サイトメトリーによるDNA損傷の検出と細胞動態解析 －リン酸化ヒストンH2AXを指標として－

抄録

Histone H2AX phosphorylated on Ser 139, defined as γH2AX, is a reporter of DNA double strand breaks (DSBs). While induction of DNA damage by genotoxic agents lead H2AX to γH2AX, it also is phosphorylated in unterated normal and tumor cells. The latter is designated as constitutive H2AX phosphorylation. We show here flow cytometric investigation of γH2AX in relation to cell cycle phase. Even in untreated exponentially growing HL-60 cells, γH2AX was detected during the cell cycle. Some of normal endothelial cells demonstrated dot-like positivity of γH2AX in their nuclei. Oxide-releasing aspirin induced DNA damage and it was attenuated in the presence of N-acetyl-L-cysteine, a scavenger of reactive oxigen species. A decrease in a cells’metabolic activity as a result of inhibition of glycolysis by 2-deoxy-D-glucose reduced DNA damage. Exposure of HL-60 cells to camptothecin led to phosphorylation of H2AX in S-phase cells. Four hours’exposure to camptothecin induced a cell population which had dramatically increased γH2AX immunofluorescence and corresponded to apoptosis. Inhibitors of DNA replication which are used to synchronize cultured cells in G1 phase caused DNA damage in S-phase cells and led to their apoptosis, i. e., the synchronization was caused by selective kill of S-phase cells through induction of DSBs. Moreover, synchronized cells in G1 phase showed DNA damage. Thus, detection of γH2AX immunohistochemically and cytometric investigation of it in relation to cell cycle phases is advantageous to estimate the effect of many agents causing DNA damage such as oxidative stress, metabolism or chemicals, and have a possibility to contribute significantly to elucidate carcinogenesis.