Original Contribution DOMINANT-NEGATIVE JUN N-TERMINAL PROTEIN KINASE (JNK-1) INHIBITS METABOLIC OXIDATIVE STRESS DURING GLUCOSE DEPRIVATION IN A HUMAN BREAST CARCINOMA CELL LINE YONG J. LEE, 1, *SANDRA S. GALOFORO,* JULIA E. SIM, † LISA A. RIDNOUR, † JINAH CHOI, ‡ HENRY JAY FORMAN, ‡ PETER M. CORRY,* and DOUGLAS R. SPITZ 1,† *Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, MI; † Section of Cancer Biology, Radiation Oncology Center, Washington University School of Medicine, St. Louis, MO; and ‡ Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA, USA (Received 10 August 1999; Revised 9 November 1999; Accepted 7 December 1999) Abstract—Signal transduction pathways involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytotoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, -glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of -glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation-induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades. © 2000 Elsevier Science Inc. Keywords—Free radicals, Glucose deprivation, JNK, Oxidative stress, Signal transduction, Glutathione metabolism, -Glutamylcysteine synthetase INTRODUCTION We have previously observed that glucose deprivation induces cell death [1], activates c-Jun N-terminal kinase 1 (JNK1) [2], and increases intracellular pro-oxidant production and oxidized glutathione content [3] in mul- tidrug-resistant human breast carcinoma cells (MCF-7/ ADR). During glucose deprivation the thiol antioxidant, N-acetylcysteine, suppresses JNK1 activation and glu- cose deprivation-induced cytotoxicity as well as sup- pressing increased pro-oxidant production and the accu- mulation of oxidized glutathione [3,4]. These results have led us to investigate the involvement of JNK1 signaling pathways in the process of glucose deprivation- induced cytotoxicity and oxidative stress in MCF-7/ADR cells. Several studies have demonstrated that JNK, also called stress-activated protein kinases (SAPK), signal transduction pathways can be activated by a diverse array of cellular stimuli. JNKs are activated in response to mitogenic signals, including growth factors [5], T cell activation and proliferation signaling [6 – 8], oncogenic Ras [9], and CD40 ligation [10,11]. JNKs are also in- Address correspondence to: Dr. Yong J. Lee, Department of Phar- macology, Cancer Institute, University of Pittsburgh, W634 Monte- fiore, University Hospital, 200 Lothrop St., Pittsburgh, PA 15213, USA; Tel: (412) 692-2320; Fax: (412) 692-2899. 1 These authors contributed equally to the preparation of the manuscript. Free Radical Biology & Medicine, Vol. 28, No. 4, pp. 575–584, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter PII S0891-5849(99)00267-1 575