Malignant transformation of imortalized hepatocytes by Hepatitis B virus DNA

Persistent infection by hepatitis B virus (HBV) is epidemiologically correlated with the prevalence of hepatocellular carcinoma (HCC), but its role in tumor development is not yet understood. The long latency period between viral infection and HCC growth suggest an indirect tumorigenic potential of HBV and its complementation by oncogenic cofactor(s). To study this hypothesis we used the non-malignant immortalized hepatocyte line FMH202-1 (Paul. D. et al., Exptl. Cell Res. 175:354 (1988)) which have been derived from transgenic mice harbouring SV40 sequences under the transcriptional control of the metallothionein I promoter. After transfection of the cells with HBV dimer sequences linked to the SV40 early promoter they acquired tumorigenic potential causing the growth of tumors in nu/nu mice. These tumors have been identified as typical infiltrating and metastasizing undifferentliated HCC's. HBV-transfected hepatocyte lines released HBV particles and support reverse transcription of H BV RNA into protein-linked HBV DNA. In tumors derived from 2 clones tested, growing in nu/nu mice, a potential extinction of HBV gene expression was observed: Remarkably, levels of HBV X-mRNA remained unchanged in the tumors and tumorigenic lines, suggesting that expression of X-protein is associated with the induction and maintenance of the malignant state in this system. An extensive rearrangement and amplification of integrated HBV sequences were observed in tumors and tumor-derived cell lines. Protooncogene expression of the silent c-fos in the hepatocyte line was strongly enhanced in HBV-transfected cells. In contrast, constitutive c-myc expression was not significantly modified after transfection, however, it was effectively repressed in the tumors. After transfection with HBV DNA activation of specific families of endogenous retroviral sequences linke IAP and Mo-MLV occured with a massive enhancement in the tumors. The results demonstrate that transfection of HBV DNA Into the i m