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S Goswami, N Sheets, BK Chauhan, J Zavadil, V Reddy, E Bottinger, M Kantorow, A Cvekl; Elucidating the Range of Oxidative Stress Pathways in Human Lens Epithelial Cells: A cDNA Microarray Study . Invest. Ophthalmol. Vis. Sci. 2002;43(13):851.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The lens has evolved multiple pathways to defend against oxidative stress and many of these pathways respond to stress through rapid changes in gene expression. Here we sought to identify the spectrum of genes responding to H2O2-induced oxidative stress (OS) in lens epithelial cells using cDNA microarrays. Methods: HLE cells (SR-01-04) were treated with 50 mM of H2O2 for 1 h in the absence of serum and cultured for 2, 3, 5 and 8 h. Recovery of the cells was monitored by examining levels of apoptosis by Annexin V binding and propidium iodine staining and FACS analysis. RNAs were prepared from treated and untreated cells at times 0,1,2,3, 5 and 8 hr. Controls included serum starved cells for 1h and serum stimulated cells. RNAs were analyzed by cDNA microarray hybridizations with the AECOM human chips containing 9,600 genes. Reference RNA was prepared by pooling multiple RNA samples. Each experiment was conducted in triplicate with a total amount of 30 individual data entries. Scanning, digitalization, normalization, and flagging of data were conducted using Axon scanner, Genepix, and GeneSpring software, respectively. Data mining procedures involved public and private (Compugen, Inc.) databases. Expression levels of selected genes were confirmed by semi-quantitative RT-PCR and western analysis. Results: A gene expression database was developed to generate time-dependent clustering profiles of 4,500 genes in response to OS. Individual clusters were analyzed and differentially expressed genes were grouped according their known and/or deduced function. Differential expression of selected genes was confirmed both on the RNA and protein levels. Conclusions: The data revealed that at least 1,400 lens epithelial genes rapidly respond to OS through increased or decreased expression at the 2 to 12-fold level. Many of these genes fall into previously identified OS responsive pathways while others point to novel chemical and heat shock pathways. Collectively, these data set the groundwork to characterize the spectrum, magnitude and functional importance of those lens pathways responding to oxidative stress. Support: NIH (EY12200 (AC), 13022 (MK) 00484 (VR)), RPB (AC), Inc. Proprietary Interests: None
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