Abstract
At least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the β-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.
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Abbreviations
- DPI:
-
diphenyleneiodonium
- Cyt c:
-
cytochrome C
- BCECF-AM:
-
2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein
- DCFH-DA:
-
2′,7′-dichlorofluorescein diacetate
- O2−:
-
superoxide
- H2O2:
-
hydrogen peroxide
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Acknowledgements
We thank Dr Roberta Gottlieb, Scripps, La Jolla, CA, USA for the generous gift of the CEM/Neo and CEM/Bcl-2 cells, and Dr Stanley Korsmeyer, Boston, MA, USA for the generously providing the pcDNA3-Bcl-2 plasmid. We also wish to acknowledge Kartini Iskander and Sun Yu for technical assistance. This work was supported by Grants R-185-000-019-213 and R-185-000-032-213 to SP, and R-364-000-013-213 to M-VC from the NMRC, Singapore and a Grant R-185-000-048-305 to SP and M-VC form the BMRC, Singapore.
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Clément, MV., Hirpara, J. & Pervaiz, S. Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria. Cell Death Differ 10, 1273–1285 (2003). https://doi.org/10.1038/sj.cdd.4401302
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DOI: https://doi.org/10.1038/sj.cdd.4401302
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