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MP-17.13: Silencing of clusterin gene with siRNA accelerates doxazosin induced apoptosis in PC-3 human prostate cancer cell lines
MODERATED POSTER SESSIONS
tate cancer cell growth and androgen receptor (AR) transcriptional activity. Results: PPAR-gamma is frequently overexpressed in androgen independent prostate cancer cell lines and 41% of human prostate cancer tissues. The effect of PPAR-gamma ligands, 15 deoxy-delta12, 14 prostaglandin J2 (15d-PGJ2) and troglitazone on the growth of a prostate cancer cell line was assessed. Both 15d-PGJ2 and troglitazone inhibited proliferation and DNA synthesis of prostate cancer cell lines in a dose-dependent manner, and increased the proportion of cells with S phase DNA content. Further, there was no significant change in the proportion of cells with G0/G1 and G2/M DNA content. PSA promoter reporter assays showed that troglitazone and 15d-PGJ2 down-regulated androgen stimulated reporter gene activity in LNCaP. The PSA promoter contains eight androgen receptor response elements. Interestingly, LNCaP with pioglitazone dramatically suppressed PSA protein expression without suppressing AR expression. Conclusions: These findings indicate that PPAR-gamma regulates AR transcriptional activity and PPAR-gamma ligand is a useful therapeutic agent for the treatment of prostate cancer. MP-17.11 Detection of TMPRSS2: ERG fusion gene in the urine of men with prostate cancer Dimitriadis E1, Arvanitakis T2, Thanos A2, Kalogeropoulos T2, Pallantzas A2, Apostolaki A3, Pandis N1 1 Department of Genetics; 2Urology Department; 3Pathology Department, Saint Savvas Anticancer Hospital, Athens, Greece Introduction: Recent studies attested that a high proportion of prostate cancer samples express fusion genes. These fusion genes occur when the 5’ region of the androgen regulated TMPRSS2 gene merges with one of the ETS family transcription factors, mainly ERG and rarely ETV1 and ETV4 (1). The TMPRSS2:ERG was also detected in the urine of patients with prostate cancer (3). The aim of this study was to investigate further the feasibility of the noninvasive detection of the fusion genes in the urine of men with prostate cancer. Methods: Urine samples were obtained following a digital rectal exam. A minimum of 30ml of urinary sample was centrifuged and the cell pellet was immediately processed for RNA extraction. Total RNA was extracted using the NucleoSpin
RNAII kit (Macherey-Nagel). The urine RNA samples were processed for cDNA synthesis using one of two methods; either SuperScript II reverse transcriptase protocol (Invitrogen) or MessageBOOSTER cDNA synthesis kit (Epicentre). NestedPCR and Real-Time PCR approaches were used for TMPRSS2:ERG and ETV1 detection, as previously described (2,3). RealTime PCR was also applied for the detection of mRNA PSA, as an indicator of prostate cells presence in the urine samples. Results: Herein, 11 samples have been analyzed so far. Six were tested positive for mRNA PSA with the SuperScript and 8 with the MessageBooster cDNA. Three out of these 8 samples tested positive for TMPRSS:ERG with the nested PCR and two (2) with the Real-Time approach. TMPRSS2 (exon1)-ERG (exon4) was the fusion transcript variant detected in all 3 positive samples. The TMPRSS2 (exon2)ERG (exon4) rearrangement was also detected in one of the samples. Conclusion: TMPRSS2:ETS fusion genes can be detected in the urine of prostate cancer patients. The nested PCR analysis combined with an RNA amplification method is suggested as the more sensitive approach in the detection of these fusion genes. Possibly, the collection of multiple urine samples of the same patient will further increase the sensitivity of this noninvasive diagnostic approach. MP-17.12 Bicalutamide promotes tumor growth in a novel androgen-dependent prostate cancer xenograft model derived from a bicalutamide-treated patient Shimizu Y, Yoshida T, Segawa T, Inoue T, Kobayashi T, Terada N, Nakamura E, Kinoshita H, Kamoto T, Ogawa O Department of Urology, Kyoto University Graduate School of Medicine, Kyoto, Japan Introduction: Although androgen ablation therapies are effective in controlling prostate cancer (CaP), virtually all CaP progress to androgen ablation-resistant cancer. Through the progression to androgen ablation-resistant CaP, some patients experience antiandrogen withdrawal syndrome (AWS). Recent studies demonstrate that the androgen receptor (AR) signaling continues to influence CaP growth even after androgen ablation. Some mutant ARs were reported to be activated by flutamide in vitro, and considered to be a potential mechanism for AWS. Here we report the development and characteriza-
UROLOGY 70 (Supplment 3A), September 2007
tion of a novel androgen-dependent CaP xenograft, KUCaP, in which mutant AR play an important role in cancer progression under bicalutamide treatment. Methods: We established KUCaP by transplantation of liver metastatic tissue of a bicalutamide-treated patient into male SCID mice. AR genes of KUCaP and clinical materials from the patient were sequenced to examine AR mutation. To investigate the role of mutant AR under bicalutamide treatment, PC-3 cells were transfected with wild-type or mutant ARs and transcriptional response to dihydrotestosterone and antiandrogens were analyzed. Mice bearing KUCaP were castrated and administered with bicalutamide. Xenograft volume and serum PSA levels were measured to examine the effect of bicalutamide treatment. Results: KUCaP had W741C mutation in the ligand-binding domain of AR and this mutation was developed in the liver metastatic tissue of the patient. Transactivation assay showed that W741C mutant AR was activated by bicalutamide. Although castration of mice bearing KUCaP repressed KUCaP growth and PSA production, bicalutamide administration promoted the growth and PSA secretion. These results suggested that W741C mutant AR which developed during bicalutamide treatment play an important role in AWS or bicalutamide treatment failure. Conclusion: We have established a novel CaP xenograft model KUCaP that may account for CaP progression during bicalutamide treatment. Development of mutant AR must be one of the important mechanism for AWS or androgen ablation-resistant CaP. Abstract Withdrawn Abstract Withdrawn MP-17.13 Silencing of clusterin gene with siRNA accelerates doxazosin induced apoptosis in PC-3 human prostate cancer cell lines Cha GS1, Yoo TK1, Youm YH1, Jo MK2, Lee C3 1 Eulji Medical Center, Seoul, Korea; 2Korea Cancer Center Hospital, Seoul, Korea; 3 Northwestern University, Chicago, IL, USA Introduction: Clusterin has been reported to be over-expressed in prostate cancer cells under stressful conditions. Expression of clusterin endows the cancer cells the ability to be resistant against apoptosis. Doxazosin, an alpha adrenoceptor
133
MODERATED POSTER SESSIONS
antagonist, is known to induce apoptosis of human prostate cells. We investigated the effect of clusterin siRNA oligonucleotides (CLU-HSS101985) alone or in combination with doxazosin on apoptosis of prostate cancer cells. Methods: PC-3 cells were cultured for 24 hr and transfected with 100 nM oligonucleotide of CLU-HSS101985. At 48 or 72 hr after transfection, cells were examined by western blot to confirm clusterin silencing. Doxazosin (25 M), either alone or in combination with CLU-HSS101985 (100nM), was treated for 12, 24 and 48 hr. Apoptosis in the cultured cells was evaluated by DNA fragmentation analysis and TUNEL assay. Clusterin expression in these cells was determined by immunohistochemical staining. Confocal microscopy image analysis was performed to evaluate apoptosis and clusterin expression. Results: Clusterin was localized mostly in TUNEL negative cells. Results of western blot analysis indicated that effective silencing of clusterin in PC-3 cells was accomplished 48 hr after siRNA treatment. In untransfected PC-3 cells, apoptosis began to appear 24 hr after treatment with doxazosin, and apoptosis was prominent at 48 hr, as determined by DNA fragmentation and TUNEL assay. On the other hands, in CLU-HSS101985-treated cells, apoptosis was evident at 12 hr after doxazosin treatment and was further increased at 48 hr. Cells treated with doxazosin and CLU-HSS101985 showed 51% more apoptosis than those treated with doxazosin alone. Conclusion: These results suggest that clusterin play an important role of protecting PC-3 cells from apoptosis. Clusterin knock-down by siRNA can effectively block the anti-apoptotic effect of clusterin. We suggest that the approach of a combination of clusterin siRNA and doxazosin treatment may be applied in the development of new treatment for advanced prostate cancer. MP-17.14 Survivin shRNA constructed in adenovirus vector and apoptosis induction of prostate cancer PC-3 cells Shan YX, Gao P, Yang DR Department of Urology, the Second Affiliated Hospital, Suzhou University, Suzhou, China Introduction: To construct adenovirus vector encoding two survivin short hairpin RNA (shRNA) and investigate its effects on hormone -refractory prostate cancer PC-3 cell line. Methods: Two shRNAs of survivin were
134
designed and synthesized respectively. Both were inserted into adenovirus DNA. The recombinant adenovirus vectors were confirmed by sequencing and agarose gel electrophoresis after restriction digestion. This recombinant construct was then linearlized and transfected into HEK 293 cell line to generate recombinant adenovirus. After transfection of it to PC-3 cells for 48 hours, the result was detected by RT-PCR Western blotting and flow cytometer. Results: The recombinant adenovirus vectors were constructed and the aim sequence was obtained. It could significantly reduce the expression of survivin mRNA and survivin protein in PC-3 cells. Meanwhile, the cells growth was inhibited and the apoptosis of PC-3 cells increased obviously. Conclusion: Suppression of survivin mRNA and survivin protein could inhibit the proliferation of PC-3 cells and increase apoptosis of PC-3 cells conspicuously.
MP-17.15 Expression of DD3PCA3 is androgen dependent Shaw G1,3, Purkis P2, Oliver T2,3, Prowse D3 1 University College Hospital NHS Foundation Trust; 2St. Bartholomew’s Hospital; 3Queen Mary and Westfield University, London, UK Introduction: PCA3Plus™ is being marketed by Bostwick Laboratories as a marker of prostate cancer, with greater specificity than serum PSA. Specific DD3PCA3 mRNA is isolated from purified prostate epithelial cells obtained from post prostate massage urine. The role of DD3PCA3 is unclear. It is a short sequence of RNA which does not code for any protein. Its function is unknown. Methods: LNCaP cells are an androgen dependent human prostate cancer cell line. LNCaP cells were grown in regular medium (containing androgen), then transferred to androgen free medium (charcoal stripped serum) for a period of 24 hours. Subsequently synthetic androgen (R1881) was added. RT-PCR using published primer sets and cycling characteristics for DD3PCA3 was performed. Housekeeper gene expression controls were used to calculate relative DD3PCA3 expression levels. Results: When these cells are placed in androgen free medium, the production of DD3PCA3 rapidly decreases to 10% of the level of expression seen in the presence of androgen within 24 hours. When R1881 (synthetic androgen) is added,
DD3PCA3 expression increases rapidly in a dose dependent manner. Conclusions: The observation that DD3PCA3 expression is androgen dependent corroborates observations made by the workers who first identified thei prostate cancer specific marker. However, this effect is not well known by those using this test and interpreting the results. Androgen dependence might be explained by the presence in the DD3PCA3 promoter of a fifteen base pair region that has high homology to the putative androgen responsive element in the KRT18 promoter. This finding may be of clinical significance and suggests that the interpretation of DD3PCA3 results should be made in the light of serum testosterone levels, particularly when patients are taking medication which might lower their serum testosterone levels. The role of DD3PCA3 expression levels in monitoring disease, particularly with reference to detection of androgen independence requires further evaluation. MP-17.16 Distinct role of DNA methyltransferases and methyl-CpG binding proteins on apoptosis, invasion, proliferation and migration of prostate cancer cell – an in-vitro study Yaqinuddin A1, Abbas F2, Qureshi S1 Departments of 1Biological and Biomedical Sciences, 2Surgery, The Aga Khan University, Karachi, Pakistan Introduction: Alterations in DNA methylation could lead to various molecular events resulting in development of cancer. Though aberrant methylation of the cytosine is known to affect expression of a lexicon of different genes, the role of DNA methyltranferases and methyl-CpG binding proteins in a variety of cellular processes including proliferation, metastasis and cell death remains poorly understood. We investigated the role of individual proteins involved in DNA methylation mechanism on cellular proliferation, apoptosis, migration and invasion of a metastatic prostate cancer cell line using an in-vitro model. Materials & Methods: By using a plasmid based RNA interference (RNAi) technology, we produced stably transfected prostate cancer lines (PC-3) with silencing of genes encoding DNA methyltransferases DNMT1 and DNMT3b, as well as methylCpG binding proteins MBD1 and MeCP2. We analyzed cellular proliferation, apoptosis, migration and invasion in these silenced cell lines compared with controls.
UROLOGY 70 (Supplment 3A), September 2007
tate cancer cell growth and androgen receptor (AR) transcriptional activity. Results: PPAR-gamma is frequently overexpressed in androgen independent prostate cancer cell lines and 41% of human prostate cancer tissues. The effect of PPAR-gamma ligands, 15 deoxy-delta12, 14 prostaglandin J2 (15d-PGJ2) and troglitazone on the growth of a prostate cancer cell line was assessed. Both 15d-PGJ2 and troglitazone inhibited proliferation and DNA synthesis of prostate cancer cell lines in a dose-dependent manner, and increased the proportion of cells with S phase DNA content. Further, there was no significant change in the proportion of cells with G0/G1 and G2/M DNA content. PSA promoter reporter assays showed that troglitazone and 15d-PGJ2 down-regulated androgen stimulated reporter gene activity in LNCaP. The PSA promoter contains eight androgen receptor response elements. Interestingly, LNCaP with pioglitazone dramatically suppressed PSA protein expression without suppressing AR expression. Conclusions: These findings indicate that PPAR-gamma regulates AR transcriptional activity and PPAR-gamma ligand is a useful therapeutic agent for the treatment of prostate cancer. MP-17.11 Detection of TMPRSS2: ERG fusion gene in the urine of men with prostate cancer Dimitriadis E1, Arvanitakis T2, Thanos A2, Kalogeropoulos T2, Pallantzas A2, Apostolaki A3, Pandis N1 1 Department of Genetics; 2Urology Department; 3Pathology Department, Saint Savvas Anticancer Hospital, Athens, Greece Introduction: Recent studies attested that a high proportion of prostate cancer samples express fusion genes. These fusion genes occur when the 5’ region of the androgen regulated TMPRSS2 gene merges with one of the ETS family transcription factors, mainly ERG and rarely ETV1 and ETV4 (1). The TMPRSS2:ERG was also detected in the urine of patients with prostate cancer (3). The aim of this study was to investigate further the feasibility of the noninvasive detection of the fusion genes in the urine of men with prostate cancer. Methods: Urine samples were obtained following a digital rectal exam. A minimum of 30ml of urinary sample was centrifuged and the cell pellet was immediately processed for RNA extraction. Total RNA was extracted using the NucleoSpin
RNAII kit (Macherey-Nagel). The urine RNA samples were processed for cDNA synthesis using one of two methods; either SuperScript II reverse transcriptase protocol (Invitrogen) or MessageBOOSTER cDNA synthesis kit (Epicentre). NestedPCR and Real-Time PCR approaches were used for TMPRSS2:ERG and ETV1 detection, as previously described (2,3). RealTime PCR was also applied for the detection of mRNA PSA, as an indicator of prostate cells presence in the urine samples. Results: Herein, 11 samples have been analyzed so far. Six were tested positive for mRNA PSA with the SuperScript and 8 with the MessageBooster cDNA. Three out of these 8 samples tested positive for TMPRSS:ERG with the nested PCR and two (2) with the Real-Time approach. TMPRSS2 (exon1)-ERG (exon4) was the fusion transcript variant detected in all 3 positive samples. The TMPRSS2 (exon2)ERG (exon4) rearrangement was also detected in one of the samples. Conclusion: TMPRSS2:ETS fusion genes can be detected in the urine of prostate cancer patients. The nested PCR analysis combined with an RNA amplification method is suggested as the more sensitive approach in the detection of these fusion genes. Possibly, the collection of multiple urine samples of the same patient will further increase the sensitivity of this noninvasive diagnostic approach. MP-17.12 Bicalutamide promotes tumor growth in a novel androgen-dependent prostate cancer xenograft model derived from a bicalutamide-treated patient Shimizu Y, Yoshida T, Segawa T, Inoue T, Kobayashi T, Terada N, Nakamura E, Kinoshita H, Kamoto T, Ogawa O Department of Urology, Kyoto University Graduate School of Medicine, Kyoto, Japan Introduction: Although androgen ablation therapies are effective in controlling prostate cancer (CaP), virtually all CaP progress to androgen ablation-resistant cancer. Through the progression to androgen ablation-resistant CaP, some patients experience antiandrogen withdrawal syndrome (AWS). Recent studies demonstrate that the androgen receptor (AR) signaling continues to influence CaP growth even after androgen ablation. Some mutant ARs were reported to be activated by flutamide in vitro, and considered to be a potential mechanism for AWS. Here we report the development and characteriza-
UROLOGY 70 (Supplment 3A), September 2007
tion of a novel androgen-dependent CaP xenograft, KUCaP, in which mutant AR play an important role in cancer progression under bicalutamide treatment. Methods: We established KUCaP by transplantation of liver metastatic tissue of a bicalutamide-treated patient into male SCID mice. AR genes of KUCaP and clinical materials from the patient were sequenced to examine AR mutation. To investigate the role of mutant AR under bicalutamide treatment, PC-3 cells were transfected with wild-type or mutant ARs and transcriptional response to dihydrotestosterone and antiandrogens were analyzed. Mice bearing KUCaP were castrated and administered with bicalutamide. Xenograft volume and serum PSA levels were measured to examine the effect of bicalutamide treatment. Results: KUCaP had W741C mutation in the ligand-binding domain of AR and this mutation was developed in the liver metastatic tissue of the patient. Transactivation assay showed that W741C mutant AR was activated by bicalutamide. Although castration of mice bearing KUCaP repressed KUCaP growth and PSA production, bicalutamide administration promoted the growth and PSA secretion. These results suggested that W741C mutant AR which developed during bicalutamide treatment play an important role in AWS or bicalutamide treatment failure. Conclusion: We have established a novel CaP xenograft model KUCaP that may account for CaP progression during bicalutamide treatment. Development of mutant AR must be one of the important mechanism for AWS or androgen ablation-resistant CaP. Abstract Withdrawn Abstract Withdrawn MP-17.13 Silencing of clusterin gene with siRNA accelerates doxazosin induced apoptosis in PC-3 human prostate cancer cell lines Cha GS1, Yoo TK1, Youm YH1, Jo MK2, Lee C3 1 Eulji Medical Center, Seoul, Korea; 2Korea Cancer Center Hospital, Seoul, Korea; 3 Northwestern University, Chicago, IL, USA Introduction: Clusterin has been reported to be over-expressed in prostate cancer cells under stressful conditions. Expression of clusterin endows the cancer cells the ability to be resistant against apoptosis. Doxazosin, an alpha adrenoceptor
133
MODERATED POSTER SESSIONS
antagonist, is known to induce apoptosis of human prostate cells. We investigated the effect of clusterin siRNA oligonucleotides (CLU-HSS101985) alone or in combination with doxazosin on apoptosis of prostate cancer cells. Methods: PC-3 cells were cultured for 24 hr and transfected with 100 nM oligonucleotide of CLU-HSS101985. At 48 or 72 hr after transfection, cells were examined by western blot to confirm clusterin silencing. Doxazosin (25 M), either alone or in combination with CLU-HSS101985 (100nM), was treated for 12, 24 and 48 hr. Apoptosis in the cultured cells was evaluated by DNA fragmentation analysis and TUNEL assay. Clusterin expression in these cells was determined by immunohistochemical staining. Confocal microscopy image analysis was performed to evaluate apoptosis and clusterin expression. Results: Clusterin was localized mostly in TUNEL negative cells. Results of western blot analysis indicated that effective silencing of clusterin in PC-3 cells was accomplished 48 hr after siRNA treatment. In untransfected PC-3 cells, apoptosis began to appear 24 hr after treatment with doxazosin, and apoptosis was prominent at 48 hr, as determined by DNA fragmentation and TUNEL assay. On the other hands, in CLU-HSS101985-treated cells, apoptosis was evident at 12 hr after doxazosin treatment and was further increased at 48 hr. Cells treated with doxazosin and CLU-HSS101985 showed 51% more apoptosis than those treated with doxazosin alone. Conclusion: These results suggest that clusterin play an important role of protecting PC-3 cells from apoptosis. Clusterin knock-down by siRNA can effectively block the anti-apoptotic effect of clusterin. We suggest that the approach of a combination of clusterin siRNA and doxazosin treatment may be applied in the development of new treatment for advanced prostate cancer. MP-17.14 Survivin shRNA constructed in adenovirus vector and apoptosis induction of prostate cancer PC-3 cells Shan YX, Gao P, Yang DR Department of Urology, the Second Affiliated Hospital, Suzhou University, Suzhou, China Introduction: To construct adenovirus vector encoding two survivin short hairpin RNA (shRNA) and investigate its effects on hormone -refractory prostate cancer PC-3 cell line. Methods: Two shRNAs of survivin were
134
designed and synthesized respectively. Both were inserted into adenovirus DNA. The recombinant adenovirus vectors were confirmed by sequencing and agarose gel electrophoresis after restriction digestion. This recombinant construct was then linearlized and transfected into HEK 293 cell line to generate recombinant adenovirus. After transfection of it to PC-3 cells for 48 hours, the result was detected by RT-PCR Western blotting and flow cytometer. Results: The recombinant adenovirus vectors were constructed and the aim sequence was obtained. It could significantly reduce the expression of survivin mRNA and survivin protein in PC-3 cells. Meanwhile, the cells growth was inhibited and the apoptosis of PC-3 cells increased obviously. Conclusion: Suppression of survivin mRNA and survivin protein could inhibit the proliferation of PC-3 cells and increase apoptosis of PC-3 cells conspicuously.
MP-17.15 Expression of DD3PCA3 is androgen dependent Shaw G1,3, Purkis P2, Oliver T2,3, Prowse D3 1 University College Hospital NHS Foundation Trust; 2St. Bartholomew’s Hospital; 3Queen Mary and Westfield University, London, UK Introduction: PCA3Plus™ is being marketed by Bostwick Laboratories as a marker of prostate cancer, with greater specificity than serum PSA. Specific DD3PCA3 mRNA is isolated from purified prostate epithelial cells obtained from post prostate massage urine. The role of DD3PCA3 is unclear. It is a short sequence of RNA which does not code for any protein. Its function is unknown. Methods: LNCaP cells are an androgen dependent human prostate cancer cell line. LNCaP cells were grown in regular medium (containing androgen), then transferred to androgen free medium (charcoal stripped serum) for a period of 24 hours. Subsequently synthetic androgen (R1881) was added. RT-PCR using published primer sets and cycling characteristics for DD3PCA3 was performed. Housekeeper gene expression controls were used to calculate relative DD3PCA3 expression levels. Results: When these cells are placed in androgen free medium, the production of DD3PCA3 rapidly decreases to 10% of the level of expression seen in the presence of androgen within 24 hours. When R1881 (synthetic androgen) is added,
DD3PCA3 expression increases rapidly in a dose dependent manner. Conclusions: The observation that DD3PCA3 expression is androgen dependent corroborates observations made by the workers who first identified thei prostate cancer specific marker. However, this effect is not well known by those using this test and interpreting the results. Androgen dependence might be explained by the presence in the DD3PCA3 promoter of a fifteen base pair region that has high homology to the putative androgen responsive element in the KRT18 promoter. This finding may be of clinical significance and suggests that the interpretation of DD3PCA3 results should be made in the light of serum testosterone levels, particularly when patients are taking medication which might lower their serum testosterone levels. The role of DD3PCA3 expression levels in monitoring disease, particularly with reference to detection of androgen independence requires further evaluation. MP-17.16 Distinct role of DNA methyltransferases and methyl-CpG binding proteins on apoptosis, invasion, proliferation and migration of prostate cancer cell – an in-vitro study Yaqinuddin A1, Abbas F2, Qureshi S1 Departments of 1Biological and Biomedical Sciences, 2Surgery, The Aga Khan University, Karachi, Pakistan Introduction: Alterations in DNA methylation could lead to various molecular events resulting in development of cancer. Though aberrant methylation of the cytosine is known to affect expression of a lexicon of different genes, the role of DNA methyltranferases and methyl-CpG binding proteins in a variety of cellular processes including proliferation, metastasis and cell death remains poorly understood. We investigated the role of individual proteins involved in DNA methylation mechanism on cellular proliferation, apoptosis, migration and invasion of a metastatic prostate cancer cell line using an in-vitro model. Materials & Methods: By using a plasmid based RNA interference (RNAi) technology, we produced stably transfected prostate cancer lines (PC-3) with silencing of genes encoding DNA methyltransferases DNMT1 and DNMT3b, as well as methylCpG binding proteins MBD1 and MeCP2. We analyzed cellular proliferation, apoptosis, migration and invasion in these silenced cell lines compared with controls.
UROLOGY 70 (Supplment 3A), September 2007