Redox regulation of caspase-3(-like) protease activity: Regulatory roles of thioredoxin and cytochrome c

Oxidative stress induces a variety of cellular responses, including apoptos is, and caspase family proteases are known to be involved in apoptosis, Cas pase-3(-like) protease activity was examined in Jurkat T cells to investiga te the mechanism of apoptosis induced by a thioloxidant, diamide, Caspase-3 was activated when cells were cultured with 200 mu M diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 mu M diami de that induced necrosis, When apoptosis was induced in cells with exposure to 200 mu M diamide, the intracellular thioredoxin (TRX) levels were maint ained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier t han the activation of caspase-3, In contrast, when cells were exposed to 50 0 mu M diamide, intracellular reactive oxygen intermediate generation was i ncreased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis, Caspase-3 activity in cell lysate precultur ed with anti-Fas Ab was suppressed dose dependently by diamide and restored by thiol-reducing agents, DTT or TRX, When cells were precultured with 5 m M of buthionine sulfoximine,an inhibitor of glutathione synthesis, intracel lular TRX levels were maintained, and as low as 20 mu M diamide could induc e apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3, These results indicate that the activation of casp ase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochr ome c release from mitochondria, and the cellular reducing environment main tained by TRX, as well as glutathione, is required for caspase-3 activity t o induce apoptosis.