Elsevier

Free Radical Biology and Medicine

Volume 39, Issue 10, 15 November 2005, Pages 1332-1341
Free Radical Biology and Medicine

Original Contribution
Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress

https://doi.org/10.1016/j.freeradbiomed.2005.06.017Get rights and content

Abstract

Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.

Section snippets

Transfection of WI-38 SV40 human fibroblasts by rat MsrA-cDNA

WI-38 SV40 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C, 5% CO2, in a humidified incubator. The rat MsrA cDNA full sequence was cloned between the EcoRI and the BamHI restriction sites in the expression vector pCDNA3.1(+) (Invitrogen, Cergy Pontoise, France) under the control of the cytomegalovirus enhancer–promoter for high level expression. WI-38 SV40

Overexpression of MsrA in WI-38 SV40 human fibroblasts and characterization of the C3 clone

WI-38 SV40 cells were transfected with the pCDNA3.1(+)/msrA plasmid for stable expression of MsrA. After selection with Geneticin, resistant clones were isolated and individually expanded. Overexpression of MsrA was verified by Western blotting using an anti-MsrA antibody. As shown in Fig. 1A, several clones produced high levels of MsrA compared with cells transfected with the empty vector pCDNA3.1(+) (Fig. 1A, lanes C1, C2, and C3). Clone 3, which presented a higher expression of MsrA, was

Discussion

In this study, we have shown that MsrA overexpression is able to protect immortalized WI-38 SV40 human fibroblasts from protein oxidative damage and cell death by, at least in part, lowering the content of intracellular ROS. We had previously demonstrated that MsrA activity, as well as mRNA and protein expression, declined in rat organs such as brain, liver, and heart during aging [12]. More recently, we reported downregulation of MsrA and MsrB2 (CBS-1), one of the members of the MsrB family,

Acknowledgments

We thank F. Groh and Dr. F. Baleux for amino acids determination and N. Kassis for FACS analysis. This work was supported by funds from the MENRT (Université Paris 7) and by LVMH–Christian Dior Parfums (Convention Cifre No. 724/2002).

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