Abstract
Viral infection of hepatitis B virus (HBV) is a major etiologic factor of hepatocellular carcinoma (HCC) in Taiwan. The expression of viral large surface protein (LHBs) is involved in HBV-related hepatocarcinogenesis. To investigate the role of LHBs in cell cycle progression, we showed that several essential mitotic regulators, including Polo-like kinase-1 (Plk1), Cyclin-dependent kinase-1 (Cdk1) and Cyclin B, were upregulated by LHBs proteins in human telomerase reverse transcriptase (hTERT)-immortalized hepatocytes. With the application of live cell imaging, we confirmed that hepatocytes carrying LHBs proteins displayed early mitotic entry. Furthermore, we demonstrated that LHBs-positive cells entered mitosis prior to the completion of DNA replication, as indicated by BrdU incorporation assay. Inhibition of topoisomerase-II completely blocked mitotic entry in control cells, but only caused a slight mitotic delay in LHBs-positive cells, suggesting that the G2/M checkpoint is attenuated in the presence of LHBs. To explore the underlying mechanism leading to this attenuation, we found that the inhibition of Plk1 decreased the phosphorylation on tyrosine-15 of Cdk1, and thereby delayed the mitotic entry. Thus, we speculate that Plk1 overexpression attenuate the G2/M checkpoint through activating of Cdc25. In searching for potential upstream regulators of these mitotic kinases, we found several heat shock proteins (HSP), including HSP70 and HSP72, were overexpressed in LHBs-expressing cells. Inhibitions of HSP70 family not only reduced the expression levels of Aurora B, Plk1 and Cdk1, but also delayed the mitotic entry and overall cell proliferation in LHBs-expressing cells. Together, these results suggest that LHBs promotes early mitotic entry through the attenuation of G2/M checkpoint, which is mediated by the HSP-dependent induction of essential mitotic kinases.