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R. Antony, M. Hardy, N.G. Bazan; Oxidative Stress Triggers the Expression of Bcl–2 Family Proteins in Retinal Pigment Epithelial Cells (RPE) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5357.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous works from our lab have shown that apoptosis is triggered in RPE cells when exposed to oxidative stress (PNAS, 101:8491–8496, 2004). Bcl–2 family proteins are expressed when cells are exposed to a variety of stimuli. Some of these proteins are anti–apoptotic and others are pro–apoptotic. Our goal in this study was to investigate which members of the Bcl–2 family proteins are triggered by oxidative stress and TNF–α in ARPE–19 cells. We also aimed at defining cellular localization of the Bcl–2 protein by immunohistochemistry. Methods: Human RPE cells were grown in a 37 °C humidified chamber with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM). Cells were grown on chamber glass slides (LAB–TEK) with 80% confluence. Cells were treated with TNF–α (10 µg/ml) and H2O2 (0.8 mM); untreated cells were controls. The incubation lasted for 16 hours. Immunostaining was done by using primary antibodies against Bcl–2, Bax, Bcl–xL, Bad (Santa Cruz), and Cytochrome C proteins at 1:100 dilution for 2 hours. Some of the antibodies were conjugated to FITC or TRITC. Cells were viewed using a Zeiss deconvolution microscope. Results: TNF–α / H2O2 triggers the induction of the Bcl–2 family proteins. Our study demonstrates that RPE cells under oxidative stress show a significant increase (10 fold) in the anti–apoptotic proteins Bcl–xL and Bcl–2. Pro–apoptotic proteins Bad and Bax expression levels also increased (at least 20 fold) with cytoplasmic condensation and nuclear shrinkage. Conclusions: Oxidative stress in RPE cells significantly increased the expression of anti–apoptotic members of the Bcl–2 family of proteins. The induction of anti–apoptotic members suggests a protective attempt to counteract the effects of oxidative stress from homeostasis. Supported by NIH EY05121
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