The DNA Damage And Repair Of Lung Cancer A549 Cells After Hypofractionated Radiotherapy

Objectives:Hypofractionated radiotherapy in lung cancer patients has achieved good local control rate,but its molecular mechanism is not very clear currently.Radiotherapy can induce DNA double-strand breaks and cells can repair the DSB by NHEJ and HR.The available reports about hypofractionated radiotherapy-induced cell injury and repair mechanisms are rare.We used lung adenocarcinoma cell line A549 as model to investigate NHEJ repair pathway under different radiation doses.Methods:1.Colony-forming experiment was performed to detect the influence of dose in A549 cell clone growth.2.We used Reverse Transcription-Polymerase Chain Reaction to detect the mRNA level of endogenous DNA-PKcs?Ku70 and Ku80 in lung cancer A549 cells which were divided into different dose groups.3.Western Blotting assays were performed to detect the influence of radiation dose and time point after radiation on the repair protein and damage protein of A549 cells.4.We used laser scanning con focal microscope to observe yH2AX and 53BP1 colocalization in A549 cells and analysis colocalization of repair protein and damage protein under different radiation dose.Results:1.With radiation dose increasing,the A549 cell colony formation ability diminished.2.There was no significant difference between dose groups in mRNA levels of DNA-PKcs,Ku70,and Ku80 in A549 cells.3.Damage protein ?H2AX and 53BP1 reached a peak at 1 hour after radiation and increased with increasing dose.The level of repair protein DNA-PKcs,Ku70,Ku80 in different dose groups was consistently high,showing time independence.DNA-PKcs pSer2056 was up to maximum 1 hour after radiotherapy,while comparing its level in group 10Gy/f with 2Gy/f,2Gy*9f,the former was slightly higher than the latter with no statistically significant.DNA-PKcs pThr2609 showed different trends over time in each group,as group lOGy/f and 2Gy*9f reached a peak 1 hour and 6 hour after radiotherapy,respectively.4.We used immunofluorescence to confirm that the damage protein yH2AX and 53BP1 of A549 cells colocalized in the nucleus.The repair protein DNA-PKcs,Ku70,Ku80 diffused in the nucleus and maintained at a high level in different dose groups.DNA-PKcs pSer2056 and DNA-PKcs pThr2609 formed focus in nuclear and colocalized with the damage protein yH2AX.DNA-PKcs pSer2056 in group 10Gy/f was slightly higher than group 2Gy/f,2Gy*9f,while there was obvious difference in DNA-PKcs pThr2609 between group 10Gy/f and group 2Gy/f,2Gy*9f(P<0.05).Conclusions:1.With radiation dose increasing,the A549 cell colony formation ability diminished.2.There was no significant difference between dose groups in mRNA levels of DNA-PKcs,Ku70,and Ku80 in A549 cells.3.DSBs and repair protein including DNA-PKcs,Ku70 and Ku80 reached a peak at 1 hour after radiation,showing dose dependence.DNA-PKcs pThr2609 showed significant differences in different dose groups.How the phosphorylation of different sites in DNA-PKcs affect cell response to injury and its mechanism is not known now,further reserch is needed.4.The damage protein yH2AX and 53BP1 colocalized in the nucleus.The repair protein DNA-PKcs,Ku70,Ku80 diffused in the nucleus and maintained at a high level in different dose groups.DNA-PKcs pSer2056 and DNA-PKcs pThr2609 formed focus in nuclear and colocalized with the damage protein yH2AX.DNA-PKcs pSer2056 in group 10Gy/f was slightly higher than group 2Gy/f,2Gy*9f,while there was obvious difference in DNA-PKcs pThr2609 between group 10Gy/f and group 2Gy/f,2Gy*9f(P<0.05).