Rapid proliferation of daughter cells lacking particular chromosomes due to multipolar mitosis promotes clonal evolution in colorectal cancer cells

Aneuploidy and chromosome instability (CIN) are hallmarks of the vast majority of solid tumors. However, the origins of aneuploid cells are unknown. The aim of this study is to improve our understanding of how aneuploidy and/or CIN arise and of karyotype evolution in cancer cells. By using fluorescence in situ hybridization (FISH) on cells after long-term live cell imaging, we demonstrated that most (> 90%) of the newly generated aneuploid cells resulted from multipolar divisions. Multipolar division occurred in mononucleated and binucleated parental cells, resulting in variation of chromosome compositions in daughter cells. These karyotypes can have the same chromosome number as their mother clone or lack a copy of certain chromosomes. Interestingly, daughter cells that lost a chromosome were observed to survive and form clones with shorter cell cycle duration. In our model of cancer cell evolution, the rapid proliferation of daughter cells from multipolar mitosis promotes colonal evolution in colorectal cancer cells.     

A quantitative study of the second meiotic metaphase in male mice (Mus musculus).

Over 11,000 second meiotic metaphase spreads stained for the pericentromeric region have been studied quantitatively in male mice of 14 strains. The sex-chromosome constitution of a cell could be judged objectively if X and Y chromosomes and ploidy were all scored. A bias arose if only Y chromosomes and ploidy were scored but could be corrected statistically. There was no sign of other forms of bias. The original contiguity of X and Y second metaphases in vivo was very occasionally evident in the preparations. Most of the subhaploid aneuploid counts were assumed to be artifactual. The incidence of truly aneuploid second metaphases in 13 strains was estimated as 0.38+/-0.12%. The estimated average rate per chromosome was 0.019+/-0.006%, with a comparable order of magnitude for the sex chromosomes alone. Simultaneous aneuploidy of two or more chromosomes of the haploid set was estimated to be very rare. Of the spreads from 13 strains, 9.6% were polyploid (2N, 3N, 4N) and showed most of the possible combinations of sex chromosomes. Nearly all the polyploid spreads were considered to arise by artifactual cell fusion at the time of second metaphase during the preparative technique, especially of the X and Y daughter-cell products of the first meiotic division. Other modes of origin (true polyploidy, accidental superposition of cells during preparation) were unlikely. The data could be accommodated by a statistical model with only four parameters. It allowed for artifactual fusion mainly between daughter cells but also between non-daughter cells, bias in one scoring method, and bias in the numbers of cells with given ploidy successfully mounted. Current techniques of chromosome preparation were thought to be wholly unsuitable for the recognition of true polyploidy. The artifactual origin of polyploid spreads was borne out by an absence of polyploid spermatozoa in 14 strains. There appeared to be a virtually constant transmission rate of paternal X and Y chromosomes from early meiosis to late blastocyst. The estimated rate of 49.05+/-0.67% with a Y chromosome also estimated the primary sex ratio. There was evidence of polymorphism in autosomal pericentromeric staining in 3 strains. No measure of the numbers of autosomes or sex chromosomes varied significantly between duplicate preparations or between duplicate males of a strain.     

Examining the link between chromosomal instability and aneuploidy in human cells

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule-chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, near-diploid cells. However, chromosome missegregation compromises the proliferation of diploid cells, indicating that phenotypic changes that permit the propagation of nondiploid cells must combine with elevated chromosome missegregation rates to generate aneuploid cells with CIN.     

Parasexual Ploidy Reduction Drives Population Heterogeneity Through Random and Transient Aneuploidy in Candida albicans

The opportunistic pathogen Candida albicans has a large repertoire of mechanisms to generate genetic and phenotypic diversity despite the lack of meiosis in its life cycle. Its parasexual cycle enables shifts in ploidy, which in turn facilitate recombination, aneuploidy, and homozygosis of whole chromosomes to fuel rapid adaptation. Here we show that the tetraploid state potentiates ploidy variation and drives population heterogeneity. In tetraploids, the rate of losing a single heterozygous marker [loss of heterozygosity (LOH)] is elevated ∼30-fold higher than the rate in diploid cells. Furthermore, isolates recovered after selection for LOH of one, two, or three markers were highly aneuploid, with a broad range of karyotypes including strains with a combination of di-, tri-, and tetrasomic chromosomes. We followed the ploidy trajectories for these tetraploid- and aneuploid-derived isolates, using a combination of flow cytometry and double-digestion restriction-site-associated DNA analyzed with next-generation sequencing. Isolates derived from either tetraploid or aneuploid isolates predominately resolved to a stable euploid state. The majority of isolates reduced to the conventional diploid state; however, stable triploid and tetraploid states were observed in ∼30% of the isolates. Notably, aneuploid isolates were more transient than tetraploid isolates, resolving to a euploid state within a few passages. Furthermore, the likelihood that a particular isolate will resolve to the same ploidy state in replicate evolution experiments is only ∼50%, supporting the idea that the chromosome loss process of the parasexual cycle is random and does not follow trajectories involving specific combinations of chromosomes. Together, our results indicate that tetraploid progenitors can produce populations of progeny cells with a high degree of genomic diversity, from altered ploidy to homozygosis, providing an excellent source of genetic variation upon which selection can act.     

Aneuploidy, the primary cause of the multilateral genomic instability of neoplastic and preneoplastic cells

Cancers have a clonal origin, yet their chromosomes and genes are non-clonal or heterogeneous due to an inherent genomic instability. However, the cause of this genomic instability is still debated. One theory postulates that mutations in genes that are involved in DNA repair and in chromosome segregation are the primary causes of this instability. But there are neither consistent correlations nor is there functional proof for the mutation theory. Here we propose aneuploidy, an abnormal number of chromosomes, as the primary cause of the genomic instability of neoplastic and preneoplastic cells. Aneuploidy destabilizes the karyotype and thus the species, independent of mutation, because it corrupts highly conserved teams of proteins that segregate, synthesize and repair chromosomes. Likewise it destabilizes genes. The theory explains 12 of 12 specific features of genomic instability: (1) Mutagenic and non-mutagenic carcinogens induce genomic instability via aneuploidy. (2) Aneuploidy coincides and segregates with preneoplastic and neoplastic genomic instability. (3) Phenotypes of genomically unstable cells change and even revert at high rates, compared to those of diploid cells, via aneuploidy-catalyzed chromosome rearrangements. (4) Idiosyncratic features of cancers, like immortality and drug-resistance, derive from subspecies within the 'polyphyletic' diversity of individual cancers. (5) Instability is proportional to the degree of aneuploidy. (6) Multilateral chromosomal and genetic instabilities typically coincide, because aneuploidy corrupts multiple targets simultaneously. (7) Gene mutation is common, but neither consistent nor clonal in cancer cells as predicted by the aneuploidy theory. (8) Cancers fall into a near-diploid (2 N) class of low instability, a near 1.5 N class of high instability, or a near 3 N class of very high instability, because aneuploid fitness is maximized either by minimally unstable karyotypes or by maximally unstable, but adaptable karyotypes. (9) Dominant phenotypes, because of aneuploid genotypes. (10) Uncertain developmental phenotypes of Down and other aneuploidy syndromes, because supply-sensitive, diploid programs are destabilized by products from aneuploid genes supplied at abnormal concentrations; the maternal age-bias for Down's would reflect age-dependent defects of the spindle apparatus of oocytes. (11) Non-selective phenotypes, e.g., metastasis, because of linkage with selective phenotypes on the same chromosomes. (12) The target, induction of genomic instability, is several 1000-fold bigger than gene mutation, because it is entire chromosomes. The mutation theory explains only a few of these features. We conclude that the transition of stable diploid to unstable aneuploid cell species is the primary cause of preneoplastic and neoplastic genomic instability and of cancer, and that mutations are secondary.     

Phaffia rhodozyma is polyploid

The ploidy of the red yeast Phaffia rhodozyma was evaluated using flow cytometric analyses of propidium iodide- stained cells and mutagenic inactivation kinetics. Our findings suggest that Phaffia rhodozyma is not haploid. Auxotrophic strains were generated at a high frequency following treatment of mutagenized cells with a combination of benomyl and ethyl acetate. Studies of an auxotrophic mutant using flow cytometry and UV inactivation indicated possible chromosome loss to an aneuploid state. Received 21 June 1998/ Accepted in revised form 25 September 1998     

Regulation of karyotype stability in tobacco tissue cultures of normal and tumorous genotypes

Summary Callus cultures of Nicotiana glauca, N. langsdorffii and of their tumor-forming hybrid plants contained a high frequency of cells with irregular chromosome numbers and chromosome aberrations (hypo-, hyper-, polyploid, aneuploid cells; bridges, polytene, broken, fragmented chromosomes, megachromosomes, etc.). Meristematic cells of shoot tips regenerated from the same cultures contained only regular chromosome numbers with normal chromosome structures. Variability in chromosome numbers is a consequence of abnormal mitoses. The data suggest genome segregation in the cultures. Cytological instability appears to be independent of genome segregation composition, genotype, tumorous condition, hormonal requirement and level of ploidy. The karyotype stability of the cultures is only dependent on the degree of organization of tissues and is regulated by factors involved in the control mechanisms of organizational processes.     

The wages of CIN

Aneuploidy and chromosome instability (CIN) are hallmarks of the majority of solid tumors, but the relationship between them is not well understood. In this issue, Thompson and Compton (Thompson, S.L., and D.A. Compton. 2008. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell. Biol. 180:665-672) investigate the mechanism of CIN in cancer cells and find that CIN arises primarily from defective kinetochore-spindle attachments that evade detection by the spindle checkpoint and persist into anaphase. They also explore the consequences of artificially elevating chromosome missegregation in otherwise karyotypically normal cells. Their finding that induced aneuploidy is rapidly selected against suggests that the persistence of aneuploid cells in tumors requires not only chromosome missegregation but also additional, as yet poorly defined events.     

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

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

Clone heterogeneity in diploid and aneuploid breast carcinomas as detected by FISH

We investigated the relationship between DNA ploidy and alterations in chromosomes 1, 8, 12, 16, 17, and 18 in 63 breast carcinoma samples by static cytofluorometry and fluorescence in situ hybridization. Thirty specimens were diploid and 33 were aneuploid. In aneuploid samples, the DNA index value ranged from 1.3 to 3.1, with a main peak near tetraploid values. Diploid clones were present in 21 of 33 aneuploid specimens. Fluorescence in situ hybridization analysis showed a heterogeneous degree of alterations in diploid specimens: one sample was normal, 16 samples had one to three chromosome alterations involving mostly chromosomes 1, 16, and 17, and 13 samples an even higher degree of alterations. The 33 aneuploid specimens showed a very high number of signals (four, five, or more). All the investigated chromosomes were affected in 23 of 33 specimens. Alterations in chromosomes 1 and 17 were detected to a similar percentage in diploid and aneuploid samples, whereas chromosome 16 monosomy was more frequent in diploid samples. Overrepresentation of chromosomes 8, 12, 16, and 18 was significantly higher in aneuploid than in diploid samples. Based on these results, we suggest that diploid and aneuploid breast carcinomas are genetically related. Chromosome 1 and 17 alterations and chromosome 16 monosomy are early changes. Allelic and chromosomal accumulations occur during progression of breast carcinoma by different mechanisms. The high clone heterogeneity found in 17 of 33 aneuploid samples could not be completely explained by endoreduplication and led to the suggestion that chromosomal instability concurs with aneuploidy development. This different evolutionary pathway might be clinically relevant because clone heterogeneity might cause metastasis development and resistance to therapy.     

A balancing act: focus on aneuploidy

Aneuploidy has emerged as a major health concern in cancer and fertility. This issue of EMBO reports features four reviews that discuss aneuploidy and its consequences from different viewpoints, and are contextualized in this editorial.EMBO reports (2012) 13, 472; doi:10.1038/embor.2012.66Faithful chromosome segregation is crucial for the viability of cells and organisms, as evidenced by the fact that in humans only one autosomic trisomy—and no autosomic monosomies—allow survival into adulthood. Cells therefore use sophisticated mechanisms to ensure that each daughter receives an intact copy of the genome during cell division. Eukaryotic chromosomes have a specialized region known as the centromere, which recruits a complex proteinaceus structure—the kinetochore—that binds spindle microtubules to enable the separation of chromosomes during mitosis. The mitotic checkpoint and the machinery that controls kinetochore–microtubule attachment ensure correct chromosome segregation. However, several processes can lead to aneuploidy—the deviation from a haploid chromosomal number—such as defects in mitotic checkpoint proteins or sister chromatid cohesion, incorrect or hyperstabilized chromosome-spindle attachments, centrosome amplification or defects in cytokinesis.Aneuploidy is a major health concern. It is the leading cause of mental retardation and spontaneous miscarriage, and the current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years [1]. Furthermore, most solid tumours and about 50% of haematopoietic cancers are aneuploid. During the past few years, the cell-cycle, cancer and fertility fields have therefore made a substantial effort to understand the causes and consequences of aneuploidy.To bring together knowledge from different viewpoints and highlight recent advances in this exciting field, this issue of EMBO reports features four reviews on aneuploidy. An article by Rolf Jessberger analyses the process of oocyte meiosis and how it becomes less accurate with age, and reviews by Holland & Cleveland, Pfau & Amon and Swanton & colleagues focus on aneuploidy in the context of cancer.An overarching theme is the importance of intact sister chromatid cohesion to ensure the fidelity of chromosome segregation. In mammalian oocytes—which remain arrested in meiosis for up to four decades in humans—cohesin is loaded onto chromosomes during development and is probably not turned over for the life of the oocyte. Progressive loss of cohesin or ‘exhaustion'' seems responsible for the dramatic increase in aneuploid eggs with age. Similarly, defects in cohesion proteins are frequently found in various types of cancer.As will become apparent in the three cancer-related reviews, it is important to distinguish between aneuploidy and chromosomal instability (CIN)—a high rate of gain or loss of chromosomes. CIN leads to aneuploidy, but stable aneuploidy can occur without CIN, which is associated with a good prognosis in cancer and occurs in normal brain and liver tissue. An outstanding question is how and whether aneuploidy and CIN predispose to tumorigenesis. Technological advances have allowed the characterization of CIN status of a variety of cancers, underscoring the prevalence of aneuploidy. However, whether aneuploidy is a driving cause of tumour formation remains unclear. Despite the extensive association of aneuploidy with tumours in vivo, extensive data from yeast, mouse and human cell culture indicate that abnormal chromosome content provides a growth disadvantage in vitro, and the presence of CIN in some tumours correlates with good prognosis: this is the so-called ‘aneuploidy paradox''.In this review series, the Cleveland, Amon and Swanton groups provide their own particular views on this paradox. CIN could endow tumour cells with extreme evolvability that is beneficial in vivo, but would be a growth disadvantage under the constant, rich conditions of cell culture. On the other hand, aneuploidy could interfere with cell proliferation—as seen in vitro—and would be selected against; further mutations or chromosomal alterations would allow cells to overcome this restriction and reveal their full tumorigenic potential. According to this view, CIN would allow cells to overcome the negative effects of aneuploidy and promote tumorigenesis below a certain threshold. However, as Swanton and colleagues discuss, the nonlinear relationship between the extent of CIN and cancer prognosis suggests that, beyond this threshold, CIN would become unfavourable owing to the accumulation of deleterious genomic alterations.An increase in genomic material is generally accompanied by an increase in the expression of proteins encoded there, leading to altered metabolic properties, imbalances in the cell proteome and proteotoxic stress due to an overloading of protein degradation pathways. These effects imply that therapeutically targetable pathways would be common in a variety of aneuploid tumour cells. Initial proof-of-principle screens show promise in this regard and, as discussed in these reviews, have led to potential drug candidates.Swanton and colleagues provide a much needed—but rare—translational perspective into the issue of aneuploidy and CIN. Their review highlights the prognostic value of CIN assessment in human tumours, evaluates the methods used to analyse CIN and provides insights into how it could be therapeutically targeted.We hope this selection of comprehensive reviews will contribute to a better understanding of the complexities of aneuploidy and its causes. The possibility of targeting this imbalanced state in cancer therapy and harnessing our increasing knowledge to alleviate fertility problems are exciting prospects. We look forward to future developments in this fast-moving field.     

Proliferation of aneuploid human cells is limited by a p53-dependent mechanism

Most solid tumors are aneuploid, and it has been proposed that aneuploidy is the consequence of an elevated rate of chromosome missegregation in a process called chromosomal instability (CIN). However, the relationship of aneuploidy and CIN is unclear because the proliferation of cultured diploid cells is compromised by chromosome missegregation. The mechanism for this intolerance of nondiploid genomes is unknown. In this study, we show that in otherwise diploid human cells, chromosome missegregation causes a cell cycle delay with nuclear accumulation of the tumor suppressor p53 and the cyclin kinase inhibitor p21. Deletion of the p53 gene permits the accumulation of nondiploid cells such that CIN generates cells with aneuploid genomes that resemble many human tumors. Thus, the p53 pathway plays an important role in limiting the propagation of aneuploid human cells in culture to preserve the diploid karyotype of the population. These data fit with the concordance of aneuploidy and disruption of the p53 pathway in many tumors, but the presence of aneuploid cells in some normal human and mouse tissues indicates that there are known exceptions to the involvement of p53 in aneuploid cells and that tissue context may be important in how cells respond to aneuploidy.     

2n or not 2n: Aneuploidy,polyploidy and chromosomal instability in primary and tumor cells

Mitotic defects leading to aneuploidy have been recognized as a hallmark of tumor cells for over 100 years. Current data indicate that ∼85% of human cancers have missegregated chromosomes to become aneuploid. Some maintain a stable aneuploid karyotype, while others consistently missegregate chromosomes over multiple divisions due to chromosomal instability (CIN). Both aneuploidy and CIN serve as markers of poor prognosis in diverse human cancers. Despite this, aneuploidy is generally incompatible with viability during development, and some aneuploid karyotypes cause a proliferative disadvantage in somatic cells. In vivo, the intentional introduction of aneuploidy can promote tumors, suppress them, or do neither. Here, we summarize current knowledge of the effects of aneuploidy and CIN on proliferation and cell death in nontransformed cells, as well as on tumor promotion, suppression, and prognosis.     

Spontaneous and induced chromosome aberrations in the bone marrow cells of different strains of mice during aging

Sensitivity of bone marrow cell chromosomes to alkylating agent thiophosphamide and to gamma-irradiation has been studied in the course of ageing in 101/H, A/He, CBA, BALB/c and C57BL/6 mice. The effects of both the kinds of mutagenic treatment and of the genotype of the animals on the age-dependent changes in sensitivity of bone marrow cell chromosomes were found. Following gamma-irradiation under our experimental conditions, no variation in the output of chromosomal aberrations was observed between the strains studied. Following thiophosphamide treatment, aged mice of strains 101/H, A/He and CBA showed an increased chromosome instability as compared to young ones. In C57BL/6 mice the level of induced chromosome aberrations was found to be age-independent. Following thiophosphamide treatment, cells with multiple chromosome lesions were found in the bone marrow. The higher instability of aged animals in some strains was mainly due to a sharp increase in the number of such cells. In the intact mice of all the strains studied no age-dependent increase in the number of cells showing structural chromosome aberrations was observed, while accumulation of aneuploid cells varied with genotype.     

Chromosomal instability in the lymphocytes of breast cancer patients

Genomic instability in the tumor tissue has been correlated with tumor progression. In the present study, chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) of breast tumor patients were studied to assess whether chromosomal instability (CIN) in PBLs correlates with aggressiveness of breast tumor (i.e., disease stage) and has any prognostic utility. Cultured blood lymphocyte metaphases were scored for aberrations in 31 breast cancer patients and 20 healthy age and sex-matched controls. A variety of CAs, including aneuploidy, polyploidy, terminal deletions, acentric fragments, double minutes, chromatid separations, ring chromosome, marker chromosome, chromatid gaps, and breaks were seen in PBLs of the patients. The CAs in patients were higher than in controls. A comparison of the frequency of metaphases with aberrations by grouping the patients according to the stage of advancement of disease did not reveal any consistent pattern of variation in lymphocytic CIN. Neither was any specific chromosomal abnormality found to be associated with the stage of cancer. This might be indicative of the fact that cancer patients have constitutional CIN, which predisposes them to the disease, and this inherent difference in the level of genomic instability might play a role in disease progression and response to treatment.     

B chromosomes and genome size in flowering plants.

B chromosomes are extra chromosomes found in some, but not all, individuals within a species, often maintained by giving themselves an advantage in transmission, i.e. they drive. Here we show that the presence of B chromosomes correlates to and varies strongly and positively with total genome size (excluding the Bs and corrected for ploidy) both at a global level and via a comparison of independent taxonomic contrasts. B chromosomes are largely absent from species with small genomes; however, species with large genomes are studied more frequently than species with small genomes and Bs are more likely to be reported in well-studied species. We controlled for intensity of study using logistic regression. This regression analysis also included effects of degree of outbreeding, which is positively associated with Bs and genome size, and chromosome number, which is negatively associated with Bs and genome size, as well as variable ploidy (more than one ploidy level in a species). Genome size, breeding system and chromosome number all contribute independently to the distribution of B chromosomes, while variable ploidy does not have a significant effect. The genome size correlates are consistent with reduced selection against extra DNA in species with large genomes and with increased generation of B sequences from large A genomes.     

FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major

The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra-strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this 'divergent' eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity.     

Checkpoint-independent stabilization of kinetochore-microtubule attachments by Mad2 in human cells

Faithful chromosome segregation is required for cell and organism viability and relies on both the mitotic checkpoint and the machinery that corrects kinetochore-microtubule (k-MT) attachment errors. Most solid tumors have aneuploid karyotypes and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). Mad2 is essential for mitotic checkpoint function and is frequently overexpressed in human tumors that are CIN. For unknown reasons, cells overexpressing Mad2 display high rates of lagging chromosomes. Here, we explore this phenomenon and show that k-MT attachments are hyperstabilized by Mad2 overexpression and that this undermines the efficiency of correction of k-MT attachment errors. Mad2 affects k-MT attachment stability independently of the mitotic checkpoint because k-MT attachments are unaltered upon Mad1 depletion and Mad2 overexpression hyperstabilizes k-MT attachments in Mad1-deficient cells. Mad2 mediates these effects with Cdc20 by altering the centromeric localization and activity of Aurora B kinase, a known regulator of k-MT attachment stability. These data reveal a new function for Mad2 to stabilize k-MT attachments independent of the checkpoint and explain why Mad2 overexpression increases chromosome missegregation to cause chromosomal instability in human tumors.     

Spontaneous chromosome loss in Saccharomyces cerevisiae is suppressed by DNA damage checkpoint functions.

Genomic instability is one of the hallmarks of cancer cells and is often the causative factor in revealing recessive gene mutations that progress cells along the pathway to unregulated growth. Genomic instability can take many forms, including aneuploidy and changes in chromosome structure. Chromosome loss, loss and reduplication, and deletions are the majority events that result in loss of heterozygosity (LOH). Defective DNA replication, repair, and recombination can significantly increase the frequency of spontaneous genomic instability. Recently, DNA damage checkpoint functions that operate during the S-phase checkpoint have been shown to suppress spontaneous chromosome rearrangements in haploid yeast strains. To further study the role of DNA damage checkpoint functions in genomic stability, we have determined chromosome loss in DNA damage checkpoint-deficient yeast strains. We have found that the DNA damage checkpoints are essential for preserving the normal chromosome number and act synergistically with homologous recombination functions to ensure that chromosomes are segregated correctly to daughter cells. Failure of either of these processes increases LOH events. However, loss of the G2/M checkpoint does not result in an increase in chromosome loss, suggesting that it is the various S-phase DNA damage checkpoints that suppress chromosome loss. The mec1 checkpoint function mutant, defective in the yeast ATR homolog, results in increased recombination through a process that is distinct from that operative in wild-type cells.     

A small-molecule inhibitor of Mps1 blocks the spindle-checkpoint response to a lack of tension on mitotic chromosomes

The spindle checkpoint prevents chromosome loss by preventing chromosome segregation in cells with improperly attached chromosomes [1, 2 and 3]. The checkpoint senses defects in the attachment of chromosomes to the mitotic spindle [4] and the tension exerted on chromosomes by spindle forces in mitosis [5, 6 and 7]. Because many cancers have defects in chromosome segregation, this checkpoint may be required for survival of tumor cells and may be a target for chemotherapy. We performed a phenotype-based chemical-genetic screen in budding yeast and identified an inhibitor of the spindle checkpoint, called cincreasin. We used a genome-wide collection of yeast gene-deletion strains and traditional genetic and biochemical analysis to show that the target of cincreasin is Mps1, a protein kinase required for checkpoint function [8]. Despite the requirement for Mps1 for sensing both the lack of microtubule attachment and tension at kinetochores, we find concentrations of cincreasin that selectively inhibit the tension-sensitive branch of the spindle checkpoint. At these concentrations, cincreasin causes lethal chromosome missegregation in mutants that display chromosomal instability. Our results demonstrate that Mps1 can be exploited as a target and that inhibiting the tension-sensitive branch of the spindle checkpoint may be a way of selectively killing cancer cells that display chromosomal instability.