874 EPIGENETIC MODULATION OF TUMOUR SUPPRESSOR GADD45 BETA EXPRESSION BY HCV LEADS TO DEFECTIVE CELL CYCLE ARREST AND MAY PLAY A ROLE IN LIVER CARCINOGENESIS

Abstract

Background and Aims: Chronic hepatitis C virus (HCV) infection is a major risk factor of hepatocellular carcinoma (HCC). Sustained oxidative stress and elevated DNA damage are considered major contributors to HCC. Gadd45b is a major mediator of the cellular response to DNA damage. Our goal was to study the effect of chronic HCV infection on Gadd45b expression. Methods: Gadd45b expression were analyzed by western blotting or quantitative RT-PCR in: (a) paired non-tumoral and tumoral human liver samples from uninfected patients, and patients chronically infected with hepatitis B virus (HBV) or HCV; (b) Huh7 cell cultures harboring the HCV genotype 1b replicon and the JFH1 infectious strain; (c) liver tissues from 18 month old wild-type (wt) and transgenic mice expressing the full-length HCV open reading frame (FL-N/35). Primary hepatocytes were isolated from 7 month old mice. Promoter hypermethylation was assessed by the effect of 5-azacytidine (a DNA methyltransferase inhibitor) on Gadd45b mRNA expression. Cell cycle arrest and mitotic indices were assessed by the presence of phospho-histone H3 after UV-C irradiation. Results: Gadd45b expression was abrogated in tumoral tissue from HBV-positive or -negative patients, whereas it was found at normal levels in non-tumoral tissues. In contrast, in HCV-infected individuals, Gadd45b was absent from both tumoral and nontumoral tissues. Gadd45b expression was also reduced in Huh7 cells exhibiting HCVRNA replication (although these cells originate from a liver tumor). A 2-fold decrease in levels of Gadd45b mRNA was observed in livers of FL-N/35 mice compared to controls, whereas amounts of related Gadd family members remained unaffected. Activation of the Gadd45b promoter was inhibited in the presence of HCV, with cells isolated from FL-N/35 mice demonstrating a 2-fold reduction in promoter activity compared to wt controls. Reduced murine Gadd45b expression was associated with decreased cell cycle arrest in response to DNA damage, and both Gadd45b expression and cell cycle arrest were restored to wt levels by treatment with 5-azacytidine. Conclusions: These data demonstrate that HCV protein expression is associated with inhibition of Gadd45b expression through hypermethylation of its promoter, adversely altering the DNA damage response and potentially promoting liver carcinogenesis.