Radiation-induced apoptosis is independent of caspase-8 but dependent on cytochrome c and the caspase-9 cascade in human leukemia HL60 cells

We investigated the role of the caspase activation cascade in apoptosis induced by ionizing radiation or hydrogen peroxide (H2O2) in human leukemia HL60 cells. Electron paramagnetic resonance (EPR) spectra revealed that hydroxyl and hydrogen radicals were generated in the culture medium after exposure to radiation or H2O2. Initial accumulation of DNA fragments at 2 h after exposure was delayed in irradiated cells compared with H2O2-treated cells, although formation of abasic sites immediately after exposure was significantly higher in irradiated cells and similar quantities of hydroxyl radicals were produced under both conditions. Activity assay of caspases revealed that caspase-3, -8 and -9 were activated 2 h after exposure to H2O2, whereas in irradiated cells caspase-3 and -9 activation occurred 4 h after exposure but increased caspase-8 activation was not observed. Release of cytochrome c into cytosol was seen at 2 h after radiation and H2O2 treatment. Radiation did not affect proapoptotic proteins (Bax and Bid), whereas H2O2 increased accumulation of Bax in the mitochondrial membrane 2 h to 6 h after treatment, independently of the truncation of Bid by activated caspase-8. Moreover, treatment with the caspase-8 inhibitor Z-IETD-FMK increased cell survival and prevented accumulation of DNA fragments in H2O2-treated cells, but not in irradiated cells. These results suggest that, unlike the caspase cascade of H2O2-induced apoptosis, cytochrome c and caspase-9 are important for the intrinsic pathway of radiation-induced apoptosis, independent of caspase-8. (author)