The objective of this proposal is to identify the mechanism(s), on the molecular level, whereby the hepatitis B virus (HBV) encoded X antigen (HBxAg) causes primary hepatocellular carcinoma (PHC). The first specific aim takes advantage of transfection experiments showing that the cloned X- region of HBV DNA alone can fully transform a nontumorigenic mouse hepatocyte cell line into one capable of forming tumors in nude mice. It will identify the protein domains of HBxAg responsible for hepatocellular transformation by testing selected X gene mutations for transformation. The ability of HBxAg to act as a substrate for phosphorylation, or mediate trans-activation, will be tested with different HBxAg mutants, to determine whether of these characteristics correlate with transformation. In this way, the function(s) of HBxAg relevant to transformation will be identified. The recent finding of HBxAg-p53 complexes in vivo and in vitro, suggest that one or more characteristics of these complexes are important for the development of PHC. Hence, the second specific aim will identify the characteristics of HBxAg-p53 complexes which correlate with hepatocellular transformation. In vitro experiments will determine whether HBxAg stabilizes p53 in cells as it does in cell free systems; whether the p53 conformation and/or phosphorylation state (important for function) is altered by complex formation; whether HBxAg binds to and alters the function of p53 mutants commonly associated with human cancers; and whether p53 affects HBxAg associated trans-activation. HBxAg mutants which fail to bind p53 and transform the established mouse hepatocyte cell line will demonstrate the mechanisms whereby of X-p53 complex formation mediates transformation. The third specific aim will identify other HBxAg binding proteins which are likely to represent additional important steps in multistep hepatocarcinogenesis. HBxAg binding proteins will be identified by screening human liver cDNA expression libraries with HBxAg, verification that the expressed protein from each clone binds HBxAg, and identifying the product by DNA sequence analysis. Alternatively, HBxAg binding proteins will be isolated from HBV infected liver tissues, hepatoma cell lines, or from HBxAg producing transgenic mice that develop PHC. The approaches used include affinity chromatography and immunoprecipitation, followed by gel electrophoresis of the binding proteins, and finally identification by sequencing. HBxAg binding proteins which are likely to be relevant to hepatocellular transformation will be those that bind wild type HBxAg, but do not bind X mutants which have lost their ability to transform FMH-202-1 mouse hepatocytes in culture. Together, these studies will define the critical biochemical pathways used by HBxAg in hepatocellular transformation, and provide opportunities for the development of logical intervention strategies for the prevention and/or treatment of PHC.
| Schinazi, Raymond F; Bassit, Leda; Clayton, Marcia M et al. (2012) Evaluation of single and combination therapies with tenofovir disoproxil fumarate and emtricitabine in vitro and in a robust mouse model supporting high levels of hepatitis B virus replication. Antimicrob Agents Chemother 56:6186-91 |
