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Proceedings of the National Academy of... Apr 1998Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca2+ concentration ([Ca2+]i). In a single bovine aortic EC, the [Ca2+]i rise started...
Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca2+ concentration ([Ca2+]i). In a single bovine aortic EC, the [Ca2+]i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca2+ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca2+ wave was unaffected by the removal of extracellular Ca2+, demonstrating its dependence on intracellular Ca2+ release. Microinjection of heparin into the cell inhibited the ATP-induced Ca2+ responses, indicating that the Ca2+ wave is at least partly mediated by the inositol 1,4, 5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca2+ waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca2+]i increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca2+ waves in ECs.
Topics: Adaptor Protein Complex alpha Subunits; Adaptor Proteins, Vesicular Transport; Adenosine Triphosphate; Animals; Calcium; Cattle; Caveolin 1; Caveolins; Cell Compartmentation; Cells, Cultured; Clathrin; Cytoplasm; Demecolcine; Endosomes; Endothelium, Vascular; Fluorescent Antibody Technique, Indirect; Inositol 1,4,5-Trisphosphate; Lasers; Membrane Proteins; Microscopy, Confocal; Transferrin
PubMed: 9560219
DOI: 10.1073/pnas.95.9.5009 -
The Journal of Biological Chemistry Feb 2004Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de...
Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.
Topics: Autoantigens; Cell Line; Cell Nucleus; Centromere; Centromere Protein B; Chromatin; Chromosomal Proteins, Non-Histone; Cloning, Molecular; DNA; DNA, Complementary; DNA-Binding Proteins; Demecolcine; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique, Indirect; G2 Phase; Gene Library; HeLa Cells; Humans; Kinetochores; Metaphase; Microscopy, Phase-Contrast; Mutation; Peptides; Plasmids; Precipitin Tests; Protein Biosynthesis; Protein Structure, Tertiary; Saccharomyces cerevisiae Proteins; Time Factors; Transcription, Genetic; Transfection; Two-Hybrid System Techniques
PubMed: 14612452
DOI: 10.1074/jbc.M306477200 -
The Journal of Biological Chemistry Jul 2004Recent studies have implicated heat shock proteins (HSP) and heat shock transcription factor 1 (HSF1) in tumor progression. We have examined the role of HSF1 in the...
Recent studies have implicated heat shock proteins (HSP) and heat shock transcription factor 1 (HSF1) in tumor progression. We have examined the role of HSF1 in the malignant phenotype of PC-3 prostate carcinoma cells. We have developed a dominant negative construct of HSF1 that antagonizes transcription from HSP promoters and results in the depletion of intracellular HSP 70. Our studies indicate that expression of DN-HSF1 dramatically alters the DNA content of PC-3 cells (derived from p53 null prostatic carcinoma) and inhibits aneuploidy in these cells. This effect is due to prolonged expression of DN-HSF1, and transient expression of the dominant negative factor from an inducible promoter failed to cause the effect. Inhibition of aneuploidy in p53 null PC-3 cells by DN-HSF1 expression was recapitulated by expression within the cells of wild type p53. Furthermore, cells expressing DN-HSF1 showed a profound inhibition in the development of aneuploidy when exposed to chemical agents that disrupt the mitotic spindle and prevent progression through metaphase. Inhibition of aneuploidy in PC-3 cells expressing DN-HSF1 was associated with delayed breakdown of cyclin B1 compared with controls, consistent with a role for wild type HSF1 in the regulation of cyclin B1 degradation, a key step in the control of mitosis. Our experiments therefore demonstrate that HSF1 plays a functional role in cancer cells under nonstress conditions and influences cell cycle behavior and progression through mitosis and promotes the development of the aneuploid state.
Topics: Aneuploidy; Antineoplastic Agents, Phytogenic; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; Cells, Cultured; Cyclin B; Cyclin B1; DNA; DNA-Binding Proteins; Demecolcine; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Genes, Dominant; Genes, Reporter; Genes, p53; Genetic Vectors; HSP72 Heat-Shock Proteins; Heat Shock Transcription Factors; Heat-Shock Proteins; Humans; Immunoblotting; Luciferases; Male; Mitosis; Mutation; Phenotype; Ploidies; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Structure, Tertiary; Resting Phase, Cell Cycle; Spectrometry, Fluorescence; Transcription Factors; Transfection
PubMed: 15152009
DOI: 10.1074/jbc.M401475200 -
Cell Division Nov 2012Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part...
BACKGROUND
Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.
METHODS
Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (-)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (-)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (-), Parthenogenetic (-) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher's exact test (p≤0.05).
RESULTS
All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.
CONCLUSIONS
We have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.
PubMed: 23173571
DOI: 10.1186/1747-1028-7-23 -
The Journal of Cell Biology Jul 1984Cell shape is known to influence the chondrogenic differentiation of cultured limb bud mesenchyme cells (Solursh, M., T. F. Linsenmayer, and K. L. Jensen, 1982, Dev....
Cell shape is known to influence the chondrogenic differentiation of cultured limb bud mesenchyme cells (Solursh, M., T. F. Linsenmayer, and K. L. Jensen, 1982, Dev. Biol., 94: 259-264). To test whether specific cytoskeletal components mediate this influence of cell shape, we examined different cytoskeleton disrupting agents for their ability to affect chondrogenesis. Limb bud cells cultured at subconfluent densities on plastic substrata normally become flattened, contain numerous cytoplasmic microtubules and actin bundles, and do not undergo spontaneous chondrogenesis. If such cultures are treated with 2 micrograms/ml cytochalasin D during the initial 3-24 h in culture, the cells round up, lose their actin cables, and undergo chondrogenesis, as indicated by the production of immunologically detectable type II collagen and a pericellular Alcian blue staining matrix. Cytochalasin D also induces cartilage formation by high-density cultures of proximal limb bud cells, which normally become blocked in a protodifferentiated state. In addition, cytochalasin D was found to reverse the normal inhibition by fibronectin of chondrogenesis by proximal limb bud cells cultured in hydrated collagen gels. Agents that disrupt microtubules have no apparent effect on the shape or chondrogenic differentiation of limb bud mesenchymal cells. These results suggest an involvement of the actin cytoskeleton in controlling cell shape and chondrogenic differentiation of limb bud mesenchyme. Interactions of the actin cytoskeleton and extracellular matrix components may provide a regulatory mechanism for mesenchyme cell differentiation into cartilage or fibrous connective tissue in the developing limb.
Topics: Actins; Animals; Benzimidazoles; Cartilage; Cell Differentiation; Cytochalasin D; Cytochalasins; Cytoskeleton; Demecolcine; Extremities; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microtubules; Nocodazole
PubMed: 6539780
DOI: 10.1083/jcb.99.1.115 -
British Journal of Pharmacology May 19981. Alpha1B-adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist....
1. Alpha1B-adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti-N-terminus-antibody (1B-N1-C, (Hirasawa et al., 1996)) facilitated the quantification of cell surface alpha1B-adrenoceptor. Also, the cellular distribution of alpha1B-adrenoceptors was visually monitored by immunocytochemical confocal microscopy. 2. Utilizing this combined approach, we have examined the molecular mechanism for cellular trafficking of alpha1B-adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist-induced internalization. The two processes were separately examined by using alpha1B-adrenoceptor inducible DDT1MF-2 cells for the sorting process and CHO cells stably expressing alpha1B-adrenoceptors for the agonist-promoted internalization. 3. We examined the effects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), bafilomycin A1 (a specific inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin-mediated endocytosis) on these processes. 4. We found that the agonist-promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery. 5. The experimental approach as exemplified in this study would provide a valuable system to study further the molecular mechanism(s) of cellular trafficking of G protein-coupled receptors.
Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-Agonists; Amino Acid Sequence; Animals; CHO Cells; Cell Line; Cell Membrane; Cricetinae; Endocytosis; Flow Cytometry; Immunohistochemistry; Microscopy, Confocal; Molecular Sequence Data; Receptors, Adrenergic, alpha-1
PubMed: 9630343
DOI: 10.1038/sj.bjp.0701795 -
The Journal of Cell Biology Mar 2010The mechanisms that maintain the orientation of cortical polarity and asymmetric division unchanged in consecutive mitoses in Drosophila melanogaster neuroblasts (NBs)...
The mechanisms that maintain the orientation of cortical polarity and asymmetric division unchanged in consecutive mitoses in Drosophila melanogaster neuroblasts (NBs) are unknown. By studying the effect of transient microtubule depolymerization and centrosome mutant conditions, we have found that such orientation memory requires both the centrosome-organized interphase aster and centrosome-independent functions. We have also found that the span of such memory is limited to the last mitosis. Furthermore, the orientation of the NB axis of polarity can be reset to any angle with respect to the surrounding tissue and is, therefore, cell autonomous.
Topics: Animals; Animals, Genetically Modified; Cell Division; Cell Polarity; Cells, Cultured; Centrioles; Demecolcine; Drosophila Proteins; Drosophila melanogaster; Larva; Microtubules; Mutation; Neurons; Spindle Apparatus; Tubulin Modulators
PubMed: 20194641
DOI: 10.1083/jcb.200905024 -
Biochimica Et Biophysica Acta Dec 2000Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of...
Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.
Topics: Actin Cytoskeleton; Biological Transport; Calcium; Egtazic Acid; Endoplasmic Reticulum; Fluorescent Antibody Technique; Golgi Apparatus; Green Fluorescent Proteins; Humans; Luminescent Proteins; Microscopy, Confocal; S100 Proteins; Signal Transduction; Thapsigargin; Transfection; Tubulin; Tumor Cells, Cultured
PubMed: 11108965
DOI: 10.1016/s0167-4889(00)00098-7 -
Chemical Research in Toxicology Feb 2010Aneuploidy and extensive chromosomal rearrangements are common in human tumors. The role of DNA damage response proteins p53 and p21(CIP1/WAF1) in aneugenesis and...
Aneuploidy and extensive chromosomal rearrangements are common in human tumors. The role of DNA damage response proteins p53 and p21(CIP1/WAF1) in aneugenesis and clastogenesis was investigated in telomerase immortalized diploid human fibroblasts using siRNA suppression of p53 and p21(CIP1/WAF1). Cells were exposed to the environmental carcinogen sodium arsenite (15 and 20 microM), and the induction of micronuclei (MN) was evaluated in binucleated cells using the cytokinesis-block assay. To determine whether MN resulted from missegregation of chromosomes or from chromosomal fragments, we used a fluorescent in situ hybridization with a centromeric DNA probe. Micronuclei were predominantly of clastogenic origin in control cells regardless of p53 or p21(CIP1/WAF1) expression. MN with centromere signals in cells transfected with NSC siRNA or Mock increased 30% after arsenite exposure, indicating that arsenite induced aneuploidy in the tGM24 cells. Although suppression of p53 increased the fraction of arsenite-treated cells with MN, it caused a decrease in the fraction with centromeric DNA. Suppression of p21(CIP1/WAF1) like p53 suppression decreased the fraction of MN with centromeric DNA. Our results suggest that cells lacking normal p53 function cannot become aneuploid because they die by mitotic arrest-associated apoptosis, whereas cells with normal p53 function that are able to exit from mitotic arrest can become aneuploid. Furthermore, our current results support this role for p21(CIP1/WAF1) since suppression of p21(CIP1/WAF1) caused a decrease in aneuploidy induced by arsenite, suggesting that p21(CIP1/WAF1) plays a role in mitotic exit.
Topics: Aneuploidy; Arsenites; Blotting, Western; Cell Proliferation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Demecolcine; Fibroblasts; Gene Knockdown Techniques; Humans; In Situ Hybridization, Fluorescence; Mitomycin; Teratogens; Tumor Suppressor Protein p53
PubMed: 20000476
DOI: 10.1021/tx900353v -
BioTechniques Jul 2000We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding...
We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.
Topics: Aminophenols; Bibenzyls; Binding Sites; Binding, Competitive; Biotinylation; Colchicine; Demecolcine; Mebendazole; Microspheres; Podophyllotoxin; Scintillation Counting; Solvents; Stilbenes; Streptavidin; Sulfonamides; Tritium; Tubulin; Yttrium
PubMed: 10907090
DOI: 10.2144/00291rr02