Absence of an immediate G1/S checkpoint in primary MEFs following γ-irradiation identifies a novel checkpoint switch
Absence of an immediate G1/S checkpoint in primary MEFs following γ-irradiation identifies a novel checkpoint switch
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DOI：
10.4161/cc.5.16.3009
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发表时间：
2006-08-15
期刊：
影响因子：
4.3
通讯作者：
Hicks, Geoffrey G.
中科院分区：
文献类型：
--
作者：
Cann, Kendra L.;Hicks, Geoffrey G.
电离辐射引起的DNA双链断裂已被证明可诱导G(1)/S-i s -期内和G(2)/M细胞周期检查点。然而，在细胞水平上对G(1)/S检查点的直接诱导的分析一直受到阻碍，因为无法区分损伤时已经复制DNA的细胞和DNA损伤后进入S期的细胞。我们开发了一种新的策略来评估异步低传代小鼠胚胎成纤维细胞培养物（mef）中G照射后G(1)/S检查点的启动，使用交错CldU/IdU双标记方案。与目前的G(1)/S检查点模型相反，我们发现65%的g1晚期原发性mef在5 Gy的γ辐照剂量后仍进入S期。延迟的p53依赖性G(1)/S检查点在这些细胞中是完整的，并且在照射后1小时内诱导的G(2)/M检查点的有效性超过90%，并维持到照射后6小时。此外，这些细胞还表现出S期内复制减慢，因为γ辐照后原代mef的S/G(2)转换频率降低。原发性mef中缺乏立即的G(1)/S检查点，这表明在G(1)晚期，这些细胞可能主要在个体复制起点水平上对DNA损伤做出反应，而不是诱导S期进入的完全关闭。
DNA double-strand breaks caused by ionizing radiation have been shown to induce G(1)/S-i intra-S-phase, and G(2)/M cell cycle checkpoints. However, analysis of the immediate induction of G(1)/S checkpoint at a cellular level has been hampered by the inability to distinguish cells that were already replicating DNA at the time of damage from cells that entered S phase following the DNA damage. We have developed a novel strategy for assessing the initiation of the G(1)/S checkpoint following g-irradiation within asynchronous, low passage, primary mouse embryonic fibroblast cultures (MEFs) using a staggered CldU/IdU double-labeling protocol. Contrary to the current model of the G(1)/S checkpoint, we found that 65% of late-G1 primary MEFs still proceeded into S phase after a gamma-irradiation dose of 5 Gy. The delayed p53-dependent G(1)/S checkpoint was intact in these cells, and a G(2)/M checkpoint that was over 90% effective was induced within one hour and maintained through six hours post-irradiation. Furthermore, these cells also exhibited an intra-S-phase replication slow-down, as there was a decrease in the S/G(2) transition frequency of primary MEFs following gamma-irradiation. The absence of an immediate G(1)/S checkpoint in primary MEFs suggests that in late G(1) these cells may predominantly respond to DNA damage at the level of individual replication origins, rather than by inducing a complete shut-down of S-phase entry.