Predicting Of Radiosensitivity Of Human Tumor Cell Lines In Vitro By Determining 4977bp Deletion In Mitochondrial DNA

Objective:To study mtDNA4977bp deletion of there different tumor cell lines,ie prostate cancer(PC-3),hepatoma cells(HepG₂)and esophageal cancer cell(EC-9706) ,induced by different single doses of X-ray irradiation,and compare the survival fractions of cells after irradiation,then discuss the feasibility of the mtDNA4977bp deletion for testing radiation sensitivity of tumor cells.Methods:To irradiate the cultured prostate cancer cells(PC-3),hepatoma cells(HepG₂)and esophageal cancer cells(EC-9706)by different doses of X-rays.(1) after irradiation,using the improved high-salt precipitation method to extract the cells' mitochondrial DNA;amplify the internal fragment of mtDNA by PCR methods,and amplify the fragments occurred mtDNA4977bp deletion by nested-PCR;check the PCR products using gel electrophoresis and acquire image,of each dose the gray value proportion of fragment with mtDNA4977bp deletion and the internal mtDNA fragment on behalf of the relative rate of the fragments occurred mtDNA4977bp deletion in the whole mtDNA.(2) acquire cell survival fraction by MTT colorimetric assay after irradiation.(3) fit dose survival curve in accordance with cell survival fraction;compare radiation-induced mtDNA4977bp deletion rates of different tumor cells,as well as the relationship between the rates and cell survival fraction by SPSS13.0 software.Results:1.The extracted mtDNA samples of three tumor cell lines with different brightness situate in the location of 15000bp,and Consistent with the location of theoretical mtDNA(16569bp) position;2.The survival fraction of tumor cells after X-ray irradiation increase with the decrease of radiation dose;in the low-dose stage(0Gy,1Gy,2Gy),the drop of cell survival fractions are lower,all the survival fractions are above 50%;in the high-dose phase,especially on the dose of 8Gy,cell survival fractions are very low; on the same dose,the survival fraction of HepG₂ is lowest,PC-3 the highest, EC-9706 between the two.3.Gel electrophoresis showed that the positions of the internal fragments ordinary PCR amplified and the fragments occurred mtDNA4977bp deletion nested-PCR amplified are consistent with the theoretical locations.With the increase in radiation dose,the radiation-induced mtDNA4977bp deletion of three tumor cells increase;on 1Gy and 2Gy,the rate of mtDNA4977bp deletion have no significant statistically difference among three tumor cells;on 4Gy,HepG₂ and EC-9706 have no significant difference,but they have statistically difference with PC-3 respectively; on 8Gy,the rate of HepG₂ and EC-9706 are still increasing,while PC-3 decreasing, the rate of radiation-induced mtDNA4977bp deletion of PC-3 is higher than the other two cells,but the rates of HepG₂ and EC-9706 have no statistical difference.4.The radiation-induced mtDNA4977bp deletion rate and with the the cell survival fraction are consistent.Conclusions:1.The cell survival curves fited According to the cell survival fractions of tumor cells are in line with the general characteristics of Multi-target click model,and results that HepG₂ has a higher radiation sensitivity, PC-3 radiation sensitivity is lowest,EC-9706 between between the two.2.There are "background" deletions in the three tumor cell lines,ionizing radiation can Induce mtDNA4977bp deletion,and have a dose-related relationship. Statistical analysis show that the radiation-induced mtDNA4977bp deletion rate of HepG₂ and EC-9706 have no difference,while PC-3 is lower than the former two.3.The results of tumor cell survival fraction and the results of ionizing radiation-induced mtDNA4977bp deletion is consistent,and after relevant analysis, the ionizing radiation mtDNA4977bp deletion of HepG₂ and EC-9706 reflect the radiation sensitivity of cells to some extent.Therefore we consider that the simple and quick detection of radiation-induced mtDNA4977bp deletion may become a way of prediction of tumor cell radiosensitivity.