Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene- induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2.
PURPOSE: To investigate the physiological role of two major alpha-class glutathione S-transferases (GSTs), hGSTA1-1 and hGSTA2-2 in protection against oxidative stress and lipid peroxidation (LPO) in human lens epithelial ( HLE B-3) cells. METHODS: Total GSTs were purified from HLE B-3 cells by glutathione (GSH)-affinity chromatography and characterized by Western blot analysis, isoelectric focusing, and kinetic studies. The relative contributions of the alpha-class GSTs and the Se-dependent glutathione peroxidase (GPx)-1 in GSH-dependent reduction of phospholipid hydroperoxide (PL-OOH) were quantitated through immunoprecipitation studies using separately the specific polyclonal antibodies against human alpha-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTA1-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the alpha-class GSTs against H2O2- and naphthalene-induced LPO and apoptosis were examined by transfecting HLE B-3 cells with cDNAs of hGSTA1 and hGSTA2. RESULTS. HLE B-3 cells expressed only the alpha and pi class GSTs. The Michaelis-Menten constant (k(m)) and turnover number (k(cat)) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 +/- 4 microM and 1.95 +/- 0.26 seconds, respectively. The alpha-class GSTs accounted for approximately 65% of the total GPx activity of HLE B-3 cells toward PC-OOH. Our results demonstrate for the first time that hGSTA1-1 and hGSTA2-2 effectively catalyzed GSH-dependent reduction of membrane PL-OOH in situ in HLE B-3 cells. Transfection with hGSTA1 or hGSTA2 protected these cells from H2O2- and naphthalene-induced LPO and attenuated H2O2- and naphthalene-induced apoptosis through inhibiting caspase 3 activation. CONCLUSIONS: These results demonstrate that the alpha-class GSTs hGSTA1-1 and hGSTA2-2 play a major role as antioxidant enzymes and are the main determinants of the levels of LPO caused by oxidative stress in human lens epithelial cells.[1]References
- Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2. Yang, Y., Sharma, R., Cheng, J.Z., Saini, M.K., Ansari, N.H., Andley, U.P., Awasthi, S., Awasthi, Y.C. Invest. Ophthalmol. Vis. Sci. (2002) [Pubmed]
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