Establishment Of A Radioresistant Subline Human Lung Cancer Cell Line And The Preliminary Study On The Mechanism Of Radioresistance

PartⅠEstablishment of a radioresistant subline of human no small lung cancer cell lineObjective: To establish a radioresistant cell subline from a human A549 lung cancer cell line.Methods: Two proposals were applied for the non-small cell lung cancer A549 cells irradiated with X-rays:A group of A549 cell line was irradiated five times, the fractionated dose was 600 cGy, and the other group was exposed 15 times, the fractionated dose was 200 cGy. After the completion of irradiation, two monoclones were obtained from the survival of cells and named the subline A549-S1 and A549-S2. The radiosensitivity and cell cycle distribution of these two clones,together with its parental A549 cells were measured by clone formation assay and flow cytometry.Three moths later, the radiosensitivity of radioresistant subline was detected to verify its formation.Results: compared to the parental A549 cells, A549-S1 cells showed significant resistance to radiation, D0,Dq and N values increased, showed a broader initial shoulder than those of the parent A549 cell line and were 1.38-fold increased in radioresistance as regards to the SF2.The A549-S1 subline also showed higher percentage of cells in S phase and G2/M phase (P<0.05),but lower percentages in G0/G1 phase. And radiosensitivity of A549-S2 slightly increased comparing with the parental cells with its D0,Dq and N values were decreased and a curve initial shoulder. the cell cycle in S and G0/G1 phase ratio dropped than its parental A549 cells, and G2/M phase ratio increased significantly(P<0.05).Three moths later, the colony formation assay showed that A549-S1 subline kept its radioresistance .Conclusions:The radioresistance of the A549 cell subline is correlated with the different irradiation program and the characteristic could go down to the future generation. The cell subline showed a different cell cycle distribution from their parental line. PartⅡThe functional mechanism of Notch1 and VEGF in the radioresistance of NSCLCObjective:To investigate the expression of Notch1 and VEGF in the process of irradiation to NSCLC cell line A549,so as to find out the potential functional mechanism of Notch signal pathway and VEGF in the radioresistance of NSCLC.Methods:The radiosensitivity and cell cycle distribution of A549 cell were measured by Clone formation assay and flow cytometry. The mRNA and protein of Notch1 and VEGF in A549 cells were determined by RT-PCR and Western blots. Then, six parallel flasks of A549 cells were randomized divided into two groups: the simplex radiation group and the radiation combined VEGF antibody group, every group was exposed to 600cGy per fraction X-rays five times. After 24h of radiation, the mRNA and protein of Notch1 and VEGF were detected. After the radiation were finished, two cell sub-line A549-S1 and A549-S3 were got, and their radiosensitivity and cell cycle distribution were measured. Linear correlations were used to analyze the relationship between Notch1 and the dose, VEGF.Result:A549-S1 showed significant resistance to radiation and higher percentage of cells in S phase. The mRNA and protein of Notch1 increased gradually as the increasing radiation dose. The correlation between Notch1 and dose is positive , and the coefficient R in the level of mRNA and protein are 0.9288 and 0.8648(P<0.05). The correlation between VEGF and dose is also positive , and the coefficient R in the level of mRNA and protein are 0.9223 and 0.9233 (P<0.05)。The expression of Notch1 and VEGF had statistically positive correlation, and the coefficient in the level of mRNA and protein are 0.9041 and 0.8782 (P<0.05),The radiosensitive and the cell cycle of A549-S3 changed unnoticed,and the expression of mRNA and protein of Notch1 and VEGF along the process of radiation had no remarkable diversity.Conclusion:The A549-S1 is more radioresistant as compare to A549, and has a different cell cycle distribution. The diversity of the radiosensitivity and the cell cycle distribution is probable owing to the activation of the Notch signal pathway induced by VEGF alone the radiation.