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.     

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.     

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.     

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.     

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.     

Karyotypic determinants of chromosome instability in aneuploid budding yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.     

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.     

Transient defects of mitotic spindle geometry and chromosome segregation errors

ABSTRACT: 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 low 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.     

Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression

Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoid^tetO-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption.     

Aneuploidy,cell delamination and tumorigenesis in Drosophila epithelia

Chromosomal instability (CIN) is a common feature in human cancer, and highly aneuploid tumors are frequently associated with poor prognosis; however, the molecular and cellular mechanisms underlying CIN-induced tumorigenesis are poorly understood. Here we review recent findings about the role of CIN in driving tumor-like growth and host invasiveness in Drosophila epithelia and discuss the commonalities of CIN-induced tumors with other Drosophila-based cancer models. We also discuss possible scenarios that can account for the participation of CIN in tumorigenesis and propose that, alternatively to the classical role of aneuploidy in promoting the accumulation of mutations in cancer cells, aneuploidy can be a source of stress that may contribute to cancer initiation and/or progression.     

Tumorigenicity analysis of heterogeneous dental stem cells and its self-modification for chromosome instability

Heterogeneity demonstrates that stem cells are constituted by several sub-clones in various differentiation states. The heterogeneous state is maintained by cross-talk among sub-clones, thereby ensuring stem cell adaption. In this study, we investigated the roles of heterogeneity on genetic stability. Three sub-clones (DF2, DF8 and DF18) were isolated from heterogeneous dental stem cells (DSCs), and were proved to be chromosome instability (CIN) after long term expansion. Cell apoptosis were not detected in sub-clones, which exhibited strong tumorigenesis tendency, coupled with weak expression of p53 and aberrant ultra-structure. However, 3 sub-clones did not overexpress tumor related markers or induce tumorigenesis in vivo. The mixed-culture study suggested that 3-clone-mixed culturing cells (DF1) presented apparent decrease in the ratio of aneuploidy. The screening experiment further proved that 3 sub-clones functioned separately in this modification procedure but only mixed culturing all 3 sub-clones, simulated heterogeneous microenvironment, could achieve complete modification. Additionally, osteogenesis capability of 3 sub-clones was partially influenced by CIN while DSCs still kept stronger osteogenesis than sub-clones. These results suggested aberrant sub-clones isolated from heterogeneous DSCs were not tumorigenesis and could modify CIN by cross-talk among themselves, indicating that the heterogeneity played a key role in maintaining genetic stability and differentiation capability in dental stem cells.     

Chromosome instability and kinetochore dysfunction

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Thus, defect in kinetochore function is a candidate source for CIN and the generation of aneuploidy. Recently, a number of kinetochore components have been shown to be mutated and/or aberrantly expressed in human cancers, which suggests an important role of kinetochore for CIN and carcinogenesis. In this article, we will discuss about how kinetochore dysfunction causes CIN and might lead to the development of cancer.     

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.     

Does Cancer Solve an Optimization Problem?

Many cancers are characterized by a high degree of aneuploidy, which isbelieved to be a result of chromosomal instability (CIN). The precise role of CIN incancer is still the matter of a heated debate. We present a quantitative framework forexamining the selection pressures acting on populations of cells and weigh the \pluses"and \minuses" of CIN from the point of view of a sel¯sh cell. We calculate the optimalrate of chromosome loss assuming that cancer is initiated by inactivation of a tumorsuppressor gene followed by a clonal expansion. The resulting rate, p* ~ ¼ 10^-2 per celldivision per chromosome, is similar to that obtained experimentally by Lengauer et al(1997). Our analysis further suggests that CIN does not arise simply because it allowsa faster accumulation of carcinogenic mutations. Instead, CIN must arise because ofalternative reasons, such as environmental factors, epigenetic events, or as a directconsequence of a tumor suppressor gene inactivation. The increased variability aloneis not a su±cient explanation for the presence of CIN in the majority of cancers.     

Somatic genomic variations in extra-embryonic tissues

In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

Loss of chromosome 13 in cultured human vascular endothelial cells

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.     

Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells

The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, we analyze the functions of six kinetochore-bound MT-associated proteins (kMAPs) using RNAi, live-cell microscopy and quantitative image analysis. We find that RNAi-mediated depletion of two kMAPs, the adenomatous polyposis coli protein (APC) and its binding partner, EB1, are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. We propose that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis.     

Genetic diagnosis in infertile men with numerical and constitutional sperm abnormalities

Infertile men having numerical or structural sperm defects may carry several genetic abnormalities (karyotype abnormalities, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations, androgen receptor gene mutations, and abnormalities seen in sperm cells) leading to this situation. First we aimed to investigate the relationship between the numerical and constitutional (morphological) sperm anomalies and the genetic disorders that can be seen in infertile males. Our other aim was to compare two different kinds of kits that we use for the detection of Y chromosome microdeletions. Sixty-three infertile males [44 nonobstructive azoospermic, 8 severe oligozoospermic, and 11 oligoasthenoteratozoospermic] were investigated in terms of somatic chromosomal constitutions and microdeletions of the Y chromosome. Sperm aneuploidy levels were analyzed by fluorescence in situ hybridization (FISH) in sperm cells obtained from the semen of six OAT patients. Microdeletion and sex chromosome aneuploidy (47,XXY) rates in somatic cells were found to be approximately 3.2% and 4.7%, respectively. Sperm aneuploidy rates were determined as 9%, 22%, and 47% in three patients out of six. Two of these three patients also had high rates of head anomalies in semen samples. High correlation was found between sperm aneuploidy rates and sperm head anomalies. Since the introduction of the assisted reproductive techniques for the treatment of severe male infertility, genetic tests and genetic counseling became very important due to the transmission of genetic abnormalities to the next generation. Thus in a very near future, for a comprehensive male infertility panel, it will be essential to include additional genetic tests, such as CFTR gene mutations, sperm mitochondrial DNA mutations, and androgen receptor gene mutations, besides the conventional chromosomal analyses, Y chromosome microdeletion detection, and sperm-FISH analyses.