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

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 H2O2-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 H2O2 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 hGSTAI-1 in hGSTA1-1 transfected cells versus background staining in control cells. Western blotting of protein extracts from cells transfected with hGSTAI-1 revealed a 26 kDa protein band which corresponds to the size of recombinant mature hGSTAI-1. The active GST present in the hGSTAI-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 H2O2 (100 and 200 mum) 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 H2O2, suggesting an important role of GST in protection against oxidative stress in RPE cells. (C) 2004 Elsevier Ltd. All rights reserved.