Ceramide: A potential mediator of apoptosis in human retinal pigment epithelial cells

PURPOSE. To investigate the signal transduction mechanisms involved in the cell death of human retinal pigment epithelial (RPE) cells after their expo sure to either hydrogen peroxide (H2O2) or tri-butyl hydroxperoxide (tBH). METHODS. Cultured human RPE (hRPE) cells were treated with the chemical oxi dants tBH and H2O2 as well as with the synthetic ceramide analogs C-2, C-6, and dihydroceramide for different time periods. Apoptosis was determined b y TUNEL staining and annexin-V labeling of phosphatidylserine exposure. Cer amide levels were quantified by the diacylglycerol kinase assay using thin- layer chromatography. RESULTS. H2O2 and tBH caused a high level of apoptosis in the hRPE cells. A t the same time, both of these oxidants induced an early and late increase in the intracellular production of ceramide, a lipid second messenger. More over, addition of C-2 and C-6 synthetic ceramides caused a high level of ap optosis in these hRPE cells. In contrast, treatment with the immediate prec ursor of ceramide, dihydroceramide, resulted in no apoptotic response. CONCLUSIONS. The results demonstrate that H2O2 and tBH induce apoptosis in hRPE cells and suggest that the underlying signaling mechanism involves cer amide generation.