== (A) Western blot shows reduction in Chk2 expression using SiRNA specific to Chk2 (SiChk2) and scrambled siRNA (SiScrb) as control in stable clones generated in 1437R lung cancer cell line. 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. == Introduction == NPRL2/Gene Hexacosanoic acid 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 component 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 apoptosisin vitroandin vivo[5]. However, the molecular events responsible for resensitization to cisplatin byNPRL2have 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 (-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 (meiotic recombination 11)-RAD50 (radiation sensitive 50)-NBS1 (Nijmegen Hexacosanoic acid breakage syndrome 1) complex[10][13]. Phospho-ATM facilitates the phosphorylation of those mediators, and the formation of nuclear foci of the activated molecules promotes transmission of the DNA damage signal to downstream targets, such as Chk1 (check point kinase 1), Chk2 (check point kinase 2), and SMC1 (structural maintenance of chromosomes 1)[14][16]. Chk1 and Chk2 are involved in various DNA-damage responses, including cell-cycle checkpoint, genome maintenance, DNA repair, and apoptosis[17]. Therefore, we hypothesized that the sensitization of non-small cell lung cancer (NSCLC) cells to cisplatin by NPRL2 may be due to the direct regulation of key components in the DNA-damage checkpoint pathway. To test this hypothesis, we analyzed effects of the ectopic expression of NPRL2 in the presence or absence of the DNA-damaging agent cisplatin on tumor cell growth and apoptosis in a panel of NSCLC cell lines with varied status of the endogenous NPRL2 gene and protein expression and cisplatin sensitivity. We Rabbit Polyclonal to CK-1alpha (phospho-Tyr294) show that reexpression of NPRL2 in NPRL2-negative and cisplatin-resistant cells significantly activates those key components, including ATM, Chk, and Hexacosanoic acid H2AX, and resensitizes lung cancer cells to cisplatin treatmentin vitroandin vivo, leading to cell cycle arrest in the G2/M phase and induction of apoptosis. The findings of the present study also lend experimental support to our hypothesis that NPRL2 may function not only as a prognostic biomarker for sensitivity to cisplatin, but also as a therapeutic agent for resensitizing nonresponding cancer cells to cisplatin. == Materials and Methods == == Cell lines and cell culture == The human NSCLC cell lines H1299[5]and H322[5], with various 3p21.3 status described previously[18],[19], were used forin vitroandin vivoexperiments. H1299 and H322 are cisplatin-resistant (50% inhibitory concentration [IC50] and IC20values for cisplatin in H1299 were 7.6 and 3.0 M, respectively,.
== (A) Western blot shows reduction in Chk2 expression using SiRNA specific to Chk2 (SiChk2) and scrambled siRNA (SiScrb) as control in stable clones generated in 1437R lung cancer cell line
