PURPOSE. To investigate the signal transduction mechanisms involved in the cell death of human retinal pigment epithelial (RPE) cells after their exposure to either hydrogen peroxide (H2O2) or tri-butyl hydroxperoxide (tBH). METHODS. Cultured human RPE (hRPE) cells were treated with the chemical oxidants tBH and H2O2 as well as with the synthetic ceramide analogs C2, C6, and dihydroceramide for different time periods. Apoptosis was determined by TUNEL staining and annexin-V labeling of phosphatidylserine exposure. Ceramide 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. At the same time, both of these oxidants induced an early and late increase in the intracellular production of ceramide, a lipid second messenger. Moreover, addition of C2 and C6 synthetic ceramides caused a high level of apoptosis in these hRPE cells. In contrast, treatment with the immediate precursor 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 ceramide generation.
|Number of pages||8|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - 2001|