Hepatitis B virus (HBV) is an agent which infects man, integrates into the genome of a specific tissue (liver), and probably causes hepatocellular carcinoma (HCC). In the United States, there are over three quarters of a million individuals with persistent HBV infection and worldwide HCC is one of the leading causes of death from cancer. The purpose of this proposal is to examine the specificities, location and effects of the integrated HBV DNA sequences within the host genome; and, to examine host and other factors regulating the response to HBV infection. Using recombinant DNA and molecular hybridization technology, four specific questions will be addressed. 1. Does the HBV genome insert within a certain region of the host genome? 2. Since males are at a greater risk for persistent HBV infection and the development of HCC, is the HBV genome susceptible to hormonal regulation? 3. Does modification of DNA sequences, such as by methylation, contribute to HBV and host DNA expression? 4. Are transforming genes activated in HBV-associatd HCC? The methodology used to investigate these questions will include: construction of recombinant DNA molecules containing integrated HBV seuqences from DNA of HCC tumors containing only one site of integrated HBV DNA; isolation and characterization of host flanking sequences with use of a single copy sequence probe to map the sites of integration to a specific chromosome in a human X rodent somatic cell hybrid panel; in vitro effects of androgens and corticosteroids on cultured hepatoma cells containing HBV DNA with examination of transcription and protein production; determination of methylation in DNA obtained from HCC cells and HBV-infected hepatocytes by paired restriction digestion with methyl sensitive and insensitive enzymes followed by Southern blot analysis; and, transfection of HCC DNA into untransformed cells by Ca phosphate precipitation with subsequent assay for transformed phenotype in recipient cells. The long-term objectives of this proposal are to understand the molecular features of a naturally occurring disease in man whereby new genes are introduced and expressed in a tissue specific manner. This biologic process is central to elucidation of the mechanism involved in carcinogenesis. Furthermore, dissection of the host factors involved in persistent HBV infection should allow the development of new therapies.
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