Localization of anti-topoisomerase IIα antibodies under normal conditions and after artificial chromosome condensation and decondensation

By means of immunofluorescence method, localization of DNA-topoisomerase IIα (Topo IIα) in interphase nuclei and chromosomes at different stages of mitosis was studied in situ under normal conditions and after treatment with condensing and decondensing solutions. In non-isolated mitotic M-HeLa cell chromosomes, Topo IIα was uniformly distributed along chromatids after fixation and permeabilization in situ. After treatment of cells with decondensing solutions (10 mM Tris; 0.1 mM CaCl₂ in 10 mM Tris; 0.3 mM CaCl₂ in 10 mM Tris; 15% DMEM; 75 mM KCl), Topo IIα was evenly distributed along chromatids in prophase, prometaphase and metaphase; its concentration was the highest in the pericentromere region. After treatment of cells with condensing solutions containing 0.7 mM, 1 mM, 2 mM or 3 mM CaCl₂ in 10 mM Tris, Topo IIα was not detected in prophase, metaphase and anaphase. However, in late telophase anti-Topo IIα antibodies were found in reforming nuclei under identical conditions. After sequential treatment with condensing and decondensing solutions, the distribution patterns of Topo IIα in chromosomes were the same as after treatment with only decondensing solutions. In anaphase and telophase, Topo IIα was evenly distributed along chromatids, while in prophase, prometaphase and metaphase it was predominantly localized in the pericentromere region. After the treatment of cells with condensing solutions chromosome staining was not observed, apparently due to “masking” of binding sites for anti-Topo IIα antibodies. Homogenous distribution of Topo IIα along chromatids in non-isolated chromosomes was preserved after the treatment of cells with hypotonic solutions; however, under these conditions Topo IIα concentration was higher in centromeres.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Evidence for Heterochromatin Involvement in Chromosome Breakage in Maize Callus Culture

ng from delayed separation of chromatids and typical bridgeswere observed in Feulgen preparations. The analysis of C-bandedanaphases showed that delayed chromatids were held togetherat heterochromatic knob sites (primary event), and the presenceof typical bridges with and without bands corresponding to knobs.These events suggest the occurrence of breakage-fusion-bridge(BFB) cycles initiated by chromosome arms broken during theprimary event. Additional evidence for such a mechanism wasthe presence of gross aberrations involving chromosome 7, detectedin several C-banded metaphases of some cultures. It is hypothesizedthat such aberrations are duplication deficiencies producedby BFB cycles and chromosome healing that would have occurredafter some cell divisions. Zea mays L.; maize; tissue culture; chromosome breakage; heterochromatin; C-banding     

Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance

Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.     

Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea

Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia.     

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.     

Cytogenetic demonstration of out-of-phase DNA synthesis in endoreduplicated CHO cells: evidence for partial endoreduplication

Out-of-phase DNA synthesis, which is demonstrated cytogenetically as premature chromosome condensation (PCC), was analyzed in endoreduplicated Chinese hamster ovary (CHO) cells induced by colchicine or vincristine. Like conventional polyploid cells, endoreduplicated cells exhibited PCC in either S or G2. The former was more frequently observed in drug-treated cultures. In addition to these two types of PCC, other mitotic figures showing out-of-phase DNA synthesis were found. Such cells contained both conventional chromosomes (monochromosomes) and diplochromosomes. Differential FPG staining of chromatids in these cells showed that diplochromosomes incorporated BrdU twice while monochromosomes did so once, indicating the occurrence of partial endoreduplication in one of the sister nuclei of multinucleate cells. The possible mechanisms underlying induction of out-of-phase DNA synthesis and production of partial endoreduplication are discussed.     

INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES AFTER FUSION WITH INTERPHASE CELLS

The process of cellular fusion induced by Sendai virus in Chinese hamster cells (Don line) afforded us the opportunity to study nuclear envelope formation around metaphase sets in the presence of interphase nuclei, when chromosome pulverization failed to occur in such multinucleate cells. Morphologically, the enveloped metaphase chromosomes resembled a normal telophase nucleus, though minor differences prompted us to call it telophase-like. Electron microscopic observations demonstrated that the membranes enveloping the chromosomes appeared to be identical with a normal nuclear envelope. The longer the cells were incubated with Colcemid before fusion, the higher was the number of cells with telophase-like nuclei and the lower the percentage of cells with pulverizations. Furthermore, the number of pulverizations bore a somewhat direct relationship to the ratio of metaphase to interphase nuclei in multinucleate cells, and the number of telophase-like nuclei was inversely proportional to this ratio. A hypothesis is advanced in which a balance between the activities of a chromosome pulverization factor and a nuclear envelope formation factor, the former in metaphase cells and the latter in interphase cells, is decisive as to the nature of morphologic events observed in virus-induced fused cells.     

Mitotic chiasmata in human diplochromosomes

Summary Three placental tissue cultures of spontaneous human abortions showed an unusually high frequency of metaphases with diplochromosomes. In 62 such cells, nine configurations were interpreted as mitotic chiasmata between the two sister chromosomes of a diplochromosome. One U-type exchange between two sister chromosomes was also found. This differs significantly from the 1:1 ratio of adjacent and alternate exchanges in translocations, thus supporting the idea that mitotic chiasmata are in principle different from chromatid translocations. The hypothesis is put forward that the frequency of homologous exchanges is determined by the intimacy of pairing which ranges from meiotic pairing through sister chromatid association, through sister chromosome association in diplochromosomes to accidental pairing of homologous regions in diploid cells.     

T-lymphocytes with 7;14 translocations: frequency of occurrence, breakpoints, and clinical and biological significance.

Among 11,915 consecutive patients and 37 normal controls who had chromosome analysis at the Mayo Clinic between 1978 and 1984, 83 had a single sporadic metaphase with a 7;14 translocation. In 81 of the translocations, the breakpoints were at 14q11 and either 7q34 (type I) or 7p13 (type II): type I translocations occurred in 42 patients, and type II, in 39. The two other translocations had different breakpoints: one was t(7;14)(q11;q32), and the other was t(7;14)(p13;q32). All type I and type II translocations occurred in phytohemagglutinin-stimulated lymphocyte cultures; their combined incidence was 4.88 X 10(-4) per metaphase (81 of 165,991 metaphases) in such cultures. No type I or II translocation was found among 6,713 fibroblast metaphases, 33,463 amniocyte metaphases, or 68,972 bone marrow and unstimulated peripheral blood metaphases. One variant 7;14 translocation occurred in a phytohemagglutinin-stimulated culture, and the other occurred in a fibroblast culture. We did not find a correlation of sporadic 7;14 translocations with any month or season of the year or with patient age or sex. Of the 83 patients, 78 had various clinical disorders, three had ataxia-telangiectasia, one was a normal control, and one was an artificial insemination donor. Follow-up studies on 64 (77%) patients indicate that, to date, none have developed any malignant process subsequent to chromosome analysis. Except for ataxia-telangiectasia, the occurrence of types I and II translocations in lymphocyte cultures may have little, if any, clinical significance. The biological significance of these translocations may be the association of genes in chromosome bands 14q11, 7p13, and 7q34 with the normal physiology of lymphocytes such as the alpha- and beta-chains for T-cell antigen receptor.     

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.     

Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam (Spisula solidissima) oocytes

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.     

Condensins: organizing and segregating the genome

Condensins are multi-subunit protein complexes that play a central role in mitotic chromosome assembly and segregation. The complexes contain 'structural maintenance of chromosomes' (SMC) ATPase subunits, and induce DNA supercoiling and looping in an ATP-hydrolysis-dependent manner in vitro. Vertebrate cells have two different condensin complexes, condensins I and II, each containing a unique set of regulatory subunits. Condensin II participates in an early stage of chromosome condensation within the prophase nucleus. Condensin I gains access to chromosomes only after the nuclear envelope breaks down, and collaborates with condensin II to assemble metaphase chromosomes with fully resolved sister chromatids. The complexes also play critical roles in meiotic chromosome segregation and in interphase processes such as gene repression and checkpoint responses. In bacterial cells, ancestral forms of condensins control chromosome dynamics. Dissecting the diverse functions of condensins is likely to be central to our understanding of genome organization, stability and evolution.     

Mutations in the Drosophila condensin subunit dCAP-G: defining the role of condensin for chromosome condensation in mitosis and gene expression in interphase

Chromosomes are dynamic structures that are reorganized during the cell cycle to optimize them for distinct functions. SMC and non-SMC condensin proteins associate into complexes that have been implicated in the process of chromosome condensation. The roles of the individual non-SMC subunits of the complex are poorly understood, and mutations in the CAP-G subunit have not been described in metazoans. Here we elucidate a role for dCAP-G in chromosome condensation and cohesion in Drosophila. We illustrate the requirement of dCAP-G for condensation during prophase and prometaphase; however, we find that alternate mechanisms ensure that replicated chromosomes are condensed prior to metaphase. In contrast, dCAP-G is essential for chromosome condensation in metaphase of single, unreplicated sister chromatids, suggesting that there is an interplay between replicated chromatids and the condensin complex. In the dcap-g mutants, defects in sister-chromatid separation are also observed. Chromatid arms fail to resolve in prophase and are unable to separate at anaphase, whereas sister centromeres show aberrant separation in metaphase and successfully move to spindle poles at anaphase. We also identified a role for dCAP-G during interphase in regulating heterochromatic gene expression.     

Studies in the Mitosis of Euglena I. On the Chromosome Cycle of Euglena viridis Ehrbg.

The chromosome cycle in the vegetative division of Euglena viridis was investigated. The seeming chromatin granules in the interphase nucleus are in reality thread structures, paired and very loosely twisted. Each component of the paired threads is called a chromatid, and consists of a fine thread of even thickness, the chromonema.
In the prophase, linear contraction and thickening of the chromatids occurs by means of the spiralization of them. In the later prophase, the coiled chromonema splits into two finer strands which show the plectonemic spiral. At the metaphase, the chromosomes are arranged in the form of an equatorial ring, encircling the median portion of the elongated endosome. Nearly all of the chromosomes have a submedian or a sub-terminal and a few of them have a terminal kinetochore. In the early anaphase, separation of the sister chromosomes takes place beginning at the kinetochore. The spindle fibres in the metaphase and anaphase were not observed. The two stranded spiral in the chromosomes is separated into distinct components by the uncoiling in the later telophase, and they are transformed, in the interphase nucleus, into the paired chromatids.     

SEPARATION OF HELA CELLS BY COLLOIDAL SILICA DENSITY GRADIENT CENTRIFUGATION : I. Separation and Partial Synchrony of Mitotic Cells

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040–1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.