1. siRNA-mediated Down-Regulation of Survivin Inhibits Bladder Cancer Cell Growth S. Fuessel, S.Ning, M. Kotzsch #, K. Kraemer, M. Kappler*, U. Schmidt, H. Taubert*, Manfred P. Wirth, and Axel Meye Department of Urology and # Institute of Pathology, Technical University of Dresden, Germany Department of Urology and # Institute of Pathology, Technical University of Dresden, Germany *Institute of Pathology, University of Halle-Wittenberg, Germany Deutsche Krebshilfe e.V. (grant number 1999.009.1/2) Tab.1Effects of treatment with siRNA SVV284 on cell cycle distribution 48h after transfection. The changes are values normalized to the luciferase siRNA control. Materials & Methods We selected two constructs: siRNA SVV284 and siRNA SVV094 complementary to the mRNA of survivin. As siRNA control we used luciferase GL3 duplex without homology to any human mRNA. All five investigated human BCa cell lines (EJ28, 5637, J82, RT112 and RT4) express moderate to high levels of survivin. Cells at 50-60% growth density were transfected for 4h with a mixture of lipofectin reagent and siRNA at a ratio of 3:1 (w/w) to yield a final siRNA concentration of 250nM. Inhibition of viability by siRNA was measured using the cell proliferation reagent WST-1 (Roche). Colony formation assays were performed to evaluate the long-term survival of treated cells. Cell cycle analysis of treated cells was carried out by flow cytometry (FACScan, BD Biosciences) using the CycleTest Plus DNA Reagent Kit (BD Biosciences). Rates of apoptosis were assessed by flow cytometry after double staining cells with propidium iodide (PI) and FITC-labeled Annexin V (Annexin V-FITC Apoptosis Dectection Kit I; BD Biosciences). The survivin mRNA was quantified by real time RT-PCR based on the TaqMan technology. Survivin protein was determined quantitatively using the survivin ELISA kit (DuoSet  IC-ELISA; R&D Systems). Results A significant reduction of viability was observed in siRNA SVV284 treated EJ28 cells and 5637 cells 72h after the start of transfection (Fig.1), but not in siRNA SVV094 treated cells (data not shown). Inhibition of proliferation is reflected by obvious prolongation of doubling times of EJ28, 5637 and J82 cells (Fig.2). Furthermore, we found a reduction of clonogenic survival in the range of 39-63% in all BCa cell lines studied resulting from transfection with siRNA SVV284 (Fig.3). siRNA SVV284 treatment resulted in a relative decrease of up to 18.5% (5637) of the number of cells entering the S phase 48h after transfection. Furthermore, we observed a remarkably increased number of cells arrested in the G2/M phase in three of five BCa cell lines (Tab.1). We found an increase of the total apoptotic cell population in siRNA treated BCa cells of 50 ‑ 60% as compared to luciferase control, e.g. in 5637 treated cells (Fig.4). Interestingly, siRNA SVV284 treatment caused a multinucleated (polyploid) cell population in EJ28 and 5637 cells in contrast to luciferase treatment (Fig.5) indicating failure of correct course of mitosis. The levels of survivin mRNA and protein in all cell lines - except for RT4 - were reduced specifically by at least 50% compared to cells treated with luciferase control in a period of 24 to 72 h after transfection (Fig.6). Conclusions siRNA SVV284 significantly reduced the growth of BCa cells and resulted in a G2/M arrest and S phase reduction. The inhibition of cell growth was associated with an induction of apoptosis and a failure of cytokinesis. These effects were accompanied by a specific, long-lasting reduction in survivin expression. The siRNA construct SVV284 that showed a remarkable efficiency of inhibition is directed at a putative single stranded region within the survivin mRNA. This region is also accessible for other nucleic acid based inhibitors as antisense oligodeoxynucleotides. The results emphasize that the anti-survivin siRNA treatment may represent a suitable adjuvant therapeutic approach to inhibit growth of BCa cells. Introduction & Objectives Survivin, a member of the inhibitor of apoptosis protein (IAP) family is accepted as a general target in cancer therapy because of its differential overexpression in the majority of tumors. In bladder cancer (BCa), its expression correlates with tumor grade, recurrence risk and survival. RNA interference (RNAi) is a recently described cellular mechanism leading to silencing of a specific gene caused by small interfering RNA duplexes (siRNAs) containing an antisense strand complementary to the appropriate target transcript. siRNAs are 21-mers to 23-mers of sense and antisense RNA oligonucleotides characterized by 3´-overhangs of two nucleotides in length. In this study, we compared the therapeutic efficiency of two survivin-specific siRNA constructs to inhibit the growth of five human BCa cell lines (EJ28, 5637, J82, RT112 and RT4). www.tu-dresden.de/meduro shuanglining@hotmail.com Fig.4 Rate of apoptosis 48h of 5637 cells after treatment with siRNA SVV284 and luciferase control (analysis of 1x10 3 cells) assessed by FITC-Annexin V- and PI-Staining. The percentages of early (lower right quadrant) and late (upper right quadrant) apoptotic events are indicated. siRNA SVV 284 luciferase control Annexin V-FITC Propidium iodide Fig.5 Morphology of EJ28 and 5637 cells after transfection with siRNA SVV284 and luciferase control. (magnification ×125). EJ28: siRNA SVV284EJ28: luciferase control 5637: luciferase control5637: siRNA SVV284 Fig.1 Effects on cellular viability (in %) after siRNA SVV284 treatment in EJ28 and 5637. The relative viability was normalized to the respective luciferase control. Data represent mean  SD of 6 experiments (**p<0.01, ***p<0.001 vs control, unpaired two-sided Student’s t-test). *** ** *** basal DT25 h 30 h 27 h 26 h 50-60 h Fig.2Relative cell duplication times (in %) after siRNA SVV284 transfection in five bladder cancer cell lines normalized to luciferase control. Fig.3 Colony formation assay 48h after transfection with siRNA SVV284 in five bladder cancer cell lines normalized to luciferase control (in %). Data represent mean ± SD of three experiments (*p<0.05, ***p<0.001 vs luciferase control, unpaired two-sided Student’s t-test). *** * A) B) Fig.6 Down-regulation of survivin expression after siRNA SVV284 transfection A)Relative survivin mRNA expression determined by quantitative RT-PCR B)Realtive expression of survivin protein determined by ELISA The data were normalized to luciferase control. Cell line TreatmentCell cycle distribution (in %) Relative change in S-phase Relative change in G2/M-phase G0/G1SG2/M EJ28 siRNA SVV28448.432.818.8 + 1.5%+ 35.3% Luciferase conrol53.832.313.9 5637 siRNA SVV28432.832.135.1 - 18.5%+ 106.5% Luciferase conrol43.639.417.0 J82 siRNA SVV28446.425.028.6 - 14.4%+ 91.9% Luciferase conrol55.929.214.9 RT112 siRNA SVV28453.330.316.4 - 0.2%- 5.3% Luciferase conrol52.330.417.3 RT4 siRNA SVV28475.916.18.1 - 5.1%+ 6.6% Luciferase conrol75.516.97.6