Enhanced expression of glutathione-S-transferase A1-1 protects against oxidative stress in human retinal pigment epithelial cells

Abstract

Glutathione-S-transferases (GSTs) play an important role in protection mechanisms against oxidative stress. We sought to determine whether over-expression of human GSTA1-1 in RPE cells is able to attenuate H(2)O(2)-induced oxidative stress. SV40-transformed human fetal RPE cells were stably transfected with pRC/hGSTA1-1 vector which carries a full-length of human GSTA1-1 cDNA. The control RPE cells were either non-transfected or transfected with control vector pRC. Expression of hGSTA1-1 protein in these cells was confirmed by Western blot and immunocytochemical analyses. The protective effects of hGSTA1-1 on cell viability and mitochondrial DNA (mtDNA) damage caused by H(2)O(2) were examined with MTT assay and quantitative PCR (QPCR), respectively. The hGSTA1-1 transfected RPE cells exhibited a similar morphology and growth rate as control RPE cells. Immunocytochemical analysis showed robust expression hGSTA1-1 in hGSTA1-1 transfected cells versus background staining in control cells. Western blotting of protein extracts from cells transfected with hGSTA1-1 revealed a 26 kDa protein band which corresponds to the size of recombinant mature hGSTA1-1. The active GST present in the hGSTA1-1 transfected cells was approximately three times higher than in control cells. The MTT assay showed a significantly greater viability of hGSTA1-1 cells in response to H(2)O(2) (100 and 200 microm) compared to control cells (p<0.05). QPCR indicated that mtDNA damage was significantly decreased in hGSTA1-1 cells than in control cells (p<0.05). Human GSTA1-1 transfection protect against RPE cell death and mtDNA damage caused by H(2)O(2), suggesting an important role of GST in protection against oxidative stress in RPE cells.