Aneuploid Cancer Cells Research Articles

Large-scale analysis of chromosomal aberrations in cancer karyotypes reveals two distinct paths to aneuploidy

BackgroundChromosomal aneuploidy, that is to say the gain or loss of chromosomes, is the most common abnormality in cancer. While certain aberrations, most commonly translocations, are known to be strongly associated with specific cancers and contribute to their formation, most aberrations appear to be non-specific and arbitrary, and do not have a clear effect. The understanding of chromosomal aneuploidy and its role in tumorigenesis is a fundamental open problem in cancer biology.ResultsWe report on a systematic study of the characteristics of chromosomal aberrations in cancers, using over 15,000 karyotypes and 62 cancer classes in the Mitelman Database. Remarkably, we discovered a very high co-occurrence rate of chromosome gains with other chromosome gains, and of losses with losses. Gains and losses rarely show significant co-occurrence. This finding was consistent across cancer classes and was confirmed on an independent comparative genomic hybridization dataset of cancer samples. The results of our analysis are available for further investigation via an accompanying website.ConclusionsThe broad generality and the intricate characteristics of the dichotomy of aneuploidy, ranging across numerous tumor classes, are revealed here rigorously for the first time using statistical analyses of large-scale datasets. Our finding suggests that aneuploid cancer cells may use extra chromosome gain or loss events to restore a balance in their altered protein ratios, needed for maintaining their cellular fitness.

Illicit survival of cancer cells during polyploidization and depolyploidization

Tetraploidy and the depolyploidization of tetraploid cells may contribute to oncogenesis. Several mechanisms have evolved to avoid the generation, survival, proliferation and depolyploidization of tetraploids. Cells that illicitly survive these checkpoints are prone to chromosomal instability and aneuploidization. Along with their replication, tetraploids constantly undergo chromosomal rearrangements that eventually lead to pseudodiploidy by two non-exclusive mechanisms: (i) multipolar divisions and (ii) illicit bipolar divisions in the presence of improper microtubule-kinetochore attachments. Here, we describe the regulation and the molecular mechanisms that underlie such a 'polyploidization-depolyploidization' cascade, while focusing on the role of oncogenes and tumor suppressor genes in tetraploidy-driven tumorigenesis. We speculate that the identification of signaling/metabolic cascades that are required for the survival of tetraploid or aneuploid (but not diploid) cancer cells may pave the way for the development of novel broad-spectrum anticancer agents.

Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase

Posttranslational histone modifications regulate both gene expression and genome integrity. Despite the dynamic nature of these modifications, appropriate real-time monitoring systems are lacking. In this study, we developed a method to visualize histone modifications in living somatic cells and preimplantation embryos by loading fluorescently labeled specific Fab antibody fragments. The technique was used to study histone H3 Ser10 (H3S10) phosphorylation, which occurs during chromosome condensation in mitosis mediated by the aurora B kinase. In aneuploid cancer cells that frequently missegregate chromosomes, H3S10 is phosphorylated just before the chromosomes condense, whereas aurora B already accumulates in nuclei during S phase. In contrast, in nontransformed cells, phosphorylated H3S10 foci appear for a few hours during interphase, and transient exposure to an aurora B-selective inhibitor during this period induces chromosome missegregation. These results suggest that, during interphase, moderate aurora B activity or H3S10 phosphorylation is required for accurate chromosome segregation. Visualizing histone modifications in living cells will facilitate future epigenetic and cell regulation studies.

Nucleation capacity and presence of centrioles define a distinct category of centrosome abnormalities that induces multipolar mitoses in cancer cells

Analysis of centrosome number and structure has become one means of assessing the potential for aberrant chromosome segregation and aneuploidy in tumor cells. Centrosome amplification directly causes multipolar catastrophic mitoses in mouse embryonic fibroblasts (MEFs) deficient for the tumor suppressor genes Brca1 or Trp53. We observed supernumerary centrosomes in cell lines established from aneuploid, but not from diploid, colorectal carcinomas; however, multipolar mitoses were never observed. This discrepancy prompted us to thoroughly characterize the centrosome abnormalities in these and other cancer cell lines with respect to both structure and function. The most striking result was that supernumerary centrosomes in aneuploid colorectal cancer cell lines were unable to nucleate microtubules, despite the presence of gamma-tubulin, pericentrin, PLK1, and AURKA. Analysis by scanning electron microscopy revealed that these supernumerary structures are devoid of centrioles, a result significantly different from observations in aneuploid pancreatic cancer cell lines and in Trp53 or Brca1 deficient MEFs. Thus, multipolar mitoses are dependent upon the ability of extra gamma-tubulin containing structures to nucleate microtubules, and this correlated with the presence of centrioles. The assessment of centrosome function with respect to chromosome segregation must therefore take into consideration the presence of centrioles and the capacity to nucleate microtubules. The patterns and mechanisms of chromosomal aberrations in hematologic malignancies and solid tumors are fundamentally different. The former is characterized by specific chromosome translocations, whose consequence is the activation of oncogenes. Most carcinomas, however, reveal variations in the nuclear DNA content. The observed genomic imbalances and gross variations in chromosome number can result from unequal chromosome segregation during mitotic cell division. It is therefore fundamental to elucidate mechanisms involved in distribution of the genome to daughter cells. Prior to cell division, the centrosome organizes microtubules and the mitotic spindle. Deciphering the consequences of alterations in centrosome number, structure, and function is an important step towards understanding how a diploid genome is maintained. Although extra centrosomes have now been observed in carcinomas and were correlated with aneuploidy, a careful functional investigation of these structures and their role in generating chromosome imbalances may lead to the identification of distinct mechanistic pathways of genomic instability. Understanding these pathways will also be important in determining whether they are potential molecular targets of therapeutic intervention.

Hybrids of aneuploid human cancer cells permit complementation of simple and complex cancer defects

Causes for the complex phenotypes of cancers, such as altered differentiation, invasion, and metastasis, are not known, and multigenic defects are likely. In contrast, well-defined deficiencies, such as those affecting DNA-repair mechanisms and enzymatic path¬ways, are simple, typically caused by one or a few gene mutations. Complemen¬tation by introducing defined genetic elements is used to study simple cancer phenotypes, while complementation by the fusion of whole cells is employed occasionally for complex ones. Hybrids formed solely from the common lines (aneuploid due to chromosomal instability, CIN) are rarely reported. We created stable hybrids of two CIN lines, producing a nearly complete genetic sum of the parental karyotypes. Complementation of a simple cancer phenotype, a Fanconi anemia pathway defective in both parental lines, occurred in all hybrids, restoring the normal drug-resistance phenotype. The grossly defective mitotic spindle checkpoint present in both parental lines was partially corrected in some hybrids, supporting a multigenic origin rather than a single gene defect. Using Affymetrix 100K SNP chips, we mapped chromosomal loci differing among the phenotypically distinct hybrid clones. Fusing CIN cell lines to form mapped hybrids offers new tools for positional cloning or classification of simple and complex cancer phenotypes, including mechanical defects and altered drug responses.

Novel approach for detecting global epigenetic alterations associated with tumor cell aneuploidy

Although aneuploidy is commonly observed in human cancers, the molecular mechanism underlying aneuploidization remains unclear. We used multiploid cancer model that had diploid and aneuploid cancer cells within the same cancerous tissue and attempted to detect specific epigenetic alterations associated with tumor cell aneuploidy. Thirty-four multiploid colorectal cancers were subjected to crypt isolation and cell sorting, and paired diploid and aneuploid cancer cells were separated from each cancerous tissue. A methylated CpG island amplification provided a considerable number of CpG sequences that showed different methylation status between the above 2 cell populations. BLAST homology search revealed 24 different candidates (11 hypermethylated and 13 hypomethylated) from these sequences. The putative promoter sequence of the SALL4 (sal-like 4, a human homolog to Drosophila spalt) gene was particularly more frequently hypermethylated in aneuploid cells (62%) than diploid ones (35%) in the 34 multiploid cancers. Moreover, such hypermethylation occurred more often in aneuploid cancers (8 of 16, 50%) than diploid cancers (3 of 18, 17%). In combination with demethylation study on cultured cells, these results implied a possible association between epigenetic silencing of SALL4 and tumor cell aneuploidy. SALL4 may be one of important key players that act as "caretakers" for chromosomal stability. Our new approach is a powerful tool for the global identification of such key players.

Temporal profile of replication of human chromosomes

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization

Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses–gains that are detected with array CGH in microdissected tissues.

Aneuploid colon cancer cells have a robust spindle checkpoint.

Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused by mismatch repair defects, the lesions responsible for CIN are unknown. The observation that CIN cells fail to undergo mitotic arrest following spindle damage suggested that mutations in spindle checkpoint genes may account for CIN. However, here we show that CIN cells do undergo mitotic arrest in response to spindle damage. Although the maximum mitotic index achieved by CIN lines is diminished relative to MIN lines, CIN cells clearly have a robust spindle checkpoint. Consistently, mutations in spindle checkpoint genes are rare in human tumours. In contrast, the adenomatous polyposis coli (APC) gene is frequently mutated in CIN cells. Significantly, we show here that expression of an APC mutant in MIN cells reduces the mitotic index following spindle damage to a level observed in CIN cells, suggesting that APC dysfunction may contribute to CIN.

Mitotic checkpoint genes hBUB1, hBUB1B, hBUB3 and TTK in human bladder cancer, screening for mutations and loss of heterozygosity.

Chromosomal instability is common in bladder cancer and could be caused by mutations of mitotic checkpoint genes. Therefore we screened for mutations of the mitotic checkpoint genes hBUB1, hBUB1B, hBUB3 and TTK in six aneuploid bladder cancer cell lines and 15 human bladder tumours. The screening was performed by sequence analysis of the entire coding regions of the four genes. No mutations were detected in any of the four genes. We detected several sequence variations in hBUB1, hBUB1B and TTK both new and previously published. The genetic stability of the four gene loci were tested by loss of heterozygosity (LOH) analysis in the 15 patient samples, showing one LOH for each of the hBUB1B, hBUB3 and TTK loci (6.7%) of the cases, all in different tumour samples. No LOH was detected at hBUB1. We conclude that both mutational inactivation, and loss of one allele, of the examined mitotic checkpoint genes are relatively uncommon.

Explaining the high mutation rates of cancer cells to drug and multidrug resistance by chromosome reassortments that are catalyzed by aneuploidy.

The mutation rates of cancer cells to drug and multidrug resistance are paradoxically high, i.e., 10(-3) to 10(-6), compared with those altering phenotypes of recessive genes in normal diploid cells of about 10(-12). Here the hypothesis was investigated that these mutations are due to chromosome reassortments that are catalyzed by aneuploidy. Aneuploidy, an abnormal number of chromosomes, is the most common genetic abnormality of cancer cells and is known to change phenotypes (e.g., Down's syndrome). Moreover, we have shown recently that aneuploidy autocatalyzes reassortments of up to 2% per chromosome per mitosis because it unbalances spindle proteins, even centrosome numbers, via gene dosage. The hypothesis predicts that a selected phenotype is associated with multiple unselected ones, because chromosome reassortments unbalance simultaneously thousands of regulatory and structural genes. It also predicts variants of a selected phenotype based on variant reassortments. To test our hypothesis we have investigated in parallel the mutation rates of highly aneuploid and of normal diploid Chinese hamster cells to resistance against puromycin, cytosine arabinoside, colcemid, and methotrexate. The mutation rates of aneuploid cells ranged from 10(-4) to 10(-6), but no drug-resistant mutants were obtained from diploid cells in our conditions. Further selection increased drug resistance at similar mutation rates. Mutants selected from cloned cells for resistance against one drug displayed different unselected phenotypes, e.g., polygonal or fusiform cellular morphology, flat or three-dimensional colonies, and resistances against other unrelated drugs. Thus our hypothesis offers a unifying explanation for the high mutation rates of aneuploid cancer cells and for the association of selected with unselected phenotypes, e.g., multidrug resistance. It also predicts drug-specific chromosome combinations that could become a basis for selecting alternative chemotherapy against drug-resistant cancer.

Mutation and expression analysis of human BUB1 and BUB1B in aneuploid breast cancer cell lines

Genetic instability is a hallmark feature of breast, colorectal and other types of cancers. One type characterized by chromosomal instability is thought to be important in the pathogenesis of many solid tumors displaying aneuploidy. Two related protein kinases and homologues of the yeast checkpoint genes, hBUB1 and hBUB1B, have been implicated in the pathogenesis of colorectal cancers. Mutations in hBUB1 have demonstrated a dominant negative effect by disrupting the mitotic checkpoint when transfected into euploid colon cancer cell lines. In Brca2 deficient murine cells, Bub1 mutants potentiate growth and cellular transformation. This would suggest that aneuploidy in solid tumors including breast, could be the result of defects in mitotic checkpoint genes and may be responsible for a chromosomal instability phenotype contributing to tumor progression. We conducted mutational analysis of 19 aneuploid breast cancer cell lines. No mutations were found but we identified nine sequence variations including five previously unreported sequence variants in hBUB1B, two of which affect restrictions sites. None of these nucleotide changes predict significant changes in the predicted protein structure. Expression analysis by Northern blot of breast cell lines showed variable expression of hBUB1 and hBUB1B genes. This suggest that while regulation of expression of these genes may be important in cancer, the lack of putative deleterious mutations in the coding sequence does not support a frequent role for mutant hBUB1 and hBUB1B alleles in the pathogenesis of breast cancer.

Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells.

Breast cancer is a heterogeneous disorder in which most tumors display some degree of aneuploidy, especially those at later stages of the disease. Aneuploidy and associated chromosome instability may be important in the progression of mammary tumorigenesis. Aneuploidy is prevented during normal cell division in part through regulation of a mitotic spindle checkpoint where mitotic arrest prevents segregation of misaligned chromosomes into daughter cells at anaphase. Mitotic arrest genes, including the MAD family, which was originally characterized in yeast, help regulate normal function of the mitotic spindle checkpoint. Decreased expression of the human gene MAD2L1 was previously reported in a breast cancer cell line exhibiting chromosome instability and aneuploidy. To explore further the potential role of MAD2L1 in breast cancer, we analyzed MAD2L1 gene expression in 13 minimally to grossly aneuploid human breast cancer cell lines and found significant differences of expression in three lines. Sequence analysis of MAD2L1 cDNA in these as well as nine additional aneuploid breast cancer and five immortalized normal human mammary epithelial cell lines revealed one heterozygous frameshift (572 del A) mutation in a cancer cell line that demonstrated a high level of transcript expression. In addition, two 3'UTR sequence variants were noted in breast cancer cell lines. The 572 del A mutation creates a truncated MAD2 protein product. Further functional studies in primary breast tumors are therefore warranted to determine the potential role MAD2L1 may play in breast cancer.

Genetic instability of cancer cells is proportional to their degree of aneuploidy.

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623-627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Molecular karyotype (amplotype) of metastatic colorectal cancer by unbiased arbitrarily primed PCR DNA fingerprinting.

Genomic instability characterizes the aneuploid cancer cell. Losses of genetic material are critical in cancer by exposing recessive mutations in tumor suppressor genes. Gains of genetic material also may lead to overexpression of genes contributing to tumor progression either in the presence or absence of mutation. However, the detection of moderate gains (such as tri-tetraploidy) has been a challenge in cancer research. Unbiased DNA fingerprinting by the arbitrarily primed PCR allows the detection of moderate gains (in addition to losses) of DNA sequences of known chromosomal localization. We have generated in this manner a molecular karyotype of metastatic colon cancer. This amplotype shows that sequences from several chromosomes undergo both losses (1, 4, 9, 14, and 18) and gains (6, 7, 12, and 20) in over half of the tumors. Moreover, gains of sequences from chromosomes 8 and 13 occurred in most tumors, indicating the existence in these chromosomes of positive regulators of cell growth or survival that are under strong positive selection during tumor progression. We conclude that overrepresentation of these chromosomal regions is a critical step for metastatic colorectal cancer. Comparative amplotype analysis from primary and metastatic tumors suggest the existence in chromosome 4 of gene(s) whose loss is specifically selected in cells that reach the metastatic stage.

Flow cytometry and feulgen cytophotometry in evaluation of effusions

Fifty-eight effusions (42 pleural and 16 ascitic fluids) from patients with and without cancer were analyzed by conventional cytology and the results compared with DNA patterns generated by flow cytometry of 10(4) nuclei and several modes of Feulgen cytophotometry. In 31 patients (24 without evidence of cancer and seven with history of cancer and cytologically negative fluids), the fluids were diploid by flow cytometry. One fluid with atypical cells from a lymphoma suspect was also diploid. Flow cytometry of 26 cytologically cancerous fluids disclosed aneuploid DNA patterns in 16 and diploid patterns in ten. Feulgen cytophotometry of 11 of these fluids (three aneuploid, eight diploid) was performed on nuclear preparations identical to those used in flow cytometry and on restrained smears used for visual analysis. The analysis was performed in two modes: as a study of 500 sequential nuclei in an automated system, mimicking flow cytometry, and visually selected large, presumably malignant nuclei. In nine of the 11 cases, the DNA content of visually selected cancer cells was aneuploid, even though this DNA pattern was not evident in the analysis of 500 sequential cells. In two cases, both diploid by flow cytometry, the Feulgen analysis confirmed the presence of cancer cells in the diploid range. In samples of 10(4) nuclei representing a mixed population of cells occurring in effusions, the presence of aneuploid cancer cells may not be disclosed by conventional flow cytometry. A larger sample of cells, a detailed analysis of DNA histograms, and perhaps sorting of select cells in the hypertetraploid range, may prove essential before flow cytometry can be accepted as a diagnostic tool in the laboratory in the assessment of effusions.

Do cells fuse in vivo?

Spontaneous or virally induced fusion between somatic cells can occur in vitro (1, 2, 3) and has provided a new and important tool for analysis of gene expression. In some instances, nuclear "mating" or hybridization is achieved; in others, the nuclei remain separate within the fusion product and a heterokaryon is formed. Under appropriate culture conditions, fusion can even take place between cells of unrelated species (3). Many ingenious experimental systems are being devised in this rapidly developing area of investigation and the differences between donor cells have been variously chosen so as to furnish models for normal differentiation or for the etiology of disease states. In a multicellular organism such as a mammal, do cells also fuse in vivo? If so, what are the consequences? Does cell hybridization occur in hematopoietic tissue, as some studies (4, 5, 6) have suggested? Are syncytia (e.g., skeletal muscle) formed by cell fusion? Do polyploidy and binucleation (e.g., in liver) ever arise by means of cell fusion, or only by endoreduplication? Cells in the body may be phagocytosed; does any of their DNA or RNA become incorporated and remain functional in the host cell? Do aneuploid cancer cells sometimes originate from cell "matings", and is this a significant factor in conversion of cells from normalcy to malignancy? In order to answer these and related questions in vivo, unequivocal genetic markers are required to distinguish the putative cellular contributors in a way that would be comparable to the tests of fusion in vitro. Moreover, it would be desirable to have the genotypically dissimilar cells associated throughout development of the intact organism, to learn what role any cell hybridization or heterokaryonization might play in differentiation, aging, or disease, as these processes ordinarily occur in situ. The allophenic mouse is a laboratory creation that fulfills these ground rules (7, 8, 9, 10, 11, 12, 13). As the name suggests, each animal contains phenotypically different subpopulations of cells within any or all tissues as a result of genotypic cellular differences. Allophenic mice in a sense owe their existence to the many innovations of modern tissue culture methodology, since each individual is produced by artificially aggregating very early embryo cells of different genotypes into one composite embryo in vitro. The aggregate, after a short culture period, is then returned to a uterine environment. The projected purpose in thus establishing genetic mosaicism throughout life was to allow complex developmental events at supracellular levels of organization to become decipherable. Owen's discovery (14) of two genetic populations of erythrocytes in dizygotic cattle co-twins with a shared placental circulation suggested to me that genetic mosaicism in all functional systems might be a useful experimental device for examining many sorts of problems in mammals. The embryologists of the nineteenth century had shown that blastomeres could be recombined in invertebrate or amphibian embryos. If cells of diverse constitution could be introduced into an early mammalian embryo, their eventual inclusion in all organs and tissues might be expected. The laboratory mouse, with its profus1 These investigations were supported by U.S.P.H.S. grants No. HD 01646 (formerly CA 05201) and CA 06927, and by an appropriation from the Commonwealth of Pennsylvania.