Loss of Hus1 sensitizes cells to etoposide-induced apoptosis by regulating BH3-only proteins - PubMed (original) (raw)

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

C L Meyerkord et al. Oncogene. 2008.

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.

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Figures

Figure 1

Figure 1

Loss of Hus1 sensitizes cells to apoptosis induced by etoposide treatment. (a) Hus1+/+_p21_-/- and _Hus1_-/- _p21_-/- MEFs were treated with increasing doses of etoposide for 24 h and viability was determined by trypan blue exclusion assay (mean ± s.d.; n=3). (b) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 6.25 μg/ml etoposide for varying time points and subjected to trypan blue exclusion assay (mean ± s.d.; n=3). (c) Cells were treated as in (a). Total cell lysate was normalized for protein content and subjected to SDS-PAGE/immunoblot analysis. (d) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 6.25 μg/ml etoposide for 0 or 48 h. Apoptosis was determined by examination of nuclear morphology. Arrows indicate apoptotic nuclei. (e) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 6.25 μg/ml etoposide for the times indicated. The percent of apoptotic cells was quantified based on nuclear morphology (mean ± s.d.; n=3).

Figure 2

Figure 2

Loss of Hus1 results in upregulation of Bim and Puma expression in response to DNA damage. (a, b, c) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with (a) 6.25 μg/ml etoposide, (b) 500 nM camptothecin (CPT) or (c) 50 μM hydroxyurea (HU) for the indicated time points. Total cell lysate was prepared and analyzed by SDS-PAGE/immunoblot using the indicated antibodies. (d) _Hus1_-/-_p21_-/- MEFs stably expressing Hus1 or GFP were treated with 6.25 μg/ml etoposide for varying time points. The expression of Bim and Puma was examined by SDS-PAGE/immunoblot analysis.

Figure 3

Figure 3

Induction of Bim and Puma expression in response to etoposide treatment is regulated at the transcriptional level. (a) _Hus1_-/-_p21_-/- MEFs were treated with control DMSO (-), 1 μg/ml actinomycin D or 5 μg/ml cycloheximide alone or in combination with 3.125 μg/ml etoposide for 24 h. Total cell lysate was prepared and the expression of Bim and Puma was analyzed by SDS-PAGE/immunoblot. (b, c) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 6.25 μg/ml etoposide for 0, 3, 6, 12, 24, 36 or 48 h. Semi-quantitative RT-PCR was used to examine the mRNA levels of (b) Bim and (c) Puma. (d, e) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 25 μg/ml etoposide for 0, 1, 2, 3 or 4 h. Semi-quantitative RT-PCR was used to examine the mRNA levels of (d) Bim and (e) Puma.

Figure 4

Figure 4

Suppression of Bim and Puma expression confers resistance to etoposide-induced apoptosis in _Hus1_-deficient cells. (a, b) _Hus1_-/-_p21_-/- MEFs were mock transfected or transiently transfected with siRNA targeting GFP or Bim. Thirty-six hours after transfection, the cells were treated with control DMSO or 6.25 μg/ml etoposide for 30 h. (a) Whole cell lysate was subjected to SDS-PAGE/immunoblot analysis with antibodies to PARP (full length PARP is shown), Bim, Puma and Tubulin. (b) Viability was determined by trypan blue exclusion assay (mean ± s.d.; n=2). The DMSO-treated mock transfected cells were used to show the basal levels of Bim expression and cell death. (c, d) _Hus1_-/-_p21_-/- MEFs were infected with lentivirus expressing shRNA targeting Bim, Puma, Bim and Puma, or a control scrambled shRNA (shScram). After selection on Puromycin, the cells were treated with 12.5 μg/ml etoposide or control DMSO for 16 h. (c) Knockdown of Bim and Puma was confirmed by SDS-PAGE/immunoblot analysis. (d) Induction of apoptosis was measured by caspase-3 activity assay. The caspase-3 activity of control DMSO treated cells was subtracted from the amount of caspase-3 activity observed in the etoposide treated cells. The data are represented as percent relative apoptosis as normalized to the control infected cells (mean ± s.d.; n=3).

Figure 5

Figure 5

Rad9 is predominantly detected in the cytoplasm of _Hus1_-deficient cells where it binds to Bcl-2 in response to DNA damage. (a) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 12.5 μg/ml etoposide for 0, 2 or 8 h and subjected to subcellular fractionation. The resulting chromatin bound and soluble fractions were analyzed by SDS-PAGE/immunoblot using antibodies specific for Rad9 and RPA as a control. (b) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 12.5 μg/ml etoposide or control DMSO for 12 h and subjected to subcellular fractionation. The resulting cytosolic (Cyt) and nuclear (Nuc) fractions, along with whole cell lysate (WCL), were analyzed by SDS-PAGE/immunoblot. (c, d) Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were treated with 12.5 μg/ml etoposide or control DMSO for 12 h. (c) The cytosolic fractions of Hus1+/+_p21_-/- and _Hus1_-/-_p21_-/- MEFs were subjected to immunoprecipitation in the absence or presence of anti-Bcl-2 monoclonal antibody. The resulting immune complexes were analyzed by SDS-PAGE/immunoblot. (d) Whole cell lysate (WCL) was subjected to immunoprecipitation with anti-Bcl-2 or control anti-Flag monoclonal antibodies. The resulting immunocomplexes and WCL were analyzed by SDS-PAGE/immunoblot. The amount of Rad9 in the immunocomplexes was quantified and normalized to cytosolic (c) or total (d) Rad9. The levels of Bcl-2 bound Rad9 are listed relative to those of untreated Hus1-/-p21-/- cells, which were set as 1.0.

Figure 6

Figure 6

Rad9 collaborates with Bim and Puma to sensitize _Hus1_-deficient cells to etoposide-induced apoptosis. (a, b) _Hus1_-/-_p21_-/- MEFs stably expressing shBim and shPuma were infected with lentivirus expressing shRNA targeting Rad9. The cells were treated with 12.5 μg/ml etoposide or control DMSO for 16 h. (a) Knockdown of Rad9, as well as Bim and Puma, was confirmed by SDS-PAGE/immunoblot analysis. (b) Induction of apoptosis was measured using a caspase-3 activity assay. (c) _Hus1_-/-_p21_-/- MEFs stably expressing control shRNA, shBim and shPuma, or shBim and shPuma plus shRad9 were treated with 12.5 μg/ml etoposide for 0, 24 or 48 h. Cell death was determined by trypan blue exclusion assay (mean ± s.d.; n=3).

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References

    1. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 2001;15:2177–96. - PubMed
    1. Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007;26:1324–37. - PMC - PubMed
    1. Bakkenist CJ, Kastan MB. Initiating cellular stress responses. Cell. 2004;118:9–17. - PubMed
    1. Bao S, Lu T, Wang X, Zheng H, Wang LE, Wei Q, et al. Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene. 2004;23:5586–93. - PubMed
    1. Bermudez VP, Lindsey-Boltz LA, Cesare AJ, Maniwa Y, Griffith JD, Hurwitz J, et al. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc Natl Acad Sci U S A. 2003;100:1633–8. - PMC - PubMed

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