Mitotic Checkpoint Protein Research Articles

The basal body gene, RPGRIP1L, is a candidate tumour suppressor gene in human hepatocellular carcinoma

Loss of heterozygosity (LOH) on chromosome 16q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC). Our previous data showed that the smallest common deleted region was between D16S415 and D16S419, encompassed approximately by a 0.75 cM region on 16q12.2, suggesting that the putative tumour suppressor genes (TSGs) at this locus might be involved in the development of HCC. Of the four genes ( CHD9, RBL2, AKTIP and RPGRIP1L) located in this region, only RPGRIP1L was downregulated in HCCs. Downregulation of RPGRIP1L was found in 91% (10/11) HCC cell lines and in 35% (14/40) HCCs, respectively. To investigate the role of RPGRIP1L in HCCs, we used the overexpression of RPGRIP1L in four HCC cell lines (HepG2, Huh6, Huh7 and Hep3B). Overexpression of RPGRIP1L suppressed colony formation of tumour cells. Conversely, expression of RPGRIP1L M (dominant negative form) in HCC cells enhanced colony formation. Furthermore, knockdown RPGRIP1L by RNA interference in SK-HepI cells promoted colony formation. Taken together, these data strongly suggest that RPGRIP1L might be the putative TSG in HCC. Moreover, we showed that Mad2, Survivin and Securin were elevated in RPGRIP1L M- HepG2 transfectants and RPGRIP1L-shRNA-SK-HepI transfectants. After knockdown of MAD2 in RPGRIP1L-shRNA-SK-HepI transfectants partly reverse cellular colony formation capability. These data suggest that RPGRIP1L suppresses anchorage-independent growth partly through the mitotic checkpoint protein Mad2.

SIRT2 downregulation confers resistance to microtubule inhibitors by prolonging chronic mitotic arrest

We previously identified SIRT2, a deacetylase for tubulin and histone H4, as a protein down-regulated in gliomas, and reported that exogenously-expressed SIRT2 arrests the cell cycle prior to entry into mitosis to prevent chromosomal instability in response to microtubule inhibitors (MTIs) such as nocodazole, characteristics previously reported for the CHFR protein. We herein investigated the effects of SIRT2 downregulation on sensitivity to MTIs using HCT116 cells, a mitotic checkpoint-proficient near-diploid cancer cell line used for studying checkpoints. We found that SIRT2 downregulation confers resistance to MTIs as well as that of BubR1, a well-characterized mitotic checkpoint protein, though by a different mechanism. While BubR1 suppression abolished spindle checkpoint functions, which is a requirement for cell death after release from the spindle checkpoint, SIRT2 downregulation prolonged chronic mitotic arrest from sustained activation of the mitotic checkpoint and consequently prevented a shift to secondary outcomes, including cell death, after release from chronic mitotic arrest. Consistent with this notion, BubR1 downregulation was dominant over SIRT2 knockdown in regard to mitotic regulation in the presence of nocodazole. These results suggest that SIRT2 functions to release chronic mitotic arrest in cells treated with MTIs, leading to other outcomes. We also found that SIRT2 downregulation caused centrosome fragmentation in response to nocodazole prior to the alteration in spindle checkpoint function, implying not only a novel function of SIRT2 for centrosome maintenance upon exposure to mitotic stress caused by MTIs, but also the existence of a centrosome-mediated signaling pathway to sustain the spindle checkpoint. Therefore, this study highlights a novel pathway leading to resistance to MTIs, in which SIRT2 downregulation participates.

Retracted: Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV‐1‐infected T cells through enhanced NF‐κB activity

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-kappaB (NF-kappaB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-kappaB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-kappaB activity in an HTLV-1-infected T-cell line by reducing IkappaB kinase beta phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-kappaB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

Oncogenic Adenomatous Polyposis Coli Mutants Impair the Mitotic Checkpoint through Direct Interaction with Mad2

The majority of colorectal tumors are aneuploid because of the underlying chromosome instability (CIN) phenotype, in which a defective mitotic checkpoint is implicated. Adenomatous polyposis coli (APC), a tumor suppressor gene that is commonly mutated in colon cancers, has been suggested in causing CIN; however, the molecular mechanism remains unresolved. In this study, we report an interaction of tumor-associated N-terminal APC fragments (N-APC) with Mad2, an essential mitotic checkpoint protein, providing a direct molecular support for linking APC mutations to the generation of CIN. N-APC interacts with Mad2 in Xenopus egg extracts, colon cancer cells, and in vitro with purified components. The interaction between N-APC and Mad2 decreases the soluble pool of Mad2, which is essential for Mad2 cycling and releasing from unattached kinetochores to produce a diffusible |P;wait anaphase|P' signal. Addition of such an N-APC mutant of egg extracts inactivates the mitotic checkpoint. Expressing a tumor-associated N-APC mutant in mammalian cells with an intact mitotic checkpoint produces premature anaphase onset with missegregated chromosomes.

Assessment of the Mitotic Spindle Assembly Checkpoint (SAC) as the Target of Anticancer Therapies

The mitotic spindle assembly checkpoint (SAC) is an essential control system of the eukaryotic cell cycle. This surveillance mechanism monitors the kinetochore, the multi-component complex that assembles on the centromeric DNA and attaches chromosomes to the microtubules of the spindle. The recruitment of mitotic checkpoint proteins to kinetochores that are not correctly attached to microtubules initiates a signalling cascade that results in the CDC20-dependent inhibition of the anaphase-promoting complex/cyclosome (APC/C). Mutations in the genes encoding for diverse SAC proteins have been identified in human tumour cells and associated with chromosome segregation and cancer progression. This work describes the current understanding on the organisation, function and structure of SAC components and shows this knowledge assists the identification of those that may constitute suitable targets for the clinical treatment of cancer.

Unattached Kinetochores Catalyze Production of an Anaphase Inhibitor that Requires a Mad2 Template to Prime Cdc20 for BubR1 Binding

Premature anaphase onset is prevented by the mitotic checkpoint through production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.

APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation

The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC also negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/CCdc20 in response to the SAC.

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4',5-trimethoxy-3'-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.

BubR1 N Terminus Acts as a Soluble Inhibitor of Cyclin B Degradation by APC/CCdc20 in Interphase

BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, we demonstrate that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. We find that the BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, our results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates.

Functional Interaction between BubR1 and Securin in an Anaphase-Promoting Complex/CyclosomeCdc20–Independent Manner

Activation of the mitotic checkpoint requires the precise timing and spatial organization of mitotic regulatory events, and ensures accurate chromosome segregation. Mitotic checkpoint proteins such as BubR1 and Mad2 bind to Cdc20, and inhibit anaphase-promoting complex/cyclosome(Cdc20)-mediated securin degradation and the onset of anaphase. BubR1 mediates the proper attachment of microtubules to kinetochores, and links the regulation of chromosome-spindle attachment to mitotic checkpoint signaling. Therefore, disruption of BubR1 activity results in a loss of the checkpoint control, chromosome instability, and/or early onset of malignancy. In this study, we show that BubR1 directly interacts with securin in vitro and in vivo. In addition, the BubR1 interaction contributes to the stability of securin, and there is a significant positive correlation between BubR1 and securin expressions in human cancer. Importantly, BubR1 competes with Cdc20 for binding to securin, and thereby the interaction between BubR1 and securin is greatly increased by the depletion of Cdc20. Our findings may identify a novel regulation of BubR1 that can generate an additional anaphase-inhibitory signal through the Cdc20-independent interaction of BubR1 with securin.

Regulation of Kinetochore Recruitment of Two Essential Mitotic Spindle Checkpoint Proteins by Mps1 Phosphorylation

Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.

Generation and Characterization of Novel Monoclonal Antibodies Against Human Aurora-A

The mitotic kinase Aurora-A is essential for mitotic progression, including centrosome maturation, mitotic spindle formation, and faithful segregation of chromosomes to daughter cells. Several lines of evidences also suggest that the mammalian aurora kinase family proteins play a role in tumorigenesis. We have previously shown that human Aurora-A was ubiquitinated and negatively regulated by an early mitotic checkpoint protein, Chfr (checkpoint protein with FHA and RING domain). Here, we established several mouse anti-Aurora-A monoclonal antibodies (MAb). GST-tagged human Aurora-A was expressed in BL21 and used as an antigen to immunize mice. Three different hybridomas were obtained and antibodies produced by these hybridomas were analyzed. The results reveal that these antibodies specifically recognize endogenous Aurora-A in both immunoblotting and immunofluroscence experiments. They are useful tools for further analysis of human Aurora-A.

Evolutionary analysis of WD40 super family proteins involved in spindle checkpoint and RNA export: molecular evolution of spindle checkpoint.

The spindle checkpoint delays sister chromatid separation until all chromosomes have undergone bipolar spindle attachment. Previous studies have revealed BUB3, as an essential spindle checkpoint protein and its extensive sequence similarity with Rae1 (Gle2), a highly conserved member of WD40 repeat protein family throughout their length which was first shown to be involved in mRNA export. However, the recent discovery of Rae1 as an essential mitotic checkpoint protein, based on the studies from mouse and drosophila, has renewed the interest in its function during cell division. Study of evolution of proteins involved in checkpoint might throw light on evolution of eukaryotic cell cycle regulation. Here we report the evolutionary relationships between these two WD40 repeat family proteins. Amino acid sequences of BUB3 and Rae1 homologs were retrieved from various databases and phylogenetic analysis was performed with the MEGA program. Multiple sequence alignments of these two protein homologues with the ClustalX software revealed specific amino acid signatures corresponding to the protein function and also few amino acids, which are conserved in BUB3 and Rae1 indicating some common overlapping function. Data indicated a common ancestral origin of these two important proteins and further suggest that, BUB3 mediated cell cycle checkpoint might have evolved with compartmentalization of genetic material into the nucleus in eukaryotes.

CHFR: A Novel Mitotic Checkpoint Protein and Regulator of Tumorigenesis

Checkpoint with FHA and RING finger domains (CHFR) was first recognized as an early mitotic checkpoint protein that delayed the cell cycle in response to microtubule-targeting drugs. It is an E3 ubiquitin ligase that ubiquitinates target proteins to direct them to the proteasome for degradation or to alter their activity. To date, however, the downstream target proteins critical to CHFR's normal cellular functions largely remain unidentified with the exception of the key mitosis regulators, and oncogenes, PLK1 and Aurora A kinases. Rapidly growing evidence in mice, primary human tumors, and mammalian cell culture models indicate that CHFR may also function as a potent tumor suppressor. Interestingly, studies reported to date suggest that CHFR both controls a novel prophase checkpoint early in mitosis and regulates chromosome segregation later in mitosis to maintain genomic stability. In addition, loss of CHFR sensitizes cancer cells to microtubule poisons, altering chemoresponsiveness to taxanes and making it a potential biomarker for chemotherapeutic response. Importantly, CHFR may be one of the few proteins that are required for regulating the cell cycle and maintaining genomic instability to inhibit tumorigenesis.

Loss of CHFR in Human Mammary Epithelial Cells Causes Genomic Instability by Disrupting the Mitotic Spindle Assembly Checkpoint

CHFR is an E3 ubiquitin ligase and an early mitotic checkpoint protein implicated in many cancers and in the maintenance of genomic stability. To analyze the role of CHFR in genomic stability, by siRNA, we decreased its expression in genomically stable MCF10A cells. Lowered CHFR expression quickly led to increased aneuploidy due to many mitotic defects. First, we confirmed that CHFR interacts with the mitotic kinase Aurora A to regulate its expression. Furthermore, we found that decreased CHFR led to disorganized multipolar mitotic spindles. This was supported by the finding that CHFR interacts with α-tubulin and can regulate its ubiquitination in response to nocodazole and the amount of acetylated α-tubulin, a component of the mitotic spindle. Finally, we found a novel CHFR interacting protein, the spindle checkpoint protein MAD2. Decreased CHFR expression resulted in the mislocalization of both MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation. Further evidence of a compromised spindle checkpoint was the presence of misaligned metaphase chromosomes, lagging anaphase chromosomes, and defective cytokinesis in CHFR knockdown cells. Importantly, our results suggest a novel role for CHFR regulating chromosome segregation where decreased expression, as seen in cancer cells, contributes to genomic instability by impairing the spindle assembly checkpoint.

Diphosphothreonine-Specific Interaction between an SQ/TQ Cluster and an FHA Domain in the Rad53-Dun1 Kinase Cascade

Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling.

Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

To improve cancer chemotherapy, we need to understand the mechanisms that determine drug sensitivity in cancer and normal cells. Here, we investigate this question across a panel of 11 cell lines at a phenotypic and molecular level for three antimitotic drugs: paclitaxel, nocodazole, and an inhibitor of kinesin-5 (also known as KSP, Eg5, Kif11). Using automated microscopy with markers for mitosis and apoptosis (high content screening), we find that the mitotic arrest response shows relatively little variation between cell types, whereas the tendency to undergo apoptosis shows large variation. We found no correlation between levels of mitotic arrest and apoptosis. Apoptosis depended on entry into mitosis and occurred both from within mitosis and after exit. Response to the three drugs strongly correlated, although paclitaxel caused more apoptosis in some cell lines at similar levels of mitotic arrest. Molecular investigations showed that sensitivity to apoptosis correlated with loss of an antiapoptotic protein, XIAP, during the drug response, but not its preresponse levels, and to some extent also correlated with activation of the p38 and c-Jun NH(2) kinase pathways. We conclude that variation in sensitivity to antimitotic drugs in drug-naive cell lines is governed more by differences in apoptotic signaling than by differences in mitotic spindle or spindle assembly checkpoint proteins and that antimitotics with different mechanisms trigger very similar, but not identical, responses.

TRAIL Inactivates the Mitotic Checkpoint and Potentiates Death Induced by Microtubule-Targeting Agents in Human Cancer Cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted interest as an anticancer treatment, when used in conjunction with standard chemotherapy. We investigated the mechanistic basis for combining low-dose TRAIL with microtubule-targeting agents that invoke the mitotic checkpoint. Treatment of T98G and HCT116 cells with nocodazole alone resulted in a robust mitotic block with initially little cell death; low levels of cell death were also seen with TRAIL alone at 10 ng/mL final concentration. In contrast, the addition of low-dose TRAIL to nocodazole was associated with maximally increased caspase-3, caspase-8, and caspase-9 activation, which efficiently abrogated the mitotic delay and markedly increased cell death. In contrast, the abrogation of mitotic checkpoint and increased cell death were blocked by inhibitors of caspase-8 and caspase-9 or pan-caspase inhibitor. The addition of TRAIL to either nocodazole or paclitaxel (Taxol) reduced levels of the mitotic checkpoint proteins BubR1 and Bub1. BubR1 mutated for the caspase cleavage sites, but not wild-type BubR1, was resistant to cleavage induced by TRAIL added to nocodazole, and partially blocked the checkpoint abrogation. These results suggest that adding a relatively low concentration of TRAIL to antimicrotubule agents markedly increases complete caspase activation. This in turn accentuates degradation of spindle checkpoint proteins such as BubR1 and Bub1, contributes to abrogation of the mitotic checkpoint, and induces cancer cell death. These results suggest that TRAIL may increase the anticancer efficacy of microtubule-targeting drugs.

Mps1 Phosphorylates Borealin to Control Aurora B Activity and Chromosome Alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Mitotic checkpoint defects in human cancers and their implications to chemotherapy

The mitotic checkpoint, also known as spindle assembly checkpoint, is to ensure accurate chromosome segregation by inducing mitotic arrest when errors occur in the spindle structure or in the alignment of the chromosomes on the spindle. Loss of mitotic checkpoint control is a common event in human cancer cells, which is thought to be responsible for chromosome instability frequently observed in cancer cells. Several reports have shown that cells with a defective mitotic checkpoint are more resistant to several types of anticancer drugs from microtubule disruptors to DNA damaging agents. In addition, inactivation of key mitotic checkpoint proteins such as BUB (budding uninhibited by benzimidazole) and MAD (mitotic arrest deficient ) is influential in drug resistance in mitotic checkpoint defective cancer cells. The mitotic checkpoint has also been linked to DNA damage response and a defective mitotic checkpoint confers cancer cells resistance to certain DNA damaging anticancer drugs. This review presents recent evidence on mitotic checkpoint defects in human cancers and their association with resistance to anticancer drugs. In addition, the clinical importance and potential therapeutic implications of targeting the mitotic checkpoint to reverse drug resistance in cancer cells are also discussed.