Delayed disruption of the telomeric links between chromosomes in experimentally induced cells with micronuclei exposed to 5-bromodeoxyuridine in the 1st S period after colcemid administration

Under a long-term administration of colcemid in the Chinese hamster cell culture some cells with micronuclei are seen to form. In the case of co-treatment with colcemid and 5-bromodeoxyuridine (5-BrdU) at metaphases of the first division of cells with micronuclei polycentric chromosomes were observed. These polycentric chromosomes occur due to delayed disruption of telomeric links, previously existing in the interphase. During colcemid treatment the cells pass through two S-periods: one in mononuclear cells, the other in cells with micronuclei. This phenomenon was tested according to the frequency of metaphases with dicentrics after 5-BrdU-treatment of cells at the first or second S-period or during the two cycles of chromosome replication. The 5-BrdU treatment during the first cycle or two cycles of replication resulted in the same frequency of cells with dicentrics--about 50%. The treatment with colcemid alone during two cycles of replication and administration 5-BrdU at the second S-period results in a considerably lower amount (%) of cells with dicentrics--about 10%. Thus, the delayed disruption of telomeric links between chromosomes may occur under the treatment with 5-BrdU at the first S-period after colcemid administration. It is also concluded that this phenomenon can be reproduced in cell with micronuclei when 5-BrdU is incorporated differentially in the sister chromatids.     

5-Chlorodeoxyuridine, more effectively than 5-bromodeoxyuridine, induces the formation of specific dicentric chromosomes in cells with micronuclei

5-chlorodeoxyuridine (5-ChldU) and 5-bromodeoxyuridine (5-BrdU) were tested in respect of the formation of specific dicentric chromosomes in cells with micronuclei. It is shown that the frequency of dicentrics is higher when 5-ChldU is used. On the ground of results of this study and of the previous data it is concluded that the activity of halogenated deoxyuridines is falling with molecular weight increasing.     

Enhanced intensity of polycentric chromosome formation in cells with micronuclei exposed to 5-bromodeoxyuridine under prolonged hyperthermia at 40 degrees C

The formation of polycentric chromosomes in metaphases of the first mitotic division of polykaryocytes containing micronuclei, under 5-BrdU treatment in the condition of a prolonged hyperthermia was studied. It was shown that the enhance of the frequency of polycentric chromosomes at 40 degrees C was statistically reliable. These data propose the existence of an enzymatic mechanism of disrupting the chromosomal ends at mitosis at the optimal condition (37 degrees C). In cells with micronuclei, under 5-BrdU treatment at 40 degrees C, this mechanism undergoes, presumably, a partial inhibition leading to the enhanced frequency of chromosomes with fused telomeres.     

Telomeric chromosome fusion in cells with micronuclei under the combined action of hyperthermia and hypothermia and halogenated analogs of thymidine]

The reaction of cells with micronuclei in respect of the induction of specific dicentric chromosomes with halogenated analogs of thymidine at various temperatures was studied. The positive correlation between the temperature and frequency of dicentrics was shown for all halogenated analogs of thymidine. The minimum frequency of dicentrics was found in the case when used 5-iododeoxyuridine and hypothermia (34 degrees C). The using of 5-bromodeoxyuridine at different temperatures displayed the intermediate results. The maximum level of dicentrics discovered under action of 5-chlorodeoxyuridine and hyperthermia (40 degrees C). In the former case the depression of mitotic chromosome condensation of micronuclei registered, in the latter one the chromosomes with portions of delayed spiralization were not found.     

The induction of specific dicentric chromosomes in CHO-6 Chinese hamster ovary cells

Under a long treatment of cells CHO-6 with 5-bromodeoxyuridine and colcemid it was discovered that dicentric chromosomes in metaphases of the first division of polykaryocytes contained micronuclei. When the cells were treated with colcemid only, the number of dicentrics was less. This phenomenon was described earlier in the Chinese hamster cell line B11d-ii FAF28. Under the similar testing of continuous cell lines of human, swine, African green monkey and Syrian hamster origin this result failed be reproduced. From the data of experiments it was testified that the phenomenon in question may be very likely the characteristics of all the Chinese hamster continuous cell lines.     

Condensation anomalies and exclusion in micronuclei of rearranged chromosomes in human fibroblasts cultured in vitro

Anomalies of chromatin condensation, such as fragmentation, uncoiling and pulverization, were observed in XP9UV25, a xeroderma pigmentosum fibroblast clone in which a high proportion of cells carried an end-to-end dicentric chromosome, dic (5;16) (p15.2;q24), that gives rise during propagation in culture to a variety of dicentric and monocentric derivatives. The coiling anomaly affected exclusively part of a rearranged chromosome, in particular the region previously involved in breakage events. The heterochromatic 16q region, which is a preferential breakpoint in the formation of dicentric and monocentric derivatives, was consistently the limit of the uncoiled or pulverized regions. This observation suggests that the anomalous chromatin behavior could derive from alteration of a region relevant for the correct condensation of the chromosome. In XP9UV25 the frequency of nuclei with associated micronuclei increased with time in culture, in parallel with that of mitoses with dicentric chromosomes. In situ hybridization with DNA probes specific for chromosomes 5 and 16 revealed hybridization signals in about 40% of micronuclei. Since the frequency of micronuclei is about ten times less than that of dicentrics, it is probable that only the rearranged chromosomes undergoing coiling anomalies are excluded in micronuclei.     

Time course study of the chromosome-type breakage-fusion-bridge cycle in maize.

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. "Healing" of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.     

Telomeric fusion of the chromosomes in cells with micronuclei during the subsequent incorporation into the DNA of thymidine halogenated analogs

As was shown elsewhere among halogenated analogs of thymidine there exist strong (5-chlorodeoxyuridine, 5-ChldU) and weak (5-iododeoxyuridine, 5-IdU) inducers of dicentric chromosomes in cells with micronuclei. Since from colcemid administration to the moment of fixation two cycles of replication are passed, a study was made of the pattern of induction of dicentric chromosomes under subsequent administration of 5-ChldU and 5-IdU, and the other way round. It was shown that the pattern of induction of dicentric chromosomes was strong or weak, and depended on the fact which analog was administered in the first S-period.     

Web-based scoring of the dicentric assay,a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0–5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.     

Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption

The effects of colcemid (0.16-1.0 microM) and taxol (10 microM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high-voltage electron microscopy of serial 0.25-micron sections. Although these drugs induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubules, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5-7 h at 37 degrees C and then form 4N "micronucleated" restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration-dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16-0.25 microM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (greater than or equal to 1.0 microM) concentrations. However, even in the presence of 1.0 microM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell-cycle-dependent assembly and disassembly of axonemal microtubules in vivo.     

NuMA is required for the proper completion of mitosis

NuMA is a 236-kD intranuclear protein that during mitosis is distributed into each daughter cell by association with the pericentrosomal domain of the spindle apparatus. The NuMA polypeptide consists of globular head and tail domains separated by a discontinuous 1500 amino acid coiled-coil spacer. Expression of human NuMA lacking its globular head domain results in cells that fail to undergo cytokinesis and assemble multiple small nuclei (micronuclei) in the subsequent interphase despite the appropriate localization of the truncated NuMA to both the nucleus and spindle poles. This dominant phenotype is morphologically identical to that of the tsBN2 cell line that carries a temperature-sensitive mutation in the chromatin-binding protein RCC1. At the restrictive temperature, these cells end mitosis without completing cytokinesis followed by micronucleation in the subsequent interphase. We demonstrate that the wild-type NuMA is degraded in the latest mitotic stages in these mutant cells and that NuMA is excluded from the micronuclei that assemble post-mitotically. Elevation of NuMA levels in these mutant cells by forcing the expression of wild-type NuMA is sufficient to restore post-mitotic assembly of a single normal-sized nucleus. Expression of human NuMA lacking its globular tail domain results in NuMA that fails both to target to interphase nuclei and to bind to the mitotic spindle. In the presence of this mutant, cells transit through mitosis normally, but assemble micronuclei in each daughter cell. The sum of these findings demonstrate that NuMA function is required during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly.     

Nuclei with protrusions--"tailed" nuclei--and radiation cytogenetic markers in a lymphocyte culture after x-ray irradiation

Frequency of the appearance of binuclear cells with nuclei having outgrowth into the cytoplasmic space and arise after first mitosis in human lymphocyte culture is linear-square dependent on the X-irradiation at doses from 0.0 to 4.0 Gy. Positive correlation between frequency of cells with "tailed" nuclei and frequency of metaphases of first mitosis having dicentrics and rings was established. Apparently, formation such "tailed" nuclei is connected with dicentrics and rings.     

Use of unstable chromosome aberrations for biological dosimetry after the first postirradiation mitosis

The loss of unstable chromosome aberrations after the first postirradiation mitosis makes their use difficult in radiation dosimetry. We describe here a method which, in a cell population observed at this stage, allows retrospective estimation of the frequencies of the unstable aberrations induced at the time of irradiation, and their use as a dosimeter. The laws controlling the behavior of unstable aberrations during mitosis were defined from a large-scale experiment on irradiated human lymphocytes. For cells undergoing the first, second, or third mitosis after irradiation, relationships were determined between the frequency, at irradiation time, of acentric fragments not arising from formation of dicentrics or rings, and the ratio of dicentrics and centric rings appearing without acentric fragments to the total number of dicentrics plus rings. On the basis of this ratio, the method described here provides an assessment of the postirradiation mitotic activity in a cell population. This assessment permitted estimation of the cell distribution and frequency of dicentrics plus centric rings, and of the frequency of acentric fragments at the time of irradiation. The use of this method for retrospective dosimetry after whole-body irradiation under various conditions of exposure is illustrated.     

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.     

Emergence of Micronuclei and Their Effects on the Fate of Cells under Replication Stress

The presence of micronuclei in mammalian cells is related to several mutagenetic stresses. In order to understand how micronuclei emerge, behave in cells, and affect cell fate, we performed extensive time-lapse microscopy of HeLa H2B-GFP cells in the presence of hydroxyurea at low concentration. Micronuclei formed after mitosis from lagging chromatids or chromatin bridges between anaphase chromosomes and were stably maintained in the cells for up to one cell cycle. Nuclear buds also formed from chromatin bridges or during interphase. If the micronuclei-bearing cells entered mitosis, they either produced daughter cells without micronuclei or, more frequently, produced cells with additional micronuclei. Low concentrations of hydroxyurea efficiently induced multipolar mitosis, which generated lagging chromatids or chromatin bridges, and also generated multinuclear cells that were tightly linked to apoptosis. We found that the presence of micronuclei is related to apoptosis but not to multipolar mitosis. Furthermore, the structural heterogeneity among micronuclei, with respect to chromatin condensation or the presence of lamin B, derived from the mechanism of micronuclei formation. Our study reinforces the notion that micronucleation has important implications in the genomic plasticity of tumor cells.     

Cell-cycle progression without an intact microtuble cytoskeleton

For mammalian somatic cells, the importance of microtubule cytoskeleton integrity during interphase cell-cycle progression is uncertain. The loss, suppression, or stabilization of the microtubule cytoskeleton has been widely reported to cause a G1 arrest in a variable, and often high, proportion of cell populations, suggesting the existence of a "microtubule damage," "microtubule integrity," or "postmitotic" checkpoint in G1 or G2. We found that when normal human cells (hTERT RPE1 and primary fibroblasts) are continuously exposed to nocodazole, they remain in mitosis for 10-48 hr before they slip out of mitosis and arrest in G1; this finding is consistent with previous reports. To eliminate the persistent effects of prolonged mitosis, we isolated anaphase-telophase cells that were just finishing a mitosis of normal duration, then we rapidly and completely disassembled microtubules by chilling the preparations to 0 degrees C for 10 minutes in the continuous presence of nocodazole or colcemid treatment to ensure that the cells entered G1 without a microtubule cytoskeleton. Without microtubules, cells progressed from anaphase to a subsequent mitosis with essentially normal kinetics. Similar results were obtained for cells in which the microtubule cytoskeleton was partially diminished by lower nocodazole doses or augmented and stabilized with taxol. Thus, after a preceding mitosis of normal duration, the integrity of the microtubule cytoskeleton is not subject to checkpoint surveillance, nor is it required for the normal human cell to progress through G1 and the remainder of interphase.     

Kinetochore analysis of micronuclei allows insights into the actions of colcemid and mitomycin C

We have induced micronuclei in two strains of diploid human fibroblasts with a known aneugen, colcemid, and a known clastogen, mitomycin C. Using immunofluorescence to detect the presence of kinetochores in micronuclei, we were able to demonstrate a 26.8-fold increase in fluorescence-positive micronuclei (aneuploidy) in colcemid-treated cells. However, colcemid also induced an increase in kinetochore-negative micronuclei. Our findings support previous reports that suggest colcemid may induce chromosome breakage in addition to its major aneugenic effect. The frequency of kinetochore-negative micronuclei (chromosome breakage) in mitomycin C-treated cells rose an average of 7.9-fold in the two test strains, a clear reflection of its clastogenic action. However, a 4-fold increase in the kinetochore-positive fraction was seen. We conclude that the fibroblast micronucleus assay, coupled with kinetochore immunofluorescence, provides a useful screening approach for genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the rates of chromosomal breakage or lag may require more detailed analysis.     

Persistence of micronuclei in the marine mussel, Mytilus galloprovincialis, after treatment with mitomycin C

The frequency of micronuclei induced by mitomycin C (MMC) in cells of the gill tissue of the marine mussel, Mytilus galloprovincialis Lmk., was determined over a long period (up to 40-52 days) following treatment. Two doses of MMC (0.5 X 10(-7) and 10(-7) M) were tested at 13 degrees C and 23 degrees C, temperatures representative of the winter and summer thermic conditions of the Mediterranean Sea. In all cases, the frequency of micronuclei was significantly increased by MMC and declined after treatment until it reached a plateau level, significantly higher than the control value. This persisted for a very long time. The frequency of micronuclei induced by a second treatment with MMC performed on the 28th day, did not differ significantly from that produced by the first treatment at the same dose. Temperature did not influence the pattern of the described phenomena to a significant extent. The reason for the persistence of an increased frequency of micronuclei is discussed, and a system is proposed for evaluating the genotoxicity of water pollutants present long before sampling.     

A comparative investigation of the antimitotic action of 2-mercaptoethanol and colcemid on V-79 Chinese hamster cells

Abstract. The current study was performed to characterize the antimitotic action of 2-mercaptoethanol (MET) on mammalian cells.
At concentrations of 2.5 × 10^-2 M, MET arrests V-79 Chinese hamster cells in metaphase. Smaller concentrations (from 5 × 10^-3 M) only produce a mitotic block after several hours, only arresting those mitoses which have gone through one cell cycle in the presence of MET. The accumulation of mitoses by MET is smaller in comparison with colcemid, explained by an effect reducing the number of cells which enter mitosis. In contrast to colcemid, MET-concentrations which do not lead to a mitotic block cause a delay in proliferation. It was shown, by means of the BUdR-labelling method that cells in the presence of colcemid concentrations which arrest mitosis again enter interphase and become polyploid, whereas MET leads to an irreversible arrest of mitosis and does not produce polyploidy in V-79 cells.     

Observations on dicentrics in living cells

Summary In previously irradiated endosperm cells of Haemanthus katherinae studied in vitro by means of micro-cinematography, two-kinetochore chromatids and dicentric chromosomes have been observed. Breaking of such dicentric chromatids and chromosomes has been analysed. Behaviour of some of the dicentric chromosomes during anaphase deserves special attention: interlocking dicentrics cut one through another and rejoin in a few minutes. In this way from a metaphase interlocking dicentric, two sister anaphase dicentrics are formed. Interlocked dicentrics can also uncoil and not break at all. In this case no activity was observed in one kinetochore of one dicentric in later stages of anaphase (two kinetochores were active in one dicentric and only one in its sister). Analysis of chromosome movements in two-kinetochore chromatids and dicentrics is also presented.