Intracellular calcium stores are not required for Bcl-2-mediated protection from apoptosis.
The ability of Bcl-2 to inhibit cell death is well documented but its mechanism of action remains elusive. Recent reports have suggested that Bcl-2 prevents apoptosis by inhibiting the release of Ca2+ from the thapsigargin-sensitive Ca2+ store. The mobilization of Ca2+ from this store has been implicated as a signal regulating apoptotic cell death induced by glucocorticoids and by interleukin-3 withdrawal. The present study was designed to determine if Bcl-2 would still inhibit apoptosis after depletion of intracellular Ca2+ stores. We compared the response of two Chinese hamster ovary cell lines (5AHSmyc and 5A300bcl-2.2) following incubation with the calcium ionophore ionomycin to deplete intracellular Ca2+ stores. Continued incubation of 5AHSmyc cells in calcium-free media induced substantial apoptotic DNA fragmentation within 4 h and >95% loss of viability within 48 h. However, 5A300bcl-2.2 cells showed no evidence of DNA fragmentation or loss of viability over the same time period. Intracellular Ca2+ was analyzed with the Ca2+-sensitive fluorescent dye INDO-1 and confirmed that ionomycin was capable of releasing Ca2+ from intracellular stores in both cell lines. These results show that depletion of intracellular Ca2+ stores induces apoptosis and that these Ca2+ stores are not required for the protection afforded by Bcl-2.
Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J E
JE
Reynolds
A
A
Eastman
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004330:Drosophila; D016259:Genes, myc; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D015759:Ionomycin; D019284:Thapsigargin; D004247:DNA; D002118:Calcium