| Research Abstract |
Hepatitis B virus (HBV) chronically infects more than 350 million people worldwide and causes serious liver diseases including cirrhosis and liver cancer, accompanied by hepatocyte death and regeneration. Among the proteins encoded by the HBV genome, the 17-kDa regulatory protein, HBx, has been shown to induce transformation of some rodent cell types and to bring about liver cancer in some transgenic mice.
Multiple cellular functions have been proposed for HBx. Although HBx is a potent transcriptional co-activator through multiple cis-acting elements, including AP-1, AP-2, ATF/CREB, NF-kB, NF-AT and C/EBP, HBx does not directly bind DNA and localizes on mitochondria. HBx also mediates several signal transduction cascades, including the RAS/MAPK-, JNK- and Src-dependent pathways. Recently, we have shown that HBx facilitated cell death induction via association with mitochondria resulting in loss of mitochondrial membrane potential. Thus, HBx exhibits both proliferating and cell death-pro
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moting activity.
In this project, we successfully introduced recombinant HBx and its mutants, an internal deletion mutant, XΔ(5-67), and a C-terminus truncation mutant, X(1-67), into all cells by employing the protein transduction method. HBx and XΔ(5-67), which were able to associate with mitochondria, inhibited cell proliferation and caused decreased cellular ATP level in a concentration dependent manner. However, a mutant X (1-67), which was unable to associate with mitochondria, did not affect cell proliferation. The generation of reactive oxygen species (ROS) was observed after incubation with HBx and XΔ(5-67), but not with X(1-67). We then analyzed mutagenic effect of HBx on the cII gene of Big Blue rat cells, where lambda shuttle vector was integrated exogenously. The mutation rate was increased over 4 times by HBx transduction. Mutation frequency was suppressed by the presence of ROS scavengers, Tiron and N-acetylcystein (NAC). The present results strongly suggest that HBx causes DNA mutation through ROS generation. Less
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