Production of intracellular superoxide mediates dithiothreitol-dependent inhibition of apoptotic cell death.

Apoptotic cell death proceeds from the activation of cysteine proteinases called caspases. As full enzymatic activity of caspases requires reduction of cysteine residues in and around the catalytic site of the proteases, cysteine- reducing agents such as dithiothreitol (DTT) are expected to facilitate caspase activity upon induction of apoptosis. However, DTT has been shown to efficiently protect cells from apoptosis. The mechanism involved in DTT-mediated inhibition of apoptosis has been attributed to its antioxidant activity. Interestingly, under physiological conditions, thiol-mediated antioxidant reaction has also been shown to result in intracellular generation of superoxide (O(2) (.-)). In line with our earlier findings implicating a slight prooxidant state in resistance to apoptosis, we set out to investigate if the death-inhibitory activity of DTT could be mediated by intracellular O2 (.-). Our results show that incubation of human melanoma cell line M14TF or human bladder carcinoma cell line T24 with DTT induced an increase in intracellular O2 (.-) with concomitant inhibition of apoptosis triggered by CD95 signaling, staurosporine, or hydrogen peroxide. Moreover, preincubation of either cells with Tiron, a specific O2 (.-) scavenger, reverted DTT-induced inhibition of apoptosis. These results show that the apoptosis-inhibitory activity of DTT may not be due to its antioxidant property, but instead linked to its ability to induce an increase in intracellular O2 (.-) level.