(12) United States Patent (10) Patent No.: US 7,807,373 B2 Srivastava Et Al

US007807373B2

(12) United States Patent (10) Patent No.: US 7,807,373 B2 Srivastava et al. (45) Date of Patent: Oct. 5, 2010

(54) PROSTATE SPECIFIC GENE, PCGEM1, AND GenBank. Accession No. ACO 13401, GI:8569780, 169700 bp DNA, METHODS OF USING PCEGM1 TO DETECT, Jul. 7, 2000. TREAT, AND PREVENT PROSTATE CANCER “AC003046.” EBI Database XPO02143197, Dec. 1, 2001. (75) Inventors: Shiv Srivastava, Potomac, MD (US); Srikantanet al., “Structure and Expression of a Novel Prostate Spe Judd W. Moul, Bethesda, MD (US); cific Gene: PCGEM1. Proc. American Assoc. Cancer Research Vasantha Srikantan, Rockville, MD Annual, XP000929230, vol. 40, p. 37 (Mar. 1999). (US); Zhiqiang Zou, Gaithersburg, MD Bussemakers et al., “A New Prostate-Specific Arker, Strongly (US) Overexpressed in Prostatic Tumors.” Urological Research, XP0020743.05, vol. 25, No. 1, p. 76 (Feb. 1997). (73) Assignee: Henry M. Jackson Foundation for the Wang et al., “Genes Regulated by Androgen in the Rat Ventral Pros Advancement of Military Medicine, tate.” Proc. Nat.1 Acad. Sci., U.S.A., vol. 94, pp. 12999-13004 (Nov. Rockville, MD (US) 1997). “ACO 13401, EBI Database XP002143200, Apr. 16, 2005. (*) Notice: Subject to any disclaimer, the term of this Srikantan et al., “PCGEM1, a Prostate-Specific Gene, Is Overex patent is extended or adjusted under 35 posed in Prostate Cancer.” Proc. Natl. Acad. Sci., vol.97, No. 22, pp. U.S.C. 154(b) by 285 days. 12216-12221 (Oct. 24, 2000). (21) Appl. No.: 12/166,723 Srikantan et al., Identification of Prostate Cancer Associated Novel Gene Expression Alterations, Proc. American Assoc. Cancer (22) Filed: Jul. 2, 2008 Research, vol.39, p. 362 (Mar. 1998). Srikantan et al., Identification of Prostate Cancer Associated Novel (65) Prior Publication Data Gene Expression Alterations, USUHS Research Day-Graduate Stu US 2010/O184O23 A1 Jul. 22, 2010 dent Colloquium, p. 164 (Apr. 1-2, 1998). Srikantan et al., Identification of Prostate Cancer Associated Novel Related U.S. Application Data Gene Expression Alterations, J. Urology, AUA 93rd Annual Meeting, (60) Division of application No. 10/802,823, filed on Mar. p. 40 (May 30-Jun. 4, 1998). 18, 2004, now abandoned, which is a continuation of Petrovics, et al. Oncogene, vol. 23, pp. 605-611 (2004). application No. 09/534,072, filed on Mar. 24, 2000, Genbank. Accession No. AC003046, GI:2583140, 22.9335bp DNA, now Pat. No. 6,828,429. Nov. 3, 1997. Moreno, J.G. et al., “Detection of Hematogenous Micrometastasis in (60) Provisional application No. 60/126,469, filed on Mar. Patients with Prostate Cancer, Cancer Research, 52.61 10-61 12 26, 1999. (Nov. 1992). (51) Int. Cl. Dixon et al., Cancer Chemother. Pharmacol., (1999)43(Suppl.):S78 CI2O I/68 (2006.01) S84. (52) U.S. Cl...... 435/6: 435/91.2:536/23.1; Genbank. Accession No. AF099810, GI:38.00891, 193824 bp DNA, 536/24.33:436/64 Oct. 28, 1998. (58) Field of Classification Search ...... 435/6, Genbank. Accession No. AC006925, GI:4417322, 176186 bp DNA, 435/91.2: 536/23.1, 24.33: 436/64 Mar. 16, 1999. See application file for complete search history. Office Action dated Dec. 20, 2005, from European Application No. (56) References Cited OO 9183732. Office Action dated Jul. 19, 2007, from European Application No. 00 U.S. PATENT DOCUMENTS 9183732. 6,828,429 B1 12/2004 Srivastava et al. 2001/005.3519 A1 12, 2001 Fodor et al. * cited by examiner Primary Examiner Stephen L Rawlings FOREIGN PATENT DOCUMENTS (74) Attorney, Agent, or Firm—MH2 Technology Law Group WO WO95/19434 7, 1995 LLP WO WO99.00498 1, 1999 WO PCT/USOO.O7906 5, 2000 (57) ABSTRACT

OTHER PUBLICATIONS A nucleic acid sequence that exhibits prostate-specific Petrovics et al. (Oncogene. Jan. 15, 2004; 23 (2): 605-611).* Fu et al. (DNA Cell Biol. Mar. 2006; 25 (3): 135-141).* expression and over-expression in tumor cells is disclosed. Ifere et al. (Recent Pat. DNA Gene Seq. Nov. 2009; 3 (3): 151-163).* The sequence and fragments thereof are useful for detecting, GenBank. Accession No. ACO 13401, GI:6289254, 163807 bp DNA, diagnosing, preventing, and treating prostate cancer and other Nov. 9, 1999. prostate related diseases. The sequence is also useful for GenBank. Accession No. ACO 13401, GI:8050939, 170695 bp DNA, measuring hormone responsiveness of prostate cancer cells. May 24, 2000. GenBank. Accession No. ACO 13401, GI:8099084, 172760 bp DNA, May 26, 2000. 7 Claims, 21 Drawing Sheets U.S. Patent Oct. 5, 2010 Sheet 1 of 21 US 7,807,373 B2

STRATEGY FORTHEIDENTIFICATION OF GENE EXPRESSIONALTERATIONS IN PROSTATE CANCER OCTEMBEDDED FROZEN PROSTATE TUMOR/NORMAL TISSUE MAKE 6 am SERIAL SECTIONS

HISTOLOGICALEXAMINATION OF H & ESLIDE

RNA PREPARATION RT-PCRAMPLIFICATIONUSING ARBITRARY AND ANCHORED PRIMER CONTAINING 5 M13 ORT7 SEQUENCES

HIGHRESOLUTIONGEL ELECTROPHORESIS (GENOMYX SYSTEM) AND EXCISION OF DIFFERENTIALLY EXPRESSEDBANDS

REAMPLIFICATIONUSING M13 AND T7 PRIMERS

PURIFICATION AND AUTOMATED DNA SEQUENCING ONAB1377 USING M13/T7 PRIMER

DNA SEQUENCE DATABASE SEARCH (NCBI, CGAP)

RT-PCR USING GENE SPECIFIC PRIMER FORTUMOR SPECIFIC ALTERATIONS TO SCREEN RNAFROMDEFINED GROUP OF PATIENTS AND CELL LINES

ANALYZE EXPRESSION PATTERN STUDY BIOLOGICAL FUNCTION FOR CLINICAL CORRELATIONS OF SELECTED GENES FIG. 1 U.S. Patent Oct. 5, 2010 Sheet 2 of 21 US 7,807,373 B2

TNT NTNT NT T-TUMOR N-NORMAL

- or t 1. 8 FIG 2 T-TUMOR St T N T N T N T N T N - No RT - N-NORMAL

: : -- CYTOKERATIN

T N T N T N T N T N - No RT

: - PCGEM 1

FIG 3 U.S. Patent Oct. 5, 2010 Sheet 3 of 21 US 7,807,373 B2

123 CD >>H O >< LLI CY

-- PCGEM 1

FIG 4 U.S. Patent Oct. 5, 2010 Sheet 4 of 21 US 7,807,373 B2

WHSOOd|

SDENTO}}_LNOO O 1 nM 10 nM O 12 HR FIG 5A U.S. Patent Oct. 5, 2010 Sheet 5 of 21 US 7,807,373 B2

K b

2.37

- 18S 1.35

FIG 5B U.S. Patent Oct. 5, 2010 Sheet 6 of 21 US 7,807,373 B2

MO&&WWNO9 - "... ONWS) TWNBOW - WHOWAL - SOON HoWA is OOO TWNS OOAH HOWWOLS - rt : SWAONW - ANOX 98 SW TWTXS - WIT SDN?. - WLNOW NW 9 - LAWH - . 3 E3 : MOOX?. OOO8 WHA (SNINI IWSOOnW) NOOO SNILSSLN TWWS - MAWAO - SISL - WISOd SWAHL - NB9TS - 2 : ; ; ; U.S. Patent Oct. 5, 2010 Sheet 7 of 21 US 7,807,373 B2

appendix lung trachoa placent

fetal fitn fatal fetal at it fit brin heart karey er open thymus ting yeast A a st an co "Poly (A) human 00 ng 100 ng 100 ng 100ng 100 gg isong a U.S. Patent Oct. 5, 2010 Sheet 8 of 21 US 7,807,373 B2

U.S. Patent Oct. 5, 2010 Sheet 9 of 21 US 7,807,373 B2

cDNA sequence of PCGEM Sed. ID No. 1 AAGGCACTCT GGCACCCAGTTTTGGAACTG CAGTTTTAAA AGTCATAAATTGAATGAAAA TGATAGCAAA 70 CCTGGAGGTTTTTAAAGAGCTATTTATAGGTCCCTGGACA GCATCTTTTTTCAATTAGGC AGCAACCTTT 140 TTGCCCTATG CCGTAACCTG TGTCTGCAAC TTCCTCTAATTGGGAAATAG TAAGCAGATTCATAGAGCT 210 CAATGATAAA ATTGAACTAC GAGATGCACT GGGACTCAAC GTGACCTTAT CAAGTGAGCA GGCTTGGTGC 280 ATTTGACAC, TCATGATATC AGCCAAAGTG GAACTAAAAA CAGCTCCTGG AAGAGGACTA TGACATCATC 350 AGGTTGGGAG TCTCCAGGGA CAGCGGACCC TTTGGAAAAG GACTAGAAAGTGTGAAATCT ATTAGTCTTC 420 CATATGAAATTCTCTGTCTC TGTAAAAGCA TTTCATATTT ACAAGACACA GGCCTACTCC TAGGGCAGCA 490 AAAAGTGGCA ACAGGCAAGC AGAGGGAAAA GAGATCATGA GGCATTTCAG AGTGCACTGT CTTTTCATAT 560 ATTTCCAAT GCCGTATGTTTGGTTTTATTTTGGCCAAGC ATAACAATCT GCTCAAGAAAAAAAAATCTG 630 (AGAAAACAA ACGTCCCTTT GCCAATGTTA TGTTTCTTTTTGACAAGCCC TGAGATTTCT GAGGGGAATT 700 CACATAAATG GGATCAGGTC ATTCATTTAC CTTGTGTGCA AATATGATTT AAAGATACAA CCTTTGCAGA 770 CAGCAIGCTTTCCTAAGGGT AGCCACGTGG ACGACTAAGG GTAAAGCATT CTTCAAGATC ACTTAATCAA 840 CAAAGGTGCT CITTGCATTC TGAAATGCCCTTGTTGCAAA. TATTGGTTATATTGATTAAA TTTACACTTA 910 ATGGAAACAA CCTTTAACTT ACAGATGAAC AAACCCACAA AAGCAAAAAATCAAAAGCCC TACCTATGAT 980 TTCATATTTT CTGTGTAACT GGATTAAAGG ATTCCTGCTT GCTTTTGGGC ATAAATGATA ATGGAATATT 1050 TCCAGGTATT GTTTAAAATC AGGGCCCATC TACAAATTCT TAGCAATACT TTGGATAATT CTAAAATTCA 1120 CCTGGACATT GTCTAATTGTTTTTTATATA CATCTTGCT AGAATTTCAA ATTTTAAGTA TCTGAATTTA 1190 CTTAATTAGCTCTCCTGATC AATTCAAAAA CATTACTTTC CTAAATTTTAGACTATGAAG GTCATAAATT 1260 CAACAAATAT, ATCTACACAT ACAATTATAG ATTGTTTTTC ATTATAATGT CTTCATCTTA ACACAATTGT 1330 CTTTGTGATT GTTTTTAGAA AACTGAGAGTTTTAATTCAT AATTACTTCA TCAAAAAATT GTGGGAACAA 1400 TCCACCATTA, ATTCTATCTG ATTGTTTTTA TCTACATAAG GACTCTTAAG CTTCGTCCCTTGAACTCTTT 1470 TCTACTTAGT CCCATGTTTA AAATTACTAC TTTATATCTA AACCATTTAT GTTTTTCAATTCAATTTACA 1540 TGATGCTAAT TATGGCAATT ATAACAAATA TTAAAGATTT CGAAATAGAAAAAAAAAAAA AAA 1603

FIG. 8 U.S. Patent Oct. 5, 2010 Sheet 10 of 21 US 7,807,373 B2

cDNA sequence of PCGEM Seg. ID No.2 GCGGCCGCGT CGACGCAACTTCCTCTAATT GGGAAATAGT TAAGCAGATT CATAGAGCTG AATGATAAAA 70 TTGTACTTCG AGATGCACTG GGACTCAACG TGACCTTATC AAGTGAGATG GAGTCTTGCC CTGTCTCCAA 140 GGCEGGAGCC CAATGGTGTG ATCTTGGCTC ACTGCAACCT CCACCTCCCA GGTTCAAACGTTTCTCCTGC 210 CTCAGCCTCC CAAGTAACTG GGATTACAGC AGGCTTGGTG CATTTGACAC TTCATGATAT CAGCCAAAGT 280 GGAACTAAAA ACAGCTCCTG GAAGAGGACT ATGACATCAT CAGGTTGGGA GTCTCCAGGG ACAGCGGACC 350 CTTTGGAAAA GGACTAGAAA GTGTGAAATC TATTAGTCTT CGATATGAAA TTCTCTGTCT CCGTAAAAGC 420 ATTTCATATT TACAAGACAC AGGCCTACTC CTAGGGCAGC AAAAAGTGGC AACAGGCAAG CAGAGGGAAA 490 ACACATCATGAGCCATTTCA GAGTGCACTG TCTTTTCATA TATTTCCAA TGCCGTATGTTTGGTTTTAT 560 TTTGGCCAAG CATAACAATC TGCTCAAAAAAAAAAAATCT GGAGAAAACA AAGGTGCCTT TGCCAATGTT 630 ATGTTTCTTTTTGACAAGCC CTGAGATTTC TGAGGGGAATTCACATAAAT GGGATCAGGT CATTCATTTA 700 CCTTGTGTGC AAATATGATT TAAAGATACA ACCTTTGCAG AGAGCATGCTTTCCTAAGGG TAGGCACGTG 770 GACGACTAAG GGTAAAGCAT TCTTCAAGAT CAGTTAATCA AGAAAGGTGC TCTTGCATT CTGAAATGCC 840 CTTGTTCCAA ATATTGGTTA TATTGATTAA ATTTACACTTAATGGAAACA ACCTTTAACTTACAGATGAA 910 CAAACCCCAC AAAACCAAAA AATCAAAAGC CCTACCTATG ATTTCATATTTTCCTGTAA CTCCATTAAA 980 GGATTCCTCC TTCCTTTTGG GCATAAATGA TAATGGAATA TTTCCAGGTATTGTTTAAAA TCAGGGCCCA 1050 TCTACAAATT CTTAGCAATA CTTTGGATAA TTCTAAAATT CAGCTGGACA TTGTCTAATT GTTTTTTATA 1120 TACATCTTTG CTAGAATTTC AAATTTTAAG TATGTGAATT TAGTTAATTA GCTGTGCTGA TCAATTCAAA 1190 AACATTACTTTCCTAAATTT TAGACTATGA AGGTCATAAA TTCAACAAAT ATATCTACAC ATACAATTAT 1260 AGATTGTTTTTCATTATAAT GTCTTCATCT TAACAGAATT GTCTTTGTGA TTGTTTTTAGAAAACTGACA 1330 GTTTTAATTCATAATTACTT GATCAAAAAA TTGTGGGAACAATCCAGCAT TAATTGTATG TGATTGTTTT 1400 TATGTACATA AGGAGTCTTA AGCTTGGTGC CTTGAACTCT TTTGTACTTA GTCCCATGTT TAAAATTACT 1470 ACTTTATATC TAAAGCATTT ATGTTTTTCA ATTCAATTTA CATGATGCTA ATTATGGCAA TTATAACAAA 1540 TATTAAAGAT TTCGAAATAG AAAAAAAAAA AAAAATCTA 1579 FIG 9

U.S. Patent Oct. 5, 2010 Sheet 12 of 21 US 7,807,373 B2

cDNA sequence of PCGEM1 Promoter Region Seg. ID No.3 TCCCTCTTGC GTTCTCCAATTTCTGAAAAA AAGATGTTTA TTCCAAAGTG ATATGAGCAC TGGAAAGGTA 70 CTAATTCCAA TTTGATTCTA ATTGGATGAC TGACATGGGT AAGCGATTCT AAGCATTTGT GTTTTTTTTA 140 GTAGTATGGA ATTTAATTAG TTCTCAGTAT GTTAGTGAAG ATGAATGAAAACATGCATAT GTTTCCATGT 210 ATTATAAATA TTTTAAAATG CAAAAAATTA TTCTAATGAA TATATAAATA TAAAGCATAA CAATAATAAT 280 ACAATACCAC CCATAAAGTC ATCATCTAAT TTAAAAACTA AAACATTAAC ACTTGAATCT CCCCCATTGC 350 AACATCTTTC CCGACTTCTG TGTTTTTTTC TTTTCCTTTT AAAATTTTTC TTTTATCATA TGTCTCCATA 420 AGATTATATA CCTTTCCTTGTTTTAAGCTTTTTAAATAAT ATATTGTAGT TATATTATTT GTCCTTTCCT. 490 TTTTTTACTT AACATTATCG TTCTAAAATT CAGTAATGTG TTGGGCATGT ATAATTTGTTTATTTTTAAT 560 CTCTTTCACA TTCGACTATATAAATTTCAGTTTGTTTATTGACTCCTTTG TCTATAGATA CTCTGCTATT 630 TCTCTTTTTC CTGTTACAAA AATAATGCTG TTTTAAATTT CATTTTGTAT ACTTTTTTGA CGCATCTGTA 700 TGACTTATTC TAAGCTAAAA AAATAAGAAAAAATTCCTGG GTTATAAGAT TGTCACATGC TCGAATTTAC 770 AAGATAATGC CAAATCATTTTTCAAAGTAA TTATACCTAT TTATACTACC GGTATGAGTA TATTGGTGCC 840 CACATAGTTC CTTGTTCTCC CAAAGTTTGG TATGATCGAA CAATAATTTTTCCCCATCAA ATGGCATAAA 910 ATAAAATCTC ACTCTCCTTT TAATTTCCAT TTTCTATCTT TAAGAATTGTTTCTTTTTTA ACCATTTATA 980 ATTTACTTTT GCTGAAATCC TTCCTTATTA TTTTTCCTCC CCATTTTTTC CTATTGGATT CCTTTTCTCA 1050 TTAATTTATA ACAATTTTAT ATCGTTTAGA TACTAATTAT TATATTACTG AAAATACCTT TATCAGTTTG 1120 TTGTGTACTT TCTACTTTAT GTCTTCTGAT GGATAAAAGTTTTAAATTGTATTGTGTTGAACTTAACATT 1190 TTTAAATTTT ATAATCAGCA TCTTTAATAA TCTCTTTMTA AAATTTTCCTTTACATAGAT GTCATAAAGA 1260 TACATCTCTATAATTTCTTA TTTTTTTGGC ATATGTTCAT TAAGTCATTT TATCATTTTT TAGTAATAAA 1330 TTGCAGTTAT TTATGAAACA AATAATTTTT AAAATTATATATGCTTTCTT TAAAAATTGA TCTTACCATG 1400 CTTCACTATGAAGCTTGAGG CTTCACTGCA CGTTGTACTG AAATTATGTATAAAACAGTG (GTTCTGAAAA 1470 TCTCTGAGTT CATGACACCTTTACTCTCTC AGGTTTTTTT GCTTTTGTTC TTGTTTTTTC TCACAAAGCA 1540 CCTAAGTTAAATAAAAACAA AGCACAAAGC TATCAGCTTC ATGTATTAAG TAGTAAGCTC CCATGTTAAC 1610 AGTTCTAACTTGCCTGGTCC CCAATAGATG TCACTCTGTTTTCCTAGAAA CTTTAAAATA TCCCTCAGTG 1680 CTCCTGTTAATTCATGGTAG TCCCCCAAGG CACTCTGGCA CCCAGTTTTG GAACTGCACT TTTAAAACTC 1750 ATAAATTGAA TGAAAATCAT AGCAAAGGTG GAGGTTTTTA AAGAGCTATT TATAGGTCCC TGGACAGCA 1819 FIG 11 U.S. Patent Oct. 5, 2010 Sheet 13 of 21 US 7,807,373 B2

cDNA sequence of PCGEMI PROBE Seg. ID No. 4 TTTTTTCAAT TAGGCAGCAA CCTTTTTGCC CTATGCCGTA ACCTGTGTCT GCAACTTCCT CAATTGGGA 70 AATAGTTAAG CAGATTCATA GACCTGAATGATAAAATTGT ACTACGAGAT GCACTGGGAC TCAACGTGAC 140 CTTATCAAGT GAGCAGGCT GGTGCATTGACACTTCATG ATATCATCCA AAGTGGAACT AAAAACAGC 210 CCTGGAAGAG GACTATGACA TCATCAGGTT GGGAGTCTCC AGGGACAGCG GACCCTTTGG AAAAGGACTA. 280 GAAAGTCTCA AATCTATTAG TCTTCGATAT CAAATTCTCT CTCTCTGTAA AAGCATTTCATATTTACAAG 350 ACACAGGCCT ACTCCTAGGG CACCAAAAAGTGGCAACAGG CAAGCAGAGGGAAAAGAGAT CATGAGGCAT 420 TTCAGAGTGC ACTGTCTTTT CATATATTTC TCAATGCCGT ATGTTTGGTTTTATTTTCGC CAAGCATAAC 490 AATCTGCTCA AGAAAAAAAA ATCTGGAGAAAACAAAGGTG CCTTTGCCAA TGTTATGTTT CTTTTTGACA 560 ACCCCTGAGA TTTCTGAGGG GAATTCACAT. AAATGGGATC AGGTCATTCA TTTACGTTCT GTGCAAATAT 630 GATTTAAAGA TACAACCTTT GCAGAGAGCA TGCTTTCCTA AGGGTAGGCA CGTGGAGGAC TAAGGGTAAA 700 GCATTCTTCA AGATCAGTTA ATCAAGAAAG GTGCTCTTTG CATTCTGAAA TGCCCTTGTT GCAAATATTG 770 GTTATATTGA TTAAATTTAC ACTTAATGGA AACAACCTTT AACTTACAGA TGAACAAACC CACAAAAGCA 840 AAAAATCAAA ACCCCTACCT ATCATTTCAT ATTTTCTGTG TAACTGGATT AAAGGATTCCTCCTTCCTTT 910 TGGGCATAAA TGATAATGGA, ATATTTCCAG GTATTGTTTA AAATGAGGGC CCATCTACAAATTCTTAGCA 980 ATACTTTGGA TAATTCTAAA ATTCACCTGG ACATTGTCTA ATTCT 1025 F.G. 12 U.S. Patent Oct. 5, 2010 Sheet 14 of 21 US 7,807,373 B2

PCGEM1. PrimerS Used for PCR

PCR PRIMER 1 (SEO ID NO. 5 Sense Primer 5' TGCCTCAGCCTCCCAAGTAAC 3' PCR PRIMER 2 (SEO ID No. 6) AntiSense Primers 5' GGCCAAAATAAAACCAAACAT 3' PCR PRIMER 3 (SEO ID No. 7) Sense Primer 5' TGGCAACAGGCAAGCAGAG 3." FIG. 13 U.S. Patent Oct. 5, 2010 Sheet 15 of 21 US 7,807,373 B2

Complete Genomic DNA sequence of PCGEM1 gene. TCCCTCTCCGTTCTCCAATTTCTGAAAAAAAGATGTTTATTGCAAAGTGATATGAGCACTGGAAAGGTACTAATTCCAA TTTGATTCTAATTGGATGAGTGACATGGGTAAGCGATTCTAAGCATTTGTGTTTTTTTTAGTAGTATGGAATTTAATTAG TTCTCAGTATGTTAGTGAAGATGAATGAAAACATGCATATGTTTCCATGTATTATAAATATTTTAAAATCCAAAAAATTA TTCTAATGAATATATAAATATAAAGCATAACAATAATAATACAATACCACCCATAAAGTCATCATCTAATTTAAAAACTA AAACATTAACACTTGAATCTCCCCCATTCCAACATCTTTCCCGACTTGTGTGTTTTTTTCTTTTCCTTTTAAAATTTTTG TTTTATCATATCTCTCCATAAGATTATATAGCTTTCCTTGTTTTAAGCTTTTTAAATAATATATTGTAGTTATATTATTT GTGCTTTCCTTTTTTTACTTAACATTATGGTTCTAAAATTCACTAATGTGTTGGGCATGTATAATTTGTTTATTTTTAAT CTCTTGACATTCGACTATATAAATTTCAGTTTGTTTATTGACTCCTTTGTCTATACATACTCTGCTATTTCTGTTTTTG CTGTTACAAAAATAATGCTCTTTTAAATTCATTTTCTATACTTTTTTGAGCCATGTGTATGAGTATTCTAACGTAAAA AAATAAGAAAAAATTGCTGGGTTATAAGATTGTCACATGCTCGAATTTACAAGATAATGCCAAATCATTTTTCAAAGTAA TTATACCTATATACTACCCCTATGACTATATTGGTCCCCACATAGTTGCTTGTTCTGCCAAAGTTTCCTATGATCGAA CAATAATTTTTGCCCACAAATCGCATAAAATAAAATCTCACTCTCCTTTTAATTTGCATTTTCTATCTTTAAGAATTGT TTCTTTTTTAACCATTTATAATTTACTTTTCCTGAAATCCTTGCTTATTATTTTTCCTCCCCATTTTTTCCTATTGGATT GCTTTTCTCATTAATTIATAACAATTTTATATGGTTTAGATACTAATTATTATATTACTGAAAATACCTTTATCAGTTTG TTGTGTACTTTCTACTTTATGTCTTGTCATGGATAAAAGTTTTAAATTGTATTGTCTTGAACTTAACATTTTTAAATTTT ATAATCACCATCTTTAATAATCTCTTTATAAAATTTTCCTTACATAGATGTCATAAAGATACATCTCTATAATTTCTTA TTTTTTTCGCATATGTTCATTAACTCATTTTATCATTTTTTAGTAATAAATTGCACTTATTTATGAAACAAATAATTTTT AAAATTATATATGCTTTCTTTAAAAATTGATCTTAGCATGCTTCACTATGAACCTTGAGCCTTCACTCCACGTTGTACTG TTGTTTTTTGTCACAAAGCACCTAAGTAAATAAAAACAAAGCACAAAGCTATCAGCTTCATGTATTAAGTAGTAAGCTC CCATGTTAACAGTTGTAACTTGCCTGGTCCCCAATAGATGTCACTCTCTTTTCCTAGAAACTTTAAAATATCCCTCAGTG CTCCTGTTAATTCATGGTAGTGCCCCAAGGCACTCTGGCACCCAGTTTTGGAACTGCAGTTTTAAAAGTCATAAATTGAA TGAAAATGATAGCAAAGGTGGAGGTTTTTAAAGAGCTATTTATACCTCCCTGGACAGCATCTTTTTTCAATTAGGCAGCA ACCTTTTTGCCTATGCCGTAACTCTCTCTCCACTTCCTCTAATTCGGGTGAGTAACAGATTTTCTTATGTATATAATAGC TAAGAATATAGTAATAATCCCTTAAATCATGGTTATTTTTAAACTACTAACATTTAGAAGACAAAATAAAAATGCTTTGA AAAGTATAGAGGTTTTAGTGTAATTAGCAGGGAATAATGAAATGATTTGATAGGGCTACTCAGTTTTGTATAACTTTGGT CCTTTAACTCTGAATGCACAGCATGGATGTTGTGATCCAGCCTTTATATGTTTTCCCTGAAGAAGATTTAATTTATTTCC CCTTTTGAGAAACACATTTGGCATTGTAATATGTTTTCCTTCCAGGTTCTATCTCCAAGGATAATTTGACAAAATCACAC ATAAATTTATTTTCAGGGCACACAGTTTCCCTTTTAGGGAACTCACAGAGGTAGAGAGTAATACAATAATCACATTTGAA TATTCAGTAAGTGAGGTCCTCATAGATCTTATGTGTATGTCACCATGTATATAATTTTCTTAATCACTAGATGTATGAGA CAACAAATTTGAGGAATCTTAACTAGAGATTAAAATCAGGGATTTAAATCAAAGAAACATTTAAATCCCTCCTTTATTAT TTAAATACCTGCATGGGAGAATCATTGAAAAAAAAATAAAAAGCATACAACTTGGGAATATTATAAACCAAGAAGAATTT GTATTCTGGTTGATTTTTTTTTCACGCTCCGCACAGCCAACTTACCTTTATCTCTTTGTGATTTTTATTTCTTGTTAAA ATATACAGAAATAGTTAAGCAGATTCATAGAGCTGAATATAAAATTTACTACGAGATGCACTGGGACTCAACGTGACCTT ATCAAGIGACTTATCAGTGAGGTGAGCATTCTTAATTCAGATAATGGAACTTATTATCATAATCTTTTCCTTATCCTATT GTTGAGCTTAACTACTTATTCATATTTGCATATGCATATTGAGATAATATCATTTCATTAATTTCACTACTGAACACTAA TCTCCTAAGAGTAATTCTGAAAGTTTCAGATTGCACTATTTTTAACTATATATCTGTATGTTATCTTCATATATGCTTGA ATAACTTATAAGCAATTGAAACTTTCAATTACAGTATACTATTGAAGCAAATCAACAAATATATACACATATCCATTAGC AATAGTAGATAATTTTTGTAAATCTCCACCACAGTTCTTCATATGTAGAGGATGTTCAAATTGGCTAAGTTCCTTTTCTC TCTTAATTATTAGTATTTTTCCTACTGCTCTTTCTATAATTATTCCTTCCTCTTTAGCTCCAATCCTTACAATCTATTCT F.G. 14 U.S. Patent Oct. 5, 2010 Sheet 16 of 21 US 7,807,373 B2

TAACATAGCAACTGGGAAGAAAGTTTTTAAACATAAACCAGATGATGTCACTCCACCCCACAAAACTTCCACTATTCTCT GTCACACATAGAAAGAAAGAAAAAAAATATTGAAAACCTACAAAGACTTGCTATGATCTGGTCCAGGCTCTCCCTAAAAT TTCATGTAATTTCCAGCCACTAGCCCTTTCTGGCTCTCCTTCAATCTCATTAGCCTTTTCACTACTACAAGTTAGACTGG GTTTTCCCCGAGGTATTTCTTTTTTTCATATTTTGCCTTTCCCTAGATTGCTCTCCAATAGATATTCACAATTGCATCA TCATTTCTATATACGTGCTAAAAGGTTTCCTTCTCCAAAATACCTTCACTGACCACCTGATCTAGAATAGTCTCGATCAA AAGTTTCTTTTCCTTTTCCTCACCACTTGATATTTATATCAAACATTTATTTGTGTAATTTATGTGTTTGTTTGTTTTCT GTACTAGCATTATGATGACCATACTATTTGATCCCCCCCAAAAAATACTTTCGAGAATGACAGGGCAAAGCTAAAATAAT TAAATTATATAATTTTGACATAGGCACTATTGACAAAAAGCAATTGATGTTATGATAGTGTTAGATCTATGAAATAGTAC TATTTAAAAGTAATTCTCTGAAATACAATTTTCTAAAACTAAAAGCAGCATATGTACATGAAACACCAAAAAACTTCCTT AIATTTATCACTGGAAGATTTAAAATAGTATAAGTAGTAACTTATTTAATATATTTTTGATTATTTAATTAATTTTATAG TATCCAACTCTAATATAATGCCACTGGTATTTCTTCAAAATATTTTAATGTTGTCTATTTATTTTTAATTTCCCTAAAAA TIATCTTAAATGAAAATTTTTGGTTAATAAATTTGAAAATACTCAAACCCTCATCTCCAGTCTCTGTGGATCCTAAAGTT TITAGTTGAGAAAATAATTTTTCCTAGAGAATGAACTAGCTTGTAAGCTTGGAGAAATTTCTGCTAAATAAATGATATT ATCAACTCTTATTTTCTTCAATACCAAATATATAAATATTTCACCTCATATATTTTTCCACGTCCTATCCITTTCCTTCC AATCATAATTTCTGACAAATATTTTGGAAGTCAAAACTTGTCTTCTATTTTGTTATTTAAAATTATATAGACTACTTTTG TAAACCTTTATACTATCAAATCATAGCCAATTCAGTTTGATTTCATTCTGGTCCAGAATATAAGTTTATCCAAGTAAAA CAGGAGTCACTTCAAAAGATTCCICCCACTGACTGAGATATTCCAAAGCCAACTTTGCAAAATTTCAGAATTAAATATTA TACTTCTTTGTACCTTCATTTTAITTGTTCAATTTTTCTTTCTGTTTCTAGAAAATTTTAATATTTTTCTCTTTTCAAGT TTTGATTTTAATTTACTACTTATAATTTTTAAAGCTAAGTTTTGTCACGCTATATTCATTATGTGTTTTGAATAAAGAC ATACAATTAATTTTGAGAACTGCAATAAAAATTATAAGACTATTAAAAATGCACTAAGTGTACTACACTTAGGCTGCTAA AAATCCAGTACCAGTAGACTACAITTAGCCTCCTTAAACTTAGTTCTTCTAAGTACCATATACTTTAAAATTTTAGCTAA TGATGGAGAACAAAGACAGAAAGACTGTGTTACCATATTCTAGTTGGCCATTTTGTTTTGTTTTGAGAGACGTCACATCA GCCTTATCATAAAAATTATTGGTTTTACCATTTTGACTGTGACCAAAATATACAGCATAATATACAAAATAAAATACA GTACATCTTCACAACTTCTTGTTTAGGATGCAATTATATATATATATATATATATATTTATTATTATACTTTAACTTCTA GGGTACATGGCACCACGTGCAGGTTGTTACATATCTATACATGTCCCATGTTCGTGTGCTCCACCCATTAACTCGTCATT TACATTAGGTGTATCTCCTAATGCTATCCCTCCCCTCTCTCCCCACCCCACAACAAGCCCCGGTGTGTGATGTTCCCCTT CCTGTCTCCATCTGTTCTCATTGTTCAATTCCCACCTATGACTGAGAACACCCACTCTTTCCTTTTTTCTCCTTGCAATA GTTTCCTGAGAATGATGGTTTCCAGCTTCATCCATGTCCCTACAAAGCACATGAACTCATCATTTTTTATGGCTGCATAG TATTCCATGCTCTATATGTGCCACCATTTTCTTAATCCGAGTCTCTCCATTCTTGTTGGACATTTCGGTTCCAATTTTCA GTTTCATGTCTAGCATCTATACCACAACCAATTAAGATTTCTTTCTTTCTCTCTTTTTTTTTTTTTTTTCTTGAAATCGA GTCTTGCCTCTCTCCAAGGCTGGAGCCCAATGGTGTGATCTTGGCTTACTGCAACCTCCACCTCCCGGGTTCAAGCGATT CTCCTGCCTCAGCCATCCGAGTAGCTGGGACTATAGGCCFGCACCACCATGCCCAGCTAATTTTTGTATTTTTAGTACAG ACGGGGTTTCACCACCGTGGCCAGGATGGTCTCAATTTCTTGACCTCATGATTCACCCGCCTTGGCCTCCCAAAGTGCTG GGATTACAGGTGTGAACCACCAAGCCCGGCCTGTCACAAGTTTTTAGTGTTCTATTTTAATACAGAAATTAGATAAATCC AAAGAGAAAGACATTTCATATGTGCGTAGAGTTGTCCGAAGAAATGAGAGTCTTATAAATAACTTTAAAAATTGTGAAGA AATAAAGCCAAAATAGTCCTATGCAGTTTGATTTAAATATATTCTTAATAAGAGCTACTTTTGTGAAACCACAATAATTG AAACATGTAGATATGGATCTTCATTAGTGACTGACATAATATATTGTATTGTTACTATTTTATTGTATCAGCCAACTAA TATTGAGTGCTTTGTGTATCCTAACCACTATCCTAAACACTGTACCACTATTACCTGATATAATCATATTAATATTTATT FIG 14(cont'd-1) U.S. Patent Oct. 5, 2010 Sheet 17 of 21 US 7,807,373 B2

ATTTCACTTTTCATATGAAAAAATTGAAGCACAGATTAAGACACTCCGAAATCATACCTCTATTGATTATCAGCACCAGG ATTGAATTGACGCACTCTGATCCAGAGAAGCTTTTGTTTCCATGAAGGCTTATGTTGGGGAAAAATAATCAAATTGCCT GTACCTCAGTTGTATAAATAAGAGGTTGGGTTGGTAGATGATTCTGGCTGATTCAGCAGAAAAGAAATTTATTCAAAGGA TATCACACAGTTTTCATAACAGTTAAGAATACAGAGGAAACAGGGCACCAGGGCTAAGTACAGACCAAAGTCCAAAACCA CTCCCAAACTTCCACCAAGGAGAACAGCACAAATTTGCTTGCTGTCACCCGCCACTAGATGCTTTTGTTTGGAGCCTTGA ACTTGACTTACACTCCCACTGACATCAGCACCAGTGCTCTCTGTGTACTAGGAGGTGGAGTTGGTGACGTTGCTGAACTA AAAGCAGATGTTTCTGCTGTGAAATAGATACCTAATACAGAACCTGATTCCTCATTCATTCCCTCCCCAAATCATATGCT TGTAGTGTCGCTAGAGTTTCTGTTTCTCCTTGGTCCAGGCAGAATTTATGAAGCTTGCTATTTATCGCCTTAAAGATTAG AAGAATATTCATAACGTATTAGATTCCCATAACGTTGAACAAATCAACATTCAACTTCAAGGATTCAACATTGTTTTGTT TTCTTTTCCGATACCTCTCCACCAGTTCAAATCTTATTTCTCCCCTTCGACAACCAGGTTTATAAATATTGCAGATTCTC CACTCACTCCTTGATCCTATCTTCTATATTTATGTATACTAATTAGCATATAATAAAAGATTATGTTACAGAATCTCAA AATTAGTAATTATGAATTGAGATGGTGTTATACAGTACACTAACATCCAAGAGACTTGTTTATTCCAAGGAAAATATTTA GAGATATTAAATGATATTTCTCATCCTTTAGACATATACATTTTTTAGCTTACAGCCTGCTTAGGCAAGCAACAGACTC TCAGGATCTGCTCCTACCAGGGTCTGAACATTTCCTCCCAGTTTTAAAGAAACAAATTCAAATAACATTGTAACCTCCAG AGGAAAGTTCAAGGTCTTTTATAGTATTGTTTAAACAGTACAGCTGAGGAAACTAAAGACAGAGAAGTTAAATGCCTTGG CACTTAGTCTAGATTTACAATAAACTCCTYTCTACTTAGGACCCACTAACAGGCGCTCCATTTACACCAAAACCATGAAG GTGGCCCAAGTCATCACTGAGAAGTAGTACAAGCACCGAGGGAATGACTTCAACAGGAACAAGAAAGCGTGGAAGGAGAT CCTAGCAGGAAGCTCCACAAGAAGATAGCATGTTACGTCTTGCATTGGATGAAGCAGGTTCAGAGAGACCAGTGACAGC TATCTCCGTCAAGGTGCAGAAGGAGAGATCATTGAATGTAGCATTTTCATCCAAAAAAAAAAATGTTGAAGTCTTTGGAC TTCGGGAGTCTGTCCAAACTGCAGGTCACTCAGCCTACAGTTGGGATGAATTTCAAAACACCAGTTGGAGCCGGTTGAAT CTTTCTGCTATGCTGTAATATTTTCAGTAAACCCAGCGCAACAACAACAACAAAACACAAAAGGAGGAGAAGCAGCCAAG TCTCTTGGTTTACACAGTAGCTCCTAATACCCCTTCCTCTCTCTCTCAAGTCCCCAATCGGAAGATAGTCAAAACAATAT TCACACCTGTGATTCATCTCTCTACATCCAGTCTCTCTCAATCTTTATATACTGCATATTAACGATCTCTCTTACAGAT AAAAACTAAAGCATTGAAGGAACTCCTTGTTTTGACTTATCAAAGTCCTTAAGAAAATACTAGAAAATTATAGCCATTGT TTCAAATTTTAGCTTTATATTATCACTTGAAATGTGATGAAATGTGGCTGATAGATAATAATTCACTGATAACCTACAGA CAATTCCCATCTTAAAATGGACCATTCGATTGAAGAATTAAATAAAATTGAGGGTTTTCCTTACATGTTTTGTCTAAAGA GCGAAGTAGAAACAACTGTTCATAGATCTTCATTGAGGATTCGCATGTGAAGTAAGTACTCCTAACATAAACAAGTGGAC TTATCAACCAAGTTCCATAAATCATGAACAAAAATATTTGTCCCCAGAGAGACTATTTTTCCACCACATCTCTTCTAATA AACACAGAGCCCAGTTCAGTTAAAATACTTTAAGGGTGGACGGTTCAGGGCCTGCTGAGTGGCACTCAGTAAGAAAACCC AGCAGAACATTACTTCTCTCTTTATTCCAGAGCATCAATGGCCAAGGCTGGAAGATCCCAGAACACTGAACAGACATTT GCTCTCTTATGGCCTGCCAATTTTCACAGTGGGTTCCAACGCTTTGGGTCAAACCAAAATAGACCTGTTAGAAAAATGTC GGTTGGAATACGCTAACAATAAGACAGAATAAATGTGATTATTTCACCTCATTTTTATAGGACTTGAGTAATTTTATTAT AACATTCTTGAGGGCTGGAAAATCTGAATGTTAGGACACCAAATATCTCCAGAAAACAAGTTTTATATTTCTAATCCTCC ATAATAAACCTGGGGCCACTGCAGGCCTCATTAATAAAAACCTAATGGTATAACAATAATGAGGAGGAAATGCCAATGCC GCACAAATCTGTTGAGACTAAAATATTTCTCACCCCAGCAGGCTTCCTCCATTTGACACTTCATGATATCAGCCAAACTG GAACTAAAAACAGCTCCTGGAAGAGGACTATGACATCATCAGGTTGGGAGTCTCCAGGGACAGCGGACCCTTTGGAAAAG GACTAGAAAGTGTGAAATCTATTAGTCTTCGATATGAAATTCTCTCTCTCTGTCAAAAGCATTTCATATTTACAAGACAC ACGCCTACTCCTAGGGCAGCAAAAAGTGGCAACAGGCAAGCAGAGGGAAAAGAGATCATGAGGCATTTCAGAGTGCACTG FIG 14(cont'd-2) U.S. Patent Oct. 5, 2010 Sheet 18 of 21 US 7,807,373 B2

TCTTTTCATATATTTCCAATGCCGTATGTTTGGTTTTATTTTCGCCAAGCATAACAATCTGCTCAAGAAAAAAAAATCT GGAGAAAACAAACGTCCCTTTCCCAATCTTATGTTTCTTTTTGACAAGCCCTGAGATTTCTGAGGGGAATTCACATAAAT GGCATCACGTCATTCATTTACCTTGTGTCCAAATATGATTTAAAGATACAACCTTTGCAGAGAGCATGCTTTCCTAAGGG TAGGCACGTGGAGGACTAAGGGTAAAGCATTCTTCAAGAATCAGTTAATCAAAGAAAGGIGCTCTTTGCATTCTGAAATG CCCTTGTTCCAAATATTGGTTATATTGATTAAATTTACACTTAATGGAAACAACCTTTAACTTACAGATGAACAAACCCA CAAAAGCAAAAAAGCAAAAGCCCGACCTATGATTTCATATTTTCTGTGTAACTGGATTAAAGGATTCCTGCTTGCTTTTG GGCATAAATGATAATCGAATATTTCCAGGTATTGTTTAAAATGAGGGCCCATCTACAAATTCTTAGCAATACTTTGGATA ATTCTAAAATTCAGCTGGACATTGTCTAATTGTTTTTTATATACATCTTTGCTAGAATTTCAAATTTTAAGTATGTGAAT TTACTTAATTAGCTCTCCTGATCAATTCAAAAACATTACTTTCCTAAATTTTAGACTATGAAGGTCATAAATTCAACAAA TATATCTACACATACAATTATAGATTGTTTTTCATTATAATGTCTTCATCTTAACAGAATTGTCTTTGTGATTGTTTTTA GAAAACTGAGAGTTTTAATTCATAATTACCTTGATCAAAAAATTGTGGGAACAATCCAGCATTAATTGTATGTGATTGTT TTTATGTACATAAGCACTCTTAAGCTTGGTCCCTTGAACTCTTTTGTACTTAGTCCCATGTTTAAAATTACTACTTATA TCTAAAGCATTTATGTTTTTCAATTCAATTTACATCATCCTAATTATGGCAATTATAACAAATATTAAAGATTTCCAAAT AGAATATGTGAATTGTTCACCATACATAGAAATGAAAAGTTCATTTCGTAAAGCAAGATGCTGGGTGAAAGAGTGCTTTT GATTGAAAGATCACTAGATTAGTAGAGGGCAAGACTTTTAGTCCCTAATCTACCCTTAATAGCCATGTCGTCACGTGTAA CTCAGTGAACCCATCTCATTCTCCTCATACTTTTTTCATCTCTAAAATCACGCTATAATTTAAGCTCGTTCATTTTTTTT TTTTTTTGAGATAGAGTTTTGCTCTTGTCACCCAGGTTGGAGTGCAATGGCACGATCTCAGCTCACTCCAACCCTCTCCT TCCTCGGTTCAAGTGATTCTCCCTGCTTCACCCTCCCAAGTGAGCCCGGGATTACAGGTCCCCGCCACCACATCTGGGCC TAGATTTTTTGTATTTTCACCATGTTGGCCAGGCTGGTCTCGAACCCCTACCTCACGTGATCCCTCGCCTCGGCCTCTCA AAGTGCTGGGATTACAGGTGTGAGCCACCACGCCCACCCCAATATCAGTTTTTCTTTTTTAACACAAGGCTAACACAATC AAAATACTAGCTAGGGGAGAAAAAAAAAATAAGGCACTGTTTATGTGTAACAGGCTCTTGTTGCAATCCACTGGGGCAGA CCAAATAAACAGTAAGAATCAAATCCTTTTCATATAATCCTTTCTTTGCAGAATACATAAAATCCCCACAAATGGCTTAT CTTCCTTTTTATCATATGTTGGAGAATTGTAGCTAAGTGACAGATATTTTGCTTGGGTGTATAGACCACAAAGGACTGTG TCTTGATGATGGTTTGCATAAAATTATACCTTAGTTTTTACTTTGTATCTTACATGTTAGATTTAGAGTATGAAAATTAG TAGGGAGGATTATTAACAAAGAACAGGGCAAGAGGAGTAGAATTAAACCTCTTCTAATACCTGTGCACAAGTAGGCTTTT CAGAAACTCTACAACCCCAACATAAACTGGATAGTTAGAAAAGCACACTCCCAAGGAAGGCGGTTATGTTTTCCAGTTTG AATCAGAAGAATAGAGCTATAGCAATCTTCATTCTATAGTAACATTAAAGAGCCTGGTTTATATTATAGCAGTCATTAAG ATTTAAAAATTTACATCTTGCCGTTCTTCTTACTCACAGATTTTCGAGAGGTAATGTAATGATCACACGAGCTGAGAATC ACTGCCTTTTATAATGCGATTAAATGCATGAACAAAGTTTCCAACAAATAACAGTAATAAAAAGAAACATGTATTAGCAC TTAATAAGCCAGGTGCTGTACGACGTGTGTTACATGCTTTCAATCCATGAACTGGTAAACTGGTACTAGTATCTCTATTG GACATGTGAGGAAACCAAATGGAGTTGATAAACAGTAGAGTTAAAAATTACTCTTCATATATTATATTGCCTCAATCTCA CAGACATCTCTGCTACCAAAAGCTATCATATCTAGACTCCA FIG 14(cont'd-3) U.S. Patent Oct. 5, 2010 Sheet 19 of 21 US 7,807,373 B2

O -N1

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C U.S. Patent Oct. 5, 2010 Sheet 20 of 21 US 7,807,373 B2

TESTCODE OF: vsnucck: 6724, 1 to: 1588 WINDOW: 200 bp MARCH 14, 1999 20:25 CDO O. O. O. do did o do d o 0 O 9 O Cood oooh O d c o ce od O 0 o oxo cxdo o OOO of n odd

14 d to O. do X to ed do odo go o O OO o o odd o o

TESTCODE OF: humoctosk.gb-pr2 ck: 9544, 1 to: 1374 WINDOW: 200 bp MARCH 14, 1999 20:23 OO O o o o O O O O 9 14 - - - ''' o

O 500 1,000 FIG. 16B

US 7,807,373 B2 1. 2 PROSTATE SPECIFIC GENE, PCGEM1, AND layer, changes in the expression of differentiation markers of METHODS OF USING PCEGM1 TO DETECT, the prostatic secretory epithelial cells, nuclear and nucleolar TREAT, AND PREVENT PROSTATE CANCER abnormalities, increased cell proliferation, DNA content alterations, and chromosomal and allelic losses (8, 9). These CROSS REFERENCE TO RELATED 5 molecular and genetic biomarkers, particularly their progres APPLICATIONS sive gain or loss, can be followed to trace the etiology of prostate carcinogenesis. Foremost among these biomarkers This is a divisional of application Ser. No. 10/802,823, filed would be the molecular and genetic markers associated with Mar. 18, 2004 (abandoned), which is a continuation of appli histological phenotypes in transition between normal pros cation Ser. No. 09/534,072, filed Mar. 24, 2000 (issued as 10 tatic epithelium and cancer. Most studies So far seem to agree U.S. Pat. No. 6,828,429), which is based upon U.S. provi that PIN and prostatic adenocarcinoma cells have a lot in sional application Ser. No. 60/126,469, filed Mar. 26, 1999, common with each other. The invasive carcinoma more often priority to which is claimed under 35 U.S.C. S 119(e). The reflects a magnification of some of the events already mani entire disclosure of U.S. provisional application Ser. No. fest in PIN. 60/126,469 is expressly incorporated herein by reference. 15 Early detection of prostate cancer is possible today because of the widely propagated and recommended blood PSA test GOVERNMENT INTEREST that provides a warning signal for prostate cancer if high levels of serum PSA are detected. However, when used alone, The invention described herein may be manufactured, PSA is not sufficiently sensitive or specific to be considered licensed, and used for governmental purposes without pay- 20 an ideal tool for the early detection or staging of prostate ment of royalties to us thereon. cancer (10). Combining PSA levels with clinical staging and Gleason scores is more predictive of the pathological stage of FIELD OF THE INVENTION localized prostate cancer (11). In addition, new molecular techniques are being used for improved molecular staging of The present invention relates to nucleic acids that are 25 prostate cancer (12, 13). For instance, reverse transcriptase expressed in prostate tissue. More particularly, the present polymerase chain reaction (RT-PCR) can measure PSA of invention relates to the first of a family of novel, androgen circulating prostate cells in blood and bone marrow of pros regulated, prostate-specific genes, PCGEM1, that is over tate cancer patients. expressed in prostate cancer, and methods of using the Despite new molecular techniques, however, as many as 25 PCGEM1 sequence and fragments thereof to measure the 30 percent of men with prostate cancer will have normal PSA hormone responsiveness of prostate cancer cells and to levels—usually defined as those equal to or below 4 nano detect, diagnose, prevent and treat prostate cancer and other grams per milliliter of blood (14). In addition, more than 50 prostate related diseases. percent of the men with higher PSA levels are actually cancer free (14). Thus, PSA is not an ideal screening tool for prostate BACKGROUND 35 cancer. More reliable tumor-specific biomarkers are needed that can distinguish between normal and hyperplastic epithe Prostate cancer is the most common solid tumor in Ameri lium, and the preneoplastic and neoplastic stages of prostate can men (1). The wide spectrum of biologic behavior (2) CaCC. exhibited by prostatic neoplasms poses a difficult problem in Identification and characterization of genetic alterations predicting the clinical course for the individual patient (3,4). 40 defining prostate cancer onset and progression is important in Public awareness of prostate specific antigen (PSA) screen understanding the biology and clinical course of the disease. ing efforts has led to an increased diagnosis of prostate cancer. The currently available TNM staging system assigns the The increased diagnosis and greater number of patients pre original primary tumor (T) to one of four stages (14). The first senting with prostate cancer has resulted in wider use of stage, T1, indicates that the tumor is microscopic and cannot radical prostatectomy for localized disease (5). Accompany- 45 be felt on rectal examination. T2 refers to tumors that are ing the rise in Surgical intervention is the frustrating realiza palpable but fully contained within the prostate gland. A T3 tion of the inability to predict organ-confined disease and designation indicates the cancer has spread beyond the pros clinical outcome for a given patient (5, 6). Traditional prog tate into Surrounding connective tissue or has invaded the nostic markers. Such as grade, clinical stage, and pretreatment neighboring seminal vesicles. T4 cancer has spread even fur PSA have limited prognostic value for individual men. There 50 ther. The TNM staging system also assesses whether the is clearly a need to recognize and develop molecular and cancer has metastasized to the pelvic lymph nodes (N) or genetic biomarkers to improve prognostication and the man beyond (M). Metastatic tumors result when cancer cells break agement of patients with clinically localized prostate cancer. away from the original tumor, circulate through the blood or As with other common human neoplasia (7), the search for lymph, and proliferate at distant sites in the body. molecular and genetic biomarkers to better define the genesis 55 Recent studies of metastatic prostate cancer have shown a and progression of prostate cancer is the key focus for cancer significant heterogeneity of allelic losses of different chro research investigations worldwide. mosome regions between multiple cancer foci (21-23). These The new wave of research addressing molecular genetic studies have also documented that the metastatic lesion can alterations in prostate cancer is primarily due to increased arise from cancer foci other than dominant tumors (22). awareness of this disease and the development of newer 60 Therefore, it is critical to understand the molecular changes molecular technologies. The search for the precursor of pro which define the prostate cancer metastasis especially when static adenocarcinoma has focused largely on the spectrum of prostate cancer is increasingly detected in early stages (15 microscopic changes referred to as “prostatic intraepithelial 21). neoplasia” (PIN). Bostwick defines this spectrum as a histo Moreover, the multifocal nature of prostate cancer needs to pathologic continuum that culminates in high grade PIN and 65 be considered (22-23) when analyzing biomarkers that may early invasive cancer (8). The morphologic and molecular have potential to predict tumor progression or metastasis. changes include the progressive disruption of the basal cell Approximately 50-60% of patients treated with radical pros US 7,807,373 B2 3 4 tatectomy for localized prostate carcinomas are found to have moting cell growth. The invention provides the isolated microscopic disease that is not organ confined, and a signifi nucleotide sequence of PCGEM1 or fragments thereof and cant portion of these patients relapse (24). Utilizing biostatis nucleic acid sequences that hybridize to PCGEM1. These tical modeling of traditional and genetic biomarkers such as sequences have utility, for example, as markers of prostate p53 and bcl-2. Bauer et al. (25-26) were able to identify cancer and other prostate related diseases, and as targets for patients at risk of cancer recurrence after Surgery. Thus, there therapeutic intervention in prostate cancer and other prostate is clearly a need to develop biomarkers defining various related diseases. The invention further provides a vector that stages of the prostate cancer progression. directs the expression of PCGEM1, and a host cell transfected Another significant aspect of prostate cancer is the key role or transduced with this vector. that androgens play in the development of both the normal 10 In another embodiment, the invention provides a method of prostate and prostate cancer. Androgen ablation, also referred detecting prostate cancer cells in a biological sample, for to as "hormonal therapy,” is a common treatment for prostate example, by using nucleic acid amplification techniques with cancer, particularly in patients with metastatic disease (14). primers and probes selected to bind specifically to the Hormonal therapy aims to inhibit the body from making PCGEM1 sequence. The invention further comprises a androgens or to block the activity of androgen. One way to 15 method of selectively killing a prostate cancer cell, a method block androgen activity involves blocking the androgen of identifying an androgen responsive cell line, and a method receptor; however, that blockage is often only successful of measuring responsiveness of a cell line to hormone-abla initially. For example, 70-80% of patients with advanced tion therapy. disease exhibit an initial Subjective response to hormonal In another aspect, the invention relates to an isolated therapy, but most tumors progress to an androgen-indepen polypeptide encoded by the PCGEM1 gene or a fragment dent state within two years (16). One mechanism proposed thereof, and antibodies generated against the PCGEM1 for the progression to an androgen-independent state involves polypeptide, peptides, or portions thereof, which can be used constitutive activation of the androgen signaling pathway, to detect, treat, and prevent prostate cancer. which could arise from structural changes in the androgen Additional features and advantages of the invention will be receptor protein (16). 25 set forth in the description which follows, and in part will be As indicated above, the genesis and progression of cancer apparent from the description, or may be learned by practice cells involve multiple genetic alterations as well as a complex of the invention. The objectives and other advantages of the interaction of several gene products. Thus, various strategies invention will be realized and attained by the sequences, cells, are required to fully understand the molecular genetic alter vectors, and methods particularly pointed out in the written ations in a specific type of cancer. In the past, most molecular 30 description and claims herein as well as the appended draw biology studies had focused on mutations of cellular proto 1ngS. oncogenes and tumor suppressor genes (TSGs) associated with prostate cancer (7). Recently, however, there has been an BRIEF DESCRIPTION OF THE DRAWINGS increasing shift toward the analysis of “expression genetics' in human cancer (27-31), i.e., the under-expression or over 35 FIG. 1 depicts the scheme for the identification of differ expression of cancer-specific genes. This shift addresses limi entially expressed genes in prostate tumor and normal tissues. tations of the previous approaches including: 1) labor inten FIG. 2 depicts a differential display pattern of mRNA sive technology involved in identifying mutated genes that obtained from matched tumor and normal tissues of a prostate are associated with human cancer; 2) the limitations of cancer patient. Arrows indicate differentially expressed experimental models with a bias toward identification of only 40 cDNAS. certain classes of genes, e.g., identification of mutant ras FIG. 3 depicts the analysis of PCGEM1 expression in genes by transfection of human tumor DNAS utilizing primary prostate cancers. NIH3T3 cells; and 3) the recognition that the human cancer FIG. 4 depicts the expression pattern of PCGEM1 in pros associated genes identified so far do not account for the diver tate cancer cell lines. sity of cancer phenotypes. 45 FIG. 5a depicts the androgen regulation of PCGEM1 A number of studies are now addressing the alterations of expression in LNCaP cells, as measured by reverse tran prostate cancer-associated gene expression in patient speci scriptase PCR. mens (32-36). It is inevitable that more reports on these lines FIG. 5b depicts the androgen regulation of PCGEM1 are to follow. expression in LNCaP cells, as measured by Northern blot Thus, despite the growing body of knowledge regarding 50 hybridization. prostate cancer, there is still a need in the art to uncover the FIG. 6a depicts the prostate tissue specific expression pat identity and function of the genes involved in prostate cancer tern of PCGEM1. pathogenesis. There is also a need for reagents and assays to FIG. 6b depicts a RNA master blot showing the prostate accurately detect cancerous cells, to define various stages of tissue specificity of PCGEM1. prostate cancer progression, to identify and characterize 55 genetic alterations defining prostate cancer onset and pro FIG. 7A depicts the chromosomal localization of gression, to detect micro-metastasis of prostate cancer, and to PCGEM1 by fluorescent in situ hybridization analysis. treat and prevent prostate cancer. FIG. 7B depicts a DAPI counter-stained chromosome 2 (left), an inverted DAPI stained chromosome 2 shown as SUMMARY OF THE INVENTION 60 G-bands (center), and an ideogram of chromosome 2 showing the localization of the signal to band 2d32(bar). The present invention relates to the identification and char FIG. 8 depicts a cDNA sequence of PCGEM1 (SEQ ID acterization of a novel gene, the first of a family of genes, NO:1). designated PCGEM1, for Prostate Cancer Gene Expression FIG.9 depicts an additional cDNA sequence of PCGEM1 Marker 1. PCGEM1 is specific to prostate tissue, is androgen 65 (SEQID NO:2). regulated, and appears to be over-expressed in prostate can FIG. 10 depicts the colony formation of NIH3T3 cell lines cer. More recent studies associate PCGEM1 cDNA with pro expressing various PCGEM1 constructs. US 7,807,373 B2 5 6 FIG. 11 depicts the cDNA sequence of the promoter region Preferred Sequences of PCGEM1 SEQID NO:3. Particularly preferred nucleotide sequences of the inven FIG. 12 depicts the cDNA of a probe, designated SEQID tion are SEQID NO:1, SEQID NO:2, and SEQID NO: 8, as NO:4. set forth in FIGS. 8,9, and 14, respectively. Two cloNA clones FIG. 13 depicts the cDNAs of primers 1-3, designated SEQ having the nucleotide sequences of SEQID NO:1 and SEQ ID NOS:5-7, respectively. ID NO:2, and the genomic DNA having the nucleotide FIG. 14 depicts the genomic DNA sequence of PCGEM1, sequence of SEQ ID NO: 8, were isolated as described in designated SEQID NO:8. Example 2. FIG. 15 depicts the structure of the PCGEM1 transcription Thus, in a particular embodiment, this invention provides unit. 10 an isolated nucleic acid molecule selected from the group FIG. 16 depicts a graph of the hypothetical coding capacity consisting of (a) the polynucleotide sequence of SEQ ID of PCGEM1. NO:1, SEQ ID NO:2, or SEQ ID NO: 8; (b) an isolated FIG. 17 depicts a representative example of in situ hybrid nucleic acid molecule that hybridizes to either strand of a ization results showing PCGEM1 expression in normal and denatured, double-stranded DNA comprising the nucleic acid tumor areas of prostate cancer tissues. 15 sequence of (a) under conditions of moderate stringency in 50% formamide and about 6xSSC at about 42°C. with wash DETAILED DESCRIPTION OF THE INVENTION ing conditions of approximately 60° C., about 0.5xSSC, and about 0.1% SDS; (c) an isolated nucleic acid molecule that The present invention relates to PCGEM1, the first of a hybridizes to either strand of a denatured, double-stranded family of genes, and its related nucleic acids, proteins, anti DNA comprising the nucleic acid sequence of (a) under con gens, and antibodies for use in the detection, prevention, and ditions of high stringency in 50% formamide and about treatment of prostate cancer (e.g., prostatic intraepithelial 6xSSC, with washing conditions of approximately 68°C., neoplasia (PIN), adenocarcinomas, nodular hyperplasia, and about 0.2xSSC, and about 0.1% SDS; (d) an isolated nucleic large duct carcinomas) and prostate related diseases (e.g., acid molecule derived by in vitro mutagenesis from SEQID benign prostatic hyperplasia), and kits comprising these 25 NO:1, SEQID NO:2, or SEQID NO:8; (e)anisolated nucleic reagents. acid molecule degenerate from SEQID NO:1, SEQID NO:2, Although we do not wish to be limited by any theory or or SEQ ID NO:8 as a result of the genetic code; and (f) an hypothesis, preliminary data suggest that the PCGEM1 isolated nucleic acid molecule selected from the group con nucleotide sequence may be related to a family of non-coding sisting of human PCGEM1 DNA, an allelic variant of human poly A+RNA that may be implicated in processes relating to 30 PCGEM1 DNA, and a species homolog of PCGEM1 DNA. growth and embryonic development (40-44). Evidence pre As used herein, conditions of moderate stringency can be sented herein Supports this hypothesis. Alternatively, readily determined by those having ordinary skill in the art PCGEM1 cDNA may encode a small peptide. based on, for example, the length of the DNA. The basic conditions are set forth by Sambrook et al. Molecular Clon Nucleic Acid Molecules 35 ing: A Laboratory Manual, 2d ed. Vol. 1, pp. 1.101-104, Cold In a particular embodiment, the invention relates to certain Spring Harbor Laboratory Press, (1989), and include use of a isolated nucleotide sequences that are substantially free from prewashing solution for the nitrocellulose filters of about contaminating endogenous material. A “nucleotide 5xSSC, about 0.5% SDS, and about 1.0 mM EDTA (pH 8.0), sequence” refers to a polynucleotide molecule in the form of hybridization conditions of about 50% formamide, about a separate fragment or as a component of a larger nucleic acid 40 6xSSC at about 42'C (or other similar hybridization solution, construct. The nucleic acid molecule has been derived from such as Stark's solution, in about 50% formamide at about DNA or RNA isolated at least once in substantially pure form 42°C.), and washing conditions of about 60°C., about 0.5x and in a quantity or concentration enabling identification, SSC, and about 0.1% SDS. Conditions of high stringency can manipulation, and recovery of its component nucleotide also be readily determined by the skilled artisan based on, for sequences by Standard biochemical methods (such as those 45 example, the length of the DNA. Generally, such conditions outlined in Sambrook et al., Molecular Cloning: A Labora are defined as hybridization conditions as above, and with tory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold washing at approximately 68°C., about 0.2xSSC, and about Spring Harbor, N.Y. (1989)). 0.1% SDS. The skilled artisan will recognize that the tem Nucleic acid molecules of the invention include DNA in perature and wash solution salt concentration can be adjusted both single-stranded and double-stranded form, as well as the 50 as necessary according to factors such as the length of the RNA complement thereof DNA includes, for example, probe. cDNA, genomic DNA, chemically synthesized DNA, DNA Additional Sequences amplified by PCR, and combinations thereof. Genomic DNA Due to the known degeneracy of the genetic code, wherein may be isolated by conventional techniques, e.g., using the more than one codon can encode the same amino acid, a DNA cDNA of SEQID NO:1, SEQID NO:2, or suitable fragments 55 sequence can vary from that shown in SEQID NO:1, SEQID thereof, as a probe. NO:2, or SEQ ID NO:8, and still encode PCGEM1. Such The DNA molecules of the invention include full length variant DNA sequences can result from silent mutations (e.g., genes as well as polynucleotides and fragments thereof. The occurring during PCR amplification), or can be the product of full length gene may include the N-terminal signal peptide. deliberate mutagenesis of a native sequence. Although a non-coding role of PCGEM1 appears likely, the 60 The invention thus provides isolated DNA sequences of the possibility of a protein product cannot presently be ruled out. invention selected from: (a) DNA comprising the nucleotide Therefore, other embodiments may include DNA encoding a sequence of SEQID NO:1, SEQID NO:2, or SEQID NO:8: soluble form, e.g., encoding the extracellular domain of the (b) DNA capable of hybridization to a DNA of (a) under protein, either with or without the signal peptide. conditions of moderate stringency; (c) DNA capable of The nucleic acids of the invention are preferentially 65 hybridization to a DNA of (a) under conditions of high strin derived from human sources, but the invention includes those gency; and (d) DNA which is degenerate as a result of the derived from non-human species, as well. genetic code to a DNA defined in (a), (b), or (c). Such US 7,807,373 B2 7 8 sequences are preferably provided and/or constructed in the Use of PCGEM1 Nucleic Acid or Oligonucleotides form of an open reading frame uninterrupted by internal In a particular embodiment, the invention relates to non-translated sequences, or introns, that are typically PCGEM1 nucleotide sequences isolated from human pros present in eukaryotic genes. Sequences of non-translated tate cells, including the complete genomic DNA (FIG. 14, DNA can be present 5' or 3' from an open reading frame, 5 SEQ ID NO: 8), and two full length cDNAs. SEQID NO:1 where the same do not interfere with manipulation or expres (FIG. 8) and SEQID NO:2 (FIG. 9), and fragments thereof. sion of the coding region. Ofcourse, should PCGEM1 encode The nucleic acids of the invention, including DNA, RNA, a polypeptide, polypeptides encoded by Such DNA sequences mRNA and oligonucleotides thereof, are useful in a variety of are encompassed by the invention. Conditions of moderate applications in the detection, diagnosis, prognosis, and treat and high Stringency are described above. 10 ment of prostate cancer. Examples of applications within the In another embodiment, the nucleic acid molecules of the scope of the present invention include, but are not limited to: invention comprise nucleotide sequences that are at least 80% amplifying PCGEM1 sequences: identical to a nucleotide sequence set forth herein. Also con detecting a PCGEM1-derived marker of prostate cancer by templated are embodiments in which a nucleic acid molecule hybridization with an oligonucleotide probe; comprises a sequence that is at least 90% identical, at least 15 identifying chromosome 2: 95% identical, at least 98% identical, at least 99% identical, mapping genes to chromosome 2: or at least 99.9% identical to a nucleotide sequence set forth identifying genes associated with certain diseases, syn herein. dromes, or other conditions associated with human Percent identity may be determined by visual inspection chromosome 2: and mathematical calculation. Alternatively, percent identity 20 constructing vectors having PCGEM1 sequences; of two nucleic acid sequences may be determined by com expressing vector-associated PCGEM1 sequences as RNA paring sequence information using the GAP computer pro and protein; gram, version 6.0 described by Devereux et al. (Nucl. Acids detecting defective genes in an individual; Res. 12:387, 1984) and available from the University of Wis developing gene therapy; consin Genetics Computer Group (UWGCG). The preferred 25 developing immunologic reagents corresponding to default parameters for the GAP program include: (1) a unary PCGEM1-encoded products; and comparison matrix (containing a value of 1 for identities and treating prostate cancer using antibodies, antisense nucleic 0 for non-identities) for nucleotides, and the weighted com acids, or other inhibitors specific for PCGEM1 parison matrix of Gribskov and Burgess, Nucl. Acids Res. Sequences. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., 30 Detecting, Diagnosing, and Treating Prostate Cancer Atlas of Protein Sequence and Structure, National Biomedi The present invention provides a method of detecting pros cal Research Foundation, pp. 353–358, 1979; (2) a penalty of tate cancer in a patient, which comprises (a) detecting 3.0 for each gap and an additional 0.10 penalty for each PCGEM1 mRNA in a biological sample from the patient; and symbol in each gap; and (3) no penalty for end gaps. Other (b) correlating the amount of PCGEM1 mRNA in the sample programs used by one skilled in the art of sequence compari- 35 with the presence of prostate cancer in the patient. Detecting son may also be used. PCGEM1 mRNA in a biological sample may include: (a) The invention also provides isolated nucleic acids useful in isolating RNA from said biological sample; (b) amplifying a the production of polypeptides. Such polypeptides may be PCGEM1 cDNA molecule; (c) incubating the PCGEM1 prepared by any of a number of conventional techniques. A cDNA with the isolated nucleic acid of the invention; and (d) DNA sequence of this invention or desired fragment thereof 40 detecting hybridization between the PCGEM1 cDNA and the may be subcloned into an expression vector for production of isolated nucleic acid. The biological sample can be selected the polypeptide or fragment. The DNA sequence advanta from the group consisting of blood, urine, and tissue, for geously is fused to a sequence encoding a suitable leader or example, from a biopsy. In a preferred embodiment, the bio signal peptide. Alternatively, the desired fragment may be logical sample is blood. This method is useful in both the chemically synthesized using known techniques. DNA frag- 45 initial diagnosis of prostate cancer, and the later prognosis of ments also may be produced by restriction endonuclease disease. This method allows for testing prostate tissue in a digestion of a full length cloned DNA sequence, and isolated biopsy, and after removal of a cancerous prostate, continued by electrophoresis on agarose gels. If necessary, oligonucle monitoring of the blood for micrometastases. otides that reconstruct the 5' or 3' terminus to a desired point According to this method of diagnosing and prognosticat may be ligated to a DNA fragment generated by restriction 50 ing prostate cancer in a patient, the amount of PCGEM1 enzyme digestion. Such oligonucleotides may additionally mRNA in a biological sample from a patient is correlated with contain a restriction endonuclease cleavage site upstream of the presence of prostate cancer in the patient. Those of ordi the desired coding sequence, and position an initiation codon nary skill in the art can readily assess the level of over (ATG) at the N-terminus of the coding sequence. expression that is correlated with the presence of prostate The well-known polymerase chain reaction (PCR) proce- 55 CaCC. dure also may be employed to isolate and amplify a DNA In another embodiment, this invention provides a vector, sequence encoding a desired protein fragment. Oligonucle comprising a PCGEM1 promoter sequence operatively otides that define the desired termini of the DNA fragment are linked to a nucleotide sequence encoding a cytotoxic protein. employed as 5' and 3' primers. The oligonucleotides may The invention further provides a method of selectively killing additionally contain recognition sites for restriction endonu- 60 a prostate cancer cell, which comprises introducing the vector cleases, to facilitate insertion of the amplified DNA fragment to prostate cancer cells under conditions Sufficient to permit into an expression vector. PCR techniques are described in selective killing of the prostate cells. As used herein, the Saiki et al., Science 239:487 (1988); Recombinant DNA phrase “selective killing' is meant to include the killing of at Methodology, Wu et al., eds. Academic Press, Inc., San least a cell which is specifically targeted by a nucleotide Diego (1989), pp. 189-196; and PCR Protocols: A Guide to 65 sequence. The putative PCGEM1 promoter, contained in the Methods and Applications, Innis et al., eds. Academic Press, 5' flanking region of the PCGEM1 genomic sequence, SEQ Inc. (1990). ID NO: 3, is set forth in FIG. 11. Applicants envision that a US 7,807,373 B2 9 10 nucleotide sequence encoding any cytotoxic protein can be ID NO: 1) and comprises nucleotides 116 to 1140 of that incorporated into this vector for delivery to prostate tissue. sequence. It has been designated SEQ ID NO. 4 and is set For example, the cytotoxic protein can be ricin, abrin, diph forth in FIG. 12. theria toxin, p53, thymidine kinase, tumor necrosis factor, When a hybridization probe binds to a target sequence, it cholera toxin, Pseudomonas aeruginosa exotoxin A, riboso forms a duplex molecule that is both stable and selective. mal inactivating proteins, or mycotoxins such as trichoth These nucleic acid molecules may be readily prepared, for ecenes, and derivatives and fragments (e.g., single chains) example, by chemical synthesis or by recombinant tech thereof. niques. A wide variety of methods are known in the art for This invention also provides a method of identifying an detecting hybridization, including fluorescent, radioactive, or androgen-responsive cell line, which comprises (a) obtaining 10 enzymatic means, or other ligands such as avidin/biotin. a cell line Suspected of being androgen-responsive, (b) incu In another aspect of the invention, these nucleic acid mol bating the cell line with an androgen; and (c) detecting ecules may be introduced into a recombinant vector. Such as PCGEM1 mRNA in the cell line, wherein an increase in a plasmid, cosmid, or virus, which can be used to transfect or PCGEM1 mRNA, as compared to an untreated cell line, transduce a host cell. The nucleic acids of the present inven correlates with the cell line being androgen-responsive. 15 tion may be combined with other DNA sequences, such as The invention further provides a method of measuring the promoters, polyadenylation signals, restriction enzyme sites, responsiveness of a prostatic tissue to hormone-ablation multiple cloning sites, and other coding sequences. therapy, which comprises (a) treating the prostatic tissue with Because homologs of SEQID NO: 1, SEQID NO: 2, and hormone-ablation therapy; and (b) measuring PCGEM1 SEQ ID NO: 8 from other mammalian species are contem mRNA in the prostatic tissue following hormone-ablation plated herein, probes based on the human DNA sequence of therapy, wherein a decrease in PCGEM1 mRNA, as com SEQID NO: 1, SEQID NO: 2, and SEQID NO: 8 may be pared to an untreated cell line, correlates with the cell line used to screen cDNA libraries derived from other mammalian responding to hormone-ablation therapy. species, using conventional cross-species hybridization tech In another aspect of the invention, these nucleic acid mol niques. ecules may be introduced into a recombinant vector. Such as 25 In another aspect of the invention, one can use the knowl a plasmid, cosmid, or virus, which can be used to transfect or edge of the genetic code in combination with the sequences transduce a host cell. The nucleic acids of the present inven set forth herein to prepare sets of degenerate oligonucle tion may be combined with other DNA sequences, such as otides. Such oligonucleotides are useful as primers, e.g., in promoters, polyadenylation signals, restriction enzyme sites, polymerase chain reactions (PCR), whereby DNA fragments multiple cloning sites, and other coding sequences. 30 are isolated and amplified. Particularly preferred primers are Probes set forth in FIG. 13 and Table I and are designated SEQ ID Among the uses of nucleic acids of the invention is the use NOS: 5-7 and 9-22, respectively. A particularly preferred of fragments as probes or primers. Such fragments generally primer pair is p518 (SEQID NO: 15) and p839 (SEQID NO: comprise at least about 17 contiguous nucleotides of a DNA 22), which when used in PCR, preferentially amplifies sequence. The fragment may have fewer than 17 nucleotides, 35 mRNA, thereby avoiding less desirable cross-reactivity with such as, for example, 10 or 15 nucleotides. In other embodi genomic DNA. ments, a DNA fragment comprises at least 20, at least 30, or Chromosome Mapping at least 60 contiguous nucleotides of a DNA sequence. As set forth in Example 3, the PCGEM1 gene has been Examples of probes or primers of the invention include those mapped by fluorescent in situ hybridization to the 232 region of SEQID NO: 5, SEQID NO: 6, and SEQID NO: 7, as well 40 of chromosome 2 using a bacterial artificial chromosome as those disclosed in Table I. (BAC) clone containing PCGEU1 genomic sequence. Thus, TABLE I Starting Primer Sequence (5'-->3') SAS Base # SEQID NO. 413 TGGCA ACAGGCAAGCAGAG S 510 DNO: 9 b414 GGCCAAAATAAAACCAAACAT AS 610 DNO: 10 489 GCAAATATGATTTAAAGATACAAC S 752 DNO: 11 3490 GGTTGTATCTTTAAATCATATTTGC AS 776 DNO: 12 3491 ACTGTCTTTTCATATATTTCTCAATGC S 559 DNO: 13 b517 AAGTAGTAATTTTAAACATGGGAC AS 1516 DNO: 14 518 TTTTTCAATTAGGCAGCA ACC S 131 DNO: 15 519 GAATTGTCTTTGTGATTGTTTTTAG S 1338 DNO: 16 S60 CAATTCACAAAGACAATTCAGTTAAG AS 1355 DNO: 17 b561 ACAATTAGACAATGTCCAGCTGA AS 1154 DNO: 18 562 CTTTGGCTGATATCATGAAGTGTC AS 322 DNO: 19 623 AACCTTTTGCCCTATGCCGTAAC S 148 DNO: 20 624 GAGACTCCCAACCTGATGATGT AS 376 DNO: 21 839 GGTCACGTTGAGTCCCAGTG AS 270 DNO: 22 SAS indicates whether the primer is Sense or AntiSense Starting Base # indicates the starting base number with respect to the sequence of SEQID O: 1.

S/AS indicates whether the primer is Sense or AntiSense all or a portion of the nucleic acid molecule of SEQID NO:1, Starting Base it indicates the starting base number with SEQ ID NO:2, and SEQ ID NO:8, including oligonucle respect to the sequence of SEQID NO:1. otides, can be used by those skilled in the art using well 65 known techniques to identify human chromosome 2, and the However, even larger probes may be used. For example, a specific locus thereof, that contains the PCGEM1 DNA. Use particularly preferred probe is derived from PCGEM1 (SEQ ful techniques include, but are not limited to, using the nucle US 7,807,373 B2 11 12 otide sequence of SEQ ID NO:1, SEQ ID NO:2, or SEID nucleotides having modified Sugar-phosphodiester back NO:8, or fragments thereof, including oligonucleotides, as a bones (or other Sugar linkages. Such as those described in probe in various well-known techniques such as radiation WO91/06629) and wherein such sugar linkages are resistant hybrid mapping (high resolution), in situ hybridization to to endogenous nucleases. Such oligonucleotides with resis chromosome spreads (moderate resolution), and Southern tant Sugar linkages are stable in vivo (i.e., capable of resisting blot hybridization to hybrid cell lines containing individual enzymatic degradation) but retain sequence specificity to be human chromosomes (low resolution). able to bind to target nucleotide sequences. For example, chromosomes can be mapped by radiation Other examples of sense or antisense oligonucleotides hybridization. First, PCR is performed using the Whitehead include those oligonucleotides which are covalently linked to Institute/MIT Center for Genome Research Genebridge4 10 organic moieties, such as those described in WO 90/10448, panel of 93 radiation hybrids. Primers are used which lie and other moieties that increases affinity of the oligonucle within a putative exon of the gene of interest and which otide for a target nucleic acid sequence, Such as poly-(L- amplify a product from human genomic DNA, but do not lysine). Further still, intercalating agents, such as ellipticine, amplify hamster genomic DNA. The results of the PCRs are and alkylating agents or metal complexes may be attached to converted into a data vector that is submitted to the White 15 sense or antisense oligonucleotides. Such modifications may head/MIT Radiation Mapping site on the internet. The data is modify binding specificities of the antisense or sense oligo scored and the chromosomal assignment and placement rela nucleotide for the target nucleotide sequence. tive to known Sequence Tag Site (STS) markers on the radia Antisense or sense oligonucleotides may be introduced tion hybrid map is provided. into a cell containing the target nucleic acid sequence by any Identifying Associated Diseases gene transfer method, including, for example, lipofection, As noted above, PCGEM1 has been mapped to the 2d 32 CaPO-mediated DNA transfection, electroporation, or by region of chromosome 2. This region is associated with spe using gene transfer vectors such as Epstein-Barr virus or cific diseases, which include but are not limited to diabetes adenovirus. mellitus (insulin dependent), and T cell leukemia/lymphoma. Sense or antisense oligonucleotides also may be intro Thus, the nucleic acids of SEQID NO: 1, SEQID NO: 2, or 25 duced into a cell containing the target nucleotide sequence by SEQ ID NO:8, or fragments thereof, can be used by one formation of a conjugate with a ligand binding molecule, as skilled in the art using well-known techniques to analyze described in WO 91/04753. Suitable ligand binding mol abnormalities associated with gene mapping to chromosome ecules include, but are not limited to, cell Surface receptors, 2. This enables one to distinguish conditions in which this growth factors, other cytokines, or other ligands that bind to marker is rearranged or deleted. In addition, nucleotides of 30 cell Surface receptors. Preferably, conjugation of the ligand SEQID NO:1, SEQID NO:2, or SEQID NO:8, or fragments binding molecule does not substantially interfere with the thereof, can be used as a positional marker to map other genes ability of the ligand binding molecule to bind to its corre of unknown location. sponding molecule or receptor, or block entry of the sense or The DNA may be used in developing treatments for any antisense oligonucleotide or its conjugated version into the disorder mediated (directly or indirectly) by defective, or 35 cell. insufficient amounts of PCGEM1, including prostate cancer. Alternatively, a sense or an antisense oligonucleotide may Disclosure herein of native nucleotide sequences permits the be introduced into a cell containing the target nucleic acid detection of defective genes, and the replacement thereof sequence by formation of an oligonucleotide-lipid complex, with normal genes. Defective genes may be detected in in as described in WO 90/10448. The sense or antisense oligo vitro diagnostic assays, and by comparison of a native nucle 40 nucleotide-lipid complex is preferably dissociated within the otide sequence disclosed herein with that of a gene derived cell by an endogenous lipase. from a person Suspected of harboring a defect in this gene. Sense-Antisense Polypeptides and Fragments. Thereof Other useful fragments of the nucleic acids include anti The invention also encompasses polypeptides and frag sense or sense oligonucleotides comprising a single-stranded 45 ments thereof in various forms, including those that are natu nucleic acid sequence (either RNA or DNA) capable of bind rally occurring or produced through various techniques such ing to target mRNA (sense) or DNA (antisense) sequences. as procedures involving recombinant DNA technology. Such Antisense or sense oligonucleotides, according to the present forms include, but are not limited to, derivatives, variants, and invention, comprise a fragment of DNA (SEQID NO:1, SEQ oligomers, as well as fusion proteins or fragments thereof. ID NO:2, or SEQID NO:8). Such a fragment generally com 50 The polypeptides of the invention include full length pro prises at least about 14 nucleotides, preferably from about 14 teins encoded by the nucleic acid sequences set forth above. to about 30 nucleotides. The ability to derive an antisense or The polypeptides of the invention may be membrane bound a sense oligonucleotide, based upon a cDNA sequence encod or they may be secreted and thus soluble. The invention also ing a given protein is described in, for example, Stein and includes the expression, isolation and purification of the Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. 55 polypeptides and fragments of the invention, accomplished (BioTechniques 6:958, 1988). by any Suitable technique. The biologic activity of PCGEM1 in assay cells and the The following examples further illustrate preferred aspects over expression of PCGEM1 in prostate cancer tissues sug of the invention. gest that elevated levels of PCGEM1 promote prostate cancer cell growth. Thus, the antisense oligonucleotides to PCGEM1 60 EXAMPLE 1. may be used to reduce the expression of PCGEM1 and, con sequently, inhibit the growth of the cancer cells. Differential Gene Expression Analysis in Prostate Binding of antisense or sense oligonucleotides to target Cancer nucleic acid sequences results in the formation of duplexes. The antisense oligonucleotides thus may be used to block 65 Using the differential display technique, we identified a expression of proteins or to inhibit the function of RNA. novel gene that is over-expressed in prostate cancer cells. Antisense or sense oligonucleotides further comprise oligo Differential display provides a method to separate and clone US 7,807,373 B2 13 14 individual messenger RNAs by means of the polymerase nificant homology with sequences in the publicly available chain reaction, as described in Liang et al., Science, 257:967 databases. Later searching of the high throughput genome 71 (1992), which is hereby incorporated by reference. Briefly, sequence (HTGS) database revealed perfect homology to a the method entails using two groups of oligonucleotide prim chromosome 2 derived uncharacterized, unfinished genomic ers. One group is designed to recognize the polyadenylate tail sequence (accession it AC 013401). of messenger RNAS. The other group contains primers that are short and arbitrary in sequence and anneal to positions in EXAMPLE 2 the messenger RNA randomly distributed from the polyade Characterization of Full Length PCGEM1 cDNA nylate tail. Products amplified with these primers can be Sequence differentiated on a sequencing gel based on their size. If 10 different cell populations are amplified with the same groups of primers, one can compare the amplification products to The full length of PCGEM1 was obtained by 5' and 3 identify differentially expressed RNA sequences. RACE/PCR from the original 530 by DD product (nucle otides 410 to 940 of PCGEM1 cDNA SEQID NO: 1) using a Differential display (“DD) kits from Genomyx (Foster normal prostate cDNA library in lambda phage (Clontech, City, Calif.) were used to analyze differential gene expres 15 CA). The RACE/PCR products were directly sequenced. Sion. The steps of the differential display technique are sum Lasergene and MacVector DNA analysis software were used marized in FIG.1. Histologically well defined matched tumor to analyze DNA sequences and to define open reading frame and normal prostate tissue sections containing approximately regions. We also used the original DD product to screen a similar proportions of epithelial cells were chosen from indi normal prostate cDNA library. Three overlapping cDNA vidual prostate cancer patients. clones were identified. Genomic DNA-free total RNA was extracted from this Sequencing of the cDNA clones was performed on an enriched pool of cells using RNAZolTM B (Tel-Test, Inc., ABI-310 sequence analyzer and a new dRhodamine cycle Friendswood, Tex.) according to manufacturer's protocol. sequencing kit (PE-Applied BioSystem, CA). The longest The epithelial nature of the RNA source was further con PCGEM1 cDNA clone, SEQ ID NO:1 (FIG. 8), revealed firmed using cytokeratin 18 expression (45) in reverse tran 25 1643 nucleotides with a potential polyadenylation site, Scriptase-polymerase chain reaction (RT-PCR) assays. Using ATTAAA, close to the 3' end followed by a poly(A)tail. As arbitrary and anchored primers containing 5' M13 or T7 noted above, although initial searching of PCGEM1 gene in sequences (obtained from Biomedical Instrumentation Cen publically available DNA databases (e.g., National Center for ter, Uniformed Services University of the Health Sciences, Biotechnology Information) using the BLAST program did Bethesda), the isolated DNA-free total RNA was amplified by 30 RT-PCR which was performed using ten anchored antisense not reveal any homology, a recent search of the HTGS data primers and four arbitrary sense primers according to the base revealed perfect homology of PCGEM1 (using cDNA of protocol provided by HieroglyphTM RNA Profile Kit 1 SEQID NO: 1) to a chromosome 2 derived uncharacterized, (Genomyx Corporation, CA). The cDNA fragments pro unfinished genomic sequence (accession it AC 013401). One of the cDNA clones, SEQID NO:2 (FIG.9), contained a 123 duced by the RT-PCR assay were analyzed by high resolution 35 by insertion at 278, and this inserted sequence showed strong gel electrophoresis, carried out by using GenomyxTM LR homology (87%) to Alu sequence. It is likely that this clone DNA sequencer and LR-OptimizedTMHR-1000TM gel formu represented the premature transcripts. Sequencing of several lations (GenomyX Corporation, CA). clones from RT-PCR further confirmed the presence of the A partial DD screening of normal/tumor tissues revealed two forms of transcripts. 30 differentially expressed cDNA fragments, with 53% 40 Sequence analysis did not reveal any significant long open showing reduced or no expression in tumor RNA specimens reading frame in both strands. The longest ORF in the sense and 47% showing over expression in tumor RNA specimen strand was 105 nucleotides (572-679) encoding 35 amino (FIG. 2). These cDNAs were excised from the DD gels, acid peptides. However, the ATG was not in a strong context reamplified using T7 and M13 primers and the RT PCR of initiation. Although we could not rule out the coding capac conditions recommended in HieroglyphTM RNA Profile Kit-1 45 ity for a very small peptide, it is possible that PCGEM1 may (Genomyx Corp., CA), and sequenced. The inclusion of T7 function as a non-coding RNA. and M13 sequencing primers in the DD primers allowed rapid The sequence of PCGEM1 cDNA has been verified by sequencing and orientation of cDNAs (FIG. 1). several approaches including characterization of several All the reamplified cDNA fragments were purified by Cen clones of PCGEM1 and analysis of PCGEM1 cDNAs ampli triconTM-c-100 system (Amicon, USA). The purified frag 50 fied from normal prostate tissue and prostate cancer cell lines. ments were sequenced by cycle sequencing and DNA We have also obtained the genomic clones of PCGEM1, sequence determination using an ABI 377 DNA sequencer. which has helped to confirm the PCGEM1 cDNA sequence. Isolated sequences were analyzed for sequence homology The complete genomic DNA sequence of PCGEM1 (SEQID with known sequences by running searches through publicly NO:8) is shown in FIG. 14. In FIG. 14 (and in the accompa available DNA sequence databases, including the National 55 Center for Biotechnology Information and the Cancer nying Sequence Listing), “Y” represents any one of the four Genome Anatomy Project. Approximately two-thirds of nucleotide bases, cytosine, thymine, adenine, or guanine. these cDNA sequences exhibited homology to previously Comparison of the cDNA and genomic sequences revealed described DNA sequences/genes e.g., ribosomal proteins, the organization of the PCGEM1 transcription unit from three mitochondrial DNA sequences, growth factor receptors, and 60 exons (FIG. 15: E, Exon; B: BamHI: H: HindIII; X: Xbal; R: genes involved in maintaining the redox state in cells. About EcoRI). one-third of the cDNAs represented novel sequences, which EXAMPLE 3 did not exhibit similarity to the sequences available in pub licly available databases. The PCGEM1 fragment, obtained Mapping the Location of PCGEM1 from the initial differential display screening represents a 530 65 base pair (nucleotides 410 to 940 of SEQID NO: 1) cDNA Using fluorescent in situ hybridization and the PCGEM1 sequence which, in initial searches, did not exhibit any sig genomic DNA as a probe, we mapped the location of US 7,807,373 B2 15 16 PCGEM1 on chromosome 2q to specific region 2q32 (FIG. mal or tumor tissue derived RNAs. Two of 16 (12.5%) tumor 7A). Specifically, a Bacterial Artificial Chromosome (BAC) specimens showed reduced expression in tumors. These clone containing the PCGEM1 genomic sequence was iso results of PCGEM1 expression in tumor tissues could be lated by custom services of Genome Systems (St. Louis, explained by the expected individual variations between Mo.). PCGEM1-Bac clone 1 DNA was nick translated using tumors of different patients. Most importantly, initial DD spectrum orange (Vysis) as a direct label and florescent in situ observations were confirmed by showing that 45% of patients hybridization was done using this probe on normal human analyzed did exhibit over expression of PCGEM1 in tumor male metaphase chromosome spreads. Counterstaining was prostate tissues when compared to corresponding normal done and chromosomal localization was determined based on prostate tissue of the same individual. the G-band analysis of inverted 4,6-diamidino-2-phenylin 10 dole (DAPI) images. (FIG. 7B: a DAPI counter-stained chro EXAMPLE 5 mosome 2 is shown on the left; an inverted DAPI stained chromosome 2 shown as G-bands is shown in the center; an In Situ Hybridization ideogram of chromosome 2 showing the localization of the signal to band 2d 32Obar) is shown on the right.) NU200 image 15 In situ hybridization was performed essentially as acquisition and registration software was used to create the described by Wilkinson and Green (48). Briefly, OCT embed digital images. More than 20 metaphases were analyzed. ded tissue slides Stored at -80° C. were fixed in 4% PFA (paraformaldehyde), digested with proteinase K and then EXAMPLE 4 again fixed in 4% PFA. After washing in PBS, sections were treated with 0.25% acetic anhydride in 0.1M triethanolamine, Analysis of PCGEM1 Gene Expression in Prostate washed again in PBS, and dehydrated in a graded ethanol Cancer series. Sections were hybridized with S-labeled riboprobes at 52° C. overnight. After washing and RNase A treatment, To further characterize the tumor specific expression of the sections were dehydrated, dipped into NTB-2 emulsion and PCGEM1 fragment, and also to rule out individual variations 25 exposed for 11 days at 4°C. After development, slides were of gene expression alterations commonly observed in tumors, lightly stained with hematoxylin and mounted for micros the expression of the PCGEM1 fragment was evaluated on a copy. In each section, PCGEM1 expression was scored as test panel of matched tumor and normal RNAs derived from percentage of cells showing S signal: 1+, 1-25%; 2+. the microdissected tissues of twenty prostate cancer patients. 25-50%; 3+, 50-75%, 4+, 75-100%. Using the PCGEM1 cDNA sequence (SEQ ID NO:1), 30 Paired normal (benign) and tumor specimens from 13 specific PCR primers (Sense primer 1 (SEQ ID NO. 5): 5' patients were tested using in situ hybridization. A represen TGCCTCAGCCTCCCAAGTAAC3" and Antisense primer 2 tative example is shown in FIG. 17. In 11 cases (84%) tumor (SEQ ID NO: 6): 5' GGCCAAAATAAAACCAAACAT 3') associated elevation of PCGEM1 expression was detected. In were designed for RT-PCR assays. Radical prostatectomy 5 of these 11 patients the expression of PCGEM1 increased to derived OCT compound (Miles Inc. Elkhart, Ind.) embedded 35 1+ in the tumor area from an essentially undetectable level in fresh frozen normal and tumor tissues from prostate cancer the normal area (on the 0 to 4+ Scale). Tumor specimens from patients were characterized for histopathology by examining 4 of 11 patients scored between 2+ (example shown in FIG. hematoxylin and eosin stained sections (46). Tumor and nor 17B) and 4+. Two of 11 patients showed focal signals with 3+ mal prostate tissues regions representing approximately score in the tumor area, and one of these patients had similar equal number of epithelial cells were dissected out of frozen 40 focal signal (2+) in an area pathologically designated as sections. DNA-free RNA was prepared from these tissues and benign. In the remaining 2 of the 13 cases there was no used in RT-PCR analysis to detect PCGEM1 expression. One detectable signal in any of the tissue areas tested. The results hundred nanograms of total RNA was reverse transcribed into indicate that PCGEM1 expression appears to be restricted to cDNA using RT-PCR kit (Perkin-Elmer, Foster, Calif.). The glandular epithelial cells. (FIG. 17 shows an example of in PCR was performed using Amplitaq Gold(R) from Perkin 45 situ hybridization of S labeled PCGEM1 riboprobe to Elmer (Foster, Calif.). PCR cycles used were: 95°C. for 10 matched normal (A) versus tumor (B) sections of prostate minutes, 1 cycle: 95°C. for 30 seconds, 55° C. for 30 seconds, cancer patients. The light gray areas are hematoxylin stained 72°C. for 30 seconds, 42 cycles, and 72°C. for 5 minutes, 1 cell bodies, the black dots represent the PCGEM1 expression cycle followed by a 4°C. storage. Epithelial cell-associated signal. The signal is background level in the normal (A), 2+ cytokeratin 18 was used as an internal control. 50 level in the tumor (B) section. The magnification is 40x.) RT-PCR analysis of microdissected matched normal and tumor tissue derived RNAs from 23 CaP patients revealed EXAMPLE 6 tumor associated overexpression of PCGEM1 in 13 (56%) of the patients (FIG. 5). Six of twenty-three (26%) patients did PCGEM1 Gene Expression in Prostate Tumor Cell not exhibit detectable PCGEM1 expression in either normal 55 Lines or tumor tissue derived RNAs. Three of twenty-three (13%) tumor specimens showed reduced expression in tumors. One PCGEM1 gene expression was also evaluated in estab of the patients did not exhibit any change. Expression of lished prostate cancer cell lines: LNCaP. DU145, PC3 (all housekeeping genes, cytokeratin-18 (FIG. 3) and glyceralde from ATCC), DuPro (available from Dr. David Paulson, Duke hyde-3-phosphate dehydrogenase (GAPDH) (data not 60 University. Durham, N.C.), and an E6/E7-immortalized pri shown) remained constant in tumor and normal specimens of mary prostate cancer cell line, CPDR1 (47). CPDR1 is a all the patients (FIG.3). These results were further confirmed primary CaP derived cell line immortalized by retroviral vec by another set of PCGEM1 specific primers (Sense Primer 3 tor, LXSN 16 E6 E7, expressing E6 and E7 gene of the human (SEQ ID NO: 7): 5’ TGGCAACAGGCAAGCAGAG 3' and papilloma virus 16. LNCaP is a well studied, androgen-re Antisense Primer 2 (SEQ ID NO: 6): 5' GGC 65 sponsive prostate cancer cell line, whereas DU145, PC3, CAAAATAAAACCAAACAT3'). Four of 16 (25%) patients DuPro and CPDR1 are androgen-independent and lack did not exhibit detectable PCGEM1 expression in either nor detectable expression of the androgen receptor. Utilizing the US 7,807,373 B2 17 18 RT-PCR assay described above, PCGEM1 expression was the prostate tissue specificity of PCGEM1 is comparable to easily detectable in LNCaP (FIG. 4). However, PCGEM1 the well known prostate marker PSA (77mer oligo probe) and expression was not detected in prostate cancer cell lines far better than two other prostate specific genes PSMA (234 DU145, PC3, DuPro and CPDR. Thus, PCGEM1 was by fragment from PCR product) and NKX3.1 (210 by expressed in the androgen-responsive cell line but not in the cDNA). For instance, PSMA is expressed in the brain (37) androgen-independent cell lines. These results indicate that and in the duodenal mucosa and a Subset of proximal renal hormones, particularly androgen, may play a key role in tubules (38). While NKX3.1 exhibits high levels of expres regulating PCGEM1 expression in prostate cancer cells. In sion in adult prostate, it is also expressed in lower levels in addition, the results suggest that PCGEM1 expression may be testis tissue and several other tissues (39). used to distinguish between hormone responsive tumor cells 10 and more aggressive hormone refractory tumor cells. To test if PCGEM1 expression is regulated by androgens, EXAMPLE 8 we performed experiments evaluating PCGEM1 expression in LNCaP cells (ATCC) cultured with and without androgens. Biologic Functions of the PCGEM1 Total RNA from LNCaP cells, treated with synthetic andro 15 gen R1881 obtained from (DUPONT, Boston, Mass.), were The tumor associated PCGEM1 overexpression suggested analyzed for PCGEM1 expression. Both RT-PCR analysis that the increased expression of PCGEM1 may favor tumor (FIG. 5a) and Northern blot analysis (FIG. 5b) were con cell proliferation. NIH3T3 cells have been extensively used to ducted as follows. define cell growth promoting functions associated with a LNCaP cells were maintained in RPMI 1640 (Life Tech wide variety of genes (40-44). Utilizing pcDNA3.1/Hygro nologies, Inc., Gaithersburg, Md.) supplemented with 10% (+/-)(Invitrogen, CA), PCGEM1 expression vectors were constructed in sense and anti-sense orientations and were fetal bovine serum (FBS, Life Technologies, Inc., Gaithers transfected into NIH3T3 cells, and hygromycin resistant burg, Md.) and experiments were performed on cells between colonies were counted 2-3 weeks later. Cells transfected with passages 20 and 35. For the studies of NKX3.1 gene expres PCGEM1 sense construct formed about 2 times more colo sion regulation, charcoal/dextran stripped androgen-free FBS 25 nies than vector alone in three independent experiments (FIG. (cFBS, Gemini Bio-Products, Inc., Calabasas, Calif.) was 10). The size of the colonies in PCGEM1 sense construct used. LNCaP cells were cultured first in RPMI 1640 with transfected cells were significantly larger. No appreciable 10% clfBS for 4 days and then stimulated with a non-metabo difference was observed in the number of colonies between lizable androgen analog R1881 (DUPONT, Boston, Mass.) at anti-sense PCGEM1 constructs and vector controls. These different concentrations for different times as shown in FIG. 30 5A. LNCaP cells identically treated but without R1881 promising results document a cell growth promoting/cell Sur served as control. Poly A+ RNA derived from cells treated vival function(s) associated with PCGEM1. with/without R1881 was extracted at indicated time points The function of PCGEM1, however, does not appear to be with RNAZolTM B (Tel-Test, Inc, TX) and fractionated (2 due to protein expression. To assess this hypothesis, we used ug/lane) by running on 1% formaldehyde-agarose gel and 35 the TestCode program (GCG Wisconsin Package, Madison, transferred to nylon membrane. Northern blots were analyzed Wis.), which identifies potential protein coding sequences of for the expression of PCGEM1 using the nucleic acid mol longer than 200 bases by measuring the non-randomness of ecule set forth in SEQID NO: 4 as a probe. The RNA from the composition at every third base, independently from the LNCaP cells treated with R1881 and RNA from control reading frames. Analysis of the PCGEM1 cDNA sequence LNCaP cells were also analyzed by RT-PCR assays as 40 revealed that, at greater than 95% confidence level, the described in Example 4. sequence does not contain any region with protein coding As set forth in FIGS. 5a and 5b, PCGEM1 expression capacity (FIG. 16A). Similar results were obtained when increases in response to androgen treatment. This finding various published non-coding RNA sequences were analyzed further supports the hypothesis that the PCGEM1 expression with the TestCode program (data not shown), while known is regulated by androgens in prostate cancer cells. 45 protein coding regions of similar size i.e., alpha actin (FIG. 16B) can be detected with high fidelity. (In FIG. 16, evalua EXAMPLE 7 tion of the coding capacity of the PCGEM1 (A) and the human alpha actin (B), is performed independently from the Tissue Specificity of PCGEM1 Expression reading frame, by using the TestCode program. The number 50 of base pairs is indicated on the X-axis, the TestCode values Multiple tissue Northern blots (Clontech, CA) conducted are shown on the Y-axis. Regions of longer than 200 base pairs according to the manufacturer's directions revealed prostate above the upper line (at 9.5 value) are considered coding, tissue-specific expression of PCGEM1. Polyadenylate RNAs under the lower line (at 7.3 value) are considered non-coding, of 23 different human tissues (heart, brain, placenta, lung, at a confidence level greater than 95%.) liver skeletal muscle, kidney, pancreas, spleen, thymus, pros 55 The Codon Preference program (GCG Wisconsin Package, tate, testis, ovary, Small intestine, colon, peripheral blood, Madison, Wis.), which locates protein coding regions in a stomach, thyroid, spinal cord, lymph node, trachea, adrenal reading frame specific manner further Suggested the absence gland and bone marrow) were probed with the 530 base pair of protein coding capacity in the PCGEM1 gene. In vitro PCGEM1 cDNA fragment (nucleotides 410 to 940 of SEQID transcription/translation of PCGEM1 cDNA did not produce NO:1). A 1.7 kilobase mRNA transcript hybridized to the 60 a detectable protein/peptide. Although we can not unequivo PCGEM1 probe in prostate tissue (FIG. 6a). Hybridization cally rule out the possibility that PCGEM1 codes for a short was not observed in any of the other human tissues (FIG. 6a). unstable peptide, at this time both experimental and compu Two independent experiments revealed identical results. tational approaches strongly suggest that PCGEM1 cDNA Additional Northern blot analyses on an RNA master blot does not have protein coding capacity. (It should be recog (Clontech, CA) conducted according to the manufacturers 65 nized that conclusions regarding the role of PCGEM1 are directions confirm the prostate tissue specificity of the speculative in nature, and should not be considered limiting in PCGEM1 gene (FIG. 6b). Northern blot analyses reveal that any way). US 7,807,373 B2 19 20 The most intriguing aspect of PCGEM1 characterization 3. Mostofi F K: Grading of prostate carcinoma. Cancer has been its apparent lack of protein coding capacity. Chemothera Rep., 59:111, 1975. Although we have not completely ruled out the possibility 4. Lu-Yao G L. McLerran D. Wasson J. Wennberg J E: An that PCGEM1 codes for a short unstable peptide, careful assessment of radical prostatectomy. Time trends, Geo sequencing of PCGEM1 cDNA and genomic clones, compu- 5 graphical Variations and Outcomes. JAMA, 269:2633 tational analysis of PCGEM1 sequence, and in vitro tran 2636, 1993. Scription/translation experiments (data not shown) strongly 5. Partin AW and Oesterling JE: The clinical usefulness of Suggest a non-coding nature of PCGEM1. It is interesting to prostate-specific antigen: update 1994, J. Urol., 152:1358 note that an emerging group of novel mRNA-like non-coding 1368, 1994. RNAs are being discovered whose function and mechanisms 10 6. Wasson J H. Cushman CC, Bruskewitz RC, Littenberg B. of action remain poorly understood (49). Such RNA mol Mulley AG, and Wennberg J E: A structured literature ecules have also been termed as “RNA riboregulators' review of treatment for localized CaP. Arch. Fam. Med., because of their function(s) in development, differentiation, 2:487-493, 1993. DNA damage, heat shock responses and tumorigenesis (40 7. Weinberg RA: How cancer arises. Sci. Ameri, 9, 62-70, 42, 50). In the context of tumorigenesis, the H19. His-1 and 15 1996. Bic genes code for functional non-coding mRNAs (50). In 8. Bostwick D G: High grade prostatic intraepithelial neopla addition, a recently reported prostate cancer associated gene, sia: The most likely precursor of prostate cancer. Cancer, DD3 also appears to exhibit a tissue specific non-coding 75:1823-1836, 1995. mRNA (51). In this regard it is important to point out that 9. Bostwick D G. Pacelli A, Lopez-Beltran A: Molecular PCGEM1 and DD3 may represent a new class of prostate Biology of Prostatic Intraepithelial Neoplasia. The Pros specific genes. The recent discovery of a steroid receptor tate, 29:117-134, 1996. co-activator as an mRNA, lacking protein coding capacity 10. Pannek J. Partin A W: Prostate specific antigen: What's further emphasizes the role of RNA riboregulators in critical new in 1997. Oncology, 11:1273-1278, 1997. biochemical function(s) (52). Our preliminary results showed 11. Partin A W. Kattan M. W. Subong EN, Walsh PC, Wojno that PCGEM1 expression in NIH3T3 cells caused a signifi 25 KJ. Oesterling J. E. Scardino PT Pearson J D: Combina cant increase in the size of colonies in a colony forming assay tion of prostate specific antigen, clinical stage, and Gleason and suggests that PCGEM1 cINA confers cell proliferation score to predict pathological stage of localized prostate and/or cell survival function(s). Elevated expression of cancer. A multi-institutional update. JAMA, 277: 1445 PCGEM1 in prostate cancer cells may represent again in 1451, 1997. function favoring tumor cell proliferation/survival. On the 30 12. Gomella LG, Raj GV, Moreno JG: Reverse transcriptase basis of our first characterization of PCGEM1 gene, we pro polymerase chain reaction for prostate specific antigen in pose that PCGEM1 belongs to a novel class of prostate tissue management of prostate cancer. J. Urol., 158:326-337, specific genes with potential functions in prostate cell biology 1997. and the tumorigenesis of the prostate gland. 13. Gao CL, Dean RC, Pinto A, Mooneyhan R. Connelly R In summary, utilizing surgical specimens and rapid differ R, McLeod D G, Srivastava, S, Moul J W: Detection of ential display technology, we have identified candidate genes PSA-expressing prostatic cells in bone marrow of radical of interest with differential expression profile in prostate can prostatectomy patients by sensitive reverse transcriptase cer specimens. In particular, we have identified a novel nucle polymerase chain reaction (RT-PCR). 1998 International otide sequence, PCGEM1, with no match in the publicly Symposium on Biology of Prostate growth, National Insti available DNA databases (except for the homology shown in 40 tutes of Health, p. 83, 1998. the high throughput genome sequence database, discussed 14. Garnick M B, Fair W. R. Prostate cancer. Sci. Amer., above). APCGEM1 cDNA fragment detected a 1.7 kb mRNA 75-83, 1998. on Northern blots with selective expression in prostate tissue. 15. Moul J.W. Gaddipati J, and Srivastava S: 1994. Molecular Furthermore, this gene was found to be up-regulated by the biology of CaB. Oncogenes and tumor Suppressor genes. synthetic androgen, R1881. Careful analysis of microdis 45 Current Clinical Oncology: CaP. (Eds. Dawson, N.A. and sected matched tumor and normal tissues further revealed Vogelzang, N.J.), Wiley-Liss Publications, 19-46. PCGEM1 over-expression in a significant percentage of pros tate cancer specimens. Thus, we have provided a gene with 16. Lalani E-N, Laniado ME and Abel PD: Molecular and broad implications for the diagnosis, prevention, and treat cellular biology of prostate cancer. Cancer and Mets. Rev. 50 16:29-66, 1997. ment of prostate cancer. 17. Shi XB, Gumerlock PH, deVere White RW: Molecular The specification is most thoroughly understood in light of Biology of CaP World J. Urol. 14, 318-328, 1996. the teachings of the references cited within the specification 18. Heidenberg HB, Bauer JJ, McLeod D G, Moul J W and which are hereby incorporated by reference. The embodi Srivastava S: The role of p53 tumor suppressor gene in ments within the specification provide an illustration of 55 CaP: a possible biomarker? Urology, 48:971-979, 1996. embodiments of the invention and should not be construed to 19. Bova G Sand Issacs W B: Review of allelic loss and gain limit the scope of the invention. The skilled artisan readily in prostate cancer. World J. Urol., 14:338-346, 1996. recognizes that many other embodiments are encompassed 20. Issacs W B and Bova G S: Prostate Cancer: The Genetic by the invention. Basis of Human Cancer. Eds. Vogelstein B, and Kinzler K W. McGraw-Hill Companies, Inc., pp. 653-660, 1998. REFERENCES 60 21. Srivastava S and Moul J W: Molecular Progression of Prostate Cancer. Advances in Oncobiology. (In Press) 1. Parker S L, Tong T. Bolden S, and Wingo PA: Cancer 1998. statistics. CA Cancer J. Clin., 46:5-27, 1996. 22. Sakr WA, Macoska JA, Benson P. Benson DJ, Wolman 2. Visakorpi T, Kallioniemi O P. Koivula T and Isola J: New 65 S R, Pontes J E, and Crissman: Allelic loss in locally prognostic factors in prostate carcinoma. Eur: Uro., metastatic, multi-sampled prostate cancer. Cancer Res., 24:438-449, 1993. 54:3273-3277, 1994. US 7,807,373 B2 21 22 23. Mirchandani D, Zheng J. Miller GL, Ghosh A K, Shibata expression in normal and malignant human tissues. 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W. and 127, 1998. 65 Srivastava S: PCGEM1: A Novel Prostate Specific Gene is 38. Silver DA, Pellicer I, Fair W R, Heston, W D, and Overexpressed in Prostate Cancer. Submitted to Proceed Cordon-Cardo C: Prostate-specific membrane antigen ings of the National Academy of Sciences.

US 7,807,373 B2 29 30

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<210s, SEQ I D NO 5 &211s LENGT H: 21 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATU RE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 5 tgcct cagcc ticc caagtaa c 21

<210s, SEQ I D NO 6 &211s LENGT H: 21 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATU RE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 6 ggccaaaata aaaccaaaca t 21

<210s, SEQ I D NO 7 &211s LENGT H: 19 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATU RE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OO > SEQUENCE: 7 tggcaa.cagg Caagcagag 19

<210s, SEQ I D NO 8 &211s LENGT H: 118O1 212. TYPE : DNA <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221 > NAMEAKEY: unsure &222s. LOCAT ION: (7470) <223> OTHER INFORMATION: n may represent any of the four nucleotide bases

<4 OOs, SEQUE NCE: 8 toccitcttgc gttctgcaat ttctgaaaaa aagatgttta ttgcaaagtg atatgagcac 6 O tggaaaggta Ctaatt CCaa tittgatticta attggatgag tgacatgggt aag.cgattct 12 O aagcatttgt gtttttittta gtag tatgga atttaattag ttct cagtat gttagtgaag 18O atgaatgaaa acatgcatat gtttccatgt attataaata ttittaaaatg Caaaaaatta 24 O ttctaatgaa tatataaata taaag cataa Caataataat acaataccac c cataaagtic 3OO at Catctaat ttaaaaacta aaaCattaac actitgaatct cc.cccattgc aaCat Cttt C 360 ccgacttgttg tgttttitt to ttttgcttitt aaaatttittg ttittatcata tgtctgcata agattatata gctitt.ccttg ttittaagctt tittaaataat at attgtagt tat attattt gtgctittgct titt tt tact t aac attatgg ttctaaaatt Cagtaatgtg ttgggcatgt 54 O ataatttgtt tatttittaat ctic tittgaca titcgactata taaattt cag tttgtttatt gact cotttg tctatagata citctgctatt tctgtttittg ctgttacaaa aataatgctg 660 ttittaaattit cattttgtat actitttittga ggcatgtgta tgagttatt c talaggtaaaa 72 O aaataagaaa aaattgctgg gttataagat tgtcacatgc tcqaatttac aagataatgc US 7,807,373 B2 31 32

- Continued caaatcattt ttcaaagtaa ttatacct at ttatact acc gg tatgagta tattggtgcc 84 O cacatagttg cittgttctgc caaagtttgg tatgatcgaa caataattitt togcc.cat caa 9 OO atgg catalaa ataaaatcto agtgtgcttt taatttgcat titt citatgtt taagaattgt 96.O ttcttttitta accatttata atttacttitt gctgaaatgc titgct tatta tttittgcticc O2O c catttitt to c tattggatt gcttittctica ttaatttata agaattittat atggtttaga O8O tactaattat tatatt actgaaaatacctt tat cagtttgttgttgtact t t ct acttitat 14 O gtc.ttgttgat ggataaaagt tittaaattgt attgttgttga agitta acatt tittaaattitt 2OO ataatcagca totttaataa tot ctittata aaattitt cott ttacatagat gtcataaaga 26 O tacatctota taattt citta ttitttittggc atatgttcat taagt cattt tat catttitt 32O tagtaataaa ttgcagtt at titatgaaaca aataatttitt aaaattatat atgctitt citt 38O taaaaattga t cittagcatg citt cactatog aagcttgagg citt cactgca cqttgtactg 44 O aaattatgta taaaac agtg gttctgaaaa totctgagtt catgacacct ttagtgtctic SOO aggtttittitt gcttttgttc ttgtttitt to tca caaag.ca cctaagttaa ataaaaacaa 560 agcacaaag.c tat cagottc atgtattaag tagtaagctic ccatgttaac agttgtaact 62O tgcc toggtgc ccaatagatgtcact ctdtt titcctagaaa ctittaaaata t cc ct cagtg 68O citcc tdttaa tt catggtag togc.cccaagg cactctggca cccagttittg gaactgcagt 74 O tittaaaagtic atalaattgaa taaaatgat agcaaaggtg gaggtttitta aagagctatt 8OO tatagg tocc toggacago at Cttttittcaa ttaggcagoa accttitttgc ctatogcc.gta 86 O actgtgtctg. cacttic ct ct aattggggtgagtaagagat tttgttatgt atataatagc 92 O taagaatata gtaataatgg cittaaatcat ggittatttitt aaact actaa catttagaag 98 O acaaaataaa aatgctttga aaagtataga ggttt tagtg taattagcag ggaataatga 2O4. O aatgatttga tagggctact cagttttgta taactittggit gctittaagtic togaatgcaga 21OO gcatggatgt tdtgat coag cctittatatgtttitc cctoga agaagattta atttatttgg 216 O ccttittgaga aacacatttg gcattgtaat atgttittgct tcc aggttct atctocaagg 222 O ataatttgac aaaatcacac ataaattitat titt cagggca cacagtttcc ctitt taggga 228O acticacagag gtagagagta atacaataat cacatttgaa tatto agitaa gtgaggit cot 234 O catagat citt atgtgtatgt cac catgitat ataattttgt taatcactag atgitatgaga 24 OO caagaaattit gaggaatctt aactagagat taaaatcagg gatttaaatc aaagaaacat 246 O ttaaatgcct c ctittatt at ttaaatacct gcatgggaga at cattgaaa aaaaaataaa 252O aag catacaa cittgggaata ttata aacca agaagaattt gttatt citgg ttgattittitt 2580 titt caggctic cqcacaggca acttacctitt atctotttgt gatttittatt tottgttaaa 264 O atatacagaa atagittaa.gc agatt catag agctgaatat aaaatttact acgagatgca 27 OO

Ctgggactica acgtgacctt at Caagtgac titat cagtga ggtgagcatt Cttaatt cag 276 O ataatggaac ttattatcat aatcttittgc titatgctatt gttgagctta act acttatt 282O catatttgca tatgcatatt gagataatat cattt catta atttcagtac tdaacactaa 288O tctic ctaaga gtaattgttga aagtttcaga ttgcactatt tittaactata tatctgtatg 294 O titat citt cat atatgcttga ataacttata agcaattgaa actittcaatt acagtatact 3 OOO attgaa.gcaa atcaactaat atatacacat atc cattagc aatagtagat aattitttgta 3 O 6 O aatgtc.ca.gc acagttctitc atatgtagag gatgttcaaa ttggctaagt toctitttctic 312 O US 7,807,373 B2 33 34

- Continued t cittaattat tag tatttitt cotactgctic tttgtataat tattoctitcc tictittagctic 318O caatcc ttac aatctatt ct taa catagca actgggaaga aagtttittaa acataaacca 324 O gatgatgtca citccaccc.ca caaaact tcc act attct ct gtcacacata gaaagaaaga 33 OO aaaaaaat at tdaaaaccta caaag acttig citatgatctg gtc.caggctic ticcictaaaat 3360 tt catgtaat titccagocac taggc ctittctggct ct cott toaatct cat tag ccttitt c 342O actact acaa gttagactgg gttittgg.ccg agg tatttct ttttitt cata ttittgcc titt 3480 gcctagattig citct tccaat agatatt cac aattgcatca to atttctat atacgtgcta 354 O aaaggttt cottgtccaaaa tagcttcagt gaccacctga tictagaatag tot coat caa 36OO aagtttcttt toctitt to ct caccacttga tatttatat c aaa catttat ttgtgtaatt 366 O tatgtgtttgtttgttittct gtact agcat tatgatgacc atact atttg atgcc.ccc.ca 372 O aaaaat actt togagaatga cagggcaaag ctaaaataat taaattatat aattittgaca 378 O tagg cact at tdacaaaaag caattgatgt tatgatagtg ttagat citat gaaatagitac 384 O tatttaaaag taattctic td aaatacaatt ttctaaaact aaaag cago a tatgtacatg 3900 aaacaccalaa aaact tcc tt at atttatca citggaagatt taaaatagta taagtag taa 396 O cittatttaat at atttittga ttatttaatt aattittatag tat coaactic taatataatg 4 O2O c cagtggitat ttgttcaaaa tattittaatgttgtctattt atttittaatt togcctaaaaa 4 O8O titat cittaaa tdaaaattitt toggittaataa atttgaaaat actgaaaccc ticatcto cag 414 O t citctgtgga t cotaaagtt tttagttgag aaaataattt ttct c tagag aatgaagtag 42OO cittgtaagct toggagaaatt totgctaaat aaatgatatt atcaact citt attitt cittca 426 O atacgaaata tataaatatt to agct cata tatttittgca ggtgctatoc ttittgct tcc 432O aat cataatt totgacaaat attittggaag toaaaacttig tottctattt tdttatttaa 438 O aattatatag act acttittg taaac ctitta tact atcaaa toataggcaa titt cagtttg 4 44 O attt cattct ggtgcagaat ataagtttat coaagtaaaa caggagt cac ttcaaaagat 4500 t cct cocact gactgagata t t c caaagcc aactittgcaa aattt cagaa ttaaat atta 456 O tact tc.tttg tacctt catt ttatttgttcaatttitt citt tdtgtttgta gaaaattitta 462O at atttittct gttittcaagt tttgattitta atttact act ttataattitt taaaggtaag 468O ttttgtgagg ctatatt cat tatgtgttitt gaataaagac atacaattaa ttittgagaac 474. O tgcaataaaa attataagac tattaaaaat gcagtaagtg tact acactt aggctgctaa 48OO aaatgcagta ccagtag act acatttaggc tigcttaaagt tagttcttct aagtaccata 486 O tactittaaaa ttittagctaa tdatggagaa caaagacaga aag actgttgt taccatatt c 492 O tagttggcca ttttgttttgttittgagaga cqt cacatca gcc titat cat aaaaattatt 498O tggttttacc attittgactg tdagcaaaat atacagdata atatacaaaa taaaatatat 5040 gtacat ctitc acaactitctt gtttaggatg caattatata tatatatata tatatattta 51OO ttattatact ttaagttcta ggg tacatgg caccacgtgc aggttgttac atatgtatac 516 O atgtgc catgttggtgtgct gcacc catta act cqt catt tacattaggt gitat citccta 522 O atgctatocc ticc cct ct ct c cccaccc.ca caacaa.gc.cc cqgtgttgttga tigttc.ccctt 528 O cctgtgtc.ca totgttct cattgttcaatt cocaccitatgagtgagaaca cqcagtgttt 534 O gctttitttgt ccttgcaata gtttgctgag aatgatggitt to cagct tca to catgtc.cc 54 OO tacaaaggac atgaact cat cattttittat ggctgcatag tatto catgg togtatatgtg 546 O ccaccatttt cittaatccga gttctgtc.cat tdttgttgga catttgggitt gcaattittga 552O US 7,807,373 B2 35 36

- Continued gttt catgtg tag catgitat agcacaacca attaagattt ctittctittct citctttittitt 558 O tttitttitttgttgaaatgga gtc.ttgcctg. it ct coaaggc tiggagcc caa tigtgtgat C 564 O ttggct tact gcaacctic ca cct cocqqgt toaa.gcgatt citcctgcctic agc catc.cga st OO gtagctggga citatagg.cgit gcaccaccat gcc cagotaa tttttgtatt tittagtacag 576. O acggggtttc accacggtgg C caggatggt Ctcaatttct tacct catg attcaccc.gc 582O

Cttggcct co caaagtgctg ggattacagg ttgaac cac caa.gc.ccggc Ctgtcacaag 588 O tttittagtgt totattittaa tacagaaatt agataaatcc aaa.gagaaag acattt cata 594 O tgtgcgtaga gttgtcggaa gaaatgagag ticttataaat aactittaaaa attgttgaaga 6 OOO aataaaggca aaatagt cct atgcagtttg atttaaatat attcttaata agagctactt 6 O6 O ttgttgaaaac cagaat attgaaacatgtag atatggat.ct t cattagtga citgacataat 612 O at attgtt at tdt tactatt ttattgtatic agccaactaa tattgagtgc tittgttgtatic 618O ctaa.gcacta togctaalacac td taccagta ttacctgata taatcatatt aatatttatt 624 O attt cactitt toatatgaaa aaattgaagc acagattaag acact cogala at catacctic 63 OO tattgattat cagcaccagg atttgaattig aggcactctg atccagagaa gottttgttt 636 O c catgaaggc titatgttggg gaaaaataat caaattgcct gtacct cagt totataaata 642O agaggttggg ttggtagatg attctggctg attcagoaga aaagaaattt attcaaagga 648 O tat cacacag titt toataac agittaagaat acagaggaala Cagggcacca gggctaagta 654 O Cagaccaaag ticcaaaacca Ctgccaaagt tdcagcaagg agaac agcac aaatttgctt 66OO gctgtcaccc gcc act agat gcttttgttt ggagccttga acttgacitta cactgcc act 666 O gacatcagca C cagtgct ct ctgttgtacta ggaggtggag ttggtgacgt totgaactic 672 O aaag cagatgtttctgctgt gaaatagata cctaatacag aacctgcttic ct catt catt 678 O. c cct coccaa at catatgct togtag tigtgg ctagagtttctgtttct cot togg to caggc 6840 agaattitatgaagcttgcta tittatcgcct taaagattag aagaatatt catalagg tatt 69 OO agattgc.cat aaggttgaac aaatcaac at t caacttcaa goattcaa.ca ttgttttgtt 696 O ttcttittggg atacct ctdc agcagttcaa at cittatttic togc ccttgga caaccaggitt 7 O2O tataaatatt gcagattct c cactgactgc tittgatccta t cittctatat titatgtatac 708 O taattagcat ataataaaag attatgttac agaat ct caa aattagtaat tatgaattga 714. O gatggtgtta tacagtacac taa catccaa gag acttgtt tatt coaagg aaaat attta f2OO gagatattaa atgatattitc. tcatc ctitta gacatataca ttttittagct tacagcctgc 726 O tittaggcaag caacagactic ticaggatctg ctic ctaccag gigtctgaac a titt cotcc.ca 732O gttittaaaga aacaaattica aataa cattg taacctic cag aggaaagttcaagct cittitt 7380 atag tattgt ttaalacagta Cagctgagga aactaaagac agagaagtta aatgccttgg 744. O cact tagt ct agatttacaa taalacticc tn totact tagg acccactaac aggggctgca 75OO tttacaccala aac catgaag gtggccCaag ticatc actga gaagtag tac aag caccgag 756 O ggaatgactt Caacaggaac aagaaag.cgt ggaaggagat cct agcagga agctic cacala 762O gaagatagca tttacgt.ct tcattggat galagcaggtt Cagagagacic tagtgacagc 768 O tat citc.cgt.c aaggtgcaga aggagagatc attgaatgta gcatttt cat gcaaaaaaaa 774. O aaatgttgaa gtctttggac titcgggagtic titccaaact gcagg to act cagcctacag 78OO ttgggatgaa tittcaaaa.ca ccagttggag ccggttgaat ctittctgcta togctgtaata 786 O

US 7,807,373 B2 39 40

- Continued caattcaatt tacatgatgc taattatggc aattataa.ca aat attaaag attitcgaaat O32O agaatatgtgaattgttcac catacataga aatgaaaagt to attitcgta aagcaagatg O38O

Ctgggtgaaa gagtgcttitt gattgaaaga t cact agatt agtagagggc alagacttitta O44 O gtcc ctaatc taccct taat agc catgtgg toacgtgtaa gtcagtgaac ccatctgatt OSOO citcc to atac titttitt catc tictaaaatga gqqtataatt taagct cott catttitttitt O560 tttittittgag atagagttitt gct cittgtca cccaggttgg agtgcaatgg cacgatcto a O62O gct cactgca accctctgct tcc toggttcaagtgattct coctogct tca gcct c ccaag O68O tgagc.ccggg attacaggtg ccc.gc.cacca catctgggcc tagattittitt g tattitt cac Of 4 O catgttggcc aggctggit ct cqaac cccta cct caggtga t coct cqcct cqgcct citca O8OO aagtgctggg attacaggtg tdagccacca cqc coagc.cc aatat cagtt tttcttttitt O 860 alacaca aggc taacacaatc aaaatact ag Ctaggggaga aaaaaaaaat aaggcactgt O92O titatgtgtaa Caggct Cttgttgcaatcca Ctgggggaga C caaataaac agtaagaatc O98O aaatcc ttitt catataatcc titt ctittgca gaatacataa aatc.cccaca aatggctitat O4 O ctitcctttitt atgatatgtt ggaga attgt agctaagtga cagatattitt gcttgggtgt 1OO atagaccaca aaggactgtg tottgatgat gigtttgcata aaattatacct tagtttitta 16 O

Ctttgtatgt tacatgttag atttagagta tdaaaattag tagggaggat tattaacaaa 22 O gaac agggca agaggagtag aatta aacct Cttctaatac Ctgtgcacaa gtaggcttitt 28O Cagaaact ct acaa.cccCaa cataaactgg at agittagaa aag cacact C C Caagga agg 34 O cggittatgtt ttgcagtttgaat cagaaga atagagctat agcaatcttic attctatagt 4 OO aacattaaag agcctggttt at attatago agt cattaag atttaaaaat ttacatc.ttg 460 cc.gttcttct tact cacaga ttitt cqagag gtaatgtaat gat cacacga gotgagaatc 52O actgcc ttitt ataatgcg at taaatgcatgaacaaagttt ccaacaaata acagtaataa 58 O aaagaaacat g tattagcac ttaataagcc aggtgctgta coacgtgttgt tacatgctitt 64 O caatccatga actggtaaac togg tactagt atctotattg gacatgtgag gaalaccalaat 7 OO ggagttgata aacagtagag ttaaaaatta ct citt catat attatattgc ct caatctoa 760 caga catc to tdctaccalaa agctatoata t c tag acticg a 8O1

<210s, SEQ ID NO 9 &211s LENGTH: 19 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 9 tggcaa.cagg Caagcagag 19

<210s, SEQ ID NO 10 &211s LENGTH: 21 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 10 ggccaaaata aaaccaaaca t 21

<210s, SEQ ID NO 11 US 7,807,373 B2 41 42

- Continued

&211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 11 gcaaatatga tittaaagata caac 24

<210s, SEQ ID NO 12 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 12 ggttgt atct ttaaat cata tittgc 25

<210s, SEQ ID NO 13 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 13 actgtc.ttitt catatattitc. tcaatgc 27

<210 SEQ ID NO 14 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 14 aagtag taat tittaalacatg ggac 24

<210s, SEQ ID NO 15 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 15 tttittcaatt aggcagdaac c 21

<210s, SEQ ID NO 16 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 16 gaattgtc.tt tdtgattgtt tittag 25

<210s, SEQ ID NO 17 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 17 US 7,807,373 B2 43 44

- Continued caatticacaa agacaattica gttaag 26

SEQ ID NO 18 LENGTH: 23 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 18 acaattagac aatgtc.ca.gc tiga 23

SEQ ID NO 19 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 19 ctittggctga tat catgaag tdtc 24

SEQ ID NO 2 O LENGTH: 23 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer < 4 OO > SEQUENCE: 2O aaccttittgc cctatogcc.gt aac 23

SEQ ID NO 21 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 21 gagacticcca acctgatgat git 22

SEQ ID NO 22 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Probe/Primer <4 OOs, SEQUENCE: 22 ggtcacgttg agt cccagtg

We claim: 55 (c) determining that there is an indication of the presence of 1. A method of detecting prostate cancer in a patient, the prostate cancer in the patient if the level of the RNA method comprising: molecule detected in the biological sample is elevated above the level of the RNA molecule present in the (a) detecting a PCGEM1 nucleic acid sequence RNA mol- 60 sample of normal prostate cells. ecule having a nucleotide sequence corresponding to 2. The method according to claim 1, wherein step (a) SEQID NO: 1 in a biological sample from the patient, includes: (a) isolating RNA from the sample; (b) amplifying wherein the biological sample comprises human pros a PCGEM1 cDNA molecule using at least two primers tate cells designed to amplify a fragment of SEQ ID NO: 1; and (c) (b) comparing the level of the RNA molecule detected in 65 detecting the amplified PCGEM1 cDNA. the biological sample to a level of the RNA molecule 3. The method according to claim 2, wherein the PCGEM1 present in a sample of normal prostate cells; and cDNA is amplified with at least two primers comprising US 7,807,373 B2 45 46 nucleotide sequences selected from SEQID NO:5, SEQ ID 5. A method according to claim 1, wherein the biological NO:6, SEQID NO:7, SEQIDNO:9, SEQID NO:10, SEQID sample is selected from blood, urine, and prostate tissue. NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, 6. The method according to claim 1, wherein the biological SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID sample is blood. NO:18, SEQID NO:19, SEQID NO:20, SEQID NO:21, and 5 7. The method of claim 1, wherein the PCGEM1 RNA SEQID NO:22. molecule is detected using a Reverse Transcriptase Poly 4. The method according to claim 2, wherein said at least merase Chain Reaction (RT-PCR). two primers include primers comprising nucleotide sequences are SEQID NO:15 and SEQID NO:22. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,807,373 B2 Page 1 of 1 APPLICATIONNO. : 12/166723 DATED : October 5, 2010 INVENTOR(S) : Shiv Srivastava et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In claim 1, column 43, lines 59-60, “PCGEM1 nucleic acid sequence RNA molecule' should read -- PCGEM1 RNA molecule --.

Signed and Sealed this Twenty-second Day of February, 2011

David J. Kappos Director of the United States Patent and Trademark Office UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,807,373 B2 Page 1 of 1 APPLICATIONNO. : 12/166723 DATED : October 5, 2010 INVENTOR(S) : Shiv Srivastava et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the Title Page, item 54, and at Column 1, line 1, in the title “using PCEGM 1 should read -- using PCGEM1 --.

Signed and Sealed this

David J. Kappos Director of the United States Patent and Trademark Office