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C.T. Schulteis, K.L. Rhoades, A. Klimova, A.G. Gutierrez, N.F. Paoni, F. Wong–Staal; AG126 Protects ARPE–19 Cells From Oxidative Stress via a Mechanism Dependent on RNA Transcription . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1630.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Oxidative stress has been implicated in retinal damage associated with both dry and exudative forms of age–related macular degeneration. In vitro experiments in ARPE–19 cells have suggested that apoptotic cell death in response to oxidative stress occurs via a ceramide–mediated signaling mechanism (Barak, et al., (2001) Inv. Ophthalmol. & Vis. Sci., 42, 247). Additional reports have demonstrated that cultured cells can be protected from oxidative stress by the tyrphostin, AG126 (Garg & Tang (2003) BMC Ophthalmology, 3, 5). It remains unclear, however, whether AG126 protection occurs via tyrosine kinase inhibition or through an alternate mechanism. In this study, AG126 protection of ARPE–19 cells from oxidative stress and ceramide–induced apoptosis is characterized. In addition, the dependence of AG126 protection on RNA transcription and tyrosine kinase inhibition is evaluated. Methods: Oxidative stress or apoptotic cell death was induced in ARPE–19 cells via treatment with H2O2, tBH, 7–ketosterol, or C2–ceramide. Cell health in the absence and presence of AG126, genestein, and/or Actinomycin D was monitored via WST–1 analysis. Results: ARPE–19 cells were protected from oxidative stress induced by H2O2 or tBH upon pretreatment with 20 to 50 µM AG126. AG126 did not protect ARPE–19 cells from oxysterol– or C2–ceramide–induced cell death. A minimum of 5 h pretreatment was required for AG126 protection, while concurrent treatment had no protective effect. Treatment with the broad range tyrosine kinase inhibitor, genestein, did not ameliorate the effects of H2O2 or tBH. Addition of actinomycin D, an RNA transcription inhibitor, blocked the protective effect of AG126. Conclusions: AG126 protects ARPE–19 cells from oxidative stress through a mechanism independent of tyrosine kinase inhibition but dependent upon gene expression. The pretreatment requirement indicates that AG126 is not acting as an antioxidant. The action of AG126 is likely to occur before the ceramide signaling cascade is triggered by H2O2 or tBH. Characterization of the intervening molecular mechanism may reveal relevant targets for therapeutic intervention in the management of both wet and dry forms of age–related macular degeneration.
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