Atomic Bomb Disease Institute, Graduate School of Biochemical Sciences, Nagasaki University, Nagasaki 852-8523, Japan.
J Radiat Res. 2011;52(6):766-74. doi: 10.1269/jrr.10164. Epub 2011 Oct 14.
Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm(2)). Cells were grown for up to 3 days, and phosphorylated ATM at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process.
细胞周期检查点是保护基因组免受 DNA 损伤剂破坏的重要细胞过程。在本研究中，我们开发了一种微集落分析方法，其中正常的人类二倍体成纤维样细胞暴露于电离辐射下，以非常低的密度（3 个细胞/cm(2))接种到盖玻片上。细胞生长长达 3 天，并用磷酸化 ATM 在 Ser1981 和 53BP1 焦点作为放大 DNA 损伤信号的标记进行分析。我们观察到非分裂细胞的比例呈剂量依赖性增加，敲低 p53 表达会损害其增加。虽然大的持续焦点主要形成在非分裂细胞中，但我们观察到一些含有大焦点的生长菌落。由于每个微集落都源自单个细胞，似乎有些细胞可以带有大焦点进行增殖。使用转染了 EGFP 标记的 53BP1 基因的 hTERT 永生化正常人类二倍体细胞的实时成像分析表明，持续大焦点的形成具有高度动态性。在 X 射线照射后 12-72 小时可视化暴露细胞时，经常观察到大焦点的出现和消失延迟。因此，我们的结果表明，放大的 DNA 损伤信号可能被忽略，这部分可以通过放大过程的动态性质来解释。
