Volume 49, Issue 1, Pages 161-168 (January 2006) p53 Protein Transduction Therapy: Successful Targeting and Inhibition of the Growth of the Bladder Cancer Cells  Miyabi Inoue, Kazuhito Tomizawa, Masayuki Matsushita, Yun-Fei Lu, Teruhiko Yokoyama, Hiroyuki Yanai, Atsushi Takashima, Hiromi Kumon, Hideki Matsui  European Urology  Volume 49, Issue 1, Pages 161-168 (January 2006) DOI: 10.1016/j.eururo.2005.08.019 Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 1 (A) Time-dependent changes of the level of transduced 11R-p53 in J82 and T24 cells. Purified 11R-p53 (0.1μM) was added to the culture medium of each cell line. After 2h, the cells were washed with PBS and were incubated in fresh medium in the absence of 11R-p53. The cells were further cultured and were harvested at each indicated time point after the addition of the protein. Western blot analysis was performed using anti-p53 antibodies. 11R-p53, 10ng purified 11R-p53; Cont, no addition of 11R-p53. (B) The localization of p53 lacking 11R (p53) and 11R-p53 in T24 cells. a and c, 11R-p53 localization (green); b and c, Merged image with 11R-p53 (green) and F-actin (red). Bars, 50μm. (C) Time-dependent changes of the transcription regulatory activity of 11R-p53 and p53 in J82 and T24 cells. A luciferase reporter vector carrying the p21/WAF1 promoter was transfected the cells. After 24h, the cells were transduced with 11R-p53 or p53. As a control (Cont.), the cells were only transfected with the luciferase reporter vector. n=6 in each group. European Urology 2006 49, 161-168DOI: (10.1016/j.eururo.2005.08.019) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 2 Effect of 11R-p53 and pAdex-p53 on the growth of J82 (A and C) and T24 (B and D) cells. A and B, Dose-dependent growth inhibition by 11R-p53. J82 and T24 cells were treated with each indicated concentration of 11R-p53 (♦, control; , 0.01μM; ■, 0.1μM; ◊, 0.5μM; ●, 1μM 11R-p53) every 24h. Cell growth was assessed every 24h by the WST assay. C and D, Comparison of the inhibitory effects of 11R-p53, 11R-GFP and pAdex-p53. Each indicated concentration of 11R-p53 or 11R-GFP was applied every 24h, while pAdex-p53 and –lacZ were transfected on Day 0. After 96h, the cells were harvested and the WST assay was performed. n=6 each, *, p<0.05; **, p<0.01; ***, p<0.001; +, p<0.0005 and #, p<0.0001 compared with the control. European Urology 2006 49, 161-168DOI: (10.1016/j.eururo.2005.08.019) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 3 Synergistic effects of 11R-p53 in combination with CDDP on the inhibition of growth (A and B) and induction of apoptosis (C and D) of J82 and T24 cells. A and B, Cell viability after each treatment was assessed using the WST assay 96h after adding CDDP. n=6 each, *, p<0.05; **, p<0.01 and #, p<0.001. C and D, The cells were treated with 11R-p53, CDDP or were co-treated with CDDP and 11R-p53 or pAdex-p53. After Hoechst staining, fluorescent signals were observed with a fluorescence microscope and apoptotic cells were identified by the presence of highly condensed or fragmented nuclei. n=50 in each treatment. *, p<0.01. European Urology 2006 49, 161-168DOI: (10.1016/j.eururo.2005.08.019) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 4 Protein transduction of 11R-GFP in bladder tumors in vivo. SCID mice were transplanted with J82 cells in the bladder. After 10 days, 11R-GFP was transurethrally transduced in the bladder. A, Immunohistochemical analysis of human epidermal keratin in the tumor-transplanted bladder. B, The expression of 11R-GFP in the bladder. C, Bright field image of the same area. D, Merged image of human epidermal keratin (red) and 11R-GFP (green). Colocalization of human epidermal keratin and 11R-GFP was observed. E, H-E staining in the boxed area in C. H-E staining revealed that the localization of the strong expression of 11R-GFP was colocalized with the tumor formation of J82 cells in SCID mice. Scale bars=100μm. European Urology 2006 49, 161-168DOI: (10.1016/j.eururo.2005.08.019) Copyright © 2005 Elsevier B.V. Terms and Conditions