Effects of intracellular calcium chelator BAPTA-AM on radiation-induced apoptosis regulated by activation of SAPK/JNK and caspase-3 in MOLT-4 cells.
OBJECTIVE
To examine the roles ofintracellular calcium in radiation-induced apoptosis of MOLT-4 cells, the effects of intracellular calcium chelator BAPTA-AM on the induction of apoptosis and the activation of apoptosis-relating enzymes SAPK/JNK and caspase-3 were studied.


METHODS
MOLT-4 cells pretreated with 5 microM BAPTA-AM were exposed to X-rays. DNA fragmentation, the expression of phosphorylated SAPK/JNK and the activation of caspase-3 and calcium concentration were measured by agarose gel electrophoresis, Western blotting and spectrofluorometry.


RESULTS
Time-dependent ladder-like DNA fragmentation was observed at 4h, 5 h and 6 h after exposure to 15 Gy of X-rays. This fragmentation was significantly attenuated by pretreatment with BAPTA-AM up to 5 h after irradiation, but the attenuation due to BAPTA-AM was no longer detectable at 6 h. Activation of SAPK/JNK and caspase-3 was observed at 1 and 4 h after X-irradiation, respectively, and BAPTA-AM retarded the activation for 2 h. The pretreatment with BAPTA-AM was found to suppress the increase of calcium concentration for 6h after irradiation.


CONCLUSIONS
These results revealed that chelation of calcium merely delayed the onset of the radiation-induced apoptosis regulated by the activation of SAPK/JNK and caspase-3, and calcium was not essential for the induction of apoptosis in X-irradiated MOLT-4 cells.
Takahashi
K
K
Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Inanami
O
O
Kuwabara
M
M
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D051017:Apoptosis Regulatory Proteins; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000528:radiation effects; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation / Q000528:radiation effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D011506:Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014965:X-Rays
D051017:Apoptosis Regulatory Proteins; D002614:Chelating Agents; D011506:Proteins; C109664:caspase-activated DNase inhibitor; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002118:Calcium