Nm23-H1 is involved in the repair of ionizing radiation-induced DNA double-strand breaks in the A549 lung cancer cell line

Ya Sheng,Xueqin Yang,Mingfang Xu,Dong Wang,Xunjie Kuang,Yanli Xiong,Yi Yang,Chongyi Li

doi:10.1186/s12885-018-4592-2

Ya Sheng, Xueqin Yang + Show 6 more

Open Access

https://doi.org/10.1186/s12885-018-4592-2

Copy DOI

Journal: BMC Cancer	Publication Date: Jul 3, 2018
Citations: 16	License type: open-access

Affiliation: Army Medical University, Daping Hospital

Abstract

BackgroundAlthough originally identified as a putative metastasis suppressor, increasing studies have confirmed a possible role for Nm23-H1 in DNA repair, through the base excision repair and nucleotide excision repair pathways. In this study, we explored whether Nm23-H1 was also involved in double-strand break repair (DSBR).Methods and resultsWe constructed a stable A549-shNm23-H1 cell line with doxycycline-regulated expression of Nm23-H1, and a A549-nNm23-H1 cell line that over expressed a nucleus-localized version of Nm23-H1. Results from both lines confirmed that Nm23-H1 participated in the repair of double-strand breaks induced by X-rays, using Comet and γ-H2AX foci assays. Subsequent studies showed that Nm23-H1 activated the phosphorylation of checkpoint-related proteins including ATM serine/threonine kinase (on S1981), tumor protein p53 (on S15), and checkpoint kinase 2 (Chk2) (on T68). We also detected interactions between Nm23-H1 and the MRE11-RAD50-NBS1 (MRN) complex, as well as Ku80. Moreover, NBS1 and Ku80 levels were comparably higher in Nm23-H1 overexpressing cells than in control cells (t = 14.462, p < 0.001 and t = 5.347, p = 0.006, respectively). As Ku80 is the keystone of the non-homologous end joining (NHEJ) pathway, we speculate that Nm23-H1 promotes DSBR through NHEJ.ConclusionsThe results indicate that Nm23-H1 participates in multiple steps of DSBR.

Highlights

Originally identified as a putative metastasis suppressor, increasing studies have confirmed a possible role for Nm23-H1 in DNA repair, through the base excision repair and nucleotide excision repair pathways
The results indicate that Nm23-H1 participates in multiple steps of double-strand break repair (DSBR)
Nm23-H1 promotes the repair of X-ray-induced double-strand breaks (DSBs) To investigate the role and mechanism of Nm23-H1 in DSBR, we constructed a stable A549-shNm23-H1 cell line with doxycycline-regulated expression of Nm23-H1.We constructed a stable A549-nNm2 3-H1 cell line that overexpressed Nm23-H1 and a nuclear localization sequence (NLS) to introduce Nm23-H1 into the nucleus, which would be beneficial to the follow-up experiment on the interaction of protein in the nucleus

Summary

Introduction

Originally identified as a putative metastasis suppressor, increasing studies have confirmed a possible role for Nm23-H1 in DNA repair, through the base excision repair and nucleotide excision repair pathways. Nm23-H1 is a multifunctional enzyme with decreased expression in certain highly metastatic cell lines and tumors, and was initially identified as a putative metastasis suppressor [1]. It possesses nucleoside diphosphate kinase (NDPK) activity, which maintains the intracellular nucleotide balance and is required for DNA synthesis [2]. DNA damage repair takes three forms, namely, base excision repair (BER), nucleotide excision repair (NER), and double strand break repair (DSBR). At almost the same time, Jarrett et al reported that human melanoma cell lines with coordinately low expression of Nm23-H1 and Nm23-H2 repaired UV-induced 6–4 photoproducts and other DNA polymerase-blocking lesions at a slower rate, and

Methods

Results

Discussion

Conclusion