Rad51 in regulating the radiosensitivity of non-small cell lung cancer with different epidermal growth factor receptor mutation status

Abstract

Background: Non-small cell lung cancer (NSCLC) harboring kinase-domain mutations in epidermal growth factor receptors (EGFR) has been observed to be sensitive to ionizing radiation (IR). We explore Rad51-dependent homologous recombination (HR) DNA repair in regulating radiosensitivity in two NSCLC cell lines with different EGFR mutation status.

Methods: NSCLC cell lines, wild-type EGFR A549 and mutant EGFR H820 with an in-frame deletion in exon 19 of EGFR (ΔE746-E750), were cultured. Radiosensitivity was estimated by colony forming assay. Rad51 expression was evaluated by quantitative real time-polymerase chain reaction and Western-blot. Lentiviral small hairpin ribonucleic acid-Rad51 and ΔE746-E750 deletion mutant EGFR were constructed and transfected into cells. Flowcytometry assay was used to analyze DNA double strand breaks, cell cycle alterations, and apoptosis.

Results: A549 had a higher survival factor (SF)2 (0.66 vs. 0.44) and lower α/β value (4.07 vs. 9.01). Compared with the A549 cell, the H820 cell exhibited defective arrest in the S-phase, a higher rate of G2/M accumulation, early apoptosis, and residual γ-H2AX. Downregulated Rad51 expression decreased SF2 (0.42 vs. 0.31) and increased the α/β ratio (7.51 vs. 10.5), G2/M accumulation, early apoptosis, and γ-H2AX in two cell lines. H820 had a low IR-induced Rad51 expression and nuclear translocation. Exogenous expression of the ΔE746-E750 deletion mutant EGFR caused the A549 cell to become more radiosensitive.

Conclusions: An EGFR mutated NSCLC cell line is sensitive to IR , which is correlated with reduced IR-induced Rad51 expression and nuclear translocation. The signaling pathway of EGFR maintaining Rad51 protein levels maybe a novel lung cancer therapeutic target to overcome radioresistance.

Keywords: Epidermal growth factor receptor; Rad51; mutation; non‐small cell lung cancer; radiosensitivity.

Figure 1

Mutant epidermal growth factor receptor‐expressing cell lines show enhanced sensitivity to radiation. (a) Colonies were counted on the 14th day following radiation, and survival fractions were plotted as a function of dose. The survival curve was obtained by the linear‐quadratic model. Error bars, standard deviation (SD) from mean of triplicate measurements. (b) The apoptosis rate induced by radiation in H820 and A549 cells. Cells were stained with AnnexinV‐ fluorescein isothiocyanate/propium iodide (PI) at 36 hours following radiation; radiation‐induced apoptosis was higher in H820 than in A549 cells. (c) Cells were fixed at 24 hours after irradiation and probed with anti‐phosphorylated histone γ‐H2AX antibody to detect DNA repair capability. H820 cells show significantly more residual γ‐H2AX , which indicates inefficient DNA repair. Normalized fold fluorescence intensity: the relative values of γ‐H2AX fluorescence intensity divided by unirradiated A549 cells. (d) A549 and H820 cells were stained with PI at 18 hours following radiation. Both cell lines revealed an increase of cells in G2/M with IR treatment. H820 cells show significantly more G2/M arrest and dismal S arrest compared with WT‐expressing cells. Error bars, SD from mean of triple independent measurements. (a) () A549, () H820; (b) () A549, () H820; (c) () A549, () H820; (d) () A549, () H820.

Figure 2

Rad51 expression levels in H820 and A549 cells following IR. (a) Rad51 protein expression levels with (10 Gy) or without irradiation at different time points (0, 1, 12, and 24 hours following irradiation). (b) Levels of Rad51 protein in two cells at 12 hours after various doses of radiation. (c) Levels of Rad51 messenger ribonulcleic acid at 12 hours after radiation. Error bars, standard deviation (SD) from mean of triple independent measurements. Normalized fold expression: the relative expression of Rad51 divided by glyceraldehyde 3‐phopshate dehydrogenase; the expression level of Rad51 in A549 cell at 0 Gy was set as 1; *P < 0.05. (d) Levels of Rad51 protein in cytosolic and nuclear fractions with (10 Gy) or without irradiation at different time points (0 and 12 hours following irradiation). Blots were probed for β‐actin and lamin B to identify cytosolic and nuclear fractions, respectively. (c) () A549, () H820.

Figure 3

Knockdown of Rad51 sensitizes non‐small cell lung cancer cells to IR. (a) Eighty‐four hours after transfection of the lentivirus, small hairpin ribonucleic acid vector successfully inhibited Rad51 expression with (10 Gy) or without irradiation (0 Gy) in H820 and A549 cells; (b) The survival fractions decreased after downregulation of Rad51; (c) Downregulation of Rad51 increased apoptotic cell fractions in both cell lines. Error bars, SD from mean of triple independent measurements. *P < 0.05; (d) Downregulation of Rad51 induced more residual γ‐H2AX , indicating shrinkage of DNA repair function. Error bars, SD from mean of triple independent measurements. Normalized fold fluorescence intensity: the relative values of γ‐H2AX fluorescence intensity divided by unirradiated A549 cells. (e) Downregulation of Rad51 increased G2/M phase arrest and decreased S phase fractions. Column, mean values of triple independent experiments. (b) () A549‐nc, () H820‐nc, () A549 si‐rad51, () H820 si‐rad51; (c) () A549‐NC, () A549‐Rad51, () H820‐NC, () H820‐Rad51; (d) () A549‐NC, () H820‐NC, () A549‐Rad51, () H820‐Rad51; (e) () G2, () S, () G1. Si, small interfering; NC, negative control.

Figure 4

Expression of ΔE746–E750 deletion mutant epidermal growth factor receptor (EGFR) sensitizes cells to radiation. (a) Levels of ΔE746–E750 mutant EGFR protein expression in four cells. (B) expression of ΔE746–E750 deletion mutant EGFR decreased clonogenic survival after IR in A549‐del cells. Error bars, standard deviation (SD) from mean of triplicate measurements. (c) Radiation induced apoptosis was increased in radiosensitive mutant EGFR‐expressing A549‐del cells. Error bars, SD from mean of triple independent measurements. *P < 0.05. (d) A549‐del cells showed significantly more G2/M arrest and dismal S arrest compared with A549‐nc cells. Column, mean values of triple independent experiments. (b) () A549‐nc, () A549‐del, () H820; (c) () A549‐nc, () A549‐del, () H820; (d) () G1, () S, () G2. NC, negative control.

Figure 5

The effects of mutant epidermal growth factor receptor (EGFR) on Rad51 messenger ribonucleic acid, protein expression, and subcellular distribution. (a) Rad51 protein expression in A549‐nc and A549‐del cells with 10 Gy irradiation at different time points (0, 12, and 24 hours following irradiation). (b) Cytosolic and nuclear fractions of Rad51 expression in A549‐nc, A549‐del, and H820 cells with 10 Gy irradiation at different time points (0, 12, and 24 hours following irradiation). Blots were probed for β‐actin and lamin B1 to identify cytosolic and nuclear fractions, respectively. NC, negative control.