Proliferative activity of endotheliocytes of growing corneal capillaries in the rabbit

The intensity of capillaries growth in the rabbit's cornea was investigated after chemical burn and exposure to colchicine. Proliferative activity was determined in endothelial cells along the course of newly formed capillaries. Growth intensity proved to be 2 times higher when burn and colchicine effects were combined, than in case of burn alone. The increase in the proliferative activity of endothelial cells and the peculiarities of their arrangement were found in both cases. The most active proliferation was observed in cells located in a zone where capillaries originated from intact vessels of the limb. The process of new vessel formation depends on endotheliocyte shift from this zone towards the site where growth-stimulating factors were applied.     

Influence of colchicine on pulmonary silicotic fibrogenesis in rats

With an aim to evaluate the antifibrotic action of colchicine in experimental model of pulmonary silicosis, the effect of colchicine on developing and developed pulmonary silicosis induced by quartz was studied in rats in vivo and on alveolar macrophages exposed to quartz particulates in vitro. A progressive increase in wet and dry weight of lungs exposed to quartz dust alone, and quartz dust and colchicine injected orally was investigated. An increase in collagen contents, with lapse in time, in animals exposed intratracheally to quartz dust, or exposed similarly to quartz dust but receiving colchicine simultaneously through oral route was observed. A blindfold evaluation of histological sections of lungs of silicotic animals with or without colchicine administration during development of lesions did not reveal any difference between two groups of silicotic rats. Administration of colchicine for 4 weeks after the lesions were developed neither inhibited nor retarded the laying down of collagen. The studies were extended to investigate the effect of colchicine on quartz-induced alveolar macrophage cytotoxicity. The presence of varying concentrations of colchicine in the culture medium did not significantly alter cytotoxic potential of quartz. The results reveal that colchicine administration during the development of and on developed silicosis does not significantly alter pathogenesis of silicotic lesions. At the cellular level colchicine does not modulate quartz-induced alveolar macrophage cytotoxicity, believed to be a significant event for the onset of pulmonary silicotic fibrogenesis.     

The effect of pH on colchicine conformation and structure.

The effect of various pH values between 0 and 14 on the structure and conformation of colchicine was examined using UV-vis spectrophotometry at a concentration of 1.7 x 10(-5) M and NMR techniques at a colchicine concentration of 0.1M. The complete interpretation of the colchicine NMR spectrum in D2O is given. A stable structure of the colchicine molecule in aqueous solutions at pH from 2 to 12 was demonstrated. However, during incubation at 40 degrees C colchicine was found to be stable only at pH values between 2 and 10. The significance of these data for reactions of cholchicine in regard to metabolism and interaction with macromolecules is discussed.     

P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries

A significant proportion of P-glycoprotein (P-gp) and caveolin was co-localized in caveolae isolated from resistant (CH(R)C5) cells overexpressing P-gp and from drug-sensitive Chinese hamster ovary cells (AuxB1). The proportion of P-gp and caveolin associated with caveolar microdomains was higher in CH(R)C5 cells grown in the presence of P-gp substrates (cyclosporin A or colchicine) than in untreated CH(R)C5 cells. Coimmunoprecipitation of P-gp and caveolin from CH(R)C5 lysates suggests that there is a physical interaction between them. Furthermore, co-localization of P-gp and caveolin was found in caveolae from brain capillaries, indicating that this association also takes place in vivo.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

A voltage-clamp study of the effects of colchicine on the squid giant axon

The effects of colchicine applied inside a squid giant axon were studied using voltage-clamp and internal perfusion techniques. It was found that colchicine selectively and reversibly suppresses the sodium conductance during excitation. The possible involvement of the microtubular structure in the functioning of the excitable channel is discussed.     

Colchicine after-effects on the absorption and utilisation of phosphates and nitrates by mycelial felts of Cunninghamella sp.

Summary Colchicine at 5 and 10 p.p.m. increased both phosphorus uptake and incorporation into organic forms (nucleoproteins or other simpler organophosphorus compounds). Continuous supply of colchicine at 20 p.p.m., almost checked phosphorus uptake during the second 24 hours of the experiment.The absorbed nitrates were utilised through the classical reduction steps. It appears also that colchicine had an inhibitory effect on the nitrite reductase that increased by increasing the concentration of the drug. Continuous supply of colchicine at its highest concentration (20 p.p.m.) checked completely the protein building during the second 24 hours of the experiment, though nitrate absorption continued; a phenomenon that caused the accumulation, in the tissue medium systems, of large amounts of peptide nitrogen.The mechanism of nitrate reduction as affected by colchicine treatment was fully discussed.     

Biochemical aspects of colchicine action on meiotic cells

Summary The effect of colchicine on various metabolic processes in meiotic cells was analyzed in order to clarify the mechanism by which the drug reduces chiasma frequency. Synthesis of DNA, RNA, and protein was either unaffected or slightly inhibited during the early stages of meiotic prophase. Such inhibition as did occur could not be related to the achiasmatic effect. DNA synthesis during the pachytene stage was very much reduced in achiasmatic cells. Such reduction appeared to be a consequence and not a cause of the achiasmatic condition. The principal biochemical effect of colchicine was in the reduction of a membrane associated DNA-binding protein which normally appears during meiotic prophase. A basis for this effect was found in the presence of a colchicine binding protein in the nuclear membrane which is distinguishable from its cytoplasmic counterpart by several physicochemical criteria.Supported by a grant from the National Science Foundation, GB 29562 and by a grant from the National Institutes of Health, USPHS HD 03015.     

Effects of colchicine on the morphology and prolactin secretion of rat anterior pituitary cells in monolayer culture

The effects of incubation of rat anterior pituitary cells in monolayer culture with 10(-6) M colchicine have been investigated during time-intervals extending from 1 to 96 hours. Prolactin release, as measured by radioimmunoassay, was rapidly inhibited by colchicine, this inhibition being accompanied by increased cellular prolactin content for up to 24 hours of treatment and followed by decreased values of cellular prolactin concentration at later time-intervals. Immunocytochemical localization showed an increased positive reaction for prolactin up to 24 hours after colchicine treatment, whereas transmission electron microscopy demonstrated, in parallel, an increased number of intracellular prolactin secretory granules during the same interval. Longer periods of treatment (24-96 hours) resulted in the appearance of more lysosomes, autophagic vacuoles and microfilaments in the cells, whereas the number of Golgi elements was decreased. Following four hours of colchicine treatment and at later stages, microtubules could no longer be observed in the sections. Scanning electron microscopic data showed that colchicine treatment induced dramatic changes in the cell surface morphology: at short time intervals (4 and 8 hours), the number of microvilli decreased and the cell surface became folded, whereas, later, "bleb"-like protrusions of variable dimensions partially covered the cell surface and seemed to be released from it. These data show a good correlation between secretory activity of prolactin-producing cells and morphological changes induced by colchicine treatment.     

Microtubule proteins in axolotl eggs and developing embryos

Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity.     

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.     

Effect of colchicine on estrogen action. II. Translocation of 17 beta-estradiol cytosol receptor complex into the nucleus

Colchicine has previously been shown in our laboratory to inhibit 17 beta-estradiol stimulation of uterine water uptake in the immature rat measured 6 h after administration of the agents. We sought to determine whether this effect was mediated through colchicine action on translocation of estradiol receptor complex into the uterine cell nucleus. The time course of estradiol effect on uterine water uptake was followed with and without concurrent colchicine administration up to 6 h after administration. At no time during this period did there appear to be any influence of colchicine on translocation of the estradiol receptor complex into the nucleus. Examination of physical chemical characteristics of the nuclear estradiol receptr complex after estradiol and estradiol plus colchicine treatments revealed no observable differences. Thus, colchicine inhibition of estradiol-stimulated uterine water retention does not appear to be mediated through inhibition of nuclear translocation of estradiol-receptor complex nor to be due to any reduced retention time of estradiol-receptor complex in uterine nuclei.     

Stress-induced blocking of cell division in the colchicine arrest technique

Previous studies have shown that procedures commonly used in studies of cell population kinetics in laboratory animals cause a transient block of the entrance of cells into mitosis. The purpose of the present study was, therefore, to examine whether the handling of animals in conjunction with the administration of colchicine affects the results obtained by the metaphase arrest technique. Groups of rats were injected with colchicine or saline at 1300 h and sacrificed at regular intervals during the following 140 min. Histological sections of the palatal mucosa were produced and the number of metaphases was assessed in the epithelium. In both the saline-treated and colchicine-treated groups the numbers of metaphases decreased immediately after injection and reached a minimum within 30 min. After that time a transient increase was observed in the saline group, whereas the number of metaphases in the colchicine-treated group increased continuously during the experimental period. These results indicate that colchicine takes effect after a delay period of 30 min and that the handling procedures in conjunction with the administration of colchicine provoke a transient block of the entry of cells into mitosis. The second part of the study was designed in such a way that the number of metaphases collected during a 2 h period under the influence of stress induced by injection of colchicine could be compared with the number of metaphases collected during the same time period without the influence of stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidences for direct involvement of microtubules in cleavage furrow formation in newt eggs

This paper aims at examining the effect of colchicine, a microtubular poison, on the process of furrow formation in whole eggs and egg fragments as well as the process of artificial induction of furrow-like dents, in eggs of the newt, Cynops pyrrhogaster. To apply colchicine locally to eggs, the eggs were slit across or along a furrow in a colchicine solution during first cleavage. When a slit was made across or in front of a growing furrow at the onset of its growth, the furrow quickly ceased growing and often regressed. Cortices containing an entire growing furrow were isolated along with a thin layer of subcortical cytoplasm immediately after the start of the first cleavage. Furrows in the cortices degenerated when the cortices were cultured in a colchicine solution, whereas they continued growing when they were cultured in Holtfreter's saline. Furrow-inducing cytoplasm was injected to a site beneath the cortex in the animal half of the egg during first cleavage. When a small slit was made close to the site of the injection in a colchicine solution, no furrow-like dent was induced. These results imply that microtubules are directly involved in the generation and growth of cleavage furrows.     

Effects of colchicine and cycloheximide on the functional and non-functional lipoprotein lipase fractions of rat heart.

In response to food deprivation, total myocardial lipoprotein lipase activity increased gradually over a period of 9 h. Although lipoprotein lipase exists in a functional and non-functional form in the myocardium, most of the increas in activity occurred in the functional (heparin-releasable) lipoprotein lipase fraction. The administration of colchicine, while having no effect on the increase seen in total lipoprotein lipase activity, did inhibit the increase in the functional fraction, while at the same time, caused a marked rise in the activity of the non-functional (non-releasable) fraction. In rats injected with colchicine after a 24-h fast, total lipoprotein lipase activity was not affected, but activity levels in the functional fraction declined while that in the non-functional fraction increased. These results suggest that the functional lipoprotein lipase is constantly being formed in sites not readily accessible to heparin (presumably the myocardial cells) and transported to its site of action, the surface of the endothelial cells of the capillaries. Cycloheximide administration to rats starved for 24 h caused a decline in activity in both the functional (half-life of about 2 h) and the non-functional (half-life of about 4 h) lipoprotein lipase fractions. These results suggest that the functional and non-functional lipoprotein lipase fractions may correspond to two distinct enzyme species.     

Impact of colchicine application during callus induction and shoot regeneration on micropropagation and polyploidisation rates in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Grasses from the genus Miscanthus have several characteristics that make them very favourable crops for efficient, low input, multifunctional and environmentally friendly biomass production. This study is aimed to improve a polyploidisation method to effectively induce polyploids in Miscanthus sinensis and Miscanthus x giganteus. Colchicine was applied for 2, 4 or 7 d in micropropagation systems using inflorescence segments at two different points: during callus induction (313 and 626 μM colchicine) and during shoot regeneration from callus (313 μM colchicine). Among the tested combinations, the most effective (up to 40%) was the 4-d colchicine treatment of a shoot-forming callus cultured 4 d before the experiment on regeneration medium under light conditions. In vitro colchicine treatment during callus induction and during shoot regeneration from callus resulted in no chimeric polyploids as well as a very low number of albinos (2.5%). Additionally, some combinations using colchicine did not significantly reduce the rates of micropropagation effectiveness. The obtained material is promising for the creation of new high-biomass-yielding forms in the Miscanthus genus. In all genotypes tested, chromosome doubling significantly increased pollen stainability. According to preliminary results, induced tetraploids are fertile and useful in hybrid production. Leaves of polyploid forms of two genotypes demonstrated significantly greater width in comparison to the controls.     

P-glycoprotein, glutathione and glutathione S-transferase increase in a colon carcinoma

The acquisition of resistance to anticancer agents used in chemotherapy is the main cause of treatment failure in malignant disorders, provoking tumours to become resistant during treatment, although they initially respond to it. The main multidrug resistance (MDR) mechanism in tumour cells is the expression of P-gly-coprotein (P-gly), that acts as an ATP-dependent active efflux pump of chemotherapeutic agents. Furthermore, an increased detoxification of compounds mediated by high levels of glutathione (GSH) and glutathione S-transferase (GST), has been found in resistant cells. We developed a study aiming to evaluate the evolution of the main drug resistance markers in tumour cells: P-gly, GSH and GST, during the acquisition of resistance to colchicine, for the purpose of studying the adaptation process and its contribution to the MDR phenomenon. A human colon adenocarcinoma cell line was exposed to colchicine during 82 days, being P-gly, GSH levels and GST activity evaluated by flow cytometry, spectrofluorimetry and spectrophotometry, during exposure time. P-gly and GSH levels increased gradually during the exposure to colchicine, reaching 2.35 and 3.21 fold each. On day 82, GST activity increased 1.84 fold at the end of the exposure period. Moreover, an increment in drug cross-resistance was obtained that ranges from 2.62 to 5.22 fold for colchicine, vinblastine, vincristine and mitomycin C. The increments obtained in P-gly, GSH and GST could probably contribute to the MDR phenomenon in this human colon adenocarcinoma cell line.     

An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene

Multidrug resistance in human cells results from increased expression of the mdr1 (P-glycoprotein) gene. Although the same gene is activated in cells selected with different drugs, multidrug-resistant cell lines can be preferentially resistant to their selecting agent. The mdr1 cDNA sequence from vinblastine-selected KB cells, which are uniformly resistant to different lipophilic drugs, was compared with the corresponding sequence from colchicine-selected KB cells preferentially resistant to colchicine. These sequences differ at three positions, resulting in a single amino acid change in P-glycoprotein. These differences result from mutations that occurred during colchicine selection. The appearance of these mutations coincides with the emergence of preferential resistance to colchicine. We have constructed biologically active mdr1 cDNA clones that express either wild-type or mutant P-glycoprotein. Multi-drug-resistant transfectants obtained with the mutant sequence were characterized by increased relative resistance to colchicine compared with transfectants obtained with wild-type sequence. mdr1 mutations are therefore responsible for preferential resistance to colchicine in multidrug-resistant KB cells.     

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.