trypan blue staining and MTT assay. The expression of specific pro- teins in the signaling pathway, Egr-1, Cdk5 and p35 were detected by immunoblotting. Subcellular localization of Cdk5 and p35 was analyzed by immunofluorescence microscopy. To identify the role of Cdk5 in ATRA-induced DU145 cells apoptosis, cells were treated with roscovi- tine, a potent and specific inhibitor of Cdk5 kinase and DNA content was measured using flow cytometry. RESULTS: Treatment with ATRA effectively inhibited the total living cell counts and the cell viability. Cdk5, p35 and Egr1 expression showed a significant increase in the treated cells. In the control cell, Cdk5 was seen primarily localized in the cytoplasma and p35 in both the nucleus and cytoplasma. After treatment with 1£gM ATRA, both Cdk5 and p35 protein expression were seen increased in the cytoplasm. Co-treatment with ATRA and the specific inhibitor of Cdk5, Roscovitine, retrieved the drop of cell viability which was induced by ATRA. We found that the accumulation of DU145 cells in sub-G1 phase was apparently increased by ATRA treatment whereas cotreatment with roscovitine could reverse this effect. It is believed that the accumulation of cells in sub-G1 phase indicates DNA fragmentation, which is a common index of apoptosis. CONCLUSIONS: Our results are the first demonstration of the biological function of Cdk5 kinase/p35 in ATRA related growth inhibition in hormone refractory prostate cancer cells. We hope that the applica- tion of this finding would help to increase the efficiency of clinical chemotherapy in hormone refractory prostate cancer in the near future. Source of Funding: This work was supported by Taiwan National Science Council (96-2628-B-005-013-MY3) and Chang Bing Show Chwan Memorial Hospital (CBSH 9905001), Taiwan, Republic of China 420 IN VIVO CASTRATION-RESISTANT ACCELERATION OF PROSTATE CANCER INHIBITED BY A SMALL MOLECULE INHIBITOR OF INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR/ INSULIN RECEPTOR TYROSINE KINASE Takamitsu Inoue*, Akita, Japan; Mahvash Zakikhani, Matei Mireuta, Marie-Jose ´ Blouin, Michael Pollak, Montreal, Canada INTRODUCTION AND OBJECTIVES: Increased serum insulin level has been reported to be associated with increased risk of prostate cancer diagnosis and recurrence. It is plausible that insulin signaling could influence prostate cancer evolution to castration resistant pros- tate cancer (CRPC). We investigated the in vivo inhibitory effect on androgen independent acceleration of LNCaP xenograft using small molecule inhibitor of insulin-like growth factor 1 receptor/insulin recep- tor tyrosine kinase in order to analyze the contribution of insulin signaling on the development of CRPC. METHODS: A total of 2  10 6 LNCaP cells were injected subcutaneously in right flank of the Balb/c nu/nu mice. When tumor volume was reached 200 mm3, mice were randomized to castrated group or non-castrated group and compared the tumor volume. When tumor volume was reached approximately 500 mm3, mice were randomized to inhibitor-treated group and non-treated group in each castrated and non- castrated group mice. Western blot analyses and RT-PCR were per- formed using the tumor tissue obtained at euthanasia. RESULTS: In the short term, the tumor volume was significantly decreased in the castrated group compared with the non-castrated group (p = 0.013); however, in the long term, the tumor volume was significantly increased in the castrated group compared to the non-castrated group (p  0.0001). In the castrated group, the tumor volume was significantly decreased by inhibitor treatment (p = 0.023) while there was no effect in the non-castrated group (p = 0.49). Western blot analyses using tumor lysate demonstrated that total IR expression was increased in castrated mice compared to non-castrated (p = 0.02), while total IGF-1R was decreased (p = 0.02). Phosphorylation of IR, which was augmentable by insulin injection, was inhibited by tyrosine kinase inhibitor. CONCLUSIONS: This experiment suggests that insulin signal- ing may be one of the pathways involved in progression of prostate cancer to castration resistance. Source of Funding: Supported by the Prostate Cancer Foundation (Santa Monica, CA, USA) and the Prostate Cancer Foundation of Canada Bladder and Urethra: Anatomy, Physiology and Pharmacology Podium Sunday, May 15, 2011 1:00 PM–3:00 PM 421 THE SURGICAL LEARNING CURVE FOR ARTIFICIAL URINARY SPHINCTER PROCEDURES COMPARED TO TYPICAL SURGEON EXPERIENCE Jaspreet Sandhu, Alexandra Maschino, Andrew Vickers*, New York, NY INTRODUCTION AND OBJECTIVES: The artificial urinary sphincter (AUS) is a well established treatment for male stress urinary incontinence. AUS surgery is a complex prosthetic procedure. It is plausible that operative outcomes improve with increasing surgeon experience. The object of this study was to study the learning curve for AUS surgery, measured in terms of reoperation rates, in contrast to the typical experience levels of surgeons in contemporary practice. METHODS: The study cohort consisted of 65,602 adult males who received an AUS after 1988. Data on reoperations were obtained from the manufacturer, which requires documentation for warranty Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011 THE JOURNAL OF UROLOGY e169