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Over-expression of HCV NS3/4A Influences Cell Apoptosis and Cellular Response to DNA Damage
Ren Laifeng1,3*, Tang Zizhi2, Wu Huiwen3, Xu Ning3, Liu Gang2, Zeng Ming2, Guo Liandi2*
1Department of Medical Laboratory Science, Fenyang College of Shanxi Medical University, Fenyang 032200, China; 2Developmental & Stem Cell Institute, West China Second University Hospital, Sichuan University, Chengdu 610041, China; 3Department of Oncology, Fenyang Hospital of Shanxi Province, Fenyang 032200, China
Abstract: NS3/4A is a proteases complex encoded by hepatitis C virus (HCV)， which is essential for the replication cycle of HCV. In this study， to investigate the role of NS3/4A in cellular apoptosis and response to DNA damage， the HCV NS3/4A gene was over-expressed in Huh7 cells. The cellular apoptosis and viability were determined by DAPI stains and MTT assay， and the results indicated that over-expression of NS3/4A could remarkably induce the apoptosis and decreased viability of Huh7 cells in vitro. The expression of γH2AX was determined by immunofluorescence (IF)， and the result showed that NS3/4A could induce DNA double-strand breaks (DSBs) in Huh7 cells. In addition， the results also showed that over-expression of NS3/4A resulted in a marked DSB repair defect after treating cells with X-ray (prolonged existence of the γH2AX foci). Furthermore， we could also detect decreased activatory phosphorylation event (Serine 1 981) of the protein kinase Ataxia-telangiectasia mutated (ATM) after Camptothecin (CPT) treatment in NS3/4A expressed Huh7 cells by Western blot. Together， these results indicate that over-expression of NS3/4A gene can lead to increased levels of DNA damage， inhibition of DSBs repair mediated by ATM， and activation of the apoptosis pathway in Huh7 cells.
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