Stimulation of plasminogen activator expression and induction of DNA synthesis by microtubule-disruptive drugs

Treatment of confluent Swiss 3T3 cells in serum-free medium with colchicine, a drug known to depolymerize microtubules, results in a dose-dependent increase in both released and cell-associated plasminogen activator levels. Other anti-microtubule drugs (vinblastine and nocodazole) are also active in stimulating plasminogen activator expression. In contrast, cytochalasin B, a microfilament-disruptive drug, has no effect. In addition, treatment with colchicine, vinblastine or nocodazole, but not cytochalasin B, also results in a dose-dependent induction of DNA synthesis in both confluent and quiescent sparse 3T3 cells in the absence of serum. Furthermore, colchicine treatment also mediates a marked morphologic change. Thus, disruption of microtubules may be sufficient to render 3T3 cells in an “activated” state characterized by morphologic alteration, enhanced plasminogen activator expression and induction of DNA synthesis.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Role of microtubules in the intracellular transport of growth hormone

Summary Pulse-chase experiments utilising(³H)leucine have been used to study the effects of colchicine and vinblastine on intracellular transport and secretion of newly synthesised growth hormone from rat anterior pituitary fragments. Growth hormone was isolated from medium and fragments by polyacrylamide gel electrophoresis. When colchicine or vinblastine, which disrupt microtubules, were added immediately after pulse labelling, inhibition of the subsequent secretion of newly synthesised growth hormone was detected throughout the succeeding 5 h. Similar inhibition was seen if the drugs were added after a 1 h delay. However, if colchicine or vinblastine were added only after a 2 h chase incubation, then no significant effect on subsequent release of labelled growth hormone was seen. The results suggest that these agents may inhibit the transport of newly formed growth hormone storage granules from the Golgi complex to the cytoplasmic pool. Microtubules do not appear to be involved in the mechanism of the final secretion of newly synthesised hormone by exocytosis.These studies were supported by grants from the Medical Research Council and British Diabetic Association     

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.     

Effect of cytochalasin B on lymphocyte stimulation induced by concanavalin A or periodate

The effects of cytochalasin B on lymphocyte stimulation induced by concanavalin A (Con A) and by periodate were investigated. At low concentrations (0.1 – 1 μg/ml) cytochalosin B greatly potentiated the responses to these two mitogens. Cytochalasin B was most effective when added with the mitogens at the beginning of incubation. The action of cytochalasin B at low concentration was suggested to be on an early process of DNA synthesis induced by these mitogens.     

Effect of phytohaemagglutinin on the nuclear RNA polymerase activity of human lymphocytes

The DNA-dependent RNA polymerase activity of isolated nuclei from human peripheral blood has been shown to increase following stimulation with phytohaemagglutinin (PHA). Using the toxin α-amanitin it has been possible to demonstrate that within 4 h of the addition of PHA there is a two-fold increase in the amanitin-resistant polymerase activity (polymerase A) with little increase in the sensitive polymerase activity (polymerase B). 24 h following PHA stimulation the amanitin-resistant activity is stimulated 4–5 fold and the amanitin-sensitive activity less than two-fold. The susceptibility of this increased amanitin-resistant activity to low doses of actinomycin D both in vivo and in vitro indicates that the amanitin-resistant enzyme is mainly engaged in ribosomal RNA precursor synthesis. These changes in DNA-dependent RNA polymerase activity closely correspond to the observed changes in ribosomal and non-ribosomal RNA synthesis following lymphocyte stimulation.The increased polymerase A activity is diminished by a 1 h incubation of the cells with cycloheximide added 24 h after PHA whereas polymerase B activity remains unaffected. This indicates that the polymerase A activity observed after transformation is dependent on continuing protein synthesis.In our incubation conditions the polymerase activity observed in isolated nuclei appeared to be almost wholly attributable to elongation of nascent RNA molecules attached to the endogenous DNA template.     

Gametogenesis in Plasmodium; the inhibitory effects of anticytoskeletal agents

Gametocytes of Plasmodium yoelii were incubated with colchicine, vinblastine sulphate and cytochalasin B and then induced to undergo gametogenesis. All the drugs inhibited gamete formation in a dose and time dependent manner. Electron microscopy revealed that colchicine and vinblastine sulphate inhibited the polymerisation of cytoplasmic- axonemal- and intranuclear mitotic- microtubules. Cytochalasin B did not inhibit microtubule assembly but blocked spindle pole separation and axoneme distribution into the microgamete. All drugs prevented escape of the gametocyte from the erythrocyte. The mechanism of mitotie spindle action is discussed.     

Inhibition of macrophage-lymphocyte interaction by cytochalasin B during antigen recognition by T lymphocytes.

The effects of cytochalasin B on functional and physical macrophage-lymphocyte interaction have been examined. Cytochalasin B, an inhibitor of a variety of membrane activities blocks antigen-dependent bindings of immune lymphocytes to macrophages and antigen-triggered lymphocytes proliferation if added at the initiation of culture. Cytochalasin B becomes progressively less inhibitory if addition is delayed by increasing intervals from the onset of culture. Under these conditions neither antigen handling by macrophages nor the proliferative response of lymphocytes to PHA is inhibited by cytochalasin B. These data are interpreted to suggest that cytochalasin B inhibits antigen-specific macrophage-lymphocyte interaction either by inhibition of an initial antigen-independent phase of macrophage-lymphocyte interaction or by interfering with a lymphocyte membrane event necessary for the interaction of the antigen-specific lymphocyte receptor with the macrophage-bound antigenic signal.     

Effects of four agents that modify microtubules and microfilaments (vinblastine,colchicine, lidocaine,and cytochalasin B) on macrophage-mediated cytotoxicity to tumor cells

Summary The effects of vinblastine, colchicine, lidocaine, and cytochalasin B on tumor cell killing by BCG-activated macrophages were examined. These four drugs were selected for their action on membrane-associated cytoskeletal components, microtubules, and microfilaments. Colchicine and vinblastine, which block microtubular synthesis, inhibit macrophage-mediated tumor-cell cytotoxicity at a concentration of 10^–6 M. Cytochalasin B, which disrupts microfilaments, enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10^–7 M. Lidocaine, which may induce the disappearance of both microtubules and microfilaments, has the same inhibiting effect as vinblastine at a concentration of 5×10^–7 M. Whereas vinblastine and lidocaine seem to act on the macrophage itself, cytochalasin B exerts its effect predominantly on the tumor cell. These results suggest that microtubules and microfilaments play a role in the destruction of tumor cells by activated macrophages.     

Inhibition of mitogen and specific antigen-induced human lymphocyte proliferation by cadmium

Cadmium chloride (CdCl2) added to human lymphocyte culture inhibits the proliferative response induced by phytohaemagglutinin (PHA), pokeweed mitogen and allogenic lymphocytes in mixed lymphocyte reaction. Minimally effective concentrations of CdCl2 were 3.3, 1.6 and 1.6 microM, respectively. The inhibition was greatest when CdCl2 was added at initiation of cultures and declined if the addition of CdCl2 was postponed. The presence of CdCl2, regardless of the presence of PHA during the first 24 h of incubation suppressed the proliferative response to subsequent stimulation with PHA, indicating that cadmium affects an early step of blastogenic transformation.     

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10^-5 M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of the cells with a microfilament-disrupting drug, cytochalasin B (10 g/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5–15 min. A morphological abnormality, microtubule paracystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.     

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.     

Involvement of polyadp-ribose polymerase in the initiation of phytohemagglutinin induced human lymphocyte proliferation

Nicotinamide (10 mM) or 3-aminobenzamide (5 mM) added at the onset of phytohemagglutinin (PHA) treated human lymphocyte cultures provoke a marked inhibition of the PHA induced DNA synthesis and cell proliferation as well as of poly(ADPR) polymerase activity. When the inhibitors of poly(ADPR) polymerase are added at a later stage of culture (48 h) no inhibition of the stimulation of DNA synthesis and cell proliferation by PHA in human lymphocyte cultures is observed. The intervention of ADP ribosylation at the initiation of DNA synthesis is suggested.     

Factors which disorganize microtubules or microfilaments increase the frequency of cell transformation by polyoma virus.

Griseofulvin, 12-O-tetradecanoyl phorbol-13-acetate, melittin, epidermal growth factor, vinblastine, cytochalasin B, podophyllotoxin, colcemid, and colchicine were unable to transform cells but could increase from 8- to 40-fold the frequency of cell transformation by polyoma virus. The 3T3-like cells were resting at confluence and were exposed to the drug only during the 1st week after viral infection. Griseofulvin, a tumor promoter, reduced or increased the frequency of transformation depending on the dose with which the infected cells were treated. The antitumor activity of tumor promoters is discussed.     

Drug-induced alterations in rat peritubular cell cytoskeleton result in proteoglycan synthesis modifications. Comparison with some intracellular signaling pathways.

The influence of phorbol myristate acetate (PMA), dibutyryl cAMP and insulin-like growth factor (IGF-1) as well as cytoskeletal disrupting drugs on morphological changes has been studied in peritubular cells isolated from immature rat testis. Morphological studies were combined with immunofluorescence investigations of cytoskeletal elements and their rearrangements by various agents. The results were correlated with modulation of proteoglycan synthesis. Peritubular cells exposed to dibutyryl cAMP or cytochalasin D were transformed from flattened, fibroblast-like into neuronal-like morphology. In such cells, destruction of actin filaments was accompanied with a 50% decrease in cell-associated proteoglycan synthesis as well as with oversulfation of total proteoglycans. On the contrary, peritubular cell shape has been slightly altered after addition of PMA, IGF-1, vinblastine or colchicine. After these treatments, destruction or rearrangement of cytoskeletal elements was observed; cell-layer proteoglycan synthesis remained either unchanged or increased while total proteoglycans were always undersulfated. IGF-1, PMA and dibutyryl cAMP modified the peritubular cell morphology, cytoskeletal organization and proteoglycan production; the cytoskeleton disrupting drugs such as vinblastine, colchicine and cytochalasin D mimicked some of these effects. These observations suggest that alterations in proteoglycan biosynthesis, after activation of tyrosine kinase, protein kinase C and protein kinase A pathways might be mediated, at least in part, by the disorganization of the cytoskeleton structure.     

Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes

We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.     

Preliminary lymphocyte activation by a mitogen as a condition for demonstrating the ability of the Fab-fragment of normal IgG to inhibit lymphocyte transformation

Peculiarities attending inhibition of the PHA-induced blast-cell transformation of human lymphocytes by F(ab')2 fragment of rabbit IgG were studied. It was shown that the fragment did not affect the intensity of blast-cell transformation if the lymphocytes were preliminarily incubated with the fragment for 24 h at 37 degrees or 4 degrees C and then transferred to the fresh medium containing PHA. However, if the fragment was added to the cells 24 or 48 h following PHA it produced a significant inhibition of the blast-cell transformation. These data may indicate that F(ab')2 fragment interferes with the lymphocyte transformation only when the cells are already activated with PHA.     

Microtubules and lymphocyte responses: effect of colchicine and taxol on mitogen-induced human lymphocyte activation and proliferation

The role of microtubules in mitogen-induced human lymphocyte activation and proliferation was examined. The effect of colchicine, a microtubule-disrupting agent, was compared with taxol, a microtubule-stabilizing drug, and with isaxonine (N-isopropyl-amino-2-pyrimidine orthophosphate), a proposed microtubular-active drug. Lymphocyte proliferation, assessed by measuring the increase in the number of cells in mitogen-stimulated cultures, was completely suppressed by both colchicine and taxol (100 nM) whereas significant inhibition by isaxonine required much higher concentrations (5 mM). In order to characterize the inhibition, initial lymphocyte blast transformation and subsequent DNA synthesis were investigated. Neither colchicine nor taxol inhibited lymphocyte blast transformation assessed by quantitating the change in volume of the stimulated cells after a 24-hour incubation. In contrast, isaxonine (2-5 mM) suppressed blast transformation. Initial DNA synthesis, evaluated by measuring the cumulative incorporation of [3H]thymidine between 30 and 48 hours of culture, was inhibited in a concentration-dependent manner by both isaxonine and colchicine but not by taxol. Electron microscopic studies confirmed that both taxol and colchicine (10 nM) arrested the responding lymphocytes in mitosis, and that isaxonine inhibited initial activation. These results suggest that normal microtubule function is only necessary for cell division and that drug effects on blast transformation and initial DNA synthesis are unrelated to microtubules.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.