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

Abstract

Background: Although 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 results: We 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.

Conclusions: The results indicate that Nm23-H1 participates in multiple steps of DSBR.

Keywords: Double-strand break repair; Lung cancer; Nm23-H1.

Fig. 1

The expression and location of Nm23-H1 in A549-shNm23-H1 cell line and A549-nNm23-H1 cell line. A549-shNm23-H1 cell line was a Dox-regulated vector system of conditional suppressing the expression of Nm23-H1, and the Nm23-H1 expression was suppressed only when doxycycline was added. A549-nNm23-H1 cell line was constructed for over expressed Nm23-H1 with nuclear located sequence (NLS) which can introduce Nm23-H1 into nucleus. a The expression of Nm23-H1 in A549-shNm23-H1 cell line detected by Western blot. b The expression and location of Nm23-H1 in A549-nNm23-H1 cell line detected by Western blot NLS-Nm23-H1 is the constructed protein(18 kDa) and Nm23-H1 is the endogenous protein (17 kDa). 1 A549; 2 A549-vector; 3 A549-nNm23-H1. c The expression and location of Nm23-H1 detected by confocal microscopy in A549-nNm23-H1 cell line. The Nm23-H1 protein is stained with green fluorescent and the nucleus is stained with blue fluorescent . 1 A549; 2 A549-vector; 3 A549-nNm23-H1

Fig. 2

Colony formation assay of the cells after irradiation. Cells were irradiated with different doses (0Gy, 2Gy, 4Gy, 6Gy and 8Gy) and then were cultured for 10–14 days. Colonies were counted after fixing and staining (*: p < 0.05)

Fig. 3

Quantification of DNA damage using Comet assay. All the cells were collected at 0 h, 1 h, 2 h, and 4 h after irradiation with 8 Gy X-rays. DNA damage was evaluated as the tail moment, combining comet tail length and the proportion of DNA migrating into the tail. a Nm23-H1-low-expressing group. b Nm23-H1-over expressing group. c The tail moment image of both groups. 1. A549-shNm23-H1; 2. DOX+ A549-shNm23-H1; 3. A549-vector; 4. A549-nNm23-H1 (*: p < 0.05; **: p < 0.01)

Fig. 4

Quantification of DNA damage using γ-H2AX foci numbers. All the cells were collected at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h after irradiation with 8 Gy X-rays. DNA damage was evaluated as γ-H2AX foci numbers. a Nm23-H1-low-expressing group. b Nm23-H1-over expressing group. c The γ-H2AX foci image of both groups. The γ-H2AX protein is stained with red fluorescent and the nucleus is stained with blue fluorescent. 1. A549-shNm23-H1; 2. DOX+ A549-shNm23-H1; 3. A549-nNm23-H1; 4. A549-vector. (*: p < 0.05; **: p < 0.01)

Fig. 5

Phosphorylation of checkpoint pathway related proteins detected by Western blot. Cells were collected at 0 h and 8 h after irradiation with 8 Gy X-rays. Checkpoint pathway related proteins were detected by Western blot. Note that radiation treatment increased the overall levels of pT68-Chk2, pS15-p53 and pS1981-ATM and the Nm23-H1 level also related to the amount of the phosphorylated protein. 1 A549; 2 A549-vector; 3 A549-nNm23-H1; 4 A549-shNm23-H1; 5 DOX+ A549-shNm23-H1. (**: p < 0.01)

Fig. 6

Proteins interacted with Nm23-H1 detected by co-immunoprecipitation. Cells were collected at 0 h and 8 h after irradiation with 8 Gy X-rays. Nm23-H1 antibody and IgG (as control) was used for pull the entire protein complex out of solution and then the binding proteins was detected by Western blot. 1 A549-nNm23-H1; 2 A549-vector; 3 A549-nNm23-H1. IR: ionizing radiation; IP: immunoprecipitation; WB: Western blot. (**: p < 0.01)

Fig. 7

The mechanism of Nm23-H1 participating in multiple steps of DSBR pathway. Nm23-H1 involved in two pathways, which was cell-cycle checkpoint signaling pathway (ATM, Chk2 and p53 phosphorylated signaling pathway) and DNA repair pathway (interaction with Ku80). Moreover, Nm23-H1 also interacted with MRN complex. MRN complex functioned as the recognition and stabilization of DSBs and was the part of the DSB-sensing machinery. Thus the interaction of Nm23-H1 with MRN complex may promote the signaling of above two pathways