Cardiolipin-specific peroxidase reactions of cytochrome C in mitochondria during irradiation-induced apoptosis.
OBJECTIVE
To determine whether cytochrome c (cyt c) content and associated cardiolipin oxidation can be determinants of cell sensitivity to irradiation-induced apoptosis.


METHODS
The small interfering RNA (siRNA) approach was used to engineer HeLa cells with lowered contents of cyt c (14%, HeLa 1.2 cells). Cells were treated by gamma-irradiation (in doses of 5-40 Gy). Lipid oxidation was characterized by electrospray ionization mass spectrometry analysis and fluorescence high-performance liquid chromatography-based Amplex Red assay. Release of a proapoptotic factor (cyt c, Smac/DIABLO) was detected by Western blotting. Apoptosis was revealed by caspase-3/7 activation and phosphatidylserine externalization.


RESULTS
Irradiation caused selective accumulation of hydroperoxides in cardiolipin (CL) but not in other phospholipids. HeLa 1.2 cells responded by a lower irradiation-induced accumulation of CL oxidation products than parental HeLa cells. Proportionally decreased release of a proapoptotic factor, Smac/DIABLO, was detected in cyt c-deficient cells after irradiation. Caspase-3/7 activation and phosphatidylserine externalization were proportional to the cyt c content in cells.


CONCLUSIONS
Cytochrome c is an important catalyst of CL peroxidation, critical to the execution of the apoptotic program. This new role of cyt c in irradiation-induced apoptosis is essential for the development of new radioprotectors and radiosensitizers.
Belikova
Natalia A
NA
Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA.
Jiang
Jianfei
J
Tyurina
Yulia Y
YY
Zhao
Qing
Q
Epperly
Michael W
MW
Greenberger
Joel
J
Kagan
Valerian E
VE
D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002308:Cardiolipins / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation; D005453:Fluorescence; D005720:Gamma Rays; D006367:HeLa Cells / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D015227:Lipid Peroxidation / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism / Q000528:radiation effects*; D024101:Mitochondrial Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010718:Phosphatidylserines / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D002308:Cardiolipins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D010718:Phosphatidylserines; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D053148:Caspase 3; D053179:Caspase 7