Proteomic analysis of oxidative stress-resistant cells: a specific role for aldose reductase overexpression in cytoprotection.

We are using a proteomic approach that combines two-dimensional electrophoresis and tandem mass spectrometry to detect and identify proteins that are differentially expressed in a cell line that is resistant to oxidative stress. The resistant cell line (OC14 cells) was developed previously through chronic exposure of a parent cell line (HA1 cells) to increasing hydrogen peroxide concentrations. Biochemical analyses of this system by other investigators have identified elevated content and activity of several classical antioxidant proteins that have established roles in oxidative stress resistance, but do not provide a complete explanation of this resistance. The proteomics studies described here have identified the enzyme aldose reductase (AR) as 4-fold more abundant in the resistant OC14 cells than in the HA1 controls. Based on this observation, the role of AR in the resistant phenotype was investigated by using a combination of AR induction with ethoxyquin and AR inhibition with Alrestatin to test the cytotoxicity of two oxidation-derived aldehydes: acrolein and glycolaldehyde. The results show that AR induction in HA1 cells provides protection against both acrolein- and glycolaldehyde-induced cytotoxicity. Furthermore, glutathione depletion sensitizes the cells to the acrolein-induced toxicity, but not the glycolaldehyde-induced toxicity, while AR inhibition sensitizes the cells to both acrolein- and glycolaldehyde-induced. These observations are consistent with a significant role for AR in the oxidative stress-resistant phenotype. These studies also illustrate the productive use of proteomic methods to investigate the molecular mechanisms of oxidative stress.