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E.A. Blakely, P.Y. Chang, K.A. Bjornstad, C.J. Rosen; Differential Gene Expression Induced in Human Lens Cells by Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2896.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To understand the molecular basis of radiation–induced cataract, it is important to identify those genes with altered expression or function due to radiation–specific oxidative stress. Several published reports have investigated global genomic or proteomic changes in lens cells exposed to hydrogen peroxide, but it is unknown if this oxidative stresssor activates a response similar to ionizing radiation. We have used microarray analyses to investigate changes in gene expression in cultured human lens cells exposed to single acute doses of 55 MeV/amu protons. Protons are a predominant component of radiation encountered in space travel. Methods: A cultured human lens cell model (Blakely et al., IOVS 41:3808, 2000) was used for these studies. Lens cells were grown on matrix–coated plastic tissue culture dishes and irradiated with 55 MeV/nucleon proton beams at Berkeley Lab. Single doses of either 0.5, 2, or 4 Gy were given. RNA and protein were harvested at different times from 2 hr to 12 hr after radiation exposure. For gene array analysis, total RNA from an unirradiated control and an irradiated sample were reverse transcribed, PCR amplified and processed per directions provided by the manufacturer (Superarray Biosciences, Corp, MD). The biotin–labeled PCR amplicons were hybridized to functional cell cycle, p53 and ECM gene profiles, and applied to ECL film for fluorescent signal detection. Background noise signals were subtracted from the signal intensities for each gene spot and normalized to the positive housekeeping controls in each filter. Replicate experiments were completed. Results: Lens epithelial cells differentiating into fiber demonstrated an approximately two–fold increase in expression in a select number of genes. Irradiation of epithelial cells with 4 Gy protons demonstrated a nearly 3–fold increase in these genes within 2 hrs after exposure, whereas lens fiber cells showed significant decreased expression of these genes. Conclusions: Exposure to the oxidative stress of ionizing radiation from proton beams triggers premature expression of a select profile of cell cycle, p53, and ECM genes in cultured human lens epithelial cells, while decreasing their expression in differentiating fiber cells. Western analyses are in progress to confirm our findings at the protein level. The potential functional consequences of these radiation–induced changes will be discussed.
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