Micronuclei with multiple copies of the X chromosome: do chromosomes replicate in micronuclei?

The formation of a micronucleus due to chromosome lagging is a well known mechanism of chromosomal loss. However, the post-mitotic fate of the micronucleus and the chromosomal DNA within it is poorly understood. We observed micronuclei (MN) that had multiple copies of the X chromosome (ranging from 4 to 10) when analyzing cultured human lymphocytes using fluorescence in situ hybridization (FISH). A possible mechanism for this observation is that the chromosome(s) or chromatid(s) contained within the micronuclei successfully completed one or more cycles of replication after their expulsion from the primary nucleus.     

Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise

The in vitro micronucleus test with Syrian hamster embryo (SHE) cells assays the induction of micronuclei by chemical agents. Both chromosome fragments and lagging chromosomes can give rise to micronuclei. Nevertheless, only limited information is available on the ultrastructure of micronuclei and the mechanisms of their formation. Diethylstilbestrol (DES), a non-mutagenic carcinogen, as well as its analogue 3.3'-DES induce micronuclei in SHE cells. A comparison of the dose response of DES-induced micronucleus formation with the previously published ones for aneuploidy and transformation shows that all 3 run in parallel. Thus, a functional relationship between these endpoints, in the SHE system, may be implied. The present study is designed to address the formation of micronuclei using supravital UV microscopy, to test for the presence of defined chromosome domains within micronuclei using immunocytochemistry, and to define aspects of their ultrastructure by electron microscopy. Supravital UV microscopy showed that 3.3'-DES induces displacement of chromosomes/chromatids during prophase/anaphase and formation of micronuclei during cytokinesis. Immunocytochemistry revealed that micronuclei contain, at high frequencies, CREST antibody-reactive kinetochores, indicating the presence of whole chromosomes or centric fragments in these structures. Moreover, transmission electron microscopy showed that micronuclei exhibit ultrastructural details typical of interphase nuclei. Specifically, micronuclei exhibited morphological evidence of a nuclear lamina and segregation of karyoplasm into euchromatic and heterochromatic regions. All micronuclei examined were enclosed by a nuclear envelope of normal morphology and showed nuclear pore complexes. Together the findings provide evidence that DES interferes with the mitotic apparatus as early as prophase, resulting in the formation of micronuclei and, as a consequence, in the loss of chromatids or chromosomes.     

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.     

The fate of micronucleated cells post X-irradiation detected by live cell imaging

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.     

Activation status of the X chromosome in human micronucleated lymphocytes

The frequency of X chromosome aneuploidy in human female peripheral blood lymphocytes has been reported by several investigators to be significantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsible for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromsome is the inactive and not the active X. Blood samples were obtained from two unrelated females, 36 and 33 years of age, each with a different X; 9 reciprocal translocation. In each, the normal X chromosome is inactive and the translocated X is active. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 year old, while 9,500 binucleated cells were scored from the 33 year old. The slides were first labeled and the kinetochore status of each micronucleus was determined. This was followed by simultaneous hybridization with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronucleated cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2%) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) contained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inactive X chromosome is preferentially included in the micronuclei, and suggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon. Received: 5 May 1995 / Revised: 15 July 1995     

Premature chromosme condensation in the bone marrow of Chinese hamster after application of bleomycin in vivo.

The Chinese hamster bone marrow was used as a test system in vivo to analyse the chromosome-danaging effect of bleomycin. Both chromosome and chromatid aberrations were found. Mitoses with aberrations (Ma) show a linear dose-effect relationship after a recovery time of 24 h, the same hold true for cells with micronuclei (Cm) and for mitoses with premature chromosome condensation (PCC). The dose-effect relationships for Ma, Cm and PCC run parallel to each other with Ma at the highest and PCC at the lowest level (Ma greater than Cm greater than PCC). The time-effect relationships for Ma, Cm and PCC show that after 12 h recovery time there are no PCCs but the highest frequencies of Ma and Cm indicating that most cells are in their first post-treatment mitoses or Gi-phases at this fixation time. In addition to the frequency determinations autoradiographic analysis were performed to clarigy the nature of the PCCs. The results are interpreted as follows: bleomycin induces chromosomal aberrations that in turn give rise to micronuclei by means of lagging chromatin, main and micronuclei eventually become asynchronous in their cell cycles and mitosing main nuclei induce PCC in the micronuclei.     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.     

Aneuploidy,centrosome activity and chromosome instability in cells deficient in homologous recombination repair

Chromosome instability and loss or gain of chromosomes are changes characteristic of many tumour cells and human disorders. However, the mechanism of these changes has not yet been fully determined. We have recently shown that hamster cell lines deficient in homologous recombination repair (HRR) genes XRCC2 and XRCC3 have an elevated frequency of aneuploidy compared with wild-type cells and mutant cells transfected with the appropriate human gene. In addition, XRCC2 and XRCC3 deficient hamster cell lines show a high frequency of multiple centrosomes and abnormal spindle formation. Cells deficient in HRR show a high frequency of both chromosome-type and chromatid-type aberrations, which could potentially lead to mis-segregation. The role of chromosome aberrations and other factors, including chromosome lagging, premature chromatid separation, and centrosome malfunctioning on chromosome mis-segregation in irs1 and irs1SF cells have been investigated. In particular, the linkage of DNA repair proteins with centrosomes suggests a key role for the centrosome in controlling cellular repair processes.     

Studies of mitotic and centromeric abnormalities in Roberts syndrome: Implications for a defect in the mitotic mechanism

Roberts syndrome is an inherited human condition that is of particular interest because separation of centromeres and constitutive heterochromatin is observed in metaphase chromosomes. In this study we investigated the frequency of other cytological abnormalities in three Roberts syndrome patients. Our findings when taken with previous cytological reports emphasize that there are other features that are equally characteristic of Roberts syndrome: (1) aneuploidy with random chromosome loss and (2) micronuclei and/or nuclear lobulations of 8%–24% of interphase cells. We observed abnormal chromosome movement involving one or all the chromosomes during anaphase. Evidence is presented suggesting that aneuploidy, micronuclei and abnormal nuclear morphology are a direct result of lagging chromosomes. The cytological features documented for Roberts syndrome indicate that this is a human mitotic mutant.by T.C. Hsu     

Relative effectiveness of HZE iron-56 particles for the induction of cytogenetic damage in vivo

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as (56)Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of (56)Fe ions (1000 MeV/nucleon) relative to low-LET gamma rays and high-LET alpha particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to (60)Co gamma rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 x 10(-4), 12 x 10(-4), and 11 x 10(-4) micronuclei/binucleated cell cGy(-1), respectively. When genetic damage induced by radiation from (56)Fe ions was compared to that from exposure to (60)Co gamma rays, (56)Fe-ion radiation was between 0.9 and 3.3 times more effective than (60)Co gamma rays. However, the HZE-particle exposures were only 10-20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by delta rays for each cell that was traversed by the primary HZE (56)Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the delta rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be "wasted" as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.     

Age-dependent inclusion of sex chromosomes in lymphocyte micronuclei of man.

Two-color centromeric FISH was used to study the inclusion of the X and Y chromosomes in micronuclei of cultured lymphocytes from 10 men representing two age groups (21-29 years and 51-55 years). In addition, pancentromeric FISH was separately performed to identify any human chromosomes in micronuclei. One hundred micronuclei per probe were examined from each donor. A higher mean frequency of Y-positive micronuclei was observed in the older men than in the younger men. In both age groups, the X chromosome was micronucleated clearly more often than expected by chance, and the Y chromosome was overrepresented in micronuclei among the older men but not among the younger men. In lymphocytes of four women, X-positive micronuclei were more frequent than they were in men, even after the fact that women have two X chromosomes was taken into account. Similar results were obtained in first-division lymphocytes identified by cytochalasin-B-induced cytokinesis block. In comparison with normal cells, these binucleate cells showed a higher frequency (per 1,000 nuclei) of X-positive micronuclei (in the older men) but a lower frequency of micronuclei harboring autosomes or acentric fragments. In conclusion, the results show that both the X chromosome and the Y chromosome are preferentially micronucleated in male lymphocytes, the Y chromosome only in older subjects. Although the X chromosome has a general tendency to be included in micronuclei, it is micronucleated much more often in women than in men, which is probably the main reason for the high micronucleus frequency in women that has been documented in many previous studies.     

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.     

The X chromosome frequently lags behind in female lymphocyte anaphase

Pancentromeric FISH and X-chromosome painting were used to characterize anaphase aberrations in 2,048 cultured lymphocytes from a healthy 62-year-old woman. Of 163 aberrant anaphases, 66.9% contained either chromosomes or their fragments that lagged behind. Characterization of 200 laggards showed that 49% were autosomes, 33. 5% were autosomal fragments, and 17.5% were X chromosomes. The X chromosome represented one-fourth of all lagging chromosomes and was involved much more often than would be expected by chance (1/23). Labeling of the late-replicating inactive X chromosome with 5-bromo-2'-deoxyuridine revealed that both X homologues contributed equally to the laggards. Among 200 micronuclei examined from interphase cells, the proportion of the X chromosome (31%) and autosomal fragments (50%) was higher than among anaphase laggards, whereas autosomes were involved less often (19%). These findings may reflect either selection or the fact that lagging autosomes, which were more proximal to the poles than were lagging X chromosomes, were more frequently included within the main nucleus. Our results suggest that the well-known high micronucleation and loss of the X chromosome in women's lymphocytes is the result of frequent distal lagging behind in anaphase and effective micronucleation of this chromosome. This lagging appears to affect the inactive and active X chromosomes equally.     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes

Micronuclei are formed from chromosomes and chromosomal fragments that lag behind in anaphase and are left outside daughter nuclei in telophase. They may also be derived from broken anaphase bridges. Nuclear buds, micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated similarly to micronuclei during nuclear division or in S-phase as a stage in the extrusion of extra DNA, possibly giving rise to micronuclei. To better understand these phenomena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelomeric DNA probes. Such information has not earlier been available for human primary cells. Surprisingly, there appears to be no previous data on the occurrence of telomeres in micronuclei (or buds) of normal human cells in general. Our results suggest that nuclear buds and micronuclei have partly different mechanistic origin. Interstitial DNA without centromere or telomere label was clearly more prevalent in nuclear buds (43%) than in micronuclei (13%). DNA with only telomere label or with both centromere and telomere label was more frequent in micronuclei (62% and 22%, respectively) than in nuclear buds (44% and 10%, respectively). Folate deprivation especially increased the frequency of nuclear buds and micronuclei harboring telomeric DNA and nuclear buds harboring interstitial DNA but also buds and micronuclei with both centromeric and telomeric DNA. According to the model we propose, that micronuclei in binucleate lymphocytes primarily derive from lagging chromosomes and terminal acentric fragments during mitosis. Most nuclear buds, however, are suggested to originate from interstitial or terminal acentric fragments, possibly representing nuclear membrane entrapment of DNA that has been left in cytoplasm after nuclear division or excess DNA that is being extruded from the nucleus.     

X chromosome inactivation and micronuclei in normal and Turner individuals

Studies on aneuploidy have shown that the X is the most frequently lost chromosome in females, and that the number of X chromosome-positive micronuclei increases with age in women. Recently, we showed that the inactive X chromosome is incorporated preferentially in micronuclei. The objectives of the current study were, firstly, to determine the incidence of X chromosome incorporation into micronuclei in males and, secondly, to determine the incidence of X chromosome incorporation into micronuclei of females with Turner syndrome. Blood samples were obtained from 18 male newborns and 35 normal adult males ranging in age from 22 to 79 years and from seven women with non-mosaic Turner syndrome aged 11–39 years. Isolated lymphocytes were cultured in the presence of cytochalasin B and 2000 binucleated cells per subject were scored for micronuclei. Cells were then hybridized with the biotinylated X centromere-specific probe, pBamX7, and visualized with fluorescein-conjugated avidin. All micronucleated cells were relocated and evaluated for the presence or absence of the X chromosome. Of the 335 micronuclei observed, 6.6% (22/335) contained an X chromosome. Analysis of variance shows a statistically significant increase, for both males and Turner females, in the number of X chromosome-positive micronuclei with age (P < 0.001). These data also show that the X chromosome is included in micronuclei from males more often than would be expected by chance (P < 0.005; χ² analysis, 15 df). Here we show that there is a tenfold difference in the frequency of X chromosome-positive micronuclei in 46,XX females compared to 46,XY males and 45,X females, providing further support to our previous finding that the X chromosome in micronuclei is the inactive chromosome. Received: 29 April 1997 / Accepted: 9 May 1997     

The effect of functional inactivation of TP53 by HPV-E6 transformation on the induction of chromosome aberrations by gamma rays in human tumor cells

The influence of expression of TP53 (formerly known as p53) on the induction of chromosome aberrations by gamma rays was examined in an isogenic pair of human tumor cell lines where TP53 expression was normal or inactivated by human papillomavirus (HPV) type 16 E6 expression. Plateau-phase cultures were exposed to 0-8 Gy gamma rays and then either immediately released by subculture or held for 24 h prior to subculture and subsequent cytogenetic analysis. Aberration frequency was determined only in cells entering their first mitosis after irradiation, and cells were sampled over a 48-h period to include cells whose progression into mitosis was delayed. While aberration frequencies were similar at early harvest times, there was evidence for a subpopulation of more heavily damaged cells in the E6-transformed cells that cycled into late mitosis. Holding cells noncycling for 24 h to allow repair of potentially lethal damage eliminated this subpopulation of more heavily damaged cells. The E6-transformed cells also had higher levels of chromatid-type aberrations and sister chromatid exchanges, consistent with an additional defect in kinetics of repair of base damage that is associated with the E6 transformation. Holding cells noncycling for 24 h eliminated the elevated levels of chromatid-type aberrations and sister chromatid exchanges. These studies demonstrate that E6 transformation of human tumor cells will influence both the frequency and types of chromosome aberrations observed after radiation exposure, and that these effects are related to the expression of potentially lethal damage.     

Non-disjunction events induced by albendazole in human cells

Albendazole (ABZ), a benzimidazole carbamate used for the treatment of several human helminthiases has high affinity for tubulin, which results in an inhibition of microtubule polymerization, blocking several vital processes in the parasites, such as motility and nutrient uptake. The ability of ABZ to act as mitotic spindle poison leads to a potential risk for aneuploidy induction in exposed human beings. ABZ, as well as albendazole sulphoxide (ABZSO), its main metabolite, induce micronuclei in human cells in a dose-dependent manner. Despite recognition that ABZ and ABZSO increase micronucleus frequency, their potential as inducers of non-disjunction in human cells, an event considered more frequent than chromosome loss, and one of the main mechanisms involved in aneuploidy induction, has not been evaluated. In the present work, we investigated the ability of ABZ and ABZSO to induce non-disjunction in cultured human lymphocytes. Non-disjunction was scored by chromosome-specific FISH using a classical or alpha satellite probe for chromosomes 1 and 7, respectively. Significant increase in non-disjunction events that involved either chromosome were observed in cells treated with ABZ or ABZSO. Both ABZ and ABZSO induced non-disjunction at lower concentrations than those at which MN were observed.     

Ring Chromosomes and rDNA Magnification in Drosophila

Tartof showed that ribosomal gene magnification in Drosophila was inhibited in a ring X chromosome. The present studies extend this observation by showing that ring X chromosomes are lost meiotically in male Drosophila undergoing ribosomal gene magnification as evidenced by the recovery of a lower number of ring-bearing progeny under magnifying conditions compared with nonmagnifying conditions. Associated with ring chromosome loss is a highly significant increase in the number of double-sized dicentric ring chromosomes in meiotic cells from magnifying males. These observations explain the failure of ring X chromosomes to magnify and imply that magnification in rod chromosomes occurs via a mechanism of unequal sister chromatid exchange. Our results support the hypothesis that the primary event of magnification is a sister chromatid exchange in the rDNA, that the frequency of sister strand exchanges is increased in magnifying flies, that a significant number of exchanges in magnifying flies occurs meiotically and that some of the exchanges are nonreciprocal. We have also found that autosomal mutations can affect both the frequency of abnormal ring structures and the ability of ring X chromosomes to magnify.     

MAPK mediates RAS-induced chromosome instability

The generation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic material. The involvement of the MAPK pathway in mediating v-ras-induced micronuclei in NIH 3T3 cells was examined by inhibiting MAPK activation. Conversely, the MAPK pathway was constitutively activated by infecting cells with a v-mos retrovirus. Micronucleus formation was inhibited by the MAPK kinase inhibitors PD98059 and U0126, but not by wortmannin, an inhibitor of the Ras/phosphatidylinositol 3-kinase pathway. Transduction of cells with v-mos resulted in an increase in micronucleus formation, also consistent with the involvement of the MAPK pathway. Staining with the anti-centromeric CREST antibody revealed that instability induced by constitutive activation of MAPK is due predominantly to aberrant mitotic segregation, since most of the micronuclei were CREST-positive, reflective of lost chromosomes. A significant fraction of the micronuclei were CREST-negative, reflective of lost acentric chromosome fragments. Some of the instability observed was due to mitotic events, consistent with the increased formation of bi-nucleated cells, which result from perturbations of the mitotic spindle and failure to undergo cytokinesis. This chromosome instability, therefore, is a consequence of mitotic aberrations, mediated by the MAPK pathway, including centrosome amplification and formation of mitotic chromosome bridges.