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A Spector, D Li, W Ma, F Sun, P Pavlidis; Gene Expression in Lens Epithelial Cell Lines Conditioned to Survive H2O2 or TBHP . Invest. Ophthalmol. Vis. Sci. 2002;43(13):852.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: An ideal approach for finding genes which are capable of contributing to cell survival is to condition the cells to survive a particular stress. Since peroxide is believed to be a major factor in oxidative stress induced cataract formation, immortal lens epithelial cell lines have been conditioned to tolerate peroxide levels which cause cataract in vitro. Such conditioned cells have a relatively constant level of gene expression allowing for examination of the changes in expression that are directly related to the response to the oxidative stress. The results are reported in this communication. Methods: αTN4-1 cells were conditioned to survive H2O2 or tertiary butyl hydroperoxide (TBHP) stress by gradually increasing the peroxide concentration over a period of 7 to 8 months (see Spector et al., IOVS 41, 832-843, 2000). Gene expression of the different cell lines was determined on isolated total RNA samples utilizing Affymetrix mouse expression chips (MG-U74v2). These chips contain suites of 25 residue oligonucleotide probes which can recognize 12,500 gene fragments and ESTs. Expression of cells conditioned to survive H2O2, H cells; TBHP, T cells; and both H2O2 and TBHP, TH cells; were examined and compared to control unconditioned C cells. Gene expression was statistically analyzed. Results: Examination of the changes in gene expression in the H line in comparison to the C line indicated that at = or ≷ ± 2X more than 1,420 genes were found. The number of detected genes decreases markedly at the 3X level to 639 genes, at 4X to 378 genes and at 5X to 256 genes. Since not all values based on fold change cutoffs are statistically meaningful, statistical analysis of expression levels and analysis utilizing other parameters such as signal log ratios were also examined and will be reported. Fourteen antioxidative genes had been found in the H versus C comparison previously reported based on version 1 Affymetrix chip analysis of 9,977 genes and ESTs with a change in expression = or ≷ ± 5 fold. In the version 2 analysis, most of these antioxidative genes had a similar fold change and only a few additional antioxidative genes were detected. The expression of some of these antioxidative genes was confirmed by real time PCR analysis. The results obtained with T and HT cell lines will also be discussed. Conclusion: Utilizing Affymetrix expression chips and statistical methodologies, as well as real time PCR analysis, a group of antioxidative genes which may protect cells against different peroxide stresses have been defined.
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