Walker D H, Adami G R, Dold K M, Babiss L E
Department of Molecular Cell Biology, Glaxo Research Institute, Research Triangle Park, North Carolina 27709, USA.
Cell Growth Differ. 1995 Sep;6(9):1053-61.
The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in checkpoint regulation between normal and transformed cells, we have compared the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G1 and G2 for in excess of 48 h after DNA damage. Surprisingly, both cyclin-dependent kinase 2 (CDK2) and cyclin A proteins were specifically down-regulated within 24 h of DNA damage. In contrast, TAg transformed cells did not down-regulate either cyclin A or CDK2 after DNA damage and showed a significantly shortened G2 arrest. To investigate the role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells fail to down-regulate both CDK2 protein and CDK2 protein kinase activity after DNA damage and also show a G2 checkpoint defect; although the cells are able to normally initiate both a G1 and a G2 arrest, they prematurely escape the G2-M arrest after DNA damage. The escape from G2 correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that the misregulation of CDK2 contributes to the failure of checkpoint control by allowing cells to enter mitosis prematurely.
细胞周期检查点的失调与肿瘤形成有关。为了深入了解正常细胞和转化细胞在检查点调控方面的差异，我们比较了正常成纤维细胞和TAg转化的成纤维细胞在γ射线照射DNA损伤后的细胞周期反应。正常细胞系在DNA损伤后，G1期和G2期均停滞超过48小时。令人惊讶的是，细胞周期蛋白依赖性激酶2（CDK2）和细胞周期蛋白A蛋白在DNA损伤后24小时内均特异性下调。相比之下，TAg转化细胞在DNA损伤后既不下调细胞周期蛋白A也不下调CDK2，并且G2期停滞明显缩短。为了研究CDK2下调在细胞周期停滞中的作用，我们构建了持续过表达CDK2的正常细胞系。这些细胞在DNA损伤后既不能下调CDK2蛋白也不能下调CDK2蛋白激酶活性，并且还表现出G2检查点缺陷；尽管这些细胞能够正常启动G1期和G2期停滞，但它们在DNA损伤后过早地逃离了G2-M期停滞。从G2期逃逸与这些细胞中细胞周期蛋白B相关蛋白激酶活性的非法激活有关。这些结果表明，CDK2的失调通过允许细胞过早进入有丝分裂而导致检查点控制失败。
