Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination
Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination
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DOI：
10.1038/onc.2012.296
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发表时间：
2013-06-13
期刊：
影响因子：
8
通讯作者：
van Vugt, M. A. T. M.
中科院分区：
文献类型：
--
作者：
Krajewska, M.;Heijink, A. M.;van Vugt, M. A. T. M.
为了应对DNA断裂，“DNA损伤反应”引发细胞周期停滞以促进DNA修复。最近的研究结果表明，细胞可以在整个细胞周期中对DNA损伤做出反应，除了在有丝分裂期间。具体地说，各种有丝分裂激酶，包括Cdk 1，Aurora A和Plk 1，显示出在细胞进入有丝分裂时对DNA损伤检查点蛋白进行关键化。因此，分裂间期有丝分裂激酶的异常激活可以调节细胞对DNA损伤的反应。在这项研究中，我们的目的是确定如何异常激活Cdk 1影响细胞的DNA损伤反应。我们使用MK-1775抑制Wee 1，以迫使Cdk 1激活，这不会导致非转化细胞的细胞毒性。相反，它加速了有丝分裂进入，并在p53缺陷的癌细胞中引起放射敏感性，但在p53熟练的癌细胞中则不然。有趣的是，我们发现，Wee 1抑制导致升高的Cdk 1活性在间期细胞。当我们随后分析了细胞中的DNA损伤反应与强迫Cdk 1激活，我们观察到一个显着的减少53 BP 1在DNA损伤位点沿着增加，在照射后的g-H2 AX染色，表明有缺陷的DNA修复。事实上，当使用体内核酸内切酶诱导的同源重组（HR）试验分析DNA修复时，证实了Wee 1抑制后受损的DNA修复。这种HR缺陷伴随着BRCA 2在Cdk 1磷酸化位点S3291的磷酸化增加。两者合计，我们的研究结果表明，Wee 1抑制导致强迫Cdk 1激活间期细胞，干扰正常的DNA损伤反应。
In response to DNA breaks, the 'DNA damage response' provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1, were shown to inactivate key DNA damage checkpoint proteins when cells enter mitosis. Aberrant activation of mitotic kinases during interphase could therefore modulate cellular responses to DNA damage. In this study, our aim was to determine how aberrant activation of Cdk1 affects the cellular responses to DNA damage. We used Wee1 inhibition, using MK-1775, to force Cdk1 activation, which did not cause cytotoxicity in non-transformed cells. Instead, it accelerated mitotic entry and caused radio sensitization in p53-defective cancer cells, but not in p53-proficient cancer cells. Interestingly, we showed that Wee1 inhibition leads to elevation of Cdk1 activity in interphase cells. When we subsequently analyzed DNA damage responses in cells with forced Cdk1 activation, we observed a marked reduction of 53BP1 at sites of DNA damage along with an increase in g-H2AX staining after irradiation, indicative of defective DNA repair. Indeed, when DNA repair was analyzed using in vivo endonuclease-induced homologous recombination (HR) assays, compromised DNA repair after Wee1 inhibition was confirmed. This defect in HR was accompanied by increased phosphorylation of BRCA2 at the Cdk1 phosphorylation site S3291. Taken together, our results indicate that Wee1 inhibition leads to forced Cdk1 activation in interphase cells, which interferes with normal DNA damage responses.