Jin Shunqian, Tong Tong, Fan Wenhong, Fan Feiyue, Antinore Michael J, Zhu Xiaocheng, Mazzacurati Lucia, Li Xianxing, Petrik Kimberly L, Rajasekaran Baskaran, Wu Min, Zhan Qimin
Department of Radiation Oncology, Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, PA 15213, USA.
Oncogene. 2002 Dec 12;21(57):8696-704. doi: 10.1038/sj.onc.1206034.
In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.
作为对DNA损伤的反应，细胞周期检查点是维持基因组保真度的重要生物学事件。Gadd45是一种受p53调控且可被DNA损伤诱导的蛋白质，最近已被证明在对DNA损伤的G2-M检查点中发挥作用。在本研究中，我们进一步研究了GADD45激活的细胞周期G2-M期阻滞所涉及的生化机制。利用四环素调控系统（tet-off），我们在HCT116（野生型p53）和Hela（p53失活状态）细胞中建立了GADD45诱导细胞系。在诱导表达Gadd45蛋白后，HCT116和Hela细胞的生长均受到强烈抑制。有趣的是，HCT116细胞出现了明显的G2-M期阻滞，但Hela细胞未能阻滞在G2-M期，这表明GADD45激活的G2-M期阻滞需要正常的p53功能。观察到GADD45诱导的G2-M期阻滞与p38激酶活性无关。重要的是，Gadd45蛋白的诱导导致核周期蛋白B1水平降低，其核定位对于G2-M期转换的完成至关重要。核周期蛋白B1水平的降低与Cdc2/周期蛋白B1激酶活性的抑制相关。此外，周期蛋白B1的过表达基本上消除了GADD45诱导的细胞生长抑制。因此，GADD45通过改变周期蛋白B1的亚细胞定位来抑制Cdc2激酶活性可能是GADD45诱导细胞周期G2-M期阻滞和生长抑制的关键步骤。
