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.     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

Use of a human X mouse hybrid cell line to detect aneuploidy induced by environmental chemicals

A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome segregation following chemical treatment. The human chromosome present in the mouse cells can be readily identified by differential staining procedures. The frequency of cells containing 0 or 2 human chromosomes in the progeny of chemically treated monochromosomal hybrid cells provided a direct measure of aneuploidy. We tested the sensitivity of the proposed system with 3 model chemicals (colcemid, cyclophosphamide and benomyl) known to induce numerical or structural changes in chromosomes. The frequency of an abnormal segregation of the human chromosome was found to be dose dependent and consistently higher than controls. This system has the capability to detect gain as well as loss of a chromosome resulting from nondisjunction or other mechanisms leading to aneuploidy.     

Analysis by BrUdR-labelling technique of induced aneuploidy in mammalian cells in culture

To study the origin of induced aneuploid cells, the BrUdR-labelling technique was applied to V79/AP4 Chinese hamster cells treated with colcemid or benomyl. In this way we were able to recognize the cells which had undergone one cellular division after the treatment since their chromosomes exhibited sister-chromatid differentiation. The results showed that the induced aneuploid cells can have either a few or numerous additional chromosomes depending on the concentrations of the drug. Moreover, it could be established that aneuploid cells with numerous additional chromosomes were obtained mainly when polyploid cells were also present in the treated population. This strongly suggests that the excess of additional chromosomes found in the aneuploid cells induced by the highest concentrations may be derived by disturbances of the whole mitotic apparatus rather than by a multiplicity of errors affecting individual chromosomes.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

The effect of hyper- and hypothermia on induced telomeric chromosome fusion]

After administration of colcemid and 5-BrdU in the cell culture, the cells pass through the first interphase to delay in mitosis. Then the cells overcome the colcemid blockade, and polykaryocytes with micronuclei are formed. The second interphase in followed by the second mitosis, during which dicentric chromosomes are observed. These dicentrics are the result of telomeric chromosome fusion. The action of hyperthermia (40 degrees C) during the whole period of colcemid and 5-BrdU treatment or that of the hyperthermia (40 degrees C) only during the first 17 hours (the first interphase and the first mitosis) lead to the increased frequency of dicentrics. Under condition of hypothermia (34 degrees C) the frequency of dicentric formation decreases. Changes in cultivation temperature during the latest 25 hr of colcemid and 5-BrdU action (the second interphase and the second mitosis) exert no influence on the dicentric formation frequency. Because there are no dicentrics in cells during the metaphase of the first mitosis, it is supposed that the temperature--sensitive period may be the latest steps of colcemid blockade, i.e. the period of formation of micronuclei.     

Induction of aneuploidy by mitotic arrestants in mouse bone marrow

Most human and animal carcinogens induce gene mutation, chromosome breakage or other types of DNA lesions. However, recent studies indicate that some carcinogens do not directly damage DNA, but may cause missegregation of chromosomes resulting in aneuploidy production. Aneuploidy-producing agents pose serious genetic hazards to the human population. Such agents may cause genomic imbalance not only in somatic cells which may result in cancer development, but also in germinal cells which may result in the production of abnormal offspring (e.g. Down's syndrome). To limit human exposure to potential aneuploidy-producing agents, such agents must first be identified in experimental animals. The present study demonstrates that vinblastine and colcemid are capable of inducing aneuploidy in bone marrow cells of treated mice. Both of these compounds are chemotherapeutic agents that arrest mitosis by interfering with the formation of spindle microtubules. Single intraperitoneal injections of vinblastine, at a dose of 9 mg/kg, were found to produce 1.5-5.2% of hyperdiploidy in all of the 10 treated mice sampled at 17-96 h after injection. Only the frequency of hyperdiploidy was determined because hypodiploid cells could result from artifactual chromosome loss during slide preparation. At 0.9 mg/kg, vinblastine was found to produce 0.5-3.5% of hyperdiploidy in 8 of the 10 treated animals. The frequency of hyperdiploid cells in animals treated with colcemid was low. A dose as high as 37 mg/kg was found to produce only 0.5-1% of hyperdiploidy in 3 of the 10 treated animals, and hyperdiploidy was observed only in animals sampled at 17-24 h. In 10 mice treated with saline alone, no hyperdiploid cells were observed. Unlike cell cultures where vinblastine and colcemid had been shown to be equally effective in producing aneuploidy, vinblastine was found in this study to be a much more potent aneuploidy inducer than colcemid in mice.     

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.     

The size of micronuclei in human lymphocytes varies according to inducing agent used

Micronuclei were induced in vitro in human lymphocytes by mitomycin C, X-rays, vincristine, and colcemid and analyzed in cells with preserved cytoplasm. The micronucleus/cell nucleus ratio was measured. It was found that micronuclei induced by mitomycin C and X-rays were significantly smaller than those formed by vincristine and colcemid. Thus, in spite of the wide size span of human chromosomes, it could be shown that it is possible to differentiate between micronuclei formed by spindle-damaging agents (vincristine and colcemid) and those induced by agents directly damaging the chromosomes (mitomycin C and X-rays). Mitomycin C-induced micronuclei were smaller than those induced by X-rays, probably because the former agent preferentially produces chromatid fragments and the latter chromosome fragments.     

Asynchrony of DNA replication and mitotic spiralization along heterochromatic portions of Chinese hamster chromosomes

Sequence of DNA synthesis and mitotic chromosome spiralization along heterochromatic portions of the sex (X₁X₂) and of some marker chromosomes in cultured Chinese hamster cells were studied, employing two methods: study of segmentation pattern caused in chromosomes with colcemid, and autoradiography with tritiated thymidine. The heterochromatic portions of all chromosomes studied were characterized by striking internal asynchrony of DNA replication. In particular, they had segments that replicated relatively early. The short arm of the X₂ chromosome, heterochromatic in female somatic cells, had at least three such segments. Replication patterns of the long arms of the X₁ and X₂ chromosomes were different. In X₁ this arm contains several segments showing relatively early replication. The long arm of X₂ had no similar segments. The possible significance of the data obtained is discussed with regard to the problem of genetic inertness of heterochromatin. At the terminal stage of the S period, H³-thymidine seems to be incorporated into condensed chromatin of interphase nuclei. On the basis of the data obtained, it is proposed that during replication of heterochromatin consecutive despiralization of parts of it takes place.     

Commitment to ploidy conversion of 3Y1 cells during metaphase arrest by colcemid

Diploid rat 3Y1 fibroblasts proliferate to a saturation density, where they are arrested with a 2N DNA content. After treatment to induce ploidy conversion, the conversion rate can be estimated by determining the fraction of cells with a 4N DNA content in the confluent culture using flow cytometry. Using this method it was found that during mitotic inhibition with colcemid, 3Y1 cells were converted to tetraploids with a high efficiency (above 80%); the optimum colcemid concentration and exposure period were 40 ng/ml and 8 hr, respectively. When metaphase cells were reseeded with 40 ng/ml of colcemid, they delayed anchorage to a dish; 6 hr was required for complete adhesion (in the absence of colcemid only 1 hr was required). When reseeded metaphase cells were exposed to 40 ng/ml of colcemid for 5 hr followed by its removal, a greater fraction of the cells anchored to the substratum were converted to tetraploids, whereas most of the floating cells were not. A greater fraction of the anchored cells had formed nuclei, whereas most of the floating cells preserved condensed metaphase chromosomes. These results indicate that the cells which have formed nuclear structure without chromosome separation during mitotic inhibition are irreversibly committed to ploidy conversion, with restoration of anchorage.     

Commitment to Ploidy Conversion of 3y1 Cells During Metaphase Arrest By Colcemid

Abstract. Diploid rat 3Y1 fibroblasts proliferate to a saturation density, where they are arrested with a 2N DNA content. After treatment to induce ploidy conversion, the conversion rate can be estimated by determining the fraction of cells with a 4N DNA content in the confluent culture using flow cytometry. Using this method it was found that during mitotic inhibition with colcemid, 3Y1 cells were converted to tetraploids with a high efficiency (above 80%); the optimum colcemid concentration and exposure period were 40 ng/ml and 8 hr, respectively. When metaphase cells were reseeded with 40 ng/ml of colcemid, they delayed anchorage to a dish; 6 hr was required for complete adhesion (in the absence of colcemid only 1 hr was required). When reseeded metaphase cells were exposed to 40 ng/ml of colcemid for 5 hr followed by its removal, a greater fraction of the cells anchored to the substratum were converted to tetraploids, whereas most of the floating cells were not. A greater fraction of the anchored cells had formed nuclei, whereas most of the floating cells preserved condensed metaphase chromosomes. These results indicate that the cells which have formed nuclear structure without chromosome separation during mitotic inhibition are irreversibly committed to ploidy conversion, with restoration of anchorage.     

Arrangement of prematurely condensed chromosomes in cultured cells and lymphocytes of the Indian muntjac

Premature chromosome condensation (PCC) was induced in order to study the arrangement of muntjac chromosomes in the interphase nuclei of proliferating and resting cells with respect to their polarity and the spatial relationship between them. The data were compared with the situation in in situ fixed and colcemid blocked metaphases. It appears that in rapidly dividing cells almost all G₁- and G₂ interphase chromosomes exhibit the Rabl type polarized orientation. This pattern still predominates in G₀ lymphocytes which may have been arrested at this stage for some months or even years. — The location of the small chromosome Y₂ was found to be central in normal metaphases but peripheral in colcemid blocked mitoses. The behavior in the premature condensed chromosome preparations was intermediate. Measurements of centromere distances between all possible pairs of chromosomes as well as on the relative position of chromosomes in circular spreads revealed no evidence for homologous somatic association during interphase and metaphase or any other suprachromosomal ordering principle. Interphase chromosome orientation seems to be solely the result of chromosome arrangement of the foregoing anaphase. Association between heterochromatic regions or the nucleolus organizers did not substantially influence this pattern. There is no support for speculations that in mammalian cells close proximity of homologoues sites is instrumental in functional cooperation.     

Effect of colcemid on the spreading of fibroblasts in culture

The effect of colcemid upon the spreading of mouse embryo fibroblast-like cells on substrates was studied with the aid of time-lapse microcinematography and scanning electron microscopy. Two types of substrates were used: flat glass and narrow strips of glass surrounded by non-adhesive lipid film; on the latter, spreading and polarization of cells proceeded simultaneously. On glass, colcemid did not prevent transition of cells into a well-attached state; however, the time required for this transition increased considerably as compared with control cultures. Similar effects were caused by two other drugs inhibiting the formation of microtubules: colchicine and vinblastine. The intermediate stages of spreading on flat glass had several abnormal features in the colcemid-containing medium: (a) the shape of cytoplasmatic outgrowths formed by the cell was altered and their distribution became less regular; (b) partial detachment of the attached parts of the cells was very frequent; (c) the spreading of various parts of the cell was not well correlated: the central part of the cell could remain unspread long after the spreading of the peripheral part. Similar effects of colcemid were observed in experiments with cells spreading on the narrow strips of the glass. In addition, colcemid prevented stabilization of the cell surface, i.e., differentiation of the cellular edge into active and stable parts. About two-thirds of the cells attached to the narrow strips of glass were completely detached from the substrate in the course of spreading in colcemid-containing medium. The possible mechanisms of the action of colcemid on spreading are discussed and it is suggested that intracellular structures sensitive to colcemid are essential for the coordination of the reactions in various parts of the cell in the course of spreading.     

Variability in the expression of the fragile site of the (fra)X chromosome in 2 consecutive cell cycles

The number and morphology of X chromosomes were analysed in tetraploid cells induced with colcemid in cultured blood lymphocytes obtained from a patient with fra(X) syndrome of mental retardation. In contrast to diploid cells containing fra(X) chromosome in 22.7% of cells, the marker X was found in 51.6% of tetraploids, each cell containing only one fragile X out of the two expected ones. The data obtained indicate an extreme lability of the expression of fragile site (X) (q 27) in consecutive lymphocyte generation.     

拟南芥（Arabidopsis thaliana）体细胞胚胎发生体系中的体细胞类减数分裂研究

在拟南芥生态型LandsbergErecta体细胞胚胎发生体系的胚性愈伤组织中观察到2种类型的体细胞减数分裂现象。一种是体细胞染色体减数分组,其中,处于前期或中期的细胞染色体分为2个或2个以上的组。其共同特点是,染色体直接分开,未观察到纺锤体,从染色体的形态也看不出纺锤体的作用。染色体减数分组较多发生于多倍体细胞中。另一种类型是体细胞减数分裂,这种类型类似于大小孢子发生过程的减数分裂,如第一次分裂前期也有染色体的联会和配对。在脱分化培养基上的胚性愈伤组织中,单倍体细胞约占3%,四倍体细胞约占4%。经体细胞类减数分裂产生的细胞都发生染色体重组。     

Behavior of the nucleolus organizer regions of the chromosomes in polykaryocytes consisting of micronuclei

The nucleolar organizer regions (NORs) of Chinese hamster chromosomes (clone 237, cell line BIId-ii-FAF28) were studied in mononuclear cells and polykaryocytes induced with colcemid. The chromosomes with NORs were marked as 1, 2, 3, 4. The activity of NORs in mononuclear cells was higher in chromosomes 1, 2, 3. The associations of NORs were observed between chromosomes I and 2 (3% of all metaphases). In polykaryocytes the chromosomal pairs 1, 2, 3 showed different NOR activity in different metaphases. The associations of NORs in pairs of chromosome I were found in 51.3% of cases. The associations of NORs in pairs of chromosome 2 were observed in 7.5% of cases. This method may be used for the estimation of association potency of NORs in chromosomes.     

Chromosome preparations of human blood lymphocytes—Evaluation of techniques

Several parameters of human lymphocyte culturing techniques and metaphase chromosome preparation procedures were studied and quantitatively evaluated in regard to their influence on the results of Q and G banding procedures. The culturing conditions were studied using³H thymidine incorporation as a parameter. A whole blood culturing technique using Ham's F12 medium was found to give optimal and consistent results. Colcemid concentration proved to be of no influence on chromosome contraction or on the number of metaphases obtained over the concentration range investigated. Prolonged exposure to colcemid was found to cause a decrease in the mean chromosome length but the absolute number of metaphases with a low degree of chromosomal contraction hardly decreased. Different spreading techniques were quantitatively analysed and factors important for the spreading of chromosomes were evaluated. Based on the results obtained, an optimal procedure is described which over a period of one year has given consistent results.     

Measurement of micronuclei by cytokinesis-block method in cultured Chinese hamster cells: comparison with types and rates of chromosome aberrations

The cytokinesis-block method of Fenech and Morley (1985) has been tested for the enumeration and characterization of micronuclei in exponentially growing Chinese hamster cells in culture. The consistent dose-response relations were obtained in cultures treated with mitomycin C, caffeine and colcemid. Comparison with the chromosome aberration frequencies indicated that approximately 30% of the acentric chromosomes are expressed as micronuclei in the mitomycin C and caffeine treated cells. The size distribution of the micronuclei suggested that the base-line frequency of micronuclei is mainly a reflection of mitotic dysfunctions rather than chromosome structural aberrations.