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

Method of detecting androgen-regulated gene Download PDF

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US20030170713A1
US20030170713A1 US10/390,045 US39004503A US2003170713A1 US 20030170713 A1 US20030170713 A1 US 20030170713A1 US 39004503 A US39004503 A US 39004503A US 2003170713 A1 US2003170713 A1 US 2003170713A1
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androgen
regulated
polynucleotide
prostate
protein
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Shiv Srivastava
Judd Moul
Linda Xu
Takehiko Segawa
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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Priority to US10/434,479 priority patent/US20090176722A9/en
Publication of US20030170713A1 publication Critical patent/US20030170713A1/en
Priority to US11/452,925 priority patent/US7537896B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • 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.
  • Prostate cancer 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).
  • 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.
  • CGH comparative genomic hybridization
  • transcripts that can be analyzed are the major limitation encountered in subtractive hybridization and differential display approaches.
  • 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.
  • hormone therapy 314
  • 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.
  • hormone therapy 314
  • 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.
  • ARGs androgen responsive genes
  • 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.
  • 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.
  • 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.
  • the present invention also relates to a polynucleotide array
  • 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.
  • the polynucleotides immobilized on the solid support include genes that are known to be involved in testosterone biosynthesis and metabolism.
  • the oligonucleotides immobilized on the solid support include genes whose expression is altered in prostate cancer or is specific to prostate tissue.
  • 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.
  • 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.
  • 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.
  • 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.
  • computer models to such cancers may be developed.
  • 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.
  • 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.
  • 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.
  • FIG. 3 shows PMEPA1 expression in CwR22 xenograft tumors. Lane 1, sample from CWR22 tumor (androgen dependent). Lanes 2-5, samples from 4 individual CW2R tumors (AR positive but androgen independent).
  • 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.
  • 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.
  • 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).
  • Table 3 genes and ESTs derived from the inventors' SAGE library that are up-regulated and down-regulated by and
  • Tables 3-8 are located at the end of the specification at the end of the “Detailed Description” section and before the “References.”
  • genes in bold type are known androgen-regulated genes based on Medline Search.
  • genes in bold type are known prostate-specific genes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ESTs expressed sequence tags
  • 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.
  • Our study provides quantitative information on about 23,000 transcripts expressed in LNCaP cells, the most common cell line used in prostate cancer research.
  • ARGs 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.
  • ARFBs AR Function Bio-indicators
  • 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).
  • ARGs including PMEPA1
  • ARGs can be used as biomarkers of AR function readout in the subset of prostate cancers that may involve abrogation of androgen signaling.
  • the newly defined ARGs have potential to identify novel targets in therapy of hormone refractory prostate cancer.
  • nucleic acid molecules encompassed in the invention include the following PMEPA1 nucleotide sequence: ATGGCGGAGC TGGAGTTTGT TCAGATCATC ATCATCGTGG TGGTGATGAT 50 (SEQ ID NO.
  • amino acid sequences of the polypeptides encoded by the PMEPA1 nucleotide sequences of the invention include: MAELEFVQII IIVVVMMVMV VVITCLLSHY KLSARSFISR HSQGRRREDA 50 (SEQ ID NO.
  • nucleic acids of the invention enable 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,
  • prostate cancer e.g
  • nucleotide sequence refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct.
  • the nucleic acid molecule has been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).
  • 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.
  • 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.
  • nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.
  • 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.
  • 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.
  • 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.
  • polypeptides encoded by such DNA sequences are encompassed by the invention.
  • conditions of moderate stringency can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA.
  • the basic conditions are set forth by (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989)), and include use of a prewashing solution for the nitrocellulose filters 5 ⁇ SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 6 ⁇ SSC at about 42° C.
  • 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.
  • 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.
  • the percent identity may be determined by visual inspection and mathematical calculation.
  • the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by (Devereux et al., Nucl. Acids Res., 12:387 (1984)) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of (Gribskov and Burgess. Nucl.
  • the invention also provides isolated nucleic acids useful in the production of polypeptides.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • Oligonucleotides that define the desired termini of the DNA fragment are employed as 5′ and 3′ primers.
  • the oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector.
  • PCR techniques are described in (Saiki et al., Science, 239:487 (1988)); (Wu et al., Recombinant DNA Methodology, eds., Academic Press, Inc., San Diego, pp. 189-196 (1989)); and (Innis et al., PCR Protocols: A Guide to Methods and Applications, eds., Academic Press, Inc. (1990)).
  • 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.
  • 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.
  • 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.
  • 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.
  • soluble forms are 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.
  • the invention also provides polypeptides and fragments of the extracellular domain that retain a desired biological activity.
  • a fragment may be a soluble polypeptide, as described above.
  • 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.
  • Variants may exhibit amino acid sequences that are at least 80% identical. Also contemplated are embodiments in which a polypeptide or fragment comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the preferred polypeptide or fragment thereof. Percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by comparing sequence information using the GAP computer program, based on the algorithm of (Needleman and Wunsch, J. Mol. Bio., 48:443 (1970)) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by (Henikoff and Henikoff Proc. Natl. Acad. Sci. USA, 89:10915 (1992)); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps.
  • Other programs used by one skilled in the art of sequence comparison may also be used.
  • 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.
  • 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.
  • Other derivatives include covalent or aggregative conjugates of the polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with oligomers. Further, fusion proteins can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in (Hopp et al., Bio/Technology, 6:1204 (1988)).
  • FLAG® 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.
  • variant polypeptides that retain the native biological activity or the substantial equivalent thereof.
  • variants 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.
  • 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.
  • a given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics.
  • 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.
  • 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.
  • 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
  • yeast or mammalian expression systems e.g., COS-1 or COS-7 cells
  • Expression of polypeptides of the invention in bacterial expression systems, such as E. Coli provides non-glycosylated molecules.
  • 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.
  • glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).
  • 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.
  • nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain.
  • 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.
  • 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.
  • 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.
  • polypeptides and fragments of the invention may be accomplished by any suitable technique, including but not limited to the following:
  • 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.
  • 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.
  • suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • 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.
  • 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.
  • a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide 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.
  • Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in (Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985)). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein.
  • Prokaryotes include gram-negative or gram-positive organisms. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium , and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • 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.
  • 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).
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978); and (Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412 (1982)).
  • a particularly useful prokaryotic host cell expression system employs a phage ⁇ P L promoter and a cI857ts thermolabile repressor sequence.
  • Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the ⁇ P L promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).
  • the polypeptides may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae ). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed.
  • yeast vectors will often contain an origin of replication sequence from a 2 ⁇ 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.
  • 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.
  • enolase glyceraldehyde-3-phosphate dehydrogenase
  • hexokinase hexokinase
  • pyruvate decarboxylase phosphofructokinase
  • glucose-6-phosphate isomerase 3-phosphoglycerate mutase
  • yeast vectors and promoters for use in yeast expression are further described in (Hitzeman, EPA-73,657).
  • Another alternative is the glucose-repressible ADH2 promoter described by (Russell et al., J. Biol. Chem. 258:2674 (1982)) and (Beier et al., Nature 300:724 (1982)).
  • Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp r gene 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 ⁇ -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., (Kurjan et al., Cell 30:933 (1982)) and (Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330 (1984)).
  • Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art.
  • a leader sequence may be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art.
  • One such protocol is described by (Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978)).
  • the Hinnen et al. protocol selects for Trp + 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.
  • 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.
  • Mammalian or insect host cell culture systems also may be employed to express recombinant polypeptides.
  • Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by (Luckow and Summers, Bio/Technology, 6:47 (1988)).
  • Established cell lines of mammalian origin also may be employed.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175 (1981)), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by (McMahan et al., EMBO J., 10: 2821 (1991)).
  • DHFR dihydrofolate reductase resistance.
  • a suitable host strain for DHFR selection can be CHO strain DX-B 11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)).
  • a plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media.
  • selectable markers 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, 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.
  • 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., 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)).
  • EASE expression augmenting sequence element
  • TPL tripartite leader
  • VA gene RNAs from Adenovirus 2
  • the internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3:295-300 (1993)); (Ramesh et al., Nucleic Acids Research 24:2697-2700 (1996)).
  • IRS internal ribosome entry site
  • 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., Cell 59:335-348 (1989)).
  • Other expression vectors for use in mammalian host cells can be constructed as disclosed by (Okayama and Berg, Mol. Cell. Biol. 3:280 (1983)).
  • a useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by (Cosman et al., Mol. Immunol. 23:935 (1986)).
  • a useful high expression vector, PMLSV N1/N4 described by (Cosman et al., Nature 312:768 (1984)), has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982, incorporated by reference herein.
  • the vectors can be derived from retroviruses.
  • pFLAG® 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.
  • DHFR dihydrofolate reductase
  • the invention also includes methods of isolating and purifying the polypeptides and fragments thereof.
  • 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.
  • 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.
  • 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.
  • the culture medium first can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • the concentrate can be applied to a purification matrix such as a gel filtration medium.
  • 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.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a chromatofocusing step can be employed.
  • a hydrophobic interaction chromatography step can be employed.
  • Suitable matrices can be phenyl or octyl moieties bound to resins.
  • 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.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups
  • RP-HPLC media e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups
  • 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.
  • 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.
  • 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.
  • 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.
  • avidin-coated beads is known in the art. See (Berenson, et al. J. Cell. Biochem., 10D:239 (1986)). Wash of unbound material and the release of the bound cells is performed using conventional methods.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • These antigenic determinants or epitopes can be either linear or conformational (discontinuous).
  • Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9, Garland Publishing Inc., 2nd ed. (1996)).
  • the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14, Garland Publishing Inc., 2nd ed. (1996)).
  • Epitopes may be identified by any of the methods known in the art.
  • 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.
  • 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.
  • both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, (Kennet et al., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, eds., Plenum Press, New York (1980); and Harlow and Land, Antibodies: A Laboratory Manual, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988)).
  • 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.
  • 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.
  • 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.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in (Riechmann et al., Nature 332:323 (1988), Liu et al., PNAS 84:3439 (1987), Larrick et al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS 14:139 (May 1993)).
  • Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein.
  • Antigen-binding fragments of the antibodies which may be produced by conventional techniques, are also encompassed by the present invention.
  • fragments include, but are not limited to, Fab and F(ab′) 2 fragments.
  • Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
  • 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.
  • 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.
  • FBS fetal bovine serum
  • 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.
  • 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.).
  • 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.
  • 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 ⁇ g/lane) by running through 1% formmaldehyde-agarose gel and transferred to nylon membrane. The cDNA probes of several ARGs were labeled with 32 P-dCTP by random priming (Stratagene Cloning Systems, La Jolla, Calif.).
  • 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 the 32 P 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.
  • 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 32 P labeled probes of the cDNA of interest.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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 (
  • the mitochondrial transcription factor 1 (mtTF1) was induced by 8 fold in response to R1881.
  • mtTF1 mitochondrial transcription factor 1
  • PGC-1 nuclear receptor superfamily
  • ear-2 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.
  • 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.
  • 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.
  • RNA processing and translation components 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FBS fetal bovine serum
  • charcoal/dextran stripped androgen-free FBS cFBS, Gemini Bio-Products, Inc., Calabasas, Calif.
  • 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 + 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.
  • R1881 DUPONT, Boston, Mass.
  • RNA specimens (2 ⁇ g/lane) were electrophoresed in a 1% formaldehyde-agarose gel and transferred to a nylon membrane.
  • Two PMEPA1 probes used for Northern blots analysis were (a) cDNA probe spanning nucleotides 3-437 of PMEPA1 cDNA sequence (See Table 1) and (b) 71-mer oligonucleotide between nucleotides 971 to 1,041 of PMEPA1 cDNA sequence (See Table 1).
  • the cDNA probe was generated by RT-PCR with primers 5′CTTGGGTTCGGGTGAAAGCGCC 3′ (SEQ ID NO. 7) (sense) and 5′GGTGGGTGGCAGGTCGATCTCG 3′ (SEQ ID NO. 8) (antisense).
  • PMEPA1 oligonucleotide and cDNA probes and glyceraldehyde phosphate dehydrogenase gene (GAPDH) cDNA probe were labeled with 32 P-dCTP using 3′ end tailing for oligonucleotides (Promega, Madison, Wis.) and random priming for cDNA (Stratagene, La Jolla, Calif.).
  • 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 the 32 P labeled probes (1 ⁇ 10 6 cpm/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.
  • CWR22 is an androgen-dependent, serially transplantable nude mouse xenograft derived from a primary human prostate cancer.
  • Transplanted CWR22 tumors are positive for AR and the growth of CWR22 is androgen dependent.
  • CWR22 tumors regress initially upon castration followed by a relapse.
  • the recurrent CWR22 tumors (CWR22R) express AR, but the growth of these tumors become androgen-independent (Gregory et al., 1998; Nagabhushan et al., 1996).
  • One CW 2 and four CWR22R tumor specimens were kindly provided by Dr. Thomas Pretlow's laboratory (Case Western Reserve University School of Medicine). Tumor tissues were homogenized and poly A + 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.
  • 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.
  • 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).
  • 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.
  • PMEPA1 also shares other features with C18orf1, e.g., similar size of the predicted protein and similar transmembrane domain as the ⁇ 1 isoform of C18orf1.
  • C18orf1 SEQ ID NO: 11
  • C18orf1 SEQ ID NO: 12
  • PMEPA1 S G E + PR DR P F QR+RF RFQPTYPY
  • 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).
  • AA280663 EST Up-regulated by Androgen U31657 KRAB-associated protein 1 Up-regulated by Androgen AI879709 EST Up-regulated by Androgen AA602190 EST Up-regulated by Androgen AF035587 Homo sapiens X-ray repair cross-complementing Up-regulated by Androgen protein 2 (XRCC2) AF151898 Homo sapiens CGI-140 protein mRNA Up-regulated by Androgen AA418786 No reliable matches, only see in two linberary (1 Up-regulated by Androgen each) AI308812 EST Up-regulated by Androgen X59408 Membrane cofactor protein (CD46, trophoblast- Up-regulated by Androgen lymphocyte cross-reactive antigen) X81817 Accessory proteins BAP31/BAP29 Up-regulated by Androgen AF071538 Homo sapiens Ets transcription factor PDEF Up-regulated by Androgen (PDEF) mRNA, complete NM_00
  • Androgen U58855 Guanylate cyclase 1, soluble, alpha 3 Up-regulated by Androgen X12794 Human v-erbA related ear-2 gene.
  • Androgen U88542 Mus musculus homeobox protein Nkx3.1 Up-regulated by Androgen D89729 Homo sapiens mRNA for CRM1 protein, complete Up-regulated by Androgen cds.
  • 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.
  • Up-regulated by Androgen AF000979 Homo sapiens testis-specific Basic Protein Y 1 Up-regulated by Androgen (BPY1) mRNA, AA889510 EST Up-regulated by Androgen AB018330 Homo sapiens mRNA for KIAA0787 protein, partial Up-regulated by Androgen cds.
  • AA026941 EST Up-regulated by Androgen AA532377
  • Chromosome 1 open reading frame 8 Up-regulated by Androgen AF010313
  • Homo sapiens Pig8 (PIG8) mRNA etoposide- Up-regulated by Androgen induced mRNA
  • L06328 Human voltage-dependent anion channel isoform 2 Up-regulated by Androgen (VDAC) mRNA
  • U41804 Human putative T1/ST2 receptor binding protein Up-regulated by Androgen precursor mRNA
  • AB020676 Homo sapiens mRNA for KIAA0869 protein, partial Up-regulated by Androgen cds.
  • AA120930 EST Up-regulated by Androgen AB002321 Human mRNA for KIAA0323 gene, partial cds Up-regulated by Androgen AF151837 Homo sapiens CGI-79 protein mRNA, complete cds Up-regulated by Androgen AA481027 EST Up-regulated by Androgen AA039343 EST Up-regulated by Androgen U09716 Human mannose-specific lectin (MR60) mRNA, Up-regulated by Androgen complete cds.
  • MR60 Human mannose-specific lectin
  • AF044773 Homo sapiens breakpoint cluster region protein 1 Up-regulated by Androgen BCRG1 mRNA U51586 Human siah binding protein 1 (SiahBP1) mRNA, Up-regulated by Androgen partial cds.
  • AI282096 EST Up-regulated by Androgen W45510 RAB7, member RAS oncogene family-like 1 Up-regulated by Androgen X16135
  • X61123 B-cell translocation gene 1 anti-proliferative Up-regulated by Androgen X63423 H.sapiens mRNA for delta-subunit of mitochondrial Up-regulated by Androgen F1F0 ATP-synthase AJ010025 Homo sapiens mRNA for unr-interacting protein. Down-regulated by Androgen AF003938 Homo sapiens thioredoxin-like protein mRNA, Down-regulated by Androgen complete cds.
  • CTBP1 C-terminal binding protein 1
  • BTF1 Human butyrophilin
  • SIII Transcription elongation factor B (SIII), polypeptide Down-regulated by Androgen 1-like M34539 FK506-binding protein 1A (12kD) Down-regulated by Androgen N43807 yy19a05.r1 Soares melanocyte 2NbHM Homo Down-regulated by Androgen sapiens cDNA clone U03269 Human actin capping protein alpha subunit (CapZ) Down-regulated by Androgen mRNA, complete AI571685 EST Down-regulated by Androgen AA010412 EST Down-regulated by Androgen L40403 Homo sapiens (clone zap3) mRNA, 3′ end of cds.
  • CapZ Human actin capping protein alpha subunit
  • RNA-binding protein 1 Down-regulated by Androgen NM_006560 CUG triplet repeat RNA-binding protein 1 Down-regulated by Androgen NM_004713
  • Serologically defined colon cancer antigen 1 Down-regulated by Androgen U36188 Clathrin-associated/assembly/adaptor protein Down-regulated by Androgen medium 1 AB020721 KIAA0914 gene product Down-regulated by Androgen T35365 EST Down-regulated by Androgen AF029789 Homo sapiens GTPase-activating protein (SIPA1) Down-regulated by Androgen mRNA, complete cds.
  • SIPA1 Homo sapiens GTPase-activating protein
  • AF070666 Homo sapiens tissue-type pituitary Kruppel- Down-regulated by Androgen associated box protein R55128 Proteasome (prosome, macropain) 26S subunit, non- Down-regulated by Androgen ATPase, 2 X75621 Tuberous sclerosis 2 Down-regulated by Androgen AA019070 EST Down-regulated by Androgen AI089867 EST Down-regulated by Androgen NM_001003 Homo sapiens ribosomal protein, large, P1 (RPLP1) Down-regulated by Androgen mRNA L05093 Ribosomal protein L18a Down-regulated by Androgen AA854176 EST Down-regulated by Androgen AI929622 Homo sapiens clone 23675 mRNA sequence Down-regulated by Androgen AI264769 ESTs, Weakly similar to ORF YDL087c Down-regulated by Androgen [ S.cerevisiae ] L09159 Ras
  • X75861 Testis enhanced gene transcript Down-regulated by Androgen AA593078 Homo sapiens PAC clone DJ0167F23 from 7p15 Down-regulated by Androgen J04058 Human electron transfer flavoprotein alpha-subunit Down-regulated by Androgen mRNA AF026292 Homo sapiens chaperonin containing t-complex Down-regulated by Androgen polypeptide 1, eta AF068754 Homo sapiens heat shock factor binding protein 1 Down-regulated by Androgen HSBP1 mRNA, NM_000172 Guanine nucleotide binding protein (G protein), Down-regulated by Androgen alpha transducing activity polypeptide 1 AI140631 Hs.1915: folate hydrolase (prostate-specific Down-regulated by Androgen membrane antigen) 1
  • Hs.118724 histidine triad nucleotide-binding protein, AJ012499, mRNA activated in tumor suppression, clone TSAP19 with polyA AA082804 zn41g02.r1
  • Stratagene endothelial cell 937223 Homo sapiens cDNA, Hs.110967: ESTs, Weakly similar to KIAA0762 protein [ H.sapiens ], Hs.5662: guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 in the sequence no tag X05332 Human mRNA for prostate specific antigen.
  • AI278854 qo42f01.x1 NCI_CGAP_Lu5
  • Homo sapiens cDNA clone IMAGE 1911193 3′, NM_004537 nucleosome assembly protein 1-like
  • NM_004540 Homo sapiens neural cell adhesion molecule 2 (NCAM2) AA151796 z139c02.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone NM_001634 Homo sapiens S-adenosylmethionine decarboxylase 1 (AMD1) NM_005013 Homo sapiens nucleobindin 2 (NUCB2)AL121913 in GenBank htgc database) and 718J7 (Accession number AL035541 AF004828 Homo sapiens rab3-GAP regulatory domain mRNA, complete cds.
  • NCAM2 neural cell adhesion molecule 2
  • AAD1 S-adenosylmethionine decarboxylase 1
  • NUCB2 Homo sapiens nucleobindin 2
  • 718J7 accesion number AL035541 AF004828 Homo sapiens rab3
  • AF165967 Homo sapiens DDP-like protein mRNA X57129 H.sapiens H1.2 gene for histone H1. AA640928 nr28d08.r1
  • NCI_CGAP_Pr3 Homo sapiens cDNA clone IMAGE:1169295, mRNA U41766 Human metalloprotease/disintegrin/cysteine-rich protein precursor
  • AF023676 Homo sapiens lamin B receptor homolog TM7SF2 (TM7SF2) mRNA, U10691 Human MAGE-6 antigen (MAGE6) gene, complete cds.
  • M22976 Human cytochrome b5 mRNA, 3′ end.
  • AI204040 qe77f05.x1 Soares_fetal_lung_NbHL19W Homo sapiens cDNA clone AA577923 nl20a01.s1 NCI_CGAP_HSC1 Homo sapiens cDNA clone IMAGE:1041192, AA569633 nm38h09.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE:1062497, U65011 Human preferentially expressed antigen of melanoma (PRAME) mRNA, U21910 Human basic transcription factor BTF2p44 mRNA, 3′ end, partial cds.
  • PRAME Human preferentially expressed antigen of melanoma
  • GATA2 Human GATA-binding protein
  • AA310157 EST181013 Jurkat T-cells V Homo sapiens cDNA 5′ end, mRNA sequence. X00948 Human mRNA for prepro-relaxin H2. AB018330 Homo sapiens mRNA for KIAA0787 protein, partial cds. AA890637 ak11e11.s1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone M64929 J05 Human protein phosphatase 2A alpha subunit mRNA, complete cds.
  • Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AF032887 Homo sapiens forkhead (FKHRL1P1) pseudogene N46609 yy48h08.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA U58855 Homo sapiens soluble guanylate cyclase large subunit (GC-S-alpha-1) AA255486 zr83d03.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:682277 AA128153 zl15c06.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AA016039 ze31c05.s1 Soares retina N2b4HR Homo sapiens cDNA clone R88520
  • Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767345 5′ AI146806 qb83h04.x1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone X82942 H.sapiens satellite 3 DNA.
  • NM_000240 Homo sapiens monoamine oxidase A (MAOA) N34126 yx76c01.r1 Soares melanocyte 2NbHM Homo sapiens cDNA clone N41339 yw68g06.r1 Soares_placenta_8to9weeks_2NbHP8to9W Homo sapiens cDNA R34783 yh87b05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone N75858 yw32a03.r1 Morton Fetal Cochlea Homo sapiens cDNA clone AA633887 ac32h04.s1 Stratagene hNT neuron (#937233) Homo sapiens cDNA clone N53723 yz06d03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA AI187365 qf29b12.x1 Soares
  • Prostate Specific PNAS 95, 300, 98 seminal plasma protein, nc50a10, microminderoprotein 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.18
  • 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
  • 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 ⁇ 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
  • 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.
  • Bova G S and Issacs W B Review of allelic loss and gain in prostate cancer. World J Urol., 14:338-346, 1996.
  • Veldscholte, J, R is-Stalpers C, Kulper G G J M, Jenster G, Berre-voets C, Classen E, Van Roooj H C J, Trapman J, Brinkmann A O, Mulder E.
  • a mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem. Biophys. Res. Commun., 173:534-540, 1990.
  • Chromosome arm 20q gains and other genomic alterations in colorectal cancer metastatic to liver, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization. Genes Chromosomes Cancer. 25: 82-90.
  • VDAC voltage-dependent anion channel

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 the nitrocellulose 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., PCR Protocols: 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 Henikoff Proc. Natl. Acad. Sci. USA, 89:10915 (1992)); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.
  • 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 as E. coli, a polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.
  • 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] Nature 275:615 (1978); and (Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412 (1982)). A particularly useful prokaryotic host cell expression system employs a phage λPL promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λPL promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, 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., Nature 300:724 (1982)). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene 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] Cell 30:933 (1982)) and (Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330 (1984)). Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • 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. USA 75:1929 (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., Cell 23:175 (1981)), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by (McMahan et al., EMBO J., 10: 2821 (1991)).
  • 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. USA 84:7413-7417 (1987)). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press (1989)). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. (Kaufman et al., Meth. in Enzymology 185:487-511 (1990)), describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B 11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
  • 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. Nature 273:113 (1978)); (Kaufman, Meth. in Enzymology (1990)). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • 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 Development 3:295-300 (1993)); (Ramesh et al., Nucleic Acids Research 24:2697-2700 (1996)). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology (1990)). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by (Mosser et al., Biotechniques 22:150-161 (1997)), and p2A5I described by (Morris et al., Animal Cell Technology, pp. 529-534 (1997)).
  • A useful high expression vector, pCAVNOT, has been described by (Mosley et al., [0101] Cell 59:335-348 (1989)). Other expression vectors for use in mammalian host cells can be constructed as disclosed by (Okayama and Berg, Mol. Cell. Biol. 3:280 (1983)). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by (Cosman et al., Mol. Immunol. 23:935 (1986)). A useful high expression vector, PMLSV N1/N4, described by (Cosman et al., Nature 312:768 (1984)), has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982, incorporated by reference herein. In yet another alternative, the vectors can be derived from retroviruses.
  • 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 Biology 3:9, Garland Publishing Inc., 2nd ed. (1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14, Garland Publishing Inc., 2nd ed. (1996)). Epitopes may be identified by any of the methods known in the art.
  • 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] Nature 332:323 (1988), Liu et al., PNAS84:3439 (1987), Larrick et al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS 14:139 (May 1993)). Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein.
  • 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] 2 fragments. 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 the 32P 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 with 32P-dCTP using 3′ end tailing for oligonucleotides (Promega, Madison, Wis.) and random priming for cDNA (Stratagene, La Jolla, Calif.). The nylon membranes were treated with hybridization buffer (10 mM Tris-HCl, pH 7.5, 10% Dextran sulfate, 40% Formamide, 5×SSC, 5× Denhardt's solution and 0.25 mg/ml salmon sperm DNA) for two hours followed by hybridization in the same buffer containing the 32P labeled probes (1×106 cpm/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 sapiens prostate-specific antigen mRNA, Up-regulated by Androgen
    complete cds.*
    AA508573 Human nucleolin gene, complete cds Up-regulated by Androgen
    AB020637 Homo sapiens mRNA for KIAA0830 protein, partial Up-regulated by Androgen
    cds.
    AA280663 EST Up-regulated by Androgen
    U31657 KRAB-associated protein 1 Up-regulated by Androgen
    AI879709 EST Up-regulated by Androgen
    AA602190 EST Up-regulated by Androgen
    AF035587 Homo sapiens X-ray repair cross-complementing Up-regulated by Androgen
    protein 2 (XRCC2)
    AF151898 Homo sapiens CGI-140 protein mRNA Up-regulated by Androgen
    AA418786 No reliable matches, only see in two linberary (1 Up-regulated by Androgen
    each)
    AI308812 EST Up-regulated by Androgen
    X59408 Membrane cofactor protein (CD46, trophoblast- Up-regulated by Androgen
    lymphocyte cross-reactive antigen)
    X81817 Accessory proteins BAP31/BAP29 Up-regulated by Androgen
    AF071538 Homo sapiens Ets transcription factor PDEF Up-regulated by Androgen
    (PDEF) mRNA, complete
    NM_003201 Transcription factor 6-like 1 (mitochondrial Up-regulated by Androgen
    transcription factor 1-like)
    U41387 Human Gu protein mRNA, partial cds. Up-regulated by Androgen
    U58855 Guanylate cyclase 1, soluble, alpha 3 Up-regulated by Androgen
    X12794 Human v-erbA related ear-2 gene. Up-regulated by Androgen
    U88542 Mus musculus homeobox protein Nkx3.1 Up-regulated by Androgen
    D89729 Homo sapiens mRNA for CRM1 protein, complete Up-regulated by Androgen
    cds.
    U75329 TMPRSS2 Up-regulated by Androgen
    AA062976 EST Up-regulated by Androgen
    L12168 Homo sapiens adenylyl cyclase-associated protein Up-regulated by Androgen
    (CAP) mRNA
    AA043945 EST Up-regulated by Androgen
    AF026291 Homo sapiens chaperonin containing t-complex Up-regulated by Androgen
    polypeptide
    1, delta
    AB002301 Human mRNA for KIAA0303 gene, partial cds. Up-regulated by Androgen
    D13643 Human mRNA for KIAA0018 gene, complete cds. Up-regulated by Androgen
    AI310341 EST Up-regulated by Androgen
    U49436 Human translation initiation factor 5 (eIF5) mRNA, Up-regulated by Androgen
    complete cds
    S79862 Proteasome (prosome, macropain) 26S subunit, non- Up-regulated by Androgen
    ATPase, 5
    M14200 Human diazepam binding inhibitor (DBI) mRNA, Up-regulated by Androgen
    complete cds.
    AA653318 FK506-binding protein 5 Up-regulated by Androgen
    L07493 Homo sapiens replication protein A 14kDa subunit Up-regulated by Androgen
    (RPA) mRNA,
    AJ011916 Homo sapiens mRNA for hypothetical protein. Up-regulated by Androgen
    AA130537 EST Up-regulated by Androgen
    D16373 Human mRNA for dihydrolipoamide Up-regulated by Androgen
    succinyltransferase, complete cds.
    AL096857 Novel human mRNA from chromosome 1 Up-regulated by Androgen
    AF007157 Homo sapiens clone 23856 unknown mRNA, partial Up-regulated by Androgen
    cds.
    AA425929 NADH dehydrogenase (ubiquinone) 1 beta Up-regulated by Androgen
    subcomplex, 10 (22kD, PDSW)
    AI357815 EST Up-regulated by Androgen
    D83778 Human mRNA for KIAA0194 gene, partial cds. Up-regulated by Androgen
    AF000979 Homo sapiens testis-specific Basic Protein Y 1 Up-regulated by Androgen
    (BPY1) mRNA,
    AA889510 EST Up-regulated by Androgen
    AB018330 Homo sapiens mRNA for KIAA0787 protein, partial Up-regulated by Androgen
    cds.
    AA026941 EST Up-regulated by Androgen
    AA532377 Chromosome
    1 open reading frame 8 Up-regulated by Androgen
    AF010313 Homo sapiens Pig8 (PIG8) mRNA (etoposide- Up-regulated by Androgen
    induced mRNA), complete cds.
    L06328 Human voltage-dependent anion channel isoform 2 Up-regulated by Androgen
    (VDAC) mRNA,
    U41804 Human putative T1/ST2 receptor binding protein Up-regulated by Androgen
    precursor mRNA,
    AB020676 Homo sapiens mRNA for KIAA0869 protein, partial Up-regulated by Androgen
    cds.
    J03503 Human pyruvate dehydrogenase E1-alpha subunit Up-regulated by Androgen
    mRNA, cds.
    AA421098 EST Up-regulated by Androgen
    AF072836 Sox-like transcriptional factor Up-regulated by Androgen
    AA115355 EST Up-regulated by Androgen
    AF118240 Homo sapiens, peroxisomal biogenesis factor 16 Up-regulated by Androgen
    (PEX16) mRNA, complete
    AA011178 EST Up-regulated by Androgen
    X15573 Human liver-type 1-phosphofructokinase (PFKL) Up-regulated by Androgen
    mRNA, complete cds.
    AA120930 EST Up-regulated by Androgen
    AB002321 Human mRNA for KIAA0323 gene, partial cds Up-regulated by Androgen
    AF151837 Homo sapiens CGI-79 protein mRNA, complete cds Up-regulated by Androgen
    AA481027 EST Up-regulated by Androgen
    AA039343 EST Up-regulated by Androgen
    U09716 Human mannose-specific lectin (MR60) mRNA, Up-regulated by Androgen
    complete cds.
    AF044773 Homo sapiens breakpoint cluster region protein 1 Up-regulated by Androgen
    (BCRG1) mRNA
    U51586 Human siah binding protein 1 (SiahBP1) mRNA, Up-regulated by Androgen
    partial cds.
    M36341 Human ADP-ribosylation factor 4 (ARF4) mRNA, Up-regulated by Androgen
    complete cds.
    AI282096 EST Up-regulated by Androgen
    W45510 RAB7, member RAS oncogene family-like 1 Up-regulated by Androgen
    X16135 Human mRNA for novel heterogeneous nuclear RNP Up-regulated by Androgen
    protein, L protein
    AF052134 Homo sapiens clone 23585 mRNA sequence, Up-regulated by Androgen
    AF052134
    D26068 Williams-Beuren syndrome chromosome region 1 Up-regulated by Androgen
    X69433 H.sapiens mRNA for mitochondrial isocitrate Up-regulated by Androgen
    dehydrogenase (NADP+).
    X61123 B-cell translocation gene 1, anti-proliferative Up-regulated by Androgen
    X63423 H.sapiens mRNA for delta-subunit of mitochondrial Up-regulated by Androgen
    F1F0 ATP-synthase
    AJ010025 Homo sapiens mRNA for unr-interacting protein. Down-regulated by Androgen
    AF003938 Homo sapiens thioredoxin-like protein mRNA, Down-regulated by Androgen
    complete cds.
    AB014536 Homo sapiens copine III (CPNE3) mRNA Down-regulated by Androgen
    AA504468 EST Down-regulated by Androgen
    NM_001273 Chromodomain helicase DNA binding protein 4 Down-regulated by Androgen
    AA015746 Homo sapiens mRNA; cDNA DKFZp586H0722 Down-regulated by Androgen
    (from clone DKFZp586H0722)
    AA552354 EST Down-regulated by Androgen
    AA025744 3-prime-phosphoadenosine 5-prime-phosphosulfate Down-regulated by Androgen
    synthase 2
    X71129 H.sapiens mRNA for electron transfer flavoprotein Down-regulated by Androgen
    beta subunit
    AA046050 EST Down-regulated by Androgen
    U57052 Human Hoxb-13 mRNA, complete cds Down-regulated by Androgen
    AA400137 EST Down-regulated by Androgen
    AA487586 EST Down-regulated by Androgen
    J04208 Human inosine-5′-monophosphate dehydrogenase Down-regulated by Androgen
    (IMP) mRNA
    M64722 Testosterone-repressed prostate message 2 Down-regulated by Androgen
    (apolipoprotein J)
    AI743483 EST Down-regulated by Androgen
    AA476914 EST Down-regulated by Androgen
    AA026691 EST Down-regulated by Androgen
    AI014986 EST Down-regulated by Androgen
    X85373 Small nuclear ribonucleoprotein polypeptide G Down-regulated by Androgen
    U07231 G-rich RNA sequence binding factor 1 Down-regulated by Androgen
    T97753 Glycogen synthase 2 (liver) Down-regulated by Androgen
    AA234050 EST Down-regulated by Androgen
    AI015143 EST Down-regulated by Androgen
    U09196 Human 1.1 kb mRNA upregulated in retinoic acid Down-regulated by Androgen
    treated HL-60 neutrophilic cells.
    AA977749 EST Down-regulated by Androgen
    NM_006451 Polyadenylate binding protein-interacting protein 1 Down-regulated by Androgen
    AI818296 EST Down-regulated by Androgen
    AI250561 EST Down-regulated by Androgen
    AA063613 EST Down-regulated by Androgen
    U59209 Hs.183596: UDP glycosyltransferase 2 family, Down-regulated by Androgen
    polypeptide B17, U59209
    Z11559 Iron-responsive element binding protein 1 Down-regulated by Androgen
    AF052578 Homo sapiens androgen receptor associated protein Down-regulated by Androgen
    24 (ARA24)
    X16312 Human mRNA for phosvitin/casein kinase II beta Down-regulated by Androgen
    subunit.
    H17890 PCTAIRE protein kinase 3 Down-regulated by Androgen
    AA192312 EST Down-regulated by Androgen
    AA043787 EST Down-regulated by Androgen
    AI052020 EST Down-regulated by Androgen
    AB014512 Homo sapiens mRNA for KIAA0612 protein Down-regulated by Androgen
    NM_001328 Homo sapiens C-terminal binding protein 1 (CTBP1) Down-regulated by Androgen
    mRNA
    M15919 Human autoimmune antigen small nuclear Down-regulated by Androgen
    ribonucleoprotein E mRNA.
    AF151813 Homo sapiens CGI-55 protein mRNA, complete cds Down-regulated by Androgen
    L41351 Protease, serine, 8 (prostasin) Down-regulated by Androgen
    AF077046 Homo sapiens ganglioside expression factor 2 (GEF- Down-regulated by Androgen
    2) homolog
    U15008 Small nuclear ribonucleoprotein D2 polypeptide Down-regulated by Androgen
    (16.5kD), AA938995
    N62491 Folate hydrolase (prostate-specific membrane Down-regulated by Androgen
    antigen) 1
    AI569591 EST Down-regulated by Androgen
    AJ131245 Secretory protein 24 (SEC24). Down-regulated by Androgen
    U90543 Human butyrophilin (BTF1) mRNA, complete cds. Down-regulated by Androgen
    Z47087 Transcription elongation factor B (SIII), polypeptide Down-regulated by Androgen
    1-like
    M34539 FK506-binding protein 1A (12kD) Down-regulated by Androgen
    N43807 yy19a05.r1 Soares melanocyte 2NbHM Homo Down-regulated by Androgen
    sapiens cDNA clone
    U03269 Human actin capping protein alpha subunit (CapZ) Down-regulated by Androgen
    mRNA, complete
    AI571685 EST Down-regulated by Androgen
    AA010412 EST Down-regulated by Androgen
    L40403 Homo sapiens (clone zap3) mRNA, 3′ end of cds. Down-regulated by Androgen
    NM_006560 CUG triplet repeat, RNA-binding protein 1 Down-regulated by Androgen
    NM_004713 Serologically defined colon cancer antigen 1 Down-regulated by Androgen
    U36188 Clathrin-associated/assembly/adaptor protein, Down-regulated by Androgen
    medium
    1
    AB020721 KIAA0914 gene product Down-regulated by Androgen
    T35365 EST Down-regulated by Androgen
    AF029789 Homo sapiens GTPase-activating protein (SIPA1) Down-regulated by Androgen
    mRNA, complete cds.
    AA427857 EST Down-regulated by Androgen
    AA910404 EST Down-regulated by Androgen
    L42379 Quiescin Q6 (bone-derived growth factor) Down-regulated by Androgen
    AL117641 cDNA DKFZp434L235 Down-regulated by Androgen
    AI688119 EST Down-regulated by Androgen
    AA688073 EST Down-regulated by Androgen
    NM_002945 Replication protein A1 (70kD) Down-regulated by Androgen
    AI797610 EST Down-regulated by Androgen
    AF086095 Homo sapiens full length insert cDNA clone Down-regulated by Androgen
    YZ88A07.
    AF070666 Homo sapiens tissue-type pituitary Kruppel- Down-regulated by Androgen
    associated box protein
    R55128 Proteasome (prosome, macropain) 26S subunit, non- Down-regulated by Androgen
    ATPase, 2
    X75621 Tuberous sclerosis 2 Down-regulated by Androgen
    AA019070 EST Down-regulated by Androgen
    AI089867 EST Down-regulated by Androgen
    NM_001003 Homo sapiens ribosomal protein, large, P1 (RPLP1) Down-regulated by Androgen
    mRNA
    L05093 Ribosomal protein L18a Down-regulated by Androgen
    AA854176 EST Down-regulated by Androgen
    AI929622 Homo sapiens clone 23675 mRNA sequence Down-regulated by Androgen
    AI264769 ESTs, Weakly similar to ORF YDL087c Down-regulated by Androgen
    [S.cerevisiae]
    L09159 Ras homolog gene family, member A, may be Down-regulated by Androgen
    androgen regulated?
    AI143187 EST Down-regulated by Androgen
    H17900 cDNA DKFZp586H051 (from clone Down-regulated by Androgen
    DKFZp586H051)
    NM_005617 Ribosomal protein S14 Down-regulated by Androgen
    L49506 Cyclin G2 Down-regulated by Androgen
    AA614448 Regulator of G-protein signalling 5 Down-regulated by Androgen
    S83390 T3 receptor-associating cofactor-1 Down-regulated by Androgen
    AA917672 EST Down-regulated by Androgen
    X52151 Arylsulphatase A Down-regulated by Androgen
    U09646 Carnitine palmitoyltransferase II Down-regulated by Androgen
    Z50853 ATP-dependent protease ClpAP (E. coli), proteolytic Down-regulated by Androgen
    subunit, human
    AB023208 MLL septin-like fusion Down-regulated by Androgen
    U92014 Human clone 121711 defective mariner transposon Down-regulated by Androgen
    Hsmar2 mRNA
    AA878293 Alpha-1-antichymotrypsin Down-regulated by Androgen
    AA554191 EST Down-regulated by Androgen
    M55618 Hexabrachion (tenascin C, cytotactin) Down-regulated by Androgen
    AA027050 EST Down-regulated by Androgen
    AF112472 Homo sapiens calcium/calmodulin-dependent protein Down-regulated by Androgen
    kinase II beta
    AA583866 EST Down-regulated by Androgen
    AA115687 EST Down-regulated by Androgen
    AA043318 EST Down-regulated by Androgen
    U90329 Poly(rC)-binding protein 2 Down-regulated by Androgen
    Y00815 Protein tyrosine phosphatase, receptor type, F Down-regulated by Androgen
    X76013 H.sapiens QRSHs mRNA for glutaminyl-tRNA Down-regulated by Androgen
    synthetase.
    X75861 Testis enhanced gene transcript Down-regulated by Androgen
    AA593078 Homo sapiens PAC clone DJ0167F23 from 7p15 Down-regulated by Androgen
    J04058 Human electron transfer flavoprotein alpha-subunit Down-regulated by Androgen
    mRNA
    AF026292 Homo sapiens chaperonin containing t-complex Down-regulated by Androgen
    polypeptide
    1, eta
    AF068754 Homo sapiens heat shock factor binding protein 1 Down-regulated by Androgen
    HSBP1 mRNA,
    NM_000172 Guanine nucleotide binding protein (G protein), Down-regulated by Androgen
    alpha transducing activity polypeptide 1
    AI140631 Hs.1915: folate hydrolase (prostate-specific Down-regulated by Androgen
    membrane antigen) 1
  • [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_NbHSF Homo sapiens cDNA, Hs.108981:
    ficolin (collagen/fibrinogen domain-containing) 1, AF201077 NADH:ubiquinone
    oxidoreductase MLRQ subunit (NDUFA4) mRNA, complete cds with polyA.
    D55953 HUM407H12B Clontech human fetal brain polyA + mRNA (#6535) Homo, Hs.118724:
    histidine triad nucleotide-binding protein, AJ012499, mRNA activated in tumor
    suppression, clone TSAP19 with polyA
    AA082804 zn41g02.r1 Stratagene endothelial cell 937223 Homo sapiens cDNA, Hs.110967: ESTs,
    Weakly similar to KIAA0762 protein [H.sapiens], Hs.5662: guanine nucleotide binding
    protein (G protein), beta polypeptide 2-like 1 in the sequence no tag
    X05332 Human mRNA for prostate specific antigen.*
    AI278854 qo42f01.x1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE:1911193 3′,
    NM_004537 nucleosome assembly protein 1-like 1 (NAPIL1), tag is at beginning of the
    gene.
    W75950 zd58b02.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone, AF151840, CGI-
    82 protein mRNA, tag is at 3′ end.
    F02980 HSC1IC062 normalized infant brain cDNA Homo sapiens cDNA clone
    M99487 Human prostate-specific membrane antigen (PSM) mRNA, complete cds.
    AL035304 H.sapiens gene from PAC 295C6, similar to rat PO44.
    AI088979 ou86f03.s1 Soares_NSF_F8_9W_OT_PA_P_S1 Homo sapiens cDNA clone
    AF186249 Homo sapiens six transmembrane epithelial antigen of prostate (STEAP1) mRNA
    C15801 C15801 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNA
    L10340 Human elongation factor-1 alpha (ef-1) mRNA, 3′ end.
    NM_004540 Homo sapiens neural cell adhesion molecule 2 (NCAM2)
    AA151796 z139c02.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    NM_001634 Homo sapiens S-adenosylmethionine decarboxylase 1 (AMD1)
    NM_005013 Homo sapiens nucleobindin 2 (NUCB2)AL121913 in GenBank htgc database) and 718J7
    (Accession number AL035541
    AF004828 Homo sapiens rab3-GAP regulatory domain mRNA, complete cds.
    X60819 X60 H.sapiens DNA for monoamine oxidase type A (14) (partial).
    AA133972 z138g12.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    M69226 Human monoamine oxidase (MAOA) mRNA, complete cds.
    AA969141 op50c11.s1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone
    AA523652 ni64d09.s1 NCI_CGAP_Pr12 Homo sapiens cDNA clone IMAGE:981617, mRNA
    AF078749 Homo sapiens organic cation transporter 3 (SLC22A3)
    AA583544 nf25h10.s1 NCI_CGAP_Pr1 Homo sapiens cDNA clone IMAGE:914851, mRNA
    AF051894 Homo sapiens 15 kDa selenoprotein mRNA, complete cds.
    AF165967 Homo sapiens DDP-like protein mRNA
    X57129 H.sapiens H1.2 gene for histone H1.
    AA640928 nr28d08.r1 NCI_CGAP_Pr3 Homo sapiens cDNA clone IMAGE:1169295, mRNA
    U41766 Human metalloprotease/disintegrin/cysteine-rich protein precursor
    AF023676 Homo sapiens lamin B receptor homolog TM7SF2 (TM7SF2) mRNA,
    U10691 Human MAGE-6 antigen (MAGE6) gene, complete cds.
    M22976 Human cytochrome b5 mRNA, 3′ end.
    L14778 Human calmodulin-dependent protein phosphatase catalytic subunit
    AF071538 Homo sapiens Ets transcription factor PDEF (PDEF) mRNA, complete
    U39840 Human hepatocyte nuclear factor-3 alpha (HNF-3 alpha) mRNA,
    AA532511 nj54d03.s1 NCI_CGAP_Pr9 Homo sapiens cDNA clone IMAGE:996293, mRNA
    X07166 Human mRNA for enkephalinase (EC 3.424.11).
    M96684 H.sapiens Pur (pur-alpha) mRNA, complete cds.
    AI204040 qe77f05.x1 Soares_fetal_lung_NbHL19W Homo sapiens cDNA clone
    AA577923 nl20a01.s1 NCI_CGAP_HSC1 Homo sapiens cDNA clone IMAGE:1041192,
    AA569633 nm38h09.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE:1062497,
    U65011 Human preferentially expressed antigen of melanoma (PRAME) mRNA,
    U21910 Human basic transcription factor BTF2p44 mRNA, 3′ end, partial cds.
    AA633187 nq07c12.s1 NCI_CGAP_Lu1 Homo sapiens cDNA clone IMAGE:1143190 3′
    AF000993 Homo sapiens ubiquitous TPR motif, X isoform (UTX) mRNA,
    W76105 zd65b04.r1 Soares_fetal_heart_NbHHI9W Homo sapiens cDNA clone
    H39906 yo54a07.r1 Soares breast 3NbHBst Homo sapiens cDNA clone
    AA971717 op95c11.s1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE:1584596 3′,
    M68891 Human GATA-binding protein (GATA2) mRNA, complete cds.
    AA310157 EST181013 Jurkat T-cells V Homo sapiens cDNA 5′ end, mRNA sequence.
    X00948 Human mRNA for prepro-relaxin H2.
    AB018330 Homo sapiens mRNA for KIAA0787 protein, partial cds.
    AA890637 ak11e11.s1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone
    M64929 J05 Human protein phosphatase 2A alpha subunit mRNA, complete cds.
    W24341 zb81h12.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA
    AA974479 od58b03.s1 NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:1372109 3′
    R31644 yh69e05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone
    AA573246 nm52c02.s1 NCI_CGAP_Br2 Homo sapiens cDNA clone IMAGE:1071842 3′,
    AA507635 ng84b02.s1 NCI_CGAP_Pr6 Homo sapiens cDNA clone IMAGE:941451, mRNA
    gb|AF008915 Homo sapiens EVI5 homolog mRNA
    AL049987 Homo sapiens mRNA; cDNA DKFZp564F112 (from clone DKFZp564F112).
    U81599 Homo sapiens homeodomain protein HOXB13 mRNA
    AA641596 nr20f05.s1 NCI_CGAP_Pr2 Homo sapiens cDNA clone IMAGE:1168545, mRNA
    D84295 Human mRNA for possible protein TPRDII
    R13859 yf65d08.r1 Soares infant brain 1NIB Homo sapiens cDNA clone
    M34840 Human prostatic acid phosphatase mRNA, complete cds.
    AA572913 nm42f12.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE:1062863,
    AA094460 cp0378.seq.F Human fetal heart, Lambda ZAP Express Homo sapiens
    AF031166 Homo sapiens SRp46 splicing factor retropseudogene mRNA.
    AA625147 af70c07.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:1047372
    T39510 ya06h07.r1 Stratagene placenta (#937225) Homo sapiens cDNA clone
    R35034 yg60h03.r1 Soares infant brain 1NIB Homo sapiens cDNA clone
    AI003674 zg01c04.s1 Soares_pineal_gland_N3HPG Homo sapiens cDNA clone
    AJ003636 AJ003636 Selected chromosome 21 cDNA library Homo sapiens cDNA
    AA601385 no16d12.s1 NCI_CGAP_Phe1 Homo sapiens cDNA clone IMAGE:1100855 3′,
    AF191339 Homo sapiens anaphase-promoting complex subunit 5 (APC5)
    AA431822 zw79d02.r1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:782403
    NM_003909 Homo sapiens copine III (CPNE3)
    AA484004 ne73f04.s1 NCI_CGAP_Ew1 Homo sapiens cDNA clone IMAGE:909919
    AA535774 nj78f08.s1 NCI_CGAP_Pr10 Homo sapiens cDNA clone IMAGE:998631, mRNA
    NM_000944.1 Homo sapiens protein phosphatase 3 (formerly 2B)
    AA702811 zi90c11.s1 Soares_fetal_liver_spleen_1NFLS_S1 Homo sapiens cDNA
    X95073 H.sapiens mRNA for translin associated protein X.
    AA029039 zk12b07.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    AF032887 Homo sapiens forkhead (FKHRL1P1) pseudogene
    N46609 yy48h08.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA
    U58855 Homo sapiens soluble guanylate cyclase large subunit (GC-S-alpha-1)
    AA255486 zr83d03.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:682277
    AA128153 zl15c06.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    AA016039 ze31c05.s1 Soares retina N2b4HR Homo sapiens cDNA clone
    R88520 ym91e09.s1 Soares adult brain N2b4HB55Y Homo sapiens cDNA clone
    M26624 Human CALLA/NEP gene encoding neutral endopeptidase, exon 20.
    AA026997 ze99e01.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone
    W48775 zc44b08.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA
    AA074407 zm15c08.r1 Stratagene pancreas (#937208) Homo sapiens cDNA clone
    L13972 Homo sapiens beta-galactoside alpha-2,3-sialyltransferase (SIAT4A)
    D14661 Human mRNA for KIAA0105 gene, complete cds.
    AA115452 zk89a08.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    AA495742 zw04b12.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:768287 5′
    R13416 yf75h09.r1 Soares infant brain 1NIB Homo sapiens cDNA clone
    AA046369 zk77h07.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    T35440 EST85129 Human Lung Homo sapiens cDNA 5′ end similar to None, mRNA
    AI075860 oz25b05.x1 Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA clone
    W56437 zc57g05.r1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone
    AI583880 tt70b02.x1 NCI_CGAP_HSC3 Homo sapiens cDNA clone IMAGE:2246091 3′
    D85181 Homo sapiens mRNA for fungal sterol-C5-desaturase homolog, complete
    AF105714 Homo sapiens protein kinase PITSLRE (CDC2L2) gene, exon 3.
    AA401802 zt60c12.r1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:726742
    AB002301 Human mRNA for KIAA0303 gene, partial cds.
    U75667 Human arginase II mRNA, complete cds.
    AA585183 JTH089 HTCDL1 Homo sapiens cDNA 5′/3′, mRNA sequence.
    AF191771 Homo sapiens CED-6 protein (CED-6) mRNA
    AA650252 ns93g05.s1 NCI_CGAP_Pr3 Homo sapiens cDNA clone IMAGE:1191224, mRNA
    R64618 yi19b09.r1 Soares placenta Nb2HP Homo sapiens cDNA clone
    N24627 yx89a09.s1 Soares melanocyte 2NbHM Homo sapiens cDNA clone
    AB028951 Homo sapiens mRNA for KIAA1028 protein
    N75608 yw37a07.r1 Morton Fetal Cochlea Homo sapiens cDNA clone
    N53899 yy98e03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA
    N46696 yy50f07.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA
    AA419522 zv03d05.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:752553
    M61906 Human P13-kinase associated p85 mRNA sequence.
    C16570 C16570 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNA
    X63105 H.sapiens tpr mRNA.
    AA315855 EST187656 Colon carcinoma (HCC) cell line II Homo sapiens cDNA 5′
    L18920 Human MAGE-2 gene exons 1-4, complete cds.
    M25161 Human Na,K-ATPase beta subunit (ATP1B) gene
    AA164865 zq41g07.r1 Stratagene hNT neuron (#937233) Homo sapiens cDNA clone
    N40094 yx98g07.r1 Soares melanocyte 2NbHM Homo sapiens cDNA clone
    N98940 yy71a07.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA
    AF049907 Homo sapiens zinc finger transcription factor (ZNF-X) mRNA,
    M78806 EST00954 Hippocampus, Stratagene (cat. #936205) Homo sapiens cDNA
    AA040819 zk47b03.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone
    C15445 C15445 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNA
    AB018309 Homo sapiens mRNA for KIAA0766 protein, complete cds.
    AJ011497 Homo sapiens mRNA for Claudin-7.
    X00949 Human mRNA for prepro-relaxin H1.
    AA418633 zv93d09.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767345 5′
    AI146806 qb83h04.x1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone
    X82942 H.sapiens satellite 3 DNA.
    AA456383 aa14f03.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:813245
    AA019341 ze57e04.s1 Soares retina N2b4HR Homo sapiens cDNA clone
    AB027466 Homo sapiens SPON2 mRNA for spondin 2
    AF038170 Homo sapiens clone 23817 mRNA sequence.
    NM_000240 Homo sapiens monoamine oxidase A (MAOA)
    N34126 yx76c01.r1 Soares melanocyte 2NbHM Homo sapiens cDNA clone
    N41339 yw68g06.r1 Soares_placenta_8to9weeks_2NbHP8to9W Homo sapiens cDNA
    R34783 yh87b05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone
    N75858 yw32a03.r1 Morton Fetal Cochlea Homo sapiens cDNA clone
    AA633887 ac32h04.s1 Stratagene hNT neuron (#937233) Homo sapiens cDNA clone
    N53723 yz06d03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA
    AI187365 qf29b12.x1 Soares_testis_NHT Homo sapiens cDNA 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 Nuclear receptor gene family
    1?
    Hs.204704 EAR1 beta Nuclear receptor gene family
    E75 Nuclear receptor gene family
    Hs.2156 ROR alpha Nuclear receptor gene family
    Hs.198481 ROR beta Nuclear receptor gene family
    Hs.133314 ROR gammma Nuclear receptor gene family
    Hs.100221 NER1 Nuclear receptor gene family
    Hs.54424 HNF4A Nuclear receptor gene family
    Hs.202659 HNF4G Nuclear receptor gene family
    Hs.108301 TR2 Nuclear receptor gene family
    Hs.520 TR4 Nuclear receptor gene family
    Hs.144630 COUP-TF1 Nuclear receptor gene family
    Hs.1255 COUP-TF2 Nuclear receptor gene family
    Hs.155286 EAR2 Nuclear receptor gene family
    Hs.1119 TR3 hormone receptor Nuclear receptor gene family
    (growth factor-
    inducible nuclear
    protein N10)
    Hs.82120 NURR1 IMMEDIATE- Nuclear receptor gene family
    EARLY RESPONSE
    PROTEIN NOT
    Hs.97196 SF1 Nuclear receptor gene family
    Hs.183123 FTF fetoprotein-alpha 1 Nuclear receptor gene family
    (AFP) transcription
    factor
    Hs.46433 DAX1 Nuclear receptor gene family
    Hs.11930 SHP Homo sapiens nuclear Nuclear receptor gene family
    hormone receptor
    (shp) gene, 3′ end of
    cds
    Hs.83623, CAR-beta Nuclear receptor gene family
    IMAGE
    1761923, or
    1868028, or
    1563505, or
    1654096
    Hs.199078 Sin3 Nuclear receptor co-repressor complex Nature, 387, 43, 97.
    Nature, 387, 49, 97
    Hs.120980 SMRT Nuclear receptor co-repressor complex Nature, 377, 454, 95
    Hs.144904 N-CoR Nuclear receptor co-repressor complex Nature, 377, 297, 95
    Hs.188055 highly homologue gene Nuclear receptor co-repressor complex
    to N-CoR in prostate and
    testis
    Hs.180686 E6-AP Angelman syndrome Nuclear receptor co-activator complex MCB, 19, 1182, 99
    associated protein
    Hs.199211?Hs. hBRM ESTs, Highly similar Nuclear receptor co-activator complex
    198296? to HOMEOTIC
    GENE
    REGULATOR
    [Drosophila
    melanogaster]
    Hs.78202 hBRG1 Nuclear receptor co-activator complex
    Hs.11861 TRAP240 DRIP250, ARCp250 Nuclear receptor co-activator complex Mol Cell, 3, 361, 99
    Hs.85313 TRAP230 ARCp240, DRIP240 Nuclear receptor co-activator complex Mol Cell, 3, 361, 99
    Hs.15589 TRAP220 RB18A, PBP, Nuclear receptor co-activator complex
    CRSP200, TRIP2,
    ARCp205, DRIP205
    Hs.21586 TRAP170 RGR, CRSP150, Nuclear receptor co-activator complex
    DRIP150,
    ARCp150chromosom
    eX
    Hs.108319 TRAP150 ESTs Nuclear receptor co-activator complex Mol Cell, 3, 361, 99
    Hs.193017 CRSP133 ARCp130, DRIP130 Nuclear receptor co-activator complex Nature, 397, 6718, 99
    Hs.23106 TRAP100 ARCp100, DRIP100, Nuclear receptor co-activator complex
    ND DRIP97 TRAP97 Nuclear receptor co-activator complex
    Hs.24441 TRAP95 ESTs Nuclear receptor co-activator complex Mol Cell, 3, 361, 99
    ND TRAP93 Nuclear receptor co-activator complex
    Hs.31659 DRIP92 ARCp92? Nuclear receptor co-activator complex
    Hs.22630 TRAP80 ARCp77, Nuclear receptor co-activator complex Mol Cell, 3, 361, 99
    CRSP77,
    DRIP80(77)?
    Hs.204045 ARCp70 CRSP70, DRIP79 Nuclear receptor co-activator complex
    ND ARCp42 Nuclear receptor co-activator complex
    ND ARCp36 Nuclear receptor co-activator complex
    Hs.184947 MED6 ARCp33 Nuclear receptor co-activator complex Mol Cell, 3, 97, 99
    Hs.7558 MED7 CRSP33, ARCp34, Nuclear receptor co-activator complex Nature, 397, 6718, 99
    DRIP36
    ND ARCp32 Nuclear receptor co-activator complex
    ND SRB10 Nuclear receptor co-activator complex
    ND SRB11 Nuclear receptor co-activator complex
    ND MED10 NUT2 Nuclear receptor co-activator complex
    Hs.27289 SOH1 (yeast?) Nuclear receptor co-activator complex Mol Cell, 3, 97, 99
    ND p26 Nuclear receptor co-activator complex
    ND p28 Nuclear receptor co-activator complex
    ND p36 Nuclear receptor co-activator complex
    ND p37 Nuclear receptor co-activator complex
    ND but 2 TRFP human homologue of Nuclear receptor co-activator complex
    IMAGE clones Drosophila TRF
    proximal protein
    ND VDR interacting subunit 180kDa, HAT Nuclear receptor co-activator complex Genes Dev, 12, 1787, 98
    activity
    Hs.143696, or Coactivator associated Nuclear receptor co-activator complex Science, 284, 2174, 99
    IMAGE:23716 methyltransferase 1
    96?
    Hs.79387 SUG1 TRIP1 Nuclear receptor co-activator complex EMBO, 15, 110, 96
    ND TRUP Nuclear receptor co-activator complex PNAS, 92, 9525, 95
    Hs.28166 CRSP34 Nuclear receptor co-activator complex Nature, 397, 6718, 99
    Hs.63667 transcriptional adaptor 3 Nuclear receptor co-activator complex
    (A
    Hs.196725 ESTs, Highly similar to Nuclear receptor co-activator complex
    P300
    Hs.131846 PCAF associated factor Nuclear receptor co-activator complex
    65 al
    Hs.155635 ESTs, Moderately Nuclear receptor co-activator complex
    similar toPCAF
    associated factor 65 beta
    Hs.26782 PCAF associated factor Nuclear receptor co-activator complex
    65 beta
    Hs.118910 tumor suscitibility Modifying AR function Cancer 15, 86, 689,
    protein 101 (99)
    Hs.82932 Cyclin D1 cyclin D1 (PRAD1: Modifying AR function Can Res, 59, 2297, 99
    parathyroid
    adenomatosis 1)
    Hs.173664 HER2/Neu v-erb-b2 avian Modifying AR function PNAS, 9, 5458, 99
    erythroblastic
    leukemia viral
    oncogene homolog 2
    Hs.77271 PKA protein kinase, Modifying AR function JBC 274, 7777, 99
    cAMP-dependent,
    catalytic, alpha
    Hs.85112 IGF1 insulin-like growth Modifying AR function Can Res, 54, 5474, 94
    factor 1
    (somatomedin C)
    Hs.2230 EGF Modifying AR function Can Res, 54, 5474, 94
    Hs.129841 MEKK1 MAPKKK1 Modifying AR function Mol Cell Biol.
    19, 5143, 99
    Hs.83173 Cyclin D3 Modifying AR function Can Res, 59, 2297, 99
    Hs.75963 IGF2 Modifying AR function
    Hs.89832 Insulin Modifying AR function
    Hs.115352 GH Modifying AR function
    Hs.1989 5 alpha reductase type2 Involved in Androgen metabolism
    Hs.76205 Cytochrome P450, Involved in Androgen metabolism
    subfamily XIA
    Hs.1363 Cytochrome P450, Involved in Androgen metabolism
    subfamily XVII, (steroid
    17-alpha-hydroxylase),
    Hs.477 Hydroxysteroid (17- Involved in Androgen metabolism
    beta) dehydrogenase 3
    Hs.75441 Hydroxysteroid (17- Involved in Androgen metabolism
    beta) dehydrogenase 4
    Hs.38586 Hydroxy-delta-5-steroid Involved in Androgen metabolism
    dehydrogenase, 3 beta-
    and steroid delta-
    isomerase 1
    Hs.46319 Sex hormone-binding Involved in Androgen metabolism
    globulin
    Hs.552 SRD5A1 Involved in Androgen metabolism
    Hs.50964 C-CAM epithelial cell Down-regulated by Androgen Oncogene, 18, 3252, 99
    adhesion molecule
    Hs.7833 hSP56 selenium binding Down-regulated by Androgen Can Res, 58, 3150, 98
    protein
    Hs.77432 EGFR epidermal growth Down-regulated by Androgen Endocrinology,
    factor receptor 139, 1369, 98
    Hs.1174 p16 Down-regulated by Androgen Can Res.57, 4511, 97
    Hs.55279 maspin Down-regulated by Androgen PNAS, 94, 5673, 97
    Hs.75789 TDD5 (mouse) Human mRNA for Down-regulated by Androgen PNAS, 94, 4988, 97
    RTP, complete cds
    Hs.75106 TRPM-2 clusterin Down-regulated by Androgen
    (testosterone-repressed
    prostate message 2,
    apolipoprotein J)
    Hs.25640 rat ventral prostate gene1 claudin3 Down-regulated by Androgen PNAS, 94, 12999, 97
    ND glutathione S-transferase Down-regulated by Androgen PNAS, 94, 12999, 97
    Hs.25647 c-fos v-fos FBJ murine Down-regulated by Androgen PNAS, 94, 12999, 97
    osteosarcoma viral
    oncogene homolog
    N.D. matrix carboxyglutamic Down-regulated by Androgen PNAS, 94, 12999, 97
    acid protein (RAT)
    Hs.2962 S100P calcium binding Down-regulated by Androgen Prostate 29, 350, 96
    prottein
    Hs.75212 ornithine decarboxilase ornithine Down-regulated by Androgen J Androl, 19, 127, 98
    decarboxylase 1
    Hs.84359 Androge withdrawal Down-regulated by Androgen
    apoptosis RVP1
    Hs.79070 c-myc v-myc avian Down-regulated by Androgen
    myelocytomatosis
    viral oncogene
    homolog
    Hs.139033 partially expressed gene Down-regulated by Androgen Mol Cell Endocrinol
    3 155, 69, (99)
    Hs.20318 PLU-1 Associated with Prostate Cancer JBC, 274, 15633, 99
    Hs.18910 POV1(PB39) unique Associated with Prostate Cancer Genomics. 51, 282, 98
    Hs.119333 caveolin Associated with Prostate Cancer Clin Can Res. 4,
    1873, 98
    ND, but 1 EST R00540(2.6kbp) = IM Associated with Prostate Cancer Urology, 50, 302, 97
    IMAGE AGE:123822
    CLONE
    Hs.184906 PTI-1 prostate tumor Associated with Prostate Cancer Can Res, 57, 18, 97,
    inducing gene, PNAS, 92, 6778, 95
    trancated and mutated
    human elongation
    factor 1 alpha
    Hs.74649 cytochrome c oxidase Associated with Prostate Cancer Can Res, 56, 3634, 96
    subunit VI c
    Hs.4082 PCTA-1 prostate carcinoma Associated with Prostate Cancer PNAS, 92, 7252, 96
    tumor antigen,
    galectin family
    ND pp32r1 Associated with Prostate Cancer Nature Medicine,
    5, 275, 99
    ND pp32r2 Associated with Prostate Cancer Nature Medicine,
    5, 275, 99
    Hs.184945 GBX2 Associated with Prostate Cancer The prostate
    journal, 1, 61, 99
    Hs.8867 Cyr61 inmmediate early Associated with Prostate Cancer Prostate, 36, 85, 98
    protein
    Hs.77899 epithelial tropomyosin actin binding protein Associated with Prostate Cancer Can Res, 56, 3634, 96
    Hs.76689 pp32 Associated with Prostate Cancer Nature Medicine,
    5, 275, 99
    Hs.10712 PTEN Associated with Prostate Cancer
    Hs.194110 KAII Associated with Prostate Cancer
    Hs.37003 H-ras Associated with Prostate Cancer
    Hs.184050 K-ras Associated with Prostate Cancer
    Hs.69855 N-ras neuroblastoma RAS Associated with Prostate Cancer
    viral (v-ras) oncogene
    homolog
    Hs.220 TGFbeta receptor 1 Associated with Prostate Cancer
    Hs.77326 IGFBP3 insulin-like growth Associated with Prostate Cancer
    factor binding protein
    3
    Hs.79241 bcl-2 Associated with Prostate Cancer
    Hs.159428 Bax Associated with Prostate Cancer
    Hs.206511 bcl-x Associated with Prostate Cancer
    Hs.86386 mcl-1 myeloid cell leukemia Associated with Prostate Cancer
    sequence 1 (BCL2-
    related)
    Hs.1846 p53 tumor protein p53 Associated with Prostate Cancer
    (Li-Fraumeni
    syndrome)
    Hs38481 CDK6 cyclin-dependent Associated with Prostate Cancer
    kinase 6
    Hs.118630 Mxi.1 Associated with Prostate Cancer
    Hs.184794 GAGE7 Associated with Prostate Cancer
    Hs.118162 fibronectin Associated with Prostate Cancer Am J Pathol
    154, 1335, 99
    Hs.128231 PAGE-1 Associated with Prostate Cancer JBC, 237, 17618, 98
    Hs.75875 UEV1 ubiquitin-conjugating Associated with Prostate Cancer Am J Pathol
    enzyme E2 variant 1 154, 1335, 99
    Hs.75663 PM5 Human mRNA for Associated with Prostate Cancer Am J Pathol
    pM5 protein 154, 1335, 99
    Hs.180842 BBC1 breast basic Associated with Prostate Cancer Am J Pathol
    conserved gene 154, 1335, 99
    Hs.198024 JC19 Associated with Prostate Cancer Can Res 57, 4075, 97
    N.D. GC79 novel gene Associated with Prostate Cancer Can Res 57, 4075, 97
    Hs.77054 B cell translocation gene Associated with Prostate Cancer Can Res 57, 4075, 97
    1
    Hs.78122 Regulatory factor X- Associated with Prostate Cancer
    associated ankyrin-
    containing protein
    Hs.3337 transmembrane 4 Associated with Prostate Cancer
    superfamily member
    1
    Hs.76698 TL5 Associated with Prostate Cancer Genebank
    Hs3776 TL7 Associated with Prostate Cancer Genebank
    Hs.170311 TL35 Associated with Prostate Cancer Genebank
    Hs.184914 Human mRNA for TI- Associated with Prostate Cancer
    227H
    Hs.62954 ferritin, heavy Associated with Prostate Cancer
    polypeptide
    Hs.71119 N33 Associated with Prostate Cancer Genomics, 35, 45(96)
  • [0164]
    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. sapiens gene from PAC 295C6, similar to rat PO44 Up-regulated by Androgen
    Hs.167531 Homo sapiens mRNA fill length insert cDNA clone EUROIMAGE 195423 Up-regulated by Androgen
    Hs.9817 arg/Abl-interacting protein ArgBP2 Up-regulated by Androgen
    Hs.28241 EST Down-regulated by Androgen
    Hs.25925 Homo sapiens clone 23860 mRNA Down-regulated by Androgen
    Hs.10319 UDP glycosyltransferase 2 family, polypeptide B7 Down-regulated by Androgen
    Hs.155995 Homo sapiens mRNA for KIAA0643 protein, partial cds Down-regulated by Androgen
    Hs.23552 EST Down-regulated by Androgen
    Hs.41693 DnaJ-like heat shock protein 40 Down-regulated by Androgen
    Hs.90800 matrix metalloproteinase 16 (membrane-inserted) Down-regulated by Androgen
    Hs.2996 sucrase-isomaltase Down-regulated by Androgen
    Hs.166019 regulatory factor X.3 (influences HLA class II expression) Down-regulated by Androgen
    Hs.27695 midline 1 (Opitz/BBB syndrome) Down-regulated by Androgen
    Hs.183738 chondrocyte-derived ezrin-like protein Down-regulated by Androgen
    Hs.75761 SFRS protein kinase 1 Down-regulated by Androgen
    Hs.197298 NS1-binding protein Down-regulated by Androgen
    Hs.149436 kinesin family member 5B Down-regulated by Androgen
    Hs.81875 growth factor receptor-bound protein 10 Down-regulated by Androgen
    Hs.75844 ESTs, Weakly similar to (define not available 5257244) [H. sapiens] Down-regulated by Androgen
    Hs.30464 cyclin E2 Down-regulated by Androgen
    Hs.198443 inositol 1, 4, 5-triphosphate receptor, type 1 Down-regulated by Androgen
    Hs.177959 a disintegrin and metalloproteinase domain 2 (fertilin beta) Down-regulated by Androgen
    Hs.44197 Homo sapiens mRNA; cDNA DKFZpS64D0462 (from clone Down-regulated by Androgen
    DKFZp564D0462)
    Hs.150423 cyclin-dependent kinase 9 (CDC2-related kinase) Down-regulated by Androgen
    Hs.78776 Human putative transmembrane protein (nma) mRNA, complete cds Down-regulated by Androgen
    Hs.25740 ESTs, Weakly similar to !!!! ALU SUBFAMILY SQ WARNING ENTRY !!!! Down-regulated by Androgen
    [H. sapiens]
    Hs.131041 EST Down-regulated by Androgen
    Hs.19222 ecotropic viral integration site 1 Down-regulated by Androgen
    Hs.9879 EST Down-regulated by Androgen
    Hs.118722 fucosyltransferase 8 (alpha (1, 6) fucosyltransferase) Down-regulated by Androgen
    Hs.47584 potassium voltage-gated channel, delayed-rectifier, subfamily S, member 3 Down-regulated by Androgen
    Hs.115945 mannosidase, beta A, lysosomal Down-regulated by Androgen
    Hs.171740 ESTs, Weakly similar to Zic2 protein [M. musculus] Down-regulated by Androgen
    Hs.32970 signaling lymphocytic activation molecule Down-regulated by Androgen
    Hs.196349 EST Down-reguiated by Androgen
    Hs.182982 Homo sapiens mRNA for KIAA0855 protein, partial cds Down-regulated by Androgen
    Hs.72918 small inducible cytokine A1 (1-309, homologous to mouse Tca-3) Down-regulated by Androgen
    Hs.84232 transcobalamin II; macrocytic anemia Down-regulated by Androgen
    Hs.10086 EST Down-regulated by Androgen
    Hs.1327 Butyrylcholinesterase Down-regulated by Androgen
    Hs.166684 serine/threonine kinase 3 (Ste20, yeast homolog) Down-regulated by Androgen
    AA558631 EST Down-regulated by Androgen
    Hs.150403 dopa decarboxylase (aromatic L-amino acid decarboxylase) Down-regulated by Androgen
    Hs.177548 postmeiotic segregation increased (S. cerevisiae) 2 Down-regulated by Androgen
    Hs.205902 IGF1-R
    Hs.21330 MDR1 P glycoprotein 1/multiple drug resistance 1
    Hs.74427 PIG11 Homo sapiens Pig11 (PIG11) mRNA, complete cds
    Hs.76507 PIG7 LPS-induced TNF-alpha factor
    Hs.8141 PIG8
    Hs.146688 PIG12
    Hs.104925 PIG10
    Hs.202673 PIG6
    Hs.80642 STAT4
    Hs.72988 STAT2
    Hs.167503 STAT5A
    Hs.738 early growth
    response 1
    Hs.85148 villin2
    Hs.109012 MAD
    Hs.75251 DEAD/H box
    binding protein 1
    Hs.181015 STAT6
    Hs.199791 SSI-3 STAT induced STAT inhibitor 3
    Hs.21486 STAT1
    Hs.142258 STAT3
    Hs.76578 PIAS3 Protein inhibitor of activated STAT3
    Hs.44439 CIS4 STAT induced STAT inhibitor 4
    Hs.50640 SSI-1 JAK binding protein
    Hs.54483 NMI N-Myc and STAT interactor
    Hs.105779 PIASy Protein inhibitor of activated STAT
    Hs.110776 STATI2 STAT induced STAT inhibitor 2
    Hs.181112 EST similar to
    STAT5A
  • [0166]
    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]
  • 1 67 1 1140 DNA Homo sapiens CDS (95)..(850) 1 tccttgggtt cgggtgaaag cgcctggggg ttcgtggcca tgatccccga gctgctggag 60 aactgaaggc ggacagtctc ctgcgaaaca ggca atg gcg gag ctg gag ttt gtt 115 Met Ala Glu Leu Glu Phe Val 1 5 cag atc atc atc atc gtg gtg gtg atg atg gtg atg gtg gtg gtg atc 163 Gln Ile Ile Ile Ile Val Val Val Met Met Val Met Val Val Val Ile 10 15 20 acg tgc ctg ctg agc cac tac aag ctg tct gca cgg tcc ttc atc agc 211 Thr Cys Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile Ser 25 30 35 cgg cac agc cag ggg cgg agg aga gaa gat gcc ctg tcc tca gaa gga 259 Arg His Ser Gln Gly Arg Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly 40 45 50 55 tgc ctg tgg ccc tcg gag agc aca gtg tca ggc aac gga atc cca gag 307 Cys Leu Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu 60 65 70 ccg cag gtc tac gcc ccg cct cgg ccc acc gac cgc ctg gcc gtg ccg 355 Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro 75 80 85 ccc ttc gcc cag cgg gag cgc ttc cac cgc ttc cag ccc acc tat ccg 403 Pro Phe Ala Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro 90 95 100 tac ctg cag cac gag atc gac ctg cca ccc acc atc tcg ctg tca gac 451 Tyr Leu Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp 105 110 115 ggg gag gag ccc cca ccc tac cag ggc ccc tgc acc ctc cag ctt cgg 499 Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg 120 125 130 135 gac ccc gag cag cag ctg gaa ctg aac cgg gag tcg gtg cgc gca ccc 547 Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala Pro 140 145 150 cca aac aga acc atc ttc gac agt gac ctg atg gat agt gcc agg ctg 595 Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala Arg Leu 155 160 165 ggc ggc ccc tgc ccc ccc agc agt aac tcg ggc atc agc gcc acg tgc 643 Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser Ala Thr Cys 170 175 180 tac ggc agc ggc ggg cgc atg gag ggg ccg ccg ccc acc tac agc gag 691 Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro Thr Tyr Ser Glu 185 190 195 gtc atc ggc cac tac ccg ggg tcc tcc ttc cag cac cag cag agc agt 739 Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln His Gln Gln Ser Ser 200 205 210 215 ggg ccg ccc tcc ttg ctg gag ggg acc cgg ctc cac cac aca cac atc 787 Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg Leu His His Thr His Ile 220 225 230 gcg ccc cta gag agc gca gcc atc tgg agc aaa gag aag gat aaa cag 835 Ala Pro Leu Glu Ser Ala Ala Ile Trp Ser Lys Glu Lys Asp Lys Gln 235 240 245 aaa gga cac cct ctc tagggtcccc aggggggccg ggctggggct gcgtaggtga 890 Lys Gly His Pro Leu 250 aaaggcagaa cactccgcgc ttcttagaag aggagtgaga ggaaggcggg gggcgcagca 950 acgcatcgtg tggccctccc ctcccacctc cctgtgtata aatatttaca tgtgatgtct 1010 ggtctgaatg cacaagctaa gagagcttgc aaaaaaaaaa agaaaaaaga aaaaaaaaaa 1070 ccacgtttct ttgttgagct gtgtcttgaa ggcaaaagaa aaaaaatttc tacagtaaaa 1130 aaaaaaaaaa 1140 2 759 DNA Homo sapiens 2 atggcggagc tggagtttgt tcagatcatc atcatcgtgg tggtgatgat ggtgatggtg 60 gtggtgatca cgtgcctgct gagccactac aagctgtctg cacggtcctt catcagccgg 120 cacagccagg ggcggaggag agaagatgcc ctgtcctcag aaggatgcct gtggccctcg 180 gagagcacag tgtcaggcaa cggaatccca gagccgcagg tctacgcccc gcctcggccc 240 accgaccgcc tggccgtgcc gcccttcgcc cagcgggagc gcttccaccg cttccagccc 300 acctatccgt acctgcagca cgagatcgac ctgccaccca ccatctcgct gtcagacggg 360 gaggagcccc caccctacca gggcccctgc accctccagc ttcgggaccc cgagcagcag 420 ctggaactga accgggagtc ggtgcgcgca cccccaaaca gaaccatctt cgacagtgac 480 ctgatggata gtgccaggct gggcggcccc tgccccccca gcagtaactc gggcatcagc 540 gccacgtgct acggcagcgg cgggcgcatg gaggggccgc cgcccaccta cagcgaggtc 600 atcggccact acccggggtc ctccttccag caccagcaga gcagtgggcc gccctccttg 660 ctggagggga cccggctcca ccacacacac atcgcgcccc tagagagcgc agccatctgg 720 agcaaagaga aggataaaca gaaaggacac cctctctag 759 3 252 PRT Homo sapiens 3 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val Val Met 1 5 10 15 Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr Lys Leu 20 25 30 Ser Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly Arg Arg Arg Glu 35 40 45 Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser Glu Ser Thr Val 50 55 60 Ser Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro 65 70 75 80 Thr Asp Arg Leu Ala Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His 85 90 95 Arg Phe Gln Pro Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro 100 105 110 Pro Thr Ile Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly 115 120 125 Pro Cys Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu Asn 130 135 140 Arg Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp 145 150 155 160 Leu Met Asp Ser Ala Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn 165 170 175 Ser Gly Ile Ser Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly 180 185 190 Pro Pro Pro Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser 195 200 205 Phe Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr 210 215 220 Arg Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp 225 230 235 240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250 4 8 PRT Artificial Sequence Description of Artificial Sequence FLAG peptide 4 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 5 24 DNA Artificial Sequence Description of Artificial Sequence Primer 5 ggcagaacac tccgcgcttc ttag 24 6 24 DNA Artificial Sequence Description of Artificial Sequence Primer 6 caagctctct tagcttgtgc attc 24 7 22 DNA Artificial Sequence Description of Artificial Sequence Primer 7 cttgggttcg ggtgaaagcg cc 22 8 22 DNA Artificial Sequence Description of Artificial Sequence Primer 8 ggtgggtggc aggtcgatct cg 22 9 20 DNA Artificial Sequence Description of Artificial Sequence Primer 9 ccttcgccca gcgggagcgc 20 10 24 DNA Artificial Sequence Description of Artificial Sequence Primer 10 caagctctct tagcttgtgc attc 24 11 249 PRT Homo sapiens 11 Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val Val Met Met 1 5 10 15 Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr Lys Leu Ser 20 25 30 Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly Arg Arg Arg Glu Asp 35 40 45 Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser Glu Ser Thr Val Ser 50 55 60 Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr 65 70 75 80 Asp Arg Leu Ala Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg 85 90 95 Phe Gln Pro Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro Pro 100 105 110 Thr Ile Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro 115 120 125 Cys Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg 130 135 140 Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu 145 150 155 160 Met Asp Ser Ala Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser 165 170 175 Gly Ile Ser Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro 180 185 190 Pro Pro Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe 195 200 205 Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg 210 215 220 Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp Ser 225 230 235 240 Lys Glu Lys Asp Lys Gln Lys Gly His 245 12 244 PRT Homo sapiens 12 Ala Glu Leu Glu Phe Ala Gln Ile Ile Ile Ile Val Val Val Val Thr 1 5 10 15 Val Met Val Val Val Ile Val Cys Leu Leu Asn His Tyr Lys Val Ser 20 25 30 Thr Arg Ser Phe Ile Asn Arg Pro Asn Gln Ser Arg Arg Arg Glu Asp 35 40 45 Gly Leu Pro Gln Glu Gly Cys Leu Trp Pro Ser Asp Ser Ala Ala Pro 50 55 60 Arg Leu Gly Ala Ser Glu Ile Met His Ala Pro Arg Ser Arg Asp Arg 65 70 75 80 Phe Thr Ala Pro Ser Phe Ile Gln Arg Asp Arg Phe Ser Arg Phe Gln 85 90 95 Pro Thr Tyr Pro Tyr Val Gln His Glu Ile Asp Leu Pro Pro Thr Ile 100 105 110 Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr 115 120 125 Leu Gln Leu Arg Asp Pro Glu Gln Gln Met Glu Leu Asn Arg Glu Ser 130 135 140 Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Ile Asp 145 150 155 160 Ile Ala Met Tyr Ser Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly 165 170 175 Ile Ser Ala Ser Thr Cys Ser Ser Asn Gly Arg Met Glu Gly Pro Pro 180 185 190 Pro Thr Tyr Ser Glu Val Met Gly His His Pro Gly Ala Ser Phe Leu 195 200 205 His His Gln Arg Ser Asn Ala His Arg Gly Ser Arg Leu Gln Phe Gln 210 215 220 Gln Asn Asn Ala Glu Ser Thr Ile Val Pro Ile Lys Gly Lys Asp Arg 225 230 235 240 Lys Pro Gly Asn 13 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 13 gccagcccag 10 14 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 14 gtgcagggag 10 15 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 15 gacaaacatt 10 16 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 16 atgactcaag 10 17 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 17 gaaaagaagg 10 18 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 18 cctgtacccc 10 19 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 19 cctgaactgg 10 20 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 20 tgacagccca 10 21 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 21 tacaaaacca 10 22 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 22 aattctccta 10 23 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 23 tgcatatcat 10 24 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 24 cttgacacac 10 25 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 25 tgtctaacta 10 26 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 26 gtggacccca 10 27 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 27 ataaagtaac 10 28 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 28 tacattttca 10 29 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 29 tcagaacagt 10 30 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 30 caacttcaac 10 31 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 31 gataggtcgg 10 32 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 32 ctaaaaggag 10 33 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 33 gtggtgcgtg 10 34 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 34 tccccgtggc 10 35 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 35 attgatcttg 10 36 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 36 agctggtttc 10 37 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 37 cctcccccgt 10 38 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 38 atgtactctg 10 39 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 39 gatgaaatac 10 40 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 40 gtgcatcccg 10 41 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 41 gaaattaggg 10 42 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 42 tttctagggg 10 43 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 43 cccagggaga 10 44 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 44 gtggcgcaca 10 45 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 45 ttgcttttgt 10 46 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 46 atgtcctttc 10 47 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 47 tgtttatcct 10 48 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 48 gctttgtatc 10 49 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 49 gttccagtga 10 50 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 50 tagcagaggc 10 51 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 51 acaaattatg 10 52 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 52 cagtttgtac 10 53 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 53 gattacttgc 10 54 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 54 ggccagccct 10 55 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 55 caattgtaaa 10 56 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 56 aaagccaaga 10 57 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 57 caactaattc 10 58 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 58 aagagctaat 10 59 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 59 cttttcaaga 10 60 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 60 gtgtgtaaaa 10 61 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 61 acaaaatgta 10 62 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 62 aaggtagcag 10 63 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 63 ggcggggcca 10 64 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 64 ggccagtaac 10 65 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 65 aacttaagag 10 66 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 66 agggatggcc 10 67 10 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 67 cttaaggatt 10

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 of claim 1, wherein the polynucleotide sequences are derived from the genes of LNCaP cells that are up-regulated or down-regulated in response to androgen.
3. The polynucleotide array of claim 2, wherein the androgen is R1881.
4. The polynucleotide array of claim 1, wherein the first set of polynuceotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 3.
5. The polynucleotide array according to claim 1, further comprising a second set of polynucleotide probes, wherein the second set of polynucleotide probes comprises polynucleotide sequences derived from genes that are involved in testosterone biosynthesis and metabolism.
6. The polynucleotide array according to claim 4, further comprising a second set of polynucleotide probes, wherein the second set of polynucleotide probes comprises polynucleotide sequences derived from genes whose expression is altered in prostate cancer or is specific to prostate tissue.
7. The polynucleotide array according to claim 6, wherein the second set of polynucleotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 6.
8. The polynucleotide array according to claim 1, further comprising a second set of polynucleotide probes, wherein the second set of polynucleotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 8.
9. The polynucleotide array according to claim 7, further comprising a third, fourth, fifth, and sixth set of polynucleotide probes, wherein the third set of polynucleotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 4, the fourth set of polynucleotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 5, the fifth set of polynucleotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 7, and the sixth set of polynucleotide probes comprises polynucleotide sequences derived from the polynucleotide sequences listed in Table 8.
10. The polynucleotide array of claim 1, wherein at least one of the genes that is up-regulated in response to androgen is the polynucleotide sequence of SEQ ID NO:2.
11. The polynucleotide array of claim 1, wherein at least one of the polynucleotide sequences comprises the polynuleotide sequence of SEQ ID NO:2
12. A method of identifying an expression profile of androgen-regulated genes in a target cell, comprising hybridizing the nucleic acids of the target cell with the polynucleotide array of claim 1 to obtain a hybridization pattern, wherein the hybridization pattern represents the expression profile of androgen-regulated genes in the target cell.
13. A method of identifying an expression profile of androgen-regulated genes in a target cell, comprising hybridizing the nucleic acids of the target cell with the polynucleotide array of claim 10 to obtain a hybridization pattern, wherein the hybridization pattern represents the expression profile of androgen-regulated genes in the target cell.
14. The method acording to claim 12, wherein the target cell is a prostate cell.
15. A method of diagnosing or prognosing prostate cancer, comprising
(a) hybridizing nucleic acids of a target cell of a patient with the polynucleotide array of claim 1 to obtain a first hybridization pattern, wherein the first hybridization pattern represents an expression profile of androgen-regulated genes in the target cell;
(b) comparing the first hybridization pattern of the target cell to a second hybridization pattern, wherein the second hybridization pattern represents an expression profile of androgen-regulated genes in prostate cancer, and
(c) diagnosing or prognosing prostate cancer in the patient.
16. A method of diagnosing or prognosing prostate cancer, comprising
(a) hybridizing nucleic acids of a target cell of a patient with the polynucleotide array of claim 10 to obtain a first hybridization pattern, wherein the first hybridization pattern represents an expression profile of androgen-regulated genes in the target cell;
(b) comparing the first hybridization pattern of the target cell to a second hybridization pattern, wherein the second hybridization pattern represents an expression profile of androgen-regulated genes in prostate cancer, and
(c) diagnosing or prognosing prostate caner in the patient.
17. The method acording to claim 15, wherein the target cell is a prostate cell.
18. An isolated nucleic acid molecule selected from:
(a) the polynucleotide sequence of SEQ ID NO:2; and
(b) an isolated nucleic acid molecule that encodes a polypeptide having an amino acid sequence of SEQ ID NO:3.
19. A recombinant vector that directs the expression of the nucleic acid molecule of claim 18.
20. A host cell transfected or transduced with the vector of claim 19.
21. The host cell of claim 20 selected from bacterial cells, yeast cells, and animal cells.
22. A method of detecting prostate cancer in a patient, comprising:
(a) detecting mRNA corresponding to the polynucleotide sequence of SEQ ID NO:2 in a biological sample from the patient; and
(b) correlating the amount of mRNA corresponding to the polynucleotide sequence of SEQ ID NO:2 in the sample with the presence of prostate cancer in the patient.
23. An isolated polypeptide, comprising the amino acid sequence of SEQ ID NO:3.
24. An isolated antibody that binds to the polypeptide of claim 23.
US10/390,045 2000-01-28 2003-03-18 Method of detecting androgen-regulated gene Abandoned US20030170713A1 (en)

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