Protein synthesis-dependent apoptotic signalling pathway in X-irradiated MOLT-4 human leukaemia cell line.
OBJECTIVE
To demonstrate whether protein synthesis is required for ionizing radiation-induced apoptosis through activation of caspases in human leukaemia cell line MOLT-4, the effects of a protein synthesis inhibitor, cycloheximide, on the apoptotic signalling pathway including the activation of caspase family and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and the expression of Fas/CD95/APO-1 (Fas) were examined in X-irradiated MOLT-4 cells.


METHODS
MOLT-4 cells pretreated with 0.5 microg/ml cycloheximide for 1h were exposed to 7.5Gy of X-rays. The appearance of apoptosis, expression of Fas, activation of caspases-3, -8, -9, SAPK/JNK and AP-1, the release of mitochondrial cytochrome-C and the formation of death-induced signalling complex (DISC) between Fas and the Fas-associated death domain (FADD) were measured by fluorescence microscopy, Western blotting, flow cytometry, gel shift assay and immunoprecipitation, respectively.


RESULTS
Nuclear fragmentation and chromatin condensation were observed at 6 h after X-irradiation and gradually increased up to 12 h. These phenomena were significantly attenuated by cycloheximide. Cycloheximide also inhibited the activation of caspases and AP-1, the expression of Fas, the formation of DISC and the release of cytochrome-C, but not the activation of SAPK/JNK in X-irradiated MOLT-4 cells.


CONCLUSIONS
These results indicate that apoptosis of X-ray-induced MOLT-4 cells is dependent on the activation of caspases regulated by de novo protein synthesis through SAPK/JNK activation.
Takahashi
Kenji
K
Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan.
Inanami
Osamu
O
Hayashi
Masanobu
M
Kuwabara
Mikinori
M
D017209:Apoptosis; D029681:Arabidopsis Proteins; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D044943:Fatty Acid Desaturases / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007938:Leukemia / Q000378:metabolism* / Q000473:pathology*; D048670:MAP Kinase Kinase 4; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D015398:Signal Transduction; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014965:X-Rays
D029681:Arabidopsis Proteins; D002843:Chromatin; D003574:Cytochrome c Group; D018808:Transcription Factor AP-1; D003513:Cycloheximide; D044943:Fatty Acid Desaturases; C084083:Fad7 protein, Arabidopsis; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases