Nested PCR for mtDNA-4977-bp deletion and comet assay for DNA damage - a combined method for radiosensitivity evaluation of tumor cells.

To identify an effective method of evaluating the radiosensitivity of human tumor cell lines , the present study adopted mtDNA-4977-bp deletion coupled with comet assay. The three human tumor cell lines applied were HepG, EC-9706 and MCF-7. The surviving fraction (SF), ratio of the mtDNA-4977-bp deletion and DNA damage were detected by MTT assay, nested polymerase chain reaction (PCR) technique and comet assay, respectively. Clearly, lower SFs were found for the HepG and EC-9706 cells as compared with the MCF-7 cells following irradiation at doses of 2, 4 and 8 Gy, indicating a higher radiosensitivity for the HepG and EC-9706 cells. Additionally, no significant differences were identified in the mtDNA-4977-bp deletions found among HepG, EC-9706 and MCF-7 cells by PCR following 1- or 4-Gy γ-ray irradiation, while increased deletion ratios of mtDNA-4977 bp were observed in HepG and EC-9706 cells following 8-Gy irradiation, in contrast to decreases in MCF-7 cells. The most notable differences among these three tumor cell lines were observed by comet assay following 8-Gy γ-ray irradiation. A combined method of nested PCR and comet assay, therefore, is the most effective and accurate method in evaluating the radiosensitivity of tumor cells.