A p53-independent Pathway for Activation of WAF1/CIP1 Expression Following Oxidative Stress

doi:10.1074/jbc.270.49.29386

A p53-independent Pathway for Activation of WAF1/CIP1 Expression Following Oxidative Stress *

From the (1) Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli “Federico II”, I-80131 Naples, Italy, the
(2) Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, the
(3) Georgetown University Medical Center, Department of Biochemistry and Molecular Biology, Washington, D. C. 20007, the
(4) Laboratory of Molecular Pharmacology, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland, 20892, and the
(5) Biology Department, Brookhaven National Laboratory, Upton, New York 11973

^¶ Supported by the AIDS Directed Anti-Viral Program of the Office of the Director of NIH. To whom correspondence should be addressed:
Laboratory of Cell Biology, NCI, NIH, Bldg. 37, Rm. 1B04, Bethesda, MD 20892
. Tel.: 301-402-4177; Fax: 301-496-7220.

Abstract

Incubating human cells in diethylmaleate (DEM) depletes the intracellular 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. Nevertheless, DEM treatment induced expression of WAF1/CIP1 but not GADD45 mRNA. The fact that N-acetylcysteine, a precursor of GSH that blocks oxidative stress, prevented WAF1/CIP1 induction by DEM suggests that WAF1/CIP1 induction probably was a consequence of the ability of DEM to reduce intracellular GSH levels. DEM induced WAF1/CIP1 expression in Saos-2 and T98G cells, both of which lack functional p53 protein. DEM treatment did not produce an increase in membrane-associated protein kinase C, but ERK2, a mitogen-activated protein kinase, was phosphorylated in a manner consistent with ERK2 activation. DEM treatment also produced a dose-dependent delay in cell cycle progression, which at low concentrations (0.25 mM) consisted of a G²/M arrest and at higher concentrations (1 mM) also involved G¹ and S phase delays. Our results indicate 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 conditions that inactivate p53.

Footnotes

↵^§ Supported by the Office of Health and Environment of the U. S. Department of Energy.
↵* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (to T. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽¹⁾ The abbreviations used are:

DEM

diethylmaleate

GSH

glutathione

MAP

mitogen-activated protein

RGC

ribosomal gene cluster

NAC

N-acetylcysteine

TPA

12-O-tetradecanoylphorbol-13-acetate

PCNA

proliferating cellular antigen.
- Received June 6, 1995.
- Revision received August 17, 1995.