Abstract
Nonhomologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1 phase of the cell cycle. Acute lymphoblastic leukemia cell line Nalm-6, like a chicken DT40 cell line, allows for high-efficiency gene targeting by homologous recombination. Thus, the Nalm-6 cell line has been used in many gene-knockout studies in human cells. Although Nalm-6 cells have been applied to establish multiple NHEJ factor-deficient cell lines, a major disadvantage is that Nalm-6 cells do not respond well to cell cycle synchronization using chemical agents, such as nocodazole and mimosine. This has prevented researchers from determining whether NHEJ-deficient Nalm-6 cells show increased IR sensitivity during the G1 phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by phosphorylated-gamma-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.
