Abstract
Oxidative stress can cause significant cell death by apoptosis. We performed studies in L-cells to explore whether prior exposure to oxidative stress ("oxidative preconditioning") can protect the cell against the apoptotic consequences of subsequent oxidative insults and to establish the mediators in the preconditioning signaling cascade. Cells were preconditioned with three 5-min exposures to H(2)O(2), followed by 10-h recovery and subsequent exposure to 600 microm H(2)O(2) for 10 h. A single 10-h exposure to H(2)O(2) induced substantial apoptotic cell death (approximately 90%), as determined by enzyme-linked immunosorbent assay, TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling), and Annexin V methods, but apoptosis was largely prevented in preconditioned cells. The degree of cytoprotection depended on the strength of preconditioning or H(2)O(2) concentration (20 approximately 600 microm). Transient increases in mitogen-activated protein kinase (MAPK), p38, and JNK/SAPK activities and sustained protein kinase B (Akt) activation, accompanied by drastically reduced caspase 3 activity, were seen after preconditioning. The expression levels of these kinases were unaltered. Inhibitors of p38 (SB203580) and phosphoinositide 3-kinase (PI3K, LY294002) pathways abolished the protection provided by preconditioning. We conclude that oxidative preconditioning protects cells against apoptosis and that this effect involves MAPK and PI3K/Akt pathways. This system may be important in regulating apoptotic cell death in development and disease states.
Highlights
Oxidative stress can cause significant cell death by apoptosis
Apoptotic Preconditioning against Oxidative Stress—Exposure of cells to H2O2 (600 M) for 10 h caused significant cell values measured by ELISA for quantification of DNA fragmentation
The purposes of this study were to examine whether cells respond to oxidative stress with apoptosis and whether and how oxidative preconditioning can prevent apoptosis due to oxidative stress
Summary
Preconditioning (PC) was achieved by three brief (5-min) exposures to H2O2 (600 M) separated by 10-min recovery periods in normal culture medium. Cells were returned to normal medium for 10 h before exposure to H2O2 for another 10 h (Fig. 1A). Other sets of cells were handled in an identical fashion to preconditioned cells, except that they received sterile water in place of the H2O2 solution during the 5-min preconditioning periods (Fig. 1B). Groups of cells were removed for study immediately prior to (“control” cells) and at the end (“H2O2” cells) of the 10-h H2O2 exposure. Cells were preincubated with the drugs for 1 h prior to preconditioning (Fig. 1C) or prolonged H2O2 exposure.
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