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A. Leon, D.R. Lopez–Osa, S.G. Barreiro, W.C. Gordon, N.G. Bazan; Oxidative Stress Up–Regulates DNA Polymerase Beta in ARPE–19 Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):173.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: RPE cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. Multiple in vitro models have been developed to simulate the RPE cellular response to oxidative stress that leads to apoptosis. The purpose of this study was to characterize DNA repair through DNA polymerase beta implicated in nuclear DNA repair in ARPE–19 cells exposed to oxidative stress with H2O2/ TNF alpha. Methods: Immunocytochemistry: ARPE–19 cells were incubated in DMEM–F12 with 10% FBS until cells on slides were 80% confluent, then stressed with H2O2/TNF alpha over a time course. Slides were fixed with 10% balanced buffered formalin at 1–hour intervals from 2 hours to 6 hours, and at 8 and 24 hours. Slides were kept at 15 degrees C. Slides were then washed with 1X PBS, exposed to cold methanol for 10 minutes, washed, blocked, and exposed to primary antibodies to DNA polymerase beta and TOM–20. Fluorescent secondary antibodies and Hoechst stain were used. Pictures were acquired by deconvolution microscopy. Acquired digital images were then analyzed to measure the area with DNA polymerase beta staining and the density of DNA polymerase staining per picture at each time point. Western blot: the same time course was followed after stressing the cells with H2O2/TNFalpha. Probing was done for DNA polymerase beta. Results: Immunohistochemistry showed the presence of DNA polymerase beta and an increase in intensity in treated cells as compared to controls. The peak occurred at 5 hours post–stimulation and then trended downward. Western blot showed increased DNA polymerase beta compared to controls that peaked at 4 hours post–stimulation. Conclusions: Oxidative stress has been implicated in RPE apoptosis. In vitro models have shown different DNA–repair mechanisms, including DNA polymerase gamma that is implicated in mitochondrial DNA repair. We show the up–regulation of DNA polymerase beta in the presence of H2O2/ TNF alpha in ARPE–19 cells. This DNA–repair mechanism is activated but not able to impede apoptosis. These observations will contribute to the identification of cell–signaling pathways in which pharmacologic intervention or genetic manipulation could impede RPE cell death and preserve visual function. Supported by NIH EY05121; DARPA HR0011–04–C–0068
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