Chromosome mis-segregation and cytokinesis failure in trisomic human cells

Nicholson, Joshua M.; Macedo, Joana Catarina; Mattingly, Aaron; Wangsa, Darawalee; Camps, Jordi; Lima, Vera; Gomes, Ana Margarida; Dória, Sofia; Ried, Thomas; Logarinho, Elsa; Cimini, Daniela

doi:10.7554/elife.05068

Cited by 95 publications

(99 citation statements)

References 74 publications

(125 reference statements)

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“…It is shown in [ 20 ] that certain karyotypes promote cytokinesis failure and thus genome duplication. In particular, it is suggested that cells with 3 or more copies of chromosome 13 have a higher genome duplication rate.…”

Section: Resultsmentioning

confidence: 99%

A Markov chain for numerical chromosomal instability in clonally expanding populations

2018

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Cancer cells frequently undergo chromosome missegregation events during mitosis, whereby the copies of a given chromosome are not distributed evenly among the two daughter cells, thus creating cells with heterogeneous karyotypes. A stochastic model tracing cellular karyotypes derived from clonal populations over hundreds of generations was recently developed and experimentally validated, and it was capable of predicting favorable karyotypes frequently observed in cancer. Here, we construct and study a Markov chain that precisely describes karyotypic evolution during clonally expanding cancer cell populations. The Markov chain allows us to directly predict the distribution of karyotypes and the expected size of the tumor after many cell divisions without resorting to computationally expensive simulations. We determine the limiting karyotype distribution of an evolving tumor population, and quantify its dependency on several key parameters including the initial karyotype of the founder cell, the rate of whole chromosome missegregation, and chromosome-specific cell viability. Using this model, we confirm the existence of an optimal rate of chromosome missegregation probabilities that maximizes karyotypic heterogeneity, while minimizing the occurrence of nullisomy. Interestingly, karyotypic heterogeneity is significantly more dependent on chromosome missegregation probabilities rather than the number of cell divisions, so that maximal heterogeneity can be reached rapidly (within a few hundred generations of cell division) at chromosome missegregation rates commonly observed in cancer cell lines. Conversely, at low missegregation rates, heterogeneity is constrained even after thousands of cell division events. This leads us to conclude that chromosome copy number heterogeneity is primarily constrained by chromosome missegregation rates and the risk for nullisomy and less so by the age of the tumor. This model enables direct integration of karyotype information into existing models of tumor evolution based on somatic mutations.

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“…Human chromosomally stable colorectal cancer cell lines (DLD1) with an extra copy of chromosome 7 or 13 show an increase in chromosome missegregation rates, as do amniotic fibroblasts that are trisomic for chromosome 13. Of interest, these cells predominantly missegregate the trisomic chromosome, suggesting that a portion of them revert to the diploid state ( Nicholson et al. , 2015 ).…”

Section: Resultsmentioning

confidence: 99%

High rates of chromosome missegregation suppress tumor progression but do not inhibit tumor initiation

Zasadil

Britigan

Ryan

et al. 2016

MBoC

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“…Although aneuploid cells are genomically unstable (Blank et al, 2015; Nicholson et al, 2015; Passerini et al, 2016; Sheltzer et al, 2011; Zhu et al, 2012), mutation-induced population heterogeneity is an unlikely cause. The system we employ examines the immediate effects of mis-segregating a specific chromosome, precluding the accumulation of suppressor mutations.…”

Section: Resultsmentioning

confidence: 99%

Aneuploidy Causes Non-genetic Individuality

Beach

Ricci-Tam

Brennan

et al. 2017

Cell

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SUMMARY Phenotypic variability is a hallmark of diseases involving chromosome gains and losses, such as Down Syndrome and cancer. Allelic variances have been thought to be the sole cause of this heterogeneity. Here, we systematically examine the consequences of gaining and losing single or multiple chromosomes to show that the aneuploid state causes non-genetic phenotypic variability. Yeast cell populations harboring the same defined aneuploidy exhibit heterogeneity in cell cycle progression and response to environmental perturbations. Variability increases with degree of aneuploidy and is partly due to gene copy number imbalances, suggesting subtle changes in gene expression impact the robustness of biological networks and cause alternate behaviors when they occur across many genes. As inbred trisomic mice also exhibit variable phenotypes, we further propose that non-genetic individuality is a universal characteristic of the aneuploid state that may contribute to variability in presentation and treatment responses of diseases caused by aneuploidy.

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“…An increased rate of gain or loss of whole chromosomes during each cell cycle is a common feature of many tumor cells, referred to as chromosome instability [12, 13]. Moreover, the rate of chromosome missegregation is higher in trisomic human cells than in euploid cells [14]. Therefore, extra copies of chromosome 21 integrating the DNA cassette may tend to be lost from the HeLa cells spontaneously under condition in which cells including HSV-tk gene are excluded.…”

Section: Resultsmentioning

confidence: 99%

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

Satō

Kato

Yamaza

et al. 2017

BioMed Research International

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Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

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“…1 A ) ( 29 , 33 ). The DLD-1 colorectal cancer cell line was chosen because it does not express EWSR1/FLI1 endogenously, and because it has a near-diploid karyotype, it has been used as a model to evaluate proteins of interest for their ability to induce aneuploidy ( 24 , 34 ). The inducible system enabled us to assess the activity of EWSR1/FLI1 in the induction of aneuploidy after a single cell cycle.…”

Section: Resultsmentioning

confidence: 99%

“…Using these three DNA constructs, we established stable Tet-on cell lines by integrating EWSR1/FLI1, EWSR1/FLI1-T79A and EWSR1/FLI1-T79D transgenes (each with an mCherry tag) into the safe-harbor AAVS1 locus of DLD-1 cells using CRISPR/Cas9 technology (Fig 1A) (29,33). The DLD-1 colorectal cancer cell line was chosen because it does not express EWSR1/FLI1 endogenously, and, because it has a near-diploid karyotype, it has been used as a model to evaluate proteins of interest for their ability to induce aneuploidy (24,34). The inducible system enabled us to assess the activity of EWSR1/FLI1 in the induction of aneuploidy after a single cell cycle.…”

Section: Ewsr1/fli1 Localizes To the Chromosomes At Prophase And Resmentioning

confidence: 99%

“…Although the tumor cells display a high incidence of aneuploidy, it is unclear whether aneuploidy is induced in the early stages of tumorigenesis and promotes the progression of the disease, or if it is a by-product of tumorigenesis and expands during tumor progression. Aneuploidy is the result of abnormal chromosome segregation, which may be caused by the failure of any of several mitotic processes (including chromosome cohesion; mitotic spindle (K-fiber) attachment to the kinetochore of the chromosome; the spindle assembly checkpoint (SAC); the amplification of centrosomes; or cytokinesis accompanied by defects in midbody structure) ( 23 , 24 ). We previously demonstrated that EWSR1/FLI1 impairs the recruitment of the key mitotic regulator Aurora B kinase to the midzone, (the network of antiparallel microtubules and midzone proteins located between segregating chromosomes during anaphase) ( 25 ).…”

mentioning

confidence: 99%

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Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

Park

Kim

Hassebroek

et al. 2021

Journal of Biological Chemistry

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Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.

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“…In other respects, incomplete or delayed cell plate formation in the mun‐1 mutant by mis‐segregation of chromosomes during the process of cell division may affect mis‐localization of PIN1, which perturbs proper auxin flow. In humans, cancer cells that display aneuploid karyotypes and mis‐segregate chromosomes show a distinctive cytokinesis failure phenotype (Nicholson et al ., ). Because the growth of the cell plate is dependent upon the phragmoplast formation in plants (Higaki et al ., ) and chromosome defects generally affect phragmoplast development, we expected the defects in cell walls in mun‐1 .…”

Section: Discussionmentioning

confidence: 97%

MUN (MERISTEM UNSTRUCTURED), encoding a SPC24 homolog of NDC80 kinetochore complex, affects development through cell division in Arabidopsis thaliana

et al. 2018

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Kinetochore, a protein super-complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured-1 (mun-1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun-1 is a weak allele because of the insertion of T-DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9-mediated mutagenesis showed zygotic embryonic lethality similar to nuf2-1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two-hybrid assay and in planta co-immunoprecipitation. MUN is co-localized at the centromere with HTR12/CENH3, which is a centromere-specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.

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“…Aneuploidy has been posited to predispose somatic cells to acquired chromosomal instability [30][31][32][33][34][35], but this hypothesis has been challenging to test in humans [36]. Most of the extant literature assessing aneuploidy-related SCINF has been derived from cancer cell lines, the latter of which often have a cascade of biological/genetic changes that can confound the interpretation of the resultant data.…”

Section: Discussionmentioning

confidence: 99%

“…Passerini, et al [46] further suggested that the observed increase in trisomy-related genomic instability was attributable to replication defects and increased sensitivity to replication stress. Other investigators have also reported increases in SCINF in trisomic cells, with several of these investigators attributing the increase in chromosomal instability/mis-segregation to anaphase lagging of a chromosome or chromatid [32,[47][48][49].…”

Section: Plos Onementioning

confidence: 98%

Trisomy 21-associated increases in chromosomal instability are unmasked by comparing isogenic trisomic/disomic leukocytes from people with mosaic Down syndrome

et al. 2021

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Down syndrome, which results from a trisomic imbalance for chromosome 21, has been associated with 80+ phenotypic traits. However, the cellular changes that arise in somatic cells due to this aneuploid condition are not fully understood. The primary aim of this study was to determine if germline trisomy 21 is associated with an increase in spontaneous somatic cell chromosomal instability frequencies (SCINF). To achieve this aim, we quantified SCINF in people with mosaic Down syndrome using a cytokinesis-blocked micronucleus assay. By comparing values in their isogenic trisomic/disomic cells, we obtained a measure of differences in SCINF that are directly attributable to a trisomy 21 imbalance, since differential effects attributable to “background” genetic factors and environmental exposures could be eliminated. A cross-sectional assessment of 69 people with mosaic Down syndrome (ages 1 to 44; mean age of 12.84 years) showed a significantly higher frequency of micronuclei in their trisomic (0.37 ± 0.35 [mean ± standard deviation]) compared to disomic cells (0.18 ± 0.11)(P <0.0001). The daughter binucleates also showed significantly higher levels of abnormal patterns in the trisomic (1.68 ± 1.21) compared to disomic (0.35 ± 0.45) cells (P <0.0001). Moreover, a significant Age x Cell Type interaction was noted (P = 0.0113), indicating the relationship between age and SCINF differed between the trisomic and disomic cells. Similarly, a longitudinal assessment (mean time interval of 3.9 years; range of 2 to 6 years) of 18 participants showed a mean 1.63-fold increase in SCINF within individuals over time for their trisomic cells (P = 0.0186), compared to a 1.13-fold change in their disomic cells (P = 0.0464). In summary, these results showed a trisomy 21-associated, age-related increase in SCINF. They also underscore the strength of the isogenic mosaic Down syndrome model system for “unmasking” cellular changes arising from a trisomy 21 imbalance.

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“…More ambiguously, the RAB18-interacting microtubule binding proteins have not previously been reported to work together but do function in compatible locations. SPG20 and CAMSAP1 each associate with mitotic spindle poles, REEP4 participates in spindle-dependent ER clearance from metaphase chromatin and BICD2 is a component of the minus-end-directed dynein-dynactin motor complex (Hendershott & Vale, 2014, Hueschen et al, 2017, Kumar et al, 2019, Lind et al, 2011, Nicholson et al, 2015, Schlaitz et al, 2013, Urnavicius et al, 2015). Our TRAPPII-dependent RAB18 interaction data indicate that different GEF complexes affect largely distinct subsets of interactions.…”

Section: Discussionmentioning

confidence: 99%

Comparative proximity biotinylation implicates RAB18 in sterol mobilization and biosynthesis

Chicoine

Abou-Khalil

et al. 2019

Preprint

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65 Mark T. Handley: m.handley@leeds.ac.uk 66 67 KEYWORDS 68 RAB18, BioID, Cholesterol, Lathosterol, EBP, ORP2 69 70 SUMMARY STATEMENT 71We used proximity biotinylation together with guanine nucleotide exchange factor 72 (GEF)-null cell lines to discriminate functional RAB18-interactions. This approach 73 revealed that RAB18 mediates lathosterol mobilization and cholesterol biosynthesis. 74 ABSTRACT 76Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro 77 syndrome. To better understand this disease, we used proximity biotinylation in 78 HEK293 and HeLa cells to generate an inventory of potential RAB18 effectors. A 79 restricted set of 25 RAB18-interactions were regulated by the binary RAB3GAP1-80 RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. These 81 included three groups of functionally interrelated proteins: a group of microtubule-82 interacting/membrane shaping proteins; a group of proteins involved in membrane 83 tethering and docking; and a group of lipid-modifying/lipid transport proteins. We 84 provide confirmatory evidence for several of the interactors (SPG20/SPART, 85 SEC22A, TMCO4). Further we provide functional evidence that RAB18 links the Δ8-86

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“…, 2009; Sheltzer et al. , 2011; Nicholson et al. , 2015) suggests that even a transient exposure to microenvironmental stresses could be sufficient to kick-start a vicious loop, generating further genetic and karyotypic variation.…”

Section: Discussionmentioning

confidence: 99%

“…To support this notion, the presence of specific aneuploid chromosomes has been shown to increase genomic instability (Zhu et al. , 2012; Nicholson et al. , 2015) and drug resistance (Lee et al.…”

Section: Introductionmentioning

confidence: 99%

Environmental stresses induce karyotypic instability in colorectal cancer cells

Tan

Chan

Chua

et al. 2019

MBoC

Self Cite

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Understanding how cells acquire genetic mutations is a fundamental biological question with implications for many different areas of biomedical research, ranging from tumor evolution to drug resistance. While karyotypic heterogeneity is a hallmark of cancer cells, few mutations causing chromosome instability have been identified in cancer genomes, suggesting a nongenetic origin of this phenomenon. We found that in vitro exposure of karyotypically stable human colorectal cancer cell lines to environmental stress conditions triggered a wide variety of chromosomal changes and karyotypic heterogeneity. At the molecular level, hyperthermia induced polyploidization by perturbing centrosome function, preventing chromosome segregation, and attenuating the spindle assembly checkpoint. The combination of these effects resulted in mitotic exit without chromosome segregation. Finally, heat-induced tetraploid cells were on the average more resistant to chemotherapeutic agents. Our studies suggest that environmental perturbations promote karyotypic heterogeneity and could contribute to the emergence of drug resistance.

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“…The two identified clusters could potentially be regulating genes on other chromosomes. Nicholson and colleagues found that the overexpression of one gene, SPG20 , expressed in cluster 13q13.3, promotes cytokinesis failure in the DLD-1 + 13 cells [19].…”

Section: Discussionmentioning

confidence: 99%

Induced Chromosomal Aneuploidy Results in Global and Consistent Deregulation of the Transcriptome of Cancer Cells

et al. 2019

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Chromosomal aneuploidy is a defining feature of epithelial cancers. The pattern of aneuploidies is cancer-type specific. For instance, the gain of chromosome 13 occurs almost exclusively in colorectal cancer. We used microcell-mediated chromosome transfer to generate gains of chromosome 13 in the diploid human colorectal cancer cell line DLD-1. Extra copies of chromosome 13 resulted in a significant and reproducible up-regulation of transcript levels of genes on chromosome 13 ( P = .0004, FDR = 0.01) and a genome-wide transcriptional deregulation in all 8 independent clones generated. Genes contained in two clusters were particularly affected: the first cluster on cytoband 13q13 contained 7 highly up-regulated genes ( NBEA, MAB21L1, DCLK1, SOHLH2, CCDC169, SPG20 and CCNA1 , P = .0003) in all clones. A second cluster was located on 13q32.1 and contained five upregulated genes ( ABCC4, CLDN10, DZIP1, DNAJC3 and UGGT2, P = .003). One gene, RASL11A, localized on chromosome band 13q12.2 , escaped the copy number-induced overexpression and was reproducibly and significantly down-regulated on the mRNA and protein level ( P = .0001, FDR = 0.002). RASL11A expression levels were also lower in primary colorectal tumors as compared to matched normal mucosa ( P = .0001, FDR = 0.0001. Overexpression of RASL11A increases cell proliferation and anchorage independent growth while decreasing cell migration in +13 clones. In summary, we observed a strict correlation of genomic copy number and resident gene expression levels, and aneuploidy dependent consistent genome-wide transcriptional deregulation.

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Chromosome mis-segregation and cytokinesis failure in trisomic human cells

Cited by 95 publications

References 74 publications

A Markov chain for numerical chromosomal instability in clonally expanding populations

High rates of chromosome missegregation suppress tumor progression but do not inhibit tumor initiation

Aneuploidy Causes Non-genetic Individuality

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

MUN (MERISTEM UNSTRUCTURED), encoding a SPC24 homolog of NDC80 kinetochore complex, affects development through cell division in Arabidopsis thaliana

Trisomy 21-associated increases in chromosomal instability are unmasked by comparing isogenic trisomic/disomic leukocytes from people with mosaic Down syndrome

Comparative proximity biotinylation implicates RAB18 in sterol mobilization and biosynthesis

Environmental stresses induce karyotypic instability in colorectal cancer cells

Induced Chromosomal Aneuploidy Results in Global and Consistent Deregulation of the Transcriptome of Cancer Cells

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