Oxidative-induced apoptosis to an immortalized ganglion cell line is caspase independent but involves the activation of poly(ADP-ribose)polymerase and apoptosis-inducing factor.
The aim of the present work was to characterize the molecular basis of oxidative-induced death, a process that has been implicated in eye diseases like glaucoma, in RGC-5 cells, an immortalized retinal ganglion cell (RGC) line. Oxidative stress was induced by treatment of RGC-5 cells with hydrogen peroxide and compared to a known effect of a light insult (1000 lx, 400-760 nm). Hydrogen peroxide causes a loss of viability of RGC-5 cells in a dose-dependent manner. Loss of cell viability was by apoptosis characterized by breakdown of DNA (TUNEL method), presence of membrane phosphatidylserine (APOPercentage method), activation of PARP-1 and AIF. Oxidative stress caused a stimulation of ROS which reached maximum levels before optimum apoptosis. Hydrogen-peroxide-induced apoptosis did not result in an activation of caspase-3 and was unaffected by the caspase inhibitor Z-VAD-fmk. However, the PARP-1 inhibitor NU-1025 counteracted the effects of hydrogen peroxide and light. Evidence is provided to show that both forms of oxidative stress caused AIF to be cleaved with the product located to the cytosolic compartment. Light-induced apoptosis was attenuated by the presence of the mitochondrial uncoupler M3778 but potentiated by the presence of cobalt. In contrast, hydrogen-peroxide-induced apoptosis was unaffected by M3778 but attenuated by cobalt. The results show that oxidative stress caused by light is dependent on functional mitochondria and that the molecular mechanisms of apoptosis caused by hydrogen peroxide or light are similar but not identical.
Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK.
Guang-Yu
GY
Li
Neville N
NN
Osborne
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D003035:Cobalt / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008027:Light / Q000009:adverse effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D012164:Retinal Diseases / Q000378:metabolism* / Q000503:physiopathology; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D014475:Uncoupling Agents / Q000494:pharmacology
C494831:Aifm1 protein, rat; D051033:Apoptosis Inducing Factor; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D016877:Oxidants; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D014475:Uncoupling Agents; D003035:Cobalt; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases