A P53-INDEPENDENT PATHWAY FOR ACTIVATION OF WAF1 CIP1 EXPRESSION FOLLOWING OXIDATIVE STRESS/

Incubating human cells in diethylmaleate (DEM) depletes the intracellu lar pool of reduced glutathione (GSH) and increases the concentration of oxidative free radicals. We found that DEM-induced oxidative stress reduced the ability of p53 to bind its consensus recognition sequence and to activate transcription of a p53-specific reporter gene. Nevert heless, DEM treatment induced expression of WAF1/CIP1 but not GADD45 m RNA. The fact that N-acetylcysteine, a precursor of GSH that blocks ox idative stress, prevented WAF1/CIP1 induction by DEM suggests that WAF 1/CIP1 induction probably was a consequence of the ability of DEM to r educe intracellular GSH levels. DEM induced WAF1/CIP1 expression in Sa os-2 and T98G cells, both of which lack functional p53 protein. DEM tr eatment did not produce an increase in membrane-associated protein kin ase C, but ERK2, a mitogen-activated protein kinase, was phosphorylate d in a manner consistent with ERK2 activation. DEM treatment also prod uced a dose-dependent delay in cell cycle progression, which at low co ncentrations (0.25 mM) consisted of a G(2)/M arrest and at higher conc entrations (1 mM) also involved G(1) and S phase delays. Our results i ndicate that oxidative stress induces WAF1/CIP1 expression and arrests cell cycle progression through a mechanism that is independent of p53 . This mechanism may provide for cell cycle checkpoint control under c onditions that inactivate p53.