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J. Sohn, J. Zhou, R. Kannan, S.J. Ryan, D.R. Hinton; The Apoptotic Effect of 4–Hydroxyphenyl Retinamide (4–HPR) in Human RPE . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2893.
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. 4–hydroxyphenyl retinamide (4–HPR, also called fenretinide) has been used as an anticancer drug because of its cytotoxicity to tumor cells. Its action has been attributed to increased ceramide production in cells. In this study, we have examined the effect of 4–HPR on apoptosis in human RPE in vitro under conditions of varying serum concentrations in the culture medium.
Confluent human RPE of early passages (3–4) grown in DMEM containing 10% FBS were switched to 0, 2 or 5% serum before exposure to 4–HPR in either chamber slides or 6–well plates. In three separate experiments, the cells were treated with 1,2,5,10 and 20 µM 4–HPR for 24h. In some experiments, confluent RPE were starved for an initial 48h before an additional 24h 4–HPR treatment with varying serum concentrations. Live and dead cells were quantitated by LIVE/DEAD cell flow cytometric assay using a commercial kit. The number of apoptotic cells in control and 4–HPR–treated RPE were counted by TUNEL staining.
Initial flow cytometric analysis showed that concentrations of 4–HPR over 10 µM caused extensive cell death probably due to drug toxicity. Therefore, in the subsequent experiments, the concentration of 4–HPR ranged between 0–10 µM. A dose–dependent decrease in cell viability in flow cytometry and an increased number of TUNEL positive cells were observed. The data suggested that the increase in number of apoptotic RPE cells was directly proportional to the percentage of serum in the medium. In starved RPE, the effect of 4–HPR on cell death was more pronounced in subsequently serum containing group as compared to non–serum group.
Our data indicate that a) 4–HPR induces cell death in human RPE in a dose–dependent fashion and b) The apoptotic action of 4–HPR requires serum components.
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