Apoptosis-inducing factor plays a critical role in caspase-independent, pyknotic cell death in hydrogen peroxide-exposed cells

It has been proposed that continuously generated hydrogen peroxide (H2O2) inhibits typical apoptosis and instead initiates an alternate, apoptosis-inducing factor (AIF)-dependent process. Aside from the role of AIF, however, the detailed morphological characterization of H2O2-induced cell death is not complete. This study examined the cellular mechanism(s) by which the continuous presence of H2O2 induces cell death. We also further analyzed the precise role of AIF by inhibiting its expression with siRNA. Exposure of cells to H2O2 generated by glucose oxidase caused mitochondrion-mediated, caspase-independent cell death. In addition, H2O2 exposure resulted in cell shrinkage and chromatin condensation without nuclear fragmentation, indicating that H2O2 stimulates a pyknotic cell death. Further analysis of AIF-transfected cells clearly demonstrated that nuclear translocation of AIF is the most important event required for nuclear condensation, phosphatidyl serine translocation, and ultimately cell death in H2O2-exposed cells. Furthermore, ATP was rapidly and severely depleted in cells exposed to H2O2 generated by glucose oxidase but not by H2O2 added as a bolus. Suppression of the H2O2-mediated ATP depletion by 3-aminobenzamide led to a significant increase of nuclear fragmentation in glucose oxidase-exposed cells. Collectively, these findings suggest that an acute energy reduction by H2O2 causes caspase-independent and AIF-dependent cell death.