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A 70 year old male developed marrow aplasia and pancytopenia after receiving 10 mg of colchicine intravenously over a period of 5 days for acute gouty arthritis. Despite a fatal outcome, evidence of recovery of hematopoiesis was observed in the bone marrow 3 days after the last dose of the drug. The pathogenetic mechanism by which colchicine induced marrow aplasia in this patient remains unclear.
In the rat tibial bone marrow cavity, following colchicine injection, there is a phase of osteogenesis in which bone trabeculae replace the necrotic bone marrow tissues and fill the marrow cavity. The newly formed bone is subsequently resorbed by osteoclasts and normal bone marrow is restored. In this study, we correlated these morphologic events with the pattern of gene expression of bone sialoprotein (BSP), an extracellular matrix protein in mineralized tissues, to elucidate the possible functions of BSP in bone formation and resorption in vivo. The expressions of osteopontin (OPN) and type I collagen were also examined. Northern hybridization of the tibia demonstrated that OPN mRNA was gradually increased and expressed at a maximal level 10 days after colchicine injection (during the bone resorption process), while BSP mRNA expression already reached a maximal level at day 6 (during the initial process of bone formation). Its expression was, thus, quite temporary at the beginning of bone formation and different from that of type I collagen, which was continually elevated from days 6 to 10. In situ hybridization of the newly formed bone induced in the tibia revealed that BSP mRNA was evenly expressed in most osteoblasts and osteocytes, moreover in interconnecting colonies of spindle-shaped cells, possibly preosteoblasts, at day 6. At day 10, however, its expression became restricted to some cells on the bone surfaces, some osteoblasts, and most osteoclasts. These observations suggest that BSP may play an important role mainly in the initiation of bone formation and is also associated with the functions of osteoclast in vivo.
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.
Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.
The expression of the c-jun, c-fos, and NGFI-A genes was studied in the rabbit retina after optic nerve crush (ONC) or an intravitreal injection of colchicine. By Northern blotting, the basal expression of c-fos and NGFI-A mRNAs were undetectable, whereas c-jun mRNA showed a low basal expression in sham-operated control retinas. Very few or no Jun- or Fos-immunoreactive nuclei were seen in control retinas. From 1 to 95 days after ONC a marked induction of JUN- but not FOS-immunoreactive neurons was seen in the ganglion cell layer peaking at 3 and 7 days. Jun-positive neurons also accumulated immunoreactive phosphorylated neurofilaments, indicating that they were ganglion cells. Northern blots demonstrated that retinal levels of c-jun mRNA, but not of c-fos or NGFI-A mRNAs, were increased 3 and 7 days after ONC. An intravitreal injection of colchicine also induced Jun-immunoreactivity within 24 hr in most of the neurons in the ganglion cell layer, but not in the inner nuclear and outer nuclear layers. The results indicate that axonal damage induces a specific pattern of IEG expression including a long-term induction of the c-jun gene in CNS neurons.
Several microorganisms were screened for their ability to biotransform colchicine, and two were selected for preparative scale fermentations. Streptomyces spectabilis and Streptomyces griseus both produced O2-demethylcolchicine and O3-demethylcolchicine but in different amounts. The 13C-NMR assignments of colchicine, O10-demethylcolchicine, and trimethylcolchicinic acid are reported and were used to help identify the structures of the metabolites.
Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.
Carbon tetrachloride inhalation in phenobarbitone treated rats caused a rapid rise in hepatic prolyl hydroxylase activity which was followed by an increase in hepatic collagen and free proline concentrations. Colchicine in a dose of 5 micrograms/day largely prevented the increase in hepatic collagen. This effect was not mediated by impairment of prolyl hydroxylase activity. Colchicine is of potential therapeutic value in preventing the progression of chronic liver disease to cirrhosis.
The posttranslational removal and readdition of tyrosine at the C-terminus of alpha-tubulin is associated with generation of microtubule populations that differ in intracellular distributions, turnover rates, and sensitivities to microtubule-depolymerization agents. Here, we compared the in vitro assembly and colchicine binding characteristics of tubulin dimer preparations composed of alpha-tubulin that had been maximally tyrosinated (approximately 40% tyrosinated) by tubulin-tyrosine ligase and maximally detyrosinated (100% detyrosinated) by carboxypeptidase A. Maximally tyrosinated and detyrosinated tubulins had similar critical concentrations for polymerization and similar association constants for colchicine binding. Microtubules polymerized from the two tubulins also had similar steady-state mean lengths and length distributions. The growing and shortening dynamics (dynamic instability parameters) of individual microtubules made from maximally tyrosinated or detyrosinated alpha-tubulin as determined by video-enhanced dark-field microscopy were similar, but subtle differences in the growing and shortening rates were found. On balance, however, the dynamicity and thus the overall kinetic stability of the two microtubule populations were indistinguishable. The results support the idea that detyrosination of alpha-tubulin does not by itself generate stable microtubules.
alpha-Tocopherol, a lipid-soluble chain-breaking antioxidant, is important in the protection of biologic membranes and is transported in the body in association with lipids. It has been estimated that the liver contains up to 29% of the alpha-tocopherol stores of the body. Piperonyl butoxide (PIP) is a widely used synthetic methylenedioxyphenyl insecticide synergist. Bile was collected for 1 hr prior to, and 5 hr following, i.p. injection of 1 g/kg PIP. Within 3 hr of PIP administration, biliary alpha-tocopherol output increased to 132% of pretreatment measurements. Hepatic alpha-tocopherol levels of PIP-treated rats decreased to 26% below those of controls at the end of the bile collection period. Biliary bile acid and phospholipid output also increased significantly following PIP treatment. In a second set of experiments, rats were pretreated with either colchicine or one of two doses of vinblastine; both colchicine and the higher dose of vinblastine prevented the PIP-induced decrease in hepatic alpha-tocopherol and the increase in biliary output of alpha-tocopherol, bile acids, and phospholipids. These results suggest that (i) PIP-induced biliary secretion of alpha-tocopherol is associated with altered biliary phospholipid output, (ii) a significant portion of hepatic alpha-tocopherol may function as a highly mobilizable source of antioxidant for possible rapid enterohepatic distribution, and (iii) microtubules may be involved in this process.
To date, only one group has reported its clinical experience with colchicine therapy. This study included a randomized clinical trial of 22 patients and an open study of 53 patients. The beneficial effect of 1 mg colchicine daily for five days a week has been promising but not convincing. Corticosteroid therapy for the treatment of alcoholic hepatitis was evaluated in nine short-term randomized clinical trials that included 150 treated and 161 control patients with a mortality rate of 37 and 44%, respectively. In these studies, encephalopathy was present in 80 treated and 76 control patients with a mortality rate of 59 and 74%, respectively. None of these differences is significant. The studies did document that patients with mild disease require no specific therapy besides abstinence and general supportive measures; those with severe disease showed a trend toward better survival on corticosteroid therapy. This trend was strong in the earlier publications, but was absent in the most recent studies, which included the largest numbers of patients.
In murine colonic epithelial cells, cell-coat glycoproteins are transported to the cell surface in vesicles that originate at the Golgi apparatus. To determine the role of microtubules in the movement of these vesicles the antimicrotubule agent colchicine was injected into mice at several time intervals prior to sacrifice. In the mice that were treated with colchicine for 4.5 h it was observed that the polarity of the cells was disturbed. The Golgi apparatus and nucleus often appeared interchanged in their positions. The glycoprotein-containing vesicles, normally located apically, were sparse in that location, but abundant near the lateral plasma membranes of the cells at the level of the nucleus and Golgi apparatus. Straining by the periodic acid-chromic acid-silver methenamine technique for glycoproteins clearly revealed the reduction of vesicles apically and accumulation of vesicles laterally. The mechanism responsible for the movement of the vesicles to this location is unclear. It is suggested that the accumulation of vesicles in the lateral region may reflect some hindrance in the fusion of the vesicles with the lateral cell membranes.
Small doses (3.5 micrograms and 7 micrograms) of colchicine injected intracisternally caused an interruption of transport of secretory material from the supraoptic and paraventricular nuclei of the hypothalamus to the neural lobe of the pituitary gland. Transport was assessed by direct measurement of the incorporation of [35S] cysteine into neurophysins, by radioimmunoassay of accumulated material in discrete areas of the system and by immunocytochemistry. The larger dose (7 micrograms) switched off transport completely during the first 24h but the system began to recover within three to four days. Colchicine had little, if any, effect on synthesis; comparison of the relationships of the apparent amounts of immunoreactive neurophysins and immunoreactive hormones in the arrested product led to the conclusion that processing of the hormone precursors continues within secretory granules which accumulated in the perikarya.
Tissues slices superfused with medium containing no ACTH released only traces of corticosterone. Addition of ACTH to the medium caused the rate of corticosterone release to increase to a maximum about 45 min after the addition of ACTH, after which time it either remained constant or started to wane slightly. The rate of release was affected by tissue thickness; the maximum rate of corticosterone release occurred when the tissue slices were 200 microns. Stimulated adrenocortical cells had large spherical nuclei, numerous mitochondria with tubular cristae, numerous lipid droplets, and a large amount of smooth endoplasmic reticulum. Many cells had an extensive network of microfilaments adjacent to the plasma membrane and some microtubules. Prolonged superfusion caused degenerative changes in some of the cells. Both cytochalasin B and cytochalasin D, dissolved in DMSO before addition to the superfusion medium, inhibited the corticotropic responsiveness in a dose-dependent manner. Control tissue samples superfused with medium containing DMSO, but no ACTH and no cytochalasin, released significantly more corticosterone than corresponding unstimulated samples. Few or no microfilaments were observed in adrenocortical cells after treatment with cytochalasin. Neither colchicine nor vinblastine had any discernible effect on the corticotropic responsiveness. After treatment with colchicine, adrenocortical cells had an ultrastructure characteristic of inner zone stimulated cells except that they were mainly devoid of microtubules.
Magnocellular neurones in the supraoptic nucleus of the homozygous Brattleboro rat, which are unable to produce vasopressin, were investigated by immunocytochemistry to identify both the oxytocin cells and the abnormal neurones, which in normal animals would produce vasopressin. The abnormal cell profiles were significantly more rounded than those of the oxytocin cells. Both cell types showed evidence of hyperactivity, but the Golgi apparatus was more extensive in the oxytocin cells, probably as a result of the failure of the abnormal cells to produce vasopressin and its neurophysin and the resultant reduction in hormone packaging. Neurosecretory granules (NSG) 160 nm in diameter were found in the oxytocin perikarya but were absent from the abnormal cell bodies. In addition, a population of small dense granules (SDG) 100 nm in diameter was observed in both types of neurone, in numbers equal to the NSG in oxytocin cells. Injection of a low, non-lethal dose of the axonal transport inhibitor colchicine resulted in a rapid and equal accumulation of both NSG and SDG in oxytocin perikarya and of SDG in the abnormal perikarya after one day. The effects of colchicine were reversed 2-3 days after administration. The SDG, which may contain a co-transmitter or co-hormone substance, are thus produced at a similar rate to NSG, and appear to be transported from the perikarya for subsequent release at the nerve endings.
Recent ultrastructural studies have revealed that differences exist in endothelial cell shape and cytoskeletal architecture between the arterial and ventricular faces of developing semilunar valves. In the present work we analyzed the morphologic response of the valvular endothelial cells of chick embryos to colchicine by light microscopy, scanning electron microscopy and transmission electron microscopy. The results show that colchicine administration during the stages of valve morphogenesis causes a very conspicuous disruption of the endothelial layer of the arterial face of the valves. The cells appear rounded and show massive surface blebbing. These alterations were not present in the endothelial cells on the ventricular face of the valves at the same stages. On the basis of these results we suggest that a difference in the degree of cell differentiation exists between the endothelial cells of the arterial and ventricular faces of the cusps and that this difference may have morphogenetic significance.
The function of the microtubules that are present in the villus enterocytes of the mammalian small intestine is virtually unknown. In order to advance our knowledge about enterocyte microtubules, a quantitative ultrastructural comparison was carried out on enterocytes from rats injected intraperitoneally with colchicine (0.5 mg/100 g body weight) in saline and from rats injected with saline alone. Our morphometric and stereologic study demonstrates that colchicine treatment results in 1) an absolute decrease in microtubules, 2) a reduction in microvilli, essentially in length, 3) an increased thickness of the terminal web, 4) an increase in total lysosomal volume, apparently by an increased number of smaller lysosomes, and 5) a decrease in the number of Golgi lamellae. These results along with those from other studies suggest to us that enterocyte microtubules are involved in the biogenesis of microvillus plasma membrane. Our morphometric data from the saline-treated rats essentially agree with comparable data from other studies. However, comparison with comparable data from hamster enterocytes demonstrates species differences.
Embryonic mouse radii were cultured in the presence of colchicine or vinblastine and subsequently examined in the electron microscope. As a result of the administration of these drugs large numbers of vacuoles accumulated in the cytoplasm of the osteoblasts. The electron-dense contents of at least many of the vacuoles most probably consisted of collagen. This conclusion was based on the results of special staining methods and on the similarity of the vacuoles to collagen vacuoles described in the literature. The observations made in this study provide morphological evidence that colchicine and vinblastine disturb the secretion of collagen.