BubR1 Expression Research Articles

Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest

Arsenic induces clinical remission in patients with acute promyelocytic leukemia and has potential for treatment of other cancers. The current study examines factors influencing sensitivity to arsenic using human malignant melanoma cell lines. A375 and SK-Mel-2 cells were sensitive to clinically achievable concentrations of arsenite, whereas SK-Mel-3 and SK-Mel-28 cells required supratherapeutic levels for toxicity. Inhibition of glutathione synthesis, glutathione S-transferase (GST) activity, and multidrug resistance protein (MRP) transporter function attenuated arsenite resistance, consistent with studies suggesting that arsenite is extruded from the cell as a glutathione conjugate by MRP-1. However, MRP-1 was not overexpressed in resistant lines and GST-π was only slightly elevated. ICP-MS analysis indicated that arsenite-resistant SK-Mel-28 cells did not accumulate less arsenic than arsenite-sensitive A375 cells, suggesting that resistance was not attributable to reduced arsenic accumulation but rather to intrinsic properties of resistant cell lines. The mode of arsenite-induced cell death was apoptosis. Arsenite-induced apoptosis is associated with cell cycle alterations. Cell cycle analysis revealed arsenite-sensitive cells arrested in mitosis whereas arsenite-resistant cells did not, suggesting that induction of mitotic arrest occurs at lower intracellular arsenic concentrations. Higher intracellular arsenic levels induced cell cycle arrest in the S-phase and G 2-phase in SK-Mel-3 and SK-Mel-28 cells, respectively. The lack of arsenite-induced mitotic arrest in resistant cell lines was associated with a weakened spindle checkpoint resulting from reduced expression of spindle checkpoint protein BUBR1. These data suggest that arsenite has potential for treatment of solid tumors but a functional spindle checkpoint is a prerequisite for a positive response to its clinical application.

Spindle Checkpoint Insufficiency in Acute Myeloid Leukemia.

Chromosomal instability and aneuploidy are hallmarks of most human malignancies. Various mechanisms have been shown to give rise to numerical chromosome aberrations. Compromised function of the spindle assembly checkpoint (SAC) is generally regarded as one of the most powerful ways to drive genome instability. The SAC is a mitotic checkpoint mechanism ensuring the equal segregation of the mitotic chromosomes onto the developing daughter cells. Unfaithful mitotic surveillance by the SAC favors chromosomal misdistribution as error-prone chromosome attachment to the mitotic spindle does not induce a strong mitotic arrest by interference with anaphase promoting complex (APC)-dependent proteolysis. The APC is an important ubiquitin ligase that triggers the transition from mitosis into G1-phase by targeted proteolysis of mitotic regulators such as cyclin B and securin. The SAC prevents the proteolysis of those regulator proteins in the presence of mitotic aberrancies by inhibition of the APC. This leads to a delayed progression through mitosis and provides time to recover from defective chromosomal spindle attachment. SAC malfunction weakens the tight control on chromosome attachment and tension across the kinetochore favoring chromosomal misdistribution. We performed expression analyses of key proteins in SAC signaling in acute myeloid leukemia (AML). We found the SAC-components Bub1 and BubR1 to be down-regulated in most of the investigated AML cell lines. Functional assays revealed a defective mitotic arrest mechanism in comparison to SAC-competent cell lines after exposure to the microtubule disrupting agent nocodazole. This finding was accompanied by the observation of a decline in cyclin B and securin levels despite severe damage to the mitotic spindle induced by nocodazole. Expression of cyclin B and securin in the presence of spindle damage could be stabilized by proteasome inhibition. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found among AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Interestingly, BubR1 knockdown was sufficient to generate a phenotype resembling the behavior of our AML cell lines. Further experiments revealed a strong relation between premature degradation of cyclin B and the degree of BubR1 downregulation. Given the potent role of BubR1 in the generation of a mitotic arrest deficient phenotype, we addressed the BubR1 expression levels in a number of patients exhibiting karyotype abnormalities. Primary myeloid blast cells were stimulated with cytokines to force the largely resting cells into an actively dividing state. The maximum expression level of BubR1 in G2/M was used to define SAC-compentent and SAC-deficient populations. Strikingly, six out of eight (6/8) primary AML samples exhibited BubR1 expression patterns resembling the BubR1-knockdown model suggesting deficient mitotic surveillance in most of the primary AML samples. Since SAC deficiency is an important mechanism in creating numerical chromosomal aberrations and genetic instability, our findings underline a role for impaired SAC function in rise and progression of AML.

P53 Activation in Response to Mitotic Spindle Damage Requires Signaling via BubR1-Mediated Phosphorylation

The mitotic spindle checkpoint plays a crucial role in regulating accurate chromosome segregation and preventing the adaptation of multiploid progeny cells. Recent reports have indicated that the induction of p53 by mitotic checkpoint activation is essential for protecting cells from abnormal chromosome ploidization caused by mitotic failure. However, although studies have shown that p53 deficiencies arrest mitosis, compromise apoptosis, and may cause profound aneuploidy, the molecular mechanisms leading to p53 induction following mitotic checkpoint activation remain unknown. Here, we show that the BubR1 mitotic checkpoint kinase interacts with p53 both in vitro and in vivo, with higher levels of interaction in mitotic cells. This interaction contributes to p53 phosphorylation. Silencing of BubR1 expression reduces the phosphorylation and stability of p53, whereas exogenous introduction of BubR1 proteins into BubR1-depleted cells recovers p53 stability. In addition, inhibition of BubR1 expression in the presence of a microtubule inhibitor accelerates chromosomal instability and polyploidy in p53-null cells. These results collectively suggest that p53 activation in response to mitotic spindle damage requires signaling via BubR1-mediated phosphorylation.

Gene expression levels of ribonucleotide reductase subunit M1 (RRM1), tyrosyl-DNA phosphodiesterase (Tdp1), nuclear factor of activated T-cells (NFAT), and BubR1 mRNA expression in completely resected chemo-naive non-small cell lung cancer (NSCLC) patients

18016 Background: A relationship between gene transcripts and survival in operable NSCLC is now emerging. RRM1 is involved in DNA repair, Tdp1 is implicated in the repair of CPT-induced topoisomerase damage, and NFAT promotes cancer invasion. BubR1 is a key spindle checkpoint gene, and altered BubR1 mRNA levels are associated with lymph node metastasis and chromosome instability. Methods: In order to identify p with a high risk of relapse, we examined the expression of these four genes in frozen resected tumors from 126 resected NSCLC p by real-time quantitative PCR. Gene expression was normalized using β-actin expression as internal reference. Results: Adenocarcinoma (adeno), 33 p; squamous cell carcinoma (SCC), 93 p. Stage: IA, 18 p; IB, 53 p; IIB, 33 p; IIIA, 22 p. Tumor transcript expression: RRM1, 2.10; Tdp1, 1.77; NFAT, 0.56; BubR1, 16.40. Expression of RRM1, Tdp1 and BubR1 was higher in SCC than in adeno (P<0.001). Median time to relapse (TTR) was longer for p with low levels of RRM1 (P=0.11), Tdp1 (P=0.86), NFAT (P=0.29), or BubR1 (P=0.44) (Table). A significant trend towards longer survival was also observed in stage I p with low RRM1 P=0.06). In a multivariate Cox model, tumor size > 4 cm and stage III predicted shorter TTR and survival. Conclusion: Increased mRNA expression of these genes is associated with shorter TTR; this knowledge could be useful for customizing adjuvant chemotherapy. [Table: see text] No significant financial relationships to disclose.

Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.

Overexpression of BUBR1 is associated with chromosomal instability in bladder cancer

BUBR1, a mitotic checkpoint protein, is a key component of the mitotic spindle checkpoint machinery. Defective BUBR1 has been proposed to contribute to chromosomal instability (CIN). To elucidate the relationship of BUBR1 expression with CIN, expression of BUBR1, numbers of centrosomes, numerical aberrations of chromosomes, and DNA ploidy were examined, and BUBR1 expression status was compared with clinicopathological parameters in 104 human urothelial bladder carcinomas. Expression of BUBR1 and numbers of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9, and 17 were evaluated by fluorescence in situ hybridization. Cancers with a large intercellular variation in centromere copy number were designated as CIN cancers. Tumors with BUBR1 overexpression were associated with CIN, DNA aneuploidy, and centrosome amplification. Array CGH revealed that BUB1B amplification and loss rarely occurred, indicating that the overexpression of BUBR1 in these bladder cancers was independent of BUB1B copy number. Overexpression of BUBR1 significantly correlated with higher histological grade, advanced pathological stage, and high cell proliferation. Overexpression of BUBR1 predicted tumor recurrence and disease progression. These data suggest that overexpression of BUBR1 is potentially a new tumor marker for estimating biological characteristics of bladder cancer.

Weakened spindle checkpoint with reduced BubR1 expression in paclitaxel-resistant ovarian carcinoma cell line SKOV3-TR30

Paclitaxel sensitivity has recently been associated with the spindle checkpoint. The aim of our study is to investigate the status of spindle checkpoint and the alteration of its major components in phenotype with acquired paclitaxel resistance in ovarian carcinoma. A paclitaxel-resistant ovarian carcinoma cell line, SKOV3-TR30, with the resistant ability of 27-fold greater than its parental cell line, was derived from SKOV3 cell line. The competence of spindle checkpoint and the expression of the major spindle checkpoint proteins (BubR1 and Mad2) in SKOV3-TR30 cells were investigated. Mitotic index and flow cytometric analysis revealed that SKOV3-TR30 cells were not arrested in M phase in contrast to SKOV3, which showed a clear mitotic arrest in the presence of paclitaxel or nocodazole. The expressions of securin and cyclin B in SKOV3-TR30 cells were significantly lower than those in SKOV3 cells, indicating the premature degradation of APC substrates in SKOV3-TR30 cells in response to spindle damagers. Chromosome spread analysis showed increased rate of premature sister chromatid segregation in SKOV3-TR30 cells. The expression of spindle checkpoint protein BubR1 was evidently lower in SKOV3-TR30 cells than that in SKOV3 cells. However, there was no significant difference in Mad2 expression between SKOV3-TR30 and SKOV3 cells. This study demonstrates that weakened spindle checkpoint with reduced expression of BubR1, but not Mad2, is associated with acquired paclitaxel resistance in ovarian carcinoma cells.

BRCA1 Downregulation Leads to Premature Inactivation of Spindle Checkpoint and Confers Paclitaxel Resistance

BRCA1 germline mutations predispose women to early onset, familial breast and ovariancancer. BRCA1 has been recently implicated in the cellular response to agents that disruptthe mitotic spindle. In this report, we studied BRCA1 contribution to paclitaxel response inMCF-7 breast cancer cells. We show that MCF-7 cells transfected with BRCA1 siRNAdisplay a significant increase in resistance to paclitaxel compared with the control cells. Wenext demonstrate that down-regulation of BRCA1 reduces the mitotic index and triggerspremature cyclin B1 degradation and decrease in Cdk1 activity following paclitaxel treatment,suggesting that BRCA1 down-regulation results in precocious inactivation of the spindlecheckpoint. These findings were confirmed by showing that BRCA1 down-regulation inducespremature sister–chromatids separation in MCF-7 cells following spindle damage.Furthermore, we show that BRCA1 up-regulates the expression of the protein kinase BubR1,essential component of the functional spindle checkpoint, whose down-regulation is known toresult in paclitaxel resistance in MCF-7 cells. Altogether, our findings support the notion thatdown-regulation of BRCA1 expression mediates paclitaxel resistance through prematureinactivation of spindle checkpoint in MCF-7 breast cancer cells. They link BRCA1 to themitotic checkpoint that plays an essential role in the maintenance of chromosomal stability.

Monoallelic BUB1B mutations and defective mitotic‐spindle checkpoint in seven families with premature chromatid separation (PCS) syndrome

Cancer-prone syndrome of premature chromatid separation (PCS syndrome) with mosaic variegated aneuploidy (MVA) is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, childhood cancer, premature chromatid separation of all chromosomes, and mosaicism for various trisomies and monosomies. Biallelic BUB1B mutations were recently reported in five of eight families with MVA syndrome (probably identical to the PCS syndrome). We here describe molecular analysis of BUB1B (encoding BubR1) in seven Japanese families with the PCS syndrome. Monoallelic BUB1B mutations were found in all seven families studied: a single-base deletion (1833delT) in four families; and a splice site mutation, a nonsense mutation, and a missense mutation in one family each. Transcripts derived from the patients with the 1833delT mutation and the splice site mutation were significantly reduced, probably due to nonsense-mediated mRNA decay. No mutation was found in the second alleles in the seven families studied, but RT-PCR of BUB1B and Western blot analysis of BubR1 indicated a modest decrease of their transcripts. BubR1 in the cells from two patients showed both reduced protein expression and diminished kinetochore localization. Their expression level of p55cdc, a specific activator of anaphase-promoting complex, was normal but its kinetochore association was abolished. Microcell-mediated transfer of chromosome 15 (containing BUB1B) into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal responses to colcemid treatment. These findings indicate the involvement of BubR1 in p55cdc-mediated mitotic checkpoint signaling, and suggest that >50% decrease in expression (or activity) of BubR1 is involved in the PCS syndrome.

Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons

Mitotic spindle poisons (e.g., Taxol and vinblastine), used as chemotherapy drugs, inhibit mitotic spindle function, activate the mitotic spindle checkpoint, arrest cells in mitosis, and then cause cell death by mechanisms that are poorly understood. By expression cloning, we identified a truncated version of human TRIP1 (also known as S8, hSug1), an AAA (ATPases associated with diverse cellular activities) family ATPase subunit of the 19S proteasome regulatory complex, as an enhancer of spindle poison-mediated apoptosis. Stable expression of the truncated TRIP1/S8/hSug1 in HeLa cells [OP-TRIP1(88-406)] resulted in a decrease of measurable cellular proteasome activity, indicating that OP-TRIP1(88-406) had a dominant-negative effect on proteasome function. OP-TRIP1(88-406) revealed an increased apoptotic response after treatment with spindle poisons or with proteasome inhibitors. The increased apoptosis coincided with a significant decrease in expression of BubR1, a kinase required for activation and maintenance of the mitotic spindle checkpoint in response to treatment with spindle poisons. Small interfering RNA (siRNA)-mediated knockdown of TRIP1/S8/hSug1 resulted in a reduction of general proteasome activity and an increase in mitotic index. The siRNA treatment also caused increased cell death after spindle poison treatment. These results indicate that inhibition of TRIP1/S8/hSug1 function by expression of a truncated version of the protein or by siRNA-mediated suppression enhances cell death in response to spindle poison treatment. Current proteasome inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results suggest that targeting the ATPase subunits in 19S regulatory complex in the proteasome may enhance the antitumor effects of spindle poisons.

Caspase-Mediated Specific Cleavage of BubR1 Is a Determinant of Mitotic Progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.

Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest inDrosophila melanogaster

The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. We show that the conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions similarly to somatic cells. To determine BubR1 function during embryogenesis, we generated a new hypomorphic semi-viable female sterile allele. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, we find that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. Our results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis.

Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis

The polo-like kinase Plk4 (also called Sak) is required for late mitotic progression, cell survival and postgastrulation embryonic development. Here we identified a phenotype resulting from Plk4 haploinsufficiency in Plk4 heterozygous cells and mice. Plk4+/- embryonic fibroblasts had increased centrosomal amplification, multipolar spindle formation and aneuploidy compared with wild-type cells. The incidence of spontaneous liver and lung cancers was approximately 15 times high in elderly Plk4+/- mice than in Plk4+/+ littermates. Using the in vivo model of partial hepatectomy to induce synchronous cell cycle entry, we determined that the precise regulation of cyclins D1, E and B1 and of Cdk1 was impaired in Plk4+/- regenerating liver, and p53 activation and p21 and BubR1 expression were suppressed. These defects were associated with progressive cell cycle delays, increased spindle irregularities and accelerated hepatocellular carcinogenesis in Plk4+/- mice. Loss of heterozygosity occurs frequently (approximately 60%) at polymorphic markers adjacent to the PLK4 locus in human hepatoma. Reduced Plk4 gene dosage increases the probability of mitotic errors and cancer development.

Transcriptional Control of BubR1 by p53 and Suppression of Centrosome Amplification by BubR1

Elimination of the regulatory mechanism underlying numeral homeostasis of centrosomes, as seen in cells lacking p53, results in abnormal amplification of centrosomes, which increases the frequency of chromosome segregation errors, and thus contributes to the chromosome instability frequently observed in cancer cells. We have previously reported that p53(-/-) mouse cells in prolonged culture undergo genomic convergence similar to that observed during tumor progression; early-passage p53(-/-) cells are karyotypically heterogeneous due to extensive chromosome instability associated with centrosome amplification, while late-passage p53(-/-) cells are aneuploid yet karyotypically homogeneous and chromosomally stable. Moreover, they contain numerically normal centrosomes. Through the microarray analysis of early- and late-passage p53(-/-) cells, we identified the BubR1 spindle checkpoint protein, which plays a critical role in suppression of centrosome amplification and stabilization of chromosomes in late-passage p53(-/-) cells. Up-regulation of BubR1 augments the checkpoint function, which effectively senses the spindle/chromosome aberrations associated with centrosome amplification. We further found that BubR1 transcription is largely controlled by p53. In early-passage p53(-/-) cells, BubR1 expression is low and the checkpoint function in response to microtubule toxin is considerably compromised. In late-passage cells, however, regaining of BubR1 expression restores the checkpoint function to mitotic aberrations caused by microtubule toxin. Our studies demonstrate the molecular aspect of genomic convergence in cultured cells, providing critical information for understanding the stepwise progression of tumors.

BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice.

Faithful segregation of replicated chromosomes is essential for maintenance of genetic stability and seems to be monitored by several mitotic checkpoints. Various components of these checkpoints have been identified in mammals, but their physiological relevance is largely unknown. Here we show that mutant mice with low levels of the spindle assembly checkpoint protein BubR1 develop progressive aneuploidy along with a variety of progeroid features, including short lifespan, cachectic dwarfism, lordokyphosis, cataracts, loss of subcutaneous fat and impaired wound healing. Graded reduction of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence. Male and female mutant mice have defects in meiotic chromosome segregation and are infertile. Natural aging of wild-type mice is marked by decreased expression of BubR1 in multiple tissues, including testis and ovary. These results suggest a role for BubR1 in regulating aging and infertility.

The promoter region of the human BUBR1 gene and its expression analysis in lung cancer

Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers.

P55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase.

Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. We have been studying genes that regulated the spindle checkpoint in human cells. Enforced expression of human BUBR1, but not a BUBR1 mutant allele, enhances accumulation of mitotic cells. Yeast two-hybrid system and GST-pulldown analyses show that p55CDC/hCdc20, a protein known to link spindle checkpoint components such as MAD2 to anaphase promoting complex (APC), interacts with BUBR1. In addition, p55CDC is capable of pulling down BUBR1 in sf-9 cells infected with both p55CDC and His6-BUBR1 recombinant baculoviruses but not in the cells infected with p55CDC baculoviruses or with the baculoviral vector alone. Moreover, immunoprecipitation followed by Western blot analyses confirmed that native p55CDC is associated with BUBR1 in HeLa cells. Spindle checkpoint activation by nocodazole treatment enhances the association between p55CDC and His6-BUBR1. In nocodazole-arrested mitotic cells, both CDC16 and hyperphosphorylated CDC27, two APC components, preferentially associate with His6-BUBR1 resins, but not the control resins. Furthermore, BUBR1 phosphorylates p55CDC in vitro, and the phosphorylation of p55CDC by BUBR1 appears to be correlated with spindle checkpoint activation. Together, our studies strongly suggest that BUBR1 may target APC via p55CDC.