the
diagnosis or prognosis of prostate cancer, such as by assessing the
expression profiles of genes, derived from biological samples such
as blood or tissues, that are up-regulated and down-regulated in
response to androgen or otherwise involved in androgen
receptor-mediated signaling. In a preferred embodiment, computer
models may be developed for the analysis of expression profiles.
Moreover, such polynucleotide arrays are useful in methods to
screen drugs for the treatment of advanced prostate cancer. In
these screening methods, the polynucleotide arrays are used to
analyze how drugs affect the expression of androgen-regulated genes
that are involved in prostate cancer.
[0029] SAGE analysis. The SAGE technology is based on three main
principles: 1) A short sequence tag (10-11 bp) is generated that
contains sufficient information to identify a transcript, thus,
each tag represents a signature sequence of a unique transcript; 2)
many transcript tags can be concatenated into a single molecule and
then sequenced, revealing the identity of multiple tags
simultaneously; 3) quantitation of the number of times a particular
tag is observed provides the expression level of the corresponding
transcript (30). The schematic diagram and the details of SAGE
procedure can be obtained from the web site:
www.genzyme.com/SAGE.
[0030] About fifty percent of SAGE tags identified by the inventors
represent ESTs which need to be further analyzed for their protein
coding capacity. The known genes up-regulated or down-regulated by
four-fold (p<0.05) were broadly classified on the basis of the
biochemical functions. SAGE tag defined ARGs were grouped under
following categories: transcriptional regulators; RNA processing
and translation regulators; protein involved in genomic maintenance
and cell cycle; protein trafficking/chaperone proteins; energy
metabolism, apoptosis and redox regulators; and signal transducers.
As determined by PubMed database searches, a majority of genes
listed in FIG. 3 have not been described as androgen regulated
before. This is the first comprehensive list of the functionally
defined genes regulated by androgen in the context of prostatic
epithelial cells.
[0031] Although promising candidate ARGs have been identified using
these approaches, much remains to be learned about the complete
repertoire of these genes. SAGE provides both quantitative and high
throughput information with respect to global gene expression
profiles of known as well as novel transcripts. We have performed
SAGE analysis of the ARGs in the widely studied hormone responsive
LNCaP prostate cancer cells treated with and without synthetic
androgen, R1881. Of course, this SAGE technique could be repeated
with hormones other than R1881, including other synthetic or
natural androgens, such as dihydroxytestosterone, to potentially
obtain a slightly different ARG expression panel. A goal of the
inventors was to identify highly induced and repressed ARGs in
LNCaP model which may define a panel of surrogate markers for the
status androgen signaling in normal as well as cancerous prostate.
Here, we report identification and analyses of a comprehensive
database of SAGE tags corresponding to well-characterized genes,
expressed sequence tags (ESTs) without any protein coding
information and SAGE tags corresponding to novel transcripts. This
is the first report describing a quantitative evaluation of the
global gene expression profiles of the ARGs in the context of
prostatic cancer cells by SAGE. We have further defined the ARGs on
the basis of their known biologic/biochemical functions. Our study
provides quantitative information on about 23,000 transcripts
expressed in LNCaP cells, the most common cell line used in
prostate cancer research. Finally, comparison of the LNCaP SAGE tag
library and 35 SAGE tag libraries representing diverse cell
type/tissues have unraveled a panel of genes whose expression are
prostate specific or prostate abundant. Utilizing the LNCaP
prostate cancer cells, the only well-characterized androgen
responsive prostatic epithelial cells (normal or cancerous), we
have identified a repertoire of androgen regulated genes by
SAGE.
[0032] Utilizing cell-culture systems and cell-signaling agents or
exogenous expression of p53 and APC genes, SAGE technology has
identified novel physiologically relevant transcriptional target
genes which have unraveled new functions of p53 and APC genes
(61-64). Our analysis of ARGs has provided identification and
quantitative assessment of induction or repression of a global
expression profile of ARGs in LNCaP cells. ARGs resulting from the
mutational defects of the AR and those ARGs unaffected by AR
mutations may be identified in this model system. Subsequent
androgen regulation analysis of the selected ARGs in AR-positive,
primary cultures of normal prostatic epithelial cells, and ARGs
expression analysis in normal and tumor tissues will clarify normal
or abnormal regulation of these ARGs. A panel of highly
inducible/repressible ARGs identified by the inventors may provide
bio-indicators of the AR transcription factor activity in
physiologic context. These AR Function Bio-indicators (ARFBs) are
useful in assessing the risk of CaP onset and/or progression.
Moreover, identification or ARGs may also help in defining the
therapeutic targets which could lead to effective treatment for
hormone refractory cancer, currently a frustrating stage of the
disease with limited therapeutic options.
[0033] Characterization of a SAGE-defined EST that exhibited the
highest level of induction in LNCaP cells responding to R1881 led
to the discovery of a novel, androgen-induced gene, PMEPA1, which
encodes a polypeptide with a type Ib transmembrane domain. A
Protein sequence similarity search showed homology to C18orf1, a
novel gene located on chromosome 18 that is mainly expressed in
brain with multiple transcriptional variants (Yoshikawa et al.,
1998). In addition to the sequence similarity, PMEPA1 also shares
other features with C18orf1, e.g., similar size of the predicted
protein and similar transmembrane domain as the .beta.1 isoform of
C18orf1. Therefore, it is likely that other isoforms of PMEPA1 may
exist.
[0034] Database searches showed that the PMEPA1 sequence matched to
genomic clones RP5-1059L7 and 718J7 which were mapped to chromosome
20q13.2-13.33. Gain of 20q has been observed in many cancer types,
including prostate, bladder, melanoma, colon, pancreas and breast
(Brothman et al., 1990; Richter et al., 1998; Bastian et al., 1998;
Kom et al., 1999; Mahlamaki et al., 1997; Tanner et al., 1996).
Chromosome 20q gain was also observed during immortalization and
may harbor genes involved in bypassing senescence (Jarrard et al.,
1999; Cuthill et al., 1999). A differentially expressed gene in
hormone refractory CaP, UEV-1, mapped to 20q13.2 (Stubbs et al.,
1999). These observations indicate that one or several genes on
chromosome 20q may be involved in prostate or other cancer
progression. Although we did not observe increased expression of
PMEPA1 in primary prostate tumors, increased PMEPA1 expression was
noted in recurrent cancers of CWR22 xenograft.
[0035] PMEPA1 expression is upregulated by androgens in a time- and
concentration-specific manner in LNCaP cells. This observation
underscores the potential of measuring PMEPA1 expression as one of
the surrogate markers of androgen receptor activity in vivo in the
epithelial cells of prostate tissue. Prostate cancer is androgen
dependent and its growth in prostate is mediated by a network of
ARGs that remains to be fully characterized. Most prostate cancers
respond to androgen withdrawal but relapse after the initial
response (Koivisto et al., 1998). The growth of the relapsed tumors
is androgen independent even though tumors are positive for the
expression of the AR (Bentel et al., 1996).
[0036] One of the hypotheses of how cancer cells survive and grow
in the low androgen environrment is the sensitization or the
activation of the AR pathway (Jenster et al., 1999). Studies have
shown increased expression of the ARGs or amplification of AR in
androgen independent prostate cancer tissues (Gregory et al., 1998;
Lin et al., 1999). We have observed that PMEPA1 was expressed in
all CWR22R tumors and increased expression in three of four
compared with CWR22 tumor. Our data support the concept that
normally AR-dependent pathways remain activated, despite the
absence of androgen in androgen-independent prostate cancer. There
are only limited studies that have addressed whether ARGs play a
role in the transition from androgen dependent tumor to androgen
independent tumors. The high level of expression only in the
prostate gland indicates that PMEPA1 might have important roles
related to prostate cell biology or physiology. On the basis of
homology of PMEPA1 to C18orf1 it is tempting to suggest that the
PMEPA1 may belong to family of proteins involved in the binding of
calcium and LDL.
[0037] Characterization of genes like PMEPA1 is a step forward in
the definition of the network of androgen regulated genes in
prostate biology and tumorigenesis. In addition, ARGs, including
PMEPA1, can be used as biomarkers of AR function readout in the
subset of prostate cancers that may involve abrogation of androgen
signaling. Furthermore, the newly defined ARGs have potential to
identify novel targets in therapy of hormone refractory prostate
cancer.
[0038] The nucleic acid molecules encompassed in the invention
include the following PMEPA1 nucleotide sequence:
1 ATGGCGGAGC TGGAGTTTGT TCAGATCATC ATCATCGTGG TGGTGATGAT 50 (SEQ ID
NO. 2) GGTGATGGTG GTGGTGATCA CGTGCCTGCT GAGCCACTAC AAGCTGTCTG 100
CACGGTCCTT CATCAGCCGG CACAGCCAGG GGCGGAGGAG AGAAGATGCC 150
CTGTCCTCAG AAGGATGCCT GTGGCCCTCG GAGAGCACAG TGTCAGGCAA 200
CGGAATCCCA GAGCCGCAGG TCTACGCCCC GCCTCGGCCC ACCGACCGCC 250
TGGCCGTGCC GCCCTTCGCC CAGCGGGAGC GCTTCCACCG CTTCCAGCCC 300
ACCTATCCGT ACCTGCAGCA CGAGATCGAC CTGCCACCCA CCATCTCGCT 350
GTCAGACGGG GAGGAGCCCC CACCCTACCA GGGCCCCTGC ACCCTCCAGC 400
TTCGGGACCC CGAGCAGCAG CTGGAACTGA ACCGGGAGTC GGTGCGCGCA 450
CCCCCAAACA GAACCATCTT CGACAGTGAC CTGATGGATA GTGCCAGGCT 500
GGGCGGCCCC TGCCCCCCCA GCAGTAACTC GGGCATCAGC GCCACGTGCT 550
ACGGCAGCGG CGGGCGCATG GAGGGGCCGC CGCCCACCTA CAGCGAGGTC 600
ATCGGCCACT ACCCGGGGTC CTCCTTCCAG CACCAGCAGA GCAGTGGGCC 650
GCCCTCCTTG CTGGAGGGGA CCCGGCTCCA CCACACACAC ATCGCGCCCC 700
TAGAGAGCGC AGCCATCTGG AGCAAAGAGA AGGATAAACA GAAAGGACAC 750
CCTCTCTAG 759
[0039] The amino acid sequences of the polypeptides encoded by the
PMEPA1 nucleotide sequences of the invention include:
2 MAELEFVQII IIVVVMMVMV VVITCLLSHY KLSARSFISR HSQGRRREDA 50 (SEQ ID
NO. 3) LSSEGCLWPS ESTVSGNGIP EPQVYAPPRP TDRLAVPPFA QRERFHRFQP 100
TYPYLQHEID LPPTISLSDG EEPPPYQGPC TLQLRDPEQQ LELNRESVRA 150
PPNRTIFDSD LMDSARLGGP CPPSSNSGIS ATCYGSGGRM EGPPPTYSEV 200
IGHYPGSSFQ HQQSSGPPSL LEGTRLHHTH IAPLESAAIW SKEKDKQKGH 250 PL*
252
[0040] The discovery of the nucleic acids of the invention enables
the construction of expression vectors comprising nucleic acid
sequences encoding polypeptides; host cells transfected or
transformed with the expression vectors; isolated and purified
biologically active polypeptides and fragments thereof; the use of
the nucleic acids or oligonucleotides thereof as probes to identify
nucleic acid encoding proteins having PMEPA1-like activity; the use
of single-stranded sense or antisense oligonucleotides from the
nucleic acids to inhibit expression of polynucleotides encoded by
the PMEPA1 gene; the use of such polypeptides and fragments thereof
to generate antibodies; the use of the antibodies to purify PMEPA1
polypeptides; and the use of the nucleic acids, polypeptides, and
antibodies of the invention to detect, prevent, and treat prostate
cancer (e.g., prostatic intraepithelial neoplasia (PIN),
adenocarcinomas, nodular hyperplasia, and large duct carcinomas)
and prostate-related diseases (e.g., benign prostatic
hyperplasia).
[0041] Nucleic Acid Molecules
[0042] In a particular embodiment, the invention relates to certain
isolated nucleotide sequences that are free from contaminating
endogenous material. A "nucleotide sequence" refers to a
polynucleotide molecule in the form of a separate fragment or as a
component of a larger nucleic acid construct. The nucleic acid
molecule has been derived from DNA or RNA isolated at least once in
substantially pure form and in a quantity or concentration enabling
identification, manipulation, and recovery of its component
nucleotide sequences by standard biochemical methods (such as those
outlined in (Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (1989)). Such sequences are preferably provided and/or
constructed in the form of an open reading frame uninterrupted by
internal non-translated sequences, or introns, that are typically
present in eukaryotic genes. Sequences of non-translated DNA can be
present 5' or 3' from an open reading frame, where the same do not
interfere with manipulation or expression of the coding region.
[0043] Nucleic acid molecules of the invention include DNA in both
single-stranded and double-stranded form, as well as the RNA
complement thereof. DNA includes, for example, cDNA, genomic DNA,
chemically synthesized DNA, DNA amplified by PCR, and combinations
thereof. Genomic DNA may be isolated by conventional techniques,
e.g., using the cDNA of SEQ ID NO: 1, or a suitable fragment
thereof, as a probe.
[0044] The DNA molecules of the invention include full length genes
as well as polynucleotides and fragments thereof. The full length
gene may also include the N-terminal signal peptide. Other
embodiments include DNA encoding a soluble form, e.g., encoding the
extracellular domain of the protein, either with or without the
signal peptide.
[0045] The nucleic acids of the invention are preferentially
derived from human sources, but the invention includes those
derived from non-human species, as well.
[0046] Preferred Sequences
[0047] The particularly preferred nucleotide sequence of the
invention is SEQ ID NO:2, as set forth above. The sequence of amino
acids encoded by the DNA of SEQ ID NO:2 is shown in SEQ ID
NO:3.
[0048] Additional Sequences
[0049] Due to the known degeneracy of the genetic code, where more
than one codon can encode the same amino acid, a DNA sequence can
vary from that shown in SEQ ID NO:2, and still encode a polypeptide
having the amino acid sequence of SEQ ID NO:3. Such variant DNA
sequences can result from silent mutations (e.g., occurring during
PCR amplification), or can be the product of deliberate mutagenesis
of a native sequence.
[0050] The invention thus provides isolated DNA sequences encoding
polypeptides of the invention, selected from: (a) DNA comprising
the nucleotide sequence of SEQ ID NO:2; (b) DNA encoding the
polypeptide of SEQ ID NO:3; (c) DNA capable of hybridization to a
DNA of (a) or (b) under conditions of moderate stringency and which
encodes polypeptides of the invention; (d) DNA capable of
hybridization to a DNA of (a) or (b) under conditions of high
stringency and which encodes polypeptides of the invention, and (e)
DNA which is degenerate as a result of the genetic code to a DNA
defined in (a), (b), (c), or (d) and which encode polypeptides of
the invention. Of course, polypeptides encoded by such DNA
sequences are encompassed by the invention.
[0051] As used herein, conditions of moderate stringency can be
readily determined by those having ordinary skill in the art based
on, for example, the length of the DNA. The basic conditions are
set forth by (Sambrook et al. Molecular Cloning: A Laboratory
Manual, 2ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory
Press, (1989)), and include use of a prewashing solution for the
nitrocellulose filters 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),
hybridization conditions of about 50% formamide, 6.times.SSC at
about 42.degree. C. (or other similar ii hybridization solution,
such as Stark's solution, in about 50% formamide at about
42.degree. C.), and washing conditions of about 60.degree. C.,
0.5.times.SSC, 0.1% SDS. Conditions of high stringency can also be
readily determined by the skilled artisan based on, for example,
the length of the DNA. Generally, such conditions are defined as
hybridization conditions as above, and with washing at
approximately 68.degree. C., 0.2.times.SSC, 0.1% SDS. The skilled
artisan will recognize that the temperature and wash solution salt
concentration can be adjusted as necessary according to factors
such as the length of the probe.
[0052] Also included as an embodiment of the invention is DNA
encoding polypeptide fragments and polypeptides comprising
inactivated N-glycosylation site(s), inactivated protease
processing site(s), or conservative amino acid substitution(s), as
described below.
[0053] In another embodiment, the nucleic acid molecules of the
invention also comprise nucleotide sequences that are at least 80%
identical to a native sequence. Also contemplated are embodiments
in which a nucleic acid molecule comprises a sequence that is at
least 90% identical, at least 95% identical, at least 98%
identical, at least 99% identical, or at least 99.9% identical to a
native sequence.
[0054] The percent identity may be determined by visual inspection
and mathematical calculation. Alternatively, the percent identity
of two nucleic acid sequences can be determined by comparing
sequence information using the GAP computer program, version 6.0
described by (Devereux et al., Nucl. Acids Res., 12:387 (1984)) and
available from the University of Wisconsin Genetics Computer Group
(UWGCG). The preferred default parameters for the GAP program
include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) for nucleotides, and the
weighted comparison matrix of (Gribskov and Burgess. Nucl. Acids
Res., 14:6745 (1986)), as described by (Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358 (1979)); (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps. Other programs used by one
skilled in the art of sequence comparison may also be used.
[0055] The invention also provides isolated nucleic acids useful in
the production of polypeptides. Such polypeptides may be prepared
by any of a number of conventional techniques. A DNA sequence
encoding a PMEPA1 polypeptide, or desired fragment thereof may be
subcloned into an expression vector for production of the
polypeptide or fragment. The DNA sequence advantageously is fused
to a sequence encoding a suitable leader or signal peptide.
Alternatively, the desired fragment may be chemically synthesized
using known techniques. DNA fragments also may be produced by
restriction endonuclease digestion of a full length cloned DNA
sequence, and isolated by electrophoresis on agarose gels. If
necessary, oligonucleotides that reconstruct the 5' or 3' terminus
to a desired point may be ligated to a DNA fragment generated by
restriction enzyme digestion. Such oligonucleotides may
additionally contain a restriction endonuclease cleavage site
upstream of the desired coding sequence, and position an initiation
codon (ATG) at the N-terminus of the coding sequence.
[0056] The well-known polymerase chain reaction (PCR) procedure
also may be used to isolate and amplify a DNA sequence encoding a
desired protein fragment. Oligonucleotides that define the desired
termini of the DNA fragment are employed as 5' and 3' primers. The
oligonucleotides may additionally contain recognition sites for
restriction endonucleases, to facilitate insertion of the amplified
DNA fragment into an expression vector. PCR techniques are
described in (Saiki et al., Science, 239:487 (1988)); (Wu et al.,
Recombinant DNA Methodology, eds., Academic Press, Inc., San Diego,
pp. 189-196 (1989)); and (Innis et al., PCR Protocols: A Guide to
Methods and Applications, eds., Academic Press, Inc. (1990)).
[0057] Polypeptides and Fragments Thereof
[0058] The invention encompasses polypeptides and fragments thereof
in various forms, including those that are naturally occurring or
produced through various techniques such as procedures involving
recombinant DNA technology. Such forms include, but are not limited
to derivatives, variants, and oligomers, as well as fusion proteins
or fragments thereof.
[0059] Polypeptides and Fragments Thereof
[0060] The polypeptides of the invention include full length
proteins encoded by the nucleic acid sequences set forth above.
Particularly preferred polypeptides comprise the amino acid
sequence of SEQ ID NO:3.
[0061] The polypeptides of the invention may be membrane bound or
they may be secreted and thus soluble. Soluble polypeptides are
capable of being secreted from the cells in which they are
expressed. In general, soluble polypeptides may be identified (and
distinguished from non-soluble membrane-bound counterparts) by
separating intact cells which express the desired polypeptide from
the culture medium, e.g., by centrifugation, and assaying the
medium (supernatant) for the presence of the desired polypeptide.
The presence of polypeptide in the medium indicates that the
polypeptide was secreted from the cells and thus is a soluble form
of the protein.
[0062] In one embodiment, the soluble polypeptides and fragments
thereof comprise all or part of the extracellular domain, but lack
the transmembrane region that would cause retention of the
polypeptide on a cell membrane. A soluble polypeptide may include
the cytoplasmic domain, or a portion thereof, as long as the
polypeptide is secreted from the cell in which it is produced.
[0063] In general, the use of soluble forms is advantageous for
certain applications. Purification of the polypeptides from
recombinant host cells is facilitated, since the soluble
polypeptides are secreted from the cells. Further, soluble
polypeptides are generally more suitable for intravenous
administration.
[0064] The invention also provides polypeptides and fragments of
the extracellular domain that retain a desired biological activity.
Such a fragment may be a soluble polypeptide, as described
above.
[0065] Also provided herein are polypeptide fragments comprising at
least 20, or at least 30, contiguous amino acids of the sequence of
SEQ ID NO:3. Fragments derived from the cytoplasmic domain find use
in studies of signal transduction, and in regulating cellular
processes associated with transduction of biological signals.
Polypeptide fragments also may be employed as immunogens, in
generating antibodies.
[0066] Variants
[0067] Naturally occurring variants as well as derived variants of
the polypeptides and fragments are provided herein.
[0068] Variants may exhibit amino acid sequences that are at least
80% identical. Also contemplated are embodiments in which a
polypeptide or fragment comprises an amino acid sequence that is at
least 90% identical, at least 95% identical, at least 98%
identical, at least 99% identical, or at least 99.9% identical to
the preferred polypeptide or fragment thereof. Percent identity may
be determined by visual inspection and mathematical calculation.
Alternatively, the percent identity of two protein sequences can be
determined by comparing sequence information using the GAP computer
program, based on the algorithm of (Needleman and Wunsch, J. Mol.
Bio., 48:443 (1970)) and available from the University of Wisconsin
Genetics Computer Group (UWGCG). The preferred default parameters
for the GAP program include: (1) a scoring matrix, blosum62, as
described by (Henikoff and Henikoff Proc. Natl. Acad. Sci. USA,
89:10915 (1992)); (2) a gap weight of 12; (3) a gap length weight
of 4; and (4) no penalty for end gaps. Other programs used by one
skilled in the art of sequence comparison may also be used.
[0069] The variants of the invention include, for example, those
that result from alternate mRNA splicing events or from proteolytic
cleavage. Alternate splicing of mRNA may, for example, yield a
truncated but biologically active protein, such as a naturally
occurring soluble form of the protein. Variations attributable to
proteolysis include, for example, differences in the N- or
C-termini upon expression in different types of host cells, due to
proteolytic removal of one or more terminal amino acids from the
protein (generally from 1-5 terminal amino acids). Proteins in
which differences in amino acid sequence are attributable to
genetic polymorphism (allelic variation among individuals producing
the protein) are also contemplated herein.
[0070] Additional variants within the scope of the invention
include polypeptides that may be modified to create derivatives
thereof by forming covalent or aggregative conjugates with other
chemical moieties, such as glycosyl groups, lipids, phosphate,
acetyl groups and the like. Covalent derivatives may be prepared by
linking the chemical moieties to functional groups on amino acid
side chains or at the N-terminus or C-terminus of a polypeptide.
Conjugates comprising diagnostic (detectable) or therapeutic agents
attached thereto are contemplated herein, as discussed in more
detail below.
[0071] Other derivatives include covalent or aggregative conjugates
of the polypeptides with other proteins or polypeptides, such as by
synthesis in recombinant culture as N-terminal or C-terminal
fusions. Examples of fusion proteins are discussed below in
connection with oligomers. Further, fusion proteins can comprise
peptides added to facilitate purification and identification. Such
peptides include, for example, poly-His or the antigenic
identification peptides described in U.S. Pat. No. 5,011,912 and in
(Hopp et al., Bio/Technology, 6:1204 (1988)). One such peptide is
the FLAG.RTM. peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, (SEQ ID
NO:4) which is highly antigenic and provides an epitope reversibly
bound by a specific monoclonal antibody, enabling rapid assay and
facile purification of expressed recombinant protein. A murine
hybridoma designated 4E 11 produces a monoclonal antibody that
binds the FLAG.RTM. peptide in the presence of certain divalent
metal cations, as described in U.S. Pat. No. 5,011,912, hereby
incorporated by reference. The 4E11 hybridoma cell line has been
deposited with the American Type Culture Collection under accession
no. HB 9259. Monoclonal antibodies that bind the FLAG.RTM. peptide
are available from Eastman Kodak Co., Scientific Imaging Systems
Division, New Haven, Conn.
[0072] Among the variant polypeptides provided herein are variants
of native polypeptides that retain the native biological activity
or the substantial equivalent thereof. One example is a variant
that binds with essentially the same binding affinity as does the
native form. Binding affinity can be measured by conventional
procedures, e.g., as described in U.S. Pat. No. 5,512,457 and as
set forth below.
[0073] Variants include polypeptides that are substantially
homologous to the native form, but which have an amino acid
sequence different from that of the native form because of one or
more deletions, insertions or substitutions. Particular embodiments
include, but are not limited to, polypeptides that comprise from
one to ten deletions, insertions or substitutions of amino acid
residues, when compared to a native sequence.
[0074] A given amino acid may be replaced, for example, by a
residue having similar physiochemical characteristics. Examples of
such conservative substitutions include substitution of one
aliphatic residue for another, such as Ile, Val, Leu, or Ala for
one another; substitutions of one polar residue for another, such
as between Lys and Arg, Glu and Asp, or Gin and Asn; or
substitutions of one aromatic residue for another, such as Phe,
Trp, or Tyr for one another. Other conservative substitutions,
e.g., involving substitutions of entire regions having similar
hydrophobicity characteristics, are well known.
[0075] Similarly, the DNAs of the invention include variants that
differ from a native DNA sequence because of one or more deletions,
insertions or substitutions, but that encode a biologically active
polypeptide.
[0076] The invention further includes polypeptides of the invention
with or without associated native-pattern glycosylation.
Polypeptides expressed in yeast or mammalian expression systems
(e.g., COS-1 or COS-7 cells) can be similar to or significantly
different from a native polypeptide in molecular weight and
glycosylation pattern, depending upon the choice of expression
system. Expression of polypeptides of the invention in bacterial
expression systems, such as E. Coli, provides non-glycosylated
molecules. Further, a given preparation may include multiple
differentially glycosylated species of the protein. Glycosyl groups
can be removed through conventional methods, in particular those
utilizing glycopeptidase. In general, glycosylated polypeptides of
the invention can be incubated with a molar excess of
glycopeptidase (Boehringer Mannheim).
[0077] Correspondingly, similar DNA constructs that encode various
additions or substitutions of amino acid residues or sequences, or
deletions of terminal or internal residues or sequences are
encompassed by the invention. For example, N-glycosylation sites in
the polypeptide extracellular domain can be modified to preclude
glycosylation, allowing expression of a reduced carbohydrate analog
in mammalian and yeast expression systems. N-glycosylation sites in
eukaryotic polypeptides are characterized by an amino acid triplet
Asn-X-Y, wherein X is any amino acid and Y is Ser or Thr.
Appropriate substitutions, additions, or deletions to the
nucleotide sequence encoding these triplets will result in
prevention of attachment of carbohydrate residues at the Asn side
chain. Alteration of a single nucleotide, chosen so that Asn is
replaced by a different amino acid, for example, is sufficient to
inactivate an N-glycosylation site. Alternatively, the Ser or Thr
can by replaced with another amino acid, such as Ala. Known
procedures for inactivating N-glycosylation sites in proteins
include those described in U.S. Pat. No. 5,071,972 and EP 276,846,
hereby incorporated by reference.
[0078] In another example of variants, sequences encoding Cys
residues that are not essential for biological activity can be
altered to cause the Cys residues to be deleted or replaced with
other amino acids, preventing formation of incorrect intramolecular
disulfide bridges upon folding or renaturation.
[0079] Other variants are prepared by modification of adjacent
dibasic amino acid residues, to enhance expression in yeast systems
in which KEX2 protease activity is present. EP 212,914 discloses
the use of site-specific mutagenesis to inactivate KEX2 protease
processing sites in a protein. KEX2 protease processing sites are
inactivated by deleting, adding or substituting residues to alter
Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of
these adjacent basic residues. Lys-Lys pairings are considerably
less susceptible to KEX2 cleavage, and conversion of Arg-Lys or
Lys-Arg to Lys-Lys represents a conservative and preferred approach
to inactivating KEX2 sites.
[0080] Production of Polypeptides and Fragments Thereof
[0081] Expression, isolation and purification of the polypeptides
and fragments of the invention may be accomplished by any suitable
technique, including but not limited to the following:
[0082] Expression Systems
[0083] The present invention also provides recombinant cloning and
expression vectors containing DNA, as well as host cell containing
the recombinant vectors. Expression vectors comprising DNA may be
used to prepare the polypeptides or fragments of the invention
encoded by the DNA. A method for producing polypeptides comprises
culturing host cells transformed with a recombinant expression
vector encoding the polypeptide, under conditions that promote
expression of the polypeptide, then recovering the expressed
polypeptides from the culture. The skilled artisan will recognize
that the procedure for purifying the expressed polypeptides will
vary according to such factors as the type of host cells employed,
and whether the polypeptide is membrane-bound or a soluble form
that is secreted from the host cell.
[0084] Any suitable expression system may be employed. The vectors
include a DNA encoding a polypeptide or fragment of the invention,
operably linked to suitable transcriptional or translational
regulatory nucleotide sequences, such as those derived from a
mammalian, microbial, viral, or insect gene. Examples of regulatory
sequences include transcriptional promoters, operators, or
enhancers, an mRNA ribosomal binding site, and appropriate
sequences which control transcription and translation initiation
and termination. Nucleotide sequences are operably linked when the
regulatory sequence functionally relates to the DNA sequence. Thus,
a promoter nucleotide sequence is operably linked to a DNA sequence
if the promoter nucleotide sequence controls the transcription of
the DNA sequence. An origin of replication that confers the ability
to replicate in the desired host cells, and a selection gene by
which transformants are identified, are generally incorporated into
the expression vector.
[0085] In addition, a sequence encoding an appropriate signal
peptide (native or heterologous) can be incorporated into
expression vectors. A DNA sequence for a signal peptide (secretory
leader) may be fused in frame to the nucleic acid sequence of the
invention so that the DNA is initially transcribed, and the mRNA
translated, into a fusion protein comprising the signal peptide. A
signal peptide that is functional in the intended host cells
promotes extracellular secretion of the polypeptide. The signal
peptide is cleaved from the polypeptide upon secretion of
polypeptide from the cell.
[0086] Suitable host cells for expression of polypeptides include
prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect
cells are generally preferred for use as host cells. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described, for example, in
(Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New
York, (1985)). Cell-free translation systems could also be employed
to produce polypeptides using RNAs derived from DNA constructs
disclosed herein.
[0087] Prokaryotic Systems
[0088] Prokaryotes include gram-negative or gram-positive
organisms. Suitable prokaryotic host cells for transformation
include, for example, E. coli, Bacillus subtilis, Salmonella
typhimurium, and various other species within the genera
Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic
host cell, such as E. coli, a polypeptide may include an N-terminal
methionine residue to facilitate expression of the recombinant
polypeptide in the prokaryotic host cell. The N-terminal Met may be
cleaved from the expressed recombinant polypeptide.
[0089] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the cloning vector pBR322
(ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells. An appropriate promoter and a DNA sequence are
inserted into the pBR322 vector. Other commercially available
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,
USA).
[0090] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al., Nature
275:615 (1978); and (Goeddel et al., Nature 281:544 (1979)),
tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res.
8:4057 (1980); and EP-A-36776) and tac promoter (Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, p. 412 (1982)). A particularly useful prokaryotic host
cell expression system employs a phage .lambda.P.sub.L promoter and
a cI857ts thermolabile repressor sequence. Plasmid vectors
available from the American Type Culture Collection which
incorporate derivatives of the .lambda.P.sub.L promoter include
plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and
pPLc28 (resident in E. coli RR1, ATCC 53082).
[0091] Yeast Systems
[0092] Alternatively, the polypeptides may be expressed in yeast
host cells, preferably from the Saccharomyces genus (e.g., S.
cerevisiae). Other genera of yeast, such as Pichia or
Kluyveromyces, may also be employed. Yeast vectors will often
contain an origin of replication sequence from a 2 .mu. yeast
plasmid, an autonomously replicating sequence (ARS), a promoter
region, sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Suitable promoter
sequences for yeast vectors include, among others, promoters for
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.
Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et
al., J. Adv. Enzyme Reg. 7:149 (1968)); and (Holland et al.,
Biochem. 17:4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
further described in (Hitzeman, EPA-73,657). Another alternative is
the glucose-repressible ADH2 promoter described by (Russell et al.,
J. Biol. Chem. 258:2674 (1982)) and (Beier et al., Nature 300:724
(1982)). Shuttle vectors replicable in both yeast and E. coli may
be constructed by inserting DNA sequences from pBR322 for selection
and replication in E. coli (Amp.sup.r gene and origin of
replication) into the above-described yeast vectors.
[0093] The yeast a-factor leader sequence may be employed to direct
secretion of the polypeptide. The .alpha.-factor leader sequence is
often inserted between the promoter sequence and the structural
gene sequence. See, e.g., (Kurjan et al., Cell 30:933 (1982)) and
(Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330 (1984)). Other
leader sequences suitable for facilitating secretion of recombinant
polypeptides from yeast hosts are known to those of skill in the
art. A leader sequence may be modified near its 3' end to contain
one or more restriction sites. This will facilitate fusion of the
leader sequence to the structural gene.
[0094] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by (Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929 (1978)). The Hinnen et al. protocol
selects for Trp.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 10 mg/ml ademne and 20 mg/ml
uracil.
[0095] Yeast host cells transformed by vectors containing an ADH2
promoter sequence may be grown for inducing expression in a "rich"
medium. An example of a rich medium is one consisting of 1% yeast
extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml
adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter
occurs when glucose is exhausted from the medium.
[0096] Mammalian or Insect Systems
[0097] Mammalian or insect host cell culture systems also may be
employed to express recombinant polypeptides. Bacculovirus systems
for production of heterologous proteins in insect cells are
reviewed by (Luckow and Summers, Bio/Technology, 6:47 (1988)).
Established cell lines of mammalian origin also may be employed.
Examples of suitable mammalian host cell lines include the COS-7
line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell
23:175 (1981)), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL
10) cell lines, and the CV1/EBNA cell line derived from the African
green monkey kidney cell line CV1 (ATCC CCL 70) as described by
(McMahan et al., EMBO J., 10: 2821 (1991)).
[0098] Established methods for introducing DNA into mammalian cells
have been described (Kaufman, R. J., Large Scale Mammalian Cell
Culture, pp. 15-69 (1990)). Additional protocols using commercially
available reagents, such as Lipofectamine lipid reagent (Gibco/BRL)
or Lipofectamine-Plus lipid reagent, can be used to transfect cells
(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)).
In addition, electroporation can be used to transfect mammalian
cells using conventional procedures, such as those in (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold
Spring Harbor Laboratory Press (1989)). Selection of stable
transformants can be performed using methods known in the art, such
as, for example, resistance to cytotoxic drugs. (Kaufman et al.,
Meth. in Enzymology 185:487-511 (1990)), describes several
selection schemes, such as dihydrofolate reductase (DHFR)
resistance. A suitable host strain for DHFR selection can be CHO
strain DX-B 11, which is deficient in DHFR (Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)). A plasmid
expressing the DHFR cDNA can be introduced into strain DX-B11, and
only cells that contain the plasmid can grow in the appropriate
selective media. Other examples of selectable markers that can be
incorporated into an expression vector include cDNAs conferring
resistance to antibiotics, such as G418 and hygromycin B. Cells
harboring the vector can be selected on the basis of resistance to
these compounds.
[0099] Transcriptional and translational control sequences for
mammalian host cell expression vectors can be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from polyoma virus, adenovirus 2, simian virus 40
(SV40), and human cytomegalovirus. DNA sequences derived from the
SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites can be used
to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell. Viral early and late
promoters are particularly useful because both are easily obtained
from a viral genome as a fragment, which can also contain a viral
origin of replication (Fiers et al. Nature 273:113 (1978));
(Kaufman, Meth. in Enzymology (1990)). Smaller or larger SV40
fragments can also be used, provided the approximately 250 bp
sequence extending from the Hind III site toward the Bgl I site
located in the SV40 viral origin of replication site is
included.
[0100] Additional control sequences shown to improve expression of
heterologous genes from mammalian expression vectors include such
elements as the expression augmenting sequence element (EASE)
derived from CHO cells (Morris et al., Animal Cell Technology, pp.
529-53.4 and PCT Application WO 97/25420 (1997)) and the tripartite
leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al.,
J. Biol. Chem. 257:13475-13491 (1982)). The internal ribosome entry
site (IRES) sequences of viral origin allows dicistronic mRNAs to
be translated efficiently (Oh and Sarnow, Current Opinion in
Genetics and Development 3:295-300 (1993)); (Ramesh et al., Nucleic
Acids Research 24:2697-2700 (1996)). Expression of a heterologous
cDNA as part of a dicistronic mRNA followed by the gene for a
selectable marker (e.g. DHFR) has been shown to improve
transfectability of the host and expression of the heterologous
cDNA (Kaufman, Meth. in Enzymology (1990)). Exemplary expression
vectors that employ dicistronic mRNAs are pTR-DC/GFP described by
(Mosser et al., Biotechniques 22:150-161 (1997)), and p2A5I
described by (Morris et al., Animal Cell Technology, pp. 529-534
(1997)).
[0101] A useful high expression vector, pCAVNOT, has been described
by (Mosley et al., Cell 59:335-348 (1989)). Other expression
vectors for use in mammalian host cells can be constructed as
disclosed by (Okayama and Berg, Mol. Cell. Biol. 3:280 (1983)). A
useful system for stable high level expression of mammalian cDNAs
in C127 murine mammary epithelial cells can be constructed
substantially as described by (Cosman et al., Mol. Immunol. 23:935
(1986)). A useful high expression vector, PMLSV N1/N4, described by
(Cosman et al., Nature 312:768 (1984)), has been deposited as ATCC
39890. Additional useful mammalian expression vectors are described
in EP-A-0367566, and in WO 91/18982, incorporated by reference
herein. In yet another alternative, the vectors can be derived from
retroviruses.
[0102] Another useful expression vector, pFLAG.RTM., can be used.
FLAG.RTM. technology is centered on the fusion of a low molecular
weight (1 kD), hydrophilic, FLAG.RTM. marker peptide to the
N-terminus of a recombinant protein expressed by pFLAG.RTM.
expression vectors. pDC311 is another specialized vector used for
expressing proteins in CHO cells. pDC311 is characterized by a
bicistronic sequence containing the gene of interest and a
dihydrofolate reductase (DHFR) gene with an internal ribosome
binding site for DHFR translation, an expression augmenting
sequence element (EASE), the human CMV promoter, a tripartite
leader sequence, and a polyadenylation site.
[0103] Purification
[0104] The invention also includes methods of isolating and
purifying the polypeptides and fragments thereof.
[0105] Isolation and Purification
[0106] The "isolated" polypeptides or fragments thereof encompassed
by this invention are polypeptides or fragments that are not in an
environment identical to an environment in which it or they can be
found in nature. The "purified" polypeptides or fragments thereof
encompassed by this invention are essentially free of association
with other proteins or polypeptides, for example, as a purification
product of recombinant expression systems such as those described
above or as a purified product from a non-recombinant source such
as naturally occurring cells and/or tissues.
[0107] In one preferred embodiment, the purification of recombinant
polypeptides or fragments can be accomplished using fusions of
polypeptides or fragments of the invention to another ii
polypeptide to aid in the purification of polypeptides or fragments
of the invention.
[0108] With respect to any type of host cell, as is known to the
skilled artisan, procedures for purifying a recombinant polypeptide
or fragment will vary according to such factors as the type of host
cells employed and whether or not the recombinant polypeptide or
fragment is secreted into the culture medium.
[0109] In general, the recombinant polypeptide or fragment can be
isolated from the host cells if not secreted, or from the medium or
supernatant if soluble and secreted, followed by one or more
concentration, salting-out, ion exchange, hydrophobic interaction,
affinity purification or size exclusion chromatography steps. As to
specific ways to accomplish these steps, the culture medium first
can be concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. Following the concentration step, the
concentrate can be applied to a purification matrix such as a gel
filtration medium. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Alternatively, a cation exchange step can be
employed. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups. In
addition, a chromatofocusing step can be employed. Alternatively, a
hydrophobic interaction chromatography step can be employed.
Suitable matrices can be phenyl or octyl moieties bound to resins.
In addition, affinity chromatography with a matrix which
selectively binds the recombinant protein can be employed. Examples
of such resins employed are lectin columns, dye columns, and
metal-chelating columns. Finally, one or more reversed-phase high
performance liquid chromatography (RP-HPLC) steps employing
hydrophobic RP-HPLC media, (e.g., silica gel or polymer resin
having pendant methyl, octyl, octyldecyl or other aliphatic groups)
can be employed to further purify the polypeptides. Some or all of
the foregoing purification steps, in various combinations, are well
known and can be employed to provide an isolated and purified
recombinant protein.
[0110] It is also possible to utilize an affinity column comprising
a polypeptide-binding protein of the invention, such as a
monoclonal antibody generated against polypeptides of the
invention, to affinity-purify expressed polypeptides. These
polypeptides can be removed from an affinity column using
conventional techniques, e.g., in a high salt elution buffer and
then dialyzed into a lower salt buffer for use or by changing pH or
other components depending on the affinity matrix utilized, or be
competitively removed using the naturally occurring substrate of
the affinity moiety, such as a polypeptide derived from the
invention.
[0111] In this aspect of the invention, polypeptide-binding
proteins, such as the anti-polypeptide antibodies of the invention
or other proteins that may interact with the polypeptide of the
invention, can be bound to a solid phase support such as a column
chromatography matrix or a similar substrate suitable for
identifying, separating, or purifying cells that express
polypeptides of the invention on their surface. Adherence of
polypeptide-binding proteins of the invention to a solid phase
contacting surface can be accomplished by any means, for example,
magnetic microspheres can be coated with these polypeptide-binding
proteins and held in the incubation vessel through a magnetic
field. Suspensions of cell mixtures are contacted with the solid
phase that has such polypeptide-binding proteins thereon. Cells
having polypeptides of the invention on their surface bind to the
fixed polypeptide-binding protein and unbound cells then are washed
away. This affinity-binding method is useful for purifying,
screening, or separating such polypeptide-expressing cells from
solution. Methods of releasing positively selected cells from the
solid phase are known in the art and encompass, for example, the
use of enzymes. Such enzymes are preferably non-toxic and
non-injurious to the cells and are preferably directed to cleaving
the cell-surface binding partner.
[0112] Alternatively, mixtures of cells suspected of containing
polypeptide-expressing cells of the invention first can be
incubated with a biotinylated polypeptide-binding protein of the
invention. Incubation periods are typically at least one hour in
duration to ensure sufficient binding to polypeptides of the
invention. The resulting mixture then is passed through a column
packed with avidin-coated beads, whereby the high affinity of
biotin for avidin provides the binding of the polypeptide-binding
cells to the beads. Use of avidin-coated beads is known in the art.
See (Berenson, et al. J. Cell. Biochem., 10D:239 (1986)). Wash of
unbound material and the release of the bound cells is performed
using conventional methods.
[0113] The desired degree of purity depends on the intended use of
the protein. A relatively high degree of purity is desired when the
polypeptide is to be administered in vivo, for example. In such a
case, the polypeptides are purified such that no protein bands
corresponding to other proteins are detectable upon analysis by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be
recognized by one skilled in the pertinent field that multiple
bands corresponding to the polypeptide may be visualized by
SDS-PAGE, due to differential glycosylation, differential
post-translational processing, and the like. Most preferably, the
polypeptide of the invention is purified to substantial
homogeneity, as indicated by a single protein band upon analysis by
SDS-PAGE. The protein band may be visualized by silver staining,
Coomassie blue staining, or (if the protein is radiolabeled) by
autoradiography.
[0114] Production of Antibodies
[0115] Antibodies that are immunoreactive with the polypeptides of
the invention are provided herein. Such antibodies specifically
bind to the polypeptides via the antigen-binding sites of the
antibody (as opposed to non-specific binding). Thus, the
polypeptides, fragments, variants, fusion proteins, etc., as set
forth above may be employed as "immunogens" in producing antibodies
immunoreactive therewith. More specifically, the polypeptides,
fragment, variants, fusion proteins, etc. contain antigenic
determinants or epitopes that elicit the formation of
antibodies.
[0116] These antigenic determinants or epitopes can be either
linear or conformational (discontinuous). Linear epitopes are
composed of a single section of amino acids of the polypeptide,
while conformational or discontinuous epitopes are composed of
amino acids sections from different regions of the polypeptide
chain that are brought into close proximity upon protein folding
(C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9, Garland
Publishing Inc., 2nd ed. (1996)). Because folded proteins have
complex surfaces, the number of epitopes available is quite
numerous; however, due to the conformation of the protein and
steric hinderances, the number of antibodies that actually bind to
the epitopes is less than the number of available epitopes (C. A.
Janeway, Jr. and P. Travers, Immuno Biology 2:14, Garland
Publishing Inc., 2nd ed. (1996)). Epitopes may be identified by any
of the methods known in the art.
[0117] Thus, one aspect of the present invention relates to the
antigenic epitopes of the polypeptides of the invention. Such
epitopes are useful for raising antibodies, in particular
monoclonal antibodies, as described in more detail below.
Additionally, epitopes from the polypeptides of the invention can
be used as research reagents, in assays, and to purify specific
binding antibodies from substances such as polyclonal sera or
supernatants from cultured hybridomas. Such epitopes or variants
thereof can be produced using techniques well known in the art such
as solid-phase synthesis, chemical or enzymatic cleavage of a
polypeptide, or using recombinant DNA technology.
[0118] As to the antibodies that can be elicited by the epitopes of
the polypeptides of the invention, whether the epitopes have been
isolated or remain part of the polypeptides, both polyclonal and
monoclonal antibodies may be prepared by conventional techniques.
See, for example, (Kennet et al., Monoclonal Antibodies,
Hybridomas: A New Dimension in Biological Analyses, eds., Plenum
Press, New York (1980); and Harlow and Land, Antibodies: A
Laboratory Manual, eds., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., (1988)).
[0119] Hybridoma cell lines that produce monoclonal antibodies
specific for the polypeptides of the invention are also
contemplated herein. Such hybridomas may be produced and identified
by conventional techniques. One method for producing such a
hybridoma cell line comprises immunizing an animal with a
polypeptide; harvesting spleen cells from the immunized animal;
fusing said spleen cells to a myeloma cell line, thereby generating
hybridoma cells; and identifying a hybridoma cell line that
produces a monoclonal antibody that binds the polypeptide. The
monoclonal antibodies may be recovered by conventional
techniques.
[0120] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such humanized antibodies may be prepared by known
techniques and offer the advantage of reduced immunogenicity when
the antibodies are administered to humans. In one embodiment, a
humanized monoclonal antibody comprises the variable region of a
murine antibody (or just the antigen binding site thereof) and a
constant region derived from a human antibody. Alternatively, a
humanized antibody fragment may comprise the antigen binding site
of a murine monoclonal antibody and a variable region fragment
(lacking the antigen-binding site) derived from a human antibody.
Procedures for the production of chimeric and further engineered
monoclonal antibodies include those described in (Riechmann et al.,
Nature 332:323 (1988), Liu et al., PNAS84:3439 (1987), Larrick et
al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS
14:139 (May 1993)). Procedures to generate antibodies
transgenically can be found in GB 2,272,440, U.S. Pat. Nos.
5,569,825 and 5,545,806 and related patents claiming priority
therefrom, all of which are incorporated by reference herein.
[0121] Antigen-binding fragments of the antibodies, which may be
produced by conventional techniques, are also encompassed by the
present invention. Examples of such fragments include, but are not
limited to, Fab and F(ab').sub.2 fragments. Antibody fragments and
derivatives produced by genetic engineering techniques are also
provided.
[0122] In one embodiment, the antibodies are specific for the
polypeptides of the present invention and do not cross-react with
other proteins. Screening procedures by which such antibodies may,
be identified are well known, and may involve immunoaffinity
chromatography, for example.
[0123] The following examples further illustrate preferred aspects
of the invention.
EXAMPLE 1
[0124] Cell Culture and Androgen Stimulation
[0125] LNCaP cells (American Type Culture Collection, Rockville,
Md.) were used for SAGE analysis of ARGs. LNCaP cells were
maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg,
Md.) supplemented with 10% fetal bovine serum (FBS, Life
Technologies, Inc., Gaithersburg, Md.) and experiments were
performed on cells between passages 20 and 30. For the studies of
androgen regulation, charcoal/dextran stripped androgen-free FBS
(cFBS, Gemini Bio-Products, Inc., Calabasas, Calif.) was used.
LNCaP cells were cultured first in RPMI 1640 with 10% cFBS for 5
days and then stimulated with 10-8 M of non-metabolizable androgen
analog, R1881 (DUPONT, Boston, Mass.) for 24 hours. LNCAP cells
identically treated but without R1881 treatment served as control.
Cells were harvested at indicated time and polyA- RNA was
double-selected with Fast Track kit (Invitrogene). The quality of
polyA+ was checked by Northern hybridization analysis.
EXAMPLE 2
[0126] SAGE Analysis
[0127] Two SAGE libraries (library LNCaP-C and library LNCaP-T)
were generated according to the procedure described previously
Velculescu et al. (30). Briefly, biotinylated oligo dT primed cDNA
was prepared from five micrograms of polyA+ RNA from R1881 treated
and control LNCaP cells and biotinylated cDNA was captured on
strepravidin coated magnetic beads (Dynal Corporation, MI). cDNA
bound to the magnetic beads were digested by NlaIII followed by
ligation to synthetic linkers containing a site for anchoring
enzyme, NlaIII and a site for tagging enzyme BsrhF1. The
restriction digestion of ligated products with BsmF1 resulted in
the capture of 10-11 bp sequences termed as "tags" representing
signature sequence of unique cDNAs. A multi-step strategy combining
ligation, PCR, enzymatic digestion and gel purification yielded two
tags linked together termed as "ditags." Ditags were
concatamerized, purified and cloned in plasmid pZero cloning vector
(Invitrogen, CA). The clones containing concatamers were screened
by PCR and sequenced. The sequence and the occurrence of each of
the SAGE tags was determined using the SAGE software kindly
provided by Dr. Kenneth W. Kinzler (Johns Hopkins University School
of Medicine, Baltimore, Md.). All the SAGE tags sequences were
analyzed for identity to DNA sequence in GenBank (National Center
for Biotechnology Information, Bethesda, Md., USA). The relative
abundance of each transcript was determined by dividing the number
of individual tags by total tags in the library. The copy number of
each gene was calculated assuming there are approximately 300,000
transcripts in a cell (Zhang et al., 1997). The differentially
expressed SAGE tags were determined by comparing the frequency of
occurrence of individual tags in the two libraries obtained from
the control (library LNCaP-C) and R1881 treated LNCaP cells
(library LNCaP-T). The results were analyzed with t test, and
p<0.05 was considered as a statistically significant difference
for a specific tag between these two libraries.
EXAMPLE 3
[0128] Kinetics of Androgen Regulation ARGs Defined by SAGE
Analysis
[0129] LNCaP cells were cultured in RPMI 1640 with 10% cFBS for 5
days, then stimulated with R1881 at 10-10, 10-8, and 10-6 M for 1,
3, 12, 24, 72, 120, 168, and 216 hours. LNCaP cells identically
treated but without R1881 served as control. The cells were
harvested at indicated time and polyA+ RNA was prepared as
described as above. The polyA+ RNA was fractionated (2 .mu.g/lane)
by running through 1% formmaldehyde-agarose gel and transferred to
nylon membrane. The cDNA probes of several ARGs were labeled with
.sup.32P-dCTP by random priming (Stratagene Cloning Systems, La
Jolla, Calif.). The nylon membranes were prehybridized for 2 hrs in
hybridization buffer (10 mM Tris-HCl, pH 7.5, 10% Dextran sulfate,
40% Formamide, 5.times.SSC, 5.times. Denhardt's solution and 0.25
mg/ml salmon sperm DNA) and hybridized to the .sup.32P labeled
probes (1.times.106 cpm/ml) in the same buffer at 40.degree. C. for
12-16 hrs. Blots were washed twice in 2.times.SSC/0.1% SDS for 20
min at room temperature followed by two high-stringency wash with
0.1.times.SSC/0.1% SDS at 50.degree. C. for 20 min. Nylon membranes
were exposed to X-ray film for autoradiography.
EXAMPLE 4
[0130] ARGs Expression Pattern in Cwr22 Model.
[0131] CWR22 (androgen dependent) and CWR22R (androgen relapsed)
tumor specimens were kindly provided by Dr. Thomas Pretlow (Case
Western Reserve University School of Medicine). The tissue samples
were homogenized and polyA+ RNA was extracted with Fast Track kit
(Invitrogen) following manufacture's protocol. Northern blots were
prepared as described in Example 3 and were hybridized with
.sup.32P labeled probes of the cDNA of interest.
[0132] Analysis of SAGE tag libraries from R1881 treated LNCaP
cells. LNCaP cells were maintained in androgen deprived growth
media for five days and were treated with synthetic androgen R1881
(10 nm) for 24 hours. Since a goal of the inventors was to identify
androgen signaling read-out transcripts, we chose conditions of
R1881 treatment of LNCaP cells showing a robust and stable
transcriptional induction of well-characterized prostate-specific
androgen regulated genes, prostate-specific antigen (PSA) and
NKX3.1 genes. A total of 90,236 tags were derived from the two SAGE
libraries. Of 90,236 tags, 6,757 tags corresponded to linker
sequences, and were excluded from further analysis. The remaining
83,489 tags represented a total of 23,448 known genes or ESTs and
1,655 tags did not show any match in the GeneBank data base. The
relative abundance of the SAGE tags varied between 0.0011% and
1.7%. Assuming that there are 18,000 transcripts per cell type and
there are about 83,489 anticipated total transcripts, the estimated
abundance of transcripts will be 0.2-308 copies per cell. This
calculation indicated that single copy genes had high chance to be
recognized by SAGE analysis in this study. The distribution of
transcripts by copy number suggests that the majority (above 90%)
of the genes in our analysis are expressed at 1 or 2 copies
level/cell. A total of 46,186 and 45,309 tags were analyzed in the
control (C) and R1881 (T) groups respectively. Unique SAGE tags
corresponding to known genes, expressed sequence tags (ESTs) and
novel transcripts were 15,593 and 15,920 in the control and
androgen treated groups respectively. About 94% of the unique SAGE
tags in each group showed a match to a sequence in the gene bank
and 6% SAGE tags represented novel transcripts. The most abundant
SAGE tags in both control and androgen treated LNCaP cells
represented proteins involved in cellular translation machinery
e.g., ribosomal proteins, translation regulators, mitochondrial
proteins involved in bio-energetic pathways.
EXAMPLE 5
[0133] Analysis of the ARGs Defined by SAGE Tags
[0134] Of about 15,000 unique tags a total of 136 SAGE tags were
significantly up-regulated in response to R1881 whereas 215 SAGE
tags were significantly down-regulated (p<0.05). It is important
to note that of 15,000 expressed sequences only 1.5% were androgen
responsive suggesting that expression of only a small subset of
genes are regulated by androgen under our experimental conditions.
The ARGs identified by the inventors are anticipated to represent a
hierarchy, where a fraction of ARGs are directly regulated by
androgens and others represent the consequence of the activation of
direct down-stream target genes of the AR. Comparison of SAGE tags
between control and R1881 also revealed that 74 SAGE tags were
significantly up-regulated (p<0.05) by four-fold and 120 SAGE
tags were significantly (p<0.05) down-regulated. Two SAGE tags
corresponding to the PSA gene sequence exhibited highest induction
(16 fold) between androgen treated (T) and control (C) groups.
Another prostate specific androgen regulated gene, NKX3.1 was among
significantly up-regulated ARGs (8 fold). Prostate specific
membrane antigen (PSMA) and Clusterin known to be down-regulated by
androgens were among the SAGE tags exhibiting decreased expression
in response to androgen (PSMA, 4 fold; Clusterin, fold). Therefore,
identification of well characterized up-regulated and
down-regulated ARGs defined by SAGE tags validates the use of LNCaP
experimental model for defining physiologically relevant ARGs in
the context of prostatic epithelial cells. It is important to note
that about 90% of up-regulated ARGs and 98% of the down-regulated
ARGs defined by our SAGE analysis were not known to be
androgen-regulated before.
EXAMPLE 6
[0135] Identification of Prostate Specific/Abundant Genes
[0136] LNCaP C/T-SAGE tag libraries were compared to a bank of 35
SAGE tag libraries (http://www.ncbi.nlm.nih.gov/SAGE/) containing
1.5 million tags from diverse tissues and cell types. Our analysis
revealed that known prostate specific genes e.g., PSA and NKX3.1
were found only in LNCaP SAGE tag libraries (this report and one
LNCaP SAGE library present in the SAGE tag bank). We have extended
this observation to the other candidate genes and transcripts. On
the basis of abundant/unique expression of the SAGE tag defined
transcripts in LNCaP SAGE tag libraries relative to other
libraries, we have now identified several candidate genes and ESTs
whose expression are potentially prostate specific or restricted
(Table 4). The utility of such prostate-specific genes includes:
(a) the diagnosis and prognosis of CaP (b) tissue specific
targeting of therapeutic genes (c) candidates for immunotherapy and
(d) defining prostate specific biologic functions.
[0137] Genes with defined functions showing at least five fold up
or down-regulation (p<0.05) were broadly classified on the basis
of their biochemical function, since our results of Northern
analysis of representative SAGE derived ARGs at 5-fold difference
showed most reproducible results. Table 9, presented at the end of
this specification immediately preceding the "References" section,
represents the quantitative expression profiles of a panel of
functionally defined ARGs in the context of LNCaP prostate cancer
cells. ARGs in the transcription factor category include proteins
involved in the general transcription machinery e.g., KAPl/TIF
.beta., CHD4 and SMRT (Douarin et al., 1998; Xu et al., 1999) have
been shown to participate in transcriptional repression. The
mitochondrial transcription factor 1 (mtTF1) was induced by 8 fold
in response to R1881. A recent report describes that another member
of the nuclear receptor superfamily, the thyroid hormone receptor,
also up-regulates a mitochondrial transcription factor expression
through a specific co-activator, PGC-1 (Wu et al., 1999). As shown
in Table 9 a thyroid hormone receptor related gene, ear-2 (Miyajima
et al., 1998) was also upregulated by R188. It is striking to note
that expression of four [NKX3.1 (He et al., 1997). HOX B13
(Sreenath et al., 1999), mtTF1 and PDEF (Oettgen et al., 2000)] of
the eight transcription regulators listed in Table 9 appear to be
prostate tissue abundant/specific based on published reports as
well as our analysis described above.
[0138] ARGs also include a number of proteins involved in cellular
energy metabolism and it is possible that some of these enzymes may
be transcriptionally regulated by mtTF1. Components of enzymes
involved in oxidative decaboxylation: dihydrolipoamide succinyl
transferase (Patel et al., 1995), puruvate dehydrogenase E-1
subunit (Ho et al., 1989), and the electron tansport chain: NADH
dehydrogenase 1 beta subcomplex 10 (Ton et al., 1997) were
upregulated by androgen. VDAC-2 (Blachly-Dyson et al., 1994), a
member of small pore forming proteins of the outer mitochondrial
membrane and which may regulate the transport of small metabolites
necessary for oxidative-phosphorylation, was also up regulated by
androgen. Diazepam binding protein (DBI), a previous reported ARG
(Swinnen et al., 1996), known to be associated with the VDAC
complex and implicated in a multitude of functions including
modulation of pheripheral benzodiaepine receptor, acyl-CoA
metabolism and mitochondrial steroidogenesis (Knudsen et al., 1993)
were also induced by androgen in our study. A thioredoxin like
protein (Miranda-Vizuete et al., 1998) which may function in
modulating the cellular redox state was down regulated by androgen.
In general, it appears that modulation of ARGs involved in
regulating cellular redox status and energy metabolism may effect
reactive oxygen species concentrations.
[0139] A number of cell proliferation associated proteins
regulating cell cycle, signal transduction and cellular protein
trafficking were upregulated by androgen, further supporting the
role of androgens in survival and growth of prostatic epithelial
cells. Androgen regulation of two proteins: XRCC2 (Cartxvrght et
al., 1998) and RPA3 (Umbricht et al., 1993) involved in DNA repair
and recombination is a novel and interesting finding. Induction of
these genes may represent a response to DNA damage due to androgen
mediated pro-oxidant shift, or these genes simply represent
components of genomic surveillance mechanisms stimulated by cell
proliferation. The androgen induction of a p53 inducible gene, PIG
8 (Umbricht et al., 1997), is another intriguing finding as the
mouse homolog of this gene, ei24 (Gu et al., 2000), is induced by
etoposide known to generate reactive oxygen species. In addition,
components of protein kinases modulated by adenyl cyclase, guanyl
cyclase and calmodulin involved in various cellular signal
transduction stimuli were also regulated by androgen.
[0140] Gene expression modulations in RNA processing and
translation components is consistent with increased protein
synthesis expected in cells that are switched to a highly
proliferative state. Of note is nucleolin, one of the highly
androgen induced genes (12 fold) which is an abundant nucleolar
protein associating with cell proliferation and plays a direct role
in the biogenesis, processing and transport of ribosomes to the
cytoplasm (Srivastava and Pollard, 1999). Another androgen
up-regulated gene, exportin, a component of the nuclear pore, may
be involved in the shuttling of nucleolin. Androgen regulation of
SiahBP 1 (Page-McCaw et al., 1999), GRSF-1 (Qian and Wilusz, 1994)
and PAIP1 (Craig et al., 1998) suggests a role of androgen
signaling in the processing of newly transcribed RNAs. Two
splicesosomal genes, snRNP-G and snRNP-E coding for small
ribo-nucleoproteins were down-regulated by androgen. The
unr-interacting protein, UNRIP (Hunt et al., 1999) which is
involved in the direct ribosome entry of many viral and some
cellular mRNAs into the translational pathway, was the most
down-regulated gene in response to androgen.
[0141] Quantitative evaluation of gene expression profiles by SAGE
approach have defined yeast transcriptome (Velculescu et al., 1997)
and have identified critical genes in biochemical pathways
regulated by p53 (Polyak et al., 1997), x-irradiation (Hermeking et
al., 1997) and the APC gene (Korinek et al., 1997). Potential tumor
biomarkers in colon (Zhang et al., 1997), lung (Hibi et al., 1998),
and breast (Nacht et al., 1999) cancers and genes regulated by
other cellular stimuli (Waard et al., 1999; Berg et al., 1999) have
also been identified by SAGE. SAGE technology has enabled us to
develop the first quantitative database of androgen regulated
transcripts. Comparison of our SAGE tag libraries to the SAGE
TagBank has also revealed a number of new candidate genes and ESTs
whose expression is potentially abundant or specific to the
prostate. We have also identified a large number of transcripts not
previously defined as ARGs.
[0142] A great majority of functionally defined genes that were
modulated by androgen in our experimental system appear to promote
cell proliferation, cell survival, gain of energy and increased
oxidative reactions shift in the cells. However, a substantial
fraction of these ARGs appears to be androgen specific since they
do not exhibit appreciable change in their expression in other
studies examining cell proliferation associated genes (Iyer et al.,
1999, genome-www.stanford.edu/serum) or estrogen regulated genes in
MCF7 cells (Charpentier et al., 2000). The interesting experimental
observation of Ripple et al. (Ripple et al., 1997) showing a
prooxidant-antioxidant shift induced by androgen in prostate cancer
cells is supported by our identification of specific ARGs
(upregulation of enzymes involved in oxidative reactions, electron
transport chain and lipid metabolism in mitochondria and down
regulation of thioredoxin like protein) that may be involved in the
induction of oxidative stress by androgen.
EXAMPLE 7
[0143] Characterization of the Androgen-Regulated Gene PMEPA1
[0144] cDNA library screening and Sequencing of cDNA clone. One of
the SAGE tags (14 bp) showing the highest induction by androgen
(29-fold) exhibited homology to an EST in the GenBank EST database.
PCR primers (5'GGCAGAACACTCCGCGCTTCTTAG3' (SEQ ID NO. 5) and
5'CAAGCTCTCTTAGCTTGTGCAT- TC3' (SEQ ID NO. 6)) were designed based
on the EST sequence (accession number AA310984). RT-PCR was
performed using RNA from R1881 treated LNCaP cells and the
co-identity of the PCR product to the EST was confirmed by DNA
sequencing. Using the PCR product as probe, the normal prostate
cDNA library was screened through the service provided by Genome
Systems (St. Louis, Mo.). An isolated clone, GS 22381 was sequenced
using the 310 Genetic Analyzer (PE Applied Biosystems, Foster
Calif.) and 750 bp of DNA sequence was defined, which included 2/3
of the coding region of PMEPA1. A GenBank search with PMEPA1 cDNA
sequence revealed one EST clone (accession number AA088767)
homologous to the 5' region of the PMEPA1 sequence. PCR primers
were designed using the EST clone (5' primer) and PMEPA1 (3'
primer) sequence. cDNA from LNCaP cells was PCR amplified and the
PCR product was sequenced using the PCR primers. The sequences were
verified using at least two different primers. A contiguous
sequence of 1,141 bp was generated by these methods.
[0145] Kinetics of androgen regulation of PMEPA1 expression in
LNCaP cells. LNCaP cells (American Type Culture Collection, ATCC,
Rockville Md.) were maintained in RPMI 1640 media (Life
Technologies, Inc., Gaithersburg, Md.) supplemented with 10% fetal
bovine serum (FBS, Life Technologies, Inc., Gaithersburg, Md.) and
experiments were performed on cells cultured between passages 20
and 30. For the studies of androgen regulation, charcoal/dextran
stripped androgen-free FBS (cFBS, Gemini Bio-Products, Inc.,
Calabasas, Calif.) was used. LNCaP cells were cultured first in
RPMI 1640 with 10% cFBS for 5 days, and then stimulated with R1881
(DUPONT, Boston, Mass.) at 10-10 M and 108 M for 3, 6, 12 and 24
hours. LNCaP cells identically treated but without R1881 served as
control. To study the effects of androgen withdrawal on PMEPA1 gene
expression, LNCaP cells were cultured in RPMI 1640 with 10% cFBS
for 24, 72 and 96 hours. Poly A.sup.+ RNA samples derived from
cells treated with or without R18881 were extracted at indicated
time points with a Fast Track mRNA extraction kit (Invitrogen,
Carlsbad, Calif.) following the manufacturer's protocol. Poly
A.sup.+ RNA specimens (2 .mu.g/lane) were electrophoresed in a 1%
formaldehyde-agarose gel and transferred to a nylon membrane. Two
PMEPA1 probes used for Northern blots analysis were (a) cDNA probe
spanning nucleotides 3-437 of PMEPA1 cDNA sequence (See Table 1)
and (b) 71-mer oligonucleotide between nucleotides 971 to 1,041 of
PMEPA1 cDNA sequence (See Table 1).
[0146] The cDNA probe was generated by RT-PCR with primers
5'CTTGGGTTCGGGTGAAAGCGCC 3' (SEQ ID NO. 7) (sense) and
5'GGTGGGTGGCAGGTCGATCTCG 3' (SEQ ID NO. 8) (antisense). PMEPA1
oligonucleotide and cDNA probes and glyceraldehyde phosphate
dehydrogenase gene (GAPDH) cDNA probe were labeled with
.sup.32P-dCTP using 3' end tailing for oligonucleotides (Promega,
Madison, Wis.) and random priming for cDNA (Stratagene, La Jolla,
Calif.). The nylon membranes were treated with hybridization buffer
(10 mM Tris-HCl, pH 7.5, 10% Dextran sulfate, 40% Formamide,
5.times.SSC, 5.times. Denhardt's solution and 0.25 mg/ml salmon
sperm DNA) for two hours followed by hybridization in the same
buffer containing the .sup.32P labeled probes (1.times.10.sup.6
cpm/ml) for 12-16 hrs at 40.degree. C. Blots were washed twice in
2.times.SSC/0.1% SDS for 20 min at room temperature followed by two
high-stringency washes with 0.1.times.SSC/0.1% SDS at 58.degree. C.
for 20 min. Nylon membranes were exposed to X-ray film for
autoradiography. The bands on films were then quantified with
N1H-Image processing software.
[0147] PMEPA1 expression analysis in CWR22 tumors. CWR22 is an
androgen-dependent, serially transplantable nude mouse xenograft
derived from a primary human prostate cancer. Transplanted CWR22
tumors are positive for AR and the growth of CWR22 is androgen
dependent. CWR22 tumors regress initially upon castration followed
by a relapse. The recurrent CWR22 tumors (CWR22R) express AR, but
the growth of these tumors become androgen-independent (Gregory et
al., 1998; Nagabhushan et al., 1996). One CW 2 and four CWR22R
tumor specimens were kindly provided by Dr. Thomas Pretlow's
laboratory (Case Western Reserve University School of Medicine).
Tumor tissues were homogenized and poly A.sup.+ RNA were extracted
as above. Poly A.sup.+ RNA blots were made and hybridized as
described above.
[0148] PMEPA1 expression analysis in multiple human tissues and
cell lines. Multiple Tissue Northern blots containing mRNA samples
from 23 human tissues and Master Dot blots containing mRNA samples
from 50 different human tissues were purchased from ClonTech (Palo
Alto, Calif.). The blots were hybridized with PMEPA1 cDNA and oligo
probes, as described above. The expression of PMEPA1 in normal
prostate epithelial cells (Clonetics, San Diego, Calif.), prostate
cancer cells PC3 (ATCC) and LNCaP cells and breast cancer cells
MCF7 (ATCC) was also analyzed by northern blot.
[0149] In situ hybridization of PMEPA1 in prostate tissues. A 430
bp PCR fragment (PCR sense primer: 5'CCTTCGCCCAGCGGGAGCGC 3', (SEQ
ID NO. 9) PCR antisense primer 5'CAAGCTCTCTTAGCTTGTGCATTC3' (SEQ ID
NO. 10) was amplified from cDNA of LNCaP cells treated by R1881 and
was cloned into a PCR-blunt II-TOPO vector (Invitrogen, Carlsbad,
Calif.). Digoxigenin labeled antisense and sense riboprobes were
synthesized using an in vitro RNA transcription kit (Boehringer
Mannheim, GMbH, Germany) and a linearized plasmid with PMEPA1 gene
fragment as templates. Frozen normal and malignant prostate tissues
were fixed in 4% paraformaldehyde for 30 min. Prehybridization and
hybridization were performed at 55.degree. C. After hybridization,
slides were sequentially washed with 2.times.SSC at room
temperature for 30 min, 2.times.SSC at 58.degree. C. for 1 hr and
0.1.times.SSC at 58.degree. C. for 1 hr. Antibody against
digoxygenin was used to detect the signal and NBT/BCIP was used as
substrate for color development (Boehringer Mannheim, GMbH,
Germany). The slides were evaluated under an Olympus BX-60
microscope.
[0150] Full-Length PMEPA1 Coding Sequence and Chromosomal
Localization
[0151] Analysis of the 1,141 bp PMEPA1 cDNA sequence (SEQ ID NO. 1)
revealed an open reading frame of 759 bp nucleotides (SEQ ID NO. 2)
encoding a 252 amino acid protein (SEQ ID NO. 3) with a predicted
molecular mass of 27.8 kDa, as set forth below in Table 1.
3TABLE 1 (SEQ ID NO. 3)
TCCTTGGGTTCGGGTGAAAGCGCCTGGGGGTTCGTGGCCATGATCCCCGAGCTGCTGGAGAACTGAAGGCGGA-
CAGTCTCCTGCGAAAC 90 .tangle-soliddn.
AGGCAATGGCGGAGCTGGAGTTTGTTCAGATCATCATCATCGTGGTGGTGATGATGGTGATGGTGGTGGTGAT-
CACGTGCCTGCTGAGCC 180 M A E L E F V Q I I I I V V V M M V M V V V I
T C L L S 28 .tangle-soliddn.
ACTACAAGCTGTCTGCACGGTCCTTCATCAGCCGGCACAGCCAGGGGCG-
GAGGAGAGAAGATGCCCTGTCCTCAGAAGGATGCCTGTGGC 270 H Y K L S A R S F I S
R H S Q G R R R E D A L S S E G C L W 58 .tangle-soliddn.
CCTCGGAGAGCACAGTGTCAGGCAACGGAATCCCAGAGCCGCAGGTCTACGCCCCGCCTCGGCCCACC-
GACCGCCTGGCCGTGCCGCCCT 360 P S E S T V S G N G I P E P Q V Y A P P
R P T D R L A V P P 88
TCGCCCAGCGGGAGCGCTTCCACCGCTTCCAGCCCACCTATCCGTACCTGCAGCACGAGATCGACCTGCCACC-
CACCATCTCGCTGTCAG 450 F A Q R E R F H R F Q P T Y P Y L Q H E I D L
P P T I S L S 118
ACGGGGAGGAGCCCCCACCCTACCAGGGCCCCTGCACCCTCCAGCTTCGGGACCCCGAGCAGCAGCTGGAACT-
GAACCGGGAGTCGGTGC 540 D G E E P P P Y Q G P C T L Q L R D P E Q Q L
E L N R E S V 148
GCGCACCCCCAAACAGAACCATCTTCGACAGTGACCTGATGGATAGTGCCAGGCTGGGCGGCCCCTGCCCCCC-
CAGCAGTAACTCGGGCA 630 R A P P N R T I F D S D L M D S A R L G G P C
P P S S N S G 178
TCAGCGCCACGTGCTACGGCAGCGGCGGGCGCATGGAGGGGCCGCCGCCCACCTACAGCGAGGTCATCGGCCA-
CTACCCGGGGTCCTCCT 720 I S A T C Y G S G G R M E G P P P T Y S E V I
G H Y P G S S 208
TCCAGCACCAGCAGAGCAGTGGGCCGCCCTCCTTGCTGGAGGGGACCCGGCTCCACCACACACACATCGCGCC-
CCTAGAGAGCGCAGCCA 810 F Q H Q Q S S G P P S L L E G T R L H H T H I
A P L E S A A 238
TCTGGAGCAAAGAGAAGGATAAACAGAAAGGACACCCTCTCTAGGGTCCCCAGGGGGGCCGGGCTGGGGCTGC-
GTAGGTGAAAAGGCAGA 900 I W S K E K D K Q K G H P L * 252 (SEQ ID NO.
1) ACACTCCGCGCTTCTTAGAAGAGGAGTGAG-
AGGAAGGCGGGGGGCGCAGCAACGCATCGTGTGGCCCTCCCCTCCCACCTCCCTGTGTAT 990
AAATATTTACATGTGATGTCTGGTCTGAATGCACAAGCTAAGAGAGCTTGCAAAAAAAAAAAGAAA-
AAAGAAAAAAAAAAACCACGTTTC 1080 .tangle-soliddn.
TTTGTTGAGCTGTGTCTTGAAGGC- AAAAGAAAAAAAATTTCTACAGTAAAAAAAAAAAAAA
1141
[0152] As indicated above, Table 1 represents the nucleotide and
predicted amino acid sequence of PMEPA1 (GenBank accession No.
AF224278). The potential initiation methionine codon and the
translation stop codons are indicated in bold. The transmembrane
domain is underlined. The locations of the intron/exon boundaries
are shown with arrows, which were determined by comparison of the
PMEPA1 cDNA sequence to the publicly available sequences of human
clones RP5-1059L7 and 718J7 (GenBank accession No. AL121913 and
AL035541).
[0153] A GenBank search revealed a sequence match of PMEPA1 cDNA to
two genomic clones, RP5-1059L7 (accession number AL121913 in the
GenBank/htgc database) and 718J7 (accession number AL035541 in the
GenBank/nr database). These two clones mapped to Chromosome
20q13.2-13.33 and Chromosome 20q13.31-13.33. This information
provided evidence that PMEPA1 is located on chromosome 20q13.
[0154] The intron/exon juctions of PMEPA1 gene were determined
based on the comparison of the sequences of PMEPA1 and the two
genomic clones. A protein motif search using ProfileScan
(http://www.ch.embnet.org/cgi-bin/- TMPRED) indicated the existence
of a type Ib transmembrane domain between amino acid residues 9 to
25 of the PMEPA1 sequence. Another GenBank search further revealed
that the PMEPA1 showed homology (67% sequence identity and 70%
positives at protein level) to a recently described novel cDNA
located on chromosome 18 (accession number NM.sub.--004338)
(Yoshikawa et al., 1998), as set forth below in Table 2. In
addition to the sequence similarity, PMEPA1 also shares other
features with C18orf1, e.g., similar size of the predicted protein
and similar transmembrane domain as the .beta.1 isoform of
C18orf1.
4TABLE 2 2 AELEFVQIIIIVVVMMVMVVVITCLLSHYKLSARSFISRH-
SQGRRREDALSSEGCLWPSE 61 PMEPA1 (SEQ ID NO: 11) AELEF QIIIIVVV V
VVVITCLL+HYK+S RSFI+R +Q RRRED L GCLWPS+ 3
AELEFAQIIIIVVVVTVMVVVIVCLLNHYKVSTRSFINRPNQSRRREDGLPQEGCLWPSD 62
C18orf1 (SEQ ID NO: 12) 62 STVSGNGIPEPQVYAPPRPTDRLAVPPFAQRERFHRFQ-
PTYPYLQHEIDLPPTISLSDGE 121 PMEPA1 S G E + PR DR P F QR+RF
RFQPTYPY+QHEIDLPPTISLSDGE 63 SAAPRLGASE--IMHAPRSRDRFTAPSFIQR-
DRFSRFQPTYPYVQHEIDLPPTISLSDGE 120 C18orf1 122
EPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLMDSARL-GGPCPPSSNSGIS 180
PMEPA1 EPPPYQGPCTLQLRDPEQQ+ELNRESVRAPPNRTIFDSDL+D A GGPCPPSSNSGIS
121 EPPPYQGPCTLQLRDPEQQMELNRESVRAPPNRTIFDSDLIDIAMYSGGPCPPSSNSGIS
180 C18orf1 181 ATCYGSGGRMEGPPPTYSEVIGHYPGSSFQHQQSSGPPSLLEGTR-
LHHTHIAPLESAAIW 240 PMEPA1 A+ S GRMEGPPPTYSEV+GH+PG+SF H Q S + G+RL
ES + 181 ASTCSSNGRMEGPPPTYSEVMGHHPGASFLHHQRS---NAHR-
GSRLQFQQ-NNAESTIVP 236 C18orf1 241 SKEKDKQKGH 250 PMEPA1 K KD++ G+
237 IKGKDRKPGN 246 C18orf1 In Table 2, a "+" denotes conservative
substitution.
[0155] Analysis of PMEPA1 Expression
[0156] Northern hybridization revealed two transcripts of
.about.2.7 kb and .about.5 kb using either PMEPA1 cDNA or oligo
probe. The signal intensity of bands representing these two
transcripts was very similar on the X-ray films of the northern
blots. RT-PCR analysis of RNA from LNCaP cells with four pairs of
primers covering different regions of PMEPA1 protein coding region
revealed expected size of bands from PCR reactions, suggesting that
two mRNA species on northern blot have identical sequences in the
protein coding region and may exhibit differences in 5' and/or
3'non-coding regions. However, the exact relationship between the
two bands remains to be established. Analysis of multiple northern
blots containing 23 human normal tissues revealed the highest level
of PMEPA1 expression in prostate tissue. Although other tissues
expressed PMEPA1, their relative expression was significantly lower
as compared to prostate (FIG. 1). In situ RNA hybridization
analysis of PMEPA1 expression in prostate tissues revealed abundant
expression in the glandular epithelial compartment as compared to
the stromal cells. However, both normal and tumor cells in tissue
sections of primary tumor tissues revealed similar levels of
expression.
[0157] Androgen Dependent Expression of PMEPA1
[0158] As discussed above, PMEPA1 was originally identified as a
SAGE tag showing the highest fold induction (29-fold) by androgen.
Androgen depletion of LNCaP cells resulted in decreased expression
of PMEPA1. Androgen supplementation of the LNCaP cell culture media
lacking androgen caused induction of both .about.2.7 and .about.5.0
bp RNA species of PMEPA1 in LNCaP cells in a dose and time
dependent fashion (FIG. 2A). Basal level of PMEPA1 expression was
detected in normal prostatic epithelial cell cultures and
androgen-dependent LNCaP cells cultured in regular medium. PMEPA1
expression was not detected in AR negative CaP cells. PC3 or in the
breast cancer cell line, MCF7 (FIG. 2B).
[0159] Evaluation of PMEPA1 Expression in Androgen Sensitive and
Androgen Refractory Tumors of CWR 22 Prostate Cancer Xenograft
Model
[0160] Previous studies have described increased expression of ARGs
in the "hormone refractory" CWR22R variants of the CWR22 xenograft,
suggesting the activation of AR mediated cell signaling in relapsed
CWR22 tumors following castration. The androgen sensitive CWR2
tumor expressed detectable level of PMEPA1 transcripts. However,
three of the four CWR22R tumors exhibited increased PMEPA1
expression (FIG. 3).
[0161] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification
which are hereby incorporated by reference. The embodiments within
the specification provide an illustration of embodiments of the
invention and should not be construed to limit the scope of the
invention. The skilled artisan readily recognizes that many other
embodiments are encompassed by the invention.
5TABLE 3 Genes Regulated by Androgen: SAGE Data Derived from CPDR
SAGE Library Accession Description Effect of Androgen AA310984 EST
Up-regulated by Androgen M26663 Homo sapiens prostate-specific
antigen mRNA, Up-regulated by Androgen complete cds.* AA508573
Human nucleolin gene, complete cds Up-regulated by Androgen
AB020637 Homo sapiens mRNA for KIAA0830 protein, partial
Up-regulated by Androgen cds. AA280663 EST Up-regulated by Androgen
U31657 KRAB-associated protein 1 Up-regulated by Androgen AI879709
EST Up-regulated by Androgen AA602190 EST Up-regulated by Androgen
AF035587 Homo sapiens X-ray repair cross-complementing Up-regulated
by Androgen protein 2 (XRCC2) AF151898 Homo sapiens CGI-140 protein
mRNA Up-regulated by Androgen AA418786 No reliable matches, only
see in two linberary (1 Up-regulated by Androgen each) AI308812 EST
Up-regulated by Androgen X59408 Membrane cofactor protein (CD46,
trophoblast- Up-regulated by Androgen lymphocyte cross-reactive
antigen) X81817 Accessory proteins BAP31/BAP29 Up-regulated by
Androgen AF071538 Homo sapiens Ets transcription factor PDEF
Up-regulated by Androgen (PDEF) mRNA, complete NM_003201
Transcription factor 6-like 1 (mitochondrial Up-regulated by
Androgen transcription factor 1-like) U41387 Human Gu protein mRNA,
partial cds. Up-regulated by Androgen U58855 Guanylate cyclase 1,
soluble, alpha 3 Up-regulated by Androgen X12794 Human v-erbA
related ear-2 gene. Up-regulated by Androgen U88542 Mus musculus
homeobox protein Nkx3.1 Up-regulated by Androgen D89729 Homo
sapiens mRNA for CRM1 protein, complete Up-regulated by Androgen
cds. U75329 TMPRSS2 Up-regulated by Androgen AA062976 EST
Up-regulated by Androgen L12168 Homo sapiens adenylyl
cyclase-associated protein Up-regulated by Androgen (CAP) mRNA
AA043945 EST Up-regulated by Androgen AF026291 Homo sapiens
chaperonin containing t-complex Up-regulated by Androgen
polypeptide 1, delta AB002301 Human mRNA for KIAA0303 gene, partial
cds. Up-regulated by Androgen D13643 Human mRNA for KIAA0018 gene,
complete cds. Up-regulated by Androgen AI310341 EST Up-regulated by
Androgen U49436 Human translation initiation factor 5 (eIF5) mRNA,
Up-regulated by Androgen complete cds S79862 Proteasome (prosome,
macropain) 26S subunit, non- Up-regulated by Androgen ATPase, 5
M14200 Human diazepam binding inhibitor (DBI) mRNA, Up-regulated by
Androgen complete cds. AA653318 FK506-binding protein 5
Up-regulated by Androgen L07493 Homo sapiens replication protein A
14kDa subunit Up-regulated by Androgen (RPA) mRNA, AJ011916 Homo
sapiens mRNA for hypothetical protein. Up-regulated by Androgen
AA130537 EST Up-regulated by Androgen D16373 Human mRNA for
dihydrolipoamide Up-regulated by Androgen succinyltransferase,
complete cds. AL096857 Novel human mRNA from chromosome 1
Up-regulated by Androgen AF007157 Homo sapiens clone 23856 unknown
mRNA, partial Up-regulated by Androgen cds. AA425929 NADH
dehydrogenase (ubiquinone) 1 beta Up-regulated by Androgen
subcomplex, 10 (22kD, PDSW) AI357815 EST Up-regulated by Androgen
D83778 Human mRNA for KIAA0194 gene, partial cds. Up-regulated by
Androgen AF000979 Homo sapiens testis-specific Basic Protein Y 1
Up-regulated by Androgen (BPY1) mRNA, AA889510 EST Up-regulated by
Androgen AB018330 Homo sapiens mRNA for KIAA0787 protein, partial
Up-regulated by Androgen cds. AA026941 EST Up-regulated by Androgen
AA532377 Chromosome 1 open reading frame 8 Up-regulated by Androgen
AF010313 Homo sapiens Pig8 (PIG8) mRNA (etoposide- Up-regulated by
Androgen induced mRNA), complete cds. L06328 Human
voltage-dependent anion channel isoform 2 Up-regulated by Androgen
(VDAC) mRNA, U41804 Human putative T1/ST2 receptor binding protein
Up-regulated by Androgen precursor mRNA, AB020676 Homo sapiens mRNA
for KIAA0869 protein, partial Up-regulated by Androgen cds. J03503
Human pyruvate dehydrogenase E1-alpha subunit Up-regulated by
Androgen mRNA, cds. AA421098 EST Up-regulated by Androgen AF072836
Sox-like transcriptional factor Up-regulated by Androgen AA115355
EST Up-regulated by Androgen AF118240 Homo sapiens, peroxisomal
biogenesis factor 16 Up-regulated by Androgen (PEX16) mRNA,
complete AA011178 EST Up-regulated by Androgen X15573 Human
liver-type 1-phosphofructokinase (PFKL) Up-regulated by Androgen
mRNA, complete cds. AA120930 EST Up-regulated by Androgen AB002321
Human mRNA for KIAA0323 gene, partial cds Up-regulated by Androgen
AF151837 Homo sapiens CGI-79 protein mRNA, complete cds
Up-regulated by Androgen AA481027 EST Up-regulated by Androgen
AA039343 EST Up-regulated by Androgen U09716 Human mannose-specific
lectin (MR60) mRNA, Up-regulated by Androgen complete cds. AF044773
Homo sapiens breakpoint cluster region protein 1 Up-regulated by
Androgen (BCRG1) mRNA U51586 Human siah binding protein 1 (SiahBP1)
mRNA, Up-regulated by Androgen partial cds. M36341 Human
ADP-ribosylation factor 4 (ARF4) mRNA, Up-regulated by Androgen
complete cds. AI282096 EST Up-regulated by Androgen W45510 RAB7,
member RAS oncogene family-like 1 Up-regulated by Androgen X16135
Human mRNA for novel heterogeneous nuclear RNP Up-regulated by
Androgen protein, L protein AF052134 Homo sapiens clone 23585 mRNA
sequence, Up-regulated by Androgen AF052134 D26068 Williams-Beuren
syndrome chromosome region 1 Up-regulated by Androgen X69433
H.sapiens mRNA for mitochondrial isocitrate Up-regulated by
Androgen dehydrogenase (NADP+). X61123 B-cell translocation gene 1,
anti-proliferative Up-regulated by Androgen X63423 H.sapiens mRNA
for delta-subunit of mitochondrial Up-regulated by Androgen F1F0
ATP-synthase AJ010025 Homo sapiens mRNA for unr-interacting
protein. Down-regulated by Androgen AF003938 Homo sapiens
thioredoxin-like protein mRNA, Down-regulated by Androgen complete
cds. AB014536 Homo sapiens copine III (CPNE3) mRNA Down-regulated
by Androgen AA504468 EST Down-regulated by Androgen NM_001273
Chromodomain helicase DNA binding protein 4 Down-regulated by
Androgen AA015746 Homo sapiens mRNA; cDNA DKFZp586H0722
Down-regulated by Androgen (from clone DKFZp586H0722) AA552354 EST
Down-regulated by Androgen AA025744 3-prime-phosphoadenosine
5-prime-phosphosulfate Down-regulated by Androgen synthase 2 X71129
H.sapiens mRNA for electron transfer flavoprotein Down-regulated by
Androgen beta subunit AA046050 EST Down-regulated by Androgen
U57052 Human Hoxb-13 mRNA, complete cds Down-regulated by Androgen
AA400137 EST Down-regulated by Androgen AA487586 EST Down-regulated
by Androgen J04208 Human inosine-5'-monophosphate dehydrogenase
Down-regulated by Androgen (IMP) mRNA M64722 Testosterone-repressed
prostate message 2 Down-regulated by Androgen (apolipoprotein J)
AI743483 EST Down-regulated by Androgen AA476914 EST Down-regulated
by Androgen AA026691 EST Down-regulated by Androgen AI014986 EST
Down-regulated by Androgen X85373 Small nuclear ribonucleoprotein
polypeptide G Down-regulated by Androgen U07231 G-rich RNA sequence
binding factor 1 Down-regulated by Androgen T97753 Glycogen
synthase 2 (liver) Down-regulated by Androgen AA234050 EST
Down-regulated by Androgen AI015143 EST Down-regulated by Androgen
U09196 Human 1.1 kb mRNA upregulated in retinoic acid
Down-regulated by Androgen treated HL-60 neutrophilic cells.
AA977749 EST Down-regulated by Androgen NM_006451 Polyadenylate
binding protein-interacting protein 1 Down-regulated by Androgen
AI818296 EST Down-regulated by Androgen AI250561 EST Down-regulated
by Androgen AA063613 EST Down-regulated by Androgen U59209
Hs.183596: UDP glycosyltransferase 2 family, Down-regulated by
Androgen polypeptide B17, U59209 Z11559 Iron-responsive element
binding protein 1 Down-regulated by Androgen AF052578 Homo sapiens
androgen receptor associated protein Down-regulated by Androgen 24
(ARA24) X16312 Human mRNA for phosvitin/casein kinase II beta
Down-regulated by Androgen subunit. H17890 PCTAIRE protein kinase 3
Down-regulated by Androgen AA192312 EST Down-regulated by Androgen
AA043787 EST Down-regulated by Androgen AI052020 EST Down-regulated
by Androgen AB014512 Homo sapiens mRNA for KIAA0612 protein
Down-regulated by Androgen NM_001328 Homo sapiens C-terminal
binding protein 1 (CTBP1) Down-regulated by Androgen mRNA M15919
Human autoimmune antigen small nuclear Down-regulated by Androgen
ribonucleoprotein E mRNA. AF151813 Homo sapiens CGI-55 protein
mRNA, complete cds Down-regulated by Androgen L41351 Protease,
serine, 8 (prostasin) Down-regulated by Androgen AF077046 Homo
sapiens ganglioside expression factor 2 (GEF- Down-regulated by
Androgen 2) homolog U15008 Small nuclear ribonucleoprotein D2
polypeptide Down-regulated by Androgen (16.5kD), AA938995 N62491
Folate hydrolase (prostate-specific membrane Down-regulated by
Androgen antigen) 1 AI569591 EST Down-regulated by Androgen
AJ131245 Secretory protein 24 (SEC24). Down-regulated by Androgen
U90543 Human butyrophilin (BTF1) mRNA, complete cds. Down-regulated
by Androgen Z47087 Transcription elongation factor B (SIII),
polypeptide Down-regulated by Androgen 1-like M34539 FK506-binding
protein 1A (12kD) Down-regulated by Androgen N43807 yy19a05.r1
Soares melanocyte 2NbHM Homo Down-regulated by Androgen sapiens
cDNA clone U03269 Human actin capping protein alpha subunit (CapZ)
Down-regulated by Androgen mRNA, complete AI571685 EST
Down-regulated by Androgen AA010412 EST Down-regulated by Androgen
L40403 Homo sapiens (clone zap3) mRNA, 3' end of cds.
Down-regulated by Androgen NM_006560 CUG triplet repeat,
RNA-binding protein 1 Down-regulated by Androgen NM_004713
Serologically defined colon cancer antigen 1 Down-regulated by
Androgen U36188 Clathrin-associated/assembly/ada- ptor protein,
Down-regulated by Androgen medium 1 AB020721 KIAA0914 gene product
Down-regulated by Androgen T35365 EST Down-regulated by Androgen
AF029789 Homo sapiens GTPase-activating protein (SIPA1)
Down-regulated by Androgen mRNA, complete cds. AA427857 EST
Down-regulated by Androgen AA910404 EST Down-regulated by Androgen
L42379 Quiescin Q6 (bone-derived growth factor) Down-regulated by
Androgen AL117641 cDNA DKFZp434L235 Down-regulated by Androgen
AI688119 EST Down-regulated by Androgen AA688073 EST Down-regulated
by Androgen NM_002945 Replication protein A1 (70kD) Down-regulated
by Androgen AI797610 EST Down-regulated by Androgen AF086095 Homo
sapiens full length insert cDNA clone Down-regulated by Androgen
YZ88A07. AF070666 Homo sapiens tissue-type pituitary Kruppel-
Down-regulated by Androgen associated box protein R55128 Proteasome
(prosome, macropain) 26S subunit, non- Down-regulated by Androgen
ATPase, 2 X75621 Tuberous sclerosis 2 Down-regulated by Androgen
AA019070 EST Down-regulated by Androgen AI089867 EST Down-regulated
by Androgen NM_001003 Homo sapiens ribosomal protein, large, P1
(RPLP1) Down-regulated by Androgen mRNA L05093 Ribosomal protein
L18a Down-regulated by Androgen AA854176 EST Down-regulated by
Androgen AI929622 Homo sapiens clone 23675 mRNA sequence
Down-regulated by Androgen AI264769 ESTs, Weakly similar to ORF
YDL087c Down-regulated by Androgen [S.cerevisiae] L09159 Ras
homolog gene family, member A, may be Down-regulated by Androgen
androgen regulated? AI143187 EST Down-regulated by Androgen H17900
cDNA DKFZp586H051 (from clone Down-regulated by Androgen
DKFZp586H051) NM_005617 Ribosomal protein S14 Down-regulated by
Androgen L49506 Cyclin G2 Down-regulated by Androgen AA614448
Regulator of G-protein signalling 5 Down-regulated by Androgen
S83390 T3 receptor-associating cofactor-1 Down-regulated by
Androgen AA917672 EST Down-regulated by Androgen X52151
Arylsulphatase A Down-regulated by Androgen U09646 Carnitine
palmitoyltransferase II Down-regulated by Androgen Z50853
ATP-dependent protease ClpAP (E. coli), proteolytic Down-regulated
by Androgen subunit, human AB023208 MLL septin-like fusion
Down-regulated by Androgen U92014 Human clone 121711 defective
mariner transposon Down-regulated by Androgen Hsmar2 mRNA AA878293
Alpha-1-antichymotrypsin Down-regulated by Androgen AA554191 EST
Down-regulated by Androgen M55618 Hexabrachion (tenascin C,
cytotactin) Down-regulated by Androgen AA027050 EST Down-regulated
by Androgen AF112472 Homo sapiens calcium/calmodulin-dependent
protein Down-regulated by Androgen kinase II beta AA583866 EST
Down-regulated by Androgen AA115687 EST Down-regulated by Androgen
AA043318 EST Down-regulated by Androgen U90329 Poly(rC)-binding
protein 2 Down-regulated by Androgen Y00815 Protein tyrosine
phosphatase, receptor type, F Down-regulated by Androgen X76013
H.sapiens QRSHs mRNA for glutaminyl-tRNA Down-regulated by Androgen
synthetase. X75861 Testis enhanced gene transcript Down-regulated
by Androgen AA593078 Homo sapiens PAC clone DJ0167F23 from 7p15
Down-regulated by Androgen J04058 Human electron transfer
flavoprotein alpha-subunit Down-regulated by Androgen mRNA AF026292
Homo sapiens chaperonin containing t-complex Down-regulated by
Androgen polypeptide 1, eta AF068754 Homo sapiens heat shock factor
binding protein 1 Down-regulated by Androgen HSBP1 mRNA, NM_000172
Guanine nucleotide binding protein (G protein), Down-regulated by
Androgen alpha transducing activity polypeptide 1 AI140631 Hs.1915:
folate hydrolase (prostate-specific Down-regulated by Androgen
membrane antigen) 1 Bold font indicates known androgen-regulated
gene based on Medline Search.
[0162]
6TABLE 4 Potential Prostate Specific/Abundant Genes Derived From
NCBI and CPDR SAGE Libraries Accession Description M88700 Human
dopa decarboxylase (DDC) gene, complete cds. W45526 zc26b04.r1
Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA, Hs.108981:
ficolin (collagen/fibrinogen domain-containing) 1, AF201077
NADH:ubiquinone oxidoreductase MLRQ subunit (NDUFA4) mRNA, complete
cds with polyA. D55953 HUM407H12B Clontech human fetal brain polyA
+ mRNA (#6535) Homo, Hs.118724: histidine triad nucleotide-binding
protein, AJ012499, mRNA activated in tumor suppression, clone
TSAP19 with polyA AA082804 zn41g02.r1 Stratagene endothelial cell
937223 Homo sapiens cDNA, Hs.110967: ESTs, Weakly similar to
KIAA0762 protein [H.sapiens], Hs.5662: guanine nucleotide binding
protein (G protein), beta polypeptide 2-like 1 in the sequence no
tag X05332 Human mRNA for prostate specific antigen.* AI278854
qo42f01.x1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE:1911193 3',
NM_004537 nucleosome assembly protein 1-like 1 (NAPIL1), tag is at
beginning of the gene. W75950 zd58b02.r1 Soares_fetal_heart_NbHH19W
Homo sapiens cDNA clone, AF151840, CGI- 82 protein mRNA, tag is at
3' end. F02980 HSC1IC062 normalized infant brain cDNA Homo sapiens
cDNA clone M99487 Human prostate-specific membrane antigen (PSM)
mRNA, complete cds. AL035304 H.sapiens gene from PAC 295C6, similar
to rat PO44. AI088979 ou86f03.s1 Soares_NSF_F8_9W_OT_PA_P_S1 Homo
sapiens cDNA clone AF186249 Homo sapiens six transmembrane
epithelial antigen of prostate (STEAP1) mRNA C15801 C15801 Clontech
human aorta polyA + mRNA (#6572) Homo sapiens cDNA L10340 Human
elongation factor-1 alpha (ef-1) mRNA, 3' end. NM_004540 Homo
sapiens neural cell adhesion molecule 2 (NCAM2) AA151796 z139c02.r1
Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone NM_001634 Homo
sapiens S-adenosylmethionine decarboxylase 1 (AMD1) NM_005013 Homo
sapiens nucleobindin 2 (NUCB2)AL121913 in GenBank htgc database)
and 718J7 (Accession number AL035541 AF004828 Homo sapiens rab3-GAP
regulatory domain mRNA, complete cds. X60819 X60 H.sapiens DNA for
monoamine oxidase type A (14) (partial). AA133972 z138g12.r1
Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone M69226 Human
monoamine oxidase (MAOA) mRNA, complete cds. AA969141 op50c11.s1
Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone AA523652 ni64d09.s1
NCI_CGAP_Pr12 Homo sapiens cDNA clone IMAGE:981617, mRNA AF078749
Homo sapiens organic cation transporter 3 (SLC22A3) AA583544
nf25h10.s1 NCI_CGAP_Pr1 Homo sapiens cDNA clone IMAGE:914851, mRNA
AF051894 Homo sapiens 15 kDa selenoprotein mRNA, complete cds.
AF165967 Homo sapiens DDP-like protein mRNA X57129 H.sapiens H1.2
gene for histone H1. AA640928 nr28d08.r1 NCI_CGAP_Pr3 Homo sapiens
cDNA clone IMAGE:1169295, mRNA U41766 Human
metalloprotease/disintegrin/cysteine-rich protein precursor
AF023676 Homo sapiens lamin B receptor homolog TM7SF2 (TM7SF2)
mRNA, U10691 Human MAGE-6 antigen (MAGE6) gene, complete cds.
M22976 Human cytochrome b5 mRNA, 3' end. L14778 Human
calmodulin-dependent protein phosphatase catalytic subunit AF071538
Homo sapiens Ets transcription factor PDEF (PDEF) mRNA, complete
U39840 Human hepatocyte nuclear factor-3 alpha (HNF-3 alpha) mRNA,
AA532511 nj54d03.s1 NCI_CGAP_Pr9 Homo sapiens cDNA clone
IMAGE:996293, mRNA X07166 Human mRNA for enkephalinase (EC
3.424.11). M96684 H.sapiens Pur (pur-alpha) mRNA, complete cds.
AI204040 qe77f05.x1 Soares_fetal_lung_NbHL19W Homo sapiens cDNA
clone AA577923 nl20a01.s1 NCI_CGAP_HSC1 Homo sapiens cDNA clone
IMAGE:1041192, AA569633 nm38h09.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA
clone IMAGE:1062497, U65011 Human preferentially expressed antigen
of melanoma (PRAME) mRNA, U21910 Human basic transcription factor
BTF2p44 mRNA, 3' end, partial cds. AA633187 nq07c12.s1 NCI_CGAP_Lu1
Homo sapiens cDNA clone IMAGE:1143190 3' AF000993 Homo sapiens
ubiquitous TPR motif, X isoform (UTX) mRNA, W76105 zd65b04.r1
Soares_fetal_heart_NbHHI9W Homo sapiens cDNA clone H39906
yo54a07.r1 Soares breast 3NbHBst Homo sapiens cDNA clone AA971717
op95c11.s1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE:1584596 3',
M68891 Human GATA-binding protein (GATA2) mRNA, complete cds.
AA310157 EST181013 Jurkat T-cells V Homo sapiens cDNA 5' end, mRNA
sequence. X00948 Human mRNA for prepro-relaxin H2. AB018330 Homo
sapiens mRNA for KIAA0787 protein, partial cds. AA890637 ak11e11.s1
Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone M64929 J05
Human protein phosphatase 2A alpha subunit mRNA, complete cds.
W24341 zb81h12.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens
cDNA AA974479 od58b03.s1 NCI_CGAP_GCB1 Homo sapiens cDNA clone
IMAGE:1372109 3' R31644 yh69e05.r1 Soares placenta Nb2HP Homo
sapiens cDNA clone AA573246 nm52c02.s1 NCI_CGAP_Br2 Homo sapiens
cDNA clone IMAGE:1071842 3', AA507635 ng84b02.s1 NCI_CGAP_Pr6 Homo
sapiens cDNA clone IMAGE:941451, mRNA gb.vertline.AF008915 Homo
sapiens EVI5 homolog mRNA AL049987 Homo sapiens mRNA; cDNA
DKFZp564F112 (from clone DKFZp564F112). U81599 Homo sapiens
homeodomain protein HOXB13 mRNA AA641596 nr20f05.s1 NCI_CGAP_Pr2
Homo sapiens cDNA clone IMAGE:1168545, mRNA D84295 Human mRNA for
possible protein TPRDII R13859 yf65d08.r1 Soares infant brain 1NIB
Homo sapiens cDNA clone M34840 Human prostatic acid phosphatase
mRNA, complete cds. AA572913 nm42f12.s1 NCI_CGAP_Pr4.1 Homo sapiens
cDNA clone IMAGE:1062863, AA094460 cp0378.seq.F Human fetal heart,
Lambda ZAP Express Homo sapiens AF031166 Homo sapiens SRp46
splicing factor retropseudogene mRNA. AA625147 af70c07.r1
Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:1047372 T39510
ya06h07.r1 Stratagene placenta (#937225) Homo sapiens cDNA clone
R35034 yg60h03.r1 Soares infant brain 1NIB Homo sapiens cDNA clone
AI003674 zg01c04.s1 Soares_pineal_gland_N3HPG Homo sapiens cDNA
clone AJ003636 AJ003636 Selected chromosome 21 cDNA library Homo
sapiens cDNA AA601385 no16d12.s1 NCI_CGAP_Phe1 Homo sapiens cDNA
clone IMAGE:1100855 3', AF191339 Homo sapiens anaphase-promoting
complex subunit 5 (APC5) AA431822 zw79d02.r1 Soares_testis_NHT Homo
sapiens cDNA clone IMAGE:782403 NM_003909 Homo sapiens copine III
(CPNE3) AA484004 ne73f04.s1 NCI_CGAP_Ew1 Homo sapiens cDNA clone
IMAGE:909919 AA535774 nj78f08.s1 NCI_CGAP_Pr10 Homo sapiens cDNA
clone IMAGE:998631, mRNA NM_000944.1 Homo sapiens protein
phosphatase 3 (formerly 2B) AA702811 zi90c11.s1
Soares_fetal_liver_spleen_1NFLS_S1 Homo sapiens cDNA X95073
H.sapiens mRNA for translin associated protein X. AA029039
zk12b07.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
AF032887 Homo sapiens forkhead (FKHRL1P1) pseudogene N46609
yy48h08.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA
U58855 Homo sapiens soluble guanylate cyclase large subunit
(GC-S-alpha-1) AA255486 zr83d03.s1 Soares_NhHMPu_S1 Homo sapiens
cDNA clone IMAGE:682277 AA128153 zl15c06.s1
Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AA016039
ze31c05.s1 Soares retina N2b4HR Homo sapiens cDNA clone R88520
ym91e09.s1 Soares adult brain N2b4HB55Y Homo sapiens cDNA clone
M26624 Human CALLA/NEP gene encoding neutral endopeptidase, exon
20. AA026997 ze99e01.r1 Soares_fetal_heart_NbHH19W Homo sapiens
cDNA clone W48775 zc44b08.r1 Soares_senescent_fibroblasts_NbHSF
Homo sapiens cDNA AA074407 zm15c08.r1 Stratagene pancreas (#937208)
Homo sapiens cDNA clone L13972 Homo sapiens beta-galactoside
alpha-2,3-sialyltransferase (SIAT4A) D14661 Human mRNA for KIAA0105
gene, complete cds. AA115452 zk89a08.r1
Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AA495742
zw04b12.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:768287 5'
R13416 yf75h09.r1 Soares infant brain 1NIB Homo sapiens cDNA clone
AA046369 zk77h07.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA
clone T35440 EST85129 Human Lung Homo sapiens cDNA 5' end similar
to None, mRNA AI075860 oz25b05.x1 Soares_total_fetus_Nb2HF8_9w Homo
sapiens cDNA clone W56437 zc57g05.r1 Soares_parathyroid_tumor_NbHP-
A Homo sapiens cDNA clone AI583880 tt70b02.x1 NCI_CGAP_HSC3 Homo
sapiens cDNA clone IMAGE:2246091 3' D85181 Homo sapiens mRNA for
fungal sterol-C5-desaturase homolog, complete AF105714 Homo sapiens
protein kinase PITSLRE (CDC2L2) gene, exon 3. AA401802 zt60c12.r1
Soares_testis_NHT Homo sapiens cDNA clone IMAGE:726742 AB002301
Human mRNA for KIAA0303 gene, partial cds. U75667 Human arginase II
mRNA, complete cds. AA585183 JTH089 HTCDL1 Homo sapiens cDNA 5'/3',
mRNA sequence. AF191771 Homo sapiens CED-6 protein (CED-6) mRNA
AA650252 ns93g05.s1 NCI_CGAP_Pr3 Homo sapiens cDNA clone
IMAGE:1191224, mRNA R64618 yi19b09.r1 Soares placenta Nb2HP Homo
sapiens cDNA clone N24627 yx89a09.s1 Soares melanocyte 2NbHM Homo
sapiens cDNA clone AB028951 Homo sapiens mRNA for KIAA1028 protein
N75608 yw37a07.r1 Morton Fetal Cochlea Homo sapiens cDNA clone
N53899 yy98e03.r1 Soares_multiple_sclerosis_2Nb- HMSP Homo sapiens
cDNA N46696 yy50f07.r1 Soares_multiple_sclerosis_- 2NbHMSP Homo
sapiens cDNA AA419522 zv03d05.r1 Soares_NhHMPu_S1 Homo sapiens cDNA
clone IMAGE:752553 M61906 Human P13-kinase associated p85 mRNA
sequence. C16570 C16570 Clontech human aorta polyA + mRNA (#6572)
Homo sapiens cDNA X63105 H.sapiens tpr mRNA. AA315855 EST187656
Colon carcinoma (HCC) cell line II Homo sapiens cDNA 5' L18920
Human MAGE-2 gene exons 1-4, complete cds. M25161 Human Na,K-ATPase
beta subunit (ATP1B) gene AA164865 zq41g07.r1 Stratagene hNT neuron
(#937233) Homo sapiens cDNA clone N40094 yx98g07.r1 Soares
melanocyte 2NbHM Homo sapiens cDNA clone N98940 yy71a07.r1
Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA AF049907 Homo
sapiens zinc finger transcription factor (ZNF-X) mRNA, M78806
EST00954 Hippocampus, Stratagene (cat. #936205) Homo sapiens cDNA
AA040819 zk47b03.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA
clone C15445 C15445 Clontech human aorta polyA + mRNA (#6572) Homo
sapiens cDNA AB018309 Homo sapiens mRNA for KIAA0766 protein,
complete cds. AJ011497 Homo sapiens mRNA for Claudin-7. X00949
Human mRNA for prepro-relaxin H1. AA418633 zv93d09.r1
Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767345 5' AI146806
qb83h04.x1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone
X82942 H.sapiens satellite 3 DNA. AA456383 aa14f03.r1
Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:813245 AA019341
ze57e04.s1 Soares retina N2b4HR Homo sapiens cDNA clone AB027466
Homo sapiens SPON2 mRNA for spondin 2 AF038170 Homo sapiens clone
23817 mRNA sequence. NM_000240 Homo sapiens monoamine oxidase A
(MAOA) N34126 yx76c01.r1 Soares melanocyte 2NbHM Homo sapiens cDNA
clone N41339 yw68g06.r1 Soares_placenta_8to9weeks_2NbHP8to9W Homo
sapiens cDNA R34783 yh87b05.r1 Soares placenta Nb2HP Homo sapiens
cDNA clone N75858 yw32a03.r1 Morton Fetal Cochlea Homo sapiens cDNA
clone AA633887 ac32h04.s1 Stratagene hNT neuron (#937233) Homo
sapiens cDNA clone N53723 yz06d03.r1 Soares_multiple_sclerosi-
s_2NbHMSP Homo sapiens cDNA AI187365 qf29b12.x1 Soares_testis_NHT
Homo sapiens cDNA clone IMAGE:1751423 Genes in bold type are known
prostate-specific genes.
[0163]
7TABLE 5 Genes/ESTs as Defined by Publications: Including Androgen
Signaling, Prostate Specificity, Prostate Cancer Association, and
Nuclear Receptors/Regulators with Potential Interaction with
Androgen Receptor Cluster ID Gene Name Description References
Hs.81988 DOC-2 deliion of ovarian Up-regulated by Androgen Ablation
Endocrinology, carcinoma 2 139, 3542, 98 Hs.155389 RAR a
Up-regulated by Androgen Ablation endocrinology, 138, 553, 97
Hs.12601 AS3 DNA binding protein Up-regulated by Androgen Ablation
J Steroid Biochem Mol Biol 68, 41, 99 Hs.181426 EST Up-regulated by
Androgen Ablation Hs.2391 apical protein Up-regulated by Androgen
Ablation Hs.109530 KGF/FGF7 keratinocyte growth Up-regulated by
Androgen BBRC 220, 858, 96, factor Can Res, 54, 5474, 94 Hs.1104
TGF beta 1 Up-regulated by Androgen Endocrinology, 137, 99, 96,
Endocrinology, 139, 378, 98 Hs.75525 Calreticulin Calreticulin
Up-regulated by Androgen Can Res 59, 1896, 99 Hs.78888 DBI/ACBP
Diazepam-binding Up-regulated by Androgen JBC, 237, 19938, 98
inhibitor/acyl-CoA binding Protein Hs.41569 Phosphatidic acid
Up-regulated byAndrogen JBC, 273, 4660, 98 phosphatase type 2a
isozyme Hs.83190 Fatty acid synthase Up-regulated by Androgen Can
Res, 57, 1086, 97 Hs.99915 Androgen Receptor Up-regulated by
Androgen Steroids 9, 531, 96 Hs.2387 prostate-restricted
Up-regulated by Androgen Biochem J 315, 901, 96 transglutaminase
Hs.78996 PCNA proliferating cell Up-regulated by Androgen Can Res
56, 1539, 96 nuclear antigen Hs.74456 GAPDH Up-regulated by
Androgen Can Res 55, 4234, 95 Hs.82004 E cadherin Up-regulated by
Androgen BBRC, 212, 624, 95 Hs.57710 AIGF Androgen-induced
Up-regulated by Androgen FEBS lett 363, 226, 95 growth factor
Hs.118618 MIC2 humanpseudoauto- Up-regulated by Androgen Mol
Carcinog, somal gene? 23, 13, 98 Hs.18420 Talin cytoskeletal
protein Up-regulated by Androgen FEBS lett 434, 66, 98 Hs. 54502
clathrin heavy chain Up-regulated by Androgen Endocrinology, 139,
2111, 98 Hs.73919 clathrin light chain b Up-regulated by Androgen
Endocrinology, 139, 2111, 98 Hs.76506 L-plastin ESTs. Moderately
Up-regulated by Androgen Am J Pathol, 150, similar to L- 2009, 97
PLASTIN [H.sapiens] Hs.82173 EGR alpha TGFB inducible early
Up-regulated by Androgen Mol Endocrinol, growth response 9, 1610,
95 ND FGF10 Up-regulated by Androgen JBC, 274, 12827, 99 Hs.107169
IGFBP5 Up-regulated by Androgen Endocrinology, 140, 237 2, 99
Hs.179665 p21 Up-regulated by Androgen Mol Endocrinol, 13, 376, 99
Hs.51117 BMP-7 Up-regulated by Androgen Prostate, 37, 236, 98
Hs.73793 VEGF vascular endothelial Up-regulated by Androgen
Endocrinol, 139, 4672, 9 growth factor 8, BBRC, 251, 287, 98 Hs.166
SREBPs sterol regulatory Up-regulated by Androgen J Steroid Biochem
Mol element binding Biol, 65, 191, 98 transcription factor 1
Hs.116577 PDF prostate Up-regulated by Androgen JBC, 273, 13760, 98
differentiation factor Hs.1905 prolactin Prolactin Up-regulated by
Androgen FEBS J, 11, 1297, 97 Hs.19192 CDK2 Up-regulated by
Androgen Can Res, 57, 4511, 97 Hs.95577 CDK4 cyclin-dependent
Up-regulated by Androgen Can Res, 57, 4511, 97 kinase 4 Hs.183596
UGT2B17 uridine Up-regulated by Androgen Endocrinology,
diphosphoglucronosyl 138, 2998, 97 transferase Hs.150207 UGT2B15
UDP- Up-regulated by Androgen Can Res 57, 4075, 97
glucronosyltransferase 2B15 ND prostate binding protein
Up-regulated by Androgen PNAS, 94, 12999, 97 C2A (RAT) ND Probasin
(RAT) Up-regulated by Androgen PNAS, 94, 12999, 97 Hs.7719
prostatein C3 (RAT) Up-regulated by Androgen PNAS, 94, 12999, 97 ND
Cystatin related protein 1 Up-regulated by Androgen PNAS, 94,
12999, 97 (RAT) ND Cystatin related protein 2 Up-regulated by
Androgen PNAS, 94, 12999, 97 (RAT) Hs.394 Adrenomedulin (RAT)
Up-regulated by Androgen PNAS, 94, 12999, 97 Hs.77393 farnesyl
diphosphate Up-regulated by Androgen PNAS, 94, 12999, 97 synthase
(famesyl pyrophosphate synthetase, dimethylallyltranstrans- ferase)
Hs.153468 LDL receptor (Rat) Up-regulated by Androgen PNAS, 94,
12999, 97 ND. Hysto-blood group A Up-regulated by Androgen PNAS,
94, 12999, 97 transferase (RAT) Hs.196604 Sex limited protein
Up-regulated by Androgen PNAS, 94, 12999, 97 (RAT) slp ND prostatic
spermine Up-regulated by Androgen Mol Cell Endocrinol, binding
protein (RAT) 108, R1, 95 Hs.76353 Protein C Inhibitor Up-regulated
by Androgen FEBS lett, 492, 263, 98 Hs.203602 enolase alpha
Up-regulated by Androgen Can Res, 58, 5718, 98 Hs.169476 tubulin
alpha Up-regulated by Androgen Can Res, 58, 5718, 98 Hs.184572 Cdk1
Up-regulated by Androgen Can Res, 58, 5718, 98 Hs.107528 EST EST
similar to Up-regulated by Androgen androgen-regulated protein
FAR-17 Hs.28309 UDP-glucose Up-regulated by Androgen Endocrinology,
dehydrogenase 140, 10, 4486, (99) Hs.194270 secretory component
Up-regulated by Androgen Mol endocrinol, gene 13, 9, 1558, (99)
Hs.76136 Thioredoxin Up-regulated by Androgen J steroid Biochem Mol
Biol, 68, 5-6, 203, (99) Hs.3561 p27 Kip1 cyclin-dependent
Up-regulated by Androgen Mol kinase inhibitor 1B Endocrinol, 12,
941, 98 (p27, Kip1) Hs.1867 progastricsin Up-regulated by Androgen
J.B.C. 271, 15175, (99) (pepsinogen C) Hs.97411 hamster Androgen-
Up-regulated by Androgen Genebank dependent Expressed Protein like
protein gene Hs.155140 Protein kinase CK2 casein kinase 2, alpha
Translocated by Androgen Can Res 59, 1146, 99 1 polypeptide
IMAGE.95326 DD3 Prostate Specific Eur Urol, 35, 408, 99 2 Hs.218366
Prostase Prostate Specific PNAS, 96, 3114, 99 Hs.20166 PSCA
prostate stem cell Prostate Specific PNAS, 95, 1735, 98 antigen
Hs.171995 PSA kallikrein 3, (prostate Prostate Specific PNAS, 95,
300, 98, specific antigen) DNA Cell Biol. 16, 627, 97 Hs.183752
PSSPP prostate-secreted Prostate Specific PNAS, 95, 300, 98 seminal
plasma protein, nc50a10, microsemnoprotein beta, PSP94 Hs.1852 PAP
prostatic acid Prostate Specific PNAS, 95, 300, 98 phosphatase
Hs.52871 SYT Prostate Specific PNAS, 95, 300, 98 Hs.158309 Homoobox
HOX D13 Prostate Specific PNAS, 95, 300, 98 Hs.1968 Semenogelin 1
Prostate Specific PNAS, 95, 300, 98 Hs.76240 Adenylate kinase
adenylate kinase 1 Prostate Specific PNAS, 95, 300, 98 isoenzyme 1
Hs.184376 SNAP23 Prostate Specific PNAS, 95, 300, 98 Hs.82186
ERBB-3 receptor Prostate Specific PNAS, 95, 300, 98 protein-tyrosin
kinase Hs.180016 Semenogelin 2 Prostate Specific Hs.1915 PSMA
folate hydrolase Prostate Specific (prostate-specific membrane
antigen) 1 Hs.181350 KLK2 Prostate Specific Hs.73189 NKX3.1
Prostate Specific HPARJ1 Prostate Specific IMAGE:56577 9 Hs.76053
p68 RNA helicase Potential interaction with AR MCB, 19, 5363, (99)
Hs.111323 ARIP3 Potential interaction with AR JBC, 274, 3700, 99
Hs.25511 ARA54 Potential interaction with AR JBC274, 8319, 99
Hs.28719 ARA55 Potential interaction with AR JBC, 274, 8570, 99
HS.999908 ARA70 Potential interaction with AR PNAS, 93, 5517, 96
Hs.29131 TIF2 transcriptional Potential interaction with AR EMBO,
15, 3667, 96, intermediary factor 2 EMBO, 17, 507, 98 Hs.66394
SNURF ring finger protein 4 Potential interaction with AR MCB, 18,
5128, 98 Hs.75770 RB retinoblastoma 1 Potential interaction with AR
(including osteosarcoma) Hs.74002 SRC-1 steroid receptor Potential
interaction with AR coactivator 1 Hs.155017 RIP140 nuclear receptor
Potential interaction with AR EMBO, 14, 3741, 95, interacting
protein 1 Mol Endocrinol, 12, 864, 98 Hs.23598 CBP CREB binding
Potential interaction with AR protein (Rubinstein- Taybi syndrome)
Hs.25272 p300 EIA binding protein Potential interaction with AR
p300 Hs.78465 c-JUN Potential interaction with AR Hs.199041 ACTR
AIB1, mouse Potential interaction with AR M.C.B, 17, 2735, 97,
GRIP1, pCIP PNAS, 93, 4948, 96 Hs.6364 TIP60 Human tat interactive
Potential interaction with AR JBC, 274, 17599, 99 protein mRNA,
complete cds Hs.32587 SRA Potential interaction with AR Cell, 97,
17, 99 Hs.155302 PCAF Potential interaction with AR Hs.10842 ARA24
Potential interaction with AR Hs.41714 BAG-1L Potential interaction
with AR JBC, 237, 11660, 98 Hs.82646 dnaJ, HSP40 DNAJ PROTEIN
Potential interaction with AR HOMOLOG 1 Hs.43697 ERM ets variant
gene 5 Potential interaction with AR JBC, 271, 23907, 96
(ets-related molecule) Hs.75772 GR Potential interaction with AR
JBC, 272, 14087, 97 Hs.77152 MCM7 Potential interaction with AR ND
NJ Potential interaction with AR ND RAF Potential interaction with
AR JBC, 269, 20622, 94 ND TFIIF Potential interaction with AR PNAS,
94, 8485, 97 Hs.90093 hsp70 Potential interaction with AR Hs.206650
hsp90 Potential interaction with AR Hs.848 hsp56(FKBP52, Potential
interaction with AR FKBP59, HBI)) Hs.143482 Cyp40(cyclophilin40)
Potential interaction with AR p23 Potential interaction with AR
Hs.84285 ubiquitin-conjugating Potential Interaction with AR
J.B.C.274, 19441(99) enzyme Hs.182237 POU domain, class 2,
Potential interaction with AR transcr Hs.1101 POU domain, class 2,
Potential interaction with AR transcr Hs.2815 POU domain, class 6,
Potential interaction with AR transcr IMAGE:14199 Potential
interaction with AR 81 Hs.227639 ARA160 Potential interaction with
AR JBC, 274, 22373(99) Hs.83623 CAR-beta Xist locus Nuclear
receptor gene family Hs.2905 PR Nuclear receptor gene family
Hs.1790 MR mineralocorticoid Nuclear receptor gene family receptor
(aldosterone receptor) Hs.1657 ER alpha Nuclear receptor gene
family Hs.103504 ER beta Nuclear receptor gene family Hs.110849
ERR1 Nuclear receptor gene family Hs.194667 ERR2 Nuclear receptor
gene family Hs.724 TR a thyroid hormone Nuclear receptor gene
family receptor, alpha (avian erythroblastic leukemia viral (v-erb-
a) oncogene homolog) Hs.121503 TR b Nuclear receptor gene family
Hs.171495 RAR b retinoic acid receptor, Nuclear receptor gene
family beta Hs.1497 RAR g retinoic acid receptor, Nuclear receptor
gene family gamma Hs.998 PPAR a Nuclear receptor gene family
Hs.106415 PPAR b Human peroxisome Nuclear receptor gene family
proliferator activated receptor mRNA, complete cds Hs.100724 PPAR g
peroxisome Nuclear receptor gene family proliferative activated
receptor, gamma Hs.100221 LXR b Nuclear receptor gene family
Hs.81336 LXR a liver X receptor, Nuclear receptor gene family alpha
Hs.171683 FXR farnesoid X-activated Nuclear receptor gene family
receptor Hs.2062 VDR vitamin D (1, 25- Nuclear receptor gene family
dihydroxyvitamin D3) receptor Hs.118138 PXR Nuclear receptor gene
family ND SXR Nuclear receptor gene family ND BXR Nuclear receptor
gene family ND CAR b? CAR a Nuclear receptor gene family Hs.196601
RXRA Nuclear receptor gene family Hs.79372 RXRB Human retinoid X
Nuclear receptor gene family receptor beta (RXR- beta) mRNA,
complete cds Hs.194730?TR EAR1 Nuclear receptor gene family 1?
Hs.204704 EAR1 beta Nuclear receptor gene family E75 Nuclear
receptor gene family Hs.2156 ROR alpha Nuclear receptor gene family
Hs.198481 ROR beta Nuclear receptor gene family Hs.133314 ROR
gammma Nuclear receptor gene family Hs.100221 NER1 Nuclear receptor
gene family Hs.54424 HNF4A Nuclear receptor gene family Hs.202659
HNF4G Nuclear receptor gene family Hs.108301 TR2 Nuclear receptor
gene family Hs.520 TR4 Nuclear receptor gene family Hs.144630
COUP-TF1 Nuclear receptor gene family Hs.1255 COUP-TF2 Nuclear
receptor gene family Hs.155286 EAR2 Nuclear receptor gene family
Hs.1119 TR3 hormone receptor Nuclear receptor gene family (growth
factor- inducible nuclear protein N10) Hs.82120 NURR1 IMMEDIATE-
Nuclear receptor gene family EARLY RESPONSE PROTEIN NOT Hs.97196
SF1 Nuclear receptor gene family Hs.183123 FTF fetoprotein-alpha 1
Nuclear receptor gene family (AFP) transcription factor Hs.46433
DAX1 Nuclear receptor gene family Hs.11930 SHP Homo sapiens nuclear
Nuclear receptor gene family hormone receptor (shp) gene, 3' end of
cds Hs.83623, CAR-beta Nuclear receptor gene family IMAGE 1761923,
or 1868028, or 1563505, or 1654096 Hs.199078 Sin3 Nuclear receptor
co-repressor complex Nature, 387, 43, 97. Nature, 387, 49, 97
Hs.120980 SMRT Nuclear receptor co-repressor complex Nature, 377,
454, 95 Hs.144904 N-CoR Nuclear receptor co-repressor complex
Nature, 377, 297, 95 Hs.188055 highly homologue gene Nuclear
receptor co-repressor complex to N-CoR in prostate and testis
Hs.180686 E6-AP Angelman syndrome Nuclear receptor co-activator
complex MCB, 19, 1182, 99 associated protein Hs.199211?Hs. hBRM
ESTs, Highly similar Nuclear receptor co-activator complex 198296?
to HOMEOTIC GENE REGULATOR [Drosophila melanogaster] Hs.78202 hBRG1
Nuclear receptor co-activator complex Hs.11861 TRAP240 DRIP250,
ARCp250 Nuclear receptor co-activator complex Mol Cell, 3, 361, 99
Hs.85313 TRAP230 ARCp240, DRIP240 Nuclear receptor co-activator
complex Mol Cell, 3, 361, 99 Hs.15589 TRAP220 RB18A, PBP, Nuclear
receptor co-activator complex CRSP200, TRIP2, ARCp205, DRIP205
Hs.21586 TRAP170 RGR, CRSP150, Nuclear receptor co-activator
complex DRIP150, ARCp150chromosom eX Hs.108319 TRAP150 ESTs Nuclear
receptor co-activator complex Mol Cell, 3, 361, 99 Hs.193017
CRSP133 ARCp130, DRIP130 Nuclear receptor co-activator complex
Nature, 397, 6718, 99 Hs.23106 TRAP100 ARCp100, DRIP100, Nuclear
receptor co-activator complex ND DRIP97 TRAP97 Nuclear receptor
co-activator complex Hs.24441 TRAP95 ESTs Nuclear receptor
co-activator complex Mol Cell, 3, 361, 99 ND TRAP93 Nuclear
receptor co-activator complex Hs.31659 DRIP92 ARCp92? Nuclear
receptor co-activator complex Hs.22630 TRAP80 ARCp77, Nuclear
receptor co-activator complex Mol Cell, 3, 361, 99 CRSP77,
DRIP80(77)? Hs.204045 ARCp70 CRSP70, DRIP79 Nuclear receptor
co-activator complex ND ARCp42 Nuclear receptor co-activator
complex ND ARCp36 Nuclear receptor co-activator complex Hs.184947
MED6 ARCp33 Nuclear receptor co-activator complex Mol Cell, 3, 97,
99 Hs.7558 MED7 CRSP33, ARCp34, Nuclear receptor co-activator
complex Nature, 397, 6718, 99 DRIP36 ND ARCp32 Nuclear receptor
co-activator complex ND SRB10 Nuclear receptor co-activator complex
ND SRB11 Nuclear receptor co-activator complex ND MED10 NUT2
Nuclear receptor co-activator complex Hs.27289 SOH1 (yeast?)
Nuclear receptor co-activator complex Mol Cell, 3, 97, 99 ND p26
Nuclear receptor co-activator complex ND p28 Nuclear receptor
co-activator complex ND p36 Nuclear receptor co-activator complex
ND p37 Nuclear receptor co-activator complex ND but 2 TRFP human
homologue of Nuclear receptor co-activator complex IMAGE clones
Drosophila TRF proximal protein ND VDR interacting subunit 180kDa,
HAT Nuclear receptor co-activator complex Genes Dev, 12, 1787, 98
activity Hs.143696, or Coactivator associated Nuclear receptor
co-activator complex Science, 284, 2174, 99 IMAGE:23716
methyltransferase 1 96? Hs.79387 SUG1 TRIP1 Nuclear receptor
co-activator complex EMBO, 15, 110, 96 ND TRUP Nuclear receptor
co-activator complex PNAS, 92, 9525, 95 Hs.28166 CRSP34 Nuclear
receptor co-activator complex Nature, 397, 6718, 99 Hs.63667
transcriptional adaptor 3 Nuclear receptor co-activator complex
(A Hs.196725 ESTs, Highly similar to Nuclear receptor co-activator
complex P300 Hs.131846 PCAF associated factor Nuclear receptor
co-activator complex 65 al Hs.155635 ESTs, Moderately Nuclear
receptor co-activator complex similar toPCAF associated factor 65
beta Hs.26782 PCAF associated factor Nuclear receptor co-activator
complex 65 beta Hs.118910 tumor suscitibility Modifying AR function
Cancer 15, 86, 689, protein 101 (99) Hs.82932 Cyclin D1 cyclin D1
(PRAD1: Modifying AR function Can Res, 59, 2297, 99 parathyroid
adenomatosis 1) Hs.173664 HER2/Neu v-erb-b2 avian Modifying AR
function PNAS, 9, 5458, 99 erythroblastic leukemia viral oncogene
homolog 2 Hs.77271 PKA protein kinase, Modifying AR function JBC
274, 7777, 99 cAMP-dependent, catalytic, alpha Hs.85112 IGF1
insulin-like growth Modifying AR function Can Res, 54, 5474, 94
factor 1 (somatomedin C) Hs.2230 EGF Modifying AR function Can Res,
54, 5474, 94 Hs.129841 MEKK1 MAPKKK1 Modifying AR function Mol Cell
Biol. 19, 5143, 99 Hs.83173 Cyclin D3 Modifying AR function Can
Res, 59, 2297, 99 Hs.75963 IGF2 Modifying AR function Hs.89832
Insulin Modifying AR function Hs.115352 GH Modifying AR function
Hs.1989 5 alpha reductase type2 Involved in Androgen metabolism
Hs.76205 Cytochrome P450, Involved in Androgen metabolism subfamily
XIA Hs.1363 Cytochrome P450, Involved in Androgen metabolism
subfamily XVII, (steroid 17-alpha-hydroxylase), Hs.477
Hydroxysteroid (17- Involved in Androgen metabolism beta)
dehydrogenase 3 Hs.75441 Hydroxysteroid (17- Involved in Androgen
metabolism beta) dehydrogenase 4 Hs.38586 Hydroxy-delta-5-steroid
Involved in Androgen metabolism dehydrogenase, 3 beta- and steroid
delta- isomerase 1 Hs.46319 Sex hormone-binding Involved in
Androgen metabolism globulin Hs.552 SRD5A1 Involved in Androgen
metabolism Hs.50964 C-CAM epithelial cell Down-regulated by
Androgen Oncogene, 18, 3252, 99 adhesion molecule Hs.7833 hSP56
selenium binding Down-regulated by Androgen Can Res, 58, 3150, 98
protein Hs.77432 EGFR epidermal growth Down-regulated by Androgen
Endocrinology, factor receptor 139, 1369, 98 Hs.1174 p16
Down-regulated by Androgen Can Res.57, 4511, 97 Hs.55279 maspin
Down-regulated by Androgen PNAS, 94, 5673, 97 Hs.75789 TDD5 (mouse)
Human mRNA for Down-regulated by Androgen PNAS, 94, 4988, 97 RTP,
complete cds Hs.75106 TRPM-2 clusterin Down-regulated by Androgen
(testosterone-repressed prostate message 2, apolipoprotein J)
Hs.25640 rat ventral prostate gene1 claudin3 Down-regulated by
Androgen PNAS, 94, 12999, 97 ND glutathione S-transferase
Down-regulated by Androgen PNAS, 94, 12999, 97 Hs.25647 c-fos v-fos
FBJ murine Down-regulated by Androgen PNAS, 94, 12999, 97
osteosarcoma viral oncogene homolog N.D. matrix carboxyglutamic
Down-regulated by Androgen PNAS, 94, 12999, 97 acid protein (RAT)
Hs.2962 S100P calcium binding Down-regulated by Androgen Prostate
29, 350, 96 prottein Hs.75212 ornithine decarboxilase ornithine
Down-regulated by Androgen J Androl, 19, 127, 98 decarboxylase 1
Hs.84359 Androge withdrawal Down-regulated by Androgen apoptosis
RVP1 Hs.79070 c-myc v-myc avian Down-regulated by Androgen
myelocytomatosis viral oncogene homolog Hs.139033 partially
expressed gene Down-regulated by Androgen Mol Cell Endocrinol 3
155, 69, (99) Hs.20318 PLU-1 Associated with Prostate Cancer JBC,
274, 15633, 99 Hs.18910 POV1(PB39) unique Associated with Prostate
Cancer Genomics. 51, 282, 98 Hs.119333 caveolin Associated with
Prostate Cancer Clin Can Res. 4, 1873, 98 ND, but 1 EST
R00540(2.6kbp) = IM Associated with Prostate Cancer Urology, 50,
302, 97 IMAGE AGE:123822 CLONE Hs.184906 PTI-1 prostate tumor
Associated with Prostate Cancer Can Res, 57, 18, 97, inducing gene,
PNAS, 92, 6778, 95 trancated and mutated human elongation factor 1
alpha Hs.74649 cytochrome c oxidase Associated with Prostate Cancer
Can Res, 56, 3634, 96 subunit VI c Hs.4082 PCTA-1 prostate
carcinoma Associated with Prostate Cancer PNAS, 92, 7252, 96 tumor
antigen, galectin family ND pp32r1 Associated with Prostate Cancer
Nature Medicine, 5, 275, 99 ND pp32r2 Associated with Prostate
Cancer Nature Medicine, 5, 275, 99 Hs.184945 GBX2 Associated with
Prostate Cancer The prostate journal, 1, 61, 99 Hs.8867 Cyr61
inmmediate early Associated with Prostate Cancer Prostate, 36, 85,
98 protein Hs.77899 epithelial tropomyosin actin binding protein
Associated with Prostate Cancer Can Res, 56, 3634, 96 Hs.76689 pp32
Associated with Prostate Cancer Nature Medicine, 5, 275, 99
Hs.10712 PTEN Associated with Prostate Cancer Hs.194110 KAII
Associated with Prostate Cancer Hs.37003 H-ras Associated with
Prostate Cancer Hs.184050 K-ras Associated with Prostate Cancer
Hs.69855 N-ras neuroblastoma RAS Associated with Prostate Cancer
viral (v-ras) oncogene homolog Hs.220 TGFbeta receptor 1 Associated
with Prostate Cancer Hs.77326 IGFBP3 insulin-like growth Associated
with Prostate Cancer factor binding protein 3 Hs.79241 bcl-2
Associated with Prostate Cancer Hs.159428 Bax Associated with
Prostate Cancer Hs.206511 bcl-x Associated with Prostate Cancer
Hs.86386 mcl-1 myeloid cell leukemia Associated with Prostate
Cancer sequence 1 (BCL2- related) Hs.1846 p53 tumor protein p53
Associated with Prostate Cancer (Li-Fraumeni syndrome) Hs38481 CDK6
cyclin-dependent Associated with Prostate Cancer kinase 6 Hs.118630
Mxi.1 Associated with Prostate Cancer Hs.184794 GAGE7 Associated
with Prostate Cancer Hs.118162 fibronectin Associated with Prostate
Cancer Am J Pathol 154, 1335, 99 Hs.128231 PAGE-1 Associated with
Prostate Cancer JBC, 237, 17618, 98 Hs.75875 UEV1
ubiquitin-conjugating Associated with Prostate Cancer Am J Pathol
enzyme E2 variant 1 154, 1335, 99 Hs.75663 PM5 Human mRNA for
Associated with Prostate Cancer Am J Pathol pM5 protein 154, 1335,
99 Hs.180842 BBC1 breast basic Associated with Prostate Cancer Am J
Pathol conserved gene 154, 1335, 99 Hs.198024 JC19 Associated with
Prostate Cancer Can Res 57, 4075, 97 N.D. GC79 novel gene
Associated with Prostate Cancer Can Res 57, 4075, 97 Hs.77054 B
cell translocation gene Associated with Prostate Cancer Can Res 57,
4075, 97 1 Hs.78122 Regulatory factor X- Associated with Prostate
Cancer associated ankyrin- containing protein Hs.3337 transmembrane
4 Associated with Prostate Cancer superfamily member 1 Hs.76698 TL5
Associated with Prostate Cancer Genebank Hs3776 TL7 Associated with
Prostate Cancer Genebank Hs.170311 TL35 Associated with Prostate
Cancer Genebank Hs.184914 Human mRNA for TI- Associated with
Prostate Cancer 227H Hs.62954 ferritin, heavy Associated with
Prostate Cancer polypeptide Hs.71119 N33 Associated with Prostate
Cancer Genomics, 35, 45(96)
[0164]
8TABLE 6 Genes/ESTs as defined by publications: Differentially
expresed genes in prostate cancer from CGAP database (NIH)
Cluster.ID Gene name Hs.179809 EST Hs.193841 EST Hs.99949
prolactin-induced protein Hs.101307 EST Hs.111256 arachidonate
15-lipoxygenase Hs.185831 EST Hs.115173 EST Hs.193988 EST Hs.159335
EST Hs.191495 EST Hs.187694 EST Hs.191848 EST Hs.193835 EST
Hs.191851 EST Hs.178512 EST Hs.222886 EST Hs.210752 EST Hs.222737
EST Hs.105775 EST Hs.115129 EST Hs.115671 EST Hs.116506 EST
Hs.178507 EST Hs.187619 EST Hs.200527 EST Hs.179736 EST Hs.140362
EST Hs.209643 EST Hs.695559 EST Hs.92323 MAT8 Hs.178391 BTK
Hs.55999 EST Hs.171185 Desmin Hs.54431 SGP28 Hs.182624 EST
Hs.112259 T cell receptor gammma Hs.76437 EST Hs.104215 EST
Hs.75950 MLCK Hs.154103 LIM Hs.9542 JM27 Hs.153179 FABP5 Hs.195850
EST Hs.105807 EST Hs.115089 EST Hs.116467 EST Hs.222883 EST
[0165]
9TABLE 7 Androgen regulated Genes Defined by CPDR Genes/ESTs
Derived from CPDR-Genome Systems ARG Database Cluster Gene Name
Description Hs.152204 TMPRSS2 Up-regulated by Androgen Hs.123107
KLK1 Up-regulated by Androgen Hs.173334 elongation factor ell2
Up-regulated by Androgen Hs.151602 epithelial V-like antigen
Up-regulated by Androgen Hs.173231 IGFR1 Up-regulated by Androgen
Hs.75746 aldehyde dehydrogenase 6 Up-regulated by Androgen Hs.97708
EST prostate and testis Up-regulated by Androgen Hs.94376
proprotein convertase subtilisin/kexin type 5 Up-regulated by
Androgen AF017635 Homo sapiens Ste-20 related kinase SPAK mRNA,
complete cds {Incyte PD: Up-regulated by Androgen 60737} Hs.2798
leukemia inhibitory factor receptor Up-regulated by Androgen Hs.572
orosomucoid 1 Up-regulated by Androgen Hs.35804 KIAA0032 gene
product Up-regulated by Androgen Hs.114924 solute carrier family 16
(monocarboxylic acid transporters), member 6 Up-regulated by
Androgen Hs.37096 zinc finger protein 145 (Kruppel-like, expressed
in promyelocytic leukemia) Up-regulated by Androgen R07295 sterol
O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 1
Up-regulated by Androgen {Incyte PD: 2961248} Hs.11899
3-hydroxy-3-methylglutaryl-Coenzyme A reductase Up-regulated by
Androgen Hs.216958 Human mRNA for KIAA0194 gene, partial cds
Up-regulated by Androgen Hs.76901 for protein disulfide
isomerase-related Up-regulated by Androgen Hs.180628 dynamin-like
protein Up-regulated by Androgen Hs.81328 nuclear factor of kappa
light polypeptide gene enhancer in B-cells inhibitor, Up-regulated
by Androgen alpha Hs.159358 acetyl-Coenzyme A carboxylase alpha
Up-regulated by Androgen N24233 IMAGE:262457 Up-regulated by
Androgen Hs.188429 EST Up-regulated by Androgen Hs.77508 glutamate
dehydrogenase 1 Up-regulated by Androgen Hs.12017 Homo sapiens
KIAA0439 mRNA Up-regulated by Androgen Hs.10494 EST Up-regulated by
Androgen Hs.20843 EST Up-regulated by Androgen Hs.153138 origin
recognition complex, subunit 5 (yeast homolog)-like Up-regulated by
Androgen Hs.79136 Human breast cancer, estrogen regulated LIV-1
protein (LIV-1) mRNA, partial Up-regulated by Androgen cds Hs.35750
anthracycline resistance-associated Up-regulated by Androgen
Hs.56729 lymphocyte-specific protein 1 Up-regulated by Androgen
Hs.17631 EST Up-regulated by Androgen Hs.46348 bradykinin receptor
B1 Up-regulated by Androgen Hs.172851 arginase, type II
Up-regulated by Androgen Hs.66744 twist (Drosophila) homolog
Up-regulated by Androgen Hs.185973 membrane fatty acid (lipid)
desaturase Up-regulated by Androgen Hs.26 ferrochelatase
(protoporphyria) Up-regulated by Androgen Hs 169341 ESTs, Weakly
similar to phosphatidic acid phosphohydrolase type-2c Up-regulated
by Androgen [H. sapiens] Hs.119007 S-phase response
(cyclin-related) Up-regulated by Androgen Hs.76285 H. sapiens gene
from PAC 295C6, similar to rat PO44 Up-regulated by Androgen
Hs.167531 Homo sapiens mRNA fill length insert cDNA clone EUROIMAGE
195423 Up-regulated by Androgen Hs.9817 arg/Abl-interacting protein
ArgBP2 Up-regulated by Androgen Hs.28241 EST Down-regulated by
Androgen Hs.25925 Homo sapiens clone 23860 mRNA Down-regulated by
Androgen Hs.10319 UDP glycosyltransferase 2 family, polypeptide B7
Down-regulated by Androgen Hs.155995 Homo sapiens mRNA for KIAA0643
protein, partial cds Down-regulated by Androgen Hs.23552 EST
Down-regulated by Androgen Hs.41693 DnaJ-like heat shock protein 40
Down-regulated by Androgen Hs.90800 matrix metalloproteinase 16
(membrane-inserted) Down-regulated by Androgen Hs.2996
sucrase-isomaltase Down-regulated by Androgen Hs.166019 regulatory
factor X.3 (influences HLA class II expression) Down-regulated by
Androgen Hs.27695 midline 1 (Opitz/BBB syndrome) Down-regulated by
Androgen Hs.183738 chondrocyte-derived ezrin-like protein
Down-regulated by Androgen Hs.75761 SFRS protein kinase 1
Down-regulated by Androgen Hs.197298 NS1-binding protein
Down-regulated by Androgen Hs.149436 kinesin family member 5B
Down-regulated by Androgen Hs.81875 growth factor receptor-bound
protein 10 Down-regulated by Androgen Hs.75844 ESTs, Weakly similar
to (define not available 5257244) [H. sapiens] Down-regulated by
Androgen Hs.30464 cyclin E2 Down-regulated by Androgen Hs.198443
inositol 1, 4, 5-triphosphate receptor, type 1 Down-regulated by
Androgen Hs.177959 a disintegrin and metalloproteinase domain 2
(fertilin beta) Down-regulated by Androgen Hs.44197 Homo sapiens
mRNA; cDNA DKFZpS64D0462 (from clone Down-regulated by Androgen
DKFZp564D0462) Hs.150423 cyclin-dependent kinase 9 (CDC2-related
kinase) Down-regulated by Androgen Hs.78776 Human putative
transmembrane protein (nma) mRNA, complete cds Down-regulated by
Androgen Hs.25740 ESTs, Weakly similar to !!!! ALU SUBFAMILY SQ
WARNING ENTRY !!!! Down-regulated by Androgen [H. sapiens]
Hs.131041 EST Down-regulated by Androgen Hs.19222 ecotropic viral
integration site 1 Down-regulated by Androgen Hs.9879 EST
Down-regulated by Androgen Hs.118722 fucosyltransferase 8 (alpha
(1, 6) fucosyltransferase) Down-regulated by Androgen Hs.47584
potassium voltage-gated channel, delayed-rectifier, subfamily S,
member 3 Down-regulated by Androgen Hs.115945 mannosidase, beta A,
lysosomal Down-regulated by Androgen Hs.171740 ESTs, Weakly similar
to Zic2 protein [M. musculus] Down-regulated by Androgen Hs.32970
signaling lymphocytic activation molecule Down-regulated by
Androgen Hs.196349 EST Down-reguiated by Androgen Hs.182982 Homo
sapiens mRNA for KIAA0855 protein, partial cds Down-regulated by
Androgen Hs.72918 small inducible cytokine A1 (1-309, homologous to
mouse Tca-3) Down-regulated by Androgen Hs.84232 transcobalamin II;
macrocytic anemia Down-regulated by Androgen Hs.10086 EST
Down-regulated by Androgen Hs.1327 Butyrylcholinesterase
Down-regulated by Androgen Hs.166684 serine/threonine kinase 3
(Ste20, yeast homolog) Down-regulated by Androgen AA558631 EST
Down-regulated by Androgen Hs.150403 dopa decarboxylase (aromatic
L-amino acid decarboxylase) Down-regulated by Androgen Hs.177548
postmeiotic segregation increased (S. cerevisiae) 2 Down-regulated
by Androgen Hs.205902 IGF1-R Hs.21330 MDR1 P glycoprotein
1/multiple drug resistance 1 Hs.74427 PIG11 Homo sapiens Pig11
(PIG11) mRNA, complete cds Hs.76507 PIG7 LPS-induced TNF-alpha
factor Hs.8141 PIG8 Hs.146688 PIG12 Hs.104925 PIG10 Hs.202673 PIG6
Hs.80642 STAT4 Hs.72988 STAT2 Hs.167503 STAT5A Hs.738 early growth
response 1 Hs.85148 villin2 Hs.109012 MAD Hs.75251 DEAD/H box
binding protein 1 Hs.181015 STAT6 Hs.199791 SSI-3 STAT induced STAT
inhibitor 3 Hs.21486 STAT1 Hs.142258 STAT3 Hs.76578 PIAS3 Protein
inhibitor of activated STAT3 Hs.44439 CIS4 STAT induced STAT
inhibitor 4 Hs.50640 SSI-1 JAK binding protein Hs.54483 NMI N-Myc
and STAT interactor Hs.105779 PIASy Protein inhibitor of activated
STAT Hs.110776 STATI2 STAT induced STAT inhibitor 2 Hs.181112 EST
similar to STAT5A
[0166]
10TABLE 9 Functional Categories of ARGs Tag T/C Access # Name,
Description Transcription Regulators GCCAGCCCAG (SEQ ID NO: 13)
11/1 H41030 KAP1/TIF1beta, KRAB-associated protein 1 GTGCAGGGAG
(SEQ ID NO: 14) 18/2 AF071538 PDEF, ets transcription factor
GACAAACATT (SEQ ID NO: 15) 8/1 NM_003201 mtTF1, mitochondrial
transcription factor 1 ATGACTCAAG (SEQ ID NO: 16) 8/1 X12794 ear-2,
v-erbA related GAAAAGAAGG (SEQ ID NO: 17) 8/1 U80669 Nkx3.1,
homeobox CCTGTACCCC (SEQ ID NO: 18) 5/1 AF072836 Sox-like
transcriptional factor CCTGAACTGG (SEQ ID NO: 19) 1/8 NM_001273
CHD4/Mi2-beta, histone acetylase/deacetylase, chromodomain helicase
TGACAGCCCA (SEQ ID NO: 20) 1/7 U81599 Hox B13, homeobox RNA
Processing and Translational Regulators TACAAAACCA (SEQ ID NO: 21)
12/1 NM_005381 NCL, Nucleolin AATTCTCCTA (SEQ ID NO: 22) 8/1 U41387
GURDB, nucleolar RNA helicase TGCATATCAT (SEQ ID NO: 23) 8/1 D89729
XPOl, exportin 1 CTTGACACAC (SEQ ID NO: 24) 14/2 AL080102 EIF5,
translation initiation factor 5 TGTCTAACTA (SEQ ID NO: 25) 5/1
AF078865 CGI-79, RNA-binding protein GTGGACCCCA (SEQ ID NO: 26)
10/2 AF190744 SiahBP1/PUF60, poly-U binding splicing factor
ATAAAGTAAC (SEQ ID NO: 27) 1/11 NM_007178 UNRIP, unr-interacting
protein. TACATTTTCA (SEQ ID NO: 28) 1/7 X85373 SNRPG, small nuclear
RNP polypeptide G TCAGAACAGT (SEQ ID NO: 29) 1/7 NM_002092 GRSF-1,
G-rich RNA binding factor 1 CAACTTCAAC (SEQ ID NO: 30) 0/5
NM_006451 PAIP1, poly A BP-interactimg protein 1 GATAGGTCGG (SEQ ID
NO: 31) 0/5 Z11559 IREBP1, Iron-responsive element BP 1 CTAAAAGGAG
(SEQ ID NO: 32) 2/10 M15919 SNRPE, small nuclear RNP polypeptide E
Genomic Maintenance and Cell Cycle Regulation GTGGTGCGTG (SEQ ID
NO: 33) 10/1 AF035587 XRCC2, X-ray repair protein 2 TCCCCGTGGC (SEQ
ID NO: 34) 7/1 D13643 KIAA0018, Dimunuto-like ATTGATCTTG (SEQ ID
NO: 35) 6/1 NM_002947 RPA3, Replication protein A 14 kDa subunit
AGCTGGTTTC (SEQ ID NO: 36) 16/3 NM_004879 PIG8, p53 induced protein
CCTCCCCCGT (SEQ ID NO: 37) 10/2 AF044773 BAF,
barrier-to-autointegration factor ATGTACTCTG (SEQ ID NO: 38) 1/7
NM_000884 IMPDH2, IMP dehydrogenase 2 GATGAAATAC (SEQ ID NO: 39)
0/5 NM_006325 ARA24, androgen receptor assoc protein 24 GTGCATCCCG
(SEQ ID NO: 40) 0/5 X16312 Phosvitin/casein kinase II beta subunit
Protein Trafficking and Chaperoning GAAATTAGGG (SEQ ID NO: 41) 12/1
AB020637 KIAA0830, similar to golgi antigen TTTCTAGGGG (SEQ ID NO:
42) 10/1 AF15189 CGI-140, lysosomal alpha B mannosidase CCCAGGGAGA
(SEQ ID NO: 43) 7/1 AF026291 CCT, chaperomin t-complex polypeptide
1 GTGGCGCACA (SEQ ID NO: 44) 13/2 S79862 26 S protease subunit 5b
TTGCTTTTGT (SEQ ID NO: 45) 15/3 NM_001660 ARF4, ADP-ribosylation
factor 4 ATGTCCTTTC (SEQ ID NO: 46) 10/2 NM_005570 LMAN1, mannose
BP involved in EPR/Golgi traffic Energy Metabolism, Apoptosis and
Redox Regulators TGTTTATCCT (SEQ ID NO: 47) 13/2 M14200 DBI,
diazepam binding inhibitor GCTTTGTATC (SEQ ID NO: 48) 6/1 D16373
dihydrolipoamide succinyltransferase GTTCCAGTGA (SEQ ID NO: 49) 6/1
AA653318 FKBP5, FK506-binding protein 5 TAGCAGAGGC (SEQ ID NO: 50)
6/1 AA425929 NDUFB10, NADH dehydrogenase 1 beta subcomplex 10
ACAAATTATG (SEQ ID NO: 51) 5/1 NM_003375 VDAC, voltage-dependent
anion channel CAGTTTGTAC (SEQ ID NO: 52) 5/1 NM_000284 PDHA1,
Pyruvate dehydrogenase El-alpha subunit GATTACTTGC (SEQ ID NO: 53)
5/1 NM_004813 PEX16, peroxisomal membrane biogenesis factor
GGCCAGCCCT (SEQ ID NO: 54) 5/1 X15573 PFKL, 1-phosphofructokinase
CAATTGTAAA (SEQ ID NO: 55) 1/10 NM_004786 TXNL, thioredoxin-like
protein AAAGCCAAGA (SEQ ID NO: 56) 2/15 NM_001985 ETFB, electron
transfer flavoprotein beta subunit CAACTAATTC (SEQ ID NO: 57) 1/7
NM_001831 CLU, Clustrin AAGAGCTAAT (SEQ ID NO: 58) 0/5 NM_004446
EPRS, glutamyl-prolyl-tRNA synthetase Signal Transduction
CTTTTCAAGA (SEQ ID NO: 59) 9/1 X59408 CD46, complement system
membrane cofactor GTGTGTAAAA (SEQ ID NO: 60) 9/1 NM_005745
BAP31/BAP29 IgD accessory proteins ACAAAATGTA (SEQ ID NO: 61) 8/1
NM_000856 GUCY1A3, Guanylate cyclase 1, alpha 3 AAGGTAGCAG (SEQ ID
NO: 62) 7/1 NM_006367 CAP, Adenylyl cyclase-associated protein
GGCGGGGCCA (SEQ ID NO: 63) 7/1 AB002301 microtubule assoc.
serine/threonine kinase GGCCAGTAAC (SEQ ID NO: 64) 6/1 AL096857
similar to HAT2, integrin receptor AACTTAAGAG (SEQ ID NO: 65) 12/2
AB018330 calmodulin-dependent protein kinase kinase .beta.
AGGGATGGCC (SEQ ID NO: 66) 5/1 NM_006858 IL1RL1LG, Putative T1/ST2
receptor CTTAAGGATT (SEQ ID NO: 67) 2/10 AF151813 CGI-55
protein
[0167] The "tag to gene" identification is based on the analysis
performed by SAGE software and/or "tag to gene" application of the
NIH SAGE Website. T/C represent the number of tags for each
transcript in androgen treated (T) and control (C) LNCaP libraries.
The differences in expression levels of genes identified by tags
shown here were statistically significant (p<0.05) as determined
by the SAGE software.
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[0298]
Sequence CWU 1
1
67 1 1140 DNA Homo sapiens CDS (95)..(850) 1 tccttgggtt cgggtgaaag
cgcctggggg ttcgtggcca tgatccccga gctgctggag 60 aactgaaggc
ggacagtctc ctgcgaaaca ggca atg gcg gag ctg gag ttt gtt 115 Met Ala
Glu Leu Glu Phe Val 1 5 cag atc atc atc atc gtg gtg gtg atg atg gtg
atg gtg gtg gtg atc 163 Gln Ile Ile Ile Ile Val Val Val Met Met Val
Met Val Val Val Ile 10 15 20 acg tgc ctg ctg agc cac tac aag ctg
tct gca cgg tcc ttc atc agc 211 Thr Cys Leu Leu Ser His Tyr Lys Leu
Ser Ala Arg Ser Phe Ile Ser 25 30 35 cgg cac agc cag ggg cgg agg
aga gaa gat gcc ctg tcc tca gaa gga 259 Arg His Ser Gln Gly Arg Arg
Arg Glu Asp Ala Leu Ser Ser Glu Gly 40 45 50 55 tgc ctg tgg ccc tcg
gag agc aca gtg tca ggc aac gga atc cca gag 307 Cys Leu Trp Pro Ser
Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu 60 65 70 ccg cag gtc
tac gcc ccg cct cgg ccc acc gac cgc ctg gcc gtg ccg 355 Pro Gln Val
Tyr Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro 75 80 85 ccc
ttc gcc cag cgg gag cgc ttc cac cgc ttc cag ccc acc tat ccg 403 Pro
Phe Ala Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro 90 95
100 tac ctg cag cac gag atc gac ctg cca ccc acc atc tcg ctg tca gac
451 Tyr Leu Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp
105 110 115 ggg gag gag ccc cca ccc tac cag ggc ccc tgc acc ctc cag
ctt cgg 499 Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln
Leu Arg 120 125 130 135 gac ccc gag cag cag ctg gaa ctg aac cgg gag
tcg gtg cgc gca ccc 547 Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu
Ser Val Arg Ala Pro 140 145 150 cca aac aga acc atc ttc gac agt gac
ctg atg gat agt gcc agg ctg 595 Pro Asn Arg Thr Ile Phe Asp Ser Asp
Leu Met Asp Ser Ala Arg Leu 155 160 165 ggc ggc ccc tgc ccc ccc agc
agt aac tcg ggc atc agc gcc acg tgc 643 Gly Gly Pro Cys Pro Pro Ser
Ser Asn Ser Gly Ile Ser Ala Thr Cys 170 175 180 tac ggc agc ggc ggg
cgc atg gag ggg ccg ccg ccc acc tac agc gag 691 Tyr Gly Ser Gly Gly
Arg Met Glu Gly Pro Pro Pro Thr Tyr Ser Glu 185 190 195 gtc atc ggc
cac tac ccg ggg tcc tcc ttc cag cac cag cag agc agt 739 Val Ile Gly
His Tyr Pro Gly Ser Ser Phe Gln His Gln Gln Ser Ser 200 205 210 215
ggg ccg ccc tcc ttg ctg gag ggg acc cgg ctc cac cac aca cac atc 787
Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg Leu His His Thr His Ile 220
225 230 gcg ccc cta gag agc gca gcc atc tgg agc aaa gag aag gat aaa
cag 835 Ala Pro Leu Glu Ser Ala Ala Ile Trp Ser Lys Glu Lys Asp Lys
Gln 235 240 245 aaa gga cac cct ctc tagggtcccc aggggggccg
ggctggggct gcgtaggtga 890 Lys Gly His Pro Leu 250 aaaggcagaa
cactccgcgc ttcttagaag aggagtgaga ggaaggcggg gggcgcagca 950
acgcatcgtg tggccctccc ctcccacctc cctgtgtata aatatttaca tgtgatgtct
1010 ggtctgaatg cacaagctaa gagagcttgc aaaaaaaaaa agaaaaaaga
aaaaaaaaaa 1070 ccacgtttct ttgttgagct gtgtcttgaa ggcaaaagaa
aaaaaatttc tacagtaaaa 1130 aaaaaaaaaa 1140 2 759 DNA Homo sapiens 2
atggcggagc tggagtttgt tcagatcatc atcatcgtgg tggtgatgat ggtgatggtg
60 gtggtgatca cgtgcctgct gagccactac aagctgtctg cacggtcctt
catcagccgg 120 cacagccagg ggcggaggag agaagatgcc ctgtcctcag
aaggatgcct gtggccctcg 180 gagagcacag tgtcaggcaa cggaatccca
gagccgcagg tctacgcccc gcctcggccc 240 accgaccgcc tggccgtgcc
gcccttcgcc cagcgggagc gcttccaccg cttccagccc 300 acctatccgt
acctgcagca cgagatcgac ctgccaccca ccatctcgct gtcagacggg 360
gaggagcccc caccctacca gggcccctgc accctccagc ttcgggaccc cgagcagcag
420 ctggaactga accgggagtc ggtgcgcgca cccccaaaca gaaccatctt
cgacagtgac 480 ctgatggata gtgccaggct gggcggcccc tgccccccca
gcagtaactc gggcatcagc 540 gccacgtgct acggcagcgg cgggcgcatg
gaggggccgc cgcccaccta cagcgaggtc 600 atcggccact acccggggtc
ctccttccag caccagcaga gcagtgggcc gccctccttg 660 ctggagggga
cccggctcca ccacacacac atcgcgcccc tagagagcgc agccatctgg 720
agcaaagaga aggataaaca gaaaggacac cctctctag 759 3 252 PRT Homo
sapiens 3 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val
Val Met 1 5 10 15 Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser
His Tyr Lys Leu 20 25 30 Ser Ala Arg Ser Phe Ile Ser Arg His Ser
Gln Gly Arg Arg Arg Glu 35 40 45 Asp Ala Leu Ser Ser Glu Gly Cys
Leu Trp Pro Ser Glu Ser Thr Val 50 55 60 Ser Gly Asn Gly Ile Pro
Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro 65 70 75 80 Thr Asp Arg Leu
Ala Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His 85 90 95 Arg Phe
Gln Pro Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro 100 105 110
Pro Thr Ile Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly 115
120 125 Pro Cys Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu
Asn 130 135 140 Arg Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe
Asp Ser Asp 145 150 155 160 Leu Met Asp Ser Ala Arg Leu Gly Gly Pro
Cys Pro Pro Ser Ser Asn 165 170 175 Ser Gly Ile Ser Ala Thr Cys Tyr
Gly Ser Gly Gly Arg Met Glu Gly 180 185 190 Pro Pro Pro Thr Tyr Ser
Glu Val Ile Gly His Tyr Pro Gly Ser Ser 195 200 205 Phe Gln His Gln
Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr 210 215 220 Arg Leu
His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp 225 230 235
240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250 4 8 PRT
Artificial Sequence Description of Artificial Sequence FLAG peptide
4 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 5 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 5 ggcagaacac tccgcgcttc
ttag 24 6 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 6 caagctctct tagcttgtgc attc 24 7 22 DNA Artificial
Sequence Description of Artificial Sequence Primer 7 cttgggttcg
ggtgaaagcg cc 22 8 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 8 ggtgggtggc aggtcgatct cg 22 9 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 9
ccttcgccca gcgggagcgc 20 10 24 DNA Artificial Sequence Description
of Artificial Sequence Primer 10 caagctctct tagcttgtgc attc 24 11
249 PRT Homo sapiens 11 Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile
Val Val Val Met Met 1 5 10 15 Val Met Val Val Val Ile Thr Cys Leu
Leu Ser His Tyr Lys Leu Ser 20 25 30 Ala Arg Ser Phe Ile Ser Arg
His Ser Gln Gly Arg Arg Arg Glu Asp 35 40 45 Ala Leu Ser Ser Glu
Gly Cys Leu Trp Pro Ser Glu Ser Thr Val Ser 50 55 60 Gly Asn Gly
Ile Pro Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr 65 70 75 80 Asp
Arg Leu Ala Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg 85 90
95 Phe Gln Pro Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro Pro
100 105 110 Thr Ile Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln
Gly Pro 115 120 125 Cys Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu
Glu Leu Asn Arg 130 135 140 Glu Ser Val Arg Ala Pro Pro Asn Arg Thr
Ile Phe Asp Ser Asp Leu 145 150 155 160 Met Asp Ser Ala Arg Leu Gly
Gly Pro Cys Pro Pro Ser Ser Asn Ser 165 170 175 Gly Ile Ser Ala Thr
Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro 180 185 190 Pro Pro Thr
Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe 195 200 205 Gln
His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg 210 215
220 Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp Ser
225 230 235 240 Lys Glu Lys Asp Lys Gln Lys Gly His 245 12 244 PRT
Homo sapiens 12 Ala Glu Leu Glu Phe Ala Gln Ile Ile Ile Ile Val Val
Val Val Thr 1 5 10 15 Val Met Val Val Val Ile Val Cys Leu Leu Asn
His Tyr Lys Val Ser 20 25 30 Thr Arg Ser Phe Ile Asn Arg Pro Asn
Gln Ser Arg Arg Arg Glu Asp 35 40 45 Gly Leu Pro Gln Glu Gly Cys
Leu Trp Pro Ser Asp Ser Ala Ala Pro 50 55 60 Arg Leu Gly Ala Ser
Glu Ile Met His Ala Pro Arg Ser Arg Asp Arg 65 70 75 80 Phe Thr Ala
Pro Ser Phe Ile Gln Arg Asp Arg Phe Ser Arg Phe Gln 85 90 95 Pro
Thr Tyr Pro Tyr Val Gln His Glu Ile Asp Leu Pro Pro Thr Ile 100 105
110 Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr
115 120 125 Leu Gln Leu Arg Asp Pro Glu Gln Gln Met Glu Leu Asn Arg
Glu Ser 130 135 140 Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser
Asp Leu Ile Asp 145 150 155 160 Ile Ala Met Tyr Ser Gly Gly Pro Cys
Pro Pro Ser Ser Asn Ser Gly 165 170 175 Ile Ser Ala Ser Thr Cys Ser
Ser Asn Gly Arg Met Glu Gly Pro Pro 180 185 190 Pro Thr Tyr Ser Glu
Val Met Gly His His Pro Gly Ala Ser Phe Leu 195 200 205 His His Gln
Arg Ser Asn Ala His Arg Gly Ser Arg Leu Gln Phe Gln 210 215 220 Gln
Asn Asn Ala Glu Ser Thr Ile Val Pro Ile Lys Gly Lys Asp Arg 225 230
235 240 Lys Pro Gly Asn 13 10 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 13 gccagcccag 10
14 10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 14 gtgcagggag 10 15 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 15 gacaaacatt 10 16 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 16
atgactcaag 10 17 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 17 gaaaagaagg 10 18
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 18 cctgtacccc 10 19 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 19 cctgaactgg 10 20 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 20
tgacagccca 10 21 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 21 tacaaaacca 10 22
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 22 aattctccta 10 23 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 23 tgcatatcat 10 24 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 24
cttgacacac 10 25 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 25 tgtctaacta 10 26
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 26 gtggacccca 10 27 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 27 ataaagtaac 10 28 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 28
tacattttca 10 29 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 29 tcagaacagt 10 30
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 30 caacttcaac 10 31 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 31 gataggtcgg 10 32 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 32
ctaaaaggag 10 33 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 33 gtggtgcgtg 10 34
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 34 tccccgtggc 10 35 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 35 attgatcttg 10 36 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 36
agctggtttc 10 37 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 37 cctcccccgt 10 38
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 38 atgtactctg 10 39 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 39 gatgaaatac 10 40 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 40
gtgcatcccg 10 41 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 41 gaaattaggg 10 42
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 42 tttctagggg 10 43 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 43 cccagggaga 10 44 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 44
gtggcgcaca 10 45 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 45 ttgcttttgt 10 46
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 46 atgtcctttc 10 47 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 47 tgtttatcct 10 48 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 48
gctttgtatc 10 49 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 49 gttccagtga 10 50
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 50 tagcagaggc 10 51 10 DNA Artificial
Sequence Description of Artificial Sequence
Synthetic oligonucleotide 51 acaaattatg 10 52 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 52 cagtttgtac 10 53 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 53
gattacttgc 10 54 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 54 ggccagccct 10 55
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 55 caattgtaaa 10 56 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 56 aaagccaaga 10 57 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 57
caactaattc 10 58 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 58 aagagctaat 10 59
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 59 cttttcaaga 10 60 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 60 gtgtgtaaaa 10 61 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 61
acaaaatgta 10 62 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 62 aaggtagcag 10 63
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 63 ggcggggcca 10 64 10 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 64 ggccagtaac 10 65 10 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 65
aacttaagag 10 66 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 66 agggatggcc 10 67
10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 67 cttaaggatt 10
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References