Mitotic Chromosome Segregation Research Articles

Abstract 3114: Mutational inactivation of STAG2 causes aneuploidy in human cancer

Abstract One of the hallmarks of cancer is chromosomal instability, which leads to aneuploidy, translocations, loss of heterozygosity, and other chromosomal aberrations. Chromosomal instability is an early event in cancer pathogenesis and is thought to help generate the large number of genetic lesions required for a cell to undergo malignant transformation. It has been hypothesized that this instability is due to inactivating mutations in genes that control the mitotic checkpoint and chromosome segregation. However, in the vast majority of human tumors the molecular basis of chromosomal instability and the aneuploidy it produces remains unknown. We have recently identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors (Science 333:1039, 2011). A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer. These findings and additional unpublished observations regarding the role of STAG2 inactivation in aneuploidy will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3114. doi:1538-7445.AM2012-3114

Sister acts ‐ coordinating DNA replication & cohesion establishment

The ring‐shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate such linkages is restricted to S phase and depends on so‐called establishment factors that are intimately connected to DNA replication. Paramount among these are the Eco1‐related family of acetyltransferases. By applying high‐resolution microscopy, somatic cell gene‐targeting, and next‐generation sequencing approaches, we have begun to delineate how human Eco1 orthologs are targeted to chromosomes, how their acetyltransferase activity is coupled to cell cycle progression, and how their acetylation of cohesin's Smc3 subunit triggers cohesion establishment.. Our data suggest a model whereby Smc3 acetylation regulates a ‘gate’ within cohesin that otherwise interferes with DNA replication fork passage, and whose closure is crucial for cohesin's stable interaction with replicated DNA molecules.

Novel E3 ligase component FBXL7 ubiquitinates and degrades Aurora A, causing mitotic arrest

Aurora family kinases play pivotal roles in several steps during mitosis. Specifically, Aurora A kinase is an important regulator of bipolar mitotic spindle formation and chromosome segregation. Like other members of the Aurora family, Aurora A kinase is also regulated by post-translational modifications. Here, we show that a previously undescribed E3 ligase component belonging to the SCF (Skp-Cullin1-F-box protein) E3 ligase family, SCFFBXL7, impairs cell proliferation by mediating Aurora A polyubiquitination and degradation. Both Aurora A and FBXL7 co-localize within the centrosome during spindle formation. FBXL7 ectopic expression led to G2/M phase arrest in transformed epithelia, resulting in the appearance of tetraploidy and mitotic arrest with circular monopolar spindles and multipolar spindle formation. Interestingly, FBXL7 specifically interacts with Aurora A during mitosis but not in interphase, suggesting a regulatory role for FBXL7 in controlling Aurora A abundance during mitosis.

The Centrosome in Cells and Organisms

The centrosome acts as the main microtubule-nucleating organelle in animal cells and plays a critical role in mitotic spindle orientation and in genome stability. Yet, despite its central role in cell biology, the centrosome is not present in all multicellular organisms or in all cells of a given organism. The main outcome of centrosome reproduction is the transmission of polarity to daughter cells and, in most animal species, the sperm-donated centrosome defines embryo polarity. Here I will discuss the role of the centrosome in cell polarity, resulting from its ability to position the nucleus at the cell center, and discuss how centrosome innovation might have been critical during metazoan evolution.

DNA breaks and chromosome pulverization from errors in mitosis

Whether whole-chromosome aneuploidy promotes tumorigenesis has been controversial, in large part because of the paucity of insight into underlying mechanisms. Here we identify a mechanism by which mitotic chromosome segregation errors generate DNA breaks via the formation of structures called micronuclei. Whole chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and frequently pulverization of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for “chromothripsis” in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.

Centrosome Loss in the Evolution of Planarians

The centrosome, a cytoplasmic organelle formed by cylinder-shaped centrioles surrounded by a microtubule-organizing matrix, is a hallmark of animal cells. The centrosome is conserved and essential for the development of all animal species described so far. Here, we show that planarians, and possibly other flatworms, lack centrosomes. In planarians, centrioles are only assembled in terminally differentiating ciliated cells through the acentriolar pathway to trigger the assembly of cilia. We identified a large set of conserved proteins required for centriole assembly in animals and note centrosome protein families that are missing from the planarian genome. Our study uncovers the molecular architecture and evolution of the animal centrosome and emphasizes the plasticity of animal cell biology and development.

14-3-3zeta cooperates with Phosphorylated Plk1 and is required for correct cytokinesis

Proteins of the 14-3-3 family are functionally conserved in eukaryotic kingdom which participates in diversified and critical cellular processes. However, the exact roles of these proteins in mitotic regulation has remained elusive. Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that plays multiple critical functions such as centrosome maturation, mitotic chromosome segregation, cytokinesis, and the DNA damage response. Here we show that 14-3-3zeta interacts and cooperates with Plk1 in mitotic progress. 14-3-3zeta is associated with the spindle at metaphase and concentrated in the midbody during cytokinesis. Using yeast two hybrid assay, we found a functional connection between 14-3-3zeta and Plk1. We demonstrate that phosphorylation of Plk1 at S330 and S597 promotes its interaction with 14-3-3zeta. Importantly, 14-3-3zeta cooperates with Plk1 in ensuring successful cytokinesis. We conclude that mitotic phosphorylation of Plk1 promotes interaction with 14-3-3zeta and this interaction is required for faithful cytokinesis. Taken together with the results of previous studies, our results suggest 14-3-3 family emerges as a novel player in mitotic regulation: cooperation with Plk1 to ensure a faithful cytokinesis.

CENP-E Kinesin Interacts with SKAP Protein to Orchestrate Accurate Chromosome Segregation in Mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation.

Transient defects of mitotic spindle geometry and chromosome segregation errors

Assembly of a bipolar mitotic spindle is essential to ensure accurate chromosome segregation and prevent aneuploidy, and severe mitotic spindle defects are typically associated with cell death. Recent studies have shown that mitotic spindles with initial geometric defects can undergo specific rearrangements so the cell can complete mitosis with a bipolar spindle and undergo bipolar chromosome segregation, thus preventing the risk of cell death associated with abnormal spindle structure. Although this may appear as an advantageous strategy, transient defects in spindle geometry may be even more threatening to a cell population or organism than permanent spindle defects. Indeed, transient spindle geometry defects cause high rates of chromosome mis-segregation and aneuploidy. In this review, we summarize our current knowledge on two specific types of transient spindle geometry defects (transient multipolarity and incomplete spindle pole separation) and describe how these mechanisms cause chromosome mis-segregation and aneuploidy. Finally, we discuss how these transient spindle defects may specifically contribute to the chromosomal instability observed in cancer cells.

Different roles for Aurora B in condensin targeting during mitosis and meiosis

Condensin complexes are essential for mitotic and meiotic chromosome segregation. Caenorhabditis elegans, like other metazoans, has two distinct mitotic and meiotic condensin complexes (I and II), which occupy distinct chromosomal domains and perform non-redundant functions. Despite the differences in mitotic and meiotic chromosome behavior, we uncovered several conserved aspects of condensin targeting during these processes. During both mitosis and meiosis, condensin II loads onto chromosomes in early prophase, and condensin I loads at entry into prometaphase. During both mitosis and meiosis, the localization of condensin I, but not condensin II, closely parallels the localization of the chromosomal passenger kinase Aurora B (AIR-2 in C. elegans). Interestingly, condensin I and AIR-2 also colocalize on the spindle midzone during anaphase of mitosis, and between separating chromosomes during anaphase of meiosis. Consistently, AIR-2 affects the targeting of condensin I but not condensin II. However, the role AIR-2 plays in condensin I targeting during these processes is different. In mitosis, AIR-2 activity is required for chromosomal association of condensin I. By contrast, during meiosis, AIR-2 is not required for condensin I chromosomal association, but it provides cues for correct spatial targeting of the complex.

Mitotic Inhibitors

Mitotic Inhibitors

Different roles for Aurora B in condensin targeting during mitosis and meiosis

Condensin complexes are essential for mitotic and meiotic chromosome segregation. Caenorhabditis elegans, like other metazoans, has two distinct mitotic and meiotic condensin complexes (I and II), which occupy distinct chromosomal domains and perform non-redundant functions. Despite the differences in mitotic and meiotic chromosome behavior, we uncovered several conserved aspects of condensin targeting during these processes. During both mitosis and meiosis, condensin II loads onto chromosomes in early prophase, and condensin I loads at entry into prometaphase. During both mitosis and meiosis, the localization of condensin I, but not condensin II, closely parallels the localization of the chromosomal passenger kinase Aurora B (AIR-2 in C. elegans). Interestingly, condensin I and AIR-2 also colocalize on the spindle midzone during anaphase of mitosis, and between separating chromosomes during anaphase of meiosis. Consistently, AIR-2 affects the targeting of condensin I but not condensin II. However, the role AIR-2 plays in condensin I targeting during these processes is different. In mitosis, AIR-2 activity is required for chromosomal association of condensin I. By contrast, during meiosis, AIR-2 is not required for condensin I chromosomal association, but it provides cues for correct spatial targeting of the complex.

Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint

Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.

Mitotic motors and chromosome segregation: the mechanism of anaphase B

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an 'on/off' switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned 'off', tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.

Misregulation of mitotic chromosome segregation in a new type of autosomal recessive primary microcephaly

Primary autosomal recessive microcephaly (MCPH) is a congenital disorder characterized by a pronounced reduction of brain size and mental retardation. We present here a consanguineous Turkish family clinically diagnosed with MCPH and without linkage to any of the known loci (MCPH1-MCPH7). Autozygosity mapping identified a homozygous region of 15.8 Mb on chromosome 10q11.23-21.3, most likely representing a new locus for MCPH. Although we were unable to identify the underlying genetic defect after extensive molecular screening, we could delineate a possible molecular function in chromosome segregation by the characterization of mitosis in the patients’ cells. Analyses of chromosome nondisjunction in T-lymphocytes and fibroblasts revealed a significantly elevated rate of nondisjunction in the patients’ cells as compared to controls. Mitotic progression was further explored by immunofluorescence analyses of several chromosome and spindle associated proteins. We detected a remarkable alteration in the anaphase distribution of Aurora B and INCENP, which are key regulators of chromosome segregation. In particular, a fraction of both proteins remained abnormally loaded on chromosomes during anaphase in MCPH patients’ cells while in cells of normal control subjects both proteins are completely transferred to the spindle midzone. We did not observe any other alterations regarding cell cycle progression, chromosome structure, or response to DNA damage. Our observations point towards a molecular role of the underlying gene product in the regulation of anaphase/telophase progression possibly through interaction with chromosomal passenger proteins. In addition, our findings represent further evidence for the proposed role of MCPH genes in the regulation of mitotic progression.

DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation

During mitosis, faithful inheritance of genetic material is achieved by chromosome segregation, as mediated by the condensin I and II complexes. Failed chromosome segregation can result in neoplasm formation, infertility, and birth defects. Recently, the germ-line-specific DEAD-box RNA helicase Vasa was demonstrated to promote mitotic chromosome segregation in Drosophila by facilitating robust chromosomal localization of Barren (Barr), a condensin I component. This mitotic function of Vasa is mediated by Aubergine and Spindle-E, which are two germ-line components of the Piwi-interacting RNA pathway. Faithful segregation of chromosomes should be executed both in germ-line and somatic cells. However, whether a similar mechanism also functions in promoting chromosome segregation in somatic cells has not been elucidated. Here, we present evidence that belle (vasa paralog) and the RNA interference pathway regulate chromosome segregation in Drosophila somatic cells. During mitosis, belle promotes robust Barr chromosomal localization and chromosome segregation. Belle's localization to condensing chromosomes depends on dicer-2 and argonaute2. Coimmunoprecipitation experiments indicated that Belle interacts with Barr and Argonaute2 and is enriched at endogenous siRNA (endo-siRNA)-generating loci. Our results suggest that Belle functions in promoting chromosome segregation in Drosophila somatic cells via the endo-siRNA pathway. DDX3 (human homolog of belle) and DICER function in promoting chromosome segregation and hCAP-H (human homolog of Barr) localization in HeLa cells, indicating a conserved function for those proteins in human cells. Our results suggest that the RNA helicase Belle/DDX3 and the RNA interference pathway perform a common role in regulating chromosome segregation in Drosophila and human somatic cells.

Aurora Kinase A expression is associated with lung cancer histological-subtypes and with tumor de-differentiation.

BackgroundAurora kinase A (AURKA) is a member of serine/threonine kinase family. Several kinases belonging to this family are activated in the G2/M phase of the cell cycle being involved in mitotic chromosomal segregation. AURKA overexpression is significantly associated with neoplastic transformation in several tumors and deregulated Aurora Kinases expression leads to chromosome instability, thus contributing to cancer progression. The purpose of the present study was to investigate the expression of AURKA in non small cell lung cancer (NSCLC) specimens and to correlate its mRNA or protein expression with patients' clinico-pathological features.Materials and methodsQuantitative real-time PCR and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLC) have been performed aiming to explore the expression levels of AURKA gene.ResultsAURKA expression was significantly up-modulated in tumor samples compared to matched lung tissue (p < 0.01, mean log2(FC) = 1.5). Moreover, AURKA was principally up-modulated in moderately and poorly differentiated lung cancers (p < 0.01), as well as in squamous and adenocarcinomas compared to the non-invasive bronchioloalveolar histotype (p = 0.029). No correlation with survival was observed.ConclusionThese results indicate that in NSCLC AURKA over-expression is restricted to specific subtypes and poorly differentiated tumors.

Regulation of microtubule-based microtubule nucleation by mammalian polo-like kinase 1

Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian polo-like kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1-Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin-MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin-MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1-Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression.

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

A fungus-specific outer kinetochore complex, the Dam1 complex, is essential in Saccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. Both Candida albicans and Schizosaccharomyces pombe have regional centromeres as opposed to the short-point centromeres of S. cerevisiae. The interaction of one microtubule per kinetochore is established both in S. cerevisiae and C. albicans early during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis in S. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle in S. cerevisiae and C. albicans but only during mitosis in S. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation in C. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochore-microtubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.

Abstract 660: Inhibition of centrosomal clustering suppresses tumor growth in vivo

Abstract Tumor cells frequently contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles (centrosomal clustering). We recently described a phenotype-based small molecule screening strategy directed to induce tumor cells with supernumerary centrosomes to undergo multipolar mitoses, thereby resulting in apoptotic cell death (Rebacz et al., Cancer Res 2007; 67: 6342-6350). We here describe the novel small molecule GF-15, a derivative of griseofulvin and a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis. We tested more than 25 cancer cell lines from hematologic malignancies (including acute and chronic leukemias, lymphomas), solid tumors (including glioblastoma, colon, cervix, and pancreatic cancers) and a wide array of myeloma cell lines. We found mean inhibitory concentrations (IC50) for proliferation and survival in the range of 1-5μM GF-15, associated with activation of caspases 8, 9, and 3. As expected, tumor cell lines displaying only limited centrosomal aberrations or microsatellite instability were less sensitive to treatment with GF-15. Importantly, non-malignant cells without supernumerary centrosomes including PBMCs, immortalized hepatocytes, and bone marrow stromal cells, did not reach their IC50 even at 30μM GF-15. Specifically, cell cycle analysis of synchronized myeloma cells showed marked G2/M arrest, followed by a dramatic increase of the sub-G1 fraction, after treatment with GF-15. In addition, treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered myeloma cell migration. Pharmakodynamic studies in vivo revealed rapid renal clearance of GF-15 and metabolites within 6 hours p.i. No acute or chronic toxicity was observed. Finally, both intraperitoneal and oral GF-15 treatment of murine xenograft models of human multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival associated with significant increase of aberrant mitoses in vivo. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of the first in class small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and strongly support its clinical evaluation to improve patient outcome in both hematologic malignancies and solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2011-660