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PubMed1976

The effect of colchicine on the induction of ornithine decarboxylase by 12-O-tetradecanoyl-phorbol-13-acetate.

The induction of mouse epidermal ornithine decarboxylase, 1 of the earliest and largest phenotypic changes following treatment of mouse skin with the tumor-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate, can be inhibited by prior administration of colchicine. Maximal inhibition of this enzyme induction was observed when colchicine was injected i.p. 90 or 120 min before promoter treatment, although time intervals up to 20 hr between colchicine and promoter treatment were effective. The effect of colchicine was dose dependent, with a dose as low as 25 nmoles/mouse causing an inhibition of 35%. Other microtubule-disrupting agents, vinblastine, vincristine, and Colcemid, had a similar effect on ornithine decarboxylase activity. However, beta, gamma-lumicolchicine, a photochemical derivative of colchicine with no antimitotic or microtubule-disrupting ability, and cytochalasin B, an inhibitor of microfilament-dependent processes, had no effect. N6, O2'-dibutyryl 3',5'-cyclic adenosine monophosphate, when administered just before colchicine, blocked the inhibitory action of colchicine. The results of these studies suggest that colchicine-sensitive structures, most likely containing microtubules, may be mediating elements between the binding of tumor promoters, perhaps to specific cell surface receptors, and the subsequent induction of ornithine decdaboxylase.

Animals↗

PubMed1988

Cell expansion and single-cell separation induced by colchicine in suspension-cultured soybean cells.

Single plant cells have been obtained without the preparation of protoplasts by culturing pieces of soybean callus tissue with colchicine. Cell expansion and separation were evoked by colchicine (1 mM) within a week of culture. Microscopic observation showed that cells took on a spherical shape in the presence of colchicine and then separated into single cells. Addition of colchicine to the culture medium did not affect the composition of cell wall polysaccharides, but a uronic acid-rich extracellular polysaccharide appeared during cell expansion and separation. Addition of microtubule stabilizers, glycerol (300 mM) or dimethyl sulfoxide [3% (vol/vol)], inhibited the secretion of the polysaccharide as well as cell expansion and separation. The extracellular polysaccharide elicited by colchicine was isolated by ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-6B from the conditioned medium of colchicine-treated soybean cells. The purified 18-kDa polysaccharide immediately enhanced cell expansion and separation when added to soybean callus tissue cultured in medium containing colchicine, even at low concentrations (0.1 mM). The polysaccharide was composed of galacturonic acid and, after digestion with a pectinase preparation, had no effect on the cells. Methylation analysis suggests that the polysaccharide consists of approximately 100 sequential alpha-1,4-galacturonic acids. The galacturonan increased the viability of separated cells cultured in medium containing colchicine, and the single cells obtained did not produce a wound-response callose. (Aminoethoxyvinyl)glycine, a specific inhibitor of ethylene production, extensively decreased the cell expansion and separation but did not inhibit the formation of the extracellular polysaccharide, suggesting that the polysaccharide may exert its effect by stimulating ethylene production.

Journal Article↗

PubMed1998

Effects of colchicine on the enucleation of erythroid cells and macrophages in the liver of mouse embryos: ultrastructural and three-dimensional studies.

Enucleation is the last event in the development of a definitive erythroid line, and extruded nuclei are phagocytosed by macrophages. Both colchicine and cytochalasin have been known to exert a great influence on the enucleation process, but the relationship between enucleation and these agents has not yet been clearly revealed in vivo. Our aim was to clarify the significance of the enucleation in liver erythropoiesis and macrophage phagocytosis by colchicine and cytochalasin administration to embryonic mice. Pregnant mice were intraperitoneally injected with colchicine or cytochalasin at 13 days of gestation. Embryonic livers were removed at intervals of 3, 6 and 12 h after injection for processing for light and electron microscopy, and, to obtain three-dimensional morphology of erythroids at enucleation, computer-aided reconstructions were performed by light microscopy. Colchicine injections had cytolytic effects on hepatocytes and macrophages, and numerous erythroblasts were observed in the process of enucleation after colchicine injection. However, the extruding nuclei were irregularly shaped, and some erythroblasts at mitosis showed extreme peripheralization of their chromosomal masses and cell membrane constriction. Enucleation behavior could also be observed in immature erythroblasts. Liver macrophages engulfed extruded nuclei and erythroblasts in mitosis. Cytochalasin injections, on the other hand, had no significant effect on embryonic livers. The progress of erythroblast mitosis was clearly stopped by colchicine injection, and numerous erythroblasts at mitosis were extruding their nuclear compartment. Following colchicine injection, erythroid enucleation also took place in immature erythroblasts, and mitotic erythroids were phagocytosed. In enucleation, more attention should be paid to hematopoietic environmental factors than to hemopoietic cell factors.

Animals↗

PubMedDate not supplied

Comparative study of colchicine and trimethylcolchicinic acid on prolonged bile duct obstruction in the rat.

The aim of this work was to compare the effects of colchicine and trimethylcolchicinic acid (TMCA) on liver damage induced by bile duct ligation (BDL) for 2 months in male Wistar rats. Colchicine was evaluated at a dose of 10 micrograms rat-1 day-1, p.o. only, because higher doses produced diarrhoea and death. Trimethylcolchicinic acid showed no toxic effects at 10, 50 or 100 micrograms rat-1 day-1, p.o. Biliary obstruction resulted in a 65% mortality, colchicine decreased it to 46% and TMCA (10 micrograms) to 33.3%. Serum markers of liver damage increased by BDL (P < 0.05), colchicine prevented it partially (P < 0.05) and TMCA did it in a dose-dependent manner. Liver peroxidation increased 10 times by BDL and both drugs prevented it. Hepatic glycogen content decreased 80% by BDL, colchicine TMCA (10 micrograms) failed to preserve it and 50 micrograms of TMCA preserved it completely. Hepatocyte and erythrocyte plasma membrane Na+/K(+)- and Ca(2+)-ATPase activities decreased after BDL (P < 0.05) and 100 micrograms of TMCA preserved normal ATPase activities. It is concluded that TMCA is better than colchicine for protecting the liver from BDL-induced cirrhosis and, due to its lower toxicity, can be used at higher and more effective doses without the common side-effects of colchicine, thus making TMCA a suitable compound to be studied in other hepatic lesions and in humans.

Animals↗

PubMed2001

Colchicine for alcoholic and non-alcoholic liver fibrosis and cirrhosis.

BACKGROUND: The majority of liver fibrosis and liver cirrhosis cases in the Western World is caused by alcohol and hepatotoxic viruses. Colchicine is an anti-inflammatory and anti-fibrotic medication. Several randomised clinical trials have addressed the question whether colchicine has any efficacy in patients with alcoholic as well as non-alcoholic fibrosis and cirrhosis. OBJECTIVES: The objectives were to assess the efficacy of colchicine on mortality, clinical symptoms and complications, liver biochemistry, liver fibrosis markers, liver histology, alcohol consumption, quality of life, and health economics in patients with alcoholic and non-alcoholic fibrosis or cirrhosis, excluding primary biliary cirrhosis and other rarer forms of fibrosis/cirrhosis. Also adverse events were assessed. SEARCH STRATEGY: The Cochrane Hepato-Biliary Group Controlled Trials Register, The Cochrane Library, MEDLINE, and full text searches were combined. SELECTION CRITERIA: Only randomised clinical trials were included studying patients with alcoholic fibrosis, alcoholic hepatitis and/or alcoholic cirrhosis as well as patients with viral induced or cryptogenic fibrosis and/or cirrhosis according to the diagnostic work-up of the individual trial. Interventions encompassed peroral colchicine at any dose versus placebo or no intervention. The trials could be double-blind, single-blind or unblinded. The trials could be unpublished or published and no language limitations were applied. DATA COLLECTION AND ANALYSIS: All analyses were performed according to the intention-to-treat method. The statistical package (RevMan and MetaView) provided by the Cochrane Collaboration was used. The methodological quality of the randomised clinical trials was evaluated by the Jadad-scale. MAIN RESULTS: Combining the results of 14 randomised clinical trials including 1150 patients demonstrated no significant effects of colchicine on mortality (Peto odds ratio (OR) 0.90, 95% confidence interval (CI) 0.63 to 1.29), liver related mortality (OR 1.05, 95% CI 0.61 to 1.80), complications (OR 1.01, 95% CI 0.63 to 1.62), liver biochemistry, liver histology, and alcohol consumption (OR 1.02, 95% CI 0.58 to 1.79). Colchicine was associated with a significantly increased risk of adverse events (OR 4.92, 95% CI 2.66 to 9.10; P< 0.001). REVIEWER'S CONCLUSIONS: Colchicine should not be used for alcoholic, viral, or cryptogenic liver fibrosis or liver cirrhosis outside randomised trials.

Colchicine↗

PubMed2005

Colchicine for alcoholic and non-alcoholic liver fibrosis and cirrhosis.

BACKGROUND: Alcohol and hepatotropic viruses cause the majority of liver cirrhosis cases in the Western World. Colchicine is an anti-inflammatory and anti-fibrotic medication. Several randomised clinical trials have addressed the question whether colchicine has any efficacy in patients with alcoholic or non-alcoholic fibrosis and cirrhosis. OBJECTIVES: To assess the beneficial and harmful effects of colchicine in patients with alcoholic or non-alcoholic fibrosis or cirrhosis, excluding primary biliary cirrhosis. SEARCH STRATEGY: The Cochrane Hepato-Biliary Group Controlled Trials Register, The Cochrane Controlled Trials Register on The Cochrane Library, MEDLINE, EMBASE, Web of Science, and full text searches were combined (September 2004). Manufacturers and researchers in the field were also contacted. SELECTION CRITERIA: We included randomised trials irrespective of blinding, language, or publication status comparing per oral colchicine with placebo or no intervention for patients with fibrosis or cirrhosis induced by either alcohol, virus, or unknown factors (cryptogenic). DATA COLLECTION AND ANALYSIS: The statistical package (RevMan Analyses) provided by The Cochrane Collaboration was used. The methodological quality of the randomised clinical trials was evaluated. MAIN RESULTS: We could include fifteen randomised clinical trials in which 1714 patients were randomised. We found no significant effects of colchicine on mortality (relative risks (RR) 1.00, 95% confidence interval (CI) 0.87 to 1.16), liver-related mortality (RR 1.08, 95% CI 0.88 to 1.33), complications (RR 1.01, 95% CI 0.74 to 1.38), liver biochemistry, liver histology, and alcohol consumption (RR 1.03, 95% CI 0.77 to 1.39). Colchicine was associated with a significantly increased risk of adverse events (RR 4.35, 95% CI 2.16 to 8.77). AUTHORS' CONCLUSIONS: Colchicine should not be used for alcoholic, viral, or cryptogenic liver fibrosis or liver cirrhosis outside randomised clinical trials.

Anti-Inflammatory Agents↗

PubMed2001

A double-blind trial of colchicine in Behçet's syndrome.

OBJECTIVE: Colchicine is a widely used treatment for Behçet's syndrome, even though in a previous 6-month controlled study, it was shown to be effective only in controlling erythema nodosum and arthralgias. We reassessed the effect of colchicine in Behçet's syndrome in a study conducted among a larger group of patients for 2 years. METHODS: We randomized 116 patients with Behçet's syndrome (60 male/56 female), who had active mucocutaneous disease without eye or major organ involvement, to receive either placebo or colchicine (1-2 mg/day, adjusted to body weight) in a double-blind trial for 2 years. The primary outcome measure was the sustained absence of any lesions during treatment (complete response). The secondary outcome measure was the difference in the number of mucocutaneous lesions or arthritic joints between the active drug and placebo arms. Women and men were analyzed separately. RESULTS: Eighty-four patients (72%; 45 male, 39 female) completed the 24-month study. Kaplan-Meier analyses showed significantly more complete responses in the colchicine treatment group in terms of reduced occurrence of genital ulcers (P = 0.004), erythema nodosum (P = 0.004), and arthritis (P = 0.033) among the women, and reduced occurrence of arthritis (P = 0.012) among the men. The mean numbers of genital ulcers (P = 0.001), erythema nodosum lesions (P = 0.002), and arthritic joints (P = 0.014) among the women were less in the colchicine group, and the mean number of arthritic joints (P = 0.026) among the men was less in the colchicine group. Adverse effects were similar in both groups. CONCLUSION: Colchicine may be useful for treating some of the manifestations of Behçet's syndrome, especially among women. This might be a reflection of less severe disease among the women.

Adolescent↗

PubMed1986

Influence of colchicine on the distribution of horseradish peroxidase in the secretory ameloblast layer in vitro.

The distribution of exogenous horseradish peroxidase (HRP) in the secretory ameloblast layer of developing rat molar tooth germs was examined in a culture system with and without colchicine. The secretory ameloblast of cultured tooth germs engulfed HRP added to the medium regardless of the presence of colchicine. The reaction product was localized in various vesicles and granules. Without colchicine in the medium, many small vesicles containing HRP were located in the Tomes' processes, whereas only a few were present with colchicine at concentrations above 5 microM. An intense reaction of HRP also appeared in the distal extracellular spaces beyond the distal junctional complexes of ameloblasts cultured without colchicine, whereas it became almost indiscernible in the tooth germs cultured with colchicine. The lack of HRP in the Tomes' processes and distal extracellular spaces of ameloblasts treated with colchicine might be attributed to the disruption of microtubules. The present study suggests that the secretory ameloblast is able to transport tracer molecules through the intracellular pathway from the proximal and lateral extracellular spaces to the distal extracellular space.

Animals↗

PubMed1996

Colchicine in breast milk of patients with familial Mediterranean fever.

OBJECTIVE: To clarify whether colchicine is excreted in breast milk, and to compare its concentrations in the serum and breast milk of lactating women who have familial Mediterranean fever (FMF). METHODS: Using a specific radioimmunoassay, we determined colchicine concentrations in the serum and breast milk of 4 patients at various time points, following oral administration of the drug. The study evaluated 4 patients with FMF who had been taking colchicine on a long-term basis. RESULTS: Colchicine was found to be excreted in breast milk. Its levels ranged between 1.9 and 8.6 ng/ml, which were similar to those found in the serum (parallel concentration time curves). However, there appeared to be a considerable variation in colchicine milk concentration among the different patients, which might be related to individual breast milk composition and, possibly, to other nutritional or metabolic factors. CONCLUSION: The extensive peripheral tissue binding and relatively low concentration of colchicine in breast milk suggests that the amount ingested by the infant is small. Furthermore, based on our clinical experience, nursing appears to be safe for lactating women with FMF who continue to take colchicine.

Administration, Oral↗

PubMed2003

Impact of Solutol HS 15 on the pharmacokinetic behaviour of colchicine upon intravenous administration to male Wistar rats.

In the current investigation, the alkaloid colchicine was administered intravenously to male Wistar rats both as a solution in isotonic sodium chloride (NaCl 0.9%, control group) and in NaCl 0.9%:Solutol HS 15 (95:5) at 1.5 mg/kg. At predetermined time points, plasma and urine were collected from the animals and analysed for colchicine and its demethylated metabolites by LC/MS-MS. In the presence of Solutol HS 15, colchicine clearance (CI) was significantly decreased and its maximum plasma concentration (c(max)) was significantly increased as compared to the control group (CI: 15.6+/-7.0 ml/min/kg vs 34.3+/-2.3 ml/min/kg; c(max) 3055.1+/-587.4 h vs 1260.1+/-223.7 h; p<0.05). Moreover, the amount of parent colchicine excreted into urine was markedly increased in the Solutol HS 15 treated group (41.50+/-3.23 vs 1.17+/-0.41% of total dose; p<0.05). By contrast, there was no statistically significant difference but a trend to lower values only in the volume of distribution (V(d) 13.3+/-2.2 l/h vs 31.4+/-17.7 l/h, p=0.35). The half-lives for the first (t(1/2 1stphase). 0.21+/-0.02 h vs 0.20+/-0.03 h) and second phase (t(1/2 2ndphase). 18.5+/-6.9 h vs 18.3+/-7.7 h) did not differ significantly in dependence on the dosing vehicle. The free fraction in rat plasma (FF), the blood/plasma (lambda) and erythrocyte/plasma concentration ratios (K(e)) were not significantly changed in the presence of different concentrations of Solutol HS 15 compared with surfactant-free incubations (overall means: 72.25+/-0.50% for FF, 0.80+/-0.02 for lambda, 0.46+/-0.04 for K(e)). In vitro, in rat hepatocytes, the clearance of colchicine was significantly reduced at 0.003% Solutol HS 15 present in the incubation medium (0.86+/-0.15 microl/min/10(-6) cells vs 1.46+/-0.06 microl/min/10(-6) cells). As colchicine exhibits a comparatively high aqueous solubility, an impact of Solutol HS 15 on the solubility of the alkaloid is very unlikely to be a reason for the observed effect. Therefore, our results indicate that the most likely reasons for the changed pharmacokinetic behaviour of colchicine in the presence of Solutol HS 15 are alterations of metabolism and/or transport as well as distribution and elimination processes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Improvement of hepatocyte-specific gene expression by a targeted colchicine prodrug.

Colchicine, an established tubulin inhibitor, interferes with the trafficking of endocytotic vesicles and thereby promotes the escape of lysosome-entrapped compounds. To improve its potency and cell specificity, a targeted prodrug of colchicine was synthesized by conjugation to a high-affinity ligand (di-N(alpha),N(epsilon)-(5-(2-acetamido-2-deoxy-beta-D-galactopyranosyloxy)pentanomido)lysine, K(GalNAc)(2)) for the asialoglycoprotein receptor on parenchymal liver cells. The resulting colchicine-K(GalNAc)(2) conjugate bound to this receptor with an affinity of 4.5 nM. Confocal microscopy studies confirmed rapid uptake and receptor dependency of a prodrug conjugated with fluorescein isothiocyanate. Colchicine-K(GalNAc)(2) substantially increased the transfection efficiency of polyplexed DNA in parenchymal liver cells in a concentration- and receptor-dependent fashion. Colchicine-K(GalNAc)(2) was found to enhance the transfection efficiency by 50-fold at 1 nM, whereas the parental colchicine was ineffective. In conclusion, this nontoxic colchicine-K(GalNAc)(2) conjugate can be a useful tool to improve the transfection efficiency of hepatic nonviral gene transfer vehicles.

Animals↗

PubMed1986

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex.

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Colchicine-binding sites of brain tubulins from an antarctic fish and from a mammal are functionally similar, but not identical: implications for microtubule assembly at low temperature.

The tubulins of Antarctic fishes possess adaptations that favor microtubule formation at low body temperatures (Detrich et al.: Biochemistry 28:10085-10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus). At temperatures between 0 and 20 degrees C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5-2.6-fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (delta H degrees = +10.6 and +7.4 kcal mol-1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (delta S degrees = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was approximately 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine-binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low-temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., "colchicine-like" molecules or MAPs) that regulate microtubule assembly.

Animals↗

PubMed1995

Comparative study of the colchicine binding site and the assembly of fish and mammalian microtubule proteins.

Isolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 +/- 1.5) x 10(5)M-1 at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 x 10(-5) to 1 x 10(-3) M), MTC induced a large amount of cold-stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10(-5) M colchicine no spirals were formed, while at 10(-4) M and 10(-3) M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2+ concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins.

Animals↗

PubMed1981

Colchicine reversibly inhibits electrical activity in arthropod mechanoreceptors.

Colchicine and some other microtubule-active agents inhibit the electrical responses of cockroach tibial spine mechanoreceptors. Lumicolchicine, a colchicine analog which does not bind to microtubule protein, does not inhibit mechanoreceptive responses. Colchicine inhibition of peripheral mechanoreceptive responses is fully reversible and dose dependent, but colchicine has no effect on conduction in leg nerve axons. Colchicine inhibition is therefore an effect on the sensory dendrites or soma. The inhibition produced by colchicine could be produced by several effects. Colchicine may inhibit because it (1) disrupts the numerous intracellular microtubules which are a part of this sensory receptor's dendrite, (2) blocks axoplasmic transport of essential materials to the sensory dendrite, or (3) binds to tubulin or other proteins in the dendritic membrane.

Animals↗

PubMed2000

The interaction of the B-ring of colchicine with alpha-tubulin: a novel footprinting approach.

Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on alpha and some supporting a binding site on beta. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the alpha-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on alpha-tubulin; these residues are located well away from the alpha/beta interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the alpha/beta interface of tubulin with the B-ring on the alpha-subunit and the A and the C-rings on the beta-subunit.

Amino Acid Sequence↗

PubMed1983

Structure-activity relationship of 1-propargyl-5-halopyrimidin-2-ones. Metaphase arresting properties and competitive inhibition of colchicine binding to tubulin.

The structure-activity relationship of 1-propargyl-5-halopyrimidin-2-ones with respect to mitotic inhibitory potential and inhibition of colchicine binding to tubulin was investigated. A correlation between accumulation in metaphase of cultured human NHIK 3025 cells and drug competition with colchicine binding to tubulin was found for each compound. Effects on division of NHIK 3025 cells were determined in stained preparations following a 6 h treatment with drugs. The four halogen-substituted compounds displayed metaphase arresting ability while the H-substituted 1-propargylpyrimidin-2-one did not. 1-Propargyl-5-fluoropyrimidin-2-one was active at 1.5 and 0.75 mM while the 5-chloro, 5-bromo and 5-iodo compounds caused metaphase arrest at 0.375 and 0.18 mM. The ability of these drugs to compete with colchicine binding to DEAE-cellulose purified tubulin was also investigated. 1-Propargylpyrimidin-2-one at 5 mM did not inhibit (3H)colchicine binding to tubulin as determined by Sephadex G-50 gel filtration. The halogen-substituted pyrimidones, however, demonstrated competitive inhibition of colchicine binding to tubulin. Five mM 1-propargyl-5-iodopyrimidin-2-one inhibited colchicine binding by 43.2%, the highest value obtained within the metahalone group. The drugs tested had no effect on vincristine binding to tubulin. With respect to the halogen substitution, the increasing order of mitotic inhibitory potential and competition with colchicine binding to tubulin is H less than less than F less than Cl less than or equal to Br less than or equal to I.

Binding, Competitive↗

PubMed1990

Influence of colchicine on the addition of a sugar to the enamel protein in secretory ameloblasts of cultured germs of rat molar tooth by 3H-galactose radioautography.

The influence of colchicine on the addition of 3H-galactose to the enamel protein in secretory ameloblasts of cultured germs of rat molar tooth was investigated by light- and electron-microscopic radioautography. In tooth germs cultured without colchicine, the reaction products of 3H-galactose were observed over Golgi cisternae at early chase times and then localized over the enamel with time. In tooth germs cultured with colchicine, the silver grains were seen over the Golgi cisternae, condensing granules and accumulated secretory granules. Some grains also appeared with time over the pale granular material precipitated in the intercellular space with colchicine treatment. In quantitative analysis with light microscopic radioautography, values of silver grain counts over the unit area (100 microns2) on ameloblasts and enamel of colchicine-treated tooth germs were significantly lower at both 0 min and 30 min chase after 30 min pulse than those of control tooth germs, respectively. This finding indicates that colchicine diminished the incorporation of 3H-galactose into the secretory ameloblast of cultured tooth germs. It is suggested that colchicine decreases the activity of the Golgi apparatus with regard to the addition of sugar to the synthesizing glycoprotein in the secretory ameloblast.

Ameloblasts↗