Abstract
Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (Δψm). Exogenous ROS and caspase-3 induced Δψm drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased Δψm, and GSH, events associated with caspase activation and cytochrome c release.
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Abbreviations
- cyt c:
-
cytochrome c
- CM-H2:
-
DCFDA
- 2′:
-
7′-dichlorodihydrofluorescein diacetate
- DEVD:
-
acetyl-asp-glu-val-asp
- DHE:
-
dihydroethidium
- fmk:
-
fluoromethyl ketone
- GSH:
-
reduced glutathione
- GSSG:
-
oxidized glutathione
- H2O2:
-
hydrogen peroxide
- IR:
-
ionizing radiation
- mAb:
-
monoclonal antibody
- PI:
-
propidium iodide
- PBS:
-
phosphate-buffered saline
- PT:
-
mitochondrial permeability transition
- ROS:
-
reactive oxygen species
- YVAD:
-
acetyl-Tyr-Val-Ala-Asp
- zVAD:
-
Benzyloxycarbonyl-Val-Ala-Asp
- Δψ m :
-
mitochondrial membrane potential
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Acknowledgements
We thank Drs. SM Srinivasula, ES Alnemri (Thomas Jefferson University) for the caspase-9-DN, and G. Nunez (University of Michigan) for the pSFFV-FLAG.Bcl-2 constructs, and Ms. C Stanko (Cleveland Clinic Flow Cytometry Core) for help with flow cytometry. This work was supported by research grants from the National Institutes of Health to AA (CA81504 and CA82858) and GMC (HL29582).
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Chen, Q., Chai, YC., Mazumder, S. et al. The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction. Cell Death Differ 10, 323–334 (2003). https://doi.org/10.1038/sj.cdd.4401148
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DOI: https://doi.org/10.1038/sj.cdd.4401148
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