Rabies virus infection of cultured rat sensory neurons.

The axonal transport of rabies virus (challenge virus strain of fixed virus) was studied in differentiated rat embryonic dorsal root ganglion cells. In addition, we observed the attachment of rabies virus to neuronal extensions and virus production by infected neurons. A compartmentalized cell culture system was used, allowing infection and manipulation of neuronal extensions without exposing the neural soma to the virus. The cultures consisted of 60% large neuronal cells whose extensions exhibited neurofilament structures. Rabies virus demonstrated high binding affinity to unmyelinated neurites, as suggested by assays of virus adsorption and immunofluorescence studies. The rate of axoplasmic transport of virus was 12 to 24 mm/day, including the time required for internalization of the virus into neurites. The virus transport could be blocked by cytochalasin B, vinblastine, and colchicine, none of which negatively affected the production of virus in cells once the infection was established. It was concluded that, for the retrograde transfer of rabies virus by neurites from the periphery to the neuronal soma, the integrity of tubulin- and actin-containing structures is essential. The rat sensory neurons were characterized as permissive, moderately susceptible, but low producers of rabies virus. These neurons were capable of harboring rabies virus for long periods of time and able to release virus into the culture medium without showing any morphological alterations. The involvement of sensory neurons in rabies virus pathogenesis, both in viral transport and as a site for persistent viral infection, is discussed.     

Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells.

Multidrug resistant (MDR) cells overexpress a 170-180 kDa membrane glycoprotein, the P-glycoprotein, which is believed to export drugs in an ATP-dependent manner. Plasma membrane vesicles from the MDR CHRC5 cell line, but not the AuxB1 drug-sensitive parent, showed uptake of [3H]colchicine and [3H]vinblastine that was stimulated by the presence of ATP and an ATP-regenerating system. Steady-state uptake of drugs was achieved by 10 min and was stable for greater than 30 min. Non-hydrolysable ATP analogues were unable to support drug uptake, indicating that ATP hydrolysis is essential for transport. ATP-stimulated drug uptake appeared to result from drug transport into inside-out vesicles, since uptake was osmotically sensitive and could be prevented by detergent permeabilization. Steady-state uptake was half-maximal at 100 microM colchicine and 200 nM vinblastine and was inhibited by a 10-100-fold excess of MDR drugs and chemosensitizers, in the order vinblastine greater than verapamil greater than daunomycin greater than colchicine. In addition to being vanadate-sensitive, drug uptake was inhibited by 10-200 microM concentrations of several sulfhydryl-modifying reagents, suggesting that cysteine residues play an important role in drug transport. Vesicular colchicine was rapidly exchanged by an excess of unlabelled drug, demonstrating that drug association is the net result of opposing colchicine fluxes across the membrane.     

Acrylamide and Glycidamide Impair Neurite Outgrowth in Differentiating N1E.115 Neuroblastoma Without Disturbing Rapid Bidirectional Transport of Organelles Observed by Video Microscopy

Abstract: The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene-bisacrylamide were weakly cytotoxic in a ⁵¹Cr-release assay, but only at ≥10 m M (order of efficacy: methylene-bis-acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100-fold lower concentrations, with similar EC₅₀ values for all three toxicants, i.e., acrylamide, 70 ± 15 μ M ; methylene-bis-acrylamide, 92 ± 31 μ M ; glycidamide, 120 ± 30 μ M . Only glycidamide (1 m M ) caused degeneration of established neurites within a period of 48 h. Video-enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of ≤48 h at 1 m M , neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. III. Inhibition by cytochalasins, colchicine, and vinblastine.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages.Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function.Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (<10^?6M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction.These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.     

Effect of colchicine, vinblastine, and cytochalasin B on cell surface anionic sites of Tritrichomonas foetus

Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic site-containing macromolecules located on the cell surface of T. foetus.     

The response of primary cultured adult mouse sensory neurons to ethanol, propanol, acetaldehyde and acrolein treatments

Primary cultures of adult mouse sensory neurons maintained for 8 days in vitro (8 div), in both the presence of non-neuronal cell (NNC) outgrowth and in NNC-reduced cultures, were exposed to doses of ethanol, propanol, acetaldehyde and acrolein. The effects on cell viability were monitored: LD50's of 600 microM acrolein and 100 mM propanol were obtained after 24 h exposures and after 48 h with 1 mM acetaldehyde and 500 mM ethanol. Morphological effects were evident by scanning electron microscopy with sub-acute doses for each agent, using both lower concentrations and shorter exposures. Membrane pitting of the perikaryon and a reduction in the proportion of neurons bearing neurites were common signs of toxic insult. The neurites of treated cells were thicker and more irregular than those of untreated cells; this proved a good indicator of specific neurotoxicity rather than merely a cytotoxic response. Fetal calf serum in the medium lessened the response of neurons to ethanol treatments. Comparison with other in vitro studies suggests these primary cultures are a more sensitive system than established cell lines of neuronal origin for use in neurotoxicity testing.     

The response of primary cultured adult mouse sensory neurons to ethanol,propanol, acetaldehyde and acrolein treatments

Primary cultures of adult mouse sensory neurons maintained for 8 days in vitro (8 div), in both the presence of non-neuronal cell (NNC) outgrowth and in NNC-reduced cultures, were exposed to doses of ethanol, propanol, acetaldehyde and acrolein. The effects on cell viability were monitored: LD50’s of 600 μM acrolein and 100 mM propanol were obtained after 24 h exposures and after 48 h with 1 mM acetaldehyde and 500 mM ethanol. Morphological effects were evident by scanning electron microscopy with sub-acute doses for each agent, using both lower concentrations and shorter exposures. Membrane pitting of the perikaryon and a reduction in the proportion of neurons bearing neurites were common signs of toxic insult. The neurites of treated cells were thicker and more irregular than those of untreated cells; this proved a good indicator of specific neurotoxicity rather than merely a cytotoxic response. Fetal calf serum in the medium lessened the response of neurons to ethanol treatments. Comparison with other in vitro studies suggests these primary cultures are a more sensitive system than established cell lines of neuronal origin for use in neurotoxicity testing.     

Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

Summary The purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.     

Modulation of P-glycoprotein phosphorylation and drug transport by sodium butyrate.

P-Glycoprotein (Pgp) expression in cell lines derived from tumors arising from cells which normally express Pgp can be increased by sodium butyrate and other differentiating agents. Although the Pgp level increased 25-fold after sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of vinblastine, adriamycin, and actinomycin D increased rather than decreased. In contrast, colchicine showed the expected decrease in accumulation, as a result of increased efflux. Likewise, treatment of a Pgp-expressing multidrug-resistant SW620 subline with sodium butyrate resulted in active interference with Pgp function. This effect was partially reversed by phorbol esters with a resulting decrease in the accumulation of vinblastine, adriamycin, and actinomycin D. Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp. Time course studies revealed a tight relationship between decreased Pgp phosphorylation and increased vinblastine accumulation after sodium butyrate treatment. Either treatment with phorbol esters or withdrawal of sodium butyrate increased Pgp phosphorylation while decreasing vinblastine accumulation. These studies suggest that the specificity of Pgp transport can be modulated by phosphorylation and that vinblastine, adriamycin, or actinomycin D transport, but not that of colchicine, is diminished after dephosphorylation by sodium butyrate.     

Microtubules are required for NF-kappaB nuclear translocation in neuroblastoma IMR-32 cells: modulation by zinc

The relevance of a functional cytoskeleton for Nuclear Factor-kappaB (NF-kappaB) nuclear translocation was investigated in neuronal cells, using conditions that led to a disruption of the cytoskeleton [inhibition of tubulin (vinblastine, colchicine), or actin (cytochalasin D) polymerization and zinc deficiency]. We present evidence that an impairment in tubulin polymerization can inhibit the formation of the complex tubulin-dynein-karyopherin alpha-p50 that is required for neuronal retrograde and nuclear NF-kappaB transport. Cells treated with vinblastine, colchicine or cytochalasin D, and zinc deficient cells, all showed a low nuclear NF-kappaB binding activity, and low nuclear concentrations of RelA and p50. The altered nuclear translocation was reflected by a decreased transactivation of NF-kappaB-driven genes. The immunocytochemical characterization of cellular RelA showed that cytoskeleton disruption can lead to an altered distribution of RelA resulting in the formation of peripheral accumuli. These results support the concept that cytoskeleton integrity is necessary for the transport and translocation of NF-kappaB required for synapse to nuclei communication. We suggest that during development, as well as in the adult brain, conditions such as zinc deficiency, that affect the normal structure and function of the cytoskeleton can affect neuronal proliferation, differentiation, and survival by altering NF-kappaB nuclear translocation and subsequent impairment of NF-kappaB-dependent gene regulation.     

The binding of vincristine, vinblastine and colchicine to tubulin

Preparations of tubulin were examined for their ability to bind vincristine, vinblastine, and colchicine, as measured by adsorption on DEAE impregnated filter paper. Vincristine and vinblastine were found to bind very rapidly with tubulin (<5 min), while colchicine took considerably longer (>4 hr). When varying concentrations of the alkaloids were employed, and the data examined on a Scatchard plot, it was found that colchicine had an association constant of 1.8 × 10⁶ liters/mole, while vinblastine and vincristine had constants of 6.0 × 10⁶ liters/mole and 8.0 × 10⁶ liters/mole respectively. In addition, it was found that the ratio of molar binding of colchicine was always twice that of vinblastine or vincristine.     

Effect of Colchicine,Vinblastine, and Cytochalasin B on Cell Surface Anionic Sites of Tritrichomonas foetus1

ABSTRACT. Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.     

Microtubules and pancreatic amylase release by mouse pancreas in vitro

The effects of vinblastine and colchicine on pancreatic acinar cells were studied by use of in vitro mouse pancreatic fragments. Vinblastine inhibited the release of amylase stimulated by bethanechol, caerulein, or ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Inhibition required preincubation with vinblastine,and maximum inhibition was observed after 90 min. Inhibition was relatively irreversible and could not be overcome by a high concentration of stimulant. Inhibition could also be produced by colchicine although longer preincubation was required and inhibition was only partial. Uptake of [3H]vinblastine and [3H]colchicine by pancreatic fragments was measured and found not to be responsible for the slow onset of inhibition by these drugs. In incubated pancreas, microtubules were present primarily in the apical pole of the cell and in association with the Golgi region. Vinblastine, under time and dose conditions that inhibited the release of stimulated amylase, also reduced the number of microtubules. The only other consistent structural effects of vinblastine were the presence of vinblastine- induced crystals and an increased incidence of autophagy. The remainder of cell structure was not affected nor were overall tissue ATP and electrolyte contents or the stimulant-induced increase in 45Ca++ efflux. It is concluded that the antisecretory effects of vinblastine and colchicine are consistent with a microtubular action, but that acinar cell microtubules are more resistant to the drugs than many other cell types.     

DOES COLCHICINE AFFECT AGGREGATION OF NORMAL AND TRANSFORMED BHK CELLS DIFFERENTLY?

The effect of colchicine and vinblastine on cell aggregation was studied, using BHK cells and their transformed derivatives (pyBHK cells). When cells were dissociated with EDTA and the assay was made in a Ca²⁺-containing medium, the aggregation of transformed cells was prevented by colchicine and vinblastine, whereas the aggregation of normal cells was unaffected. When a Ca²⁺-free medium was used for aggregation, neither type of cell was influenced by these drugs. BHK and pyBHK cells, dissociated by trypsin in the presence of Ca²⁺, can aggregate only in the Ca²⁺-containing medium and the aggregation of both cell types was equally prevented by colchicine and vinblastine. Based on these results, it was concluded that colchicine and vinblastine inhibited the Ca²⁺-dependent mechanism of cell adhesion, but not the Ca²⁺-independent one which occurs in the Ca²⁺-free aggregation medium.     

Use of antibodies to probe membrane glycoproteins associated with drug-resistant J774.2 cells

Three drug-resistant sublines of the murine macrophage-like cell line J774.2 were selected in vitro for their ability to grow in high concentrations of either taxol, vinblastine, or colchicine. Each contains a major plasma membrane glycoprotein (130-150 kDa), which is barely seen in the drug-sensitive parental cell line. Polyclonal antibodies, raised against the glycoproteins present in the colchicine- and vinblastine-resistant cells, were used to probe for relationships among the three glycoproteins. Our observations suggest that the glycoproteins from the different drug-resistant cell lines share many common domains but are not identical.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

Neurite Extension by Mouse Neuroblastoma: Evidence for Two Modes of Induction

The induction of neurite extension by mouse neuroblastoma cells in vitro can be reversed by adding colchicine or cytochalasin B (CB) to the culture medium. The relative sensitivity of the neurites to retraction is a function of the method used to induce extension. Cells incubated in the absence of serum or in the presence of serum and dibutyryl cyclic-AMP are sensitive to both colchicine and CB; cells incubated in serum-free medium containing either cycloheximide or dimethylsulphoxide are sensitive only to colchicine.     

Effects of microtubule inhibitors and cytochalasin B on thyroid metabolism in vitro.

Mitotic spindle inhibitors (colchicine, vinblastine, vincristine, 020, ethanol) and cytochalasin B inhibit the phagocytosis of colloid by thyroid cells and the secretion of thyroid hormones. This inhibition has been linked to interferences with the microtubular microfilament system of the follicular cell. In order to test the possibility of using such inhibitors to selectively block secretion, the action of suppressing or highly inhibitory concentrations on other metabolic parameters has been studied on dog thyroid slices in vitro: glucose oxidation, lactate formation, iodide binding to protein, cyclic 3'5' AMP accumulation. It is shown that at a concentration of 10 mM colchicine is entirely non specific as it greatly inhibits all facets of metabolism and all the stimulatory effects of cyclic 3'5' AMP and thyrotropin. The other mictrotubule inhibitors, although affecting thyroid metabolism in various ways were more specified. The enhancement by vineblastine of glucose oxidation ald iodine binding to proteins suggests an activation of they thyroid H2O2 generating system. D2O on the other hand selectively inhibits secretion and the binding of iodide to proteins. Cytochalasin B, presumably by inhibiting hexose transport, decreased glycolysis and the uptake of iodide. However this effect cannot account for the complete inhibition of thyroid secretion.     

Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons

A large body of evidence indicates that microtubules (MTs) conduct organelle transport in axons, but recent studies on extruded squid axoplasm have suggested that actin microfilaments (MFs) may also play a role in this process. To investigate the separate contributions to transport of each class of cytoskeletal element in intact vertebrate axons, we have monitored mitochondrial movements in chick sympathetic neurons experimentally manipulated to eliminate MTs, MFs, or both. First, we grew neurons in the continuous presence of: (a) cytochalasin E to create neurites which had never contained MFs; or (b) nocodazole or vinblastine to produce neurites which had never contained MTs. Mitochondria moved bidirectionally at normal velocities along the length of neurites which contained MTs and lacked MFs, but did not even enter neurites grown without MTs but containing MFs. In a second approach, we treated established neuronal cultures with cytoskeletal drugs to disrupt either MTs or MFs in axons already containing mitochondria. In cytochalasin-treated cells, which retained MTs but lacked MFs, average mitochondrial velocity increased in both directions, but net directional transport decreased. In vinblastine- treated cells, which lacked MTs but retained essentially normal levels of MFs, mitochondria continued to move bidirectionally but the average mitochondrial velocity and excursion length were reduced for both directions of movement, and the mitochondria spent threefold as much time moving in the retrograde as in the anterograde direction, resulting in net retrograde transport. Treatment of established cultures with both drugs produced neurites lacking MTs and MFs but still rich in neurofilaments; these showed a striking absence of any mitochondrial motility. These data indicate that axonal organelle transport can occur along both MTs and MFs in vivo, but with different velocities and net transport properties.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose

Previous studies have shown that colchicine and vinblastine inhibit secretion in many cell types by interrupting the normal intracellular migration of secretory products. In the present work, radioautography has been used to study the effects of these drugs on migration of membrane and secretory glycoproteins in a variety of cell types. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for light microscope radioautography. Examination of secretory cell types such as ameloblasts and thyroid follicular cells in control animals revealed reactions of approximately equal intensity over the Golgi region and over extracellular secretion products, while in drug-treated rats most of the reaction was confined to the Golgi region. In a variety of other cell types, including endocrine cells (e.g., hepatocytes) and cells generally considered as nonsecretory (e.g., intestinal columnar cells), reaction in control animals occurred both over the Golgi region and over various portions of the cell surface. In drug-treated animals, a strong Golgi reaction was present, but reaction over the cell surface was weak or absent. These results indicate that in many cell types, colchicine and vinblastine inhibit migration out of the Golgi region not only of secretory glycoproteins, but also of membrane glycoproteins destined for the plasma membrane.