Meyerkord C L, Takahashi Y, Araya R, Takada N, Weiss R S, Wang H-G
Drug Discovery Program, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
Oncogene. 2008 Dec 11;27(58):7248-59. doi: 10.1038/onc.2008.336. Epub 2008 Sep 15.
The Rad9-Rad1-Hus1 (9-1-1) cell cycle checkpoint complex plays a key role in the DNA damage response. Cells with a defective 9-1-1 complex have been shown to be sensitive to apoptosis induced by certain types of genotoxic stress. However, the mechanism linking the loss of a functional 9-1-1 complex to the cell death machinery has yet to be determined. Here, we report that etoposide treatment dramatically upregulates the BH3-only proteins, Bim and Puma, in Hus1-deficient cells. Inhibition of either Bim or Puma expression in Hus1-knockout cells confers significant resistance to etoposide-induced apoptosis, whereas knockdown of both proteins results in further resistance, suggesting that Bim and Puma cooperate in sensitizing Hus1-deficient cells to etoposide treatment. Moreover, we found that Rad9 collaborates with Bim and Puma to sensitize Hus1-deficient cells to etoposide-induced apoptosis. In response to DNA damage, Rad9 localizes to chromatin in Hus1-wild-type cells, whereas in Hus1-deficient cells, it is predominantly located in the cytoplasm where it binds to Bcl-2. Taken together, these results suggest that loss of Hus1 sensitizes cells to etoposide-induced apoptosis not only by inducing Bim and Puma expressions but also by releasing Rad9 into the cytosol to augment mitochondrial apoptosis.
Rad9-Rad1-Hus1（9-1-1）细胞周期检查点复合物在DNA损伤反应中起关键作用。已证明具有缺陷的9-1-1复合物的细胞对某些类型的基因毒性应激诱导的凋亡敏感。然而，将功能性9-1-1复合物的缺失与细胞死亡机制联系起来的机制尚未确定。在此，我们报告依托泊苷处理显著上调Hus1缺陷细胞中仅含BH3结构域的蛋白Bim和Puma。在Hus1基因敲除细胞中抑制Bim或Puma的表达可赋予对依托泊苷诱导的凋亡的显著抗性，而敲低这两种蛋白则导致进一步的抗性，表明Bim和Puma协同作用使Hus1缺陷细胞对依托泊苷处理敏感。此外，我们发现Rad9与Bim和Puma协同作用使Hus1缺陷细胞对依托泊苷诱导的凋亡敏感。响应DNA损伤时，Rad9在Hus1野生型细胞中定位于染色质，而在Hus1缺陷细胞中，它主要位于细胞质中并与Bcl-2结合。综上所述，这些结果表明Hus1的缺失使细胞对依托泊苷诱导的凋亡敏感，不仅通过诱导Bim和Puma的表达，还通过将Rad9释放到细胞质中以增强线粒体凋亡。
