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 (H ₂O₂) 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 H ₂O₂-induced cell death is not complete. This study examined the cellular mechanism(s) by which the continuous presence of H ₂O₂ 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, H ₂O₂ exposure resulted in cell shrinkage and chromatin condensation without nuclear fragmentation, indicating that H₂O ₂ 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 H ₂O₂ added as a bolus. Suppression of the H ₂O₂-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 H ₂O₂ causes caspase-independent and AIF-dependent cell death.