Despite the extensive studies on the roles of hepatitis B virus X protein (HBx), the effects of HBx on the important cellular processes such as cell growth, cell transformation and apoptosis remain controversial. Our previous study showed that the balance between p53‐dependent activation and p53‐independent repression by HBx determines the expression level of cyclin‐dependent kinase inhibitor p21. In the present study, we further demonstrate that HBx natural variants have differential effects on p21 expression. The critical sites in HBx were identified as residues Ser‐101 for activation and Met‐130 for repression, respectively. The HBx variants with Ser‐101 instead of Pro‐101 stabilized p53 more efficiently, probably by protecting it from the MDM2‐mediated degradation. On the other hand, the Met‐130‐containing HBx strongly repressed p21 expression by inhibiting Sp1 activity. Overall, the effect of HBx on p21 expression seems to be determined by the balance between the opposite activities. Depending on their potentials to regulate p21 expression, HBx variants showed different effects on the cell cycle progression, and eventually on the cell growth rate, implicating its biological significance. The present study may provide a clue to explaining the contradictory results related to cell growth regulation by HBx as well as to understanding the progression of hepatic diseases in HBV‐positive patients.

Despite the extensive studies on the roles of hepatitis B virus (HBV) X protein (HBx) in the development of hepatocellular carcinomas (HCCs), the mechanisms by which HBx contributes to HCC remain controversial. In this study, the effect of HBx on the G₁–S checkpoint control depending on the status of p53 was compared. Transcription of p21^waf1/cip1 was activated by HBx in the presence of functional p53 in a dose-dependent manner. However, it was repressed by HBx when p53 was absent or present at a low level. Furthermore, the growth rate of the HBx-expressing NIH3T3 cell lines compared with that of the parental cells was decreased when p53 was upregulated by a DNA-damaging agent, cisplatin, whereas it increased approximately twofold when p53 was present at a very low level. Thus, the opposite effects of HBx on the regulation of the cell cycle depending on the status of p53 might be important to understand the progression of hepatic diseases in HBV-positive patients.

Aberrant promoter methylation of retinoic acid receptor-β 2 (RAR-β 2) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR-β 2 via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G1-checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR-β 2 in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-2′-deoxycytidine. As RAR-β 2 is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis.

The increased proliferation rate of hepatocytes is one of the major risk factors for the development of hepatocellular carcinoma. In this study, we investigated the mechanism by which hepatitis C virus (HCV) core protein represses transcription of the universal cyclin-dependent kinase inhibitor p21 gene in murine fibroblast NIH 3T3 cells. From the transient reporter assays of p21 promoter, we found that the TGF-β-responsive element (TβRE) located between −83 and −74 of the p21 promoter is responsible for the effect. The TGF-β-induced p21 promoter activity was specifically decreased by HCV core protein and in the presence of the inhibitory Smad7 the repression effect was almost completely abolished. Furthermore, HCV core protein stimulated the growth rate of NIH 3T3 cells and could overcome growth arrest by TGF-β but not by butyrate, suggesting that HCV core protein stimulates cell cycle progression by repressing p21 transcription through a TGF-β pathway.