Parallel pathways in RAF-induced senescence and conditions for its reversion
Parallel pathways in RAF-induced senescence and conditions for its reversion
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DOI：
10.1038/onc.2011.481
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发表时间：
2012-06-01
期刊：
影响因子：
8
通讯作者：
Mann, C.
中科院分区：
文献类型：
--
作者：
Jeanblanc, M.;Ragu, S.;Mann, C.
为了研究RAF对人成纤维细胞的衰老作用，我们建立了克隆细胞株WI-38hTERT/GFP-RAF1-ER。通过加入4-羟基他莫昔芬激活GFP-RAF1-ER激酶，在一个群体倍增中导致了强烈的衰老诱导，伴随着异染色斑的组装。至少有两条途径同时导致细胞周期蛋白依赖性蛋白激酶(CKI)的p15、p16、p21和p27抑制因子的积累。RAF1K激活后进入S期的细胞经历了复制应激，表现为H2AX和Chk2的磷酸化以及p21的合成。然而，大约一半的细胞在没有通过S阶段的情况下被阻断，也没有显示出DNA损伤检查点的激活。当细胞在5%的氧气中培养时，RAF1的激活产生最少的活性氧物种，但即使在存在抗氧化剂或DNA检查点通路抑制剂的情况下，RAF诱导的衰老也是有效的。尽管存在异染色灶，但同时敲除p16和p21并失活GFP-RAF1-ER激酶会导致衰老状态的快速逆转，大多数细胞变得能够长期增殖。这些结果表明，复制和氧化应激不是RAF诱导的衰老所必需的，这种衰老很容易在失去CKI后逆转。Oncogene(2012)31,3072-3085；doi：10.1038/onc.2011.481；2011年10月24日在线发布
We developed a clonal WI-38hTERT/GFP-RAF1-ER immortal cell line to study RAF-induced senescence of human fibroblasts. Activation of the GFP-RAF1-ER kinase by addition of 4-hydroxy-tamoxifen led to a robust induction of senescence within one population doubling, accompanied by the assembly of heterochromatic foci. At least two pathways contribute in parallel to this senescence leading to the accumulation of p15, p16, p21 and p27 inhibitors of cyclin-dependent kinases (CKIs). Cells that traversed S phase after RAF1 kinase activation experienced a replicative stress manifested by phosphorylation of H2AX and Chk2 and synthesis of p21. However, about half the cells in the population were blocked without passing through S phase and did not show activation of DNA-damage checkpoints. When the cells were cultivated in 5% oxygen, RAF1 activation generated minimal reactive oxygen species, but RAF-induced senescence occurred efficiently in these conditions even in the presence of anti-oxidants or inhibitors of DNA checkpoint pathways. Despite the presence of heterochromatic foci, simultaneous knockdown of p16 and p21 with inactivation of the GFP-RAF1-ER kinase led to rapid reversion of the senescent state with the majority of cells becoming competent for long-term proliferation. These results demonstrate that replicative and oxidative stresses are not required for RAF-induced senescence, and this senescence is readily reversed upon loss of CKIs. Oncogene (2012) 31, 3072-3085; doi: 10.1038/onc.2011.481; published online 24 October 2011