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. To examine whether the defective cell-cycle arrest in BubR1 þ /À cells was accompanied by a weakened DNA damage response, we analysed the level of phospho- histone 2AX (p-H2AX), a marker tightly associated with the extent of DNA damage (Redon et al., 2002), in paired MEFs treated with doxorubicin for various intervals. Whereas little p-H2AX was detectable in untreated wild-type or BubR1 þ /À MEFs, much stronger H2AX phosphorylation was induced by doxorubicin treatment (Figure 2a). The magnitude of increase in p- H2AX signals was more pronounced in wild-type MEFs than that of BubR1 þ /À MEFs. In fact, p-H2AX was barely inducible in BubR1 þ /À cells by doxorubicin (Figure 2a). Similar results were also obtained with paired MEFs exposed to UV irradiation. In wild-type MEFs, phospho-H2AX was detectable 12 h after UV exposure and increased significantly by 24h; however, little phospho-H2AX was detected in BubR1 þ /À MEFs even 24h after UV irradiation (Figure 2b). The tumor suppressor protein p53 plays an important role in DNA damage responses (Zhou and Elledge, 2000) and it also interacts with securin (Hamid and Kakar, 2003). Securin is a ubiquitylation target of APC/ C whose E3 ligase activity is, in turn, regulated by the spindle checkpoint (Lengauer, 2003; Bharadwaj and Yu, 2004). We then examined the level of p53 in paired MEFs. The basal level of p53 was significantly lower in þ /À Figure 2 Weakened DNA damage responses in BubR1 þ /À MEFs. BubR1 MEFs than that in wild type; consistently, the þ / þ þ /À þ /À (a) BubR1 (W, wild type) and BubR1 (H, heterozygous) basal level of p21 was also lower in untreated BubR1 MEFs were treated with doxorubicin (Dox) for various times. An MEFs (Figure 2c). Upon exposure to doxorubicin, equal amount of cell lysates was analysed for phospho-histone expression of both p53 and p21 in wild type, but not in H2AX (p-H2AX) and b-actin by Western blotting. (b) BubR1 þ / þ BubR1 þ /À MEFs, was significantly induced (Figure 2c). (W) and BubR1 þ /À (H) MEFs were exposed to UV irradiation. At Interestingly, expression of p21, but not p53, was various time intervals post irradiation, cells were collected for lysis and equal amounts of cell lysates were blotted for p-H2AX and b- drastically reduced 12 h after doxorubicin treatment actin. (c) BubR1 þ / þ and BubR1 þ /À MEFs were treated with þ /À in BubR1 cells (Figure 2c). To further confirm the doxorubicin (0.5 mM) for various time intervals. Equal amounts of observation that BubR1 deficiency compromises p53 cell lysates were blotted for p53, p21, and b-actin. (d) BubR1 þ / þ activation after DNA damage, we exposed paired MEFs and BubR1 þ /À MEFs exposed to UV irradiation for various times were lysed and equal amounts of cell lysates were blotted for p53, to UV and then analysed p53 and p21 expression at p21 and b-actin. The MEFs used in this series of experiments were various time intervals post UV exposure. Consistently, in their fourth passage. Each experiment was repeated for at least UV treatment caused a significant accumulation of both three times. Representative results were shown. p53 and p21 in wild-type MEFs; on the other hand, no significant induction of p53 and p21 was detected in BubR1 þ /À MEFs until 24h after UV exposure reduced p53 and p21 levels in 293 cells (Figure 3b). We (Figure 2d). also noticed that doxorubicin treatment significantly We next examined expression of p21, as well as reduced viability of both wild-type and BubR1 þ /À MEF securin and cyclin B1, in HeLa cells (which express no and the cell viability was inversely correlated with the detectable levels of p53) depleted of BubR1 via RNAi. concentrations of the DNA damage drug used in the We demonstrated that p21 expression was also greatly assays; however, BubR1 þ /À MEFs survived significantly reduced after knockdown of BubR1; as expected, securin better than the wild-type MEFs when they were exposed and cyclin B1 levels were significantly decreased to the same concentrations of doxorubicin (Figure 3c), (Figure 3a). To further confirm the correlation of strongly suggesting the involvement of BubR1 in BubR1 knockdown with impaired p53 function in modulation of cellular responses to DNA damage. human cells, we transfected 293 cells (which express We next asked whether DNA damage would affect both p53 and p21) with BubR1 siRNA as well as control BubR1 levels because it has been shown that an intact siRNA. Consistently, BubR1 knockdown significantly spindle-checkpoint function is required for mitotic

Oncogene BubR1 deﬁciency and DNA damage checkpoint Y Fang et al 3601

Figure 4 Genotoxic stresses induce BubR1 degradation. (a) BubR1 þ / þ and BubR1 þ /À MEFs were treated with doxorubicin for various times as indicated. Cell lysates from various treatments were blotted for BubR1 and actin. (b) HeLa cells were treated with doxorubicin for various times and cell lysates were blotted for BubR1 and Mad2. (c) BubR1 þ / þ and BubR1 þ /À MEFs were treated with UV for various times. Cell lysates from various treatments were blotted for BubR1 and actin. Phosphorylated BubR1 was indicated as p-BubR1. MEFs used were in their fourth passage.

BubR1 was proteasome-dependent, we treated HeLa Figure 3 BubR1 deficiency results in downregulation of p21, cells with MG132 with doxorubicin in the presence or correlating with a better survival rate. (a) HeLa cells were absence of the proteasome inhibitor MG132. We transfected with BubR1 or luciferase (Luc) siRNA duplex for observed that neither MG132 alone nor in combination 48 h. Equal amounts of cell lysates from the transfected cells, as with doxorubicin further induced BubR1 (Figure 4c). well as from the non-transfected controls cells, were blotted for Interestingly, MG132 preferentially stabilized the acti- p21, securin, cyclin B1, and b-actin. (b) Human 293 cells were transfected with BubR1 or luciferase (Luc) siRNA duplex for 48 h. vated, phosphorylated form of BubR1 (p-BubR1 in Equal amounts of cell lysates from the transfected cells, as well as Figure 4c). from the non-transfected controls cells, were blotted for p53, p21, To understand the molecular basis by which BubR1 and b-actin. Each experiment was repeated for at least two times modulates the DNA damage response, experiments were with independent lines of primary MEFs. Representative results designed to screen and identify proteins that were not were shown. (c) BubR1 þ / þ and BubR1 þ /À MEFs of the fourth passage were treated with various concentrations of doxorubicin, only involved in DNA damage responses but also as indicated. At 24h after treatment with the DNA damage agent, interacted with BubR1. To this end, BubR1 antibody, the cell viability was measured by the MTT assay. The data were as well as the control antibody, was immobilized to summarized from three independent experiments. protein A/G resin, which was subsequently used to enrich proteins from HeLa cell lysates. After thorough washings, proteins bound to BubR1 antibody resin, as catastrophe induced as a result of DNA damage (Nitta well as to the control antibody resin, were eluted and et al., 2004). Doxorubicin treatment for 18 h or longer analysed by SDS-PAGE followed by silver staining. We significantly decreased BubR1 levels in both wild-type observed that BubR1 antibody, but not the control and BubR1 þ /À MEFs (Figure 4a). The same decrease in antibody, was capable of pulling down distinct proteins BubR1 levels was also observed when BubR1 þ /À MEFs from both interphase and mitotic lysates (Figure 5a). were exposed to UV (data not shown). In addition, The identity of BubR1 bands (both phosphorylated and when HeLa cells were treated with doxorubicin, BubR1 non-phosphorylated) in eluents was confirmed by levels were also decreased; the DNA damage-induced immunoblotting. More than a dozen of the visible downregulation of BubR1 appeared to be specific bands were excised from the gel and subjected to protein because MAD2 levels were not affected by doxorubicin identification by mass spectrometric analysis. Among treatment of HeLa cells (Figure 4b). To examine two dozen of the proteins identified, we noticed several whether DNA damage-induced downregulation of hits of PARP-1, which is a protein known to interact

Oncogene BubR1 deﬁciency and DNA damage checkpoint Y Fang et al 3602

Figure 5 PARP-1 interacts with BubR1. (a) Mitotic or interphase cell lysates were immunoprecipitated with BubR1 or preimmune IgGs. Immunoprecipitates were thoroughly washed and analysed by SDS-PAGE followed by silver staining. Mitotic and interphase lysates were also blotted for conﬁrmation of the position of phospho- and non-phospho-BubR1 bands in immunoprecipitates. Arrows denote some of the silver-stained bands whose identities were revealed by mass spectrometric analysis. (b) Mitotic (M) and interphase (I) cell lysates were immunoprecipitated with PARP-1 IgGs or with control IgGs. Immunoprecipitates, along with cell lysates from mitotic and interphase cells, were then blotted for BubR1. (c) Mitotic (M) and interphase (I) cell lysates were immunoprecipitated with BubR1 IgGs. Immunoprecipitates as well as cell lysates were blotted for PARP-1.

with kinetochore components and involved in DNA Figure 6 Functional interaction between BubR1 and PARP-1. (a) damage responses as well (Menissier et al., 2003; Wieler HeLa cells cultured on chamber slides were stained with antibodies to BubR1 (red) and PARP-1 (green). DNA was stained with DAPI et al., 2003). (blue). Indirect immunofluorescence microscopy was performed. A We then confirmed the physical interaction between representative prophase cell was shown. (b) BubR1 þ / þ and PARP-1 and BubR1 via reciprocal co-immunoprecipi- BubR1 þ /À MEFs of the fourth passage were treated with tation. PARP-1 antibody, but not the control antibody, doxorubicin for various times. Equal amounts of lysates from the treated cells, as well as from untreated control cells, were blotted was capable of pulling down BubR1 from both for PARP-1 and b-actin. The position of p89, the major interphase and mitotic cell lysates (Figure 5b). Likewise, degradation product of PARP-1 was also indicated. (c) BubR1 þ / þ BubR1 antibody was able to precipitate p89 fragment of and BubR1 þ /À MEFs of the fourth passage were exposed to UV PARP-1 (Figure 5c). A recent study has shown that irradiation. At 24h post UV exposure, cells were collected and Poly(ADP-ribose) (PAR) is an essential component of lysed. Equal amounts of control and treated cell lysates were blotted for PARP-1, p89, and b-actin. These experiments were the mitotic spindle that is involved in bipolar spindle repeated with three independent lines of primary MEFs (in the assembly and function (Chang et al., 2004). Given the third or fourth passage) and similar results were obtained. (d) HeLa observation that PARP-1 interacts with kinetochore cells were transfected with BubR1 or luciferease (Luc) siRNA for 2 components such as CENP-A, CENP-B, and Bub3 days after which cells were lysed and equal amounts of cell lysates (Saxena et al., 2002), we asked whether PARP-1 shares in duplicates were blotted for BubR1, PARP-1, and b-actin. the same subcellular localization as BubR1 during early mitosis when BubR1 is activated. Indirect immuno- fluorescent microscopy revealed that PARP-1 coloca- doxorubicin treatment. DNA damage induced by lized with BubR1 at kinetochores during prophase doxorubicin resulted in accumulation of p89 in both (Figure 6a), suggesting a potentially functional inter- wild-type and BubR1 þ /À MEFs, which was correlated action between these two proteins. with the concomitant decrease in PARP-1 levels; As it has also been shown that PARP-1 interacts with however, the accumulation of p89 in BubR1 þ /À cells p53 and positively regulates p53 activity (Wieler et al., was significantly attenuated compared with that of wild- 2003), we then examined the level of PARP-1 and its type MEFs (Figure 6b), indicating that haplo-insuffi- degradation product p89 in BubR1 þ /À MEFs before and ciency of BubR1 enhances degradation of PARP-1. after doxorubicin treatment. There was a low basal level Moreover, when paired MEFs were exposed to UV for of PARP-1 in BubR1 þ /À MEFs before initiation of 24h, the levels of both PARP-1 and p89 in BubR1 þ /À

Oncogene BubR1 deficiency and DNA damage checkpoint Y Fang et al 3603 MEFs were also significantly decreased compared with MEFs contained little, if any, p21 although a significant that of wild-type cells (Figure 6c). The enhanced amount of p53 was detected in these cells (Figure 2c). In degradation of PARP-1 owing to BubR1 deficiency addition, BubR1 knockdown via RNAi caused a drastic was also demonstrated in HeLa cells. After BubR1 reduction of p21 in HeLa cells that do not have knockdown via RNAi, the basal level of PARP-1 was detectable levels of p53 before or after DNA damage significantly reduced as compared with that of untrans- (data not shown). As p21 is also degraded by the fected cells or the cells transfected with luciferase siRNA proteasome-mediated pathway (Bloom et al., 2003), it is (Figure 6d). interesting to know whether elevated APC/CCdc20 activity would modulate its poly-ubiquitylation in BubR1- deficient cells. A recent study shows that APC/CCdh1 participates in the regulation of SCF(Skp2-Cks1) Discussion activity, which in turn is responsible for the degradation of p21 and p27 (Bashir et al., 2004). As Skp2 is a target In this report, we demonstrate that an impaired spindle of APC/CCdh1, it would be interesting to test whether checkpoint owing to BubR1 deficiency also compromises there is a cross-talk between APC/CCdc20 and SCF. The the cell’s ability to respond to DNA damage induced by APC/CCdc20 activity is directly affected by an impaired both UV and the double-strand break mimetic drug spindle checkpoint owing to BubR1 deficiency. Thus, it doxorubicin. Specifically, BubR1 þ /À cells failed to is natural to observe that securin levels are decreased in undergo significant mitotic arrest in the presence of BubR1 þ /À cells (Figure 3a). Here we show that p53 and DNA damage. These cells also had a reduced ability to PARP-1 protein levels are also decreased in BubR1- accumulate phosphorylated H2AX, a marker strongly deficient cells. Given that both p53 and PARP-1 associated with DNA repair and apoptosis. Expression play important role in DNA repair and that BubR1 þ /À of both p53 and p21 was significantly compromised in mice are prone to the development of cancers it is BubR1-deficient cells before and after DNA damage. tempting to propose that haplo-insufficiency of BubR1 Moreover, BubR1 deficiency facilitated degradation of can cause genetic instabilities at levels of both PARP-1, especially after DNA damage. BubR1 coloca- DNA repair and chromosomal segregation, deregula- lized with PARP-1 in the cell and physically interacted tion of which will inevitably result in malignant with this protein. Given that both p53 and PARP-1 are transformation. important components in DNA damage checkpoint We have shown that both BubR1 and PARP-1 were activation (Huber et al., 2004), our studies strongly degraded after DNA damage (Figures 4and 6). suggest that spindle-checkpoint impairment owing to Although PARP-1 cleavage is mediated by caspase 3 BubR1 deficiency weakens cell’s ability to cope with (Affar et al., 2001), the molecular entity responsible genotoxic stresses. for degradation of BubR1 after genotoxic stresses It is known that p53 levels are kept low in the cells remains unknown. As inhibition of proteasome by through ubiquitylation and proteasomal degradation MG132 can partially block the degradation of BubR1, (Yang et al., 2004). Upon various stresses, p53 is especially the phosphorylated form (Figure 5), we stabilized and activated through diverse pathways that speculate that the proteasomal pathway is involved in include inhibition of ubiquitylation. As defects in regulating the stability of BubR1 after DNA damage. It p53 are present in more than half of all human tumors, is interesting to note that MAD2 is not degraded it would be logical to ask whether spindle-checkpoint after DNA damage although it functions in the same defects would affect the level of p53. Our studies way in inhibition of APC/C activity as BubR1 show that compared with wild-type MEFs, p53 was (Bharadwaj and Yu, 2004), suggesting that not all greatly reduced in BubR1 þ /À MEFs before and spindle checkpoint components are regulated by the after DNA damage; the decreased level of p53 was same molecular pathway. correlated with reduced expression of p21 (Figure 2c A recent study shows that PAR is enriched at the and d). It has been shown that p53 is physically mitotic spindle and essential for spindle function (Chang associated with securin (Hamid and Kakar, 2003), et al., 2004). Polymerization of PAR on target proteins whose degradation is enhanced owing to an elevated is catalyzed by PARPs (Chang et al., 2004). Early activity of APC/CCdc20. Although it remains unclear at studies have shown that PARP-1 and PARP-2 localize present whether APC/CCdc20 direclty poly-ubiquitylates to centrosomes and interact with kinetochore proteins p53, it is reasonable to speculate that accelerated such as CENP-A, CENP-B, and Bub3 (Saxena et al., degradation of securin by the proteasomal pathway in 2002). In addition, cellular PAR concentrations BubR1 þ /À MEFs may facilitate degradation of those are greatly elevated in mitosis owing to an increased proteins associated with it. PARP activity (Pirrotta, 2004). All of these studies Expression of p53 is induced in wild-type MEFs, suggest the importance of PAR and PARP enzymes which was accompanied by enhanced p21 expression during mitotic progression. Our observations that (Figure 2c), consistent with the fact that p21 is a BubR1 interacts with PARP-1 and that it colocalizes transcriptional target of p53. On the other hand, p21 with PARP-1 during prophase also strongly support the expression in BubR1 þ /À MEFs appears to be less notion that PARPs may play a critical role in regulating coordinated with p53 expression. For example, after chromosomal segregation as well as DNA damage treatment with doxorubicin for more than 6 h, BubR1 þ /À responses.

Oncogene BubR1 deficiency and DNA damage checkpoint Y Fang et al 3604 Materials and methods concentrations of doxorubicin was added to a final volume of 200 ml. After 24h incubation, 20 ml of MTT (Sigma, 2 mg/ml in MEF cells PBS) was then added to each well. After the cells were Primary BubR1 þ /À and wild-type MEF cells were derived from incubated at 371C for 4h, the medium was removed. MTT embryonic day 14.5 (E14.5) embryos produced from BubR1 þ /À formazan precipitates were dissolved in 100 ml DMSO with intercrosses. MEF cells were cultured under 5% CO2 in mechanical shaking for 10 min. The optical density of Dulbecco minimum essential medium (DMEM) supplemented dissolved samples was measured at 570 nm using a plate with 0.1 mM b-mercaptoethanol, 15% fetal bovine serum reader. (FBS), and antibiotics (100 mg/ml penicillin and 50 mg/ml streptomycin sulfate). MEFs of the third to fifth passages RNA interference were used for experiments. HeLa and 293 cells were cultured in Double-stranded RNAs of 21 nucleotides in length were DMEM supplemented with various components as above. synthesized by Dharmacon Research. The targeting sequence was 50-AAGGGAAGCCGAGCUGUUGAC-30, correspond- Indirect immunofluorescence microscopy and flow cytometry ing to the coding region of 1281–1301 in human BubR1 Immunoflouresence microscopy was essentially as described (accession no., AF068760) relative to the first nucleotide of (Wang et al., 2002). Briefly, cells were fixed in paraformalde- ATG start codon. The control siRNA targets luciferase hyde and then treated with 0.1% Triton X-100. After washing mRNA (accession no., X65324) of the firefly (Photinus pyralis) three times with phosphate-buffered saline (PBS), the cells and the targeting sequence was 50-UUCCTACGCTGAGTAC were blocked in 2.0% bovine serum albumin (BSA) for 15 min TTCGA-30 (GL-3, Dharmacon Research). Double nucleotides on ice and incubated with antibodies to BubR1 or PARP-1 (dTdT) were added at the 30-end of each strand. RNA duplexes (Cell Signaling) for 1 h. After additional washing with PBS, the were transfected into HeLa or 293 cells via the Lipofectamine cells were incubated with appropriate secondary antibodies 2000 approach (Invitrogen). Cells transfected with BubR1 and conjugated with Rhodamine-Red-X or fluorescein (Jackson luciferase siRNAs for 2 days were collected and lysed, and an Immuno-Research) at 41C for 1 h in the dark. Cells were then equal amount of cell lysates were blotted for expression of 0 briefly (5 min) stained with 4 ,6-diamidino-2-phenylindole BubR1, p21, securin, cyclin B1, PARP-1, and b-actin via (DAPI, Fluka). Fluorescence microscopy was performed on immunoblotting. a Nikon microscope and images were captured using a digital camera (Optronics) using Optronics MagFire, Image-Pro Plus softwares. For flow cytometric analysis, primary MEFs were Immunoprecipitation and mass spectrometry fixed in suspension in 70% ethanol, then stained with 1 mg/ml HeLa cells were cultured in the presence or absence of of DAPI dissolved in PBS, and intensity of their blue nocodazole (0.5 mg/ml) for 16 h. Lysates were prepared from fluorescence was measured using Elite.ESP flow cytometer/ mitotic as well as interphase cells immediately. After pre- sorter (Beckman Coulter). The DNA content frequency clearing with protein A/G beads (Santa Cruz), protein lysates histograms were deconvoluted with MultiCycle software of interphase and mitotic stages (10 mg each) were incubated (Phoenix Flow Systems). with anti-BubR1 antibody (40 mg) overnight at 41C. Protein A/ G beads were then added to the lysates and the incubation Western blotting continued at room temperature for 1 h. After extensive Primary MEFs or HeLa cells were treated with doxorubicin washing with the lysis buffer, protein A/G beads were 2 suspended in SDS-PAGE sample buffer. BubR1 and BubR1- (0.5 mM, unless otherwise specified) or UVC (25 J/m ). MEFs or HeLa cells were lysed in a lysis buffer as described (Ouyang interacting proteins were analysed on a 6% denaturing et al., 1997) and an equal amount of cell lysates were subjected polyacrylamide gel, which was subsequently subjected to silver to SDS-PAGE followed by immunoblotting analysis with staining using a kit from Bio-Rad. Major bands were excised antibodies to BubR1, p53 (Cell Signaling), PARP-1 (Cell and subjected to protein identification using a tandem MS/MS Signaling), securin (MBL), cyclin B1 (Cell Signaling), or b- mass spectrometer (Proteomics Core facility at Rockefeller actin (Sigma). Specific signals were detected with horseradish- University). peroxidase-conjugated goat-anti-rabbit secondary antibodies (Sigma) and enhanced chemiluminescence reagents (Amer- Acknowledgements sham Pharmacia Biotech). We thank Xuan Huang for her assistance in working with Cell viability/survival assay phospho-H2AX antibody. We are also grateful to Lisa Buerle Cell viability was assayed by the MTT method. Primary MEFs for administrative assistance and to co-workers in the (2 Â 104 cells/well) were seeded on 96-well plate in triplicates. laboratory for various discussions. The work is supported in Following a 24-h culture at 371C, the culture medium was part by a grant from the National Institutes of Health (RO1- aspirated and a fresh medium supplemented with various CA90658) to WD.

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