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Bubr1 Is Involved in Regulation of DNA Damage Responses

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Bubr1 Is Involved in Regulation of DNA Damage Responses Oncogene (2006) 25, 3598–3605 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE BubR1 is involved in regulation of DNA damage responses Y Fang1, T Liu1, X Wang1, Y-M Yang1, H Deng2, J Kunicki3, F Traganos3, Z Darzynkiewicz4, LLu5 and W Dai1,4 1Division of Molecular Carcinogenesis, New York Medical College, Valhalla, NY 10595, USA; 2Proteomics Core Facility, Rockefeller University, New York, NY 10021, USA; 3Department of Medicine, New York Medical College, Valhalla, NY 10595, USA; 4Brander Cancer Institute, New York Medical College, Valhalla, NY 10595, USA and 5Division of Molecular Medicine, Harbor-UCLA Medical Center, University of California Los Angeles, Torrance, CA 90502, USA Defective mitotic spindles or an impaired spindle–kine- Introduction tochore interaction activates the spindle checkpoint. We have previously shown that BubR1 haplo-insufficiency The spindle checkpoint functions to prevent premature results in enhanced genomic instability and tumorigenesis anaphase entry until each of every condensed chromo- in mice. Here we report that BubR1 deficiency also leads some successfully attaches to bipolar spindle micro- to a compromised response to DNA damage. Following tubules. BubR1 is a key molecule mediating spindle- treatment with doxorubicin, BubR1 þ /À murine fibroblast checkpoint activation, during which it is extensively cells (MEF) were defective in undergoing G2/M arrest. phosphorylated (Li et al., 1999). A loss of spindle- Thus, whereas in the presence of DNA damage BubR1 þ / þ checkpoint function inevitably results in chromosomal MEF cells remained arrested in mitosis, BubR1 þ /À MEFs instability and aneuploidy (Lengauer, 2003; Bharadwaj rapidly exited from mitosis and divided. The impaired and Yu, 2004). Given that aneuploidy is prevalent in mitotic arrest of BubR1 þ /À MEFs was associated with low many types of cancers it is believed that spindle- levels of phospho-histone H2AX, p53, and p21 after DNA checkpoint failure may be at least partly responsible damage caused by treatment with both doxorubicin and for the development of cancer (Lengauer et al., 1998). ultraviolet light (UV). The impaired expression of p53 and Mutations in BubR1 have been detected in colonic p21 was also confirmed in human cell lines with BubR1 cancer (Cahill et al., 1998). Our mouse genetic study knockdown via RNA interference. Affinity pull-down shows that haplo-insufficiency of BubR1 results in coupled with mass spectrometry identified Poly(ADP- enhanced genomic instability and development of cancer ribose) polymerase 1 (PARP-1) as one of the proteins in lung and colon (Dai et al., 2004; Rao et al., 2005). interacting with BubR1. Reciprocal co-immunoprecipita- Consistently, a recent study shows that a compromised tion analysis confirmed the physical interaction between BubR1 activity due to specific germline mutations BubR1 and PARP-1. Our further study revealed that the causes aneuploidy, infertility and/or early onset of ability of retaining intact PARP-1 or its cleavage product malignancies (Hanks et al., 2004), strongly suggesting þ /À p89 was compromised in BubR1 MEFs upon treatment that spindle checkpoint failure is an underlying cause for with doxorubicin or UV. Given that PARP-1 mediates the development of certain diseases. DNA damage responses and regulates the activity of p53, The signaling pathways that underlie the cellular our studies suggest that there exists a cross-talk between response to DNA damage (genotoxic stress) consist of the spindle checkpoint and the DNA damage checkpoint sensors, signal transducers, and effectors (Zhou and and that BubR1 may play an important role in mediating Elledge, 2000; Sancar et al., 2004). Although the the cross-talk. identities of the damage sensors remain unknown, Oncogene (2006) 25, 3598–3605. doi:10.1038/sj.onc.1209392; the molecular entities responsible for transducing the published online 30 January 2006 damage signals to specific effectors are relatively well Keywords: BubR1; DNA damage; checkpoint; p53; p21; characterized. ATM (mutated in ataxia telangiectasia) PARP and its homologue ATR (ATM-related) function early in the signaling pathways and are central to the DNA damage response. Downstream targets (substrates) of ATM and ATR include the protein kinases Chk1, Chk2, and Plks (Smits et al., 2000; Sancar et al., 2004; Xie et al., 2005). Effector molecules that execute the DNA damage response include p53. ATM and ATR, together Correspondence: Professor W Dai, Division of Molecular Carcino- with Chk1, Chk2, and Plk3, are thought to mediate the genesis, Department of Medicine, New York Medical College, Basic specific cellular responses to different types of DNA Science Building, A22, Valhalla, NY 10595, USA. E-mail: [email protected] damage in mammalian cells. Received 1 May 2005; revised 7 November 2005; accepted 14November Securin is a major molecular target of anaphase- 2005; published online 30 January 2006 promoting complex/cyclosome (APC/C) (Lew and BubR1 deficiency and DNA damage checkpoint Y Fang et al 3599 Burke, 2003; Bharadwaj and Yu, 2004) and it also interacts with p53 (Bernal et al., 2002; Hamid and Kakar, 2003). It has been proposed that the oncogenic effect of increased expression of securin may result from modulation of p53 functions (Bernal et al., 2002). On the other hand, securin is, in turn, a transcriptional target of p53 (Zhou et al., 2003). Given the observed transform- ing ability of securin (Hamid and Kakar, 2003), it is speculated that securin may negatively regulate p53 in a manner similar to that of MDM2. In this study, we show that BubR1 þ /À murine fibroblasts (MEFs) exhib- ited a compromised mitotic arrest after DNA damage, which was accompanied by significantly reduced levels of phospho-H2AX, p53 and p21. In addition, we demonstrate that Poly(ADP-ribose) polymerase 1 (PARP-1) interacted and colocalized with BubR1 and that PARP-1 degradation was enhanced when cellular BubR1 levels were reduced. Results DNA damage frequently induces G2/M arrest (Sancar et al., 2004). Existing evidence indicates that certain DNA damage results in defective interaction between chromosomes and kinetochores that in turn activates the spindle checkpoint (Mikhailov et al., 2002; Nitta et al., 2004). It has been shown that suppression of spindle- checkpoint function by downregulation of BubR1 and Mad2 via RNA interference causes abnormal mitosis, even in the presence of DNA damage (Nitta et al., 2004). To study whether spindle-checkpoint defects would compromise DNA damage responses, we examined BubR1-deficient (BubR1 þ /À) cells for their cell-cycle status after DNA damage. Paired BubR1 þ /À and wild- type (BubR1 þ / þ ) MEF cells, as well as HeLa cells, were þ /À treated with doxorubicin, a DNA topoisomerase 2 Figure 1 Compromised cell cycle G2/M arrest of BubR1 MEFs inhibitor causing DNA double-strand breaks, for after DNA damage. (a) BubR1 þ / þ MEFs, BubR1 þ /À MEFs, and HeLa cells were treated with adriamycin (ADR, 0.5 mM) for various time intervals, and cells were then fixed and various intervals as indicated (h). Treated cells were then collected, stained with DAPI for analysis by flow cytometry. As fixed, stained with DAPI, and processed for cell-cycle analysis by expected, an increasing proportion of HeLa cells were flow cytometry. These paired MEFs were in their third passage. (b) BubR1 þ / þ and BubR1 þ /À MEFs of the third passage were treated arrested at G2/M stages of the cell cycle after exposure to doxorubicin (Figure 1a). Similar to HeLa cells, a with nocodazole for 16 h. The cells were then released from nocodazole block and cultured in the presence of doxorubicin for significant fraction of wild-type MEFs were also rapidly various times, as indicated. Cells were fixed, stained, and processed arrested in G2/M after DNA damage; on the other for cell-cycle analysis by flow cytometry. (c) BubR1 þ / þ and þ /À þ /À hand, G2/M arrest was not apparent with BubR1 BubR1 MEFs of the third passage were treated with nocodazole MEFs even 9 h after exposure to the drug (Figure 1a), for 16 h. The cells were then released from nocodazole block and suggesting that a defective spindle checkpoint weakens cultured in the presence or absence of doxorubicin for various times. The percent of G2/M cells were calculated. These experi- cell’s ability to undergo G2/M arrest after DNA ments were repeated for at least two times with independent lines of damage. To further determine whether cells with DNA primary MEFs and representative data were presented. damage were incapable of undergoing mitotic arrest in BubR1 þ /À MEFs, we treated BubR1 þ /À and wild-type MEFs for 16 h with nocodazole that induces mitotic have divided and then exited G1 phase of the cycle. In arrest. The mitotically arrested cells were then cultured contrast, a majority of BubR1 þ / þ MEFs remained in in fresh medium free of nocodazole but supplemented G2/M 9 h post release (Figure 1b). We then quantified þ /À with doxorubicin. We observed that mitotic BubR1 the G2/M fraction of paired MEFs at various time MEFs gradually exited from mitosis and only a minor intervals post nocodazole release in the presence of fraction of cells remained in G2/M 9 h after release. In doxorubicin. After DNA damage, a large fraction of þ /À þ / þ fact, the presence of a distinct cohort of BubR1 cells BubR1 MEFs remained at G2/M stages; in the synchronized in early S-phase indicates that the cells absence of doxorubicin, the G2/M population signifi- Oncogene BubR1 deficiency and DNA damage checkpoint Y Fang et al 3600 cantly decreased 6 h after nocodazole release (Figure 1c). þ /À On the other hand, BubR1 MEFs exited from G2/M phases in a similar kinetics regardless of the DNA damage status (Figure 1c), thus consistent with the notion that BubR1 deficiency compromised G2/M arrest induced by DNA damage.
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