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 (H(2)O(2)) 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 H(2)O(2). Initial accumulation of DNA fragments at 2 h after exposure was delayed in irradiated cells compared with H(2)O(2)-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 H(2)O(2), 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 H(2)O(2) treatment. Radiation did not affect proapoptotic proteins (Bax and Bid), whereas H (2)O(2) 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 H(2)O(2)-treated cells, but not in irradiated cells. These results suggest that, unlike the caspase cascade of H(2)O(2)-induced apoptosis, cytochrome c and caspase-9 are important for the intrinsic pathway of radiation-induced apoptosis, independent of caspase-8.
Hosokawa
Yoichiro
Y
Department of Dental Radiology, School of Dentistry, Health Sciences University of Hokkaido, Japan. hosokawa@hoku-iryo-u.ac.jp
Sakakura
Yasunori
Y
Tanaka
Likinobu
L
Okumura
Kazuhiko
K
Yajima
Toshihiko
T
Kaneko
Masayuki
M
D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism*; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000008:administration & dosage*; D011829:Radiation Dosage; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects
D045304:Cytochromes c; D004247:DNA; D006861:Hydrogen Peroxide; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases