bcl-2 protects HL-60 cells from apoptosis by stabilizing their intracellular calcium pools.
bcl-2 has been shown to enhance cell survival by inhibiting apoptosis induced under different circumstances. In this study we investigated the effects of bcl-2 overexpression on the homeostasis of subcellular organelles such as ER and mitochondria. In our study, HL-60/bcl-2 and control HL-60/neo cells were obtained by transfection of bcl-2 cDNA or the neomycin-resistant gene, respectively. Apoptosis was evaluated by both DNA fragmentation and flow cytometry qualitatively and quantitatively, and the intracellular calcium by Fura-2/AM. Thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump, and Br-A23187, a calcium ionophore, were used in this study. Our results showed that overexpression of bcl-2 significantly blocked TG- and Br-A23187-induced apoptosis in calcium containing buffer. Measurement of intracellular calcium showed that bcl-2 overexpression could reduce sustained elevation of cytosolic Ca2+ induced by these agents. However, in calcium-free medium, bcl-2 overexpression maintained Ca2+ uptake in ER of both TG- and Br-A23187-treated cells. Moreover, the depletion of Ca2+ by EGTA enhanced TG- and Br-A23187-induced apoptosis, and reduced the anti-apoptotic action of bcl-2, suggesting that cytosolic Ca2+ elevation may be required for optimal ER pool refilling. These findings suggest that bcl-2 facilitates and maintains the replenishment of Ca2+ in intracellular stores and, as a result, influences the intracellular calcium, thus protecting the cells from death. In addition, there were no cytochrome c release from mitochondria into the cytosol in TG- and Br-A23187- induced apoptosis, suggesting that cytochrome c release is not a universal phenomenon in the apoptotic process.
Zhang
Q H
QH
Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, PR China.
Sheng
H P
HP
Loh
T T
TT
D017209:Apoptosis; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000172:deficiency / Q000378:metabolism*; D053143:Caspase 2; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D019254:Genes, bcl-2 / Q000502:physiology; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007476:Ionophores / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D003574:Cytochrome c Group; D007476:Ionophores; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D000001:Calcimycin; D004533:Egtazic Acid; D019284:Thapsigargin; D053143:Caspase 2; D020169:Caspases; D002118:Calcium