Apoptosis induced by hepatitis B virus X protein in a CCL13-HBx stable cell line.

The hepatitis B virus X protein (HBx) critically modulates cell growth by inducing apoptosis or proliferation. We sought to clarify whether HBx-mediated apoptosis in a CCL13 stable cell line (Chang-HBx) with inducible HBx expression proceeds through the extrinsic (death receptor-mediated) and/or intrinsic (mitochondrial-mediated) pathways of apoptosis. We used western blotting, cell viability assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, caspase activity assays, JC-1 staining and DNA fragmentation analysis to study the role of HBx in apoptosis. The expression of the pro-apoptotic proteins Bax and Bad and the release of cytochrome c also increased slightly upon HBx induction. JC-1 staining showed a loss of mitochondrial membrane potential upon HBx induction. Additionally, induction of HBx increased the levels of cleaved caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway) and the common effector caspase-3 as measured by western blotting. This elevation of cleaved caspase-8 or caspase-3 and caspase-9 or caspase-3 decreased in the presence of caspase-8 inhibitor Z-IETD-FMK or caspase-9 inhibitor Z-LEHD-FMK, respectively. Both inhibitors also rescued cell growth, and the caspase-8 inhibitor Z-IETD-FMK prevented apoptotic phenomena including the TUNEL signal. DNA fragmentation analysis showed that these phenomena were not detected in the presence of higher concentration of inhibitors. Our data suggest that HBx induces apoptosis through both extrinsic and intrinsic pathways.