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CD8+, CD25+, or GITR+ cells. Various immunological parameters (such as in vitro and in vivo cross-presentation, and in vivo CTL activity), tumor growth, and animal survival were measured after treatment.

RESULTS: When given intratumorally to mice bearing Rencatumors, Ad5-mTRAIL only minimally prolonged survival and induced low

in vivocell proliferation and CTL activity, and prolonged the survival of Rencatumor-bearing mice. Interestingly, depletion of CD4+ or CD25+ cells prior to therapy further enhanced survival and in vivo CTL activity. In addition, tumor-free mice depleted of CD4+ cells were able to reject a subsequent challenge of Renca cells, but not MHC-matched RM-11 prostate tumor cells, demonstrating the existence of immunologic memory.

CONCLUSIONS: These results collectively show that local treatment with Ad5-mTRAIL and CpG ODN can augment tumor antigen cross-presentation resulting in T cell proliferation, enhanced CTL activity, and increased animal survival.

Source of Funding: NCI (CA109446).

110INTERLEUKIN-21 ACTIVATES BOTH CYTOTOXIC T LYMPHOCYTE AND NATURAL KILLER CELL TO GENETATE ANTITUMOR RESPONSE IN MOUSE RENAL CELL CARCINOMAMasafumi Kumano*, Hideaki Miyake, Atsushi Takenaka, Sadao Kamidono, Masato Fujisawa. Kobe, Japan.

INTRODUCTION AND OBJECTIVE: Interleukin (IL)-21, a type 1 cytokine family member involved in humoral and cell mediated immune responses, has been demonstrated to enhance antitumor activities, including the induction of cytotoxic T lymphocyte differentiation and increased cytotoxic activation of NK cells. However, it remains largely unknown whether IL-21 exerts antitumor activity against renal cell carcinoma (RCC), one of the most sensitive malignancies to immunotherapy. The objectives of this study were to evaluated the antitumor effects of IL-21 gene transfer into mouse RCC cells, RenCa,so that cells could spontaneously secrete IL-21, and to investigate the mechanisms underlying this antitumor effect.

METHODS: The IL-21 gene was introduced into RenCa cells by the liposome-mediated method. The in vivo antitumor effect of IL-21-secreting RenCa cells (RenCa/IL-21) was assessed by subcutaneous injection into syngeneic BALB/c mice. Mechanisms underlying the antitumor effects were investigated in syngeneic mice in which CD8 T, CD4 T or natural killer (NK) cells had been depleted using the corresponding antibody. The cytotoxic activity of splenocytes in mice injected with RenCa/IL-21 cells was determined using non-radioactive cytotoxicity assay. Immunohistochemical examinations were performed

study was also carried out. RESULTS: RenCa/IL-21 cells were almost all rejected following

subcutaneous injection into syngeneic mice. The antitumor effect of RenCa/IL-21 remained in mice in which CD4 T cells had been depleted, but was totally abrogated in mice depleted of CD8 T cells or NK cells. Cytotoxic activities of splenocytes were higher in RenCa/IL-21 rejected mice than in mice bearing parental RenCa. Immunohistochemical study also supported the involvement of CD8 T cells and NK cells in antitumor effect of RenCa/IL-21. Moreover, mitomycin C (MMC) treated RenCa/IL-21 inhibited tumor growth of parental RenCa at distant site.

CONCLUSIONS: IL-21 secreting RenCa cells could be rejected in syngeneic mice by activation of CD8 T cells as well as NK cells. Moreover, MMC treated RenCa/IL-21 cells can effectively work

important role of IL-21 as an immunocytokine therapy for patients with advanced RCC.

Source of Funding: None

111TUMOR-SPECIFIC IMMUNITY INDUCED BY CRYOABLATION IN A MURINE RENAL CELL CARCINOMA RE-CHALLENGE MODELYoung Hwii Ko*, Seok Ho Kang, Je Hyun Bae, Du Geon Moon, Hong Seok Park, Jun Cheon, Jung Gu Lee, Duck Ki Yoon, Je Jong Kim. Seoul, Republic of Korea.

INTRODUCTION AND OBJECTIVE: To evaluate the

and immunologic responses of cryoablation with that of surgical excision in a tumor re-challenge model.

METHODS: Sixty BALB/c mice with RENCA tumors that

excision. The mice successfully treated were re-challenged with RENCA

observed. To assess the immunologic response of each treatment modality, the FASC assay and the cytotoxicity assay using a 51Cr releasing assay were performed.

RESULTS: After re-inoculation of the RENCA cells, the

higher in cryoablation group (p=0.001). However, a re-inoculation trial using another cell line, CT26 cells, showed that the tumor growth rate

cryosurgery group showed an elevated CD3, CD4, CD8 T, and NK cell

increased cytotoxicity in the cryoablation group was manifested by an E:T ratio of 33:1, and was maintained at 0.4:1 (Fig 1). The cytotoxicity of the surgical excision group was similar to that of the tumor control group.

CONCLUSIONS: These results showed that cryosurgery, compared to surgical resection, was more effective in preventing tumor growth after re-challenge with RENCA cells and that this response was

Source of Funding: None

112HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE INHIBITOR (“STATIN”), ATORVASTATIN, TREATMENT PREVENTS AMINOGLYCOSIDE ASSOCIATED RENAL DAMAGE IN RATS THROUGH INHIBITION OF NFkB AND p38 MAPK PATHWAYSAbdulmutalip Simsek, Levent Ozcan, Mesut Cilli, Emre Can Polat, Adnan Somay, Mustafa Cekmen, Cevper Ersoz, Ahmet Arslan, Emin Ozbek*. Istanbul, Turkey.

INTRODUCTION AND OBJECTIVE: Aminoglycoside antibiotics including gentamicine are widely used in the treatment of gram- negative infections. Nephrotoxicity is the major side effect of these agents. In our previous study we have shown that nuclear factor kapa b (NFkB) pathway is involved in gentamicine induced nephrotoxicity

40 THE JOURNAL OF UROLOGY® Vol. 179, No. 4, Supplement, Sunday, May 18, 2008

in rats. Here we aimed to investigate the role of NFkB and p38 MAPK(mitogene -activated protein kinase) pathways and the protection of statin therapy of nephrotoxicity in rats.

METHODS: Adult male Sprague-Dawley rats were divided into three equal groups. In group 1 (n=6) the rats were control, in group 2 (n=6) injected with gentamicine ten consequitive days, 100 mg /kg/ d, ip and group 3 (n=6) treated gentamicine plus atorvastatin,15 mg/ kg/d, nasogastric gavage. At the 24 h after the last injection,

parts for biochemical analysis and light microscopic examination. Blood samples were also taken to assess the serum levels of urea,

malondialdehyde (MDA), end product of lipid peroxidation, reduced

for tubular necrosis on light microscopy and NFkB and p38MAPK ativities using immunohistochemistry. Results were compared using the Mann-Whitney U test.

RESULTS: Na+ and K+ concentrations among three groups were similar. However,Serum levels of urea and creatinine concentrations

with control and atorvastatin treated groups (p<0.05). In gentamicine treated rat kidneys there were wide tubular necrosis, but necrosis was minimal or absent in statin treated rats. NFkB and p38MAPK activities

comparable with control group. CONCLUSIONS: Statin therapy ameliorates aminoglycoside

associated renal damage presumeably antioxydant as well as NFkBand p38MAPK inhibitor properties. Statins also can be used clinically to prevent nephrotoxicity in high risk patients.

Source of Funding: None

113GROWTH INHIBITION OF RENAL CANCER CELLS BY HISTONE DEACETYLASE INHIBITORS, SUBEROYLANILIDE HYDROXAMIC ACID (SAHA) AND PXD101Gyeong Eun Min, Jung Jin Hwang, Changhee Yoo, Mi-Joung Kim, Yong-Sook Kim, Kanghyon Song, Seong Cheol Kim*, Taek Min Kwon, Jong Yeon Park, Tai Young Ahn, Choung-Soo Kim. Seoul, Republic of Korea.

INTRODUCTION AND OBJECTIVE: Histone acetylation controls transcription of many genes, including tumor suppressor genes and cell cycle regulators. Hydroxamate-based histone deacetylase inhibitors (HDACIs), such as SAHA and PXD101, cause the accumulation of acetylated histones. As a result, HDACIs induce differentiation and/or apoptosis in transformed cell and inhibit tumor growth in xenograft models. Therefore, HDACIs, expected to have anti-cancer effect, have been studied as single or in combination with various chemotherapeutic agents against various cancers. Because SAHA and PXD101 have growth-inhibitory effect on multidrug resistant cancer cells, these agents may be effective for the treatment of renal cancer that is one of the most common drug resistant tumors.

METHODS: For proliferation assay, we used CellTiter96® AQueous One Solution Proliferation Assay from Promega. Cell cycle

of caspases were examined by Western Bolt. RESULTS: In this study, we examined anticancer effect of

HDACIs alone or combination with 5-FU on three renal cell carcinoma cell lines, CAKI-1, SN12C, and UO-31. First, we examined acetylation of histone 3 proteins by HDACIs, SAHA and PXD101 in each cell line. SAHA or PXD101 increased acetylation of histone 3 in a dose dependent manner and inhibited growth of three cell lines with IC50s in the range 4.7-5.8 uM and 0.8-2.0 uM, respectively. To determine the mechanism

G2-M populations. In addition, caspase-9 and caspase-3 were cleaved by treatment with HDACIs and pro-caspase-8 was decreased. These results indicate that HDACIs arrest cell cycle to G2-M phase and induce apoptosis via activation of intrinsic mitochondria-dependent death

pathway and extrinsic death receptor pathways in renal cell carcinoma cells.

CONCLUSIONS: While further studies are needed, these results suggest that HDAC inhibitors alone or combination with 5-FUmay be considered as a therapeutic modality for renal cancer.

Source of Funding: None

114TARGETING SPHINGOSINE-1-PHOSPHATE RECEPTORS AS ANTI-TUMOR AND ANTI-ANGIOGENIC THERAPY IN RENAL CELL CARCINOMATeresa Sanchez*, Anna Pappalardo, Mei-Hong Li, Timothy Hla, Fernando Ferrer. Farmington, CT, and Hartford, CT.

INTRODUCTION AND OBJECTIVE: Sphingosine-1-phosphate (S1P), regulates a wide variety of cellular and physiological functions (i.e. migration, invasion, proliferation and angiogenesis), through the interaction with its G protein coupled receptors (GPCR)S1P1R-5R. These receptors are distinctly coupled to Gi, Gq and G12families of heterotrimeric G proteins. At the cellular level, S1P1R and S1P2R mediate opposite effects on cell migration due to the coupling of S1P1R to the Gi-phosphatidylinositol-3-kinase (PI3K) pathway and the strong coupling of S1P2R to the G12/13-Rho pathway. In addition, we have recently shown that S1P2R, unlike S1P1R, activates the tumor suppressor PTEN, accounting for the inhibitory effect of S1P2R on cell migration. Several studies, including ours, indicate that the ability of S1P to regulate migration and invasion in a given tumor cell line depends on the balance of expression of the pro-migratory S1P1R and the anti-migratory S1P2R. In the present study we investigated the regulation of migration, invasion and VEGF expression by S1P in the renal cell carcinoma (RCC) cell lines ACHN and 786-O.

METHODS: The RCC cell lines ACHN, derived from pleural effusion, and 786-O (von Hippel Lindau mutant) were used in this study. Total RNA was isolated and S1P1R, S1P2R, S1P3R and VEGFmRNA levels were determined by reverse transcription and real time PCR. Migration and invasion studies were performed under normoxic or

RESULTS: While S1P1R was predominantly expressed in ACHN, compared to S1P2R and S1P3R, 786-O cells expressed similar levels of S1P1R, S1P2R and S1P3R. Physiological concentrations of S1P (1µM) induced migration in ACHN and 786-O both under normoxic

antagonist VPC04461. Interestingly, S1P induced ACHN and 786-O cell invasion (2-3 fold induction) just in a hypoxic environment. S1P-induced invasion was also inhibited by VPC04461. Finally, we report the novel upregulation of VEGF expression by S1P in the ACHN cell line under normoxia.

CONCLUSIONS: Our data indicates that S1P, present in the tumor microenvironment, can induce RCC cell migration and invasion in a S1P1R-dependent way, as well as VEGF expression. Therefore, S1PR can potentially be targeted as antitumor and antiangiogenic therapy in RCC. S1P1R antagonists are currently being evaluated in a murine model of RCC.

Source of Funding: NIH K08DK070468A and SERAPHFundation.

115CD3+ LYMPHOCYTE APOPTOSIS AND GANGLIOSIDE GM2 POSITIVITY: POTENTIAL NOVEL PROGNOSTIC BIOMARKERS FOR RENAL CELL CARCINOMAMatthew N Simmons*, Amy Richmond, Soumika Biswas, Steven C Campbell, Andrew C Novick, James H Finke. Cleveland, OH.

INTRODUCTION AND OBJECTIVE: Renal cell carcinoma (RCC) tumor cells secrete ganglioside GM2, a glycosphingolipid that induces apoptosis and inhibits activation of CD3+ T cells [Cancer Research (2006)and GM2 positivity would be increased in peripheral circulating CD3+

METHODS: Venous peripheral blood samples were collected according to an IRB-approved protocol from January 2005 to October