Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria

Previous studies have shown that under certain conditions some thiol-contai ning compounds can cause apoptosis in a number of different cell lines. Her ein, we investigated the apoptotic pathways in HL-60 cells triggered by dit hiothreitol (DTT), used as a model thiol compound, and tested the hypothesi s that thiols Cause apoptosis via production of hydrogen peroxide (H2O2) du ring thiol oxidation, The results show that, unlike H2O2, DTT does not indu ce apoptosis via a mitochondrial pathway. Th is is demonstrated by the abse nce of early cytochrome c release from mitochondria into the cytosol, the l ack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis, The first caspase activity dete ctable in DTT treated cells is caspase 3, which is increased significantly 1-2 h after the start of DTT treatment. This was shown by following the cle avage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known a s initiator caspases, does not start until 3-4 h after DTT exposure,well af ter caspase 3 has become active and at a time when apoptosis is in late sta ges, as shown by the occurrence of DNA fragmentation to oligonucleosomal-si zed pieces. Although oxidizing DTT can produce H2O2, data presented here in dicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.