Manganese superoxide dismutase protects from TNF-α-induced apoptosis by increasing the steady-state production of H₂O₂

Manganese superoxide dismutase (SOD2) has been well established to be essential for protection from a variety of apoptotic stimuli. Here we demonstrate that the antiapoptotic effects of SOD2 are attributed to its ability to generate H₂O₂ and that its efficient removal resensitizes cells to tumor necrosis factor (TNF)-α-induced apoptosis. SOD2 overexpression in HT-1080 cells leads to a decrease in the fluorescence of the superoxide-sensitive fluorophore, dihydroethidium, and a concomitant increase in oxidation of the H₂O₂-sensitive dye, dichlorodihydrofluorescein diacetate (DCFDA). The rate of aminotriazole- inhibited catalase activity also was increased when SOD2 is overexpressed and reflects a 1.6-fold increase in the steady-state production of H ₂O₂. The increase in H₂O₂ was associated with decreased sensitivity to TNF-α-mediated apoptosis, as measured by monitoring the loss of mitochondrial membrane potential (MMP), caspase activation, poly-ADP ribose polymerase (PARP) cleavage, and accumulation of hypodiploid DNA content. Both the increase in H₂O₂ and resistance to TNF-mediated apoptosis were reversed by coexpression of catalase. The lipid hydroperoxide scavengers, β-hydroxytoluene and trolox, and the iron chelator, desferroxamine, showed partial recovery of TNF-induced apoptosis. These findings indicate that increases in the intracellular steady-state production of H₂O₂ by SOD2 can block the activation of key processes fundamental to the process of programmed cell death.