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

S Ueda, H Nakamura, H Masutani… - The Journal of …, 1998 - journals.aai.org
S Ueda, H Nakamura, H Masutani, T Sasada, S Yonehara, A Takabayashi, Y Yamaoka…
The Journal of Immunology, 1998journals.aai.org
Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase
family proteases are known to be involved in apoptosis. Caspase-3 (-like) protease activity
was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a
thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 μM
diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 μM
diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 …
Abstract
Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase family proteases are known to be involved in apoptosis. Caspase-3 (-like) protease activity was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 μM diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 μM diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 μM diamide, the intracellular thioredoxin (TRX) levels were maintained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier than the activation of caspase-3. In contrast, when cells were exposed to 500 μM diamide, intracellular reactive oxygen intermediate generation was increased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis. Caspase-3 activity in cell lysate precultured 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 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, intracellular TRX levels were maintained, and as low as 20 μM diamide could induce apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3. These results indicate that the activation of caspase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochrome c release from mitochondria, and the cellular reducing environment maintained by TRX, as well as glutathione, is required for caspase-3 activity to induce apoptosis.
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