Regulation of G 1 Arrest and Apoptosis in Hypoxia by PERK and GCN 2-Mediated eIF 2 α Phosphorylation 1 , 2

Hypoxia is a common microenvironment in solid tumors and is correlated with tumor progression by regulating cancer cell survival. Recent studies suggest that activation of double-stranded RNA-dependent protein kinase–like endoplasmic reticulum–related kinase (PERK) and phosphorylation of α subunit of eIF2 (eIF2α) confer cell adaptation to hypoxic stress. However, eIF2α is still phosphorylated at a lowered level in PERK knockout cells under hypoxic conditions. The mechanism for eIF2α kinase(s) (EIF2AK)–increased cell survival is not clear. In this report, we provide evidence that another EIF2AK, the amino acid starvation–dependent general control of amino acid biosynthesis kinase (GCN2), is also involved in hypoxia-induced eIF2α phosphorylation. We demonstrate that both GCN2 and PERK mediate the cell adaptation to hypoxic stress. High levels of eIF2α phosphorylation lead to G1 arrest and protect cells from hypoxia-induced apoptosis. Reduced phosphorylation of eIF2α by knocking out either PERK or GCN2 suppresses hypoxia-induced G1 arrest and promotes apoptosis in accompany with activation of p53 signal cascade. However, totally abolishing phosphorylation of eIF2α inhibits G1 arrest without promoting apoptosis. On the basis of our results, we propose that the levels of eIF2α phosphorylation serve as a “switch” in regulation of G1 arrest or apoptosis under hypoxic conditions. Neoplasia (2010) 12, 61–68 Introduction Cells respond to external stimuli by rapid changes in their translational capacity. Stress, such as growth factor depletion, heat shock, and virus infection, rapidly inhibits protein synthesis through phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α) [1,2]. Four kinases, the double-stranded RNA-dependent protein kinase (PKR), the hemin-regulated inhibitor (HRI), the amino acid starvation–dependent general control of amino acid biosynthesis kinase (GCN2), and the PKR-like endoplasmic reticulum–related kinase (PERK), have been identified to phosphorylate eIF2α and reduce translation initiation in response to stress [2]. Recently, PERK-mediated phosphorylation of eIF2α has been shown to suppress protein synthesis and cell growth on hypoxia [3–6]. In vivo studies show that hypoxiainduced activation of PERK inhibits protein synthesis, decreases cell growth, and promotes tumor adaptation [2–6]. Adaptation to hypoxia could also be regulated by hypoxia-inducible factor 1 (HIF-1), which associates with tumor progression and resistance to radiotherapy and chemotherapy [2,7,8]. HIF-1 is a key mediator in hypoxia [9] and regulates the expressions of more than 70 genes [10] that facilitate metabolic adaptation, cell proliferation, cell cycle arrest, apoptosis, angiogenesis, angioinvasion, and metastasis [11,12]. Abbreviations: eIF2α, the α subunit of the eukaryotic initiation factor-2; EIF2AK, eIF2α kinase; GCN2, amino acid starvation–dependent general control of amino acid biosynthesis kinase;HIF-1α, hypoxia-inducible factor 1α; HRI, hemin-regulated inhibitor;Mdm2, murine double minute 2; MEF, mouse embryonic fibroblast; PERK, PKR-like endoplasmic reticulum–related kinase; PKR, double-stranded RNA-dependent protein kinase Address all correspondence to: Shiyong Wu, Ph.D., Edison Biotechnology Institute and Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701. E-mail: wus1@ohio.edu This work was partially supported by the National Institutes of Health grants RO1 CA86928 (to S.W.) and R56 CA086928 (to S.W.). This article refers to supplementary material, which is designated by Figure W1 and is available online at www.neoplasia. com. Received 11 August 2009; Revised 30 September 2009; Accepted 30 September 2009 Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1522-8002/10/$25.00 DOI 10.1593/neo.91354 www.neoplasia. com Volume 12 Number 1 January 2010 pp. 61–68 61