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.     

Various chemical agents can induce premature chromosome condensation in Vicia faba

A number of chemical agents known to influence the key cell cycle regulatory factors were used to assess the requirements of hydroxyurea-treated root meristem cells of Vicia faba for premature condensation of chromosomes (PCC). These included caffeine and 2-aminopurine (inhibitors of ATM/ATR sensor kinases activated by DNA damage or stalled replication forks), inhibitors of protein kinases (staurosporine and wortmannin), inhibitors of protein phosphatases (sodium vanadate and calyculin A), and other compounds like 1,2-dioctyl-sn-glycerol, an activator of protein kinase C, 5-azacytidine, an inhibitor of DNA methyltransferase, dithiothreitol and N-etylmaleimide, capable to up- and down-regulate the activity of Cdc25 phosphatase. Cytological parameters used to evaluate quantitative aspects of PCC allowed us to discriminate various phenotypes of cells and, consistent with the extent of chromosomal fragmentation, to classify them as S- or G2-PCC. Two significant aspects relevant to the induction of premature mitosis in plants seem to emerge: one concerns the inverse relationship between the incidence of mitotic and PCC events, the other refers to the extent with which a variety of chemical agents may activate mechanisms that override the S-M replication checkpoint. 1,2-dioctyl-sn-glycerol, an activator of protein kinase C in animal cells proved extremely effective in stimulation of PCC, in spite of evident lack of molecular targets in plants.     

Chromosome or chromatin condensation leads to meiosis or apoptosis in stationary yeast (Saccharomyces cerevisiae) cells

When starved of essential nutrients, yeast cells cease mitotic division and enter an alternative state called the 'stationary phase'. In this paper, we report that stationary cells enter two major pathways: meiosis and apoptosis. Using transmission electron microscopy, five types of cell were identified in the stationary phase: (1) cells with chromosome condensed nuclei; (2) cells with normal, homogeneously stained nuclei; (3) sporulated cells; (4) apoptotic cells, in which chromatin, but not individual chromosomes, was condensed; and (5) dead cells, in which nuclei and cytoplasm were degraded. Further evidence using live cell imaging and mutation analysis suggested that cells with condensed chromosomes underwent meiosis, whereas chromatin condensed cells underwent apoptotic cell death. Cells with homogeneous nuclei are believed to be in the true resting state and undergo cell death when starvation continues. Chromosome or chromatin condensation may serve as a hallmark of life or death for stationary cells.     

The superstructure of chromatin and its condensation mechanism

Model calculations on the superstructure of uncondensed and condensed chromatin are presented. It is found that agreement between the calculated X-ray solution scattering patterns and the experimental observations can be reached with the assumptions that: a) The uncondensed chromatin fibre in solution has a helix-like structure, with a pitch of ca. 33.0 nm, a helical diameter of ca. 20.0 nm and 2.75–3.25 nucleosomes per turn. b) The most condensed state of the chromatin fibre in solution is best represented by a helix-like structure with ca. 2.56 nucleosomes per turn, a pitch of ca. 3.0 nm and a helical diameter of ca. 27.0 nm.     

Dense chromatin plates in metaphase chromosomes

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (~6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young’s modulus is ~0.2 GPa and the stress required for surface penetration is ~0.03 GPa in the presence of Mg²⁺ (5–20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.     

The cytogenetic effect of bleomycin on human peripheral lymphocytes in vitro and in vivo.

The cytogenetic effect of bleomycin (BLM) in human lymphocytes was studied after exposure to different doses during the G0 and G2 phases. BLM produced a marked specific effect on the cell cycle. The main aberration types after exposure in tg0 were dicentrics and deletions; and after exposure in G2, open chromatid breaks. A linear dose--response was calculated for all these aberration types as well as for the number of aberrant cells. In the G2 experiments, partially and totally pulverized cells also increased linearly with dose. The intercellular distributions of the most frequent aberration types after exposure in G0 and G2--the dicentrics and chromatid breaks, respectively--showed over-dispersion. These results show that the cytogenetic effect of BLM may be compared with that of densely ionizing irradiation. Preliminary results of chromosome analysis of three cancer patients in the course of BLM therapy showed effects similar to those in the G0 experiments.     

Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes

The present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two-, four-, or eight-cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear-like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non-PCC (NPCC; 52.9%) embryos fused with the two-cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus-like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four- and eight-cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley-Liss, Inc.     

Defective removal of O6-methylguanine (O6-MeG) from the DNA of lymphocytes treated with N-methyl-N-nitrosourea (MNU) in autoimmune diseases

Asynchronously growing Chinese hamster cells (M3-1) were UV-irradiated (λ = 254 nm) and then incubated with/without caffeine (2 mM) for 20 h. Microscopic evaluation of metaphase spreads revealed that after UV-irradiation alone (5.0 J/m²) and caffeine treatment alone, the percentage of cells with condensed chromatin appearing fragmented and/or pulverized (‘GCS-like’ cells; GCS, Generalized Chromosome Shattering) was very low while it was high following the combined treatment. Cytogenetic and flow cytometric analysis of cells obtained by mechanical shaking cultures treated with UV and caffeine indicated that ‘GCS-like’ cells have the same DNA content as untreated cells in G2 phase and mitosis.     

Formation of multilayer rosettes in phytohemagglutinin-stimulated lymphocytes: correlation with chromatin condensation conformation in premature chromosome condensation

We found that the formation of multilayer rosettes by transformed human blood lymphocytes after phytohemagglutinin (PHA) stimulation is correlated with conformational changes of the chromatin as seen by premature chromosome condensation (PCC). The frequency distribution of grades of PCC and multilayer rosette formation suggests that changes in chromatin are a prerequisite for rosette formation. Rosette formation was most pronounced for 24-h and 48-h cultures. Chromatin decondensation and rosette formation showed identical patterns. The possibility that multilayer rosette formation is directly dependent on conformational changes of chromatin is discussed.     

Differential condensation of mouse DNA families in chromatin: accessibility to nuclease probes

A number of minor, previously unidentified GC-rich mouse DNA families have been observed in CsCl gradients. Since these DNA families are found in the DNA of mouse nuclei which is most resistant to both micrococcal nuclease and DNAase I, they must occur in highly condensed chromatin. Fractionation of high molecular weight nuclease digested DNA by sequential polyethylene glycol (PEG) precipitation demonstrates differential enrichment of these DNA families implying a differential condensation of these DNA fractions in chromatin.     

Caffeine-induced uncoupling of mitosis from DNA replication in tobacco BY-2 cells

Caffeine induced a mitosis-like state in cultured tobacco (Nicotiana tabacum L.) BY-2 cells after DNA synthesis had been arrested by aphidicolin. Cells were synchronized upon removal of aphidicolin. When aphidicolin was readded, the cell cycle was again interrupted and caffeine, when added with aphidicolin, induced the mitosis-like state in 5–10% of cells.     

Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage

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Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

The superstructure of chromatin and its condensation mechanism

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and the polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb and Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chloride and alkylammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg^-- concentration at which precipitation occures on chromatin concentration and confirm the differences between cations observed by X-ray scattering.The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.     

Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina

The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.     

Human chromokinesin KIF4A functions in chromosome condensation and segregation

Accurate chromosome alignment at metaphase and subsequent segregation of condensed chromosomes is a complex process involving elaborate and only partially characterized molecular machinery. Although several spindle associated molecular motors have been shown to be essential for mitotic function, only a few chromosome arm--associated motors have been described. Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation. HKIF4A localizes in the nucleoplasm during interphase and on condensed chromosome arms during mitosis. It accumulates in the mid-zone from late anaphase and localizes to the cytokinetic ring during cytokinesis. RNA interference--mediated depletion of HKIF4A in human cells results in defective prometaphase organization, chromosome mis-alignment at metaphase, spindle defects, and chromosome mis-segregation. HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture. Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells

A new method is described to visualize chromosome damage in interphase cells immediately after exposure to mutagenic agents. This method involves the fusion of treated interphase cells with untreated mitotic cells which results in the induction of premature chromosome condensation (PCC). Chinese hamster ovary (CHO) cells were treated with X-rays and chromosome aberrations were scored in G2-PCC and the mitotic chromosomes. The incidence of aberrations was significantly higher in PCC than that observed in the mitotic chromosomes of the treated cells. Post-irradiation incubation for I h before fusion allowed the repair of some of the chromosome damage. Data are also presented which indicate that the extent of radiation damage visualized in PCC is inversely proportional to the degree of chromosome condensation. These results indicate that the PCC method has a greater senstivity in the detection of induced chromosome damage than the standard method of scoring metaphase chromosomes.     

Lasonolide A,a potent and reversible inducer of chromosome condensation

Lasonolide A (LSA) is a natural product with high and selective cytotoxicity against mesenchymal cancer cells, including leukemia, melanomas and glioblastomas. Here, we reveal that LSA induces rapid and reversible premature chromosome condensation (PCC) associated with cell detachment, plasma membrane smoothening and actin reorganization. PCC is induced at all phases of the cell cycle in proliferative cells as well as in circulating human lymphocytes in G₀. It is independent of Cdk1 signaling, associated with cyclin B downregulation and induced in cells at LSA concentrations that are three orders of magnitude lower than those required to block phosphatases 1 and 2A in vitro. At the epigenetic level, LSA-induced PCC is coupled with histone H3 and H1 hyperphosphorylation and deacetylation. Treatment with SAHA reduced LSA-induced PCC, implicating histone deacetylation as one of the PCC effector mechanisms. In addition, PCC is coupled with topoisomerase II (Top2) and Aurora A hyperphosphorylation and activation. Inhibition of Top2 or Aurora A partially blocked LSA-induced PCC. Our findings demonstrate the profound epigenetic alterations induced by LSA and the potential of LSA as a new cytogenetic tool. Based on the unique cellular effects of LSA, further studies are warranted to uncover the cellular target of lasonolide A (“TOL”).