Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo

Oxidants such as H2O2 can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H2O2 also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H2O2 causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H2O2 can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H2O2 of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H2O2 to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H2O2 is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H2O2 to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H2O2 acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H2O2 can induce caspase-9 and -3 activation and cause apoptosis. (C) 2000 Elsevier Science Inc.