Abstract
Abstract: :
Purpose: Recently, this laboratory developed immortal murine lens epithelial cell lines which survive stress when subjected to either H2O2 (H), tertiary butylhydroperoxide (TBHP) (T), or a combination of both stresses (HT). These lines were developed since it is believed that peroxides are major contributors to the development of maturity onset cataract and other eye diseases. As part of the effort to further characterize these cell lines, cells were withdrawn from the stressing environment and their characteristics examined. Methods: Murine immortal lens epithelial cells, αTN4-1 were conditioned to survive either 125 µM H2O2, 135 µM TBHP or a combination of both stresses. To test the effect of withdrawal of stress, the peroxides were removed from the cell environment for six weeks producing H- and T- lines. Such cells were then subjected to stress in the usual manner, i.e., approximately 200-220 x 103 cells in 4 ml of a medium containing a given peroxide. Results: It was previously found that the T line survives exposure to not only TBHP but surprisingly to H2O2 as well while the H line is killed by TBHP. Now following a withdrawal of the peroxide stress, the T line gradually loses its ability to survive TBHP but retains resistance to H2O2. In contrast, the H- line retains its characteristics being resistant to H2O2 but not TBHP. Furthermore, the T- cells could be reconditioned to survive 130 µM TBHP in 4 days, rather than the 6 to 8 months required for conditioning control cells, indicating a modified system to resist TBHP. Examination of the activities of a number of antioxidative enzymes and SDS-PAGE polypeptide profiles indicated certain differences between the T and T- or the H and H- lines particularly in GSH-S-transferase. Changes in gene expression were analyzed with Affymetrix microarrays which contain probe sets for 12,422 genes and ESTs. 619 genes were found to have significant changes in expression in T vs T- based on T test and Affymetrix and Microsoft software analysis. A comparison of H and H- expression indicated 706 genes with significant change. Among the previously reported 28 antioxidative defense genes found by comparing the peroxide cell lines, a small group was found to be particularly responsive to the presence of TBHP. The overall results of these microarray analyses will be summarized. Conclusions: Examination of various conditioned cell lines and the effect of withdrawal of their peroxide stress indicates a dynamic system in which the expression of a large number of genes is changed. However, the results pinpoint a small group of genes that may be important in contributing to the specific antioxidative defense of the cell.
Keywords: oxidation/oxidative or free radical damage • molecular biology • gene microarray