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ESTABLISHMENT OF A NEW ANDROGEN-DEPENDENT AND HIGHLY TUMORIGENIC HUMAN PROSTATE CANCER CELL LINE, ADMTN
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
survival in vivo as long as the duration of the off cycle remains similar to that in standard IAA (about 10 days in our studies) - when the off F\FOHV DUH DOORZHG WR EH PD[LPDOO\ H[WHQGHG WKH VXUYLYDO EHQH¿W LV lost. Therefore, we proposed to examine the gene expression changes WKDWRFFXULQWKH¿UVWIHZGD\VRIWKHRIIF\FOHLQ,$$DQGK\SRWKHVL]HG WKDWWKHDGGLWLRQRIGXWDVWHULGH ' GXULQJWKHµRIIF\FOH¶RI,$$VKRXOG differentially enhance the expression of androgen responsive growth suppressing genes relative to standard IAA. METHODS: After establishment of LNCaP tumors, nude mice (Balb/C- Nu) were castrated and followed up for 7-10 days before being UDQGRPL]HGWR&RQWLQXRXVDQGURJHQDEODWLRQ &$$ ,$$&$$'RU IAA + D. Testes-intact animals with or without dutasteride were kept as FRQWUROV36$7DQG'+7OHYHOVZHUHDVVD\HG0LFHZHUHVDFUL¿FHG GD\VDIWHUUDQGRPL]DWLRQDQGWXPRUJHQHH[SUHVVLRQZDVVWXGLHGXVLQJ real time reverse transcriptase PCR. 5(68/76 $IWHU WKUHH GD\V RI µRII F\FOH¶ LQWHUYHQWLRQ WKH expressions of EAF2/U19 and calereticulin (both androgen responsive growth suppressive genes) were higher in the IAA+D group as compared WR WKH RWKHU LQWHUYHQWLRQ JURXSV ,$$' 9V ,$$ IROG DQG IROG UHVSHFWLYHO\S )LJVDDQGE 7KHH[SUHVVLRQRI36$GLGQRW show any difference. &21&/86,216 ĮUHGXFWDVHLQKLELWLRQWRWKHRIIF\FOHRI ,$$ XSUHJXODWHV JURZWK VXSSUHVVLYH JHQHV DIWHU GD\V RI µRII F\FOH¶ treatment. These results provide an insight into the possible mechanisms WKURXJK ZKLFK RII F\FOH ĮUHGXFWDVH LQKLELWLRQ LPSURYHV VXUYLYDO Further studies need to focus on the time course of such events so as WREHVWGH¿QHWKHGXUDWLRQRIWKHRIIF\FOHWKDWPD[LPL]HVWKHTXDOLW\ RIOLIHEHQH¿WVRIDQGURJHQVZLWKRXWVDFUL¿FLQJWKHVXUYLYDODGYDQWDJH FRQIHUUHGE\,$$RIIF\FOHĮUHGXFWDVHLQKLELWLRQ
Source of Funding: This investigation was supported in part by NIH grants R01 CA 108675, 5 R01 DK51993, 1 P50 CA90386.
540 ESTABLISHMENT OF A NEW ANDROGEN-DEPENDENT AND HIGHLY TUMORIGENIC HUMAN PROSTATE CANCER CELL LINE, ADMTN Ayush Dagvadorj*, Shyh-Han Tan, Lucianne R Cavalli, Bassem R Haddad, Marja T Nevalainen. Philadelphia, PA, and Washington, DC. INTRODUCTION AND OBJECTIVE: We have established DQGFKDUDFWHUL]HGDQHZKXPDQSURVWDWHFDQFHUFHOOOLQH$'071IURP a primary CWR22 human prostate xenograft tumor. Primary CWR22 tumors are re-transplantable human prostate xenograft tumors in nude mice and they were originally established from a Gleason score 9 primary cancer with bone metastases ( Pretlow et al., 1993). The primary CWR22 prostate tumor is highly responsive to androgen deprivation with marked tumor regression after castration, mimicking the course of human disease. No cell lines displaying the growth characteristics of the original primary CWR22 tumors have existed until now. METHODS: Androgen regulation of the cell growth and tumor formation was tested in vitro and in vivo as subcutaneous tumor in nude mice. Androgen receptor (AR) expression and androgen regulation of SURVWDWHVSHFL¿FDQWLJHQ 36$ ZHUHWHVWHGLQSURWHLQDQG51$OHYHO Activation status of transcription factor Stat5a/b and Stat3 and Akt and 0$3.VLJQDOLQJSDWKZD\VZHUHDQDO\]HGE\:HVWHUQEORWWLQJ
THE JOURNAL OF UROLOGY®
189
RESULTS: The growth of ADMTN cells in culture was reduced by six-fold if grown in the absence of DHT with a marked effect of androgen deprivation on the cell morphology. ADMTN cells express high levels of AR and PSA. AR in ADMTN has H874Y mutation in the ligandbinding domain which is identical to that in primary CWR22 xenograft tumor. When inoculated s.c. into nude mice supplemented with DHTpellets, big tumors formed rapidly in 20/20 mice, whereas no tumors developed in mice without circulating DHT. Importantly, PSA expression in ADMTN cells is regulated by androgens and the serum levels correlate with the ADMTN tumor volumes when grown as subcutaneous tumors in nude mice. In ADMTN cells, transcription factors Stat5a/b and Stat3, which are highly activated in prostate cancers of high histological grade, are activated by prolactin and IL-6 respectively. Akt and MAPK signaling pathways are active in ADMTN cells. CONCLUSIONS: This newly established androgen-regulated and tumorigenic human prostate cancer cell line, ADMTN, is likely to SURYLGHWKHSURVWDWHFDQFHUUHVHDUFK¿HOGDYDOXDEOHWRROIRUVWXGLHV on androgen-regulated growth mechanisms of prostate cancer and for development of new therapies and diagnostics for prostate cancer. Source of Funding: This work was supported by American Cancer Society (RSG-04-196-01-MGO), DOD Department of Defense Prostate Cancer Grants (W81XWH-05-01-0062 and W81XWH-07-1-0411) and NIH NCI (1RO1CA113580-01A1).
541 ANDROGEN ABLATION INDUCES SENESCENCE CHARACTERISTICS IN VIVO AND IN VITRO Timo Laurila*, Jonathan A Ewald, Dawn R Church, Nima Almassi, David F Jarrard. Madison, WI. INTRODUCTION AND OBJECTIVE: Cellular senescence is a response to sub-lethal stressors that imparts a terminal growth arrest and characteristic phenotype. Androgen ablation is known to induce apoptosis in prostate cells, but whether it generates a senescent phenotype is unknown. METHODS: In vitro, androgen-dependent prostate cancer cell OLQHV/1&D3DQG59ZHUHFXOWXUHGLQHLWKHUDQGURJHQGH¿FLHQWRU replete media. Cells were harvested after 1, 3 and 5 days. SenescenceDVVRFLDWHG JHQH H[SUHVVLRQ ZDV DQDO\]HG E\ T3&5 WHFKQLTXH DQG VHQHVFHQFHDVVRFLDWHGEHWDJDODFWRVLGDVH 6$ȕJDO H[SUHVVLRQZDV determined. Morphology and cell cycle analysis was performed. In vivo, 45 sexually mature 3 month old male B6 mice were surgically castrated. Dorsolateral and ventral prostates (DLP and VP, respectively) were harvested after 3, 6 and 10 days. Control prostate tissues were harvested 10 days after a mock-surgery. Samples were evaluated for 6$ȕJDOH[SUHVVLRQDVZHOODVVHQHVFHQFHDVVRFLDWHGKHWHURFKURPDWLQ SURWHLQJDPPD +3Ȗ 5(68/76 ,Q YLWUR SDUWLDO DQGURJHQ GH¿FLHQF\ UHVXOWHG LQ viable cells by PI analysis, as well increased side-scatter consistent with senescence. At 5 days after androgen ablation, the expression of the senescence markers GLB1, BRAK, CSPG2 and IGF2 increased in LNCaP, and a subset increased in 22RV1. In vivo, no induction of 6$ȕJDO ZDV VHHQ LQ 93 WLVVXHV ,Q FRQWUDVW WKH '/3 GHPRQVWUDWHV LQFUHDVHG6$ȕJDODFWLYLW\GD\VDIWHUFDVWUDWLRQ$XJPHQWHGQXFOHDU +3ȖIRFLDUHREVHUYHGDIWHUFDVWUDWLRQLQWKH'/3,QFUHDVHGFOHDYHG poly-ADP ribosyl polymerase (PARP), a marker of apoptosis is seen in both prostate tissues after castration. &21&/86,216 $QGURJHQGH¿FLHQF\LQGXFHVFKDUDFWHULVWLFV of cellular senescence in a subset of prostate cells in vivo, and in prostate cancer cell lines in vitro. Previous studies have shown the DLP is less androgen sensitive than the VP. These results suggest that mechanisms associated with senescence induction are involved in the response of the prostate to androgen ablation. Source of Funding: None
542 CONSERVATION OF NKX BINDING SITES WITHIN THE TMPRSS2 PROMOTER AND HORMONE REGULATION OF THE ERG GENE EXPRESSION IN PROSTATE CANCER Albert Dobi*, Rajesh Thangapazham, Shiv Srivastava. Rockville, MD. INTRODUCTION AND OBJECTIVE: Frequent overexpression of the ERG protooncogene is a hallmark of prostate cancer. Elevated
survival in vivo as long as the duration of the off cycle remains similar to that in standard IAA (about 10 days in our studies) - when the off F\FOHV DUH DOORZHG WR EH PD[LPDOO\ H[WHQGHG WKH VXUYLYDO EHQH¿W LV lost. Therefore, we proposed to examine the gene expression changes WKDWRFFXULQWKH¿UVWIHZGD\VRIWKHRIIF\FOHLQ,$$DQGK\SRWKHVL]HG WKDWWKHDGGLWLRQRIGXWDVWHULGH ' GXULQJWKHµRIIF\FOH¶RI,$$VKRXOG differentially enhance the expression of androgen responsive growth suppressing genes relative to standard IAA. METHODS: After establishment of LNCaP tumors, nude mice (Balb/C- Nu) were castrated and followed up for 7-10 days before being UDQGRPL]HGWR&RQWLQXRXVDQGURJHQDEODWLRQ &$$ ,$$&$$'RU IAA + D. Testes-intact animals with or without dutasteride were kept as FRQWUROV36$7DQG'+7OHYHOVZHUHDVVD\HG0LFHZHUHVDFUL¿FHG GD\VDIWHUUDQGRPL]DWLRQDQGWXPRUJHQHH[SUHVVLRQZDVVWXGLHGXVLQJ real time reverse transcriptase PCR. 5(68/76 $IWHU WKUHH GD\V RI µRII F\FOH¶ LQWHUYHQWLRQ WKH expressions of EAF2/U19 and calereticulin (both androgen responsive growth suppressive genes) were higher in the IAA+D group as compared WR WKH RWKHU LQWHUYHQWLRQ JURXSV ,$$' 9V ,$$ IROG DQG IROG UHVSHFWLYHO\S )LJVDDQGE 7KHH[SUHVVLRQRI36$GLGQRW show any difference. &21&/86,216 ĮUHGXFWDVHLQKLELWLRQWRWKHRIIF\FOHRI ,$$ XSUHJXODWHV JURZWK VXSSUHVVLYH JHQHV DIWHU GD\V RI µRII F\FOH¶ treatment. These results provide an insight into the possible mechanisms WKURXJK ZKLFK RII F\FOH ĮUHGXFWDVH LQKLELWLRQ LPSURYHV VXUYLYDO Further studies need to focus on the time course of such events so as WREHVWGH¿QHWKHGXUDWLRQRIWKHRIIF\FOHWKDWPD[LPL]HVWKHTXDOLW\ RIOLIHEHQH¿WVRIDQGURJHQVZLWKRXWVDFUL¿FLQJWKHVXUYLYDODGYDQWDJH FRQIHUUHGE\,$$RIIF\FOHĮUHGXFWDVHLQKLELWLRQ
Source of Funding: This investigation was supported in part by NIH grants R01 CA 108675, 5 R01 DK51993, 1 P50 CA90386.
540 ESTABLISHMENT OF A NEW ANDROGEN-DEPENDENT AND HIGHLY TUMORIGENIC HUMAN PROSTATE CANCER CELL LINE, ADMTN Ayush Dagvadorj*, Shyh-Han Tan, Lucianne R Cavalli, Bassem R Haddad, Marja T Nevalainen. Philadelphia, PA, and Washington, DC. INTRODUCTION AND OBJECTIVE: We have established DQGFKDUDFWHUL]HGDQHZKXPDQSURVWDWHFDQFHUFHOOOLQH$'071IURP a primary CWR22 human prostate xenograft tumor. Primary CWR22 tumors are re-transplantable human prostate xenograft tumors in nude mice and they were originally established from a Gleason score 9 primary cancer with bone metastases ( Pretlow et al., 1993). The primary CWR22 prostate tumor is highly responsive to androgen deprivation with marked tumor regression after castration, mimicking the course of human disease. No cell lines displaying the growth characteristics of the original primary CWR22 tumors have existed until now. METHODS: Androgen regulation of the cell growth and tumor formation was tested in vitro and in vivo as subcutaneous tumor in nude mice. Androgen receptor (AR) expression and androgen regulation of SURVWDWHVSHFL¿FDQWLJHQ 36$ ZHUHWHVWHGLQSURWHLQDQG51$OHYHO Activation status of transcription factor Stat5a/b and Stat3 and Akt and 0$3.VLJQDOLQJSDWKZD\VZHUHDQDO\]HGE\:HVWHUQEORWWLQJ
THE JOURNAL OF UROLOGY®
189
RESULTS: The growth of ADMTN cells in culture was reduced by six-fold if grown in the absence of DHT with a marked effect of androgen deprivation on the cell morphology. ADMTN cells express high levels of AR and PSA. AR in ADMTN has H874Y mutation in the ligandbinding domain which is identical to that in primary CWR22 xenograft tumor. When inoculated s.c. into nude mice supplemented with DHTpellets, big tumors formed rapidly in 20/20 mice, whereas no tumors developed in mice without circulating DHT. Importantly, PSA expression in ADMTN cells is regulated by androgens and the serum levels correlate with the ADMTN tumor volumes when grown as subcutaneous tumors in nude mice. In ADMTN cells, transcription factors Stat5a/b and Stat3, which are highly activated in prostate cancers of high histological grade, are activated by prolactin and IL-6 respectively. Akt and MAPK signaling pathways are active in ADMTN cells. CONCLUSIONS: This newly established androgen-regulated and tumorigenic human prostate cancer cell line, ADMTN, is likely to SURYLGHWKHSURVWDWHFDQFHUUHVHDUFK¿HOGDYDOXDEOHWRROIRUVWXGLHV on androgen-regulated growth mechanisms of prostate cancer and for development of new therapies and diagnostics for prostate cancer. Source of Funding: This work was supported by American Cancer Society (RSG-04-196-01-MGO), DOD Department of Defense Prostate Cancer Grants (W81XWH-05-01-0062 and W81XWH-07-1-0411) and NIH NCI (1RO1CA113580-01A1).
541 ANDROGEN ABLATION INDUCES SENESCENCE CHARACTERISTICS IN VIVO AND IN VITRO Timo Laurila*, Jonathan A Ewald, Dawn R Church, Nima Almassi, David F Jarrard. Madison, WI. INTRODUCTION AND OBJECTIVE: Cellular senescence is a response to sub-lethal stressors that imparts a terminal growth arrest and characteristic phenotype. Androgen ablation is known to induce apoptosis in prostate cells, but whether it generates a senescent phenotype is unknown. METHODS: In vitro, androgen-dependent prostate cancer cell OLQHV/1&D3DQG59ZHUHFXOWXUHGLQHLWKHUDQGURJHQGH¿FLHQWRU replete media. Cells were harvested after 1, 3 and 5 days. SenescenceDVVRFLDWHG JHQH H[SUHVVLRQ ZDV DQDO\]HG E\ T3&5 WHFKQLTXH DQG VHQHVFHQFHDVVRFLDWHGEHWDJDODFWRVLGDVH 6$ȕJDO H[SUHVVLRQZDV determined. Morphology and cell cycle analysis was performed. In vivo, 45 sexually mature 3 month old male B6 mice were surgically castrated. Dorsolateral and ventral prostates (DLP and VP, respectively) were harvested after 3, 6 and 10 days. Control prostate tissues were harvested 10 days after a mock-surgery. Samples were evaluated for 6$ȕJDOH[SUHVVLRQDVZHOODVVHQHVFHQFHDVVRFLDWHGKHWHURFKURPDWLQ SURWHLQJDPPD +3Ȗ 5(68/76 ,Q YLWUR SDUWLDO DQGURJHQ GH¿FLHQF\ UHVXOWHG LQ viable cells by PI analysis, as well increased side-scatter consistent with senescence. At 5 days after androgen ablation, the expression of the senescence markers GLB1, BRAK, CSPG2 and IGF2 increased in LNCaP, and a subset increased in 22RV1. In vivo, no induction of 6$ȕJDO ZDV VHHQ LQ 93 WLVVXHV ,Q FRQWUDVW WKH '/3 GHPRQVWUDWHV LQFUHDVHG6$ȕJDODFWLYLW\GD\VDIWHUFDVWUDWLRQ$XJPHQWHGQXFOHDU +3ȖIRFLDUHREVHUYHGDIWHUFDVWUDWLRQLQWKH'/3,QFUHDVHGFOHDYHG poly-ADP ribosyl polymerase (PARP), a marker of apoptosis is seen in both prostate tissues after castration. &21&/86,216 $QGURJHQGH¿FLHQF\LQGXFHVFKDUDFWHULVWLFV of cellular senescence in a subset of prostate cells in vivo, and in prostate cancer cell lines in vitro. Previous studies have shown the DLP is less androgen sensitive than the VP. These results suggest that mechanisms associated with senescence induction are involved in the response of the prostate to androgen ablation. Source of Funding: None
542 CONSERVATION OF NKX BINDING SITES WITHIN THE TMPRSS2 PROMOTER AND HORMONE REGULATION OF THE ERG GENE EXPRESSION IN PROSTATE CANCER Albert Dobi*, Rajesh Thangapazham, Shiv Srivastava. Rockville, MD. INTRODUCTION AND OBJECTIVE: Frequent overexpression of the ERG protooncogene is a hallmark of prostate cancer. Elevated