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The Journal of Cell Biology Oct 1992Previous studies on glucocorticoid receptors have suggested the existence of interactions between the receptor and microtubule or actin networks. It was hypothesized... (Comparative Study)
Comparative Study
Previous studies on glucocorticoid receptors have suggested the existence of interactions between the receptor and microtubule or actin networks. It was hypothesized that such interactions may contribute to the guidance of steroid hormone receptors towards the nucleus. We used a permanent L cell line expressing the delta 638-642 progesterone receptor. This mutant has all the characteristics of the wild type receptor except that the deletion of five amino acids inactivates the constitutive karyophilic signal. Consequently, the receptor is cytoplasmic in the absence of hormone but is shifted into the nucleus when administration of hormone activates the second karyophilic signal. Optical microscopy and confocal laser microscopy were used in intact cells or in cells depleted of soluble elements by permeabilization with detergents. By immunofluorescence, the receptor was found to be mainly concentrated in the perinuclear area. A small fraction of progesterone receptor (PR) persisted in this region after Triton X100 treatment. These observations suggested that the receptor could interact with some insoluble constituent(s) of the cytoplasm. However, careful colocalization studies showed that this heterogenous distribution was not due to interactions with microtubule, microfilament, or intermediate filament networks. Functional involvement of these networks in the translocation of the receptor into the nucleus was studied after cell treatment with cytoskeletal drugs such as nocodazole, demecolcine and cytochalasin. None of these compounds prevented or even delayed the hormone-dependent transfer of delta 638-642 PR into the nucleus. Similar conclusions were reached with the wild type receptor expressed by transfection in Cos-7 cells. PR was shifted from the nucleus into the cytoplasm by administration of energy-depleting drugs. After disruption of the various cytoskeletal networks normal nuclear reaccumulation of the receptor was observed when these drugs were removed. The results thus suggest that the progesterone receptor is not colocalized with the main cytoskeletal components. Disruption of the cytoskeletal networks does not prevent its nuclear translocation. Thus, karyophilic signals and interactions with the nuclear pore seem to be the primary determinants of the cellular traffic of the progesterone receptor.
Topics: Animals; Biological Transport; Cell Nucleus; Cytochalasin B; Cytoplasm; Cytoskeletal Proteins; Cytoskeleton; Demecolcine; Energy Metabolism; Fluorescent Antibody Technique; L Cells; Mice; Mutation; Nocodazole; Progesterone; Receptors, Progesterone; Transfection
PubMed: 1400578
DOI: 10.1083/jcb.119.2.337 -
British Journal of Pharmacology Oct 1975
Topics: Animals; Axonal Transport; Axons; Demecolcine; Microscopy, Electron; Rats; Spinal Nerve Roots
PubMed: 53078
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1979Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol...
Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Colchicine, Colcemid, and vinblastine (1.0 muM) caused a marked reduction in HMG-CoA reductase activity and, as a consequence, the rate of cholesterol synthesis in these cells. No effect was observed with lumicolchicine, a mixture of colchicine isomers with no effect on microtubules. The effect of colchicine was apparent within 1 h after addition to the culture medium, and, after 6 h, HMG-CoA reductase activity in treated cells was only approximately 15 to 30% of that in untreated cells. Reductase activity was very sensitive to the concentration of drug added, i.e. cells treated with just 0.1 muM colchicine for 6 h exhibited a 50% lower enzymatic activity than did untreated cells. The lack of a generalized, nonspecific toxic effect on the cells was indicated by the finding of no change in the activities of fatty acid synthetase and NADPH-cytochrome c reductase and the rate of total protein synthesis in cells treated with colchicine (1 muM) for 6 h. A close temporal and quantitative correlation was observed between the effects of colchicine on HMG-CoA reductase and on a parameter of microtubular function, i.e. maintenance of glial cell shape. The data suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in C-6 glial cells.
Topics: Animals; Cell Line; Cholesterol; Colchicine; Demecolcine; Fatty Acid Synthases; Glioma; Hydroxymethylglutaryl CoA Reductases; Kinetics; Microtubules; NADPH-Ferrihemoprotein Reductase; Rats; Vinblastine
PubMed: 107160
DOI: No ID Found -
The Journal of Cell Biology Nov 1990We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the...
The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells.
We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM-26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26-induced G2 arrest of the cell.
Topics: Animals; Aphidicolin; Cell Cycle; Cells, Cultured; Chromosomes; DNA Damage; DNA Topoisomerases, Type II; Demecolcine; Diterpenes; G1 Phase; G2 Phase; Histones; Metaphase; Mitosis; Nuclear Envelope; Phosphorylation; Protamine Kinase; Teniposide; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors
PubMed: 2172257
DOI: 10.1083/jcb.111.5.1753 -
Journal of Radiation Research Mar 2000The frequency of chromosome aberrations in circulating lymphocytes is accepted as being the most reliable indicator of the absorbed dose of radiation. Researches done to... (Review)
Review
The frequency of chromosome aberrations in circulating lymphocytes is accepted as being the most reliable indicator of the absorbed dose of radiation. Researches done to improve the accuracy of cytogenetic analysis are described in this review. These include investigations of in vitro factors that affect the yield of radiation-induced aberrations and of in vivo factors that affect the chromosomal radiosensitivity of individuals. Improved chromosome-painting methods for accurate judgment of dicentrics and translocations are introduced. The practicality of these advanced cytogenetic techniques is shown by examinations of individuals exposed in the radiation accident at Tokaimura in 1999.
Topics: Accidents, Occupational; Chromosome Aberrations; Chromosome Painting; Chromosomes, Human; Demecolcine; Dose-Response Relationship, Radiation; Humans; Japan; Lymphocytes; Radiation Tolerance; Radioactive Hazard Release; Radiometry; Uranium
PubMed: 10838805
DOI: 10.1269/jrr.41.1 -
Proceedings of the National Academy of... Mar 1977In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor...
In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.
Topics: Antigen-Antibody Complex; Antigens, Neoplasm; Blood Platelets; Cell Line; Cell Membrane; Demecolcine; Diffusion; Podophyllotoxin; Receptors, Concanavalin A; Receptors, Drug; Solubility; Vinblastine
PubMed: 265557
DOI: 10.1073/pnas.74.3.1110 -
Journal of Neuroscience Research Mar 2004Human neural progenitor cells (hNPCs) can be recovered from postmortem human brains and used to study the molecular basis of neurogenesis. Human NPCs are being used to... (Comparative Study)
Comparative Study
Human neural progenitor cells (hNPCs) can be recovered from postmortem human brains and used to study the molecular basis of neurogenesis. Human NPCs are being used to investigate the molecular basis of cell fate determination during stem cell divisions, based on comparison with the Drosophila model system. Drosophila neuroblasts and sensory organ precursors undergo well-defined asymmetric cell divisions (ACD), under the control of a genetically defined set of apical and basal determinants that are localized tightly and dynamically during division. We show by indirect immunofluorescence, confocal microscopy, and time-lapse video-microscopy that LGN and AGS3, two human homologs of the Drosophila ACD determinant Pins, have distinct patterns of localization in hNPCs. When cells are grown under conditions favoring proliferation, LGN is distributed asymmetrically in a cell cycle-dependent manner; it localizes to one side of the dividing cell and segregates into one of the daughter cells. When the cells are grown under conditions favoring differentiation, LGN accumulates in double foci similar to those containing the mitotic apparatus protein NuMA, and in a pattern shown previously for LGN and NuMA in differentiated cells. AGS3, a slightly more distant Pins homolog than LGN, does not show asymmetric localization in these cells. The progenitor cell marker nestin also localizes asymmetrically in colcemid-treated hNPCs and colocalizes with LGN. The results suggest that hNPCs undergo ACD and that similar molecular pathways may underlie these divisions in Drosophila and human cells.
Topics: Animals; Antigens, Nuclear; Brain; Carrier Proteins; Cell Count; Cell Cycle; Cell Cycle Proteins; Cell Division; Cells, Cultured; DNA; Demecolcine; Drosophila; Drosophila Proteins; GTP-Binding Protein alpha Subunits, Gi-Go; Green Fluorescent Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Microscopy, Confocal; Nerve Tissue Proteins; Nestin; Neurons; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Stem Cells; Structural Homology, Protein; Time Factors; Transfection; Tubulin; Videodisc Recording
PubMed: 14994339
DOI: 10.1002/jnr.10874 -
The Journal of Cell Biology Jul 1983Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of...
Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two-dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.
Topics: Alkaloids; Animals; Cell Division; Cell Line; Cricetinae; DNA; Demecolcine; Deuterium; Deuterium Oxide; Dose-Response Relationship, Drug; Female; Griseofulvin; Hybrid Cells; Mutation; Paclitaxel; Phenotype; Protein Biosynthesis; Water
PubMed: 6134736
DOI: 10.1083/jcb.97.1.22 -
Cell Structure and Function 2015Although most cell lines undergo mitotic arrest after prolonged exposure to microtubule inhibitors, some cells subsequently exit this state and become tetraploid. Among...
Although most cell lines undergo mitotic arrest after prolonged exposure to microtubule inhibitors, some cells subsequently exit this state and become tetraploid. Among these cells, limited numbers of rodent cells are known to undergo multinucleation to generate multiple small independent nuclei, or micronuclei by prolonged colcemid treatment. Micronuclei are thought to be formed when cells shift to a pseudo G1 phase, during which the onset of chromosomal decondensation allows individual chromosomes distributed throughout the cell to serve as sites for the reassembly of nuclear membranes. To better define this process, we used long-term live cell imaging to observe micronucleation induced in mouse A9 cells by treating with the microtubule inhibitor colcemid. Our observations confirm that nuclear envelope formation occurs when mitotic-arrested cells shift to a pseudo G1 phase and adopt a tetraploid state, accompanied by chromosome decondensation. Unexpectedly, only a small number of cells containing large micronuclei were formed. We found that tetraploid micronucleated cells proceeded through an additional cell cycle, shifting to a pseudo G1 phase and forming octoploid micronucleated cells that were smaller and more numerous compared with the tetraploid micronucleated cells. Our data suggest that micronucleation occur when cells shift from mitotic arrest to a pseudo G1 phase, and demonstrate that, rather than being a single event, micronucleation is an inducible recurrent process that leads to the formation of progressively smaller and more numerous micronuclei.
Topics: Animals; CHO Cells; Cell Cycle; Cell Nucleus; Chromosomes; Cricetinae; Cricetulus; Demecolcine; G1 Phase; Humans; Mice; Microtubules; Mitosis; Molecular Imaging; Nuclear Envelope; Ploidies
PubMed: 25736016
DOI: 10.1247/csf.14005 -
Cytometry Nov 1986In this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional...
In this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional plots. We show that the results of a single experiment, when reduced to the percentages of cells in G1, S, and G2M phases, can be completely expressed with a single point in a G1-G2 plan where iso-S lines are also drawn. By the use of this plot, the time course of the complete kinetics of a cell population may be shown in a single drawing. The second plot, we suggest, integrates information on cell cycle, represented by the distribution mean, with cell number.
Topics: Cell Cycle; Colonic Neoplasms; DNA; Demecolcine; Flow Cytometry; Humans; Mathematics
PubMed: 3780363
DOI: 10.1002/cyto.990070618