Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G 1 arrest and senescence-associated b-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-29-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.
INTRODUCTIONHepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma (HCC; Tsai & Chung, 2010). HBV X protein (HBx) is encoded by the smallest ORF of the HBV genome, termed X, which is the most frequently integrated viral sequence found in HCCs (Paterlini et al., 1995). As a multifunctional regulatory protein, it can activate several transcription factors, including AP-1, NF-kB, CREB, Oct-1, Myc and TBP (Benn et al., 1996;Maguire et al., 1991;Qadri et al., 1995). In addition, HBx has been implicated in the activation of several cytoplasmic signalling pathways, including PKC, STAT, PI3K, JNK, MAPK and Wnt (Benn et al., 1996;Lee & Yun, 1998; Shih et al., 2000). Moreover, HBx is able to induce HCC in transgenic mice (Kim et al., 1991). Despite current molecular evidence suggesting that HBx plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial.Various genetic and biochemical data suggest that cellular senescence is an important tumour-suppression process, preventing damaged cells from undergoing aberrant proliferation (Campisi, 2005;Ozturk et al., 2009;Schmitt, 2007;Yan & Wajapeyee, 2010). It is characterized by irreversible cell-cycle arrest that can be triggered by many types of intrinsic and extrinsic stress. Other senescence markers include a large flat morphology, induction of a senescence-associated b-galactosidase activity (SA b-gal) (Dimri et al., 1995) and the formation of several heterochromatin domains called senescence-associated heterochromatin foci (SAHF...
