(combination indexes 1, n = 6). Colony formation was also sup- pressed significantly (P 0.05). The combination decreased the ex- pression of cyclin D1 and CDK4 and increased the expression of p21, thus leading to the accumulation of the cells in the sub-G1 fraction. Mechanistically, the combination induced ER stress, as evidenced by the increased expression of glucose-regulated protein 78 and heat shock protein 70, and caused ubiquitinated protein accumulation syn- ergistically: 10 nM bortezomib caused ER stress and ubiquitinated protein accumulation only when combined with 50 M ritonavir. Inter- estingly, we found that the combination also enhanced histone acety- lation synergistically by decreasing the expression of HDACs. This histone acetylation would be another important mechanism of action and is thought to have caused the increased p21 expression. CONCLUSIONS: The combination of ritonavir and bortezomib induces apoptosis and inhibits the growth of bladder cancer cells by inducing ER stress and ubiquitinated protein accumulation. Histone acetylation is another important mechanism of the combination’s tu- mor-toxic action. Source of Funding: None 594 SPECIFIC INHIBITON OF NOTCH-2 AS A NOVEL THERAPY FOR INVASIVE BLADDER CANCER Tetsutaro Hayashi*, Kilian M. Gust, Wolfgang Jaeger, Shannon Awrey, Na Li, Manuel Altamirano-Dimas, Ralph Buttyan, Estelle Li, Ladan Fazli, Peter C. Black, Vancouver, Canada INTRODUCTION AND OBJECTIVES: Notch signaling guides decisions regarding cell fate, establishment of cell lineages, stem cell maintenance and differentiation during early development. Notch is active in many adult malignancies where it has been shown to regulate tumor cell proliferation and progression to aggressive disease. Previ- ously, we showed that Notch is active in certain bladder cancer cells and that active Notch2 signaling mediates cell migratory and invasive behaviors. Here we report outcomes of Notch2 knockdown or treatment with a unique Notch2 inhibitor, NRR2Mab, on these and other aggres- sive behaviors in our bladder cancer cell models. METHODS: UMUC3, UMUC13 and UMUC16 cells, represent- ing invasive bladder cancer cells with high Notch2 expression, were transduced with Notch2 or non-targeting shRNA in a lentiviral vector and were stably propogated. A Notch2-inactivating monoclonal anti- body (Mab), NRR2Mab, and an isotype-matched, non-targeting Mab were kindly provided by Genentech, Inc. (San Francisco, CA). Cells were grown under adherent (AH) or anchorage-independent conditions (AI) and the effects of Notch2 silencing or Mab inactivation were quantitatively assessed for changes in proliferation, migration and invasive activities using in vitro assays and, for expression of Notch2 or other common stem cell-related genes using quantitative RT-PCR, Western blotting and immunohistochemistry. In vivo effects were eval- uated in an orthotopic bladder cancer xenograft model with biolumines- cence imaging. RESULTS: Cells transduced with the Notch2 shRNA or treated with NRR2Mab demonstrated markedly decreased Notch2 expression. Stable silencing of Notch2 or treatment with NRR2Mab in UMUC3 and 13 diminished cell proliferation under AI but not AH conditions. Like- wise, when grown in AI conditions, UMUC3 and 13 were found to be enriched for expression of stem cell genes but Notch2 silencing or NRR2Mab diminished the expression of these genes. Coincidentally, Notch silencing and NRR2Mab treatment significantly inhibited cell migration and invasion of these cells. In vivo, stable Notch2 knockdown cells significantly inhibited xenograft growth for all 3 cell lines. NRR2Mab treatment of host mice also inhibited UMUC13 xenograft growth. CONCLUSIONS: Our results provide preclinical evidence that Notch2 may be a useful target to block aggressive behaviors and to prevent growth and/or progression of bladder cancer. Notch2 inhibition with a specific inhibitory monoclonal antibody is practical and warrants further investigation as a novel treatment strategy for bladder cancer. Source of Funding: AUA Foundation Canadian Cancer Society Research Institute Vancouver Coastal Health Research Institute. 595 GOLD NANOPARTICLE CAN SERVE AS AGENTS FOR IMMUNOMODULATION IN MURINE BLADDER TUMOR MODELS BY CAUSING THE CONFORMATIONAL CHANGES AND ATTENUATING THE BIOLOGICAL FUNCTION OF TGF-1 PROTEIN Yuh-Shyan Tsai*, Yu-Hung Chen, Pai-Chiao Cheng, Hsin-Tzu Tsai, Ai-Li Shiau, Tzong-Shin Tzai, Chao-Liang Wu, Tainan, Taiwan INTRODUCTION AND OBJECTIVES: Gold nanoparticles (AuNPs) have been widely used as carriers due to their great bio- compatibility and unique physical properties and potentially as thera- peutic agents for inactivating vascular endothelial growth factor (VEGF). Like VEGF, transforming growth factor-beta 1 (TGF-1) is a member of the cysteine-knot structural superfamily and plays a pivotal role in many diseases and is known as an immunosuppressive agent. The study is aimed to investigate whether AuNPs can serve as an intratumoral immunomodulation and its mechanism. METHODS: Murine bladder tumor-2 cells (MBT2), normal mu- rine mammary gland cells (NMuMg), C3H/HeN and NOD-SCID mice were used for in vitro and in vivo studies, including cell growth assay and tumor growth assay. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were used for exploring the mecha- nism of AuNP-TGF1 conjugation. RESULTS: The results reflected strong interactions between TGF-1 and the surface of AuNPs when incubated with serum-con- taining medium, demonstrating a time- and dose-dependent pattern from both immunoassays and Western blotting studies. Compared with other serum proteins which also can bind to AuNP surface, AuNP- TGF1 conjugate is a thermodynamically favored compound. Epithelial cells underwent epithelial-mesenchymal transition (EMT) upon treat- ment with TGF-1; however, treatment with AuNPs reversed this effect, as detected by cell morphology and expression levels of EMT markers. TGF-1 was found to bind to AuNPs through S-Au bonds by X-ray photoelectron spectroscopy. To understand why the activity of TGF-1 was attenuated after conjugating to AuNPs, Fourier transform infrared spectroscopy was employed to analyze the conformational changes of TGF-1 on the surface of AuNPs. The results indicated that TGF-1 underwent significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, resulting in the deactivation of TGF-1 protein. An in vivo experiment also showed that addition of AuNPs attenuated the growth of TGF-1- secreting murine bladder tumor-2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD-SCID mice, and this was asso- ciated with an increase in the number of tumor-infiltrating CD4+ and CD8+ T lymphocytes, and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. CONCLUSIONS: AuNPs may be a promising agent for modu- lating tumor immunity through inhibiting immunosuppressive TGF-1 signaling. Source of Funding: None 596 A NOVEL AND EFFICIENT GENE THERAPY FOR THE TREATMENT OF UROTHELIAL CARCINOMA Eduardo Landerer*, Maximiliano Bendek, Lorena Lobos, Miguel A ´ vila, Alexis Rivas, Vincenzo Borgna, Luis O. Burzio, Carlos Gonza ´ lez, Jaime Villegas, Santiago, Chile INTRODUCTION AND OBJECTIVES: The epidemiological data related to bladder cancer recurrence and mortality, support the Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013 THE JOURNAL OF UROLOGY e243