Enhanced Glutathione–S–Transferase (GST) Expression Protects Against Oxidative Stress In Retinal Pigment Epithelial (RPE) Cells

Abstract

Abstract: : Purpose: Oxidative stress may be one of the causative factors in age–related macular degeneration (AMD). Our previous studies showed that transient exposure of low dose of hydrogen peroxide (H₂O₂) to cultured RPE cells up–regulated GST expression and activity, suggesting that this may be a primary response to low–level oxidative stress. The present study aims to test the efficacy of GST overexpression in mitigating oxidative stress in RPE cells. Methods: SV40–transformed fetal human RPE cells (RPE28) were transfected with pET30a/CMV–hGSTA1–1 or with control vector using liposome–based TransFast transfection reagent. Stable transfectants were selected by using genetecin (G418). Expression of hGSTA1–1 by these cells was detected with Western blot and immunostaining. Confluent cells were exposed to H₂O₂ (100 or 200 uM) for 1 hr, and cell viability and mitochondrial DNA (mtDNA) damage were examined with the MTT reduction assay and quantitative PCR, respectively. Results: GST–transfected cells exhibited similar morphology and growth rates (doubling time approximately 24 hrs) to cells transfected with control vector and non–transfected cells. In GST–transfected RPE cells, strong immunostaining for hGSTA1–1 was observed as compared to faint staining in control vector and non–transfected cells. Prominent immunoreactive bands (26 kDa) were consistently detected in protein extracts from GST–transfected cells, indicating a mature hGSTA1–1 protein. Although all cell lines demonstrated a dose–dependent decrease in cell viability in response to H₂O₂treatment, GST cells showed a significantly higher rates of viability (p<0.05). H₂O₂–induced mtDNA damage was significantly less in GST–transfected cells (p<0.05) compared to those in control vector and non–transfected cells. Conclusions: Overexpression of GST protects RPE cells from oxidatively mediated cell death and mtDNA damage. These data suggest that this enzyme plays an important role in the oxidative defense mechanism in RPE cells, and overexpression may be a novel therapeutic approach in diseases such as AMD.

Keywords: oxidation/oxidative or free radical damage • retina • age–related macular degeneration