Chronic infection with hepatitis B virus (HBV) has been linked by extensive epidemiological evidence with the incidence of hepatocellular carcinoma (HCC) in man. HBV sequences are found integrated in host cellular DNA from tumor biopsies and from several HCC-derived cell lines, including Hep 3B (containing one copy of HBV) and PLC/PRF/5 (containing 6-8 copies). Both cell lines synthesize the HBV surface antigen and synthesis appears to be regulated according to the growth state of the cells. The proposed research will investigate the integrated state of HBV in HCC cell lines, and in particular in the Hep 3B line, whose single integration site will greatly simplify analysis of the relationship to HCC. The HBV integration sites in this and other cell lines will be chromosomally mapped to determine if integration occurs on a common chromosome and if there is correlation with karyotypic abnormalities seen in HCC lines. The Hep 3B integration site will also be isolated by genomic cloning to determine the integrated configuration of viral sequences. Flanking host sequences from the genomic clone will be used as probes of Southern blots to study how integration occurred and Northern blots to determine whether any host transcription is affected. The proposed research will also study gene expression by the integrated HBV sequences. Potential regulatory mechanisms will be examined by DNase I sensitivity and methylation analysis, and these results will be examined by DNase I sensitivity and methylation analysis, and these results will be compared in cells grown under conditions known to alter HBsAg expression. Transfection experiments will then be undertaken to examine whether this regulation is controlled by native viral sequences or if an integrated configuration is required. A transfection assay will also be used to precisely map the HBsAg promoter, with particular attention to the possibility of regulatory sequences. These results, along with a detailed analysis of HBV in its integrated form, should increase our understanding of the mode of action of this potentially oncogenic virus.
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