The overall goal of this proposal is to understand the molecular basis of hepatocellular carcinoma in hepadnavirus carriers. The first two specific aims of the proposal will involve and in-depth study of a new system we have developed to study the role of topoisomerase activity in viral replication and integration.
Specific Aim 1 will focus on the mechanism by which Camptothecin (CPT), a specific Topoisomerase I inhibitor, inhibits viral replication in cytoplasmic capsids in a chicken hepatoma cell line (LMH) transfected with a mutant (IS) DHBV genome which accumulates cytoplasmic capsid particles and recycles DHBV DNA into the nucleus. In addition to time course and dose response experiments, site specific mutagenesis of DHBV DNA and immune precipitation experiments will establish the nature of the topoisomerase activity and provide new genetic mapping information of the DHBV Pol gene. We will also express the spacer domain of the DHBV Pol gene and test it for topoisomerase activity """"""""in vitro."""""""" Specific Aim 2 will study the effect of CPT on the transport of DHBV DNA to the nucleus and its processing in the nucleus. We will determine whether CPT treatment induces integrated molecules using an integration assay based on inverse PCR technology. The studies in Specific Aim 3 will focus on gene expression in precancerous nodules of the woodchuck. We will attempt to understand the mechanism of N-myc activation in precancerous nodules. By studying the activity of a group of selected genes by """"""""in situ"""""""" hybridization we will gain insight into molecular mechanisms associated with precancerous lesions. In conjunction with this, we will utilize two woodchuck liver epithelial cell lines to directly test the effects of IL1, IL6, and TNF-alpha on N-myc expression.
Specific Aim 4 will directly test the effect of IGF-II in hepatocarcinogenesis using transgenic mouse mating experiments. Using the first transgenic mouse line to produce IGF-II in the adult mouse liver (developed by us) we will determine if IGF-II functions synergistically with HBV envelope proteins in inducing HCC. In a reverse approach, we will also test whether knocking out the endogeneous IGF-II gene prevents or inhibits the HBsAg lesion from progressing to HCC. Finally, we will utilize our transgenic mouse models (HBsAg line 50-4, and SV40 T Ag) to determine whether the maternally imprinted IGF-II allele can be activated during hepatocarcinogenesis. This experiment will increase our general understanding of a possible tumor suppressor, or cancer protective, role of gene imprinting.
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