scholarly journals Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

2019 ◽  
Author(s):  
Suganthi Chittaranjan ◽  
Jungeun Song ◽  
Susanna Y. Chan ◽  
Stephen Dongsoo Lee ◽  
Shiekh Tanveer Ahmad ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Chromosomal Instability ◽  
Spindle Assembly Checkpoint ◽  
Tumour Suppressor ◽  
Cell Cycle Regulators ◽  
Loss Of Function ◽  
Wide Range ◽  
Cancer Types

AbstractBackgroundCIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts.ResultsUsing mammalian cells lacking functional CIC, we found that CIC deficiency was associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability.ConclusionsOur study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC’s increasingly apparent role as a “pan-cancer” tumour suppressor.

scholarly journals p21 Is a Critical CDK2 Regulator Essential for Proliferation Control in Rb-deficient Cells

1998 ◽  
Vol 141 (2) ◽  
pp. 503-514 ◽  
Author(s):  
James Brugarolas ◽  
Roderick T. Bronson ◽  
Tyler Jacks
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Tumor Development ◽  
Tumor Formation ◽  
Cell Cycle Regulators ◽  
Loss Of Function ◽  
Extrinsic Factors ◽  
Cyclin Dependent Kinases ◽  
Inhibition Of Growth

Proliferation in mammalian cells is controlled primarily in the G1-phase of the cell cycle through the action of the G1 cyclin–dependent kinases, CDK4 and CDK2. To explore the mechanism of cellular response to extrinsic factors, specific loss of function mutations were generated in two negative regulators of G1 progression, p21 and pRB. Individually, these mutations were shown to have significant effects in G1 regulation, and when combined, Rb and p21 mutations caused more profound defects in G1. Moreover, cells deficient for pRB and p21 were uniquely capable of anchorage-independent growth. In contrast, combined absence of pRB and p21 function was not sufficient to overcome contact inhibition of growth nor for tumor formation in nude mice. Finally, animals with the genotype Rb+/−;p21−/− succumbed to tumors more rapidly than Rb+/− mice, suggesting that in certain contexts mutations in these two cell cycle regulators can cooperate in tumor development.

scholarly journals The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 489-503 ◽  
Author(s):  
Karen E Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Growth Defects ◽  
Genes Encoding ◽  
Specificity Factor

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

scholarly journals SAT-LB57 The Spectrum of Genomic and Transcriptomic Alterations in ACTH-Producing and ACTH-Silent Corticotroph Adenomas

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Antonio Marcondes Lerario ◽  
David Meredith ◽  
Joseph Castlen ◽  
Lauren M Johnson ◽  
Michael Catalino ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosomal Instability ◽  
Chromosomal Abnormalities ◽  
Dominant Role ◽  
Telomere Maintenance ◽  
High Morbidity ◽  
Disease Course ◽  
Loss Of Function ◽  
Aggressive Disease ◽  
Corticotroph Adenomas

Abstract Corticotroph adenomas (CA) are rare pituitary tumors that impose several challenges in clinical management - CA are difficult to diagnose, often recur, and are associated with high morbidity and mortality. CA are characteristically Tpit-positive and PIT1-negative and comprise ACTH-producing (Cushing’s disease (CD)) and ACTH-silent (AS) classes. The molecular programs contributing to disease pathogenesis in CA are still poorly characterized, largely restricted to the identification of somatic mutations in USP8 in 40-60% of CD adenomas. To more fully characterize the mutational and transcriptional landscape driving both classes of CA, we performed whole-exome sequencing and RNA-seq in 19 CD and 16 AS adenomas. We identified USP8 mutations in 53% of CD (10/19) and 6% of AS (1/16) samples. Strikingly, in 19% of AS tumors (3/16), all exhibiting an unusually aggressive disease course, including two cases with brain metastases, we identified recurrent somatic pathogenic mutations in TP53 and novel loss-of-function mutations in telomere maintenance genes DAXX and ATRX. Furthermore, while all tumors with USP8 mutations (regardless of CD/AS status) exhibited no chromosomal abnormalities as measured by copy-number variation (CNV) and loss of heterozygosity (LOH) analysis, 33% of CD (4/12, including 1 tumor with a DAXX mutation) and 36% of AS (4/11, including all DAXX/ATRX-mutated cases) samples exhibited profound chromosomal instability, characterized by hyperdiploidy, widespread whole-chromosome LOH events, and arm-level breakpoints. Using transcriptome analysis (n=22), we identified three classes of tumors (C1-C3), reflecting these distinct somatic alteration profiles. C1 tumors (n=6) are characterized by chromosomal stability, includes exclusively USP8-mutated CD, and exhibits upregulation of genes involved in metabolic processes and protein acetylation. C2 tumors (n=10) are comprised exclusively of AS (including all TP53- and/or DAXX/ATRX-mutated cases), are characterized by chromosomal instability, and exhibits concordant upregulation of cell cycle programs. Finally, C3 (n=6) contains a mixture of AS and CD cases (including CD without mutations in USP8) and features an expression profile that partly overlap with C1 tumors, but also exhibit higher expression of inflammatory genes. Taken together, our data suggest that CD and AS are distinct molecular subtypes of CA, highlighting the dominant role of USP8 mutations in driving a unique transcriptional program and illustrate for the first time that unlike most cases of CD, AS cases are characterized by profound genomic instability and cell cycle activation, features associated with a more aggressive disease course.

Evidence that the spindle assembly checkpoint does not regulate APCFzy activity in Drosophila female meiosis

Genome ◽  
2012 ◽  
Vol 55 (1) ◽  
pp. 63-67 ◽  
Author(s):  
Osamah Batiha ◽  
Andrew Swan
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Female Meiosis ◽  
Chromosome Attachment ◽  
Spindle Microtubules ◽  
Made In

The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APCFzy-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APCFzy inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APCFzy-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APCFzy to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.

scholarly journals PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

2014 ◽  
Vol 206 (7) ◽  
pp. 833-842 ◽  
Author(s):  
Antonio Espert ◽  
Pelin Uluocak ◽  
Ricardo Nunes Bastos ◽  
Davinderpreet Mangat ◽  
Philipp Graab ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Eukaryotic Cells ◽  
Aurora B ◽  
Safeguard Mechanism

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.

Unscheduled Sister-Chromatid-Separation in the Presence of Spindle Disruption in Acute Myelogenous Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3114-3114
Author(s):  
Dominik Schnerch ◽  
Julia Felthaus ◽  
Monika Engelhardt ◽  
Ralph M. Waesch
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Sister Chromatid ◽  
Spindle Assembly Checkpoint ◽  
Burkitt’S Lymphoma ◽  
Lymphoma Cell ◽  
Myelogenous Leukemia ◽  
Burkitt's Lymphoma ◽  
Cell Cycle Regulators ◽  
Sister Chromatid Separation

Abstract Mitosis is known to be one of the most critical events in the cell cycle. The spindle assembly checkpoint (SAC) is required for proper chromosome segregation during mitosis. The SAC serves as a mitotic surveillance mechanism responsible for detection of misassembly of chromosomes to the mitotic spindle. Lack of chromosome attachment and spindle tension generate a specific „wait-anaphase-signal“. This particular signal interferes with proteolysis, depending on the ubiquitin-ligase APCCdc20, thereby inhibiting mitotic progression through stabilization of mitotic regulators. We found several AML cell lines to be incapable of properly accumulating in mitosis upon nocodazole-induced spindle disruption when compared to a set of Burkitt’s lymphoma cell lines. This result was further supported by the degradation of the mitotic regulators Cyclin B and Securin in synchronized Kasumi-1 cells in the presence of nocodazole shortly after entering mitosis. Interestingly, the SAC proteins BubR1 and Bub1 were found at low expression levels in AML cell lines in comparison to Burkitt’s lymphoma cell lines. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found in AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Our findings support the view that BubR1 repression alone is sufficient to confer SAC deficiency. To determine the frequency of BubR1 repression in patient-derived primary cells, AML blasts were cytokine-stimulated to enter the cell cycle. Flow cytometry-based G2/M-specific expression analysis of BubR1 in primary AML blasts revealed lower expression in most analyzed cell populations. To further test the hypothesis that AML cells override the metaphase-to-anaphase-transition despite spindle damage, we performed Giemsa staining of cells that were incubated in nocodazole containing growth medium. In AML cell lines, unlike the analyzed Burkitt’s lymphoma cell lines, a significant number of metaphase-like cells contained single chromatids, suggesting premature sister chromatid separation in the presence of spindle damage. Premature sister-chromatid-separation in the presence of chromosomal misalignment would lead to aneuploidy and favor the onset of genomic instability. Our recent efforts focus on high-throughput automated live cell scanning, promising a better understanding of cell division and chromosome separation in the context of different challenges, such as spindle damage. This powerful tool allows a more precise characterization of our knockdown phenotypes in the double-knockdown system, which is a prerequisite for comparison of our model system with AML cell lines. Finally, this new technique might also prove useful to extend our analyses to patient derived AML blasts. As we observed deregulation of SAC protein levels in AML cell lines and primary AML blasts, our findings of premature degradation of cell cycle regulators and unscheduled sister-chromatid-separation suggest an important role for SAC malfunction in the development of AML with karyotypic abnormalities. Mitotic kinases, such as Plk1 and Aurora, are already promising targets for modern antineoplastic therapies. A deeper understanding of mitotic control in AML might contribute to even more sophisticated targeted therapeutic approaches.

scholarly journals A Novel Mammalian, Mitotic Spindle–associated Kinase Is Related to Yeast and Fly Chromosome Segregation Regulators

1997 ◽  
Vol 138 (3) ◽  
pp. 643-656 ◽  
Author(s):  
Ganesan Gopalan ◽  
Clarence S.M. Chan ◽  
Peter J. Donovan
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Proximal Part ◽  
Yeast Cells ◽  
Mutant Form ◽  
Wild Type ◽  
Protein Levels ◽  
M Phase

We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases—Ipl1 and aurora, respectively—mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.

scholarly journals Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

2015 ◽  
Vol 35 (14) ◽  
pp. 2448-2463 ◽  
Author(s):  
Matthew Jessulat ◽  
Ramy H. Malty ◽  
Diem-Hang Nguyen-Tran ◽  
Viktor Deineko ◽  
Hiroyuki Aoki ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Large Scale ◽  
Spindle Assembly Checkpoint ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Cell Cycle Regulators ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Content Type

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.

scholarly journals TheSac3Homologueshd1Is Involved in Mitotic Progression in Mammalian Cells

2004 ◽  
Vol 279 (44) ◽  
pp. 46182-46190 ◽  
Author(s):  
Sefat-e- Khuda ◽  
Mikoto Yoshida ◽  
Yan Xing ◽  
Tatsuya Shimasaki ◽  
Motohiro Takeya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Fluorescent Protein ◽  
Spindle Assembly ◽  
Centrosome Duplication ◽  
Cycle Progression ◽  
M Phase ◽  
Green Fluorescent

SaccharomycesSac3 required for actin assembly was shown to be involved in DNA replication. Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression. SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to α-tubulin, at M phase. RNA interference suppression of endogenousshd1caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei. Conversely, increased expression of SHD1 by DNA transfection withshd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells. These results demonstrated thatshd1is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function.

scholarly journals Dominant Alleles of Saccharomyces cerevisiae CDC20 Reveal Its Role in Promoting Anaphase

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 599-610
Author(s):  
Eric J Schott ◽  
M Andrew Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Limiting Factor ◽  
Mutant Form ◽  
Loss Of Function ◽  
Cycle Progression ◽  
Inhibit Cell Cycle Progression ◽  
M Phase

Abstract We identified an allele of Saccharomyces cerevisiae CDC20 that exhibits a spindle-assembly checkpoint defect. Previous studies indicated that loss of CDC20 function caused cell cycle arrest prior to the onset of anaphase. In contrast, CDC20-50 caused inappropriate cell cycle progression through M phase in the absence of mitotic spindle function. This effect of CDC20-50 was dominant over wild type and was eliminated by a second mutation causing loss of function, suggesting that it encodes an overactive form of Cdc20p. Overexpression of CDC20 was found to cause a similar checkpoint defect, causing bypass of the preanaphase arrest produced by either microtubule-depolymerizing compounds or MPS1 overexpression. CDC20 overexpression was also able to overcome the anaphase delay caused by high levels of the anaphase inhibitor Pds1p, but not a mutant form immune to anaphase-promoting complex- (APC-)mediated proteolysis. CDC20 overexpression was unable to promote anaphase in cells deficient in APC function. These findings suggest that Cdc20p is a limiting factor that promotes anaphase entry by antagonizing Pds1p. Cdc20p may promote the APC-dependent proteolytic degradation of Pds1p and other factors that act to inhibit cell cycle progression through mitosis.

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