Di Micco Raffaella, Cicalese Angelo, Fumagalli Marzia, Dobreva Miryana, Verrecchia Alessandro, Pelicci Pier Giuseppe, di Fagagna Fabrizio d'Adda
IFOM Foundation-FIRC Institute of Molecular Oncology Foundation, Milan, Italy.
Cell Cycle. 2008 Nov 15;7(22):3601-6. doi: 10.4161/cc.7.22.7152. Epub 2008 Nov 8.
Primary mouse embryonic fibroblasts (MEFs) are a popular tool for molecular and cell biology studies. However, when MEFs are grown in vitro under standard tissue culture conditions, they proliferate only for a limited number of population doublings (PD) and eventually undergo cellular senescence. Presently, the molecular mechanisms halting cell cycle progression and establishing cellular senescence under these conditions are unclear. Here, we show that a robust DNA damage response (DDR) is activated when MEFs undergo replicative cellular senescence. Senescent cells accumulate senescence-associated DDR foci (SDFs) containing the activated form of ATM, its phosphorylated substrates and gammaH2AX. In senescent MEFs, DDR markers do not preferentially accumulate at telomeres, the end of linear chromosomes. It has been observed that proliferation of MEFs is extended if they are cultured at low oxygen tension (3% O(2)). We observed that under these conditions, DDR is not observed and senescence is not established. Importantly, inactivation of ATM in senescent MEFs allows escape from senescence and progression through the S-phase. Therefore, MEFs undergoing cellular senescence arrest their proliferation due to the activation of a DNA damage checkpoint mediated by ATM kinase. Finally, we observed that spontaneously immortalized proliferating MEFs display markers of an activated DDR, indicating the presence of chromosomal DNA damage in these established cell lines.
原代小鼠胚胎成纤维细胞（MEFs）是分子生物学和细胞生物学研究中常用的工具。然而，当MEFs在标准组织培养条件下进行体外培养时，它们仅能增殖有限次数的群体倍增（PD），最终会进入细胞衰老状态。目前，在这些条件下阻止细胞周期进程并导致细胞衰老的分子机制尚不清楚。在此，我们表明，当MEFs经历复制性细胞衰老时，会激活强大的DNA损伤反应（DDR）。衰老细胞会积累含有ATM激活形式、其磷酸化底物和γH2AX的衰老相关DDR病灶（SDFs）。在衰老的MEFs中，DDR标记物并非优先在线粒体末端（线性染色体的末端）积累。据观察，如果在低氧张力（3% O₂）下培养MEFs，其增殖会延长。我们观察到，在这些条件下，未观察到DDR且未建立衰老状态。重要的是，衰老MEFs中ATM的失活允许其逃离衰老并通过S期进展。因此，经历细胞衰老的MEFs由于ATM激酶介导的DNA损伤检查点的激活而停止增殖。最后，我们观察到自发永生化的增殖性MEFs显示出激活的DDR标记，表明这些已建立的细胞系中存在染色体DNA损伤。
