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 γ-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.
Highlights
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]
We show that reexpression of NPRL2 in NPRL2negative and cisplatin-resistant cells significantly activates those key components, including ataxia telangiectasia mutated (ATM), Chk, and H2AX, and resensitizes lung cancer cells to cisplatin treatment in vitro and in vivo, leading to cell cycle arrest in the G2/M phase and induction of apoptosis
NPRL2 treatment resulted in accumulation of cells at the G1-S and G2-M checkpoints associated with inactivation of Cdc25A and Cdc25C and degradation of the cyclin-Cdk complex
Summary
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 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]. The molecular events responsible for resensitization to cisplatin by NPRL2 have not been identified. We attempt to understand the molecular link between NPRL2 and cisplatin in overcoming drug resistance
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