Loss of Hus1 sensitizes cells to etoposide-induced apoptosis by regulating BH3-only proteins

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Abstract
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.