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1.
A. Miller-Faurès N. Michel A. Aguilera A. Blave A. O. A. Miller 《Cell proliferation》1981,14(5):501-514
The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border. 相似文献
2.
It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research. 相似文献
3.
Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2) 总被引:33,自引:15,他引:18
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The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco-2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface. 相似文献
4.
5.
Pronuclear migration in Fucus distichus spp. edentatus (de la Pyl.) Powell is blocked by incubation of fertilized eggs in colchicine (1 mg/ml) and Nocodazole (2 μg/ ml). Rhizoids form prior to decondensation of the sperm chromatin in eggs in which pronuclear fusion is blocked. This occurs during continuous colchicine incubation as well as in eggs recovering from a short treatment with either drug following fertilization. During recovery of the cells, the sperm and egg chromosomes condense, and the sperm chromosomes migrate toward the egg pronucleus. The delay in migration following removal of colchicine is as much as 24 h and is even slower following removal of Nocodazole. The egg chromosomes form a metaphase plate in treated cells while the sperm chromosomes are still distant in the cytoplasm. This suggests that egg centrioles are important in the mitotic division of the zygote, not sperm centrioles. The effect of colchicine treatment on the mitotic plane and cytokinesis is also discussed. 相似文献
6.
The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis 总被引:2,自引:2,他引:0
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The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis. 相似文献
7.
Experimental modification of PC12 neurite shape with the microtubule- depolymerizing drug Nocodazole: a serial electron microscopic study of neurite shape control 总被引:2,自引:2,他引:0
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The microtubule-depolymerizing drug Nocodazole has been used to experimentally manipulate the form of PC12 neurites. Both time-lapse photography and serial electron microscopy demonstrate that microtubule depolymerization leads to varicosity formation due to a clustering of membranous organelles in young neurites (nerve growth factor activated within 7 d). Neurites that have been nerve growth factor activated 7 or more d before Nocodazole application are resistant to microtubule depolymerization. These data and data from previous papers has been combined in an attempt to predict quantitatively the volume and the shape of a neurite. The relationship is described mathematically by Vn = 4.52 Vo + 0.0054 MTl, where Vn is local neurite volume, Vo is organelle volume, and MTl is MT length (the constant, 0.0054 is micron2), and 4.52 is the obligatory volume constant derived from serial electron microscopic studies. The equation predicts the total volume of neurites despite alterations of morphology due to Nocodazole and despite changes in morphology during development. 相似文献
8.
The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border. 相似文献
9.
Can Nocodazole,an Inhibitor of Microtubule Formation,Be Used to Synchronize Mammalian Cells? 总被引:6,自引:0,他引:6
Abstract Nocodazole, a temporary inhibitor of microtubule formation, has been used to partly synchronize Ehrlich ascites tumour cells growing in suspension. the gradual entry of cells into mitosis and into the next cell cycle without division during drug treatment has been studied by flow cytometric determination of mitotic cells, analysing red and green fluorescence after low pH treatment and acridine orange staining. Determination of the mitotic index (MI) by this method has been combined with DNA distribution analysis to measure cell-cycle phase durations in asynchronous populations growing in the presence of the drug. With synchronized cells, it was shown that in the concentration range 0.4–4.0 μg/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 μg/ml, for about 5 hr. After these time intervals, the DNA content in nocodazole-blocked cells was found to be increased, and, in parallel, the ratio of red and green fluorescence was found to have changed, showing entry of cells into a next cell cycle without division (polyploidization). It was therefore only possible to partially synchronize an asynchronous population by nocodazole. However, a presynchronized population, e.g. selected G1 cells or metabolically blocked G1/S cells, were readily and without harmful effect resynchronized in M phase by a short treatment (0.4 μg/ml, 3–4 hr) with nocodazole; after removal of the drug, cells divided and progressed in a highly synchronized fashion through the next cell cycle. 相似文献
10.
Kingo Endo 《FEBS letters》2010,584(11):2387-2392
We investigated the fate of budding yeast treated with nocodazole, a microtubule-depolymerizing drug. Cells died after mitotic arrest while staying in mitosis, suggesting that mitotic cell death, but not mitotic slippage, mainly occurs in nocodazole-treated cells. Nocodazole-treated cells showed features of apoptotic-like cell death, but not those of cell lysis or autophagy. Consistently, mitochondria-dependent production of reactive oxygen species was involved in the cell death. Similar cell death was also seen in cells after mitotic arrest by perturbation of the anaphase-promoting complex/cyclosome. In addition, caspase activity was found in nocodazole-treated cells, which was independent of the metacaspase, Mca1. Our results suggest that budding yeast can be a model to study mitotic cell death in cancer treatment with antimitotic drugs. 相似文献
11.
Edwin V. Gaffney Edward G. McElwain 《In vitro cellular & developmental biology. Plant》1973,9(1):56-63
Summary A method has been developed to obtain synchronous populations from a human cell line which previously resisted the use of
the selective harvest technique. A concentration of Colcemid was determined which reversibly enriched the mitotic population
but avoided delays in cell cycle progression. Mitotic cells were then detached from monolayer cultures by brief treatment
with hypotonic salt solutions. The resulting populations of line A244 were shown to be viable and syntchronous by following
attachment efficiency and cycle time and by monitoring mitotic index and deoxyribonucleic acid synthesis. Hypotonic solutions
offer no advantage in the selection of mitotic L-929 cells, a line commonly synchronized by selective harves. However, their
use with both CV-1 and A244 cells provided large populations highly synchronized with respect to mitosis. This technique might
be applied successfully to cell types which do not demonstrate a selective advantage at division. 相似文献
12.
Summary. Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules
radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont
very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair
transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.
Correspondence and reprints: Department of Cytology and Histology, Faculty of Biology and Soil Sciences, Saint Petersburg
State University, Universitetskaya naberezhnaya 7/9, 199034 Saint Petersburg, Russia. 相似文献
13.
Nocodazole inhibition of the vasopressin-induced water permeability increase in toad urinary bladder
Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 μ/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an inhibition of 50% seen with a 3-h exposure of the tissue to colchicine (0.1 mM) prior to stimulation with vasopressin. Application of nocodazole (1 μ/ml) 3 min after hormonal stimulation shows no inhibition of the response at one-half hour past stimulation. These data support the view that microtubules are involved in the vasopressin-induced increase in water permeability in toad bladder and also indicate that this involvement is limited to the period prior to or directly after stimulation. 相似文献
14.
Sachiko Terui Kuninori Suzuki Hidenori Takahashi Ryuuichi Itoh Tsuneyoshi Kuroiwa 《Journal of phycology》1995,31(6):958-961
A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions . 相似文献
15.
Chaimovitsh D Rogovoy Stelmakh O Altshuler O Belausov E Abu-Abied M Rubin B Sadot E Dudai N 《Plant biology (Stuttgart, Germany)》2012,14(2):354-364
The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ‐tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral’s effect on microtubules was both dose‐ and time‐dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ‐tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP‐Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral’s effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules. 相似文献
16.
目的:多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程(如恶性肿瘤)的发生有密切关系。我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR/S6K1参与多倍体细胞周期的调控。本课题主要研究roTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控。方法:诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR/4E—BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E—BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果:诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E—BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2/M期。结论:mTOR/4E-BP1通路参与多倍体细胞周期的调控。 相似文献
17.
Serena Giovinazzi Dhruv Bellapu Viacheslav M Morozov Alexander M Ishov 《Cell cycle (Georgetown, Tex.)》2013,12(16):2598-2607
Microtubule-poisoning drugs, such as Paclitaxel (or Taxol, PTX), are powerful and commonly used anti-neoplastic agents for the treatment of several malignancies. PTX triggers cell death, mainly through a mitotic arrest following the activation of the spindle assembly checkpoint (SAC). Cells treated with PTX slowly slip from this mitotic block and die by mitotic catastrophe. However, cancer cells can acquire or are intrinsically resistant to this drug, posing one of the main obstacles for PTX clinical effectiveness. In order to override PTX resistance and increase its efficacy, we investigated both the enhancement of mitotic slippage and the block of mitotic exit. To test these opposing strategies, we used physiological hyperthermia (HT) to force exit from PTX-induced mitotic block and the anaphase-promoting complex/cyclosome (APC/C) inhibitor, proTAME, to block mitotic exit. We observed that application of HT on PTX-treated cells forced mitotic slippage, as shown by the rapid decline of cyclin B levels and by microscopy analysis. Similarly, HT induced mitotic exit in cells blocked in mitosis by other antimitotic drugs, such as Nocodazole and the Aurora A inhibitor MLN8054, indicating a common effect of HT on mitotic cells. On the other hand, proTAME prevented mitotic exit of PTX and MLN8054 arrested cells, prolonged mitosis, and induced apoptosis. In addition, we showed that proTAME prevented HT-mediated mitotic exit, indicating that stress-induced APC/C activation is necessary for HT-induced mitotic slippage. Finally, HT significantly increased PTX cytotoxicity, regardless of cancer cells’ sensitivity to PTX, and this activity was superior to the combination of PTX with pro-TAME. Our data suggested that forced mitotic exit of cells arrested in mitosis by anti-mitotic drugs, such as PTX, can be a more successful anticancer strategy than blocking mitotic exit by inactivation of the APC/C. 相似文献
18.
We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells. 相似文献
19.
Walen KH 《Cell biology international》2005,29(12):1057-1065
Cell changes to accelerated rates of growth are determined by random, new gene mutations that have their origin in vitro in a morphologically visible process of cell alterations. It is a continuous process that, step by step, transforms the normal cell into extended life (EL) cells. These latter cells with limited life spans can further transform to immortalized cells (i.e., cell lines) by the same sequence of morphological cell changes. In contrast to human epithelial cells, fibroblasts in culture have not given rise spontaneously to EL cells. Therefore, it was assumed that some of the cell changes (i.e., cell indicators of the process of transformation) might not be present in near senescent fibroblast cell cultures. As a positive control to normal fibroblast expansion to senescence, the same cells were stressed by inadequate nutrition and confluence. Another positive control was cell indicators induced by SV40 infections. The consecutive sequence of the cell indicators in the transformation process reported previously were: (1) large polyploid cells with nuclei that contained more than two genomes, (2) fragmentation/amitosis of the polyploid nuclei to bi- and multinucleated cells (MNCs), and (3) nuclear budding (i.e., karyoplasts) from MNCs that gave rise to EL cell colonies with various longevities beyond the senescent phase. The present study shows that MNCs and karyoplasts were present in the near-senescent fibroblast cell cultures. Furthermore, new data on the following aspect of the cell transformation process are presented: (1) association of the nuclear fragmentation process with death of cells, (2) cytological markers that distinguish between fragmentation-MNCs versus MNCs from cell fusion, (3) cytological changes of karyoplasts that go through mitotic division to produce daughter cells, and (4) presence of two centrosomes (spindle polar bodies) in the budded karyoplasts. These new findings are discussed in regard to the nuclear fragmentation process in polyploid cells which gives rise to smaller, viable nuclei in MNCs with reduced numbers of whole genomes. 相似文献
20.
Tetraploid induction by inhibiting mitosis I with heat shock (32, 35, and 38°C), cold shock (1, 4, and 7°C), and nocodazole
(0.02 to 1.6 mg/L) was investigated in the hard clam Mercenaria mercenaria. All treatments were applied to fertilized eggs about 5 min before the first cell division at 22 to 23°C, and lasted for
10, 15, and 20 min. Three replicates were produced for each treatment with different parents. The ploidy of resultant larvae
and juveniles was determined with flow cytometry. Heat shock of 35 and 38°C was effective in inhibiting mitosis I, producing
54% to 89% tetraploid larvae. Heat shock of 32°C accelerated embryonic development without inhibiting mitosis or producing
tetraploids. In all heat-shock groups, the survival to D-stage larvae was lower than in controls, suggesting that heat-shock
treatments and tetraploidy were detrimental to larval development. At the juvenile stage, survivors from heat-shock groups
contained no tetraploids. Cold shocks suspended the first cell division during the treatment, but produced no tetraploids
in the 4°C and 7°C treatment groups. Cold shock of 1°C produced 31% tetraploid larvae in one replicate, with none surviving
to juvenile stage. Nocodazole inhibited mitosis I at concentrations of 0.04 mg/L or higher, but did not produce tetraploids.
This study indicates that heat shock is most effective in inducing tetraploids through mitosis I inhibition, although none
of the induced tetraploids survived to juvenile stage. 相似文献