Thoracic Oncology West China Hospital Sichuan University Chengdu China; State Key Laboratory of Biotherapy West China Hospital Sichuan University Chengdu China.
State Key Laboratory of Biotherapy West China Hospital Sichuan University Chengdu China.
Thorac Cancer. 2016 Jan;7(1):50-60. doi: 10.1111/1759-7714.12274. Epub 2015 May 19.
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.
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.
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.
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.
表皮生长因子受体（EGFR）激酶结构域突变的非小细胞肺癌（NSCLC）对电离辐射（IR）敏感。我们探索了在两种 EGFR 突变状态不同的 NSCLC 细胞系中，Rad51 依赖性同源重组（HR）DNA 修复对放射敏感性的调节作用。
培养野生型 EGFR A549 和 EGFR 外显子 19 中框内缺失（ΔE746-E750）的突变 EGFR H820 的 NSCLC 细胞系。通过集落形成试验评估放射敏感性。通过定量实时聚合酶链反应和 Western-blot 评估 Rad51 表达。构建并转染慢病毒小发夹 RNA-Rad51 和 ΔE746-E750 缺失突变 EGFR 到细胞中。流式细胞术分析 DNA 双链断裂、细胞周期改变和细胞凋亡。
A549 的生存因子（SF）2 更高（0.66 对 0.44），α/β 值更低（4.07 对 9.01）。与 A549 细胞相比，H820 细胞在 S 期的阻滞缺陷，G2/M 期积累率更高，早期凋亡和残留的 γ-H2AX。下调 Rad51 表达降低了 SF2（0.42 对 0.31）并增加了 α/β 比值（7.51 对 10.5）、G2/M 期积累、早期凋亡和 γ-H2AX 在两种细胞系中。H820 中 IR 诱导的 Rad51 表达和核易位减少。外源性表达 ΔE746-E750 缺失突变 EGFR 使 A549 细胞对 IR 更敏感。
一种 EGFR 突变的 NSCLC 细胞系对 IR 敏感，这与 IR 诱导的 Rad51 表达和核易位减少有关。维持 Rad51 蛋白水平的 EGFR 信号通路可能是克服放射抗性的一种新的肺癌治疗靶点。
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