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[Effect of some drugs on colchicine uptake by colchicine-sensitive and resistant cells].

The effect of several agents on 3H-colchicine, uptake by L cells and resistant to colcemide and colchicine L-53 cells was studied. Vinblastin to which L-53 cells are cross-resistant increases labeled colchicine uptake by L and L-53 cells 3- and 8-fold, respectively. The substances which decrease ATP level in the cells (olygomycin, etc.) enhance colchicine uptake by L and L-53 cells 2--4-fold. In the presence of these substances colchicine uptake by resistant cells is more intensive than by sensitive L cells. The structural analogue of colchicine, lumicolchicine, inactive in binding the microtubular protein tubulin enhances colchicine uptake by L and L-53 cells to about equal degree.

Adenosine Triphosphate

In vitro effect of colchicine on neutrophil granulocyte locomotion. Assessment of the effect of colchicine on chemotaxis, chemokinesis and spontaneous motility, using a modified reversible Boyden chamber.

The effect of colchicine on human neutrophil granulocyte chemotaxis, chemokinesis and spontaneous motility was examined, using a modified reversible Boyden chamber. Colchicine was shown to inhibit the attraction of neutrophils to casein and to a bacterial chemotactic factor at concentrations as low as 10(-7) M. Experiments in which the absolute concentrations and the concentration gradients of the chemotactic agent were varied, revealed that colchicine inhibited chemokinesis rather than chemotaxis. The spontaneous motility measured in the absence of chemotactic agents was not inhibited by colchicine. Pre-incubation of the cells with a bacterial chematactic factor did not change the sensitivity of the cells to colchicine. It is concluded that the integrity of microtubule function is not necessary for the ability of the cells to discern a concentration gradient or to react to this with directional locomotion. Thus the inhibitory effect of colchicine on neutrophil granulocyte chemokinesis may not depend on its inhibition of microtubule function. It is suggested that colchicine may block the still unidentified membrane mechanism involved in the translation of the recognition signal into an appropriate locomotory cell response.

Caseins

Mechanism of action of colchicine. III. Antiinflammatory effects of colchicine compared with phenylbutazone and indomethacin.

Colchicine suppresses the development of carrageenan-induced edema in the rat with a minimum effective oral dose of 6.0 mg/kg. The slope of the dose-response regression line for colchicine differs significantly from that of indomethacin and phenylbutazone. Based on the dosages required to achieve a 50% suppression of this inflammation, colchicine is 0.6 and 1.5 times as potent as indomethacin and phenylbutazone, respectively. In the reversed passive Arthus reaction in the rat, the suppressive activity of colchicine is at least 50 times that of indomethacin and 100 times that of phenylbutazone. The possible significance of these results with regard to the unique effectiveness of colchicine in the treatment of gout is discussed.

Animals

Mechanism of action of colchicine. I. Effect of colchicine and its analogs on the reversed passive Arthus reaction and the carrageenan-induced hindpaw edema in the rat.

Colchicine and N-desacetyl-N-methylcolchicine suppressed both the reversed passive Arthus reaction and the carrageenan-induced edema in the rat. Colchicine, 2-desmethyl-colchicine glucoside and trimethylcolchicine acid had no effect on either model of inflammation. The ability or inability of these compounds to suppress the development of experimental inflammation correlated with their antimitotic activities. The findings lend support to the hypothesis that the anti-inflammatory and the antimitotic effects of colchicine may depend on the same basic, biophysical mechanism of action, i.e., the disruption of the microtubules.

Administration, Oral

Mechanism of action of colchicine. II. Effects of colchicine and its analogs on phagocytosis and chemotaxis in vitro.

Colchicine, desacetylmethylcolchicine and colchiceine suppress the phagocytosis of starch granules by rabbit peritoneal polymorphonuclear leukocytes in vitro. Trimethylcolchicinic acid and 2-desmethylcolchicine glucoside have no effect on phagocytosis. Colchicine, at concentrations as high as 1 X 10(-4) M, has no effect on chemotaxis of polymorphonuclear leukocytes in response to immune complex-activated chemotactic factors or on the motility of these cells. These results do not support the contention that the suppression of phagocytosis or chemotaxis plays a significant role in the anti-inflammatory activity of colchicine.

Animals

Metabolic transformation of colchicine, IV[1]. On the interaction of colchicine and colchiceine with sulfhydryl compounds.

The interaction of colchicine and one of its metabolites, O10-demethylcolchicine (colchiceine), with sulfhydryl compounds was studied in several experimental systems. Colchiceine protects reduced glutathione from oxidation by atmospheric oxygen. Inactivation of enzymes with catalytically essential sulfhydryl groups is prevented by colchiceine. In affinity chromatographic experiments, Agarose-bound colchiceine adsorbs sulfhydryl enzymes which may be eluted specifically by SH-compounds like mercaptoethanol. No such effects are shown by colchicine. The data demonstrate a novel type of biochemical reactivity in the metabolite colchiceine with potential biological relevance.

Adenosine Deaminase

Effect of colchicine on the antibody response. II. Demonstration of the inactivation of suppressor cell activities by colchicine.

The simultaneous administration of colchicine (CC) with a T-independent antigen, e.g. 2,4,6-trinitrophenyl-keyhold limpet hemocyanin-Sepharose, to intact animals effectively enhanced their hapten-specific plaque-forming cell (PFC) response. However, in congenitally athymic nude mice in which T-cell regulation was absent, CC was ineffective in producing enhancement. These observations suggest that the target cell acted upon by CC is most likely thymus-derived. Furthermore, the injection of CC with the co-polymer of L-glutamic acid50-L-tyrosine50 (GT) abolished GT-specific suppression of the PFC response to GT-methylated bovine serum albumin. Spleen cells from CC-treated and GT-primed hosts could no longer transfer suppressive activity to normal recipients. These results provide evidence that CC is capable of inactivating or eliminating suppressor cells or their precursors. Thus, CC-induced enhancement of the antibody response may be explained, at least in part, by its antimitotic, and hence lethal effect on dividing suppressor T cells.

Animals

Colchicine binding to bovine anterior pituitary slices and inhibition of growth-hormone release.

The uptake of [ring C-methoxyl-3H]colchicine into bovine anterior pituitary slices was studied. The data suggest that more than one site exists for the binding of colchicine. At low concentrations colchicine binds to saturable trypsin-sensitive site(s), with a dissociation constant of 3.1 +/- 0.69 mug. The binding capacity of these sites is 8.58 +/- 0.60 pmol of colchicine/mg of wet pituitary. At higher colchicine concentrations binding occurs predominantly to sites which exhibit non-saturation kinetics. Subcellular fractionation of colchicine-labelled slices shows that 90% of the saturable sites are present in the fraction containing cytosol, where the binding protein has a molecular weight of about 11.9 x 10(4) and constitutes 0.7% of the protein present. The nuclear fraction contains 10% of the saturable sites, and the mitochondria and granule fraction contain only non-saturable sites. The rate of colchicine uptake was studied at 0.84 mm- and 2mum-colchicine. At both concentrations the colchicine space exceeded the total tissue water within 10 min. Equilibration with the saturable binding sites was complete in 120 min at 2mum-colchicine. A concentration of colchicine (13.4 mum) which would give 81% maximum binding was found to decrease the length of observable microtubules in tissue fixed at 37 degrees C in glutaraldehyde by 83 +/- 4%. The colchicine-binding protein could be partially purified by using a standard procedure for isolation of brain tubulin. Colchicine inhibits the release of growth hormone in the presence of 3-isobutyl-1-methylxanthine (0.1 mm), but does not alter basal release. The concentration-dependence of colchicine inhibition is similar to that of colchicine binding, but maximum inhibition is only 35%.

Animals

Colchicine and Longitudinal Dynamics of Clonal Hematopoiesis: An Exploratory Substudy of the LoDoCo2 Trial.

BACKGROUND: Clonal hematopoiesis (CH) is an aging-related hematologic condition associated with increased risk for cardiovascular events. Larger CH clones associate more strongly with cardiovascular risk. Preclinical data indicate that inflammatory signaling drives expansion of CH clones and CH-associated cardiovascular disease. However, the effect of anti-inflammatory therapies on CH clonal dynamics in humans is unclear. OBJECTIVES: The goal of this study was to test the association of randomization to colchicine vs placebo with CH growth in participants with chronic coronary artery disease. It also assessed the association of colchicine use with change in inflammatory biomarkers over time according to CH status. METHODS: In this exploratory substudy of the LoDoCo2 (Low-Dose Colchicine 2) trial, high-coverage targeted sequencing was used to detect CH driver mutations and to quantify variant allele frequency at 4 timepoints: baseline, after a 30-day open-label colchicine run-in phase (0.5 mg daily), 1 year postrandomization to colchicine or placebo, and at end of study (median follow-up of 25.0 months). Clonal dynamics were assessed by using a generalized linear mixed model. High-sensitivity C-reactive protein and interleukin-6 were additionally measured at baseline, randomization, and 1 year postrandomization. RESULTS: In total, 854 participants contributed 2,047 observations across 4 timepoints, including before and after the prerandomization colchicine run-in period. Randomization to placebo was associated with a 14.9% annual increase in CH clone size (βtime = 0.14; 95% CI: 0.08 to 0.21) vs a nonsignificant 6.3% increase with colchicine (βtime on colchicine: 0.06; 95% CI: -0.01 to 0.14), although this difference between treatment arms was not statistically significant (Pinteraction = 0.13). Compared with placebo, colchicine was associated with attenuated clonal growth in TET2 CH (βtime on colchicine: 0.09 [95% CI: -0.04 to 0.22]; βtime placebo: 0.27 [95% CI: 0.16 to 0.37]; Pinteraction= 0.04). Among individuals with non-DNMT3A CH, interleukin-6 levels increased to a lesser extent in those receiving colchicine vs placebo over 1 year (30.0% vs 98.1% increase, respectively; Pinteraction = 0.01). CONCLUSIONS: In this exploratory analysis, treatment with low-dose colchicine was associated with attenuated clonal expansion in TET2 CH. These findings suggest the potential for colchicine to curb the proliferative advantage of key CH driver mutations and to mitigate their associated risk of cardiovascular disease. Further validation in prospective studies is warranted.

Humans

Colchicine inhibition of stalk elongation in Carchesium sp.: effect of Ca2+ and Mg2+.

The effect of colchicine on stalk elongation in the colonial peritrich ciliate Carchesium sp. has been investigated by growing this protozoon in colchicine-containing media. The length of the stalk in control cultures was 0-4-0-9 mm. In the presence of 2-5-12-5 mM colchicine, stalk elongation was inhibited, and stalk length was inversely proportional to colchicine concentration. At concentrations above 7-5 mM colchicine, stalks measured less than 0-1 mm, and sometimes contained imperfect myonemes. The rate of cell fission was retarded in colchicine-containing media, but nevertheless short-stalked colonies with apparently normal zooids were formed. On transfer of such colonies to media without colchicine normal growth was resumed, but only the newly formed branches were of normal length and contractility. The inhibitory effect of colchicine was annulled by Ca2+ and Mg2+ at 10(-3) and 10(-4) M, respectively. At lower concentrations of Mg2+, but in the presence of Ca2+, the effect of colchicine was less conspicuous than at low Ca2+ concentration in presence of Mg2+. Lowering Mg2+ concentration at low Ca2+ concentration, increased the inhibitory effect of colchicine. It is concluded that colchicine-sensitive, probably tubulin-like proteins, participate in the formation of the contractile stalk of Carchesium. Ca2+ and Mg2+ probably compete with colchicine for a common site in these proteins, or they might reduce the cell's permeability to this drug.

Animals

Treatment of cirrhosis with colchicine. A double-blind randomized trial.

As part of a double-blind, randomized, controlled trial to evaluate the effect of colchicine on liver cirrhosis, 43 cirrhotic patients were assigned to either a placebo (20 patients) or a colchicine (23 patients) treatment group. Colchicine 1 mg and an indistinguishable placebo were administered orally on a daily dose 5 days a week. In the colchicine group, 12 were males and 11 females, while in the control group 13 were males and 7 females. The time elapsed between diagnosis and inclusion in the study was 14.1 mo for the controls and 14.5 mo for the patients on colchicine. Mortality related to the liver disease occurred in 4 patients on colchicine and 8 patients on placebo. Although the probability of surviving in the colchicine group was greater than that of the placebo, the difference did not reach statistically significant levels. Of the colchicine-treated patients, in three a remarkable decrease in liver fibrosis was observed in serial biopsies. In two other patients, carcinoma of the liver developed. Six of the survivors on colchicine have improved clinically, noticing disappearance of ascites and edema, as well as a decrease in the size of the spleen. All the survivors on placebo continue to show clinical deterioration. In contrast to the usual drop of serum albumin seen in the cirrhotic patients, those receiving colchicine increased and maintained their serum albumin levels throughout the study. Serum proline values were elevated only in the alcohol cirrhotic patients. Serum alkaline phosphatase increased only in those patients receiving colchicine. The results indicate that in some cases, liver fibrosis could be modified by treatment with antifibrotic drugs. The use of colchicine at present should remain within controlled studies.

Adult

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism.

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [14C]leucine into protein and had a decreased uptake of [14C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would be consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [14C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.

Animals

The role of microtubules in chick blastoderm expansion--a quantitative study using colchicine.

Since their discovery, cytoplasmic microtubules have been much studied in the context of cell movement and cell shape change. Much of the work has used drugs, particularly colchicine and its relatives, which break down microtubules- the so-called anti-tubulins. Colchicine and its relatives, which break down microtubules- the so-called anti-tubulins. Colchicine inhibits the orientated movements of many cell types in vitro, and disrupts cell shape change in several morphogenetic situations. The investigatiion reported here used chick blastoderm expansion in New culture in an attempt to quantify the colchicine effect on orientated cell movement. However, although colchicine could halt blastoderm expansion entirely, a simple interpretation was not possible. (1) Colchicine at concentrations capable of blocking mitosis, and of disrupting all or most of the cytoplasmic microtubules of the cells studied, inhibited blastoderm expansion, often resulting in an overall retraction of the cell sheet. (2) Though blastoderm expansion does normally involve considerable cell proliferation, the colchicine effect could not be ascribed to a block on cell division since aminopterin, which stops cell division without affecting microtubules, did not inhibit expansion. (3) Blastoderm expansion is effected by the locomotion of a specialized band of edge cells at the blastoderm periphery. These are the only cells normally attached to the vitelline membrane - the substrate for expansion. When most of the blastoderm was excised, leaving the band of edge cells, and the cultures then treated with colchicine, expansion occurred normally. The colchicine effect on blastoderm expansion could not therefore be ascribed to a direct effect on the edge cells. (4) An alternative site of action of the drug is the remaining cells of the blastoderm. These normally become progressively flatter as expansion proceeds. If flattening in these cells is even partially dependent on their cytoplasmic microtubules, disruption of these microtubules might result in the inherent contractility of the cells resisting and eventually halting edge cell migration. That cell shape in these cells is dependent on microtubules was demonstrated by treating flat blastoderm fragments with colchicine. On incubation, the area occupied by these fragments decreased by 25-30% more than controls. The significance of these results in the general context of orientated cell movements and cell shape determination is discussed, with particular emphasis on the analogous system of Fundulus epiboly.

Aminopterin

Colchicine uptake and binding by human platelets.

The uptake and binding of antimitotic alkaloid colchicine has been demonstrated in washed preparations of human pletelets. A silicone oil technique was adapted so that both uptake and binding of 14C-colchicine were examined in the same platelet preparations. The time dependence and amount of colchicine taken up and bound by different pletelet preparations during a 90 to 120 min incubation period were highly reproducible. Both colchicine uptake and binding by intact platelets, and colchicine binding by preparations of lysed platelets were specific and temperature dependent. Colchicine uptake was slowly reversible. Magnesium and GTP enhanced colchicine binding by lysed platelet preparations but calcium decreased binding. exposure of platelets to either cold (4 degrees C) or to thrombin, which disrupt platelet microtubules, produced significant increases in colchicine uptake and binding. The thrombin effect was maximal at 37 degrees C and resulted in a greater increase in uptake and binding than that produced by either cold treatment alone or, by cold treatment followed by incubation with thrombin at 37 degrees C. The amount of increase in uptake and binding produced by thrombin was independent of both thrombin (1--5 Units/10(9) platelets) and colchicine concentrations (1--50 X 10(-6) M). It is postulated that thrombin may initiate the formation, or make available, colchicine binding sites (microtubule subunits) within platelets.

Blood Platelets

Effects of colchicine on cyclic AMP levels in human leukocytes.

The increase in human leukocyte adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels seen in response to various substances was markedly potentiated by colchicine and other agents that affect microtubule assembly. Addition of dl-isoproterenol (2 muM) or prostaglandin E(1) (10 muM), together with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM), caused a much greater increase in cyclic AMP in colchicine-pretreated cells than in control cells. With isoproterenol (2 muM) plus isobutylmethylaxanthine (1 mM), cyclic AMP levels rose about 3-fold but, in combination with colchicine, these drugs caused a more than 15-fold increase in cyclic AMP. The effects of colchicine were both time- and dose-dependent; they could be seen within 1 min after addition of colchicine or at concentrations as low as 10 nM. In addition to its potentiation of hormonally induced increases in cyclic AMP levels, colchicine also potentiated the effect of isobutylmethylxanthine alone on leukocyte cyclic AMP levels. Vinblastine, vincristine, podophyllotoxin, and oncodazole all had effects similar to those of colchicine but lumicolchicine did not. The data suggest that cytoplasmic microtubules interact with the leukocyte plasma membrane to impose constraints on the expression of hormone-sensitive adenylate cyclase; the therapeutic effects of colchicine may depend in part upon the relaxation of such constraints. Moreover, the synergism described here between colchicine-like agents and hormones is of potential therapeutic importance in clinical conditions in which either alkaloid or hormone has been useful separately.

Colchicine