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Biophysical Journal Jan 2000The effect of various drugs affecting the integrity of different components of the cytoskeleton on the elasticity of two fibroblast cell lines was investigated by...
The effect of various drugs affecting the integrity of different components of the cytoskeleton on the elasticity of two fibroblast cell lines was investigated by elasticity measurements with an atomic force microscope (AFM). Disaggregation of actin filaments always resulted in a distinct decrease in the cell's average elastic modulus indicating the crucial importance of the actin network for the mechanical stability of living cells. Disruption or chemical stabilization of microtubules did not affect cell elasticity. For the f-actin-disrupting drugs different mechanisms of drug action were observed. Cytochalasins B and D and Latrunculin A disassembled stress fibers. For Cytochalasin D this was accompanied by an aggregation of actin within the cytosol. Jasplakinolide disaggregated actin filaments but did not disassemble stress fibers. Fibrous structures found in AFM images and elasticity maps of fibroblasts could be identified as stress fibers by correlation of AFM data and fluorescence images.
Topics: 3T3 Cells; Actins; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Colchicine; Cytochalasin B; Cytochalasin D; Cytoskeleton; Demecolcine; Depsipeptides; Elasticity; Fibroblasts; Kinetics; Marine Toxins; Mice; Microscopy, Atomic Force; Microscopy, Fluorescence; Microtubules; Paclitaxel; Peptides, Cyclic; Rats; Stress, Mechanical; Thiazoles; Thiazolidines
PubMed: 10620315
DOI: 10.1016/S0006-3495(00)76614-8 -
Proceedings of the National Academy of... Mar 1977Intracellular and extracellular levels of 3':5'-cyclic GMP and 3':5'-cyclic AMP were studied in synchronized Novikoff rat hepatoma cells. Intracellular levels of cyclic...
Intracellular and extracellular levels of 3':5'-cyclic GMP and 3':5'-cyclic AMP were studied in synchronized Novikoff rat hepatoma cells. Intracellular levels of cyclic GMP increased spontaneously from 2-fold (without colcemid) to 10-fold (with colcemid), in proportion to the number of cells in mitosis. As cells entered mitosis, cellular cyclic AMP declined simultaneously with the rise in cyclic GMP. These reciprocal changes in cyclic nucleotide levels were reversed as cells passed out of metaphase and through anaphase. Maximum cyclic AMP and minimum cyclic GMP concentrations occurred during G-1. Less marked reciprocal fluctuations in both cyclic nucleotides were also found in S-phase and early G-2, where the ratio of cyclic AMP to cyclic GMP concentrations first fell and then increased. These changes in cyclic nucleotide ratios were closely correlated with major cell-cycle transitions at the boundaries between G-1/S-phase, S-phase/G-2, G-2/prophase, and metaphase/anaphase. Most, but not all, of the extracellular cyclic nucleotides were extruded when cells traversed mitosis. Colcemid or vinblastine completely prevented the appearance of extracellular cyclic AMP but augmented the appearance of extracellular cyclic GMP in parallel with the accumulation of mitotic cells. These results reflected changes in intracellular cyclic nucleotides and indicated that increased intracellular turnover of cyclic GMP and cyclic AMP occurred before and after metaphase, respectively. Elevated cyclic GMP levels during mitosis and S-phase are consistent with potential modulatory roles for this cyclic nucleotide in proliferation.
Topics: Bleomycin; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cyclic AMP; Cyclic GMP; Demecolcine; Liver Neoplasms; Mitosis; Neoplasms, Experimental; Vinblastine
PubMed: 66682
DOI: 10.1073/pnas.74.3.1052 -
The Journal of Biological Chemistry Apr 1981The dimerization of colchicine is demonstrated using the technique of concentration difference spectra. The difference spectra are characterized by an isosbestic point...
The dimerization of colchicine is demonstrated using the technique of concentration difference spectra. The difference spectra are characterized by an isosbestic point at 372 nm, a positive peak at 387 nm, and two negative peaks at 360 and 330 nm. The study of the concentration dependence and the effect of temperature allowed the determination of the molar extinction change and the dimerization equilibrium constant at four temperatures. The van't Hoff plot is linear, and the following thermodynamic parameters are calculated: standard enthalpy change, delta Ho/kJ . mol-1 = -31.0; and standard entropy change, delta So/J . mol-1 . K-1 = -69.5. For colcemid, similar results are obtained. The difference spectra show an isosbestic point at 385 nm and a positive peak at 397 nm. The thermodynamic parameters obtained are delta Ho/kJ . mol-1 = -26.8, and delta So/J . mol-1 . K-1 = -61.1. These thermodynamic parameters are comparable to the values obtained for the dimerization of polar dyes and the stacking of nucleotide bases.
Topics: Chemical Phenomena; Chemistry; Colchicine; Coloring Agents; Demecolcine; Structure-Activity Relationship; Thermodynamics
PubMed: 7204402
DOI: No ID Found -
FEBS Letters Dec 1986We report that the mitotic inhibitor, vinblastine (VLB), is highly toxic to the malarial parasite, Plasmodium falciparum. In cultures in vitro growth is inhibited by 50%...
We report that the mitotic inhibitor, vinblastine (VLB), is highly toxic to the malarial parasite, Plasmodium falciparum. In cultures in vitro growth is inhibited by 50% at a VLB level of about 28 nM, and totally abolished at a level of 100 nM. By tests on synchronized cultures we have found that the effect of VLB takes place at the trophozoite stage. Colcemid also inhibits schizogony with somewhat different kinetics. By mutagenesis with nitrosoguanidine followed by VLB selection we have isolated a VLB-resistant mutant which exhibits cross-resistance to vincristine. These data suggest a critical role of microtubules in the asexual schizogonic cycle of P. falciparum.
Topics: Animals; Chloroquine; Demecolcine; Kinetics; Mitosis; Plasmodium falciparum; Vinblastine; Vincristine
PubMed: 3542560
DOI: 10.1016/0014-5793(86)81077-8 -
Cell Structure and Function Oct 1996To obtain insight into the molecular dynamics and involvement of microtubules and the related signal molecules in the regulation of cell locomotion, we studied the...
To obtain insight into the molecular dynamics and involvement of microtubules and the related signal molecules in the regulation of cell locomotion, we studied the influence of microtubule disruption on actin stress fibers and focal adhesion assembly in addition to cell morphology. We found that all microtubule-disrupting drugs including colcemid and vinblastine rapidly and reversibly induce the formation of actin stress fibers and focal adhesions containing vinculin, accompanied by activated cell motility in serum-starved Balb/c 3T3 cells. In contrast, taxol, a microtubule-stabilizing drug, completely inhibited these effects of the microtubule-disrupting drugs. A microinjection of C3 ADP-ribosyltransferase, a specific inhibitor of rho GTPase, blocked the stress fiber and focal adhesion assembly induced by the microtubule disruption. These results suggested that microtubules contain signal molecules that regulate the formation of stress fibers and focal adhesions by activating the rho signal cascade. We postulate that microtubule-releasing and stress fiber-inducing factors link the intrinsically variable and irregular actin filament dynamics to coordinated and directional locomotion in the process of cell movement.
Topics: 3T3 Cells; ADP Ribose Transferases; Actins; Animals; Botulinum Toxins; Cell Adhesion; Cell Movement; Demecolcine; GTP-Binding Proteins; Insulin; Mice; Microtubules; Paclitaxel; Signal Transduction; Vinblastine; rho GTP-Binding Proteins
PubMed: 9118237
DOI: 10.1247/csf.21.317 -
The Journal of Veterinary Medical... Aug 1998Crude or dehydrated bulbs of autumn crocus (Colchicum autumnale L.) were fed to eleven calves. All the calves developed severe diarrhea and died or euthanized within 63...
Crude or dehydrated bulbs of autumn crocus (Colchicum autumnale L.) were fed to eleven calves. All the calves developed severe diarrhea and died or euthanized within 63 hr. At necropsy, the gastro-intestinal mucosa was edematous and hemorrhagic. Histologically, necrosis and degeneration with karyopyknosis and karyorrhexis were shown in the basal cell layer of the tongue, esophagus, forestomach, renal pelvis, urinary bladder, neck cell layer of the abomasal gastric glands, and intestinal cryps. These findings were also seen in Kupffer cells, renal tubular epithelial cells, and lymphocytes in the lymphoid and hemopoietic systems. The lesion of the present acute crocus poisoning of cattle closely resembled those reported in humans with colchicine intoxication. Refined acetone extract of organs of poisoned cattle proved to contain colchicine and demecolcine by high performance liquid chromatography.
Topics: Animals; Bone Marrow; Cattle; Cattle Diseases; Colchicum; Female; Foodborne Diseases; Intestinal Mucosa; Intestine, Small; Male; Plants, Medicinal; Spleen; Tongue
PubMed: 9764409
DOI: 10.1292/jvms.60.949 -
PloS One 2013An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface...
An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.
Topics: Apoptosis; Bone Marrow Cells; Cell Cycle; Cell Line; Demecolcine; Flow Cytometry; Humans; Immunophenotyping; Lymphocytes; Tubulin Modulators
PubMed: 23935867
DOI: 10.1371/journal.pone.0068425 -
Journal of Veterinary Science 2014Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences...
Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.
Topics: Animals; Biomarkers; Cloning, Organism; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Nuclear Transfer Techniques; Oocytes; Pregnancy; Real-Time Polymerase Chain Reaction; Swine
PubMed: 23820223
DOI: 10.4142/jvs.2014.15.1.73 -
Frontiers in Oncology 2013The chromosomal instability of polyploid cells, which leads to the formation of aneuploid cells, is causally related to carcinogenesis in human tissues. However, the...
The chromosomal instability of polyploid cells, which leads to the formation of aneuploid cells, is causally related to carcinogenesis in human tissues. However, the precise link between the chromosomal instability of polyploid cells and oncogenic transformation of them remains elusive. This is partly because we lack an experimental model in which non-transformed polyploid human cells can propagate in vitro. In a previous report, we demonstrated that proliferative tetraploid cells can be established from TIG-1 human fibroblasts by treatment with the spindle poison demecolcine (DC, colcemid) for 4 days. However, this procedure could not be applied to other human fibroblast strains because the resulting cells proliferated as a mixture of diploid and tetraploid populations. Here, we report a modified procedure to establish proliferative tetraploid cells from human fibroblasts of the BJ strain with minimum contamination by diploid cells. In the modified procedure, DC-arrested mitotic cells were collected by mitotic shake-off and treated with DC for an additional 3 days. DC-treated cells restarted proliferation as tetraploid cells after several days of growth arrest and showed similar growth to that of untreated diploid cells. The MDM2 antagonist Nutlin-3a activated p53 in established tetraploid cells and suppressed their growth, indicating that these cells have functional p53. These results contradicted the hypothesis that p53 functions as the tetraploidy checkpoint and prevents proliferation of tetraploid cells. Tetraploid cells established by our method could be a valuable model for the study of chromosomal instability and the oncogenic potential of polyploid cells.
PubMed: 23914348
DOI: 10.3389/fonc.2013.00198 -
Cell Proliferation Jun 2007Establishment of tetraploid ES cells.
OBJECTIVE
Establishment of tetraploid ES cells.
MATERIALS AND METHODS
Mouse H-1 (ES) cells were polyploidized by demecolcine and released from the drug.
RESULTS
A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid-tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent.
CONCLUSION
A pluripotent tetraploid cell line (4nH1 cells) was established.
Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Culture Techniques; Cell Line; Demecolcine; Embryonic Stem Cells; Mice; Mice, Inbred C3H; Pluripotent Stem Cells; Polyploidy; Stem Cell Transplantation; Teratocarcinoma
PubMed: 17531078
DOI: 10.1111/j.1365-2184.2007.00442.x