DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.
DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.
复制标题
DOI：
10.1007/s00412-016-0590-9
复制
发表时间：
2017-03
期刊：
影响因子：
1.6
通讯作者：
Scherthan H
中科院分区：
文献类型：
--
作者：
Ahmed EA;Vélaz E;Rosemann M;Gilbertz KP;Scherthan H
非循环和终末分化（TD）细胞显示辐射敏感性和DNA损伤反应的差异。与其他TD细胞不同，Sertoli细胞在体内表达增殖诱导剂和抑制剂的混合物，并且可以重新进入细胞周期。TD Sertoli细胞处于G1期样细胞周期阶段，预期通过易错的非同源末端连接途径（NHEJ）修复DSB。最近，我们提供了证据表明，Ku依赖性NHEJ参与保护睾丸细胞免受DNA损伤，如Ku 70缺陷小鼠TD支持细胞中DNA双链断裂（DSB）修复蛋白磷酸化-H2 AX，53 BP 1和磷酸化-ATM的持续病灶所示。在这里，我们分析了动力学53 BP 1灶诱导和衰减后12小时0.5戈伊γ射线照射DNA-PKcs缺陷（Prkdc scid）和野生型支持细胞。在未照射的小鼠和Prkdc scid中，支持细胞在约12%的细胞中显示出持续的DSB病灶，并且相对于野生型，这些DSB DNA损伤相关病灶的数量增加了五倍。在辐照小鼠中，Prkdc scid Sertoli细胞在电离辐射（IR）暴露后12 h，82%的细胞显示出升高水平的DSB指示病灶，而辐照野生型Sertoli细胞为52%。这些数据表明，支持细胞在体内响应并修复IR诱导的DSB，野生型中的修复动力学缓慢，Prkdc scid中的修复动力学无效。将相同剂量的IR应用于Prdkc −/−和Ku −/−小鼠胚胎成纤维细胞（MEF），发现Prdkc −/−细胞中IR后5分钟延迟诱导53 BP 1 DSB指示灶。在DNA-PKcs缺陷细胞中，IR后7小时DSB修复效率低下，但在Ku −/− MEFs中则不然。我们的数据表明，静止的支持细胞修复基因毒性DSB的DNA-PKcs依赖NEHJ在体内与较慢的动力学相对于体细胞DNA-PKcs缺陷细胞在体外，而DNA-PKcs缺陷引起低效的DSB修复后的时间点IR在这两种条件下。这些观察结果表明，DNA-PKcs有助于通过NHEJ快速和缓慢修复DSB。本文的在线版本（doi：10.1007/s 00412 -016-0590-9）包含补充材料，可供授权用户使用。
Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid. Applying the same dose of IR to Prdkc −/− and Ku −/− mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc −/− cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku −/− MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ. The online version of this article (doi:10.1007/s00412-016-0590-9) contains supplementary material, which is available to authorized users.