Raf-1 and protein kinase B regulate cell survival through the activation of NF-kappaB in hepatitis B virus X-expressing cells.

We previously demonstrated that activation of NF-kappaB by the hepatitis B virus X (HBx) gene plays an important role in cell survival. In the present study, we explored the upstream mediators of NF-kappaB activation and their correlations with cell survival. XTT assays and colony generation assays revealed that inhibition of NF-kappaB activation indeed increased cell death in HBx-expressing cells. Utilizing inactivating mutants of signal transducers, we showed that dominant negative mutants of stress-activated protein kinase/extracellular signal-regulated kinase (SEK1) or PKCalpha significantly diminished the HBx-mediated NF-kappaB activation. However, neither of these mutants significantly affected the cell survival in colony generation assays. In contrast, inactivating mutants of Raf-1 or PKB (protein kinase B)/Akt abrogated the HBx-mediated NF-kappaB activation and also suppressed the cell survival. Our results suggest that the Raf-1 or PKB-mediated NF-kappaB activation promotes cell survival in HBx-expressing cells.

Centrosome amplification and multinuclear phenotypes are Induced by hydrogen peroxide.

Multinucleated cells resulted from mitosis defect have been noted in pathophysiological states of the cells such as inflammation, senescence and cancer. Since oxidative stress has been known to correlate with these pathophysiological conditions, we tested the effect of H2O2 on the cell cycle progression and formation of multinucleated cells. H2O2 induced a significant delay in cell cycle progression in Chang liver cells. Interestingly, H2O2 actively induced hyperamplification of centrosomes (n>or=3) and multipolar spindle formation during mitosis and subsequently increased the generation of multinucleated cells. A significant increase of the phospho-ERK level was observed upon H2O2 treatment but PD98059, an MEK1/2 inhibitor, didn't reduce the frequency of cells with hyperamplified centrosomes. On the other hand, treatment of either H2O2 or adriamycin increased intracellular ROS levels and multinucleated cells, which were significantly suppressed by antioxidants, N-acetylcysteine and PDTC. Thus, our results suggest that oxidative stress can trigger centrosome hyperamplification and multinucleated cell formation, which may promote pathophysiological progression.