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Mitotic checkpoints: From yeast to cancer
Abstract
Separation of chromosomes during mitosis is monitored by a checkpoint that leads to cell-cycle arrest if the chromosomes are not properly attached to the mitotic spindle. Molecular mechanisms controlling this checkpoint have been identified. In addition, loss of this checkpoint has been shown to result in chromosome missegregation in higher eukaryotes and may contribute to the genomic instability observed in human cancers.
... The mitotic spindle checkpoint is a highly conserved mechanism that ensures that sister chromatids are aligned at the metaphase plate and do not separate prior to the bipolar attachment of all duplicated chro-mosomes, providing equally divided sister chromatids during cell division [14,17,18,34]. ...
... Yeast cells defective in the mitotic checkpoint mechanism lose chromosomes at elevated rates and are hypersensitive to mitotic-spindle inhibitors [33,34]. In humans, although mutations in known spindle checkpoint genes are extremely rare [1,2,[11][12][13]20,23,24,35,36,38], several studies have shown evidence that mutations and/or reduced levels of mitotic checkpoint proteins can cause checkpoint malfunction and chromosomal instability (CIN), and thereby contribute to tumor formation [10,16,21,33]. ...
Background : A defective mitotic checkpoint has been proposed to contribute to chromosomal instability (CIN). We have previously shown that expression changes of the mitotic arrest deficiency (MAD) gene family plays a role in renal cell cancer (RCC) characterized by numerical chromosomal changes, namely papillary and chromophobe carcinomas, but nothing is known about the expression of mitotic checkpoint genes in the clear cell histotype (ccRCC).
Methods : We analyzed the mRNA expression levels of the major mitotic checkpoint genes of the budding uninhibited by benzimidazole family ( BUB1 , BUBR1 , BUB3 ) and of the MAD gene family ( MAD1 , MAD2L1 , MAD2L2 ) by real-time quantitative PCR in 39 ccRCC and in 36 normal kidney tissue samples.We have additionally analyzed these tumors by comparative genomic hybridization (CGH) in order to evaluate the relationship between mitotic checkpoint defects and the pattern of chromosome changes in this subset of RCC.
Results : BUB1 , BUBR1 , MAD1 and MAD2L1 showed significant expression differences in tumor tissue compared to controls ( BUB1 , BUBR1 and MAD2L1 were overexpressed, whereas MAD1 was underexpressed). Overexpression of BUB1 and BUBR1 was significantly correlated with the number of genomic copy number changes ( p < 0.001 for both genes) and with Furhman grade of the tumors ( p = 0.006 and p = 0.005, respectively).
Conclusions : We conclude that BUB1 and BUBR1 overexpression plays a role in cytogenetic and morphologic progression of ccRCC.
... However, compromised spindle checkpoint mechanism may result in faulty separation of sister chromatids even in the presence of misaligned chromosomes that could be a cause for chromosomal instability (CIN) and hence result in gain or loss of chromosomes called as aneuploidy, a striking feature in human cancer [14]. Significantly, many tumors are known with weakened spindle checkpoint function, thus lack of sustenance of signal for repair of errors [15,16]. Hence, an impaired spindle checkpoint may directly lead to chromosomal instability and tumorigenesis in human cancer [15]. ...
... Significantly, many tumors are known with weakened spindle checkpoint function, thus lack of sustenance of signal for repair of errors [15,16]. Hence, an impaired spindle checkpoint may directly lead to chromosomal instability and tumorigenesis in human cancer [15]. ...
... In a subset of human cancers with chromosomal instability, mutations have been identified in genes that encode mitotic checkpoint proteins, such as Bub1, BubR1, and Mad2 (Cahill et al., 1998; Imai et al., 1999; Gemma et al., 2001). The mitotic checkpoint is a partly resolved intricate molecular network that helps to ensure faithful segregation of duplicated chromosomes during mitosis (Burke, 2000; Shah and Cleveland, 2000; Kitagawa and Hieter, 2001; Nigg, 2001; Wassmann and Benezra, 2001). Kinetochores of chromosomes that are not yet aligned at the metaphase plate and attached to microtubules activate the mitotic checkpoint, leading to inhibition of the anaphasepromoting complex (APC)* (Morgan, 1999; Peters, 2002). ...
... Because Rae1 has a high degree of sequence similarity to Bub3 and can interact with Bub1 (Taylor et al., 1998; Martinez-Exposito et al., 1999; Wang et al., 2001 ), it was of interest to determine whether Rae1 would be required in mitosis . Others have recently shown that HCT116 cells with only one copy of the mitotic checkpoint gene Mad2 fail to arrest in prometaphase and exit mitosis without cytokinesis when cultured in the presence of the microtubule-depolymerizing drug nocodazole (Michel et al., 2001 ), a response that is typical for cells with a defective mitotic checkpoint (Wassmann and Benezra, 2001 ). This information prompted us to (D) In vitro growth of representative Rae1 / and Rae1 / embryos. ...
The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.
... Thus, recent efforts have started to link sensitivity of tumor cells to taxane treatment with genetic defects in the cell cycle checkpoints in association with cancer chemotherapy. It has been suggested that inactivation of mitotic checkpoint proteins can contribute to the selective response of taxane treatment in vivo (Wassmann and Benezra, 2001). However, mutations in known checkpoint proteins occur rather rarely (Cahill et al., 1998;Haruki et al., 2001); thus broader studies are necessary to search for novel molecular targets of taxane therapy. ...
... Several molecular targets were reported, starting with mutations in α-and β-tubulin that affect drug binding, increased expression of tubulin genes, and changes in the synthesis or activity of tubulin interacting proteins (Hari et al., 2003a;Hari et al., 2003b;Wang and Cabral, 2005). Recently, a new c of potential targets are being studied after it was suggested that inactivation of mitotic proteins can contribute to the selective response of taxane treatment in vivo (Wassmann and Benezra, 2001). Thus, development of new genomic prognosis factors and in-depth understanding of dr activity on both a cellular and organismal level are needed for optimization of adjuvant therapy and proper patient stratification. ...
... CHFR (checkpoint with FHA and RING nger) is an early mitotic checkpoint gene locating at chromosome 12q24.33 [2], which delays the entry into metaphase in response to mitotic stress induced by microtubule inhibitors such as paclitaxel [3] and prevents errors in chromosome segregation [4]. Cells that do not express CHFR could bypass mitotic arrest and might be sensitive to microtubule inhibitors, with subsequent cell death resulting from impaired checkpoint function. ...
Purpose: CHFR (checkpoint with FHA and RING finger) methylation is a promising biomarker of treatment response and cancer prognosis, whereas the predictive role of it remains controversial. Thus, this meta-analysis aimed to quantify the predictive impact of this biomarker in cancer patients.
Methods: PubMed, Embase, Cochrane Library and ClinicalTrials.gov. were searched to identify studies that assessed the association between CHFR methylation and clinical outcomes in patients with cancer. Treatment response was the primary endpoint, and overall survival (OS) and recurrence were the secondary endpoints.
Results: Thirteen studies with 2185 cancer patients met the inclusion criteria were included. Our study showed that CHFR methylation was not associated with the chemotherapy response (odds ratio = 0.93, 95% confidence interval [CI]: 0.38–2.31, P =0.88). As for OS, we found no statistically significant association either (hazard ratio [HR] = 1.14; 95% CI: 0.86–1.50, P = 0.36), except in patients treated with surgery alone (HR = 1.37; 95% CI: 1.06-1.77, P = 0.02). Moreover, CHFR methylation was significantly associated with recurrence (HR = 2.01; 95% CI: 1.25–3.25, P=0.004).
Conclusion: Our study indicates that CHFR methylation cannot be a predictive factor for response to chemotherapy, but might be a biomarker of OS and recurrence in cancer patient. Future studies should be conducted to reduce confounding and explore the mechanism.
... From prophase to pro-metaphase to metaphase, the microtubules bind to the kinetochores of the chromosomes. [58] As the cells enter anaphase, the sister chromatids are pulled apart, leading to equal distribution of genetic material under normal conditions. The checkpoint proteins involved during mitosis are the spindle assembly checkpoint (SAC) and the anaphase-promoting complex (APC). ...
Hormones are essential components in the body and their imbalance leads to pathological consequences. T2DM, insulin resistance and obesity are the most commonly occurring lifestyle diseases in the past decade. Also, an increased cancer incidence has been strongly associated with obese and T2DM patients. Therefore, our aim was to study the influence of high insulin levels in accumulating DNA damage in in vitro models and patients, through the induction of oxidative stress. The primary goal of this study was to analyze the genotoxicity induced by the combined action of two endogenous hormones (insulin and adrenaline) with in vitro models, through the induction of micronuclei and to see if they cause an additive increase in genomic damage. This is important for multifactorial diseases having high levels of more than one hormone, such as metabolic syndrome and conditions with multiple pathologies (e.g., T2DM along with high stress levels). Furthermore, the combination of insulin and the pharmacological inhibition of the tumor suppressor gene: PTEN, was to be tested in in vitro models for their genotoxic effect and oxidative stress inducing potential. As the tumor suppressor gene: PTEN is downregulated in PTEN associated syndromes and when presented along with T2DM and insulin resistance, this may increase the potential to accumulate genomic damage. The consequences of insulin action were to be further elucidated by following GFP-expressing cells in live cell-imaging to observe the ability of insulin, to induce micronuclei and replicative stress. Finally, the detrimental potential of high insulin levels in obese patients with hyperinsulinemia and pre-diabetes was to be studied by analyzing markers of oxidative stress and genomic damage. In summary, the intention of this work was to understand the effects of high insulin levels in in vitro and in patients to understand its relevance for the development of genomic instability and thus an elevated cancer risk.
... When these genes are abnormally expressed, they will cause chromosome mismatch and other genetic problems during mitosis (Ooi et al., 2012). What is worse is that the unstable gene expression will eventually lead to cancer (Wassmann and Benezra, 2001;Weaver and Cleveland, 2005). Clinical studies had shown that DLGAP5 was related to the invasion and migration of CRC (Branchi et al., 2019); besides, DLGAP5 expression was also related to overall survival and lymph node metastasis but had no correlation with disease-free survival. ...
Colorectal cancer (CRC) is one of the most common neoplastic diseases worldwide. With a high recurrence rate among all cancers, treatment of CRC only improved a little over the last two decades. The mortality and morbidity rates can be significantly lessened by earlier diagnosis and prompt treatment. Available biomarkers are not sensitive enough for the diagnosis of CRC, whereas the standard diagnostic method, endoscopy, is an invasive test and expensive. Hence, seeking the diagnostic and prognostic biomarkers of CRC is urgent and challenging. With that order, we screened the overlapped differentially expressed genes (DEGs) of GEO (GSE110223, GSE110224, GSE113513) and TCGA datasets. Subsequent protein–protein interaction network analysis recognized the hub genes among these DEGs. Further functional analyses including Gene Ontology and KEGG pathway analysis and gene set enrichment analysis were processed to investigate the role of these genes and potential underlying mechanisms in CRC. Kaplan–Meier analysis and Cox hazard ratio analysis were carried out to clarify the diagnostic and prognostic role of these genes. In conclusion, our present study demonstrated that CCNA2, MAD2L1, DLGAP5, AURKA, and RRM2 are all potential diagnostic biomarkers for CRC and may also be potential treatment targets for clinical implication in the future.
... BubR1 is a spindle assembly checkpoint kinase, encoded by Bub1b, that is critical for the accurate separation of duplicated chromosomes during cell division and maintaining genomic stability (Wassmann and Benezra, 2001;Cleveland et al., 2003). Reduced BubR1 expression induces aneuploidy, which affects genomic stability (Baker et al., 2004). ...
The adult central nervous system (CNS) contains resident stem cells within specific niches that maintain a self-renewal and proliferative capacity to generate new neurons, astrocytes, and oligodendrocytes throughout adulthood. Physiological aging is associated with a progressive loss of function and a decline in the self-renewal and regenerative capacities of CNS stem cells. Also, the biggest risk factor for neurodegenerative diseases is age, and current in vivo and in vitro models of neurodegenerative diseases rarely consider this. Therefore, combining both aging research and appropriate interrogation of animal disease models towards the understanding of the disease and age-related stem cell failure is imperative to the discovery of new therapies. This review article will highlight the main intrinsic and extrinsic regulators of neural stem cell (NSC) aging and discuss how these factors impact normal homeostatic functions within the adult brain. We will consider established in vivo animal and in vitro human disease model systems, and then discuss the current and future trajectories of novel senotherapeutics that target aging NSCs to ameliorate brain disease.
... There are also checkpoints during S, G2 and M phases, which monitor the accuracy of DNA replication and correct chromosomal alignment and segregation, ensuring that each daughter cell receives a full complement of genetic information identical to the parental cell (for review and other references on checkpoints see Clarke and Gimenez-Abian, 2000;Pines and Rieder, 2001;Wassmann and Benezra, 2001). ...
The repair of cardiac lesions in adult mammals is limited to compensatory hypertrophy and fibrosis, because cardiomyocytes withdraw from the cell cycle soon after birth. In contrast, adult amphibian ventricular cardiomyocytes, such as those from the newt, proliferate locally after direct injury to the ventricle. This is paralleled by other newt differentiated cell types, which contribute to the regeneration of structures such as limbs and lens, by cell cycle re-entry and reversal of differentiation. I have established and characterised a system for primary culture of adult newt ventricular cardiomyocytes. The plasticity of the adult newt cardiomyocyte, defined here as the ability to enter into S phase and divide, was maintained in these cultures. Foetal bovine serum promoted newt cardiomyocyte entry into S phase. To address the cellular regulation of newt cardiomyocyte plasticity, I quantitatively characterised the proliferative potential of these cells at the single cell level by lineage tracing and time-lapse video microscopy. These experiments showed that 75% of adult newt ventricular cardiomyocytes enter S phase, 60% of the total imdergo mitosis and approximately half of these undergo cytokinesis, during the first 18 days in culture. This ability to divide is clearly distinct from that of adult mammalian ventricular cardiomyocytes, which have rarely been observed to undergo mitosis, and never to undergo cytokinesis. Other experiments showed that inactivation of pRb-family proteins is an endpoint of the serum stimulation pathway in newt cardiomyocytes. These results suggest that regulation of these proteins is important for the plasticity of urodele differentiated cells.
... Previous studies have already described an association between DLGAP5 deregulation and abnormal chromosomal rearrangement [5,14]. Rearrangement errors due to spindle assembly deregulation are well-known causes of genomic instability [15,16]. Thus, DLGAP5 overexpression may contribute to carcinogenesis. ...
Purpose
DLG7 (disc large homolog 7) is a microtubule-associated protein encoded by DLGAP5 (DLG associated protein 5) gene and has an important role during spindle assembly. Spindle assembly deregulation is a well-known cause of genomic instability. The aim of this study was to investigate the influence of DLGAP5 expression on survival and to evaluate its potential use as a biomarker in colorectal cancer (CRC).
Methods
DLGAP5 expression was measured in the primary tumor and corresponding normal mucosa samples from 109 patients with CRC and correlated to clinical and pathological data. The results were validated in a second, publically available patient cohort. Molecular effects of DLG7/DLGAP5 in CRC were analyzed via functional assays in knockdown cell lines.
Results
DLGAP5 downregulation led to a significant reduction of the invasion and migration potential in CRC. In addition, DLGAP5 expression correlates with nodal status and advanced UICC stage (III–IV).Subgroup analyses revealed a correlation between DLGAP5 overexpression and poor survival in patients with non-metastatic disease (M0). Furthermore, overexpression of DLGAP5 is associated with worse overall survival in distinct molecular CRC subtypes.
Conclusions
The results of this study suggest the importance of DLGAP5 in defining a more aggressive CRC phenotype. DLG7/DLGAP5 represents a potential biomarker for CRC in molecular subgroups of CRC.
... Une fois Scc1 clivée, l'anneau de cohésines qui maintient les chromatides soeurs ensemble est détruit [19]. Au cours de la division mitotique, un mécanisme de contrôle appelé point de contrôle du fuseau empêche le déclenchement de l'anaphase tant que tous les chromosomes ne sont pas attachés au fuseau mitotique et alignés sur la plaque équatoriale sous tension [20]. ...
... MCC or SAC complex prevents the errors in chromosome segregation, aneuploidy, tumor progression, and cell death when it is activated. Hence premature segregation can happen only when SAC is not activated (35,36). Previous reports have demonstrated that suppression of MAD2, a SAC component, and Securin, which inhibits Separase-mediated proteolysis of Cohesin that tether chromatids, leads to premature chromosomal segregation. ...
Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein, which plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and -blotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G2/M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-CDC20 - mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-CDC20 that maintains the temporal stability of cyclin B1 during the G2/M transition and thereby controls mitosis and cell division.
... Microtubule-targeting agents can affect the microtubule cytoskeleton and prevent the normal division course of cells, which eventually cause mitotic arrest and cell death by activating the checkpoint protein in mitosis [25,26]. CCNB1, a regulatory protein, plays an important role in pushing the cell cycle from G2 to M phase. ...
Purpose:
MBRI-001 is a novel synthetic derivative of plinabulin. In this study, our purpose is to investigate the inhibition effects of MBRI-001 on human hepatocellular carcinoma as monotherapy or in combination with sorafenib.
Methods:
HCCLM3 and Bel-7402 cell lines were used for activity evaluation in vitro. The anti-proliferative activity of MBRI-001 was assessed by MTT assay. The morphological change of microtubules was determined by immunofluorescence assay. The cell cycle was measured by flow cytometer. The expression of cyclin B1 (CCNB1) was analyzed by RT-qPCR and western blotting assays. The antitumor activities in vivo were evaluated with human HCC xenograft mice model.
Results:
Our data demonstrated that MBRI-001 had better anti-proliferative activities than that of plinabulin against HCCLM3 and Bel-7402 cell lines. MBRI-001 inhibited the formation of microtubules and induced G2/M arrest with the downregulation of CCNB1. In vivo orthotopic mice model demonstrated that MBRI-001 significantly inhibited the growth of HCCLM3 with the apoptosis and necrosis observed in tumor. The combination treatment of MBRI-001 with sorafenib in subcutaneous mice model exhibited a higher antitumor inhibition rate at 72.0%, in comparison with MBRI-001 or sorafenib as monotherapy at 40.7% or 47.7%, respectively.
Conclusion:
MBRI-001 had better inhibition effects on microtubules and human hepatocellular carcinoma than that of plinabulin. The combination treatment of MBRI-001 and sorafenib exhibited a higher antitumor effect, which could provide a new strategy to treat HCC in the future.
... The regulatory mechanism responsible for delaying anaphase in this manner is known as the spindle assembly checkpoint (SAC) [48,49]. The checkpoint protein Mad2 catalyzes the sequestration of the APC/C Cdc20 activator protein to block its ability to ubiquitinylate either securin or cyclin B by the Cdc20/Mad2 complex binding to the APC/C when unattached, mono-oriented, or syntelically attached kinetochores exist [50,51]. Depletion of Mad2 through the injection of morpholino antisense oligonucleotides results in aneuploidy and advances the onset of cyclin B and securin destruction [52]. ...
Aneuploidy is a leading genetic cause of birth defects and lower implantation rates in humans. Most errors in chromosome number originate from oocytes. Aneuploidy in oocytes increases with advanced maternal age. Recent studies support the hypothesis that cohesion deterioration with advanced maternal age represents a leading cause of age-related aneuploidy. Cohesin generates cohesion, and is established only during the premeiotic S phase of fetal development without any replenishment throughout a female’s period of fertility. Cohesion holds sister chromatids together until meiosis resumes at puberty, and then chromosome segregation requires the release of sister chromatid cohesion from chromosome arms and centromeres at anaphase I and anaphase II, respectively. The time of cohesion cleavage plays an important role in correct chromosome segregation. This review focuses specifically on the causes and effects of age-related cohesion deterioration in female meiosis.
... Mitotic spindle formation and the spindle checkpoint are critical for the maintenance of cell division and chromosome segregation (4). A number of mitotic spindle-associated proteins have been implicated in multiple malignancies, including lung cancer (5,6). ...
Deregulation of mitotic spindle genes has been reported to contribute to the development and progression of malignant tumours. The aim of the present study was to explore the association between the expression profiles of Aurora kinases (AURKA, AURKB and AURKC), cytoskel-eton-associated protein 5 (CKAP5), discs large-associated protein 5 (DLGAP5), kinesin-like protein 11 (KIF11), micro-tubule nucleation factor (TPX2), monopolar spindle 1 kinase (TTK), and β-tubulins (TUBB) and (TUBB3) genes and clinicopathological characteristics in human non-small cell lung carcinoma (NSCLC). Reverse transcription-quantitative polymerase chain reaction-based RNA gene expression profiles of 132 NSCLC and 44 adjacent wild-type tissues were generated, and Cox's proportional hazard regression was used to examine associations. With the exception of AURKC, all genes exhibited increased expression in NSCLC tissues. Of the 10 genes examined, only AURKA was significantly associated with prognosis in NSCLC. Multivariate Cox's regression analysis demonstrated that AURKA mRNA expression [hazard ratio (HR), 1.81; 95% confidence interval (CI), 1.16-2.84; P=0.009], age (HR, 1.03; 95% CI, 1.00-1.06; P=0.020), pathological tumour stage 2 (HR, 2.43; 95% CI, 1.16-5.10; P=0.019) and involvement of distal nodes (pathological node stage 2) (HR, 3.14; 95% CI, 1.24-7.99; P=0.016) were independent predictors of poor prognosis in patients with NSCLC. Poor prognosis of patients with increased AURKA expression suggests that those patients may benefit from surrogate therapy with AURKA inhibitors.
... This causes the cells to slip through the pro-metaphase arrest and continue mitosis without satisfying the SAC, which is termed uncoupling of the SAC from apoptosis (Brito and Rieder, 2006;Kurokawa and Kornbluth, 2010). Incorrect SAC functionality is generally linked to aneuploidy, and is thought to be one of the driving mechanisms behind the genome instability seen in cancers (Wassmann and Benezra, 2001). Therefore, it is not surprising that it has been hypothesized that the high frequency of chromosomal mis-segregation in human embryos may be caused by a SAC impairment at least during the cleavage stages (Wei et al., 2011;Vera-Rodriguez et al., 2015). ...
Study question:
Is the spindle assembly checkpoint (SAC) active during human preimplantation development?
Summary answer:
Mitotic spindle disruption during mitosis activates the SAC from at least Day 3 of human preimplantation development, but this does not lead to apoptosis until Day 5.
What is known already:
Human preimplantation embryos frequently acquire chromosomal abnormalities, but the mechanisms behind this are poorly understood. It has been speculated that a dysfunctional SAC could be responsible. Although research has shown that the SAC components are present during early human development, functional studies are lacking.
Study design, size, duration:
In vitro study using human preimplantation embryos in a university research laboratory. We studied a total of 38 Day-3, 38 Day-4, 29 Day-5 and 21 Day-6 human preimplantation embryos, donated for research, during 16 h of incubation.
Participant/materials, setting, methods:
We cultured human preimplantation embryos overnight in a time-lapse imaging system, in control or in a nocodazole-containing medium that prevents the formation of a proper mitotic spindle. The embryos were subsequently fixed and analysed by immunocytochemistry for tubulin or mitotic and apoptotic markers, or by FISH.
Main results and the role of chance:
All embryos showed an increase in M-phase cells from 4.1-8.8% to 21.4-53.5% when exposed to nocodazole (P < 0.05; two-way ANOVA for all groups except Day-4 embryos, P = 0.128) suggesting SAC functionality. Apoptosis, which was rarely detected between Day 3 and Day 6 in good-quality control embryos, increased from Day 5 onwards in nocodazole-treated embryos and became statistically different from Day 6 (P < 0.01; two-way ANOVA). The FISH data suggest that in compacted Day-4 embryos, approximately one in six cells started a polyploid new cell cycle rather than to go in apoptosis after the failure to maintain the SAC-mediated M-phase arrest. These results suggest that during early embryo development, blastomeres with unresolved chromosome misalignments during M-phase can escape SAC-mediated apoptosis, continue cell division which can then result in aneuploid daughter cells.
Large scale data:
Not applicable.
Limitations, reasons for caution:
This study used nocodazole to inhibit microtubule polymerization, a drug that is regularly used to induce metaphase arrest and SAC activation. Results should be extrapolated to naturally occurring chromosome misalignments with care.
Wider implications of the findings:
Our results provide functional data that can help explain the high aneuploidy rates seen in human cleavage-stage embryos and suggest that this is due to their unusual cell cycle control.
Study funding/competing interest(s):
This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) and the Methusalem grant to Karen Sermon of the Research Council of the Vrije Universiteit Brussel. The authors declare no competing financial interests.
... This discrepancy might be explained by telomerase activity status because CRCCs that have high telomerase activity during the early stages of tumor development may be genetically stable and may yield more favorable outcomes than results from those with low or no telomerase activity CRCC ( Hara et al., unpublished data ) . However, it is possible that CRCCs with high telomerase activity may develop genomic instability at later stages due to another mechanism, such as destruction of the mitotic apparatus [ 43 ] . In contrast, CRCCs with low or no telomerase activity that may not be immortal at an early stage could express high levels of telomerase activity later, resulting in stabilization of their gross genomic aberrations that would permit tumor progression as Rudolph et al. [ 44 ] reported recently. ...
Malignant tumors have telomerase activity, which is thought to play a critical role in tumor growth. However, the relation between telomerase activity and genomic DNA status in tumor cells is poorly understood. In the present study, we examined telomerase activity in 13 clear cell type renal cell carcinomas (CRCCs) with similar clinicopathologic features by telomeric repeat amplification protocol assay (TRAP). Based on TRAP assay results, we divided the CRCCs into two groups: a high telomerase activity group and a low/no telomerase activity group. We then analyzed genomic aberration, DNA ploidy, and telomere status in these two groups by comparative genomic hybridization (CGH), laser scanning cytometry (LSC), and telomere-specific fluorescence in situ hybridization (T-FISH), respectively. CGH showed the high telomerase activity group to have fewer genomic changes than the low/no telomerase activity group, which had many genomic aberrations. Moreover, with LSC, DNA diploid cells were found more frequently in the high telomerase activity group than in the low/no telomerase activity group. In addition, T-FISH revealed strong telomere signal intensity in the high telomerase activity group compared with that of the low/no telomerase activity group. These results suggest that telomerase activity is linked to genomic DNA status and that high telomerase activity is associated with genomic stability, DNA ploidy, and telomere length in CRCC.
... Because pole-proximal misaligned chromosomes are expected to possess syntelic or monotelic attachments, the inter-kinetochore tension of these sister chromatids would be weakened during formation of the mitotic spindle. The spindle assembly checkpoint (SAC) machinery detects the aberrant attachment between microtubules and kinetochores and blocks the onset of anaphase until the kinetochores of each duplicated chromosome pair have achieved a bipolar attachment to the mitotic spindle [34][35][36]. The next step was to determine whether chromosome misalignment by Cmpd-A causes SAC-mediated mitotic arrest. ...
Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.
... Chromosomal instability leading to an abnormal number of chromosomes (aneuploidy) is a hallmark of cancers [1,2]. This observation has triggered a series of research for molecular determinants and mechanism that regulate proper chromosome segregation and their abnormalities as mediators of tumorigenesis [1,3]. ...
NDC80/Hec1, one of four proteins of the outer kinetochore NDC80 complex, is involved in the tumorigenesis of a variety of cancers. In this study, we focused on that NDC80 is overexpressed in human pancreatic cancer and investigates the role of NDC80-knockdown in pancreatic cancer cells proliferation.
We determined the expression levels of NDC80 on both mRNA and protein levels in fresh pancreatic cancer tissues and cells by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Furthermore, protein level of NDC80 was identified using immunochemistry in paraffin-embedded tumor specimen, with correlation between NDC80 expression and various clinicopathological parameters evaluated. The role of NDC80 in pancreatic cancer cells (Panc-1) growth was investigated by lentivirus-mediated silencing of NDC80. The effect of NDC80 deletion on cell proliferation was analyzed by MTT assay and clone formation assay, while cell cycle distributions and apoptosis were analyzed by flow cytometry.
The mRNA and protein of NDC80 were overexpressed in pancreatic cancer tissues and cells. The statistical analysis based on immunohistochemical evaluation suggested that NDC80 overexpression was signifi cantly associated with clinicopathological parameters including pathological T staging and N staging, which may be served as an predictor for poor outcomes. The silencing of NDC80 in Panc-1 cells could suppress cell proliferation and colony formation. Furthermore, the NDC80-siRNA infected Panc-1 cells lead to cell cycle arrest at G2/M phase and induction of apoptosis.
These results demonstrated that NDC80 plays an essential role in the tumorigenesis of pancreatic cancer, and might serve as potential prognostic and therapeutic target for treatment of pancreatic cancer.
... During mitosis, the duplicated chromosome pairs are accurately split and subsequently segregated into daughter cells. The mitotic checkpoint ensures this faithful chromosomal seg-regation by delaying the onset of anaphase until both kinetochores on each duplicated chromosome pair have properly attached to the spindle microtubule and are under tension [11][12][13][14][15][16]. For the successful execution of this checkpoint function, several molecular components of mitotic checkpoint machinery are known to be necessary; these include Mps1, Mad1-3, Bub1, Bub3, BubR1 (a homologue of Mad3 and Bub1), and CENP-E (a microtubule-dependent motor protein). ...
During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.
... During mitosis the replicated genetic material is segregated, with one copy of each chromosome going to each daughter cell with fidelity. Mitosis is monitored by several checkpoint systems (7,8). Recently, Scolnick and Halazonetis (9) defined a new mitotic checkpoint which delays chromosome condensation in response to mitotic stress induced by taxol or nocodazole, and also identified a gene, named checkpoint with FHA and ring finger (Chfr), that seems to be required for delaying prophase in human cells. ...
... Nonetheless the simple singlecell organism has had a major impact in the cancer field. [79][80][81] The same applies to studying specific forms of learning and memory in animal models of intellectual disability 82 , even though patients are often affected in numerous cognitive domains. Future studies exploring additional hallmarks of ADHD, most importantly defects in attention, for example by using an optomotor maze or other attention-like performance assays 39 , would be highly useful complements to our study. ...
Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder with hyperactivity as one of the hallmarks. Aberrant dopamine signaling is thought to be a major theme in ADHD, but how this relates to the vast majority of ADHD candidate genes is illusive. Here we report a Drosophila dopamine-related locomotor endophenotype that is shared by pan-neuronal knockdown of orthologs of the ADHD-associated genes Dopamine transporter (DAT1) and Latrophilin (LPHN3), and of a gene causing a monogenic disorder with frequent ADHD comorbidity: Neurofibromin (NF1). The locomotor signature was not found in control models and could be ameliorated by methylphenidate, validating its relevance to symptoms of the disorder. The Drosophila ADHD endophenotype can be further exploited in high throughput to characterize the growing number of candidate genes. It represents an equally useful outcome measure for testing chemical compounds to define novel treatment options.Molecular Psychiatry advance online publication, 12 May 2015; doi:10.1038/mp.2015.55.
... The resulting aneuploidy can influence tumor evolution. [3][4][5][6][7] Continuous chromosomal reshuffling and high mutation rates lead to heterogeneity, which can produce cells with a growth advantage, adaptation to the environment and resistance to conventional chemotherapies. 5,8 CIN is also linked to metastasis and low survival rates in cancer patients. ...
Chromosomal INstability (CIN), a hallmark of cancer, refers to cells with an increased rate of gain or loss of whole chromosomes or chromosome parts. CIN is linked to the progression of tumors with poor clinical outcomes such as drug resistance. CIN can give tumors the diversity to resist therapy, but it comes at the cost of significant stress to tumor cells. To tolerate this, cancer cells must modify their energy use to provide adaptation against genetic changes as well as to promote their survival and growth. In this study, we have demonstrated that CIN induction causes sensitivity to metabolic stress. We show that mild metabolic disruption that does not affect normal cells, can lead to high levels of oxidative stress and subsequent cell death in CIN cells because they are already managing elevated stress levels. Altered metabolism is a differential characteristic of cancer cells, so our identification of key regulators that can exploit these changes to cause cell death may provide cancer-specific potential drug targets, especially for advanced cancers that exhibit CIN.
... An insufficiently performing spindle assembly checkpoint is also implicated in the production of aneuploidy [257]. Mice with reduced levels of Mad2 [258], BubR1 [259] or Bub3 [260] have a defective SAC and are prone to the acquisition of aneuploidy. ...
... The heterodimer of tubulin consists of a/b-tubulin and forms fibers of microtubules. Apoptotic cell death can be induced by antimicrotubule agents that are known to result in the aberrant formation of the mitotic spindle and blockage of the cell cycle in G2/M phase [37]. Microtubule-targeting agents that destabilize (nocodazole, colchicine) or stabilize (paclitaxel, docetaxel) tubulin are the most frequently used anti-cancer drugs [38]. ...
Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.
... The spindle assembly checkpoint ensures that progression toward anaphase does not happen prematurely. It is thought that throughout spindle formation and establishment of kinetochore-microtubule interactions, the status of the kinetochoremicrotubule attachments can be sensed and reflected in the levels of soluble "waitanaphase" signaling complexes (Rieder and Salmon, 1998;Wassmann and Benezra, 2001), the presence of which delays activation of APC. Indeed, unattached kinetochores or low-tension kinetochores undergo phosphorylation and act as sites where spindle 5 checkpoint Mad2-Cdc20 protein complexes are assembled and released to abrogate APC activation (Chan and Yen, 2003;Hardwick, 2005;Yu, 2002). ...
Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. The goal of my dissertation research is to identify the MEK kinase responsible for activation of the MAPK cascade during mitosis and to elucidate the biochemical mechanisms that regulate its activity. In the described herein work I purified and characterized the MEK kinase activity present in M-phase arrested Xenopus egg extracts. I demonstrate that B-Raf is the critical MEK kinase required for activation of the MAPK pathway at mitosis. Consistent with this, I show that B-Raf is activated in an M-phase dependent manner. Further, I provide data linking Cdk1/cyclin B to mitotic activation of B-Raf.
Cdk1/cyclin B associates with and phosphorylates B-Raf in M-phase arrested extracts and directly targets Xenopus B-Raf in vitro at a conserved Ser-144 residue. Phosphorylation at Ser-144 is critical for M-phase dependent activation of B-Raf and for B-Raf's ability to trigger activation of the MAPK cascade at mitosis. Finally, I demonstrate that mitotic B-Raf undergoes feedback phosphorylation by MAPK at its conserved C-terminal SPKTP motif. Mutation of both phosphorylation sites within the SPKTP sequence to alanines increases activity of mitotic B-Raf. Further, inhibition or over-activation of MAPK during mitosis enhances or diminishes B-Raf activity, respectively. These results indicate that MAPK-mediated feedback phosphorylation negatively regulates B-Raf activity. Additionally, I show that active mitotic B-Raf exists in large multi-protein complex(s). By utilizing a proteomics approach I identify a set of proteins, which potentially associate with B-Raf at M-phase.
Future studies are necessary to elucidate the involvement of these proteins in regulating B-Raf mitotic functions. In summary, my dissertation studies demonstrate that B-Raf activates MAPK signaling at mitosis and undergoes an M-phase dependent regulation. I propose that B-Raf has important functions at mitosis that contributes to its overall role in promoting cell proliferation.
... Along with the microtubules, centrosomes regulate the formation of the mitotic spindle and the polarity of cell division being therefore, crucial for chromosome segregation and cytokinesis (Brinkley, 2001). Centrosome abnormalities are a frequent finding in many tumors (Duensing & Munger, 2001) and they have been implicated in chromosome missegregation and the generation of aneuploid cells (Wassmann & Benezra, 2001). Centrosome defects are thought to drive chromosomal instability via their ability to mediate multipolar spindle formation (Fig. 3). ...
... Along with the microtubules, centrosomes regulate the formation of the mitotic spindle and the polarity of cell division being therefore, crucial for chromosome segregation and cytokinesis (Brinkley, 2001). Centrosome abnormalities are a frequent finding in many tumors (Duensing & Munger, 2001) and they have been implicated in chromosome missegregation and the generation of aneuploid cells (Wassmann & Benezra, 2001). Centrosome defects are thought to drive chromosomal instability via their ability to mediate multipolar spindle formation (Fig. 3). ...
The development of Oral Squamous Cell Carcinoma (OSCC) is characterized by a multistep row of events governed by the early detectable, gradual accumulation of genetic changes in genes transcribing key upstream and downstream regulatory proteins. The combination of genetic pre- disposition and epigenetic/environmental factors (gene methylation, tobacco and alcohol use, HPV infection) exerts, moreover, a modifying influence on the genetic background. Despite re- cent updates in our knowledge of OSCC developing process and therapeutic improvements, the prognosis of this malignancy still remains poor. Ultimately, we can envision that the demarcation of events underlying the oral malignant transformation may offer better prediction of biological behavior, more accurate clinical prognosis estimation and new targeted treatment options. This review focuses on the critical genetic alterations that characterize oral cancer and elucidates their proposed mechanism of contribution to the tumorigenic process. Emphasis has been given to the role of loss of heterozygosity in stem (progenitor) cells, the “field” cancerization hypothesis of OSCC genesis, and furthermore to data regarding the cell cycle dysregulation and cell aneu- ploidization. Disruption of certain signaling pathways (EGFR, NFkB, STAT, Wnt/‚-catenin, TGF-‚, and PI3K-AKT-mTOR) plays, also, a documented role in OSCC pathogenesis and cer- tain molecules have been suggested as fine targets for molecular-targeted therapy.
Cellular function is highly dependent on genomic stability, which is mainly ensured by two cellular mechanisms: the DNA damage response (DDR) and the Spindle Assembly Checkpoint (SAC). The former provides the repair of damaged DNA, and the latter ensures correct chromosome segregation. This review focuses on recently emerging data indicating that the SAC and the DDR proteins function together throughout the cell cycle, suggesting crosstalk between both checkpoints to maintain genome stability.
Molecular mechanisms underlying estrogen-induced micronucleus formation in breast cancer cells Aneuploidy, or numerical changes of chromosomes, has been documented in almost all solid tumours and the frequency of micronucleus formation is commonly taken as a biomarker of aneuploidy. An increasing number of observations suggest that exposure to physiologically relevant concentrations of steroidal estrogens gives rise to chromosomal impairments in estrogen receptor competent cells. Although the action of estrogenic compounds is well explained by their ability to bind and activate the estrogen receptor, there are still many uncertainties surrounding the mechanism underlying the ability of steroidal compounds to stimulate micronuclei formation. This has prompted us to investigate whether these effects are linked to activation of the estrogen receptor alpha. Co-administration of tamoxifen and the pure estrogen receptor antagonist ICI 182,780 to breast cancer MCF-7 cells with estrogenic agents did not lead to significant reductions in micronucleus frequencies. Since these anti-estrogens interfere with the transcriptional activity of the estrogen receptor and block promotion of estrogen receptor-dependent gene expression, it appears that this process is not involved in micronucleus formation. In addition to the classical activation of the estrogen receptor at the nuclear level, it is now accepted that estrogenic compounds can rapidly and transiently trigger a number of second-messenger signaling pathways, such as the MAPK cascade Erkl/2 and even their upstream effectors, such as the kinase Src and Raf. Therefore, we wanted to evaluate if the alternative mechanisms of estrogen receptor activation could be involved in the formation of micronuclei by estrogenic compounds. When MCF-7 cells were exposed to estrogenic agents in combination with the specific kinase inhibitors PP2 and PD 98059, reductions in micronucleus frequencies occurred. These findings suggest that the Src/Raf/Erk pathway plays a role in micronucleus formation by estrogenic compounds. Enhanced activation of the Src/Raf/Erk cascade disturbs the localisation of Aurora B kinase to kinetochores, leading to a defective spindle checkpoint with chromosome malsegregation. Further on, using anti-kinetochore CREST antibody staining, a high proportion of micronucleus containing kinetochores was observed when the breast cancer cells were treated with compounds able to activate non-genomic signaling pathways, indicating that such processes are relevant to the induction of micronuclei by estrogens. Our results suggest that estrogens induce micronuclei by causing improper chromosome segregation, possibly by interfering with kinase signaling that controls the spindle checkpoint, or by inducing centrosome amplification. Our findings may have some relevance in explaining the effects of estrogens in the later stages of breast carcinogenesis.
Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore–microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation.
Renal cancer is not a single disease; it comprises several different types of cancer, each with a different histology, a different clinical course, and different genetic changes¹ (Table 2.1). For each of these different tumour types, hereditary, familial, and sporadic forms exist. Most of our current knowledge was originally derived from studies on familial forms of the disease, but subsequent studies of sporadic cancers found similar changes² (Table 2.2). Recent developments in genetics and molecular biology have led to an increasing knowledge of the origin and biology of renal cell carcinoma (RCC), which will be discussed separately for sporadic and inherited forms.
Based on the results obtained from a computational study on the suitability of the isouronium and N-hydroxyguanidinium cations as hydrogen bond donors/acceptors, the DNA binding of a series of isouronium derivatives was assessed by DNA thermal denaturation experiments and compared to related N-hydroxyguanidines. Due to the poor DNA binding observed, the nature of the diaromatic linker was explored by preparing the corresponding amide-linked bis-isouronium derivative and measuring its DNA affinity. Next, the inhibitory effects of the isouronium derivatives on cell viability were evaluated in two different cancer cell lines providing IC50 values in the range of 36.9–57.4 μM (HL-60, leukemia), and 17.3–33.9 μM (Kelly, neuroblastoma). These values are comparable to those previously found for the N-hydroxyguanidine series. Compounds with the –S- linker (3, 6, and 10) proved to be considerably active in the HL-60 cells and even more active in the Kelly cell lines. No correlation was found between DNA minor groove binding and cell growth inhibition; hence, activity may depend on different modes of action. Further studies into the apoptotic potential of these compounds indicated that, besides inhibiting cell viability and proliferation, derivatives 9 and 10, are significant apoptosis-inducers in both cell lines. Results obtained with HL-60 cells suggest that G2/M arrest and subsequent apoptosis induced by compound 10 are associated with microtubular depolymerisation, loss of mitochondrial membrane potential and activation of the caspase cascade. Moreover, the effects of compound 10 on cell viability and apoptosis in two non-cancereous cell lines (NIH3T3 and MCF-10A) indicate none or minimal toxicity.
Cancer is a multifaceted disease, but a common feature of most tumours is that they harbour one or more genetic mutations that allow them to proliferate outside their normal growth restraints. Proliferation is normally restrained through control of the cell division cycle, which in turn, is regulated by the Cdk family of serine/threonine kinases and their regulatory partners the cyclins. Here, the roles of the Cdk/cyclin complexes in cell cycle control are described followed by a review of the genetic lesions in these and associated proteins which may contribute to tumour progression.
Brody disease was first described as a benign pseudo-myotonic disorder with muscular stiffness, which increased with exercise. Biochemical and genetic studies have pointed out its close relationship to a functional defect of the fast-twitch sarcoplasmic reticulum Ca(++) ATPase pump (SERCA1) encoded by the ATP2A1 gene located on chromosome 16. The histopathological features in this form of myopathy were generally described as non-specific, i.e. moderate degree of type 2 fibre atrophy and excess of internal nuclei. We here present the clinical and histopathological features of a patient with Brody disease over a 19-year follow-up period. This patient had two heterozygous ATP2A1 mutations and complained about muscle stiffness immediately after effort. He had suffered from this since early childhood and exhibited clinical symptoms mimicking myotonia. Histological, ultrastructural and cytogenetic analyses revealed morphologically abnormal nuclei with polyploidy. In this report, we discuss the possible links between the consequences of the genetic abnormality and the peculiar aspect of the nuclei.
Copyright © 2015 Elsevier B.V. All rights reserved.
Celiac disease is an intestinal disease which shows different symptoms and clinical manifestations among pediatric and adult patients. These variations could be imputable to age-related changes in gut architecture and intestinal immune system, which could be characterized by gene expression differences possibly regulated by miRNAs. We analyzed a panel of miRNAs and their target genes in duodenal biopsies of Marsh 3AB and 3C pediatric celiac patients, compared to controls. Moreover, to assess variation of expression in plasma samples, we evaluated circulating miRNA levels in controls and patients at diagnosis or on gluten-free diet. We detected a decreased miR-192-5p expression in celiac patients, but no variations in NOD2 and CXCL2, targets previously identified in adults. Conversely, we detected a significant increase in mRNA and protein levels of another target, MAD2L1, protein related to cell cycle control. miR-31-5p and miR-338-3p were down-regulated and their respective targets, FOXP3 and RUNX1, involved in Treg function, resulted up-regulated in celiac patients. Finally, we detected, in celiac patients, an increased expression of miR-21-5p, possibly caused by a regulatory loop with its putative target STAT3, which showed an increased activation in Marsh 3C patients. The analysis of plasma revealed a trend similar to that observed in biopsies, but in presence of gluten-free diet we could not detect circulating miRNAs values comparable to controls. miRNAs and their gene targets showed an altered expression in duodenal mucosa and plasma of celiac disease pediatric patients, and these alterations could be different from adult ones.
The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations-misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours.
Human papillomavirus (HPV) infection leads to aneuploidy, a numerical chromosomal aberration that is caused by dysregulation of chromosomal segregation. We previously found that the E7 proteins of high-risk HPVs, but not of low-risk HPVs, could bind to centromere protein-C (CENP-C). In this study, we first found that CENP-C could bind centromere α-satellite DNAs using ChIP analysis and HA-tagged CENP-C/nuc transfected 293T cells. We then investigated if HA-CENP-C/nuc binding to α-satellite DNAs was affected by the E7 proteins of high- or low-risk HPVs. We found that transfection of the FLAG tagged HPV 18 E7 inhibited the binding of HA-CENP-C/nuc to α-satellite DNAs. This finding was confirmed in HeLa S3 cells transfected with siRNA targeted to HPV18 E7 expression. We therefore speculate that altered function of kinetochores as a result of inhibition of CENP-C and α-satellite DNA binding may be associated with the chromosomal abnormalities observed in HPV18-positive cancers.
Copyright © 2015 Elsevier B.V. All rights reserved.
We examine features of intracellular networks that make errors less probable and beneficial responses more probable. In a false negative (F⁻) error, a network does not respond to input. A network is reliable if it operates with a low probability of a F⁻ error. Features that promote reliability include fewer reactions in sequence, more alternative pathways, no side reactions and negative feedback. In a false positive (F⁺) error, a network produces output without input. Here, a network is specific if it has a low probability of a F⁺ error. Conjunctions of signals within or between pathways can improve specificity through sigmoid steady-state response curves, kinetic proofreading and checkpoints. Both reliability and specificity are important in networks that regulate the fate of a cell and in networks with hubs or modules, and this includes scale-free networks. Some networks discriminate among several inputs by responding to each input through a different combination of pathways.
Zu Beginn des 19. Jahrhunderts stand die klinische Beschreibung einzelner Patienten mit kutanen Lymphomen im Vordergrund. Basierend auf diesen klinischen Beschreibungen und der morphologischen histopathologischen Untersuchung der „2. Dimension” der Haut konnten in der Folgezeit verschiedene Unterformen der kutanen Lymphome herausgearbeitet werden. Im Rahmen von Konsensuskonferenzen wurden dann Ende der 70er-Jahre des 20. Jahrhunderts basierend auf der morphologischen Beschreibung klinische und histologische Kriterien für die Diagnose erarbeitet und der übergeordnete Begriff des kutanen T-Zell-Lymphoms (CTCL) definiert. Schließlich konnte am Ende des 20. Jahrhunderts mit Hilfe der immunhistologischen Marker eine genaue Klassifikation der CTCL im Rahmen der EORTC und WHO vorgenommen werden. Die neuen Erkenntnisse der Molekularbiologie (TCR-Rearrangement) des ausgehenden 20.Jahrhunderts erhöhen die diagnostische Sicherheit bei dieser Erkrankung. Mit Hilfe neuer Erkenntnisse zur „genetischen Morphologie” der CTCL (Molekulare Genetik, Mikroarray-Technologie) wird es in naher Zukunft möglich sein, die Diagnostik, Prognostik und Therapie dieser Hauterkrankung weiter zu verbessern.
The purpose of this research is to classify BRCA1 variants for which cancer association is not known (unclassified variants UCV). To approach this problem we hypothesized that poorly characterized but conserved domains in BRCA1 directly participate in its tumor suppression function. To test this hypothesis we choose a global approach analyzing several BRCA1 domains and point mutants in functions that have previously been attributed to BRCA1: long term survival after irradiation, early G2/M checkpoint, intra S phase checkpoint, and spindle assembly checkpoint. We successfully optimized conditions for expression of full-length BRCA1 mutants in two different cell lines by electroporation. We also optimized all the checkpoints assays in our laboratory and are now ready to analyze BRCA1 mutants proposed in this grant. This will have a significant impact not only to understand BRCA1 role as a tumor suppressor in breast cancer but also to help patients that are carriers of BRCA1 mutation to make informed clinical decisions.
During anaphase of mitosis, sister chromatids are separated by the mitotic spindle. The spindle assembly checkpoint protects the integrity of the genome by initiating a cell cycle delay if chromosomes are not properly attached to the spindle. Cells lacking a functional spindle checkpoint may gain or lose genetic information, which can cause cell death or predispose cells to cancer. For example, loss of checkpoint function has been observed in human cancer cell lines, and decreased expression of the checkpoint component, hsMAD2, has been demonstrated in human breast cancers. Most human spindle checkpoint components were identified by their similarity to yeast checkpoint proteins that were discovered through genetic screens. Many aspects of spindle checkpoint function are not yet understood, and genetic evidence indicates there are additional checkpoint proteins that have not been identified. This project aims to use genetic screens in fission yeast to identify and characterize novel components of the yeast and animal spindle checkpoint pathways and novel mutant alleles of known yeast spindle checkpoint genes. To date, mutations in three known yeast spindle checkpoint genes and a mutation in a potentially novel component of the yeast checkpoint pathway have been identified. A second genetic screen has been initiated to identify mouse cDNAs which induce a metaphase arrest in fission yeast and may encode spindle checkpoint proteins. The genes identified by these studies will be used to further elucidate the mechanism of spindle checkpoint function.
The most common feature of human cancer cells is aneuploidy and a defective mitotic checkpoint is thought to be responsible. Since a group of anticancer drugs target the mitotic checkpoint, this review will discuss the association of mitotic checkpoint with chemotherapeutic drug sensitivity and suggest a novel therapeutic tool to achieve chemosensitization in human cancer cells.
Histone deacetylase inhibitors exhibit pleiotropic effects on cell functions, both in vivo and in vitro. One of the more dramatic effects of these drugs is their ability to disrupt normal mitotic division, which is a significant contributor to the anticancer properties of these drugs. The most important feature of the disrupted mitosis is that drug treatment overcomes the mitotic spindle assembly checkpoint and drives mitotic slippage, but in a manner that triggers apoptosis. The mechanism by which histone deacetylase inhibitors affect mitosis is now becoming clearer through the identification of a number of chromatin and nonchromatin protein targets that are critical to the regulation of normal mitotic progression and cell division. These proteins are directly regulated by acetylation and deacetylation, or in some cases indirectly through the acetylation of essential partner proteins. There appears to be little contribution from deacetylase inhibitor-induced transcriptional changes to the mitotic effects of these drugs. The overall mitotic phenotype of drug treatment appears to be the sum of these disrupted mechanisms.
Chromosomal rearrangements occur during mitosis as a result of chromosomal breakage and rejoining. Such chromosomal rearrangement occurs more frequently in cancer cells than during embryogenesis or postnatally, usually as a result of environmental exposures and/or mutations resulting in DNA repair defects.
Keywords:
chromosomal instability;
breakage–fusion–bridge cycle;
gene amplification;
chromosomal rearrangement;
cancer
Kinetochores are complexes of centromere DNA and proteins. Spindle microtubules bind to chromosomes at the kinetochore, and these complexes are required for accurate chromosome segregation during mitosis. The human kinetochore is a supramolecular complex of DNA and proteins that is involved in various mitotic activities.
Keywords:
kinetochore;
centromere;
mitosis;
chromosome segregation;
spindle checkpoint
Mammalian cells are charged with the task of evenly distributing 46 chromosomes to each of two daughter cells during every mitotic cell cycle. Abnormal function of any one of the numerous genes involved in this process can result in the aberrant transmission of chromosomes from the parental cell to its progeny.
Keywords:
aneuploidy;
centrosome;
checkpoint;
chromosome;
mitosis
Aneuploidy is a characteristic of most solid tumors, often associated with negative prognosis. It can arise from two principal mechanisms: from a tetraploid intermediate state, or directly from errors at cell division. The control of cell division, crucial to maintain genomic stability, is still poorly understood in its relationship to aneuploidy. Here we show that the TAp73alpha isoform induces polyploidy when overexpressed. This is possibly due to the interaction of TAp73alpha with kinetochore-related proteins leading to the alteration of mitotic checkpoint abilities. TAp73alpha but not p53 or any of the other p73 isoforms binds Bub1 and Bub3. Since TAp73alpha is frequently overexpressed in cancer, this interaction may contribute to the aneuploidy observed in cancer progression. Our results suggest a novel molecular mechanism leading to aneuploidy involving interference of TAp73alpha with Bub1 and Bub3 resulting in an altered mitotic checkpoint.
Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1.
During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.
During somatic cell division, faithful chromosomal segregation must follow DNA replication to prevent aneuploidy or polyploidy. Damage to the mitotic spindle is one potential mechanism that interferes with chromosomal segregation. The accumulation of aneuploid or polyploid cells resulting from a disrupted mitotic spindle is presumably prevented by cell cycle checkpoint controls. In the course of studying cells that overexpress the apoptosis-inhibiting protein Bcl-xL, we found that these cells have an increased rate of spontaneous tetraploidization, suggesting that apoptosis may play an important role in eliminating cells that fail to complete mitosis properly. When cells expressing Bcl-xL are treated with mitotic spindle inhibitors, a significant percentage reinitiate DNA replication and become polyploid. Nevertheless, the majority of cells expressing Bcl-xL undergo a prolonged p53-dependent cell cycle arrest following mitotic spindle damage. Unexpectedly, p53 expression is not induced in mitosis, nor does it influence M-phase arrest. Instead, cells with mitotic spindle damage only transiently arrest in M phase, and despite failing to complete mitosis, appear to proceed to G1. During this subsequent growth factor-dependent phase, p53 is induced and mediates cell cycle arrest. In cells that do not overexpress Bcl-xL, elimination of the p53-dependent growth arrest with a dominant negative mutant also results in polyploidy after mitotic spindle damage, but under these conditions most cells die by apoptosis. Expression of Bcl-xL and abrogation of p53 cooperate to allow rapid and progressive polyploidization following mitotic spindle damage. Our results suggest that suppression of apoptosis by bcl-2-related genes and loss of p53 function can act cooperatively to contribute to genetic instability.
Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK1 cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 +/- 6.7 (n = 94), at late metaphase 24.3 +/- 4.9 (n = 62), and at early anaphase 27.8 +/- 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 microM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 +/- 7. 4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.
The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle. Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation. Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism. One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation. We have isolated mad2, a spindle checkpoint component in fission yeast, and shown that mad2 overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition. In addition to the observation that mad2-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APC-dependent proteolysis.
Mad2 is a component of the spindle checkpoint, which delays the onset of anaphase until all chromosomes are attached to the spindle. Mad2 formed a complex with Slp1, a WD (tryptophan-aspartic acid)-repeat protein essential for the onset of anaphase. When the physical interaction between the two proteins was disrupted, the spindle checkpoint was no longer functional. Post-anaphase events such as chromosome decondensation and the next round of DNA replication were not delayed even when the spindle assembly was incomplete. This relief of dependence appears to be a result of deregulation of ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex.
The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.
In searching for cellular targets of the HTLV-I oncoprotein Tax, we identified TXBP181, which we characterized as the human homolog of yeast mitotic checkpoint MAD1 protein. Evidence supporting TXBP181 as HsMAD1 includes sequence conservation with yeast MAD1, hyperphosphorylation during S/G2/M phases and upon treatment of cells with nocodazole, and binding to HsMAD2. HsMAD1 functions as a homodimer. It localizes to the centrosome during metaphase and to the spindle midzone and the midbody during anaphase and telophase. Expression of either Tax or a transdominant-negative TXBP181 results in multinucleated cells, a phenotype consistent with a loss of HsMAD1 function. We propose a model of viral transformation in which Tax targets TXBP181, thereby abrogating a mitotic checkpoint.
A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301-1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242-246; Li, Y., and R. Benezra. Science. 274: 246-248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK1 cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929-939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK1 cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after approximately 12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.
We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
A feedback control mechanism, or cell cycle checkpoint, delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. Previously, we showed that the murine homologue of Bub1 is not only required for checkpoint response to spindle damage, but also restrains progression through a normal mitosis (Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). Here, we describe the identification of a human homologue of Bub3, a 37-kD protein with four WD repeats. Like Bub1, Bub3 localizes to kinetochores before chromosome alignment. In addition, Bub3 and Bub1 interact in mammalian cells. Deletion mapping was used to identify the domain of Bub1 required for binding Bub3. Significantly, this same domain is required for kinetochore localization of Bub1, suggesting that the role of Bub3 is to localize Bub1 to the kinetochore, thereby activating the checkpoint in response to unattached kinetochores. The identification of a human Mad3/Bub1-related protein kinase, hBubR1, which can also bind Bub3 in mammalian cells, is described. Ectopically expressed hBubR1 also localizes to kinetochores during prometaphase, but only when hBub3 is overexpressed. We discuss the implications of the common interaction between Bub1 and hBubR1 with hBub3 for checkpoint control.
Activation of the mitotic checkpoint pathway in response to mitotic spindle damage in eukaryotic cells delays the exit from mitosis in an attempt to prevent chromosome missegregation. One component of this pathway, hsMad2, has been shown in mammalian cells to physically associate with components of a ubiquitin ligase activity (termed the anaphase promoting complex or APC) when the checkpoint is activated, thereby preventing the degradation of inhibitors of the mitotic exit machinery. In the present report, we demonstrate that the inhibitory association between Mad2 and the APC component Cdc27 also takes place transiently during the early stages of a normal mitosis and is lost before mitotic exit. We also show that Mad2 associates with the APC regulatory protein p55Cdc in mammalian cells as has been reported in yeast. In contrast, however, this complex is present only in nocodazole-arrested or early mitotic cells and is associated with the APC as a Mad2/p55Cdc/Cdc27 ternary complex. Evidence for a Mad2/Cdc27 complex that forms independent of p55Cdc also is presented. These results suggest a model for the regulation of the APC by Mad2 and may explain how the spindle assembly checkpoint apparatus controls the timing of mitosis under normal growth conditions.
The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules.
The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1(+) is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Deltabub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1(+ )function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase-anaphase transition.
We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.
We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins. Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore. MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin. In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively). Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore. The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.
We have characterized the Drosophila mitotic checkpoint control protein Bub1 and obtained mutations in the bub1 gene. Drosophila Bub1 localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element–induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants.
We have also investigated the relationship between Bub1 and other kinetochore components. We show that Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies.
Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment. When unattached kinetochores are present, anaphase is delayed and the time available for chromosome-microtubule capture increases. Genes required for this delay first were identified in budding yeast (the MAD and BUB genes), but it is not yet known how the checkpoint senses unattached chromosomes or how it signals cell-cycle arrest. We report the isolation and analysis of a murine homologue of BUB3, a gene whose deletion abolishes mitotic checkpoint function in Saccharomyces cerevisiae. mBub3 belongs to a small gene family that has been highly conserved through evolution. By expressing recombinant proteins in insect cells, we show that mBub3, like yeast Bub3p, binds to Bub1 to form a complex with protein kinase activity. During prophase and prometaphase, preceding kinetochore-microtubule attachment, Bub3 localizes to kinetochores. High levels of mBub3 remain associated with lagging chromosomes but not with correctly aligned chromosomes during metaphase, consistent with a role for Bub3 in sensing microtubule attachment. Intriguingly, the number of lagging chromosomes with high Bub3 staining increases dramatically in cells treated with low (and pharmacologically relevant) concentrations of the chemotherapeutic taxol and the microtubule poison nocodazole.
The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.
The mitotic checkpoint is thought to be essential for ensuring accurate chromosome segregation by implementing mitotic delay in response to a spindle defect. To date, however, very little data has become available on the defects of the mitotic checkpoint in human cancer cells. In the present study, impaired mitotic checkpoint was found in four (44%) of nine human lung cancer cell lines. To our knowledge, this is the first demonstration of frequent impairment of the mitotic checkpoint in this leading cause of cancer deaths. As an initial step towards elucidation of the underlying mechanism, we further undertook a search for mutations in a key component of the mitotic checkpoint, known as hsMAD2, and its immediate downstream molecule, p55CDC. No such mutations were found, however, in either 21 lung cancer cell lines or 25 primary lung cancer cases, although we could identify silent polymorphisms and the transcribed and processed hsMAD2 pseudogene that was subsequently mapped at 14q21-q23. The present observations appear to warrant further investigations, such as search for alterations in other components, to better understand the molecular pathogenesis of this fatal disease, and warn against potential misinterpretation when performing mutational analyses for other cancer types based on cDNA templates.
Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386-396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.
The spindle-assembly checkpoint ensures that, during mitosis and meiosis, chromosomes do not segregate until they are properly attached to the microtubules of the spindle. Here we show that mdf-1 and mdf-2 are components of the spindle-assembly checkpoint in Caenorhabditis elegans, and are essential for the long-term survival and fertility of this organism. Loss of function of either of these genes leads to the accumulation of a variety of defects, including chromosome abnormalities, X-chromosome non-disjunction or loss, problems in gonad development, and embryonic lethality. Antibodies that recognize the MDF-2 protein localize to nuclei of the cleaving embryo in a cell-cycle-dependent manner. mdf-1, a gene encoding a product that interacts with MDF-2, is required for cell-cycle arrest and proper chromosome segregation in premeiotic germ cells treated with nocodazole, a microtubule-depolymerizing agent. In the absence of mdf gene products, errors in chromosome segregation arise and accumulate, ultimately leading to genetic lethality.
A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast
and the Cut2p protein of fission yeast. The vSecurin protein bound to a vertebrate homolog of yeast separins Esp1p and Cut1p
and was degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif. Furthermore,
expression of a stableXenopus securin mutant protein blocked sister-chromatid separation but did not block the embryonic cell cycle. The vSecurin proteins
share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts.
Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells. The oncogenic nature of increased
expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation.
Mad2 is a component of the spindle checkpoint, which delays the onset of anaphase until all chromosomes are attached to the
spindle. Mad2 formed a complex with Slp1, a WD (tryptophan–aspartic acid)–repeat protein essential for the onset of anaphase.
When the physical interaction between the two proteins was disrupted, the spindle checkpoint was no longer functional. Post-anaphase
events such as chromosome decondensation and the next round of DNA replication were not delayed even when the spindle assembly
was incomplete. This relief of dependence appears to be a result of deregulation of ubiquitin-dependent proteolysis mediated
by the anaphase-promoting complex.
The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In
the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact
with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle.
The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized
spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that
were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle
checkpoint.
During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.
Genetic instability was one of the first characteristics to be postulated to underlie neoplasia. Such genetic instability occurs in two different forms. In a small fraction of colorectal and some other cancers, defective repair of mismatched bases results in an increased mutation rate at the nucleotide level and consequent widespread microsatellite instability. In most colorectal cancers, and probably in many other cancer types, a chromosomal instability (CIN) leading to an abnormal chromosome number (aneuploidy) is observed. The physiological and molecular bases of this pervasive abnormality are unknown. Here we show that CIN is consistently associated with the loss of function of a mitotic checkpoint. Moreover, in some cancers displaying CIN the loss of this checkpoint was associated with the mutational inactivation of a human homologue of the yeast BUB1 gene; BUB1 controls mitotic checkpoints and chromosome segregation in yeast. The normal mitotic checkpoints of cells displaying microsatellite instability become defective upon transfer of mutant hBUB1 alleles from either of two CIN cancers.
While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition.
Every time a cell divides, the chromosomes must be distributed accurately to the daughter cells. Errors in distribution arise if chromosomes are improperly attached to the mitotic spindle. Improper attachment is detected by a cell-cycle checkpoint in many cells and the completion of cell division is delayed, allowing time for error correction. How is an improperly attached chromosome detected? An absence of tension from mitotic forces is one possibility. Here we test this possibility directly by applying tension to an improperly attached chromosome with a micromanipulation needle. In the absence of tension, the entry into anaphase and the completion of mitosis was delayed by 5-6 hours. When the misattached chromosome was placed under tension, however, the cell entered anaphase in 56 minutes, on average. Tension from mitotic forces or from a micromanipulator's needle evidently signals to the checkpoint that all is in order and that cell division can proceed.
Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints
to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement,
we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with
a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified
genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here
we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3
are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two.
Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses
kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1
was found to localize to the cell nucleus.
The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation. The protein kinase
Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification
of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles. Spindle assembly checkpoint mutants
overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest
of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional. Ectopic
activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor
cells and thus improve the selectivity of chemotherapy.
In Saccharomyces cerevisiae, MAD2 is required for mitotic arrest if the spindle assembly is perturbed. The human homolog of MAD2 was isolated and shown to be a necessary component of the mitotic checkpoint in HeLa cells by antibody electroporation experiments.
Human, or Homo sapiens, MAD2 (hsMAD2) was localized at the kinetochore after chromosome condensation but was no longer observed at the kinetochore
in metaphase, suggesting that MAD2 might monitor the completeness of the spindle-kinetochore attachment. Finally, T47D, a
human breast tumor cell line that is sensitive to taxol and nocodazole, had reduced MAD2 expression and failed to arrest in
mitosis after nocodazole treatment. Thus, defects in the mitotic checkpoint may contribute to the sensitivity of certain tumors
to mitotic spindle inhibitors.
The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle. The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when
microtubules are depolymerized. A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts. XMAD2 protein
associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores
at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores.
This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying
the role of the spindle assembly checkpoint in the genetic instability of tumors.
When cells divide, the chromosomes must be delivered flawlessly to the daughter cells. Missing or extra chromosomes can result
in birth defects and cancer. Chance events are the starting point for chromosome delivery, which makes the process prone to
error. Errors are avoided by diverse uses of mechanical tension from mitotic forces. Tension stabilizes the proper chromosome
configuration, controls a cell cycle checkpoint, and changes chromosome chemistry.
Pathogenesis of tumor formation in the anterior pituitary has been intensively studied, but the common mechanism involved in pituitary cell transformation and tumorigenesis remains elusive. In this study, we used mRNA differential display PCR to identify mRNAs that are differentially expressed in rat pituitary tumor cells compared with normal pituitary tissue. An mRNA exclusively expressed in pituitary tumor but not in normal pituitary was characterized. Using this pituitary tumor-specific PCR product as a probe to screen a cDNA library constructed from rat pituitary tumor GH4 cells, a cDNA of 974 bp was isolated. This cDNA encodes a novel protein of 199 amino acids, which contains no well characterized functional motifs. The mRNA of this cDNA is detected in normal adult testis and in embryonic liver, where the transcript is about 300 bp shorter and expressed at a much lower level than that detected from pituitary tumor cells. Overexpression of this protein in mouse 3T3 fibroblasts shows that it inhibits cell proliferation and induces cell transformation in vitro. Injection of transfected 3T3 cells into athymic nude mice resulted in tumor formation within 3 weeks in all animals. These results indicate that pituitary tumor cells express a unique and potent transforming gene (PTTG), which may play a role in pituitary tumorigenesis.
The mitotic checkpoint ensures proper chromosome segregation by delaying anaphase until chromosomes are aligned on the spindle. Following prolonged spindle damage, however, cells eventually exit mitosis and undergo apoptosis. We show here that a murine homolog of the yeast mitotic checkpoint gene BUB1 localizes to the kinetochore during mitosis. By expressing a dominant-negative mutant, we show that mBub1 is not only required for the checkpoint response to spindle damage, but acts in the timing of a normal mitosis. In addition, when mBub1 function is compromised, cells escape apoptosis and continue cell cycle progression, despite leaving mitosis with a disrupted spindle. These data demonstrate a role for kinetochore-associated mBub1 in regulating exit from mitosis, and suggest functional links between the mitotic checkpoint and subsequent apoptotic events in G1.
Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.
Vertebrate homologs of yeast spindle assembly checkpoint proteins are localized to kinetochores and may act as a sensor for proper chromosome attachment to the mitotic spindle.
The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G
1
following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53
+/+
and p53
−/−
murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G
1
cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21
−/−
fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.
Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly actingBUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression
of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly
but prior to anaphase spindle elongation. Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing
MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, includingBUB2 and BUB3 and MAD1,MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together
at an early step in generating the spindle damage signal.
Despite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).
Prior to sister-chromatid separation, the spindle checkpoint inhibits cell-cycle progression in response to a signal generated by mitotic spindle damage or by chromosomes that have not attached to microtubules. Recent work has shown that the spindle checkpoint inhibits cell-cycle progression by direct binding of components of the spindle checkpoint pathway to components of a specialized ubiquitin-conjugating system that is responsible for triggering sister-chromatid separation.
The BUB/MAD signaling pathway monitors attachment of chromosomes to spindle poles in mitotic cells. Mutations of the human BUB1 locus were identified in cancer cells exhibiting an unstable chromosomal complement. We report that the human BUB3 gene maps to a site on chromosome 10 subject to frequent modification in cancers. Thus, defects in BUB/MAD signaling may contribute to genetic instability and to cancer progression. In vitro, BUB1 and BUB3 proteins form a complex of monomers of each protein. These proteins interact with the human MAD1 gene product, a target of the HTLV-1 tax oncogene. This multiprotein complex exhibits a kinase activity with a requirement for lysine 821 in the BUB1 kinase motif, resulting in BUB1 autophosphorylation and phosphorylation of associated MAD1.
Aneuploidy is a characteristic of the majority of human cancers, and recent work has suggested that mitotic checkpoint defects play a role in its development. To further explore this issue, we isolated a novel human gene, MAD2B (MAD2L2), which is homologous to the spindle checkpoint gene MAD2 (MAD2L1). We determined the chromosomal localization of it and other spindle checkpoint genes, including MAD1L1, MAD2, BUB3, TTK (MPS1L1), and CDC20. In addition, we resolved the genomic intron-exon structure of the human BUB1 gene. We then searched for mutations in these genes in a panel of 19 aneuploid colorectal tumors. No new mutations were identified, suggesting that genes yet to be discovered are responsible for most of the checkpoint defects observed in aneuploid cancers.
A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. The vSecurin protein bound to a vertebrate homolog of yeast separins Esp1p and Cut1p and was degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif. Furthermore, expression of a stable Xenopus securin mutant protein blocked sister-chromatid separation but did not block the embryonic cell cycle. The vSecurin proteins share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts. Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells. The oncogenic nature of increased expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation.
The APC/C was found by searching for the apparatus responsible for destroying mitotic cyclins at the metaphase-to-anaphase transition. This apparatus is a key part of the regulatory network that generates oscillations in the activity of mitotic CDKs. Studies of how the various forms of APC/C are regulated promise to provide a deep understanding of how eukaryotic cells generate CDK waves. However, the APC/C turns out to have a much more fundamental role in the eukaryotic cell cycle than merely being a counterweight to cyclin B synthesis. It mediates the separation of sister chromatids and is the target of regulatory mechanisms whose role is to ensure that daughter cells inherit one (usually two) complete copies of the genome. The control of sister chromatid separation appears to have become more and more important as organisms became more complicated and as their genomes grew in size. Bacteria, for example, barely regulate sister chromatid separation. Indeed, they commence to separate sister chromatids almost as soon as replication has been initiated. At the other extreme are mammalian cells, in which a surveillance mechanism merely needed for high fidelity chromosome transmission in yeast has clearly become an integral part of mitosis. It was the discovery of the APC/C (and SCF) and the key roles that they have in eukaryotic cell reproduction that established once and for all the importance of ubiquitin mediated proteolysis in eukaryotic cell biology. Once perceived as a system exclusively involved in removing damaged proteins from the cell, ubiquitination is now perceived as a universal regulatory mechanism whose importance approaches that of protein phosphorylation. The irreversibility of proteolysis is utilized by cells to give the cell cycle directionality. Once CKIs have been destroyed by SCF, it is very difficult for cells to inactivate Cdks. Their activity drives the onset of DNA replication and entry into mitosis. Likewise, the destruction of cyclin B and Pds1 triggers the separation of sister chromatids and the inactivation of Cdk1. By utilizing the same apparatus to degrade mitotic cyclins and anaphase inhibitors, eukaryotic cells ensure that preparations for chromosome rereplication cannot normally precede the separation of sister chromatids generated by a previous round of DNA replication. The recent discovery that destruction of IκB and β-catenin are mediated by SCF suggests that this formula is not restricted to cell division but also has a key role in signal transduction. The persistence of APC/C in fully differentiated quiescent cells suggests that its powerful substrate recognition apparatus is not confined to proteins involved in cell cycle progression. That proteolysis should have a key role in cell cycle regulation is in retrospect possibly not too surprising. What is still less clear is why so much of the proteolysis that orders the cell cycle should be mediated by large ubiquitin ligase complexes as opposed to more conventional proteases. A key advantage of these ligases is that they can, with great specificity, promote the complete annihilation of a protein without the need for cleavage sites within functional domains of that protein. This then may be one of the key advantages of separating the marking process from proteolysis itself. It is interesting in this regard that the proteolytic program that mediates apoptosis in contrast utilizes proteolytic cleavage. Ubiquitination requires ATP, which is in short supply in dying cells. The cell cycle ends in an orgy of protein degradation that sets the scene for a new round of duplication. With the discovery of the apparatus responsible for this cleanup, we have embarked on a whole new area of cell biology.
A mitotic checkpoint arrests cell cycle progression in response to spindle damage. It now appears that this checkpoint has two separate arms, one that prevents anaphase and a second that prevents cytokinesis and DNA re-replication.
The events of late mitosis, from sister-chromatid separation to cytokinesis,
are governed by the anaphase-promoting complex (APC), a multisubunit assembly
that triggers the ubiquitin-dependent proteloysis of key regulatory proteins.
An intricate regulatory network governs APC activity and helps to ensure that
late mitotic events are properly timed and coordinated.
The proteolysis of key regulatory proteins is thought to control progress
through mitosis. Here we analyse cyclin B1 degradation in real time and find
that it begins as soon as the last chromosome aligns on the metaphase plate,
just after the spindle-assembly checkpoint is inactivated. At this point,
cyclin B1 staining disappears from the spindle poles and from the chromosomes.
Cyclin B1 destruction can subsequently be inactivated throughout metaphase
if the spindle checkpoint is reimposed, and this correlates with the reappearance
of cyclin B1 on the spindle poles and the chromosomes. These results provide
a temporal and spatial link between the spindle-assembly checkpoint and ubiquitin-mediated
proteolysis.