The effect of catalase amplification on immortal lens epithelial cell lines.
Utilizing a human beta-actin promoter, a catalase cDNA expression vector was constructed. This construct was used to transfect two immortal cell lines, mouse alpha TN4-1 and rabbit N/N 1003A. The catalase activity was increased about 3.4 fold in the alpha TN4-1 cells and 38 fold in the N/N 1003A cells. Some changes in other enzyme activities were also observed as a result of the transfections. Surprisingly, the ability to degrade H2O2 in the extracellular 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 decreased in the non-transfected cells when they were subjected to 300 microM H2O2. However, in both transfected cell lines, these parameters remained in the normal range during H2O2 stress. The results obtained upon observing aspects of DNA metabolism were more complicated. While on H2O2 stress, non-transfected cell lines showed a marked decrease in thymidine incorporation, only 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 significant 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 cells, suggesting that the high GSH peroxidase activity observed in the rabbit 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 development caused by H2O2 stress.
Yang
Y
Y
Department of Ophthalmology, Columbia University, New York, NY 10032, USA.
Spector
A
A
Ma
W
W
Wang
R R
RR
Larsen
K
K
Kleiman
N J
NJ
D000818:Animals; D001692:Biological Transport; D002374:Catalase / Q000378:metabolism*; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D011817:Rabbits