| The members of Hepadnavirus family are the etiological agents of viral hepatitis in human, ground squirrel, pekin duck, and woodchuck. Hepatitis B virus (HBV) infects about 200 million people worldwide and causes serious hepatic disorders including acute and chronic hepatitis and cirrhosis with mortality rate of 1 to 2 million per year. Furthermore, recent epidemiological data and results of animal experiments indicate that there is a close link between chronic HBV infection and hepatocellular carcinoma (HCC). Although much is known about the HBV life cycle and replication mode, the molecular mechanism for hepatocarcinogenesis is poorly understood. The objective of this study is to investigate the role of HBV in hepatocarcinogenesis.; The study was conducted in two parts. In the first part, a cell culture system was developed to measure the transactivity of the X-gene of HBV. The assay system consisted of a X-gene expression plasmid and a series of reporter plasmids containing regulatory regions of viral genome, the c-jun, c-fos, and {dollar}alpha{dollar}-fetoprotein genes (AFP). The result of the CAT assay demonstrated that the X-gene can transactivate the expression of c-jun and AFP genes as much as 7.2 and 20.3 fold, respectively. Although the precise mechanism of the activation of c-jun and AFP genes are not known, there is evidence that the AP-1 factor binding site of the c-jun is the target site for the X-gene transactivation. The results of c-jun activation by the X-gene strongly support the previous hypothesis that the X-gene may play a critical role in the development of HCC.; In the second part of this study a woodchuck system was used and the role of HBV in hepatocarcinogenesis was examined. A subtracted library was constructed from woodchuck normal and HCC liver to identify the gene(s) that are differentially expressed in HCC cells. Seven clones with different levels of expression were identified. Further sequencing analysis of two clones (HC 34 and HC49) indicates that these genes are closely related to hemopexin (HPX) and {dollar}alpha{dollar}-1-acid glycoprotein (AGP). Interestingly, the results of transfection and the following dot blot analysis suggest that the HC49 gene is significantly transactivated by the X-gene. Therefore, these differentially expressed genes identified by the subtraction method should provide useful tool to elucidate the mechanism of hepatocarcinogenesis which is linked to the HBV infection. In addition, they may be useful tumor markers for the diagnosis/prognosis of HCC. |
