NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway Gitanjali Jayachandran 1. , Kentaro Ueda 1,3. , Bingbing Wang 1 , Jack A. Roth 1,2 , Lin Ji 1,2 * 1 Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America, 2 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas, United States of America, 3 School of Medicine, Wakayama Medical University, Wakayama, Japan Abstract NPRL2, one of the tumor suppressor genes residing in a 120-kb homozygous deletion region of human chromosome band 3p21.3, has a high degree of amino acid sequence homology with the nitrogen permease regulator 2 (NPR2) yeast gene, and mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Previously, we showed that restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment in vitro and in vivo. In this study, we show that sensitization of non-small cell lung cancer (NSCLC) cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway. NPRL2 can phosphorylate ataxia telangiectasia mutated (ATM) kinase activated by cisplatin and promote downstream c-H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular- weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Activated Chk1 and Chk2 increase the expression of cell cycle checkpoint proteins, including Cdc25A and Cdc25C, leading to higher levels of G2/ M arrest in tumor cells treated with NPRL2 and cisplatin than in tumor cells treated with cisplatin only. Our results therefore suggest that ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2- negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2/M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy. Citation: Jayachandran G, Ueda K, Wang B, Roth JA, Ji L (2010) NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway. PLoS ONE 5(8): e11994. doi:10.1371/journal.pone.0011994 Editor: Kerstin Borgmann, University Medical Center Hamburg-Eppendorf, Germany Received December 7, 2009; Accepted July 9, 2010; Published August 5, 2010 Copyright: ß 2010 Jayachandran et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partially supported by grants from the National Cancer Institute, National Institutes of Health, SPORE P50CA70907, R01CA116322, and MMHCC U01CA10535201; grants from the Department of Defense Lung Cancer Program PROSPECT (DAMD17-02-1-0706); The University of Texas M. D. Anderson Cancer Center Support Core Grant (CA-16672); and a grant from the Tobacco Settlement Funds as appropriated by the Texas State Legislature. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lji@mdanderson.org . These authors contributed equally to this work. Introduction NPRL2/Gene 21 (GenBank accession #AF040707), which is 1351 bp long and encodes a protein of 380 amino acid residues, is one of the tumor suppressor genes identified in a 120-kb homozygous deletion region on human chromosome band 3p21.3 [1,2]. The frequent and early loss of heterozygosity and the overlapping homozygous deletions observed in the 3p21.3 region in lung and breast cancers suggest a critical role of one or more 3p21.3 genes in the molecular pathogenesis of these cancers [1,3]. The nitrogen permease regulator 2 (NPR2) yeast gene (GenBank accession #P39923) was identified as a novel com- ponent involved in cell killing triggered by cisplatin. Because disruption of NPR2 was shown to confer resistance to cisplatin, it was believed that NPRL2 may use a similar mechanism to mediate the cytotoxicity of anticancer drugs [4]. We recently found that the reexpression of NPRL2 in NPRL2-negative and cisplatin-resistant cells significantly resensitized the response of these cells to cisplatin treatment, as evidenced by reduced cell viability and increased apoptosis in vitro and in vivo [5]. However, the molecular events responsible for resensitization to cisplatin by NPRL2 have not been identified. In this study, we attempt to understand the molecular link between NPRL2 and cisplatin in overcoming drug resistance. The effects of cisplatin are mediated through high levels of DNA damage, leading to programmed cell death or cell cycle arrest [6]. The double-strand DNA breaks induced by cisplatin are mediated through a central DNA damage-signaling pathway controlled by the ataxia telangiectasia mutated (ATM) kinase as well as several other DNA damage-responsive kinases [7,8]. The phosphorylation of ATM at serine-1981 has been shown to phosphorylate histone H2AX [9], and the phosphorylation of histone H2AX at serine- 139 (c-H2AX) is essential for the recruitment of mediators such as MDC1 (mediator of DNA damage checkpoint protein 1), 53BP1 (p53 binding protein 1), BRCA1 (breast cancer 1), and MRE11 PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e11994