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

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

稀土与铜对鲹鲦鱼红细胞微核及核异常的影响

研究了稀土元素镧与铜离子单独或联合作用时,对鲹鲦红细胞微核及核异常的影响。结果表明:处理24h,0.01～0.5mg/L的La3 能显著降低红细胞微核及核异常的发生,不具遗传毒性.但当浓度为1～50mg/L时则能诱发红细胞微核及核异常的发生,与对照组相比,相差显著或极显著(P<0.05或P<0.01),具较强的遗传毒性;铜能显著诱导红细胞微核及核异常的发生;联合作用时,微核率及核异常率明显低于Cu2 而高于La3 单独作用时的微核率和核异常率,提示一定浓度的稀土元素能减轻其他重金属元素引起的遗传毒性。     

Quantitative estimation of nuclear buds and micronuclei in bovine cells transformed by Rous sarcoma and SV 40 viruses

Summary A study of nuclear-budding and micronuclei formation has been performed on bovine fibroblastic cells in serial tissue culture. A comparison was made between control cells and cells morphologically transformed by Rous sarcoma virus and SV 40 virus. It was found that no obvious differences in the frequency of nuclear buds and micronuclei existed between RSV transformed and control cells whereas SV 40 transformed cells showed a high frequency of nuclear buds and micronuclei. There are indications that the frequency of nuclear buds and micronuclei is correlated to chromosomal abnormalities.Supported by grants from the Medical Faculty of the University of Uppsala.     

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

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

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.     

Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.     

Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test

In this work, we have measured both micronuclei and other nuclear abnormalities in renal erythrocytes from European minnow Phoxinus phoxinus and mollie Poecilia latipinna, with the aim to contribute to the standardisation of the micronucleus test for fish species. Intraperitoneal injections of colchicine (10 mg/kg), cyclophosphamide (40 mg/kg), or mitomycin C (20 mg/kg) for 24 h induced diverse nuclear abnormalities in minnow erythrocytes, therefore nuclear abnormalities should be added to micronuclei as genotoxicity indicators in fish micronucleus tests. The adequacy of administration protocols based on intraperitoneal injections has been evaluated by injecting saline solution to both species: single or double injections have not induced neither micronuclei nor other nuclear abnormalities in any case. Finally, the differential sensitivity of both species to toxic heavy metals was evaluated by exposing individuals of both species to different doses (0.17, 1.7, 2x1.7, and 3.4 mg/kg) of cadmium and mercury for 24 h; we concluded that the mollie is sensitive to both metals whereas the minnow is not sensitive to mercury.     

Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare x H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.     

Nuclear envelope defects impede a proper response to micronuclear DNA lesions

When damage is inflicted in nuclear DNA, cells activate a hierarchical plethora of proteins that constitute the DNA damage response machinery. In contrast to the cell nucleus, the ability of micronuclear DNA lesions to activate this complex network is controversial. In order to determine whether the DNA contained in micronuclei is protected by the cellular damage response system, we studied the recruitment of excision repair factors to photolesions inflicted in the DNA of radiation-induced micronuclei. To perform this analysis, primary human dermal fibroblasts were exposed to UV-C light to induce photolesions in nuclear and micronuclear DNA. By means of immunofluorescence techniques, we observed that most micronuclei were devoid of NER factors. We conclude that UV photoproducts in micronuclei are mostly unable to generate an effective DNA damage response. We observed that the micronuclear envelope structure is a determinant factor that influences the repair of the DNA lesions inside micronuclei. Therefore, our results allow us to conclude that photolesions in radiation-induced micronuclei are poorly processed because the repair factors are unable to reach the micronuclear chromatin when a micronucleus is formed or after a genotoxic insult.     

Electron Microscopy of the Nuclear Events During Binary Fission in Paramecium multimicronucleatum

SYNOPSIS. From the interphase to the early stage of binary fission in Paramecium multimicronucleatum , when the micro-nuclei are situated close to the macronucleus, the microtubules in the cytoplasm seem to connect the nuclear pores of macro- and micronuclei. During the 1st half of macronuclear division, the microtubules are formed outside the macronucleus, while during the latter half of division, numerous microtubules appear inside it. Chromatin bodies and nucleoli remain unchanged during macro-nuclear division, but the latter show temporory irregularity in shape. In late prophase of micronuclear division, spindle micro-tubules are formed, and a polar structure, composed of randomly dispersed twisting filaments, is formed at each pole of the micro-nucleus at anaphase. Spindle microtubules terminate on the surface of this structure. The nuclear envolope of the macro-and micronuclei remains intact thruout division. The envelope of the daughter micronuclei is derived from the pre-existing one.     

Induction of micronucleated and binucleate cells in Chinese hamster ovary (CHO) cells by cis-diamminedichloroplatinum (II): a morphological and morphometric study

The mutagenicity and cytotoxicity of cis-diamminedichloroplatinum (II) (cisplatin) at doses of 5, 10 and 20 micrograms/ml in Chinese hamster ovary (CHO) cells have been examined. A morphological characterization of several cell types induced by cisplatin was carried out. The frequencies of both cells with micronuclei and binucleate cells as a time-dependent parameter have also been studied. Whilst the number of cells with micronuclei was found to decrease with time, the number of binucleate cells increased. The possible kinetic mechanism for the production of binucleate cells and cells with micronuclei is discussed. A morphometric analysis was also performed. The nuclear area in both treated and control nuclei was measured with the IBAS image analysis system. The results of this analysis show that a continuous reduction in the nuclear size in the control cells is produced. However the size of the treated cells increased after treatment.     

三氧化二砷对黄鳝毒理学效应

以黄鳝为实验动物,研究了不同剂量的三氧化二砷对黄鳝的毒理学效应。采用鱼类致突变实验、鱼类红、白血球计数等方法,测定外周血白细胞数、红细胞微核和核异常以及肝脏过氧化氢酶（CAT）活性。结果发现：在低剂量范围内,随着三氧化二砷剂量的增加红细胞的微核率及核异常率逐渐增加、CAT活性升高,白细胞数则下降;至0．50mg／kg剂量时红细胞的微核率、核异常率,TLCAT活性均达最大值,而白细胞数达最低值;随着剂量的进一步增加红细胞的微核率、核异常率以及CAT活性均反而下降,白细胞数则上升。结果表明一定剂量三氧化二砷能明显影响黄鳝外周血细胞及肝脏CAT活性。     

四种除草剂对中华大蟾蜍蝌蚪红细胞微核及核异常的影响

研究了除草剂精克草星、苯磺隆、使它隆和乙草胺单独及联合作用时对中华大蟾蝌蚪红细胞微核及核异常的影响。结果表明,处理２４小时时,单独作用的除草剂均可不同程度地引起微核率及核异常细胞率等遗传指标的上升。而且,在一定的浓度下各指标分别有高峰期,其中微核率与对照组差异极显著（Ｐ〈０．０１）或显著（Ｐ〈０．０５）。微核率与除草剂浓度之间无显著相关,不表现出剂量－效应关系。四种除草剂联合作用的微核率等指标反而     

Telomere dysfunction and chromosome structure modulate the contribution of individual chromosomes in abnormal nuclear morphologies

The cytokinesis-block micronucleus assay has emerged as a biomarker of chromosome damage relevant to cancer. Although it was initially developed to measure micronuclei, it is also useful for measuring nucleoplasmic bridges and nuclear buds. Abnormal nuclear morphologies are frequently observed in malignant tissues and short-term tumour cell cultures. Changes in chromosome structure and number resulting from chromosome instability are important factors in oncogenesis. Telomeres have become key players in the initiation of chromosome instability related to carcinogenesis by means of breakage–fusion–bridge cycles. To better understand the connection between telomere dysfunction and the appearance of abnormal nuclear morphologies, we have characterised the presence of micronuclei, nucleoplasmic bridges and nuclear buds in human mammary primary epithelial cells. These cells can proliferate beyond the Hayflick limit by spontaneously losing expression of the p16^INK4a protein. Progressive telomere shortening leads to the loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter into breakage–fusion–bridge cycles generating massive chromosomal instability. In human mammary epithelial cells, different types of abnormal nuclear morphologies were observed, however only nucleoplasmatic bridges and buds increased significantly with population doublings. Fluorescent in situ hybridisation using centromeric and painting specific probes for chromosomes with eroded telomeres has revealed that these chromosomes are preferentially included in the different types of abnormal nuclear morphologies observed, thus reflecting their common origin. Accordingly, real-time imaging of cell divisions enabled us to determine that anaphase bridge resolution was mainly through chromatin breakage and the formation of symmetric buds in daughter nuclei. Few micronuclei emerged in this cell system thus validating the scoring of nucleoplasmic bridges and nuclear buds for measuring chromosome instability in telomere-dysfunction cell environments.     

A comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures ofNicotiana plumbaginifolia

The effects of the spindle toxins colchicine, oryzalin and amiprophos-methyl (APM) on metaphase arrest, chromosome scattering, and on the induction and yield of micronuclei were compared in suspension cells ofNicotiana plumbaginifolia (kanamycin-resistant “Doba” line). The inhibition of spindle formation is stronger with oryzalin and APM than with colchicine, which resulted in a more efficient accumulation of meta-phases with well-scattered chromosomes, allowing the isolation of single chromosomes. Further, APM and oryzalin treatments resulted in a higher frequency of micro-nucleated cells and greater yield of micronuclei than after colchicine treatment. The different actions of the chemicals on the functioning of the spindle, development of nuclear membranes around the chromosomes, formation of micronuclei and fusion of micronuclei, resulting in restitution nuclei, are discussed.     

Reorganisation du Type Endomixique chez les Loxodidae et chez les Centrophorella

New investigations of the nuclear cycle in the gymnostome ciliate Loxodes rostrum verify the long-neglected hypothesis proposed by Bütschli that in this multinucleated ciliate the macronuclei never divide. The consequences of this "caryosterose" are compensated for by a process of endomixis, that is, by the transformation of a certain number of the micronuclei, during vegetative multiplication, into new macronuclei.
The nuclear cycle in the gymnostome Centrophorella fistulosa does not show any aspect of nuclear division. Interpretation of this supposes the existence of polyenergid nuclei which fragment, during the interdivisional period, into subnuclei; among these some evolve in the somatic and macronuclear direction, before disappearance by karyolysis; others, corresponding to the micronuclei, grow by endomitosis, and reconstitute the polyploid and polyenergid nuclei. Thus the very particular nuclear cycle of C. fistulosa is believed to exhibit a new aspect in the endomictic process of nuclear reorganization.     

污染水域鲫鱼外周血细胞形态和数量的变化

为探讨污染环境对鱼类外周血细胞形态及数量的影响,本研究选择相对无污染的刘家峡水库和污染较严重的黄河白银段为研究地点,以鲫鱼(Carassius auratus)为研究对象,常规方法制血涂片,Giemsa染色,统计红细胞微核率、核异常率和各类血细胞数量,并用带有数码采集头(Motic B5 Professional Series)的显微镜拍照各类细胞。结果表明,与刘家峡水库相比,黄河白银段鲫鱼红细胞数量显著减少(P<0·05),核异常率(P<0·05)和微核率(P<0·01)显著增加;白细胞总数和淋巴细胞数量显著增加(P<0·05),而嗜中性粒细胞(P<0·01)和血栓细胞(P<0·05)数量显著减少,单核细胞数量虽有减少趋势但两地差异不显著(P>0·05)。表明黄河白银段污染对鲫鱼外周血细胞数量及红细胞微核率和核异常率具有显著的影响。