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 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.
Liang
Fong-Qi
FQ
Retina Foundation of the Southwest, 9900 N. Central Expressway, Suite 400, Dallas, TX 75231, USA.
Alssadi
Rajiha
R
Morehead
Preston
P
Awasthi
Yogesh C
YC
Godley
Bernard F
BF
D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000032:analysis*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D004249:DNA Damage / Q000502:physiology; D004272:DNA, Mitochondrial / Q000502:physiology; D004847:Epithelial Cells / Q000502:physiology; D005136:Eye Proteins / Q000032:analysis*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005982:Glutathione Transferase; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D018384:Oxidative Stress / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods
D002352:Carrier Proteins; D004272:DNA, Mitochondrial; D005136:Eye Proteins; C484555:GSTA1 protein, human; D005982:Glutathione Transferase