Evaluation of antioxidant defense systems in H4IIE cells infected with a retroviral vector

Retroviral gene transfer technology is frequently used to establish stable transgenic cell lines. However, no studies to date have evaluated antioxidant defense systems in cells infected with retroviral particles. In the present study, we examined the effects of retroviral infection on antioxidant defense systems using H4IIE cells infected with a retrovirus that overexpresses green fluorescent protein (retro-H4IIE cells). Total oxyradical scavenging capacity and glutathione (GSH), malondialdehyde, and peroxide levels were not significantly altered in retro-H4IIE cells; however, retro-H4IIE cells showed a higher resistance against cytotoxicity, GSH depletion, and malondialdehyde elevation under H2O2-induced oxidative stress conditions. Immunoblot analysis showed that alpha-class GSH S-transferase (GST) was increased 2.5-fold in retro-H4IIE cells as compared with H4IIE cells; however, catalase, GSH peroxidase-1, peroxiredoxin-1, and thioredoxin-1 remained unaltered or slightly decreased. l-Buthionine-(S,R)-sulfoximine, a GSH synthesis inhibitor, and 1-chloro-2,4-dinitrobenzene, a GST substrate and competitive inhibitor, decreased the difference in H2O2 responses between the two cell types. These results support the hypothesis that the resistance of retro-H4IIE cells to H2O2 can be attributed to an increase in alpha-class GST expression, as levels of GSH and GSH peroxidase-1 were not altered. The present study suggests that antioxidant enzyme expression may change during the establishment of stable transformed cell lines using retroviral techniques.