The Hepatitis B Virus X Protein Sensitizes HepG2 Cells To Chemotherapeutic Agent Bleomycin-induced DNA Double Strand Break Damage

Many evidences showed that the hepatitis B virus (HBV) is a major etiologic factor in hepatocarcinogenesis. The hepatitis B virus X protein (HBVx) is one of the four proteins translated from the HBV genome. the HBVx as a cofactor or tumor promoter, has been implicated in the process of liver oncogenesis. A highly probable tumor-initiating event is DNA damage, HBx can stimulate reactive oxygen species (ROS) generation in the mitochondria to cause DNA damage, and X protein may evoke malignant transformation by facilitating the culmination of the transformation process in the cell. However, the effect of HBx on host cell DNA double strand break (DSB) damage repair mechanisms remains to be further studied.Objective We used bleomycin to generate DSB which possesses a complex structure similar to that produced by oxidative processes and ionizing radiation, and investigated the DSB repair impact of HBx in HepG2 cells on G2/M checkpoint induced by bleomycin. We try to explore new ways to investigate HBV related HCC and the progress of the carcinogenesis.Methods To establish a HBVx stably expressing HepG2 cell line and the control HepG2 cell line in vitro, the recombinant plasmid and empty vector was transferred into the HepG2 cells by lipofection 2000. The hepatitis B virus X gene transferred cells were selectively cultured with G418 and identified by PCR and Western blot assay. Flow cytometric analysis was performed using a FACS Calibur ?ow cytometer to determine cell cycle profiles. Apoptotic cell death was also assessed by flow cytometry after staining the cells when the DNA content and cell granularity were assessed.at the same times, the expression of the tumor suppressor protein CtIP in the HBVx stably expressing HepG2 cells and the controlled cells were assayed with western blot,and the mRNA expression were detected by real-time PCR.Results We found that the HBVx stably expressing HepG2 cells exhibited increased sensitivity to DNA DSB damage, resulting in the increase of G2/M arrest and apoptosis of these cells compared to the control cell line transformed with empty vector. Additionally, by means of western blot and real-time PCR assay, HBVx decreased the expression levles of the tumor suppressor protein CtIP that controls the decision to repair DSB damage by homologous recombination (HR), and these cells displayed a reduced DNA repair capacity in response to DNA damage.Conclusions The research suggests that HBVx may affect DNA-repair pathways by disrupting the tumor suppressor protein CtIP, and at the same times, the initial increased G2/M arrest of the HBVx stably expressing HepG2 cells and the sustained increased apoptosis of these cells after treatment with Bleomycin suggest that the repair mechanism in the HBVx stably expressing HepG2 cells maybe impaired, hence predisposing these cells toward the apoptotic pathway, and on the other hand, resulted in accumulation of genomic instability in hepatocarcinogenesis.