Yukawa Masashi, Oda Shoji, Mitani Hiroshi, Nagata Masao, Aoki Fugaku
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.
Biochem Biophys Res Commun. 2007 Jun 29;358(2):578-84. doi: 10.1016/j.bbrc.2007.04.162. Epub 2007 May 4.
DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by gamma-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX (gamma-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to gamma-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of gamma-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.
DNA双链断裂（DSBs）是由各种环境压力引起的，如电离辐射和DNA损伤剂。当发生DSBs时，细胞周期检查点机制发挥作用，使细胞周期停止，直到所有DSBs都得到修复；H2AX的磷酸化在这一过程中起重要作用。小鼠植入前阶段的胚胎对电离辐射高度敏感，经X射线照射的小鼠受精卵在第一个细胞周期的G2期停滞。为了研究小鼠植入前胚胎在G2期对DNA损伤的反应机制，我们检测了这些胚胎中的G2/M检查点和DNA修复机制。大多数经γ射线照射诱导产生DSBs的单细胞和双细胞胚胎在卵裂时出现延迟，并在囊胚期之前停止发育。在这些胚胎中，通过免疫细胞化学在单细胞或双细胞阶段未检测到磷酸化的H2AX（γ-H2AX），尽管在植入前发育的双细胞阶段之后检测到了它。这些结果表明，G2/M检查点和DNA修复机制在单细胞和双细胞胚胎中的功能不足，导致对γ射线照射高度敏感。此外，在单细胞和双细胞阶段以及其他植入前阶段的胚胎中检测到了使H2AX磷酸化的磷酸化共济失调毛细血管扩张突变蛋白和DNA蛋白激酶催化亚基，这表明单细胞和双细胞胚胎中γ-H2AX的缺失取决于这些激酶以外的某些因素。
