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THE PROSTATIC EPITHELIAL SPECIFIC ANDROGEN RECEPTOR KNOCKOUT TRAMP MICE: AN ANIMAL MODEL FOR ANDROGEN INDEPENDENT PROSTATIC TUMOR
188
THE JOURNAL OF UROLOGY®
type and mutated ARs in the presence of a physiological concentration (1.0 nM) of DHT. It was found that the 17-glutamine tract elevated the luciferase activity of four AR mutants having partial function (PF). Co-expression of SRC-1 or TIF2 with wild-type or a mutated AR UHVXOWHGLQDVLJQL¿FDQWLQFUHDVHLQOXFLIHUDVHDFWLYLW\1H[WZHWHVWHG whether a combination of the 17-glutamine tract and TIF2 affected the transactivation of AR mutants. Two AR mutants having 17 or 25 CAG repeats were separately cotransformed with TIF2 into the reporter yeast and luciferase activity was measured. Compared to the 25-glutatime tract and TIF2 cotransformatants, luciferase expression was enhanced by 24-26% in the 17-glutamine and TIF2 cotranfectants in the presence of 1.0 nM DHT, by 65-95% in the presence of castration level (0.1 nM) of androgen, and by 25-29% in response to 1.0 uM DHEA. CONCLUSIONS: Our yeast results have shown that these AR modulators can synergistically modulate the transactivational capacity of the mutant ARs in response to a low concentration of DHT, or to '+($2XU¿QGLQJVVXJJHVWWKDW&D3FHOOVH[SUHVVLQJDPXWDWHG$5 in an environment containing multiple AR modulators will be more likely to progress to androgen independence. Source of Funding: NCI RO1 CA92069.
537 PROLONGATION OF ‘OFF CYCLE’ DURATION OF INTERMITTENT $1'52*(1$%/$7,21%<Į5('8&7$6(,1+,%,7,21'2(6 NOT IMPROVE SURVIVAL IN VIVO Yujuan Wang, Shubham Gupta*, Daniel Shevrin, Joel B Nelson, Zhou Wang. Pittsburgh, PA, and Evanston, IL. INTRODUCTION AND OBJECTIVE: Intermittent Androgen Ablation (IAA) is under investigation as a means of improving quality of life and retarding progression to androgen independence. The decisions WRWHUPLQDWHDQGUHLQVWLWXWHDQGURJHQZLWKGUDZDO µ2Q¶DQGµ2II¶F\FOH GXUDWLRQV DUHEDVHGRQWKHVHUXP3URVWDWHVSHFL¿FDQWLJHQ 36$ OHYHOV :HKDYHSUHYLRXVO\VKRZQWKDWDGGLQJ¿QDVWHULGH ) GXULQJWKHRIIF\FOH RI,$$FDQLPSURYHVXUYLYDOLQYLYRE\XVLQJ¿[HGGXUDWLRQVRIRIIF\FOH treatments (Eggener etal, 2006). A recent retrospective study showed WKDWDGGLWLRQRI¿QDVWHULGHGRXEOHGWKHGXUDWLRQRIWKHRIIF\FOHZLWKRXW FKDQJLQJSURJUHVVLRQWRDQGURJHQLQGHSHQGHQFH 6FKRO]HWDO In view of the above, we attempted to determine the extent to which off F\FOHĮUHGXFWDVHLQKLELWLRQFRXOGSURORQJWKHRIIF\FOHDQGWKHHIIHFW such prolongation would have on overall survival in a murine model. METHODS: Subcutaneous LNCaP tumors were established LQQXGHPLFH %DOE&1X $IWHUWKHWXPRUVUHDFKHGDVL]HRIFP the mice were castrated and followed up for 2 weeks after which they ZHUH UDQGRPL]HG WR UHFHLYH HLWKHU 7HVWRVWHURQH 7 >,$$ JURXS@ RU 7 ) >,$$) JURXS@ RU QRWKLQJ >FRQWLQXRXV DQGURJHQ DEODWLRQ &$$ JURXS@7KHRIIF\FOHZDVGLVFRQWLQXHGZKHQWKHWXPRUVL]HVLQFUHDVHG by 40%. Subsequent cycles were carried out similarly. Testes-intact animals were kept as controls. PSA, T, and Dihydrotestosterone levels were assayed. 5(68/76 2II &\FOH ¿QDVWHULGH GRXEOHG WKH ¿UVW RII F\FOH duration (Fig 1a). This increase in the duration of the off cycle did QRW WUDQVODWH LQWR DQ LQFUHDVH LQ RYHUDOO VXUYLYDO )LJ E UDWKHU WKH survival advantage of IAA+F over IAA that we previously reported was eliminated. &21&/86,216 2II&\FOH¿QDVWHULGHLPSURYHVVXUYLYDOZKHQ WKHµRII F\FOHVDUHVLPLODUUHODWLYHWRVWDQGDUG,$$:KHQWKHµRIIF\FOH¶ is allowed to increase maximally, the survival advantage is lost. Taken together, these results argue that maximum possible lengthening of the off cycle may diminish survival and may not be advisable. Further studies should focus on delineating the nature and the time-course of the pathways that improve survival in the IAA+F group, so as to best GH¿QHWKHGXUDWLRQRIWKHRIIF\FOH
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
Source of Funding: This investigation was supported in part by NIH grants R01 CA 108675, 5 R01 DK51993, 1 P50 CA90386.
538 THE PROSTATIC EPITHELIAL SPECIFIC ANDROGEN RECEPTOR KNOCKOUT TRAMP MICE: AN ANIMAL MODEL FOR ANDROGEN INDEPENDENT PROSTATIC TUMOR Yuanjie Niu*, Ruifa Han, Shuyuan Yeh, Edward M Messing, Chawnshang Chang. Tianjin, China, and Rochester, NY. INTRODUCTION AND OBJECTIVE: To study androgen receptor (AR) functions in prostate tumor epithelium, we developed the SURVWDWH HSLWKHOLDO VSHFL¿F$5 NQRFNRXW WUDQVJHQLF DGHQRFDUFLQRPD mouse prostate mice (pes-ARKO-TRAMP). 0(7+2'6 $5 ZDV VSHFL¿FDOO\ NQRFNHG RXW LQ SURVWDWH HSLWKHOLXPLQZKROHSURVWDWHRI75$03PLFHE\ÀR[$5DQGSUREDVLQ &UHVWUDWHJ\3UREDVLQ&UHVSHFL¿FH[SUHVVLRQLQSHV$5.275$03 mice was determine by mating this pes-ARKO-TRAMP mouse with ROSA26R-B-Gal mouse. Pes-ARKO-TRAMP and Wt TRAMP littermate PLFH ZHUH VDFUL¿FHG LQ GLIIHUHQW WLPH SRLQWV IURP ZNV WR ZNV WR determine the tumor growth and lymph node metastasis. CK8/18 and CK5 were used as cell markers for luminal and basal epithelial cells respectively. The growth and apoptosis of luminal and basal cells were detected by double staining cell markers with BrdU incorporation or TUNEL assay. Mechanical investigation using cDNA microarray and Q-PCR were carried out. RESULTS: Laser capture microdissection Q-PCR and Immunohistochemical analyses of prostate tumors using the C-19 DQWLERG\ VSHFL¿F WR WKH$5¶V &WHUPLQDO UHJLRQ VKRZHG WKDW$5 ZDV knockdown in epithelial, but not stromal cells. Probasin-cre was expressed in most of epithelial luminal cells and near 45% of epithelial basal cells. Loss of AR in prostate epithelium of pes-ARKO-TRAMP mice result in increased apoptosis in CK8/18 positive luminal epithelial cells, increased proliferating in CK5 positive basal epithelial cells and increased CK5/CK8 double positive intermediate cells. Mice lacking the prostate epithelial AR developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that AR might modulate many metastasis-related JHQHV VXFK DV 003 ,/ ,*) &2; 1(3 S DQG 71)Į YLD 7*)ȕVLJQDOLQJ CONCLUSIONS: Pes-ARKO-TRAMP mice is a good animal model for androgen-independent prostate tumor experiments to develop new drugs. Source of Funding: None
539 Į5('8&7$6(,1+,%,7,21'85,1*7+(2))&<&/(2) INTERMITTENT ANDROGEN ABLATION UPREGULATES TUMOR SUPPRESSIVE ANDROGEN RESPONSE GENES Shubham Gupta*, Yujuan Wang, Daniel Shevrin, Zhou Wang. Pittsburgh, PA, and Evanston, IL. INTRODUCTION AND OBJECTIVE: Previous studies suggest that relative to dihydrotestosterone (DHT), testosterone (T) is a weak inducer of proliferation but a potent inducer of differentiation during UHJURZWK RI UHJUHVVHG SURVWDWH )XUWKHUPRUH DGGLWLRQ RI ¿QDVWHULGH GXULQJWKHµRIIF\FOH¶RI,QWHUPLWWHQW$QGURJHQ$EODWLRQ ,$$ FDQLPSURYH
THE JOURNAL OF UROLOGY®
type and mutated ARs in the presence of a physiological concentration (1.0 nM) of DHT. It was found that the 17-glutamine tract elevated the luciferase activity of four AR mutants having partial function (PF). Co-expression of SRC-1 or TIF2 with wild-type or a mutated AR UHVXOWHGLQDVLJQL¿FDQWLQFUHDVHLQOXFLIHUDVHDFWLYLW\1H[WZHWHVWHG whether a combination of the 17-glutamine tract and TIF2 affected the transactivation of AR mutants. Two AR mutants having 17 or 25 CAG repeats were separately cotransformed with TIF2 into the reporter yeast and luciferase activity was measured. Compared to the 25-glutatime tract and TIF2 cotransformatants, luciferase expression was enhanced by 24-26% in the 17-glutamine and TIF2 cotranfectants in the presence of 1.0 nM DHT, by 65-95% in the presence of castration level (0.1 nM) of androgen, and by 25-29% in response to 1.0 uM DHEA. CONCLUSIONS: Our yeast results have shown that these AR modulators can synergistically modulate the transactivational capacity of the mutant ARs in response to a low concentration of DHT, or to '+($2XU¿QGLQJVVXJJHVWWKDW&D3FHOOVH[SUHVVLQJDPXWDWHG$5 in an environment containing multiple AR modulators will be more likely to progress to androgen independence. Source of Funding: NCI RO1 CA92069.
537 PROLONGATION OF ‘OFF CYCLE’ DURATION OF INTERMITTENT $1'52*(1$%/$7,21%<Į5('8&7$6(,1+,%,7,21'2(6 NOT IMPROVE SURVIVAL IN VIVO Yujuan Wang, Shubham Gupta*, Daniel Shevrin, Joel B Nelson, Zhou Wang. Pittsburgh, PA, and Evanston, IL. INTRODUCTION AND OBJECTIVE: Intermittent Androgen Ablation (IAA) is under investigation as a means of improving quality of life and retarding progression to androgen independence. The decisions WRWHUPLQDWHDQGUHLQVWLWXWHDQGURJHQZLWKGUDZDO µ2Q¶DQGµ2II¶F\FOH GXUDWLRQV DUHEDVHGRQWKHVHUXP3URVWDWHVSHFL¿FDQWLJHQ 36$ OHYHOV :HKDYHSUHYLRXVO\VKRZQWKDWDGGLQJ¿QDVWHULGH ) GXULQJWKHRIIF\FOH RI,$$FDQLPSURYHVXUYLYDOLQYLYRE\XVLQJ¿[HGGXUDWLRQVRIRIIF\FOH treatments (Eggener etal, 2006). A recent retrospective study showed WKDWDGGLWLRQRI¿QDVWHULGHGRXEOHGWKHGXUDWLRQRIWKHRIIF\FOHZLWKRXW FKDQJLQJSURJUHVVLRQWRDQGURJHQLQGHSHQGHQFH 6FKRO]HWDO In view of the above, we attempted to determine the extent to which off F\FOHĮUHGXFWDVHLQKLELWLRQFRXOGSURORQJWKHRIIF\FOHDQGWKHHIIHFW such prolongation would have on overall survival in a murine model. METHODS: Subcutaneous LNCaP tumors were established LQQXGHPLFH %DOE&1X $IWHUWKHWXPRUVUHDFKHGDVL]HRIFP the mice were castrated and followed up for 2 weeks after which they ZHUH UDQGRPL]HG WR UHFHLYH HLWKHU 7HVWRVWHURQH 7 >,$$ JURXS@ RU 7 ) >,$$) JURXS@ RU QRWKLQJ >FRQWLQXRXV DQGURJHQ DEODWLRQ &$$ JURXS@7KHRIIF\FOHZDVGLVFRQWLQXHGZKHQWKHWXPRUVL]HVLQFUHDVHG by 40%. Subsequent cycles were carried out similarly. Testes-intact animals were kept as controls. PSA, T, and Dihydrotestosterone levels were assayed. 5(68/76 2II &\FOH ¿QDVWHULGH GRXEOHG WKH ¿UVW RII F\FOH duration (Fig 1a). This increase in the duration of the off cycle did QRW WUDQVODWH LQWR DQ LQFUHDVH LQ RYHUDOO VXUYLYDO )LJ E UDWKHU WKH survival advantage of IAA+F over IAA that we previously reported was eliminated. &21&/86,216 2II&\FOH¿QDVWHULGHLPSURYHVVXUYLYDOZKHQ WKHµRII F\FOHVDUHVLPLODUUHODWLYHWRVWDQGDUG,$$:KHQWKHµRIIF\FOH¶ is allowed to increase maximally, the survival advantage is lost. Taken together, these results argue that maximum possible lengthening of the off cycle may diminish survival and may not be advisable. Further studies should focus on delineating the nature and the time-course of the pathways that improve survival in the IAA+F group, so as to best GH¿QHWKHGXUDWLRQRIWKHRIIF\FOH
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
Source of Funding: This investigation was supported in part by NIH grants R01 CA 108675, 5 R01 DK51993, 1 P50 CA90386.
538 THE PROSTATIC EPITHELIAL SPECIFIC ANDROGEN RECEPTOR KNOCKOUT TRAMP MICE: AN ANIMAL MODEL FOR ANDROGEN INDEPENDENT PROSTATIC TUMOR Yuanjie Niu*, Ruifa Han, Shuyuan Yeh, Edward M Messing, Chawnshang Chang. Tianjin, China, and Rochester, NY. INTRODUCTION AND OBJECTIVE: To study androgen receptor (AR) functions in prostate tumor epithelium, we developed the SURVWDWH HSLWKHOLDO VSHFL¿F$5 NQRFNRXW WUDQVJHQLF DGHQRFDUFLQRPD mouse prostate mice (pes-ARKO-TRAMP). 0(7+2'6 $5 ZDV VSHFL¿FDOO\ NQRFNHG RXW LQ SURVWDWH HSLWKHOLXPLQZKROHSURVWDWHRI75$03PLFHE\ÀR[$5DQGSUREDVLQ &UHVWUDWHJ\3UREDVLQ&UHVSHFL¿FH[SUHVVLRQLQSHV$5.275$03 mice was determine by mating this pes-ARKO-TRAMP mouse with ROSA26R-B-Gal mouse. Pes-ARKO-TRAMP and Wt TRAMP littermate PLFH ZHUH VDFUL¿FHG LQ GLIIHUHQW WLPH SRLQWV IURP ZNV WR ZNV WR determine the tumor growth and lymph node metastasis. CK8/18 and CK5 were used as cell markers for luminal and basal epithelial cells respectively. The growth and apoptosis of luminal and basal cells were detected by double staining cell markers with BrdU incorporation or TUNEL assay. Mechanical investigation using cDNA microarray and Q-PCR were carried out. RESULTS: Laser capture microdissection Q-PCR and Immunohistochemical analyses of prostate tumors using the C-19 DQWLERG\ VSHFL¿F WR WKH$5¶V &WHUPLQDO UHJLRQ VKRZHG WKDW$5 ZDV knockdown in epithelial, but not stromal cells. Probasin-cre was expressed in most of epithelial luminal cells and near 45% of epithelial basal cells. Loss of AR in prostate epithelium of pes-ARKO-TRAMP mice result in increased apoptosis in CK8/18 positive luminal epithelial cells, increased proliferating in CK5 positive basal epithelial cells and increased CK5/CK8 double positive intermediate cells. Mice lacking the prostate epithelial AR developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that AR might modulate many metastasis-related JHQHV VXFK DV 003 ,/ ,*) &2; 1(3 S DQG 71)Į YLD 7*)ȕVLJQDOLQJ CONCLUSIONS: Pes-ARKO-TRAMP mice is a good animal model for androgen-independent prostate tumor experiments to develop new drugs. Source of Funding: None
539 Į5('8&7$6(,1+,%,7,21'85,1*7+(2))&<&/(2) INTERMITTENT ANDROGEN ABLATION UPREGULATES TUMOR SUPPRESSIVE ANDROGEN RESPONSE GENES Shubham Gupta*, Yujuan Wang, Daniel Shevrin, Zhou Wang. Pittsburgh, PA, and Evanston, IL. INTRODUCTION AND OBJECTIVE: Previous studies suggest that relative to dihydrotestosterone (DHT), testosterone (T) is a weak inducer of proliferation but a potent inducer of differentiation during UHJURZWK RI UHJUHVVHG SURVWDWH )XUWKHUPRUH DGGLWLRQ RI ¿QDVWHULGH GXULQJWKHµRIIF\FOH¶RI,QWHUPLWWHQW$QGURJHQ$EODWLRQ ,$$ FDQLPSURYH