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JR Warren, CM H Colitz, A Whittington; Measurement of GADD45 in Canine Lens Epithelial Cells in Response to Oxidative Stress . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2360.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:We previously demonstrated that lens epithelial cells (LEC) have telomerase activity (TA) and we hypothesized that TA serves to protect LEC from senescence resulting from oxidative damage. GADD45, a DNA repair gene, is altered in response to DNA damage caused by oxidative stress. Therefore, since oxidative stress is implicated in cataractogenesis, the purpose of these experiments was to determine how exposure of LEC to tertiary butyl-hydroperoxide (TBHP) induced oxidative stress affects TA and GADD45 expression. Methods:Freshly excised canine lenses were incubated in media with serum. In the first experiment, lenses were exposed to media with or without 0.5mM TBHP for varying time periods (8 lenses per group; 15, 25, 40, 60, 120 minutes and overnight) In the second experiment, lenses were exposed to media containing 0.5 mM TBHP for one hour and then allowed to recover in media without TBHP for varying time periods (8 lenses per group; 5, 60, 180 minute and overnight recoveries, no recovery, control group without treatment). Following treatments (tx), protein was extracted from LEC. GADD45 levels were measured by standard ELISA. TA was measured by TRAP-ELISA. Oxidized glutathione measured oxidative stress by enzyme recycling. All data is in OD units. Results:GADD45 was decreased in the first experiment (0.158-0.222) at all time points vs controls (0.275, p<0.05). TA was increased only at 120 min (p<0.01) and GSSG was elevated at all times (1.92-2.50) vs controls (0.473 p<0.0001). In the second experiment, GADD45 levels were elevated in all recovery groups except 15 minutes (0.180-0.264) vs controls (0.153, p<0.01). TA was elevated at the 15, 60 and 180 min recovery times (2.173-2.268) vs controls (1.547, p<0.05) GSSG levels initially increased sixfold vs control in the no recovery group, then decreased gradually to approximate control after 60 min recovery. Conclusion:GADD45 is a DNA-damage inducible growth arrest gene that functions at both the G1-S and G2-M checkpoints. In the first experiment, elevated GSSG indicated that the cells were stressed at all tx times. While there was no change in TA, except at the 2 hour time point, GADD45 was significantly decreased. GADD45 is usually up-regulated by oxidative stress, but can sometimes be down-regulated by c-myc under stress-induced conditions in some cell types. In the second/recovery experiment, GSSG returned to normal by 60 minutes and both TA and GADD45 were up-regulated. It is possible that GADD45 is down-regulated during acute stress and then when the stress is removed, it is up-regulated along with TA as part of the recovery/repair process.
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