US20030170713A1 - Method of detecting androgen-regulated gene - Google Patents

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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
• 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. §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.[0001]
GOVERNMENT INTEREST
• [0002] The invention described herein may be manufactured, licensed, and used for governmental purposes without payment of royalties to us thereon.
FIELD OF THE INVENTION
• 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.[0003]
BACKGROUND
• 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.[0004]
• 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.[0005]
• 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).[0006]
• 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[0007]⁻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).
• 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.[0008]
• 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.[0009]
• 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).[0010]
• 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).[0011]
• 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.[0012]
• 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.[0013]
SUMMARY OF THE INVENTION
• 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.[0014]
• 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.[0015]
• 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.[0016]
• 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.[0017]
• 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.[0018]
• 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.[0019]
• 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.[0020]
• 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.[0021]
BRIEF DESCRIPTION OF THE DRAWINGS
• 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.[0022]
• 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.[0023]
• FIG. 3 shows PMEPA1 expression in CwR22 xenograft tumors.[0024]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
• 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.[0025]
• 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.[0026]
• 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.[0027]
• 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.[0028]
• 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.[0029]
• 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.[0030]
• 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.[0031]
• 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.[0032]
• 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 β1 isoform of C18orf1. Therefore, it is likely that other isoforms of PMEPA1 may exist.[0033]
• 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.[0034]
• 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).[0035]
• 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.[0036]
• 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.[0037]
• The nucleic acid molecules encompassed in the invention include the following PMEPA1 nucleotide sequence:[0038]

  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

• The amino acid sequences of the polypeptides encoded by the PMEPA1 nucleotide sequences of the invention include:[0039]

  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

• 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).[0040]
• Nucleic Acid Molecules[0041]
• 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.,[0042]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.
• 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.[0043]
• 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.[0044]
• The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.[0045]
• Preferred Sequences[0046]
• 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.[0047]
• Additional Sequences[0048]
• 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.[0049]
• 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.[0050]
• 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.[0051]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 thenitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 6×SSC at about 42° C. (or other similar ii hybridization solution, such as Stark's solution, in about 50% formamide at about 42° C.), and washing conditions of about 60° C., 0.5×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° C., 0.2×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.
• 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.[0052]
• 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.[0053]
• 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.,[0054]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.
• 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.[0055]
• 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.,[0056]Science,239:487 (1988)); (Wu et al.,Recombinant DNA Methodology,eds., Academic Press, Inc., San Diego, pp. 189-196 (1989)); and (Innis et al., PCRProtocols: A Guide to Methods and Applications,eds., Academic Press, Inc. (1990)).
• Polypeptides and Fragments Thereof[0057]
• 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.[0058]
• Polypeptides and Fragments Thereof[0059]
• 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.[0060]
• 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.[0061]
• 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.[0062]
• 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.[0063]
• 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.[0064]
• 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.[0065]
• Variants[0066]
• Naturally occurring variants as well as derived variants of the polypeptides and fragments are provided herein.[0067]
• 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,[0068]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 HenikoffProc. 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.
• 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.[0069]
• 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.[0070]
• 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.,[0071]Bio/Technology,6:1204 (1988)). One such peptide is the FLAG® 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® 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® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.
• 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.[0072]
• 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.[0073]
• 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.[0074]
• 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.[0075]
• 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[0076]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).
• 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.[0077]
• 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.[0078]
• 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.[0079]
• Production of Polypeptides and Fragments Thereof[0080]
• 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:[0081]
• Expression Systems[0082]
• 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.[0083]
• 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.[0084]
• 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.[0085]
• 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.[0086]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.
• Prokaryotic Systems[0087]
• Prokaryotes include gram-negative or gram-positive organisms. Suitable prokaryotic host cells for transformation include, for example,[0088]E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such asE. 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.
• 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).[0089]
• Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al.,[0090]Nature275:615 (1978); and (Goeddel et al.,Nature281:544 (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 λP_Lpromoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λP_Lpromoter include plasmid pHUB2 (resident inE. colistrain JMB9, ATCC 37092) and pPLc28 (resident inE. coliRR1, ATCC 53082).
• Yeast Systems[0091]
• Alternatively, the polypeptides may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g.,[0092]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 μ 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.,Nature300:724 (1982)). Shuttle vectors replicable in both yeast andE. colimay be constructed by inserting DNA sequences from pBR322 for selection and replication inE. coli(Amp^rgene and origin of replication) into the above-described yeast vectors.
• The yeast a-factor leader sequence may be employed to direct secretion of the polypeptide. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., (Kurjan et al.,[0093]Cell30:933 (1982)) and (Bitter et al.,Proc. Natl. Acad. Sci. USA81:5330 (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.
• Yeast transformation protocols are known to those of skill in the art. One such protocol is described by (Hinnen et al.,[0094]Proc. Natl. Acad. Sci. USA75:1929 (1978)). The Hinnen et al. protocol selects for Trp⁺ 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.
• 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.[0095]
• Mammalian or Insect Systems[0096]
• 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,[0097]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.,Cell23:175 (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)).
• Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J.,[0098]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. USA84:7413-7417 (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 Enzymology185:487-511 (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. USA77:4216-4220 (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.
• 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,[0099]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.Nature273:113 (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.
• 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.,[0100]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 Development3:295-300 (1993)); (Ramesh et al.,Nucleic Acids Research24:2697-2700 (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)).
• A useful high expression vector, pCAVNOT, has been described by (Mosley et al.,[0101]Cell59:335-348 (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.,Nature312:768 (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.
• Another useful expression vector, pFLAG®, can be used. FLAG® technology is centered on the fusion of a low molecular weight (1 kD), hydrophilic, FLAG® marker peptide to the N-terminus of a recombinant protein expressed by pFLAG® 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.[0102]
• Purification[0103]
• The invention also includes methods of isolating and purifying the polypeptides and fragments thereof.[0104]
• Isolation and Purification[0105]
• 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.[0106]
• 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.[0107]
• 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.[0108]
• 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.[0109]
• 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.[0110]
• 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.[0111]
• 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.[0112]J. Cell. Biochem.,10D:239 (1986)). Wash of unbound material and the release of the bound cells is performed using conventional methods.
• 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.[0113]
• Production of Antibodies[0114]
• 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.[0115]
• 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,[0116]Immuno Biology3:9, 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 Biology2:14, Garland Publishing Inc., 2nd ed. (1996)). Epitopes may be identified by any of the methods known in the art.
• 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.[0117]
• 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.,[0118]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)).
• 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.[0119]
• 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.,[0120]Nature332:323 (1988), Liu et al.,PNAS84:3439 (1987), Larrick et al.,Bio/Technology7:934 (1989), and Winter and Harris,TIPS14:139 (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.
• 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′)[0121]₂fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
• 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.[0122]
• The following examples further illustrate preferred aspects of the invention.[0123]
EXAMPLE 1
• Cell Culture and Androgen Stimulation[0124]
• 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.[0125]
EXAMPLE 2
• SAGE Analysis[0126]
• 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.[0127]
EXAMPLE 3
• Kinetics of Androgen Regulation ARGs Defined by SAGE Analysis[0128]
• 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 μg/lane) by running through 1% formmaldehyde-agarose gel and transferred to nylon membrane. The cDNA probes of several ARGs were labeled with[0129]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×SSC, 5× Denhardt's solution and 0.25 mg/ml salmon sperm DNA) and hybridized to the³²P labeled probes (1×106 cpm/ml) in the same buffer at 40° C. for 12-16 hrs. Blots were washed twice in 2×SSC/0.1% SDS for 20 min at room temperature followed by two high-stringency wash with 0.1×SSC/0.1% SDS at 50° C. for 20 min. Nylon membranes were exposed to X-ray film for autoradiography.
EXAMPLE 4
• ARGs Expression Pattern in Cwr22 Model.[0130]
• 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[0131]³²P labeled probes of the cDNA of interest.
• 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.[0132]
EXAMPLE 5
• Analysis of the ARGs Defined by SAGE Tags[0133]
• 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.[0134]
EXAMPLE 6
• Identification of Prostate Specific/Abundant Genes[0135]
• 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.[0136]
• 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 β, 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.[0137]
• 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:[0138]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.
• 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.[0139]
• 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.[0140]
• 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.[0141]
• 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.[0142]
EXAMPLE 7
• Characterization of the Androgen-Regulated Gene PMEPA1[0143]
• 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′CAAGCTCTCTTAGCTTGTGCATTC3′ (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.[0144]
• 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[0145]⁺ 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⁺ RNA specimens (2 μg/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).
• The cDNA probe was generated by RT-PCR with[0146]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 with³²P-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×SSC, 5× Denhardt's solution and 0.25 mg/ml salmon sperm DNA) for two hours followed by hybridization in the same buffer containing the³²P labeled probes (1×10⁶cpm/ml) for 12-16 hrs at 40° C. Blots were washed twice in 2×SSC/0.1% SDS for 20 min at room temperature followed by two high-stringency washes with 0.1×SSC/0.1% SDS at 58° 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.
• 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[0147]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⁺ RNA were extracted as above. Poly A⁺ RNA blots were made and hybridized as described above.
• 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.[0148]
• In situ hybridization of PMEPA1 in prostate tissues. A 430 bp PCR fragment (PCR sense primer: 5′[0149]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° C. After hybridization, slides were sequentially washed with 2×SSC at room temperature for 30 min, 2×SSC at 58° C. for 1 hr and 0.1×SSC at 58° 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.
• Full-Length PMEPA1 Coding Sequence and Chromosomal Localization[0150]
• 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.[0151]

  TABLE 1
  (SEQ ID NO. 3)

  TCCTTGGGTTCGGGTGAAAGCGCCTGGGGGTTCGTGGCCATGATCCCCGAGCTGCTGGAGAACTGAAGGCGGACAGTCTCCTGCGAAAC	90
  ▾
  AGGCAATGGCGGAGCTGGAGTTTGTTCAGATCATCATCATCGTGGTGGTGATGATGGTGATGGTGGTGGTGATCACGTGCCTGCTGAGCC	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
  ▾
  ACTACAAGCTGTCTGCACGGTCCTTCATCAGCCGGCACAGCCAGGGGCGGAGGAGAGAAGATGCCCTGTCCTCAGAAGGATGCCTGTGGC	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
  ▾
  CCTCGGAGAGCACAGTGTCAGGCAACGGAATCCCAGAGCCGCAGGTCTACGCCCCGCCTCGGCCCACCGACCGCCTGGCCGTGCCGCCCT	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
  TCGCCCAGCGGGAGCGCTTCCACCGCTTCCAGCCCACCTATCCGTACCTGCAGCACGAGATCGACCTGCCACCCACCATCTCGCTGTCAG	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
  ACGGGGAGGAGCCCCCACCCTACCAGGGCCCCTGCACCCTCCAGCTTCGGGACCCCGAGCAGCAGCTGGAACTGAACCGGGAGTCGGTGC	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
  GCGCACCCCCAAACAGAACCATCTTCGACAGTGACCTGATGGATAGTGCCAGGCTGGGCGGCCCCTGCCCCCCCAGCAGTAACTCGGGCA	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
  TCAGCGCCACGTGCTACGGCAGCGGCGGGCGCATGGAGGGGCCGCCGCCCACCTACAGCGAGGTCATCGGCCACTACCCGGGGTCCTCCT	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
  TCCAGCACCAGCAGAGCAGTGGGCCGCCCTCCTTGCTGGAGGGGACCCGGCTCCACCACACACACATCGCGCCCCTAGAGAGCGCAGCCA	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
  TCTGGAGCAAAGAGAAGGATAAACAGAAAGGACACCCTCTCTAGGGTCCCCAGGGGGGCCGGGCTGGGGCTGCGTAGGTGAAAAGGCAGA	900
  I  W  S  K  E  K  D  K  Q  K  G  H  P  L  *   252

  (SEQ ID NO. 1)

  ACACTCCGCGCTTCTTAGAAGAGGAGTGAGAGGAAGGCGGGGGGCGCAGCAACGCATCGTGTGGCCCTCCCCTCCCACCTCCCTGTGTAT	990
  AAATATTTACATGTGATGTCTGGTCTGAATGCACAAGCTAAGAGAGCTTGCAAAAAAAAAAAGAAAAAAGAAAAAAAAAAACCACGTTTC	1080
  ▾
  TTTGTTGAGCTGTGTCTTGAAGGCAAAAGAAAAAAAATTTCTACAGTAAAAAAAAAAAAAA   1141

• 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).[0152]
• 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.[0153]
• 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[0154]_—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 β1 isoform of C18orf1.

  TABLE 2
  2	AELEFVQIIIIVVVMMVMVVVITCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPSE	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	STVSGNGIPEPQVYAPPRPTDRLAVPPFAQRERFHRFQPTYPYLQHEIDLPPTISLSDGE	121	PMEPA1
  	S     G  E  +   PR  DR   P F QR+RF RFQPTYPY+QHEIDLPPTISLSDGE
  63	SAAPRLGASE--IMHAPRSRDRFTAPSFIQRDRFSRFQPTYPYVQHEIDLPPTISLSDGE	120	C18orf1
  122	EPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLMDSARL-GGPCPPSSNSGIS	180	PMEPA1
  	EPPPYQGPCTLQLRDPEQQ+ELNRESVRAPPNRTIFDSDL+D A   GGPCPPSSNSGIS
  121	EPPPYQGPCTLQLRDPEQQMELNRESVRAPPNRTIFDSDLIDIAMYSGGPCPPSSNSGIS	180	C18orf1
  181	ATCYGSGGRMEGPPPTYSEVIGHYPGSSFQHQQSSGPPSLLEGTRLHHTHIAPLESAAIW	240	PMEPA1
  	A+   S GRMEGPPPTYSEV+GH+PG+SF H Q S   +   G+RL        ES  +
  181	ASTCSSNGRMEGPPPTYSEVMGHHPGASFLHHQRS---NAHRGSRLQFQQ-NNAESTIVP	236	C18orf1
  241	SKEKDKQKGH   250 PMEPA1
  	K KD++ G+
  237	IKGKDRKPGN   246 C18orf1

• Analysis of PMEPA1 Expression[0155]
• Northern hybridization revealed two transcripts of ˜2.7 kb and ˜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.[0156]
• Androgen Dependent Expression of PMEPA1[0157]
• 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 ˜2.7 and ˜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).[0158]
• Evaluation of PMEPA1 Expression in Androgen Sensitive and Androgen Refractory Tumors of CWR 22 Prostate Cancer Xenograft Model[0159]
• 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).[0160]
• 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.[0161]

  TABLE 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 sapiensprostate-specific antigen mRNA,	Up-regulated by Androgen
  	complete cds.*
  AA508573	Human nucleolin gene, complete cds	Up-regulated by Androgen
  AB020637	Homo sapiensmRNA for KIAA0830 protein, partial	Up-regulated by Androgen
  	cds.
  AA280663	EST	Up-regulated by Androgen
  U31657	KRAB-associatedprotein 1	Up-regulated by Androgen
  AI879709	EST	Up-regulated by Androgen
  AA602190	EST	Up-regulated by Androgen
  AF035587	Homo sapiensX-ray repair cross-complementing	Up-regulated by Androgen
  	protein 2 (XRCC2)
  AF151898	Homo sapiensCGI-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 sapiensEts 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 musculushomeobox protein Nkx3.1	Up-regulated by Androgen
  D89729	Homo sapiensmRNA for CRM1 protein, complete	Up-regulated by Androgen
  	cds.
  U75329	TMPRSS2	Up-regulated by Androgen
  AA062976	EST	Up-regulated by Androgen
  L12168	Homo sapiensadenylyl cyclase-associated protein	Up-regulated by Androgen
  	(CAP) mRNA
  AA043945	EST	Up-regulated by Androgen
  AF026291	Homo sapienschaperonin containing t-complex	Up-regulated byAndrogen
  	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-bindingprotein 5	Up-regulated by Androgen
  L07493	Homo sapiensreplication 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 fromchromosome 1	Up-regulated by Androgen
  AF007157	Homo sapiensclone 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 sapienstestis-specificBasic Protein Y 1	Up-regulated by Androgen
  	(BPY1) mRNA,
  AA889510	EST	Up-regulated by Androgen
  AB018330	Homo sapiensmRNA for KIAA0787 protein, partial	Up-regulated by Androgen
  	cds.
  AA026941	EST	Up-regulated byAndrogen
  AA532377	Chromosome
  1 open reading frame 8	Up-regulated by Androgen
  AF010313	Homo sapiensPig8 (PIG8) mRNA (etoposide-	Up-regulated by Androgen
  	induced mRNA), complete cds.
  L06328	Human voltage-dependentanion 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 sapiensmRNA 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 sapiensCGI-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 sapiensbreakpointcluster 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 sapiensclone 23585 mRNA sequence,	Up-regulated by Androgen
  	AF052134
  D26068	Williams-Beurensyndrome chromosome region 1	Up-regulated by Androgen
  X69433	H.sapiensmRNA for mitochondrial isocitrate	Up-regulated by Androgen
  	dehydrogenase (NADP+).
  X61123	B-cell translocation gene 1, anti-proliferative	Up-regulated by Androgen
  X63423	H.sapiensmRNA for delta-subunit of mitochondrial	Up-regulated by Androgen
  	F1F0 ATP-synthase
  AJ010025	Homo sapiensmRNA for unr-interacting protein.	Down-regulated by Androgen
  AF003938	Homo sapiensthioredoxin-like protein mRNA,	Down-regulated by Androgen
  	complete cds.
  AB014536	Homo sapienscopine 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 sapiensmRNA; 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.sapiensmRNA 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-interactingprotein 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-responsiveelement binding protein 1	Down-regulated by Androgen
  AF052578	Homo sapiensandrogen 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 sapiensmRNA for KIAA0612 protein	Down-regulated by Androgen
  NM_001328	Homo sapiensC-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 sapiensCGI-55 protein mRNA, complete cds	Down-regulated by Androgen
  L41351	Protease, serine, 8 (prostasin)	Down-regulated by Androgen
  AF077046	Homo sapiensganglioside 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 2NbHMHomo	Down-regulated by Androgen
  	sapienscDNA 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-bindingprotein 1	Down-regulated by Androgen
  NM_004713	Serologically definedcolon cancer antigen 1	Down-regulated by Androgen
  U36188	Clathrin-associated/assembly/adaptor protein,	Down-regulated byAndrogen
  	medium
  1
  AB020721	KIAA0914 gene product	Down-regulated by Androgen
  T35365	EST	Down-regulated by Androgen
  AF029789	Homo sapiensGTPase-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 sapiensfull 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 sapiensribosomal 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 sapiensclone 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 sapienscalcium/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.sapiensQRSHs mRNA for glutaminyl-tRNA	Down-regulated by Androgen
  	synthetase.
  X75861	Testis enhanced gene transcript	Down-regulated by Androgen
  AA593078	Homo sapiensPAC clone DJ0167F23 from 7p15	Down-regulated by Androgen
  J04058	Human electron transfer flavoprotein alpha-subunit	Down-regulated by Androgen
  	mRNA
  AF026292	Homo sapienschaperonin containing t-complex	Down-regulated byAndrogen
  	polypeptide
  1, eta
  AF068754	Homo sapiensheat shockfactor binding protein 1	Down-regulated by Androgen
  	HSBP1 mRNA,
  NM_000172	Guanine nucleotide binding protein (G protein),	Down-regulated by Androgen
  	alphatransducing activity polypeptide 1
  AI140631	Hs.1915: folate hydrolase (prostate-specific	Down-regulated by Androgen
  	membrane antigen) 1

• [0162]

  TABLE 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_NbHSFHomo sapienscDNA, 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 937223Homo sapienscDNA, 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_Lu5Homo sapienscDNA 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_NbHH19WHomo sapienscDNA clone, AF151840, CGI-
  	82 protein mRNA, tag is at 3′ end.
  F02980	HSC1IC062 normalized infant brain cDNAHomo sapienscDNA clone
  M99487	Human prostate-specific membrane antigen (PSM) mRNA, complete cds.
  AL035304	H.sapiensgene from PAC 295C6, similar to rat PO44.
  AI088979	ou86f03.s1 Soares_NSF_F8_9W_OT_PA_P_S1Homo sapienscDNA clone
  AF186249	Homo sapienssix transmembrane epithelial antigen of prostate (STEAP1) mRNA
  C15801	C15801 Clontech human aorta polyA + mRNA (#6572)Homo sapienscDNA
  L10340	Human elongation factor-1 alpha (ef-1) mRNA, 3′ end.
  NM_004540	Homo sapiensneural cell adhesion molecule 2 (NCAM2)
  AA151796	z139c02.r1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  NM_001634	Homo sapiensS-adenosylmethionine decarboxylase 1 (AMD1)
  NM_005013	Homo sapiensnucleobindin 2 (NUCB2)AL121913 in GenBank htgc database) and 718J7
  	(Accession number AL035541
  AF004828	Homo sapiensrab3-GAP regulatory domain mRNA, complete cds.
  X60819 X60	H.sapiensDNA for monoamine oxidase type A (14) (partial).
  AA133972	z138g12.r1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  M69226	Human monoamine oxidase (MAOA) mRNA, complete cds.
  AA969141	op50c11.s1 Soares_NFL_T_GBC_S1Homo sapienscDNA clone
  AA523652	ni64d09.s1 NCI_CGAP_Pr12Homo sapienscDNA clone IMAGE:981617, mRNA
  AF078749	Homo sapiensorganic cation transporter 3 (SLC22A3)
  AA583544	nf25h10.s1 NCI_CGAP_Pr1Homo sapienscDNA clone IMAGE:914851, mRNA
  AF051894	Homo sapiens15 kDa selenoprotein mRNA, complete cds.
  AF165967	Homo sapiensDDP-like protein mRNA
  X57129	H.sapiensH1.2 gene for histone H1.
  AA640928	nr28d08.r1 NCI_CGAP_Pr3Homo sapienscDNA clone IMAGE:1169295, mRNA
  U41766	Human metalloprotease/disintegrin/cysteine-rich protein precursor
  AF023676	Homo sapienslamin 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 sapiensEts transcription factor PDEF (PDEF) mRNA, complete
  U39840	Human hepatocyte nuclear factor-3 alpha (HNF-3 alpha) mRNA,
  AA532511	nj54d03.s1 NCI_CGAP_Pr9Homo sapienscDNA clone IMAGE:996293, mRNA
  X07166	Human mRNA for enkephalinase (EC 3.424.11).
  M96684	H.sapiensPur (pur-alpha) mRNA, complete cds.
  AI204040	qe77f05.x1 Soares_fetal_lung_NbHL19WHomo sapienscDNA clone
  AA577923	nl20a01.s1 NCI_CGAP_HSC1Homo sapienscDNA clone IMAGE:1041192,
  AA569633	nm38h09.s1 NCI_CGAP_Pr4.1Homo sapienscDNA 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_Lu1Homo sapienscDNA clone IMAGE:1143190 3′
  AF000993	Homo sapiensubiquitous TPR motif, X isoform (UTX) mRNA,
  W76105	zd65b04.r1 Soares_fetal_heart_NbHHI9WHomo sapienscDNA clone
  H39906	yo54a07.r1 Soares breast 3NbHBstHomo sapienscDNA clone
  AA971717	op95c11.s1 NCI_CGAP_Lu5Homo sapienscDNA clone IMAGE:1584596 3′,
  M68891	Human GATA-binding protein (GATA2) mRNA, complete cds.
  AA310157	EST181013 Jurkat T-cells VHomo sapienscDNA 5′ end, mRNA sequence.
  X00948	Human mRNA for prepro-relaxin H2.
  AB018330	Homo sapiensmRNA for KIAA0787 protein, partial cds.
  AA890637	ak11e11.s1 Soares_parathyroid_tumor_NbHPAHomo sapienscDNA clone
  M64929 J05	Human protein phosphatase 2A alpha subunit mRNA, complete cds.
  W24341	zb81h12.r1 Soares_senescent_fibroblasts_NbHSFHomo sapienscDNA
  AA974479	od58b03.s1 NCI_CGAP_GCB1Homo sapienscDNA clone IMAGE:1372109 3′
  R31644	yh69e05.r1 Soares placenta Nb2HPHomo sapienscDNA clone
  AA573246	nm52c02.s1 NCI_CGAP_Br2Homo sapienscDNA clone IMAGE:1071842 3′,
  AA507635	ng84b02.s1 NCI_CGAP_Pr6Homo sapienscDNA clone IMAGE:941451, mRNA
  gb|AF008915	Homo sapiensEVI5 homolog mRNA
  AL049987	Homo sapiensmRNA; cDNA DKFZp564F112 (from clone DKFZp564F112).
  U81599	Homo sapienshomeodomain protein HOXB13 mRNA
  AA641596	nr20f05.s1 NCI_CGAP_Pr2Homo sapienscDNA clone IMAGE:1168545, mRNA
  D84295	Human mRNA for possible protein TPRDII
  R13859	yf65d08.r1 Soares infant brain 1NIBHomo sapienscDNA clone
  M34840	Human prostatic acid phosphatase mRNA, complete cds.
  AA572913	nm42f12.s1 NCI_CGAP_Pr4.1Homo sapienscDNA clone IMAGE:1062863,
  AA094460	cp0378.seq.F Human fetal heart, Lambda ZAP ExpressHomo sapiens
  AF031166	Homo sapiensSRp46 splicing factor retropseudogene mRNA.
  AA625147	af70c07.r1 Soares_NhHMPu_S1Homo sapienscDNA clone IMAGE:1047372
  T39510	ya06h07.r1 Stratagene placenta (#937225)Homo sapienscDNA clone
  R35034	yg60h03.r1 Soares infant brain 1NIBHomo sapienscDNA clone
  AI003674	zg01c04.s1 Soares_pineal_gland_N3HPGHomo sapienscDNA clone
  AJ003636	AJ003636 Selected chromosome 21 cDNA libraryHomo sapienscDNA
  AA601385	no16d12.s1 NCI_CGAP_Phe1Homo sapienscDNA clone IMAGE:1100855 3′,
  AF191339	Homo sapiensanaphase-promoting complex subunit 5 (APC5)
  AA431822	zw79d02.r1 Soares_testis_NHTHomo sapienscDNA clone IMAGE:782403
  NM_003909	Homo sapienscopine III (CPNE3)
  AA484004	ne73f04.s1 NCI_CGAP_Ew1Homo sapienscDNA clone IMAGE:909919
  AA535774	nj78f08.s1 NCI_CGAP_Pr10Homo sapienscDNA clone IMAGE:998631, mRNA
  NM_000944.1	Homo sapiensprotein phosphatase 3 (formerly 2B)
  AA702811	zi90c11.s1 Soares_fetal_liver_spleen_1NFLS_S1Homo sapienscDNA
  X95073	H.sapiensmRNA for translin associated protein X.
  AA029039	zk12b07.s1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  AF032887	Homo sapiensforkhead (FKHRL1P1) pseudogene
  N46609	yy48h08.r1 Soares_multiple_sclerosis_2NbHMSPHomo sapienscDNA
  U58855	Homo sapienssoluble guanylate cyclase large subunit (GC-S-alpha-1)
  AA255486	zr83d03.s1 Soares_NhHMPu_S1Homo sapienscDNA clone IMAGE:682277
  AA128153	zl15c06.s1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  AA016039	ze31c05.s1 Soares retina N2b4HRHomo sapienscDNA clone
  R88520	ym91e09.s1 Soares adult brain N2b4HB55YHomo sapienscDNA clone
  M26624	Human CALLA/NEP gene encoding neutral endopeptidase, exon 20.
  AA026997	ze99e01.r1 Soares_fetal_heart_NbHH19WHomo sapienscDNA clone
  W48775	zc44b08.r1 Soares_senescent_fibroblasts_NbHSFHomo sapienscDNA
  AA074407	zm15c08.r1 Stratagene pancreas (#937208)Homo sapienscDNA clone
  L13972	Homo sapiensbeta-galactoside alpha-2,3-sialyltransferase (SIAT4A)
  D14661	Human mRNA for KIAA0105 gene, complete cds.
  AA115452	zk89a08.r1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  AA495742	zw04b12.r1 Soares_NhHMPu_S1Homo sapienscDNA clone IMAGE:768287 5′
  R13416	yf75h09.r1 Soares infant brain 1NIBHomo sapienscDNA clone
  AA046369	zk77h07.r1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  T35440	EST85129 Human LungHomo sapienscDNA 5′ end similar to None, mRNA
  AI075860	oz25b05.x1 Soares_total_fetus_Nb2HF8_9wHomo sapienscDNA clone
  W56437	zc57g05.r1 Soares_parathyroid_tumor_NbHPAHomo sapienscDNA clone
  AI583880	tt70b02.x1 NCI_CGAP_HSC3Homo sapienscDNA clone IMAGE:2246091 3′
  D85181	Homo sapiensmRNA for fungal sterol-C5-desaturase homolog, complete
  AF105714	Homo sapiensprotein kinase PITSLRE (CDC2L2) gene,exon 3.
  AA401802	zt60c12.r1 Soares_testis_NHTHomo sapienscDNA clone IMAGE:726742
  AB002301	Human mRNA for KIAA0303 gene, partial cds.
  U75667	Human arginase II mRNA, complete cds.
  AA585183	JTH089 HTCDL1Homo sapienscDNA 5′/3′, mRNA sequence.
  AF191771	Homo sapiensCED-6 protein (CED-6) mRNA
  AA650252	ns93g05.s1 NCI_CGAP_Pr3Homo sapienscDNA clone IMAGE:1191224, mRNA
  R64618	yi19b09.r1 Soares placenta Nb2HPHomo sapienscDNA clone
  N24627	yx89a09.s1 Soares melanocyte 2NbHMHomo sapienscDNA clone
  AB028951	Homo sapiensmRNA for KIAA1028 protein
  N75608	yw37a07.r1 Morton Fetal CochleaHomo sapienscDNA clone
  N53899	yy98e03.r1 Soares_multiple_sclerosis_2NbHMSPHomo sapienscDNA
  N46696	yy50f07.r1 Soares_multiple_sclerosis_2NbHMSPHomo sapienscDNA
  AA419522	zv03d05.r1 Soares_NhHMPu_S1Homo sapienscDNA clone IMAGE:752553
  M61906	Human P13-kinase associated p85 mRNA sequence.
  C16570	C16570 Clontech human aorta polyA + mRNA (#6572)Homo sapienscDNA
  X63105	H.sapienstpr mRNA.
  AA315855	EST187656 Colon carcinoma (HCC) cell line IIHomo sapienscDNA 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 sapienscDNA clone
  N40094	yx98g07.r1 Soares melanocyte 2NbHMHomo sapienscDNA clone
  N98940	yy71a07.r1 Soares_multiple_sclerosis_2NbHMSPHomo sapienscDNA
  AF049907	Homo sapienszinc finger transcription factor (ZNF-X) mRNA,
  M78806	EST00954 Hippocampus, Stratagene (cat. #936205)Homo sapienscDNA
  AA040819	zk47b03.r1 Soares_pregnant_uterus_NbHPUHomo sapienscDNA clone
  C15445	C15445 Clontech human aorta polyA + mRNA (#6572)Homo sapienscDNA
  AB018309	Homo sapiensmRNA for KIAA0766 protein, complete cds.
  AJ011497	Homo sapiensmRNA for Claudin-7.
  X00949	Human mRNA for prepro-relaxin H1.
  AA418633	zv93d09.r1 Soares_NhHMPu_S1Homo sapienscDNA clone IMAGE:767345 5′
  AI146806	qb83h04.x1 Soares_fetal_heart_NbHH19WHomo sapienscDNA clone
  X82942	H.sapienssatellite 3 DNA.
  AA456383	aa14f03.r1 Soares_NhHMPu_S1Homo sapienscDNA clone IMAGE:813245
  AA019341	ze57e04.s1 Soares retina N2b4HRHomo sapienscDNA clone
  AB027466	Homo sapiensSPON2 mRNA forspondin 2
  AF038170	Homo sapiensclone 23817 mRNA sequence.
  NM_000240	Homo sapiensmonoamine oxidase A (MAOA)
  N34126	yx76c01.r1 Soares melanocyte 2NbHMHomo sapienscDNA clone
  N41339	yw68g06.r1 Soares_placenta_8to9weeks_2NbHP8to9WHomo sapienscDNA
  R34783	yh87b05.r1 Soares placenta Nb2HPHomo sapienscDNA clone
  N75858	yw32a03.r1 Morton Fetal CochleaHomo sapienscDNA clone
  AA633887	ac32h04.s1 Stratagene hNT neuron (#937233)Homo sapienscDNA clone
  N53723	yz06d03.r1 Soares_multiple_sclerosis_2NbHMSPHomo sapienscDNA
  AI187365	qf29b12.x1 Soares_testis_NHTHomo sapienscDNA clone IMAGE:1751423

• [0163]

  TABLE 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		Nuclearreceptor 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 sapiensnuclear	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 ProstateCancer
  	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]

  TABLE 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]

  TABLE 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. sapiensgene 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]

  TABLE 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 β
  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

• 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.[0167]
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• [0298]
• 16711140DNAHomo sapiensCDS(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 11402759DNAHomo 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 7593252PRTHomo 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 25048PRTArtificial SequenceDescription of Artificial Sequence FLAG peptide 4 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5524DNAArtificial SequenceDescription of Artificial Sequence Primer 5 ggcagaacac tccgcgcttc ttag 24624DNAArtificial SequenceDescription of Artificial Sequence Primer 6 caagctctct tagcttgtgc attc 24722DNAArtificial SequenceDescription of Artificial Sequence Primer 7 cttgggttcg ggtgaaagcg cc 22822DNAArtificial SequenceDescription of Artificial Sequence Primer 8 ggtgggtggc aggtcgatct cg 22920DNAArtificial SequenceDescription of Artificial Sequence Primer 9 ccttcgccca gcgggagcgc 201024DNAArtificial SequenceDescription of Artificial Sequence Primer 10 caagctctct tagcttgtgc attc 2411249PRTHomo 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 24512244PRTHomo 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 Asn1310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 13 gccagcccag 101410DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14 gtgcagggag 101510DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15 gacaaacatt 101610DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 16 atgactcaag 101710DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17 gaaaagaagg 101810DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18 cctgtacccc 101910DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19 cctgaactgg 102010DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20 tgacagccca 102110DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 21 tacaaaacca 102210DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 22 aattctccta 102310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23 tgcatatcat 102410DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24 cttgacacac 102510DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25 tgtctaacta 102610DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 26 gtggacccca 102710DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 27 ataaagtaac 102810DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 28 tacattttca 102910DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 29 tcagaacagt 103010DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 30 caacttcaac 103110DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 31 gataggtcgg 103210DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 32 ctaaaaggag 103310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 33 gtggtgcgtg 103410DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 34 tccccgtggc 103510DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 35 attgatcttg 103610DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 36 agctggtttc 103710DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37 cctcccccgt 103810DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 38 atgtactctg 103910DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39 gatgaaatac 104010DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40 gtgcatcccg 104110DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 41 gaaattaggg 104210DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 42 tttctagggg 104310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 43 cccagggaga 104410DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 44 gtggcgcaca 104510DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 45 ttgcttttgt 104610DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 46 atgtcctttc 104710DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 47 tgtttatcct 104810DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 48 gctttgtatc 104910DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 49 gttccagtga 105010DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 50 tagcagaggc 105110DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 51 acaaattatg 105210DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 52 cagtttgtac 105310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 53 gattacttgc 105410DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 54 ggccagccct 105510DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 55 caattgtaaa 105610DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 56 aaagccaaga 105710DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 57 caactaattc 105810DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 58 aagagctaat 105910DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 59 cttttcaaga 106010DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 60 gtgtgtaaaa 106110DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 61 acaaaatgta 106210DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 62 aaggtagcag 106310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 63 ggcggggcca 106410DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 64 ggccagtaac 106510DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 65 aacttaagag 106610DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 66 agggatggcc 106710DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 67 cttaaggatt 10

Claims (24)

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 ofclaim 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 ofclaim 2, wherein the androgen is R1881.

4. The polynucleotide array ofclaim 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 toclaim 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 toclaim 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 toclaim 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 toclaim 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 toclaim 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 ofclaim 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 ofclaim 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 ofclaim 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 ofclaim 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 toclaim 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 ofclaim 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 ofclaim 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 toclaim 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 ofclaim 18.

20. A host cell transfected or transduced with the vector ofclaim 19.

21. The host cell ofclaim 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 ofclaim 23.

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