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

Abstract

It has been proposed that continuously generated hydrogen peroxide (H(2)O(2)) 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(2)O(2)-induced cell death is not complete. This study examined the cellular mechanism(s) by which the continuous presence of H(2)O(2) induces cell death. We also further analyzed the precise role of AIF by inhibiting its expression with siRNA. Exposure of cells to H(2)O(2) generated by glucose oxidase caused mitochondrion-mediated, caspase-independent cell death. In addition, H(2)O(2) exposure resulted in cell shrinkage and chromatin condensation without nuclear fragmentation, indicating that H(2)O(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 H(2)O(2)-exposed cells. Furthermore, ATP was rapidly and severely depleted in cells exposed to H(2)O(2) generated by glucose oxidase but not by H(2)O(2) added as a bolus. Suppression of the H(2)O(2)-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(2)O(2) causes caspase-independent and AIF-dependent cell death.

Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death

“…Cdk5 activation is increased by cleavage of its regulatory protein p35 to p25 in response to neurotoxic insults. 37,38 Given the emerging link between both Cdk5 and tAIF and oxidative stress-induced neuronal cell death, [39][40][41][42] we hypothesized that oxidative stress-induced Cdk5 activation triggers neuronal cell death by inhibiting CHIP-mediated degradation of tAIF. In primary cultured Figure 3 Cdk5 negatively regulates CHIP-mediated tAIF degradation via UPS.…”

Over-expression of JunB inhibits mitochondrial stress and cytotoxicity in human lymphoma cells exposed to chronic oxidative stress

“…Previously, we found that the continuous presence of H2O2 induces mitochondrial stress and subsequent cell death in a time-and dose-dependent manner (9). To determine the role of JunB in H2O2-induced cell death, we transfected cells with JunB-specific siRNA.…”

“…Whole cell lysates were made in a lysis buffer as described previously (9,11), after which the protein content was quantified according to the Bradford method (28). Nuclear and mitochondrial fractions were prepared as described elsewhere http://bmbreports.org (9).…”

“…Nuclear and mitochondrial fractions were prepared as described elsewhere http://bmbreports.org (9). An equal amount of protein (30 μg) per sample was separated by 12% SDS-PAGE and then blotted onto PVDF membranes before probing with primary and secondary antibodies.…”

“…siRNA-mediated silencing of the JunB gene was performed as described elsewhere, with slight modification (6,9). Briefly, cells were transfected with 30 nM siRNA duplexes against JunB and GFP using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.…”