| Background and PurposeLung cancer remains the most common cause of cancer death with the majority cases presented with advanced, unresectable or metastatic NSCLC. Radiotherapy has been reserved for about64.3%patients with NSCLC. Radiotherapy for the treatment of cancer can cause a wide range of cellular effects, the most biologically potent of which is the double-strand break(DSBs) in DNA. There are two main pathways existed for DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR). The ability of cancer cells to effectively repair DSBs significantly influences the outcome of therapeutic regimens.In several cancers, including non-small cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) is an important determinant of tumorigenesis and cancer progression. EGFR is an important target for cancer therapies. Recent discoveries have linked somatic mutations in the tyrosine kinase domain (TKD) of EGFR gene in a subset of NSCLC to tumor responsiveness to the EGFR tyrosine kinase inhibitor. Expression and activity of EGFR are important determinants of radioresponse in NSCLCs, and anti-EGFR therapy has been shown to sensitize tumor cells to radiation. Recently, several study reported that NSCLC cell lines that harbored somatic, activating mutations in the TKD of EGFR exhibit a marked sensitivity to IR.However, there is a lack of information on the effect of mutant EGFR-associated radiosenstivity and DSBs repair.This study will focus on the effect of mutant EGFR-associated radiosenstivity and DSBs repair. The consequences of this interference on IR-induced DSB resolution kinetics, DSBs repair capability, and clonogenic survival are examined.MethodsSurvival fraction at2Gy (SF2), a and β values of two tumor cell lines (including A549expressed the WT receptors and H1975had the L858R replacement in the exon21of the EGFR) were acquired by clone formation array. γH2AX,Rad51foci and EGFR determination after6MV X-ray irradiation were analyzed by flow cytometry analysis, and Western Blot was applied to detect the expressions of EGFR, DNA-PKcs and Rad51protein.Results1. Effect of IR on clonogenic survivalIn the clonogenic cell survival assay, the survival fraction in the H1975cell line was lower than that in the X-ray group at different doses. Surviving fraction of2Gy (SF2) were0.62and0.89for H1975and A549cell lines, respectively, α/β values were4.00 and0.19for H1975and A549cell lines, respectively.2. Effect of IR on DSBs repairUsing γH2AX foci as a surrogate for DSBs, we compared rates of DSB repair between A549cell line and H1975cell line. The mutant EGFR-expressing cell lines(H1975) show a significantly lower rate of DSB resolution and retain nearly70%of γH2AX foci at3hours and25%foci at24hours following IR. By contrast, WT EGFR NSCLC cell lines (A549) have higher rates of γH2AX resolution and eliminate80%of their foci by3hours. The data indicate that mutant EGFR expression is often associated with delayed DSB repair kinetics.3. The potential mechanism of IR induced DNA DSBs repairImmunostaining for confocal microscopy suggested that EGFR translocated into the nucleus within1h after4Gy of6MV X-ray in A549cell line, but stayed in the cytoplasma in the H1975cell line. In A549cell line within15min of exposure to4Gy IR, DNA-PKcs was detected in immune complexes precipitated with an anti-EGFR antibody. The level of DNA-PKcs-EGFR interactions peaked within60min after IR, gradually decreased over the next12h, and returned to basal levels by the24h time point. By contrast, The L858R-expressing H1975was undetectable in immunoprecipitates of the EGFR. In A549cell line nucleus, Rad51gradually increased and peaked in6h after8Gy IR.ConclusionPulmonary Adenocarcinoma cell lines that harbored somatic, activating mutations in the tyrosine kinase domain (TKD) of EGFR exhibit a marked sensitivity to IR and pronounced delays in the repair of IR-induced DNA DSBs. Decreased the repair of DNA DSBs, including non-homologous end joining and homologous recombination. |
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