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Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells
J Cai1, M Wu, K C Nelson
1Department of Biochemistry, School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
Purpose:
To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE).
Methods:
Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t-butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtdelta psi) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis.
Results:
t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL-positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtdelta psi was observed before caspase activation, together with the release of mitochondrial cyt c.
Conclusions:
Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.