Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells.
Non-homologous 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. 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 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.
Iwabuchi
Kuniyoshi
K
Department of Biochemistry, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku-gun, Ishikawa 920-0293, Japan. kuni-kmu@kanazawa-med.ac.jp
Hashimoto
Mitsumasa
M
Matsui
Tadashi
T
Kurosawa
Aya
A
Adachi
Noritaka
N
Date
Takayasu
T
D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D016193:G1 Phase / Q000187:drug effects / Q000528:radiation effects*; D006367:HeLa Cells; D006657:Histones / Q000032:analysis; D006801:Humans; D011836:Radiation Tolerance / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D014965:X-Rays
C468783:H2AX protein, human; D006657:Histones