THE EFFECT OF MITOTIC INHIBITORS ON MYOGENESIS IN VITRO

The effect of colchicine and other antimitotic drugs was studied in cultures of 11-day chick embryo breast muscle. Exposure of such cultures to 10^-6 M colchicine results in fragmentation of the elongate myotubes into rounded, cytoplasmic sacs (myosacs) containing various numbers of nuclei. Comparison of the dose-response relation between myotube fragmentation and metaphase arrest suggests that the underlying mechanism may be similar in both cases. Low temperature does not duplicate the effects of colchicine. Glycerinated myotubes are not affected by the mitotic inhibitors. The effect of colchicine on myotubes is reversible. Myosacs elongate within several days after removal from colchicine. However, the regenerated myotubes fail to incorporate additional mononucleated cells. Colchicine does not interfere with the process of fusion itself, but the metaphase block prevents cells from entering that phase of the cell cycle during which fusion can occur. Cells arrested in mitosis by colchicine do not recover when incubated in normal medium. Colcemid-induced arrest is reversible and does not prevent subsequent fusion of the cells.     

Ultrastructural immunocytochemical distribution of tubulin in cultured cells treated with microtubule inhibitors.

Microtubules in tissue cultured cells are stained immunocytochemically with the PAP-method using a purified antitubulin antibody. Treatment of the cells with microtubule inhibitors (colchicine, nocodazole, vinblastine) results in the disappearance of microtubules. The diffuse cytoplasmic staining is strongly increased in the cells by colchicine and nocodazole. Vinblastine produces paracrystalline aggregates that are strongly stained and macrotubules that are unstained. The diffuse staining is much less in vinblastine-treated cells. The bundles of intermediate filaments that are induced by all microtubule inhibitors do not bind the antitubulin antibody.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Selective 1H-NMR relaxation investigations of membrane-bound drugs in vitro. 1. Colchicine

Binding of colchicine to dipalmitoylphosphatidylcholine bilayer vesicles was detected by measuring the 1H-NMR selective spin-lattice relaxation rates of the low-field protons of colchicine. From the temperature dependence of the selective rates, preferential binding was observed above the temperature of transition. In the same way, binding of colchicine to red blood cells was detected and the equilibrium constant determined. Binding to the lipid matrix of red blood cells accounted only partially for the binding of colchicine to whole cells.     

Novel sulfonate analogues of combretastatin A-4: potent antimitotic agents

Sulfonate analogues of combretastatin A-4 have been prepared. These compounds compete with colchicine and combretastatin A-4 for the colchicine binding site on tubulin and are potent inhibitors of tubulin polymerization and cell proliferation. Importantly, these compounds also inhibit the proliferation of P-glycoprotein positive (+) cancer cells, which are resistant to many other antitumor agents.     

Inhibition of receptor-mediated endocytosis immunoglobulin G by enterocytes isolated from the neonatal rat jejunum

The effect of inhibitors of respiration (NaN3 and DNP), glycolysis (2DG, IAA and NaF) and the microtubular-microfilament system (colchicine and cytochalasin B) on the uptake of rat immunoglobulin G (IgG) by enterocytes isolated from the neonatal rat gut has been assessed. After a 1 hour incubation, NaN3, and DNP had significantly reduced IgG uptake by between 32% and 35% of the control, IAA and 2DG were less effective and NaF, colchicine and cytochalasin B had no effect at all. The findings show that IgG is internalised by isolated enterocytes in vitro and that this internalisation is under metabolic control, that inhibitors of respiration are more effective in blocking uptake than inhibitors of glycolysis.     

Use of fluorescent dyes as molecular probes for the study of multidrug resistance

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.     

Fast axonal transport: Effect of antimitotic drugs and inhibitors of energy metabolism on the rate and amount of transported protein in frog sciatic nerves

Mitosis inhibitors, drugs affecting the energy metabolism, heavy water and ouabain were used to partially inhibit fast axonal transport in frog sciatic nerves. Effects on the rate and on the amount of pulse labeled protein could be separated. The pulse of labeled protein, released after a cold-block, rapidly reached a maximum height which indicated that the transport system was saturated in the nerve segment occupied by the pulse. Both the rate and the amount were reduced by the mitosis inhibitors colchicine, vinblastine, and griseofulvin. Colchicine had a differential effect and reduced the rate of material migrating in the advancing front of the pulse less than the rate of that moving in the peak. Preincubation at low temperature potentiated the effects of colchicine. Two inhibitors of energy metabolism, NaCN and IAA, reduced the amount of labeled material in the pulse. The slope of the pulse was markedly reduced and multiple peaks appeared. The distance covered by the migrating pulse was largely unaffected, but some retardation of late components might have occurred. In contrast, 2.4-DNP reduced the rate without any effects on the amount of migrating material. Heavy water uniformly reduced the rate of the migrating pulse, whereas the main effect of ouabain was a diminished amount and multiple peaks as with NaCN and IAA. All drugs were tested for their effects on the electrical activity of sciatic nerves. The compound action potential was not affected by the mitosis inhibitors and heavy water, but was depressed by the inhibitors of energy metabolism and abolished by ouabain. The results indicate that the effects of various transport inhibitory drugs can be differentiated if both the rate and the amount are considered.     

Agglunation of a transformed mouse cell line and a variant subline with concanavalin A: Effect of temperature and time of lectin incubation

Colchicine treatment enhanced Con A-mediated agglutination of erythrocytes to LM cells (LM is a “spontaneously” transformed mouse line) incubated for brief periods with Con A at 22° C. Longer incubations with Con A at 22° C rendered colchicine treated cells less agglutinable than untreated cells. Even short incubation times with Con A at higher temperature (37° C) rendered colchicine treated LM cells less agglutinable than their untreated counterparts. Below 15° C, colchicine treated cells remained more agglutinable than untreated cells even after long periods of Con A treatment. Cells of a variant clone (Rl) isolated from LM by negative selection with concanavalin A exhibited increased substratum adhesiveness and an absolute serum requirement. LM and variant cells exhibited a differential reponse to colchicine treatment, the variant subline reguiring longer periods of colchicine treatment to elicit changes in morphology and agglutinability.     

Podophyllotoxin,Colcemid and Cold Temperature Interfere with Cilia Regeneration in Stentor*

SYNOPSIS Stentor coeruleus, induced to shed their ciliary membranellar bands, regenerate these and associated oral structures within a few hours after treatment. In cells placed in media containing optimal concentrations of mitotic spindle inhibitors, regeneration of the ingestive organelles is reversibly inhibited. Inhibitory effects of Colcemid, podophyllotoxin, and cold temperature reported here are compared with previous results using colchicine, griseofulvin and isopropyl-n-phenyl carbamate on regenerating oral membranellar cilia and cell growth.     

Drug resistance and membrane alteration in mutants of mammalian cells.

Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicate that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.     

Structural and metabolic correlation for Bacillus megaterium ACBT03 in response to colchicine biotransformation

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.     

Structural and metabolic correlation for <Emphasis Type="Italic">Bacillus megaterium</Emphasis> ACBT03 in response to colchicine biotransformation

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

Anion-induced increases in the affinity of colcemid binding to tubulin

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.     

Effects of caffeine and inhibitors of DNA synthesis on chromatid-type aberrations induced by acetaldehyde in root-tip cells

Root-tip cells of Allium cepa were exposed to acetaldehyde (AA) and post-treated with caffeine and 3 inhibitors of DNA synthesis, namely hydroxyurea (HU), 5-fluorodeoxyuridine (FdUrd), and arabinofuranosylcytosine (araC). Caffeine strongly potentiated the frequency of chromatid-type aberrations when given immediately after the AA treatment or as a 5-h treatment starting 10 h before the addition of colchicine. In contrast, no enhancement was observed when caffeine was present for the last 2.5 h, simultaneously with colchicine. The inhibitors of DNA synthesis were given following this last schedule. Both HU and FdUrd clearly enhanced the yield of AA-induced chromatid aberrations, while no enhancement fo chromosone damage was observed after exposure in araC.     

Externalization of transferrin receptor in established human cell lines

The externalization of transferrin receptors was found in established human tumor cell lines at the rate of 10-35 ng/hour/10(6) cells, when they were incubated with transferrin at 37 degrees C. This externalization is inhibited by lowering the incubation temperature to 4 degrees C or eliminating the ligand from the culture medium. Metabolic inhibitors such as sodium azide, colchicine, cytochalasin B and chloroquine also decreased the rate of externalization. Almost 95% of released transferrin receptors were precipitated by centrifugation at 100,000 x g for 30 min, suggesting that transferrin receptor is externalized into the medium as a vesicular form.     

The inhibition of systrophe in different organisms

Summary The phenomenon of systrophe following different stimuli was comparatively investigated in the cells of different plant organisms. In all cases systrophe could be inhibited by metabolic inhibitors. The influence of colchicine, cytochalasin B, vinblastine and APM (amiprophos-methyl) varied.     

Expression of phenotypic traits following modulation of colchicine resistance in J774.2 cells

Development of resistance to colchicine in the mouse macrophage-like cell line J774.2 coincides with the expression of a variety of phenotypic traits. A cloned subline (J7/CLC-20), maintained in 20 microM colchicine, exhibits reduced steady-state association with drug, increased presence of a 140,000-145,000 dalton (140-145 kD) phosphoglycoprotein associated with the plasma membrane, double minute chromosomes and cross-resistance to other drugs. While similar phenotypic traits are observed in J774.2 cells resistant to taxol and vinblastine, differences in the electrophoretic mobilities of the resistance-specific glycoproteins in each of the three sublines suggest that multi-drug resistant sublines exhibit specificity for individual drugs. In an attempt to elucidate the relationships between the phenotypic traits associated with colchicine resistance, the degree of colchicine resistance in J7/CLC-20 cells was modulated and the levels of expression of the phenotypic traits were quantitated. In the absence of colchicine in the growth medium, J7/CLC-20 cells reverted to drug sensitivity within 35 days. A decrease in the level of resistance coincided with coordinate changes in both the quantity of the resistance-specific glycoprotein and the average number of double minute chromosomes. We propose that the emergence and disappearance of the resistance-specific glycoprotein and double minute chromosomes may be closely linked. However, J7/CLC-20 cells which had regained their drug sensitivity after growth in drug-free medium maintained a reduced level of steady-state drug association. The persistence of reduced drug association in cells that have reverted to a drug-sensitive state suggests that this phenomenon, although related to colchicine resistance, need not be the primary or only mechanism of drug resistance.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.