The observation of mitotic division aberrations in mammalian cells exposed to chemical and radiation treatments

The fidelity of chromosome segregation and the maintenance of the integrity of the chromosome karyotype of eukaryotiic cells is dependent upon the synthesis and functioning of division-related structures such as the nuclear spindle and events such as the attachment of chromosomes to the spindle and their subsequent movement to the poles of the dividing cell. Chemical and physical treatment which modify the synthesis and functioning of division-related events may potentially lead to the production of cells with abnormal chromosome numbers (of both whole chromosome sets and of individual chromosomes).

The ability of environmental agents to modify division-related structures in mammalian cells has been assessed by morphological examination of exposed mitotically dividing cells using staining techniques which identify spindle and chromosome structure and by the analysis of the characteristics of microtubule polymerisation in vitro. Such techniques have been used to identify the spindle-modifying effects of chemicals such as the synthetic hormone diethylstilboestrol and modifications of chromosome to spindle attachment in cells exposed to both UV- and X-irradiation. Such modifications of cell-division-related activities may lead to alterations in the fidelity of division events leading to the production of chromosomally abnormal daughter cells with aneuploid or polyploid karyotypes.     

Preferential occurrence of chromosome 21 malsegregation in peripheral blood lymphocytes of Alzheimer disease patients

To further investigate our finding of high levels of spontaneous aneuploidy in somatic cells of Alzheimer's disease (AD) patients (Migliore et al. 1997), we studied the molecular cytogenetics of eight patients with sporadic AD and six healthy controls of similar age. Cytochalasin B-blocked binucleated peripheral blood lymphocytes from the AD patients and unaffected controls were used to measure micronucleus induction or other aneuploidy events, such as the presence of malsegregation in interphase nuclei (representing chromosome loss and gain). Dual-color fluorescence in situ hybridization (FISH) with differential labeled DNA probes was applied. We used a probe specific for the centromeres of chromosomes 13 and 21 combined with a single cosmid for the Down's syndrome region (21q22.2) to obtain information on spontaneous chromosome loss and gain frequencies for both chromosomes (13 and 21). FISH data showed that AD lymphocytes had higher frequencies of chromosome loss (evaluated as fluorescently labeled micronuclei) for both chromosomes, as well as higher frequencies of aneuploid interphase nuclei, again involving both chromosomes, compared to control lymphocytes. However, aneuploidy for chromosome 21 was more frequent than for chromosome 13 in AD patients. This preferential occurrence of chromosome 21 in malsegregation in somatic cells of AD patients raises the hypothesis that mosaicism for trisomy of chromosome 21 could underlie the dementia phenotype in AD patients, as well as in elderly Down's syndrome patients.     

Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts

The susceptibility of human fibroblast cells in culture to neoplastic transformation by chemical carcinogens is appreciably lower than that of rodent fibroblasts. We have proposed that a key step in the neoplastic progression of Syrian hamster embryo fibroblasts is the induction of aneuploidy by carcinogens. It is possible that the different sensitivity to neoplastic transformation of Syrian hamster versus human cells is due to a difference in genetic stability following treatment with chemicals inducing aneuploidy. Therefore, we measured the induction of numerical chromosome changes in normal human fibroblasts and Syrian hamster fibroblasts by 4 specific aneuploidogens. Dose- and time-dependent studies were performed. Nondisjunction, resulting in aneuploid cells with a near-diploid chromosome number, in up to 14-28% of the hamster cells was induced by colcemid (0.1 microgram/ml), vincristine (30 ng/ml), diethylstilbestrol (DES) (1 microgram/ml) or 17 beta-estradiol (10 micrograms/ml). In contrast, human cells displayed far fewer aneuploid (near-diploid) cells, i.e., 8% following treatment with colcemid (0.02 micrograms/ml) or vincristine (10 ng/ml) and only 3% following treatment with DES (6 micrograms/ml) or 17 beta-estradiol (20 micrograms/ml). The doses at which the maximum effect was observed are given. Treatment of human cells induced a higher incidence of cells with a near-tetraploid chromosome number, which was similar to the level observed in treated hamster cells except at the highest doses. These results indicate that human cells respond differently from hamster cells to agents that induce aneuploidy. In particular, nondisjunction yielding aneuploid human fibroblasts with a near-diploid chromosome number was less frequent. The magnitude of the observed species differences varied with different chemicals. The difference in aneuploidy induction may contribute, in part, to species differences in susceptibility of fibroblasts to neoplastic transformation.     

Aneuploidy,polyploidy and ploidy reversal in the liver

Polyploidy has been described in the liver for over 100 years. The frequency of polyploid hepatocytes varies by age and species, but up to 90% of mouse hepatocytes and approximately 50% of human hepatocytes are polyploid. In addition to alterations in the entire complement of chromosomes, variations in chromosome copy number have been recently described. Aneuploidy in the liver is pervasive, affecting 60% of hepatocytes in mice and 30–90% of hepatocytes in humans. Polyploidy and aneuploidy in the liver are closely linked, and the ploidy conveyor model describes this relationship. Diploid hepatocytes undergo failed cytokinesis to generate polyploid cells. Proliferating polyploid hepatocytes, which form multipolar spindles during cell division, generate reduced ploidy progeny (e.g., diploid hepatocytes from tetraploids or octaploids) and/or aneuploid daughters. New evidence suggests that random hepatic aneuploidy can promote adaptation to liver injury. For instance, in response to chronic liver damage, subsets of aneuploid hepatocytes that are differentially resistant to the injury remain healthy, regenerate the liver and restore function. Future work is required to elucidate the mechanisms regulating dynamic chromosome changes in the liver and to understand how these processes impact normal and abnormal liver function.     

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.     

Trichlorfon effects on mouse oocytes following in vivo exposure

Trichlorfon (TCF) is a widely used pesticide, which according to some epidemiological and experimental data, is suspected of being aneugenic in human and mouse cells. In particular, in vitro studies in mouse oocytes showed the induction of aneuploidy and polyploidy at the first meiotic division and of severe morphological alterations of the second meiotic spindle. We have tested the hypothesis that an acute treatment of mice with TCF might similarly affect chromosome segregation in maturing oocytes. Superovulated MF-1 mice were intraperitoneally injected with 400mg/kg TCF or orally administered with 600mg/kg TCF either at the time of or 4h after human chorionic gonadotrophin (HCG) injection. Oocytes were harvested 17h after HCG and metaphase II chromosomes were cytogenetically analyzed. No significant increase of aneuploid or polyploid cells was detected at any treatment condition. A significant (p<0.001) decrease of metaphases showing premature chromatid separation or premature anaphase II in all TCF-treated groups with respect to controls suggested that TCF treatment may have delayed the first meiotic division. To evaluate possible effects of the pesticide upon the second meiotic division, a group of females orally treated with 600mg/kg TCF at resumption of meiosis was mated with untreated males and zygotes were collected for cytogenetic analysis. No evidence of aneuploidy induction was obtained, but the frequency of polyploid zygotes was increased fivefold over the control level (p<0.01). Such polyploid embryos might have arisen from fertilization of oocytes that were either meiotically delayed and still in metaphase I at fertilization or progressed through anaphase II without cytokinesis. These findings show that in vivo studies on aneuploidy induction in oocytes may yield results different from those obtained by in vitro experiments and that both kinds of data may be necessary for risk assessment of environmentally relevant exposures.     

Flow cytometric and karyotypic analyses of androgen-responsive and androgen-independent Shionogi mouse mammary tumours

The androgen-responsive (AR) Shionogi mouse mammary carcinoma is a heterogeneous tumour composed of AR and androgen-independent (AI) cells. We characterized the cells of the AR tumour and those of its AI derivative by flow cytometric analysis of DNA content and karyotypic analysis of metaphase spreads. Both tumours had diploid and near tetraploid populations of cells. However, the AR and AI malignant cells of these tumours both appeared to be polyploid. A decrease in the polyploid population of the AR tumour accompanied tumour regression following castration, but this population was restored when tumour growth resumed. Although karyotypic analysis of metaphase spreads showed wide variations in chromosome numbers among the polyploid population, the range, 55-88 chromosomes, was found in both AR and AI tumours. In addition, the same chromosome anomalies, including a marker chromosome, were identified in both tumours. Since the AR and AI malignant cells could not be distinguished on the basis of their DNA content or karyotype, the cell types may not represent genetically distinct populations of cells. The AR cells may undergo alterations in gene expression in adapting to their androgen-free environment.     

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.     

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.     

The Role of Chromosome Missegregation in Cancer Development: A Theoretical Approach Using Agent-Based Modelling

Many cancers are aneuploid. However, the precise role that chromosomal instability plays in the development of cancer and in the response of tumours to treatment is still hotly debated. Here, to explore this question from a theoretical standpoint we have developed an agent-based model of tissue homeostasis in which to test the likely effects of whole chromosome mis-segregation during cancer development. In stochastic simulations, chromosome mis-segregation events at cell division lead to the generation of a diverse population of aneuploid clones that over time exhibit hyperplastic growth. Significantly, the course of cancer evolution depends on genetic linkage, as the structure of chromosomes lost or gained through mis-segregation events and the level of genetic instability function in tandem to determine the trajectory of cancer evolution. As a result, simulated cancers differ in their level of genetic stability and in their growth rates. We used this system to investigate the consequences of these differences in tumour heterogeneity for anti-cancer therapies based on surgery and anti-mitotic drugs that selectively target proliferating cells. As expected, simulated treatments induce a transient delay in tumour growth, and reveal a significant difference in the efficacy of different therapy regimes in treating genetically stable and unstable tumours. These data support clinical observations in which a poor prognosis is correlated with a high level of chromosome mis-segregation. However, stochastic simulations run in parallel also exhibit a wide range of behaviours, and the response of individual simulations (equivalent to single tumours) to anti-cancer therapy prove extremely variable. The model therefore highlights the difficulties of predicting the outcome of a given anti-cancer treatment, even in cases in which it is possible to determine the genotype of the entire set of cells within the developing tumour.     

Aneuploid IMR90 cells induced by depletion of pRB,DNMT1 and MAD2 show a common gene expression signature

Chromosome segregation defects lead to aneuploidy which is a major feature of solid tumors. How diploid cells face chromosome mis-segregation and how aneuploidy is tolerated in tumor cells are not completely defined yet. Thus, an important goal of cancer genetics is to identify gene networks that underlie aneuploidy and are involved in its tolerance. To this aim, we induced aneuploidy in IMR90 human primary cells by depleting pRB, DNMT1 and MAD2 and analyzed their gene expression profiles by microarray analysis. Bioinformatic analysis revealed a common gene expression profile of IMR90 cells that became aneuploid. Gene Set Enrichment Analysis (GSEA) also revealed gene-sets/pathways that are shared by aneuploid IMR90 cells that may be exploited for novel therapeutic approaches in cancer. Furthermore, Protein-Protein Interaction (PPI) network analysis identified TOP2A and KIF4A as hub genes that may be important for aneuploidy establishment.     

Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells.

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.     

Ploidy variation as an adaptive mechanism in human pathogenic fungi

Changes in ploidy have a profound and usually negative influence on cellular viability and proliferation, yet the vast majority of cancers and tumours exhibit an aneuploid karyotype. Whether this genomic plasticity is a cause or consequence of malignant transformation remains uncertain. Systemic fungal pathogens regularly develop aneuploidies in a similar manner during human infection, often far in excess of the natural rate of chromosome nondisjunction. As both processes fundamentally represent cells evolving under selective pressures, this suggests that changes in chromosome number may be a concerted mechanism to adapt to the hostile host environment. Here, we examine the mechanisms by which aneuploidy and polyploidy are generated in the fungal pathogens Candida albicans and Cryptococcus neoformans and investigate whether these represent an adaptive strategy under severe stress through the rapid generation of large-scale mutations. Insights into fungal ploidy changes, strategies for tolerating aneuploidies and proliferation during infection may yield novel targets for both antifungal and anticancer therapies.     

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.     

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4?days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2?weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3?days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.     

The origin and impact of embryonic aneuploidy

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter.     

DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.     

The mitotic checkpoint in cancer and aging: what have mice taught us?

The spindle assembly checkpoint is a cellular surveillance mechanism that functions to ensure faithful chromosome segregation during mitosis. Failure of this checkpoint can result in aneuploidy, a state of having abnormal numbers of chromosomes. Most human cancers consist of aneuploid cells, but it is unclear if the aneuploidy is a cause or a consequence of tumorigenesis. Over recent years, mouse models for spindle assembly checkpoint failure have been generated to investigate the biological relevance of the different spindle assembly checkpoint genes and the pathologies associated with chromosome number instability. Most of these models exhibit susceptibility to carcinogenesis. Moreover, one model has led to the identification of the spindle checkpoint protein BubR1 as a regulator of the normal aging process.     

Chromosomal composition of aneuploid clones with different DNA contents in head and neck squamous cell carcinomas as determined by combined flow cytometry and fluorescence in situ hybridization.

Studies with DNA flow cytometry (FCM) have shown that DNA contents of aneuploid tumour clones vary in a wide range. The aim of this study was to analyse whether homologous chromosomal changes exist despite the individual differences that may be of general relevance for the development of gross aneuploidy in squamous cell carcinomas of the head and neck. Fluorescence in situ hybridization (FISH) with 13 centromere-specific DNA probes was applied to 3 diploid and 11 aneuploid tumours with DNA indices ranging between 0.8 and 2.2. Disomic and monosomic cell populations were prevalent findings in DNA-diploid tumours. Polysomies were common in aneuploid tumours. Different degrees of aneusomy for identical chromosomes were recurrent features in aneuploid tumours. FISH signal heterogeneity was identified for all chromosomes. The mean number of aneusomic cell populations identified for DNA-aneuploid tumours ranged between 1.6 for chromosome 17 and 3.1 for chromosome 3. Inconsistencies between FISH and FCM data may indicate that centromere-specific DNA probes identify gains and losses of marker DNA due to complex karyotypic rearrangements rather than absolute changes in chromosome numbers. Overall, there was no evidence of the critical involvement of particular chromosomes in the development of different DNA contents.     

Inhibition of the M r 70,000 S6 kinase pathway by rapamycin results in chromosome malsegregation in yeast and mammalian cells

The antifungal and immunosuppressive drug rapamycin arrests the cell cycle in G1-phase in both yeast and mammalian cells. In mammalian cells, rapamycin selectively inhibits phosphorylation and activation of p70 S6 kinase (p70^S6K), a protein involved in the translation of a subset of mRNAs, without affecting other known kinases. We now report that rapamycin causes chromosome malsegregation in mammalian and yeast cells. Chromosome malsegregation was determined by metaphase chromosome analysis of human lymphocytes and lymphoblasts, detection of CREST-positive micronuclei in human lymphoblasts and Chinese hamster embryonic fibroblast (CHEF) cells, and selection of doubly prototrophic cells in a specially constructed yeast strain. The number of ana-telophases with displaced chromosomes and interphase and mitotic cells with an irregular number of centrosomes was also determined in CHEF cells. In quiescent mammalian cells (human lymphocytes and CHEF cells) induced with growth factor to re-enter the cell cycle, rapamycin was effective when cells were exposed at the time of p70^S6K activation. In yeast, rapamycin was more effective when treatment was started in G1- than in G2-synchronized cells. Cells from ataxia telangiectasia (A-T) patients are characterized by chromosome instability and have recently been found to be resistant to the growth-inhibiting effect of rapamycin. We found that an A-T lymphoblastoid cell line was also resistant to the induction of chromosome malsegregation by rapamycin, but the level of spontaneous aneuploidy was higher than in normal cells. In yeast, the induction of chromosome malsegregation was dependent on the presence of a wild-type TUB2 gene, encoding the β-subunit of tubulin. The finding that rapamycin acts in different cell types and organisms suggests that the drug affects a conserved step important for proper segregation of chromosomes. One or more proteins required for chromosome segregation could be under the control of the rapamycin-sensitive pathway. Received: 3 August 1998 / Accepted: 20 August 1998