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PubMed1983

Copolymerization of tubulin-colchicine complex and unliganded tubulin in a nonmicrotubular polymer.

We have shown previously [Saltarelli, D., & Pantaloni, D. (1982) Biochemistry 21, 2996-3006] that the tubulin-colchicine complex is able to polymerize in vitro into peculiar "curly" polymers, under the solution conditions permitting polymerization of unliganded tubulin into microtubules. Here it is further demonstrated that unliganded tubulin can be incorporated into these "curly" polymers. The partial critical concentration of tubulin-colchicine is decreased upon incorporation of unliganded tubulin into the copolymer. GTP hydrolysis occurs on unliganded tubulin upon incorporation in the copolymer. Tubulin-podophyllotoxin does not copolymerize with tubulin-colchicine to form a large polymer but interacts with it, preventing tubulin-colchicine polymerization. The data have been analyzed within a model of random copolymerization of unliganded tubulin and tubulin-colchicine into "curly" polymers. A corollary is that unliganded tubulin is virtually able to self-assemble into curly polymers with a critical concentration 10-fold higher than the critical concentration found for microtubule assembly. Consequently, these peculiar tubulin homopolymers cannot be observed except as transients at high concentrations, or when microtubule assembly is inhibited. Kinetic measurements of the T-TC copolymerization process and associated GTP hydrolysis at different T/TC ratios provide supplementary information about some privileged interactions between tubulin and tubulin-colchicine molecules. A comprehensive phase diagram of the various possible polymers formed in the presence of tubulin and tubulin-colchicine is presented.

Animals↗

PubMed1990

Effect of tubulin binding and self-association on the near-ultraviolet circular dichroic spectra of colchicine and analogues.

Near-UV circular dichroic (CD) spectra of three colchicine analogues that differ at the C-10 position have been obtained in the presence and absence of tubulin. All three colchicine analogues show dramatic alterations in the low-energy near-UV CD band upon tubulin binding that cannot be mimicked by solvent, but in no event does the rotational strength of the CD band decrease to nearly zero as in the case of colchicine [Detrich, H. W., III, Williams, R. C., Jr., Macdonald, T. L., & Puett, D. (1981) Biochemistry 20, 5999-6005]. The effect of self-association of colchicine and one of the C-10 analogues, thiocolchicine, on the near-UV CD band was also investigated. A qualitative similarity was seen between the near-UV CD spectra of colchicine and thiocolchicine dimers and the spectra of these molecules bound to tubulin. These observations support the previous suggestion that ligands bound to the colchicine site on tubulin may be interacting with an aromatic amino acid in the colchicine binding site [Hastie, S. B., & Rava, R. P. (1989) J. Am. Chem. Soc. 110, 6993-7001].

Animals↗

PubMed1982

Interaction of tubulin with single ring analogues of colchicine.

Simple analogues of the tropolone and trimethoxyphenyl moieties of colchicine have been used as probes for the colchicine binding site of purified calf brain tubulin. [3H]Tropolone methyl ether was found to bind to one site per tubulin molecule with equilibrium constant of (2.2 +/- 0.2) x 10(3) M-1 at 0 degree C, with the interaction having delta H0app = -8.3 +/- 1.0 kcal mol-1 and delta S0app = -15.2 +/- 3.6 eu. The binding of tropolone methyl ether and colchicine was inhibited by each other. Both tropolone and its methyl ether inhibited tubulin polymerization into microtubules in vitro. N-[3H]Acetylmescaline bound to tubulin with a K congruent to 4 x 10(2) M-1 at 37 degrees C. This interaction was inhibited by colchicine and at lower temperatures was below the sensitivity of the measuring method employed. [14C]Mescaline interacted with higher affinity site(s) not related to the colchicine site. Both mescaline and N-acetylmescaline inhibited partially the microtubule assembly at 10(-3) M concentrations. No linkage was observed between the binding of tropolone methyl ether and N-acetylmescaline. The relatively weak interactions of both the two separate parts of colchicine can account quantitatively for the much tighter binding of the complete drug to tubulin within a proposed model which takes into account the entropic advantage of colchicine as a bifunctional ligand.

Colchicine↗

PubMed1993

Synthesis and tubulin binding of novel C-10 analogues of colchicine.

A series of novel C-10 derivatives of colchicine have been prepared and evaluated for inhibition of in vitro microtubule assembly and of [3H]colchicine binding to tubulin. The C-10 substituent of colchicine was replaced by halogens, alkyl and alkoxy groups, and hydrogen. Many of these compounds are available by nucleophilic substitution of 10-fluoro-10-demethoxycolchicine (9) without concomitant formation of ring contraction products. Compound 9 is prepared by reaction of (diethylamino)sulfur trifluoride with colchiceine. Unlike most reactions of colchiceine, the colchicine rather than the isocolchicine regiosiomer is the predominant product of this reaction. It was found that modification of the C-10 substituent of colchicine had a relatively minor effect on the potency of the colchicinoids. The electronic nature of the substituent had no significant effect on the efficacy of the compound, indicating that hydrogen bonding or polar interactions between the C-10 substituent of colchicinoids and an amino acid in the colchicine binding site on tubulin are not present in the colchicine-tubulin complex. A decrease in activity was observed with increasing length of the alkyl chain bonded to the C-10 position, but potency was less affected when the alkyl groups were positioned in close proximity to the C-10 carbon of the tropone ring. It is concluded that the steric rather than the electronic properties of the C-10 substituent are the predominant determinants of activity in this series.

Binding Sites↗

PubMed1998

Stress and colchicine do not induce the release of galanin from the external zone of the median eminence.

The aim of the present study was to verify the hypothesis that stress exposure modifies the content and release of galanin in the hypothalamic paraventricular nucleus and the median eminence. Colchicine and immobilization served as stress stimuli, and the changes in galanin immunoreactivity were compared with those in corticotropin-releasing hormone and vasopressin. In control animals, a limited number of galanin perikarya were identified in the paraventricular nucleus. The high dose (75 microg) of colchicine enhanced galanin in both parvicellular and magnocellular subdivisions, as analysed 72 h later. In the median eminence, galanin accumulated only in the external zone. High-dose colchicine did not affect galanin, while corticotropin-releasing hormone and vasopressin were depleted from the median eminence. Immobilization (120 min) neither alone nor in combination with colchicine influenced galanin immunoreactivity in the external zone. The low dose of colchicine induced an unexpected accumulation of galanin in the internal zone of the median eminence, which was further increased by subsequent immobilization. In the external zone, low-dose colchicine induced a complete disappearance of vasopressin, substantial depletion of corticotropin-releasing hormone and no changes in galanin immunoreactivity. The present studies demonstrate that galanin in the external zone of the median eminence is not influenced by colchicine or by immobilization stress.

Animals↗

PubMed1993

Colchicine disposition in human leukocytes after single and multiple oral administration.

Inasmuch as leukocytes were reported to be an active pharmacologic compartment, colchicine disposition was determined in plasma, granulocytes, and mononuclear cells in healthy volunteers after 1 mg oral single and multiple doses. After the single dose, maximal colchicine concentration was observed at 1 hour in plasma and 47 hours later in leukocytes. This delay was confirmed by the slow accumulation of colchicine by lymphocytes in culture. In the multiple-dose study, mean granulocyte colchicine concentration (20 to 53 ng/10(9) cells) were twofold higher than in mononuclear cells (9 to 24 ng/10(9) cells). Mean predicted colchicine multiple-dose granulocyte and mononuclear cell concentrations were 2.5-fold and ninefold higher, respectively, than those measured. After the last dose, colchicine decreased, with half-life values between 41 and 46 hours for leukocytes and 49 hours for plasma. This study validates leukocytes as a microcompartment whose kinetics correlates with colchicine biologic effects.

Administration, Oral↗

PubMed1978

Characterization of colchicine-binding activity in Dictyostelium discoideum.

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.

Carrier Proteins↗

PubMed1986

The effect of colchicine on the development of lithocholic acid-induced cholestasis. A study of the role of microtubules in intracellular cholesterol transport.

The pathogenesis of lithocholic acid (LCA-Na)-induced cholestasis involves a rapid accumulation of cholesterol in the bile canalicular membrane. Since microtubules play an important role in the intracellular transport of many materials, including cholesterol, the present study was undertaken to assess the extent to which they participate in the development of LCA-Na-induced cholestasis. Rats were pretreated with either colchicine (0.2 mumol/100 g body wt.) or saline solution 90 min before injection with LCA-Na (12 mumol/100 g body wt.). Colchicine, although not increasing bile flow by itself, significantly reduced the cholestasis caused by LCA-Na (57-32% reduction in bile flow) without affecting its metabolism into less toxic bile acids or its distribution in blood, liver or bile. Bile canalicular membranes isolated from animals treated with a combination of colchicine and LCA-Na contained less cholesterol than those treated with LCA-Na alone. However, membranes obtained from rats treated with colchicine alone contained much less cholesterol than did controls. It was found that the total amount of cholesterol accumulated within the bile canalicular membrane following LCA-Na treatment (LCA-Na + colchicine versus colchicine alone compared with LCA-Na versus controls) was unchanged by colchicine treatment. In view of these findings it is suggested that the total amount of cholesterol present within the bile canalicular membrane determines the extent of LCA-Na-induced cholestasis, LCA-Na probably moves cholesterol to the bile canalicular membrane via a microtubule independent pathway, and microtubules are unlikely to function in the transcellular transport of LCA-Na.

Animals↗

PubMed1995

The phosphorylation state of the microtubule-associated protein tau as affected by glutamate, colchicine and beta-amyloid in primary rat cortical neuronal cultures.

The effects of the excitatory amino acid glutamate, the microtubule destabilizing agent colchicine, and beta 25-35-amyloid peptide on the phosphorylation state of tau were studied in rat cortical neurons in primary culture. Using immunocytochemistry and Western-blot analysis, we demonstrated that a proportion of tau in these cultures is normally highly phosphorylated, but most of this tau fraction is dephosphorylated after treatment of the cultures with glutamate or colchicine, but not with beta-amyloid; the glutamate- and colchicine-induced changes in tau phosphorylation commenced before cell death, as assessed by release of lactate dehydrogenase. Dephosphorylation of tau was readily revealed by using the monoclonal antibodies Tau.1 and AT8, which have phosphate-sensitive epitopes that both centre around serine-199 and -202 (numbering of the largest tau isoform). On Western blots and by immunocytochemistry, AT8 labelling strongly decreased after glutamate and colchicine treatments, whereas Tau.1 staining was more intense. Neurofilament monoclonal antibodies, including RT97, 8D8, SMI31 and SMI310, all additionally known to recognize tau in a phosphorylation-dependent manner, also demonstrated that glutamate and colchicine treatments of the cultures induced a dephosphorylation of tau. We also showed immunocytochemically that there is an increase in tau immunoreactivity in neuronal perikarya in response to glutamate and colchicine treatment, and this occurs concomitantly with the dephosphorylation of tau. Treatment of the primary rat cortical neuronal cultures with beta 25-35-amyloid peptide, under conditions which induce neuronal degeneration, did not induce a change in tau phosphorylation, and failed to act synergistically with glutamate to produce an increase in dephosphorylation of tau over that produced by glutamate treatment alone. These findings demonstrate that glutamate and colchicine induce tau dephosphorylation, as opposed to increased tau phosphorylation, which would be more indicative of Alzheimer-type neurodegeneration.

Amyloid beta-Peptides↗

PubMed1999

Cytochrome c release and caspase-3 activation during colchicine-induced apoptosis of cerebellar granule cells.

The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal populations including cerebellar granule cells (CGCs). In this study we investigated the involvement of cytochrome c release and caspase-3 activation during colchicine-induced CGC apoptosis. Treatment of rat CGCs with 1 micrometer colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II caspases (which includes caspase-3) was demonstrated by the proteolytic degradation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchicine. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alpha-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which was blocked with a specific, peptide-based, aldehyde inhibitor of group II caspases, i. e. DEVD-CHO. We also observed a time-dependent proteolysis of caspase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptosis may be mediated by cytochrome c since there was a close correlation between the time courses of cytochrome c release from the mitochondria and of caspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone.

Amino Acid Chloromethyl Ketones↗

PubMed2002

A randomized controlled trial to evaluate the slow-acting symptom modifying effects of a regimen containing colchicine in a subset of patients with osteoarthritis of the knee.

OBJECTIVE: As crystals may contribute to inflammation in osteoarthritis (OA), it is hypothesized that colchicine may have symptom/disease modifying effects in OA. The objective of this study was to evaluate the symptomatic benefit of addition of colchicine to a regimen of intraarticular steroids and piroxicam in patients with knee OA with inflammation. DESIGN: 39 patients with OA of the knee with persisting inflammation, despite at least 2 weeks of piroxicam, were subjected to intraarticular steroid injection and randomly assigned to receive colchicine 0.5 mg twice daily or placebo in a randomized, double blind, placebo-controlled trial over 5 months. RESULTS: VAS for index knee pain (VAS-pain) and total KGMC score (a modified WOMAC index) at 16 and 20 weeks were significantly better in the colchicine group than the corresponding scores in controls. The benefit persisted on multivariate analysis at 16 weeks (Hotellings T(2)=18.6, F(5,33)=3.3154, P=0.015). The proportion of patients who had 30% or greater response at 16 weeks was significantly higher in the colchicine group in VAS-pain (69% vs 15%) and total KGMC scores (74% vs 45%) and the significance persisted on combined analysis using Mantel-Haenszel test (M-H Risk=5.9, 95% C.I.: 2.08 to 16.73). At 20 weeks, benefit of colchicine therapy was seen on pooled analysis only (M-H risk=3.71, 95% C.I.: 1.07=8.02). CONCLUSION: The addition of colchicine produced significantly greater symptomatic benefit at 16 and 20 weeks than intraarticular steroid and piroxicam alone in patients with knee OA with inflammation.

Analysis of Variance↗

PubMed2002

Improvement of the bioavailability of colchicine in rats by co-administration of D-alpha-tocopherol polyethylene glycol 1000 succinate and a polyethoxylated derivative of 12-hydroxy-stearic acid.

Two surface-active formulation ingredients, a water-soluble derivative of vitamin E (D-alpha-tocopherol polyethylene glycol 1000 succinate, vitamin E-TPGS) as well as a polyethoxylated derivative of 12-hydroxy-stearic acid (Solutol HS 15) were investigated in rats for their potential to increase the oral bioavailability of the p-glycoprotein (p-gp) and cytochrome P450 substrate colchicine. D-alpha-Tocopherol polyethylene glycol 1000 succinate and the polyethoxylated derivative of 12-hydroxy-stearic acid will be referred to as "surfactant 1" and "surfactant 2" in the following. Colchicine was administered to the animals at a dose level of 5 mg/kg in each 10% surfactant containing formulation. A solution of colchicine in isotonic saline was selected as a reference formulation. It was found that the administration of colchicine in the surfactant containing formulations resulted in significantly higher systemic exposures as compared to the aqueous reference vehicle (2-fold increase in AUC in the presence of surfactant 1 and 4-fold increase in AUC in the presence of surfactant 2). The aqueous solubility of colchicine was about 16.7 mg/ml, and the increase in solubility in the presence of 1% surfactant 1 or surfactant 2 to about 20.5 and 18.5 mg/ml was not considered to significantly affect the oral bioavailability. In summary, it was demonstrated that both surfactants are suitable formulation ingredients to improve the systemic exposure of colchicine in the rat. Due to the high aqueous solubility of colchicine the most likely reasons for these findings are inhibition of p-gp and/or metabolism as well as permeability enhancement by interactions of the surfactants with the intestinal membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Colchicine decreases apoptotic cell death in chronic cyclosporine nephrotoxicity.

Colchicine has been shown to prevent kidney injury in chronic cyclosporine nephrotoxicity; however, the mechanisms of its action are undetermined. The purpose of this study was to clarify whether colchicine prevents cyclosporine-induced kidney injury by decreasing kidney-cell apoptosis. We also sought to determine whether such an antiapoptotic effect was related to Bcl-2/Bax protein and caspase3 activity. Adult male Sprague-Dawley rats kept on a salt-depleted diet (0.05% sodium) were treated daily for 28 days with cyclosporine (15 mg/kg in 1 mL/kg olive-oil vehicle), colchicine (30 microg/kg in 100% ethanol, diluted with sterile saline solution to a final concentration of 30 microg/mL), or both cyclosporine and colchicine. Kidney function, histomorphologic findings, in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end-labeling assay, expressions of Bcl-2 and Bax proteins, and caspase-3 enzymatic activity were compared for the different treatment groups. Compared with the vehicle-treated rats, rats given cyclosporine showed a decline in creatinine clearance rate, an increase in serum creatinine concentration, tubulointerstitial fibrosis, and an increase in the number of apoptotic cells (all P <.01). Concomitant administration of colchicine significantly reversed all the above parameters (all P <.05). The decreased expression of Bcl-2 and the ratio of Bcl-2 to Bax protein seen in cyclosporine-treated rat kidneys were significantly increased after colchicine treatment, accompanying a suppression of caspase-3 activity (P <.05). Furthermore, the decreased apoptotic cell death was closely correlated with improved renal tubulointerstitial fibrosis (r = 0.583, P <.05). These findings strongly suggest that a renoprotective effect of colchicine on cyclosporine-induced nephrotoxicity is coassociated with a decrease in apoptotic cells.

Animals↗

PubMed2005

Daily variations in colchicine-induced apoptosis in duodenal crypts.

Apoptotic cell death can be induced by several agents, among them colchicine, a microtubule disrupting-drug that affects continuously renewing cell populations, such as the intestinal crypt enterocytes. The objectives of this investigation were (1) to confirm in vivo colchicines-inductive effect and (2) to determine the existence of 24 h variations in the crypt enterocytes apoptotic indices. The study was done on C3H/S male adult mice housed under standardized conditions. Starting at midnight until the end of a circadian period, subgroups of mice were sacrificed after having been injected with colchicine or saline i.p. 4h beforehand. Duodenal samples were processed for hematoxylin-eosin staining and TUNEL technique. In order to score the number of apoptosis, the longitudinal sections of the crypts were divided into three regions comprised, respectively, of tiers 1-4, 5-12, and 13-20, proceeding from the bottom to the top of the crypt. Values of each lot were expressed as mean +/- SEM. A highly significant statistical difference in apoptotic indices was found for colchicine-treated animals. The 24 h curve for colchicine-induced apoptosis displayed qualitative and quantitative differences compared to other inducer agents. Highest apoptotic indices were found in the deepest crypt regions. Daily variations were observed in all the crypt sectors of the colchicine-treated animals and in tiers 5-12 of the saline controls. The present work demonstrates that the colchicine cytotoxicity due to its apoptotic-inducing effect depends on the dosing time during the 24 h in this mouse strain.

Animals↗

PubMed1999

The effects of colchicine treatment on wound healing in a caustic murine injury model.

OBJECTIVE: The present study determined the effects of colchicine on wound repair in a murine model of dermal chemical injury. METHODS: Standardized 4.15 cm2 circular lesions were induced on the dorsum of adult male mice with NaOH. Five minutes of IN or 3N caused lesions of graded depth. Mice received colchicine (1 mg/kg) or phosphate-buffered saline intraperitoneally in equivalent volumes. Treatment began immediately postinjury and was continued on an alternating day schedule for 14 days. Wound size was measured every third day postinjury. On day 15 postinjury, wounds were histologically graded for depth of injury, degree of fibrosis, inflammatory cell response, and revascularization. All histological determinations and wound measurements were performed in a blinded fashion. RESULTS: All mice had a similar initial wound size and completed the experimental protocol. In dermal wounds of superficial depth, colchicine-treated mice (n = 20) had a larger wound size during days 6 through 9 of the experimental trial when compared to phosphate-buffered saline-treated mice (n = 18). In the deeper wounds, there were no significant differences in wound size between colchicine and phosphate-buffered saline-treated groups. Colchicine (n = 54) did not affect the degree of fibrosis at all depths of injury vs phosphate-buffered saline treatment in mice (n = 55). The degree of wound revascularization was less in colchicine-treated mice (n = 54) than phosphate-buffered saline-treated mice (n = 55). CONCLUSION: Colchicine did not improve wound healing in an alkaline-induced dermal injury.

Animals↗

PubMed1976

Effect of colchicine on rat mast cells.

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

Animals↗

PubMed1982

Microtubules and protein secretion in rat lacrimal glands: localization of short-term effects of colchicine on the secretory process.

The pathway and kinetics of the secretory protein transport in rat lacrimal exorbital gland have been established by an in vitro time-course radioautographic study of pulse-labeled protein secretion. The colchicine-sensitive steps have been localized by using the drug at various times with respect to the pulse labeling of proteins. Colchicine (10 microM) does not block any step of the secretory protein transport, but when introduced before the pulse it decreases the transfer of labeled proteins from the rough endoplasmic reticulum to the Golgi area, suppressing their temporary accumulation in the Golgi area before any alteration of this organelle is detectable. Moreover, colchicine inhibits protein release only from the secretory granules formed in its presence because the peroxidase discharge is diminished 1 h after colchicine addition, and the secretion of newly synthesized proteins is strongly inhibited only when colchicine is introduced before secretory granule formation. Morphometric studies show that there is a great increase of secondary lysosomes, related to crinophagy, as early as 40-50 min after colchicine is added. However, changes in lysosomal enzymatic activities remained biochemically undetectable. We conclude that: (a) the labile microtubular system does not seem indispensable for protein transport in the rough endoplasmic reticulum-Golgi area but may facilitate this step, perhaps by maintaining the spatial organization of this area; and (b) in the lacrimal gland, colchicine inhibits protein release not by acting on the steps of secretion following the secretory granule formation, but by acting chiefly on the steps preceding secretory granule formation, perhaps by making the secretory granules formed in its presence incapable of discharging their content.

Animals↗

PubMed1982

Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells.

The PC12 line of nerve growth factor (NGF)-responsive rat pheochromocytoma cells was used as a model system to determine whether properties of microtubules change during neurite growth and maturation. In the absence of NGF, PC12 cells lack processes. After several days with NGF, PC12 cells begin extending neurites and, by 2-3 wk with NGF, PC12 cells have long (approximately 1 mm), highly branched neurites. We examined the effect of colchicine on microtubules of PC12 cells grown without NGF or with NGF for 1 or 21 d. PC12 cells grown under the various conditions were exposed to 50 microM colchicine for 1 or 6 h, and were then assayed for their content of polymerized tubulin using a biochemical assay. Microtubule levels in drug-treated cultures were compared to those in non-drug-treated control sister cultures. PC12 cells grown without NGF or with NGF for 1 d were depleted of MT by 1 h with colchicine. In contrast, microtubule levels in long-term NGF-treated cells exposed to colchicine for 6 h were reduced to only approximately 57% of those in control cells. Control experiments indicated that the observed differential susceptibility to colchicine was not due to differences in colchicine uptake or to the effects of colchicine on cell viability. These observations suggest that microtubules of PC12 cells grown without NGF or with NGF for 21 d differ in their properties. Such differences may be related to one or more of the changes in structure and/or motility that result from treatment with NGF.

Animals↗