The effect of catalase amplification on immortal lens epithelial cell lines

Utilizing a human beta-actin promoter, a catalase cDNA expression vector wa s constructed. This construct was used to transfect two immortal cell lines , mouse alpha TN4-1 and rabbit N/N 1003 A. The catalase activity was increa sed about 3.4 fold in the alpha TN4-1 cells and 38 fold in the N/N 1003A ce lls. Some changes in other enzyme activities were also observed as a result of the transfections. Surprisingly, the ability to degrade H2O2 in the ext racellular environment of the cells did not markedly change as a result of the catalase amplification. However, the ability to resist H2O2 stress was dramatically altered. Non-protein thiol (NP-SH) levels, choline uptake and glyceraldehyde phosphate dehydrogenase (GPD) activity were all markedly dec reased in the non-transfected cells when they were subjected to 300 mu M H2 O2. However, in both transfected cell lines, these parameters remained in t he normal range during H2O2 stress. The results obtained upon observing asp ects of DNA metabolism were more complicated. While on H2O2 stress, nontran sfected cell lines showed a marked decrease in thymidine incorporation, onl y the transfected alpha TN4-1 line remained in the normal range. Thymidine incorporation in transfected rabbit N/N 1003A cells was decreased compared to normal cells. In contrast, studies on single strand DNA breaks indicated that transfected rabbit cells had little damage compared to the significan t DNA damage observed in the normal cells. The normal N/N 1003A cells were also much more susceptible to H2O2 induced damage than normal alpha TN4-1 c ells, suggesting that the high GSH peroxidase activity observed in the rabb it cells may be detrimental since the low glutathione reductase activity in such cells results in an accelerated depletion of glutathione. The overall results suggest that augmenting lens catalase may prevent cataract develop ment caused by H2O2 stress. (C) 1998 Academic Press.