Elevated Tolerance to Aneuploidy in Cancer Cells: Estimating the Fitness Effects of Chromosome Number Alterations by In Silico Modelling of Somatic Genome Evolution

An unbalanced chromosome number (aneuploidy) is present in most malignant tumours and has been attributed to mitotic mis-segregation of chromosomes. However, recent studies have shown a relatively high rate of chromosomal mis-segregation also in non-neoplastic human cells, while the frequency of aneuploid cells remains low throughout life in most normal tissues. This implies that newly formed aneuploid cells are subject to negative selection in healthy tissues and that attenuation of this selection could contribute to aneuploidy in cancer. To test this, we modelled cellular growth as discrete time branching processes, during which chromosome gains and losses were generated and their host cells subjected to selection pressures of various magnitudes. We then assessed experimentally the frequency of chromosomal mis-segregation as well as the prevalence of aneuploid cells in human non-neoplastic cells and in cancer cells. Integrating these data into our models allowed estimation of the fitness reduction resulting from a single chromosome copy number change to an average of ≈30% in normal cells. In comparison, cancer cells showed an average fitness reduction of only 6% (p = 0.0008), indicative of aneuploidy tolerance. Simulations based on the combined presence of chromosomal mis-segregation and aneuploidy tolerance reproduced distributions of chromosome aberrations in >400 cancer cases with higher fidelity than models based on chromosomal mis-segregation alone. Reverse engineering of aneuploid cancer cell development in silico predicted that aneuploidy intolerance is a stronger limiting factor for clonal expansion of aneuploid cells than chromosomal mis-segregation rate. In conclusion, our findings indicate that not only an elevated chromosomal mis-segregation rate, but also a generalised tolerance to novel chromosomal imbalances contribute to the genomic landscape of human tumours.     

Numerical chromosome variation and mitotic segregation defects in the adult brain of teleost fish

Teleost fish are distinguished by their enormous potential for the generation of new cells in both the intact and the injured adult brain. Here, we present evidence that these cells are a genetic mosaic caused by somatic genomic alteration. Metaphase chromosome spreads from whole brains of the teleost Apteronotus leptorhynchus revealed an euploid complement of 22 chromosomes in only 22% of the cells examined. The rate of aneuploidy is substantially higher in brain cells than in liver cells, as shown by both metaphase chromosome spreads and flow cytometric analysis. Among the aneuploid cells in the brain, approximately 84% had fewer, and the remaining 16% more, than 22 chromosomes. Typically, multiple chromosomes were lost or gained. The aneuploidy is putatively caused by segregation defects during mitotic division. Labeling of condensed chromosomes of M-phase cells by phosphorylated histone-H3 revealed laggards, anaphase bridges, and micronuclei, all three of which indicate displaced mitotic chromosomes. Quantitative analysis has shown that in the entire brain on average 14% of all phosphorylated histone-H3-labeled cells exhibit such signs of segregation defects. Together with the recent discovery of aneuploidy in the adult mammalian brain, the results of the present investigation suggest that the loss or gain of chromosomes might provide a mechanism to regulate gene expression during development of new cells in the adult vertebrate brain.     

Chromosome Copy Number Variation and Control in the Ciliate Chilodonella uncinata

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.     

TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation

A cancer is a robustly evolving cell population originating from a normal diploid cell. Improper chromosome segregation causes aneuploidy, a driving force of cancer development and malignant progression. Telomeric repeat binding factor 1 (TRF1) has been established as a telomeric protein that negatively regulates telomere elongation by telomerase and promotes efficient DNA replication at telomeres. Intriguingly, overexpression of a mitotic kinase, Aurora-A, compromises efficient microtubule-kinetochore attachment in a TRF1-dependent manner. However, the precise role of TRF1 in mitosis remains elusive. Here we demonstrate that TRF1 is required for the centromeric function of Aurora-B, which ensures proper chromosome segregation. TRF1 depletion abolishes centromeric recruitment of Aurora-B and loosens sister centromere cohesion, resulting in the induction of merotelic kinetochore attachments, lagging chromosomes, and micronuclei. Accordingly, an absence of TRF1 in human and mouse diploid cells induces aneuploidy. These phenomena seem to be telomere independent, because a telomere-unbound TRF1 mutant can suppress the TRF1 knockdown phenotype. These observations indicate that TRF1 regulates the rigidity of the microtubule-kinetochore attachment, contributing to proper chromosome segregation and the maintenance of genomic integrity.     

Low level chromosome instability in embryonic cells of primary aneuploid mice

Karyotypic analyses of Down syndrome patients have identified a low level of chromosome mosaicism, suggesting that the primary aneuploid status of the cells promotes further chromosomal segregation errors. Sycp3-null female mice produce aneuploid oocytes, which after fusion with normal haploid sperm, result in offspring with systemic whole chromosome, aneuploid embryo cells. Using the Sycp3-null female as a model, we observe an increase in the number of embryonic cells at E7.0 that exhibit abnormal chromosomal bridges at the anaphas estage of mitosis. This result suggests that global changes in gene expression patterns resulting from primary aneuploidy can affect mitotic chromosome segregation, resulting in a low level of chromosomal instability. The increased level of chromosomal instability could in the absence of mitotic checkpoints, lead to chromosomal mosaicism within the adult organism, as seen in Down syndrome patients.     

Comparison of the rates of aneuploidy occurrence in quiescent and dividing cells of humans exposed to adverse environmental factors

Fluorescence in situ hybridization (FISH) was used to compare aneuploidy rates in four autosomes and two sex chromosomes in interphase nuclei of noncultivated (quiescent) and cultivated (induced to divide with phytohemagglutinin (PHA)) leukocytes in people engaged in nuclear-chemical industry and in a control group of people not exposed to mutagenic factors occupationally or at home. The overall rates of numerical chromosome aberrations for all of the six chromosomes studied showed little difference, although a higher rate of loss of the X- and Y-chromosomes was observed in the exposed group. In individuals exposed to several adverse environmental factors, the overall rate of numerical chromosome aberrations in cultivated cells after at least one DNA replication cycle exceeded that in noncultivated cells by 52% (P = 0.01), whereas only a trend for its increase was observed in the control group (23%, P = 0.25). Thus, the effect of adverse environmental factors in humans caused more than a twofold increase in the difference between the rates of aneuploid cells in cultivated and noncultivated leukocytes in the exposed group as compared to control. It is conjectured that cell division is accompanied by the expression of potential damage of mitotic chromatid segregation apparatus accumulated in vivo. These defects, realized during cell division, bring about numerical chromosome aberrations.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 391–395.Original Russian Text Copyright © 2005 by Nazarenko, Timoshevsky.     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Chromosome errors at mitotic anaphase.

Errors in mitotic divisions were assayed using various satellite DNAs as probes, hybridized in situ, to show that they included nondisjunction, chromosome and chromatid lagging, chromatid malsegregation, and monopolar segregations. The total rates of error were 1.7, 1.1, and 0.6% for chromosomes X, 17, and 18, respectively. Lagging was the most common error for all chromosomes and chromatid malsegregation, a source of 3:1 segregations occurred at about the same frequency as nondisjunction. In some cells, lagging of both X chromatids occurred and there were several cells where both X chromosomes showed errors in segregation. The disjunction of chromosomes was shown to be independent of their segregation and is speculated to involve a different mechanism.     

Whole-chromosome aneuploidy analysis in human oocytes: focus on comparative genomic hybridization

The study of aneuploidy in human oocytes, discarded from IVF cycles, has provided a better understanding of the incidence of aneuploidy of female origin and the responsible mechanisms. Comparative genomic hybridization (CGH) is an established technique that allows for the detection of aneuploidy in all chromosomes avoiding artifactual chromosome losses. In this review, results obtained using CGH in single cells (1PB and/or MII oocytes) are included. The results of oocyte aneuploidy rates obtained by CGH from discarded oocytes of IVF patients and of oocyte donors are summarized. Moreover, the mechanisms involved in the aneuploid events, e.g. whether alterations occurred due to first meiotic errors or germ-line mitotic errors are also discussed. Finally, the incidence of aneuploid oocyte production due to first meiotic errors and germ-line mitotic errors observed in oocytes coming from IVF patients and IVF oocyte donors was assessed.     

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.     

Genetic bottlenecks and the hazardous game of population reduction in cell line based research

Established tumour cell lines are ubiquitous tools in research, but their representativity is often debated. One possible caveat is that many cell lines are derived from cells with genomic instability, potentially leading to genotype changes in vitro. We applied SNP-array analysis to an established tumour cell line (WiT49). Even though WiT49 exhibited chromosome segregation errors in 30% of cell divisions, only a single chromosome segment exhibited a shift in copy number after 20 population doublings in culture. In contrast, sub-populations derived from single cells expanded for an equal number of population doublings showed on average 5.8 and 8.9 altered segments compared to the original culture and to each other, respectively. Most copy number variants differentiating these single cell clones corresponded to pre-existing variations in the original culture. Furthermore, no sub-clonal variation was detected in any of the populations derived from single cells. This indicates that genetic bottlenecks resulting from population reduction poses a higher threat to genetic representativity than prolonged culture per se, even in cell lines with a high rate of genomic instability. Genetic bottlenecks should therefore be considered a potential caveat in all studies involving sub-cloning, transfection and other conditions leading to a temporary reduction in cell number.     

Chromosome segregation and double-strand break repair - a complex connection

Genome stability requires correct chromosome segregation and DNA repair. Failure of these processes leads to cell death or accumulation of chromosomal aberrations, as often observed in tumor cells. An increasing number of observations indicate that segregation and DNA double-strand break (DSB) repair are functionally connected by the Cohesin and Smc5/6 protein complexes. Through their interaction with the duplicated genome, these complexes play essential roles in both chromosome segregation and repair by sister chromatid recombination. Both are also recruited to DSBs, and their chromosomal association is similarly regulated. Interestingly, recent studies of Cohesin suggest that DSB formation could promote proper mitotic chromosome segregation. This is reminiscent of segregation in meiotic cells, which is facilitated by break-induced chromosomal tethering.     

Inducing segmental aneuploid mosaicism in the mouse through targeted asymmetric sister chromatid event of recombination

Loss or gain of whole chromosomes, or parts of chromosomes, is found in various pathological conditions, such as cancer and aneuploidy, and results from the missegregation of chromosomes during cellular division or abnormal mitotic recombination. We introduce a novel strategy for determining the consequences of segmental aneuploid mosaicism, called targeted asymmetric sister chromatin event of recombination (TASCER). We took advantage of the Cre/loxP system, used extensively in embryonic stem cells for generating deletions and duplications of regions of interest, to induce recombination during the G2 phase. Using two loxP sites in a Cis configuration, we generated in vivo cells harboring microdeletions and microduplications for regions of interest covering up to 2.2 Mb. Using this approach in the mouse provides insight into the consequences of segmental aneuploidy for homologous regions of the human chromosome 21 on cell survival. Furthermore, TASCER shows that Cre-induced recombination is more efficient after DNA replication in vivo and provides an opportunity to evaluate, through genetic mosaics, the outcome of copy number variation and segmental aneuploidy in the mouse.     

Micromechanics of human mitotic chromosomes

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.     

Classification of chromosome segregation errors in cancer

Abnormal chromosome segregation at mitosis is one way by which neoplastic cells accumulate the many genetic abnormalities required for tumour development. In this paper, a straightforward morphology-based classification of chromosome segregation errors in cancer is suggested. This classification distinguishes between abnormalities in spindle symmetry (spindle multipolarity, size-asymmetry of ana-telophase poles) and abnormalities in sister chromatid segregation (chromosome bridges, chromatid bridges, chromosome lagging, acentric fragment lagging). Often, these categories of errors must be combined to accurately describe the events in a single abnormal mitotic cell. The suggested categories can to some extent be distinguished by standard chromatin staining. However, labelling of abnormal mitotic figures by fluorescence in situ hybridization and immunofluorescence enhances the accuracy of classification and also allows visualisation of the segregation of individual chromosomes, making it possible to detect non-disjunction also in the absence of gross alterations in mitotic morphology. Further characterisation of the molecular alterations leading to abnormal chromosome segregation together with the current developments in nano-level and real-time imaging will undoubtedly lead to an improved understanding of chromosome dynamics in cancer cells. Any morphology-based classification of chromosome segregation errors in cancer must therefore be taken as provisional, anticipating a satisfactory integration of morphology and molecular biology.     

Unpicking neurodegeneration in a dish with human pluripotent stem cells

Eukaryotic cell division is an orderly and timely process involving the error-free segregation of chromosomes and cytoplasmic components to give rise to two separate daughter cells. Defects in genome maintenance mechanisms such as cell cycle checkpoints and DNA repair can impact the segregation of the genome during mitosis leading to multiple chromosomal imbalances. In mammals, the DNA damage checkpoint effector Checkpoint Kinase 1 (Chk1) is essential for responses to DNA replication errors, external DNA damage, and chromatin breaks. We reported recently that Chk1 also was essential for chromosome segregation and completion of cytokinesis to prevent genomic instability. Our studies demonstrated that Chk1 deficiency in mitotic cells causes chromosome mis-alignment, lagging chromosomes, chromosome mis- segregation, cytokinetic regression, and binucleation. In addition, abrogation of Chk1 resulted in aberrant localization of mitotic Aurora B kinase at the metaphase plate, anaphase spindle midzone, and cytokinetic midbody as studied both in various cell lines and in a mouse model. Therefore, inappropriate regulation of Chk1 levels during cell cycle progression will result in failed cell division and enhanced genomic instability.     

The Analysis of Mutant Alleles of Different Strength Reveals Multiple Functions of Topoisomerase 2 in Regulation of Drosophila Chromosome Structure

Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.     

Report on the Eleventh International Workshop on the Identification of Transcribed Sequences 2001. November 9-11, 2001. Washington, DC, USA

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-microm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.     

Chromosome segregation in<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis, a Gram-positive bacterium commonly found in soil, is an excellent model organism for the study of basic cell processes, such as cell division and cell differentiation, called sporulation. In B. subtilis the essential genetic information is carried on a single circular chromosome, the correct segregation of which is crucial for both vegetative growth and sporulation. The proper completion of life cycle requires each daughter cell to obtain identical genetic information. The consequences of inaccurate chromosome segregation can lead to formation of anucleate cells, cells with two chromosomes, or cells with incomplete chromosomes. Although bacteria miss the classical eukaryotic mitotic apparatus, the chromosome segregation is undeniably an active process tightly connected to other cell processes as DNA replication and compaction. To fully understand the chromosome segregation, it is necessary to study this process in a wider context and to examine the role of different proteins at various cell life cycle stages. The life cycle of B. subtilis is characteristic by its specific cell differentiation process where, two slightly different segregation mechanisms exist, specialized in vegetative growth and in sporulation.