Adenovirus-mediated downregulation of the ubiquitin ligase RNF8 sensitizes bladder cancer to radiotherapy

Abstract

The ubiquitin ligase RNF8 promotes the DNA damage response (DDR). We observed that the expression of RNF8 was increased in bladder cancer cells and that this change in RNF8 expression could be reversed by adenovirus-mediated shRNA treatment. Moreover, we found that RNF8 knockdown sensitized bladder cancer cells to radiotherapy, as demonstrated by reduced cell survival. Additionally, the absence of RNF8 induced a high rate of apoptosis and impaired double-strand break repair signaling after radiotherapy. Furthermore, experiments on nude mice showed that combining shRNF8 treatment with radiotherapy suppressed implanted bladder tumor growth and enhanced apoptotic cell death in vivo. Altogether, our results indicated that RNF8 might be a novel target for bladder cancer treatment.

Keywords: RNF8; adenovirus; bladder cancer; gene therapy; radiotherapy.

Figure 1. RNF8 is highly expressed in bladder cancer cells and accumulates at DSB sites

A. Western blotting analysis of T24, BIU87, 5637 and SV-HUC-1 cell lines for RNF8. B. Western blotting analysis of bladder cancer patient carcinoma and normal control bladder tumor-adjacent tissues for RNF8. The numbers 1 to 4 and 1C to 4C represent the four randomly selected bladder cancer samples and their controls, respectively. C. Quantitative analysis of the Western blotting bands corresponding to RNF8. NBC: normal bladder cells; BCC: bladder cancer cells; NBT: normal bladder tissues; BCT: bladder cancer tissues. * indicates statistical significance compared to the control (P < 0.05). D. Immunofluorescence staining for RNF8 in the T24, BIU87, and 5637 cell lines. Cells were stained using an anti-RNF8 antibody and counterstained with DAPI. Scale bar, 5 μm. E. After irradiation (2 Gy), the cells were fixed, stained using anti-RNF8 and anti-γ-H2AX antibodies and counterstained with DAPI. Scale bar, 5 μm.

Figure 2. Knockdown of RNF8 impairs the radioresistance of bladder cancer cells

A. Western blotting for RNF8 expression after shRNF8 transfection. shNull, transfected with empty vector; shRNF8, transfected with the shRNF8-harboring vector. B.-D. Colony formation assay of cells after exposure to different doses of IR. T24, BIU87 and 5637 cells transfected with the empty adenoviral vector or the shRNF8-harboring adenoviral vector were irradiated at the indicated doses. The numbers of colonies formed in each plate were normalized to the number of colonies in the empty vector-infected, untreated cells, and all experiments were performed in triplicate. Error bars, SD. * indicates statistical significance compared to the control (*P < 0.05, **P < 0.01).

Figure 3. Knockdown of RNF8 leads to increased IR-induced apoptosis in bladder cancer cells

A. TUNEL assay of T24 cells. The cells were transfected with empty vector or the shRNF8-harboring vector and were exposed to the indicated doses of IR. Scale bar, 50 μm. B. Quantification of TUNEL-positive cells among the T24 cells. An average of 20 fields were counted at 60&times; magnification. The data are representative of 3 different experiments. * indicates statistical significance compared to the control (**P < 0.01). C. AO/PI double staining of T24 cells. The cells were transfected with the shRNF8-harboring vector and exposed to the indicated doses of IR. Scale bar, 10 μm. D. Quantification of PI-positive cells among the T24 cells. The data are representative of 3 different experiments. * indicates statistical significance compared to the control (***P < 0.001).

Figure 4. shRNF8 disrupts the DDR post-irradiation in bladder cancer cells

A. The formation of γ-H2AX foci was observed by fluorescence microscopy. T24 cells transfected with empty vector or the shRNF8-harboring vector were irradiated with 5 Gy X-ray irradiation and then allowed to recover for the indicated number of hours. Afterwards, the cells were fixed and stained using an anti-γ-H2AX antibody. Scale bar, 5 μm. B. The numbers of γ-H2AX foci at the indicated time points post-irradiation. At least 100 cells were quantified for each experiment. The data are presented as the means&plusmn;SD of at least 3 independent experiments. * indicates statistical significance (*P < 0.05; Student's t-test). C. T24 cells with or without RNF8 knockdown were treated with IR (5Gy) or were untreated. Then, the cells were analyzed by Western blotting for Ub-H2A and Ub-H2B expression. H4 was used as a loading control.

Figure 5. RNF8-related factors in the DSB repair pathway

A.-D. T24 cells transfected with shRNF8 or shNull were mock-treated (0 Gy) or irradiated (5 Gy). The cells were fixed 1 h post-IR and stained with anti-MDC1 (A), anti-53BP1 (B), anti-BRCA1 (C) or anti-RAP80 (D) antibodies and counterstained with DAPI. All experiments were independently repeated multiple times.

Figure 6. The effect of RNA interference to silence RNF8 on implanted T24 cell-based tumors in nude mice

A. Western blotting for RNF8 expression after injection of adenovirus in vivo. shNull, transfected with empty vector; shRNF8, transfected with the shRNF8-harboring vector. B. Growth curve of the subcutaneously implanted tumors in nude mice. The tumor size was compared between each group. A significant decrease in tumor volume in the shRNF8 group was observed due to IR. The arrow indicates the time point of IR treatment. C. Post-treatment photographs of nude mice injected with T24 cells followed by shNull- or shRNF8-harboring adenovirus administration combined with radiotherapy. D. Photographs of the subcutaneous tumors excised from the nude mice shown in B. The tumor sizes were measured and compared. E. H&E staining of the implanted tumors after IR treatment. Tumors from the shRNF8 group showed more necrosis and apoptosis than those from the shNull group. Scale bar, 50 μm.