The effects of vinblastine on acinar cells of the exorbital lacrimal gland of the rat

Summary The effects of vinblastine treatment on acinar cells of the rat exorbital lacrimal gland were studied by electron microscopy. Experimental animals of both sexes were given single intraperitoneal injections of (1) vinblastine (4mg/kg body weight) at 1 to 24 h before sacrifice; (2) pilocarpine (20 mg/kg b.w.) for 1 h; or (3) vinblastine for l h followed by pilocarpine for 1 h.Vinblastine treatment caused a number of changes including autophagocytosis, formation of intracisternal granules, and alteration of secretory granules. These changes varied in extent and onset between male and female rats. In addition, the Golgi apparatus was reduced in size and dispersed throughout the cytoplasm. Mitotic figures were commonly observed. Moreover, vinblastine inhibited the pilocarpine-stimulated degranulation of the acinar cells.In view of the known anti-microtubular action of vinblastine, these results suggest that microtubules are involved in various aspects of the transport, packaging, and secretion of exportable proteins in the lacrimal gland. Additionally, autophagocytosis and alteration of secretory granules may partially result from the interaction of vinblastine with membranes.The authors thank Mr. Steve Coriell and Mr. Steve Floyd for preparing the micrographs. Robert Kelly also thanks Dr. George Chapman for his support during the initial phase of this project.     

Vinblastine-induced microtubular paracrystals in prolactin cells of anterior pituitary gland of lactating rats.

Vinblastine sulfate in physiological saline was injected directly into the pituitary glands of lactating rats. Injections were made through the ear canal using a syringe equipped with a 24-gauge needle. The animals were killed at 2, 4, or 6 hours after the injections. When the anterior pituitary glands were examined by electron microscopy, many microtubular paracrystalline deposits were seen in the prolactin and growth hormone cells. The usual cytoplasmic microtubules and microfilaments were not seen in the cytoplasm of these cells. Granular extrusion (exocytosis) was markedly depressed, and an accumulation of secretory granules was definitely observed in the prolactin cells after the administration of vinblastine.     

Induction of aneuploidy by mitotic arrestants in mouse bone marrow

Most human and animal carcinogens induce gene mutation, chromosome breakage or other types of DNA lesions. However, recent studies indicate that some carcinogens do not directly damage DNA, but may cause missegregation of chromosomes resulting in aneuploidy production. Aneuploidy-producing agents pose serious genetic hazards to the human population. Such agents may cause genomic imbalance not only in somatic cells which may result in cancer development, but also in germinal cells which may result in the production of abnormal offspring (e.g. Down's syndrome). To limit human exposure to potential aneuploidy-producing agents, such agents must first be identified in experimental animals. The present study demonstrates that vinblastine and colcemid are capable of inducing aneuploidy in bone marrow cells of treated mice. Both of these compounds are chemotherapeutic agents that arrest mitosis by interfering with the formation of spindle microtubules. Single intraperitoneal injections of vinblastine, at a dose of 9 mg/kg, were found to produce 1.5-5.2% of hyperdiploidy in all of the 10 treated mice sampled at 17-96 h after injection. Only the frequency of hyperdiploidy was determined because hypodiploid cells could result from artifactual chromosome loss during slide preparation. At 0.9 mg/kg, vinblastine was found to produce 0.5-3.5% of hyperdiploidy in 8 of the 10 treated animals. The frequency of hyperdiploid cells in animals treated with colcemid was low. A dose as high as 37 mg/kg was found to produce only 0.5-1% of hyperdiploidy in 3 of the 10 treated animals, and hyperdiploidy was observed only in animals sampled at 17-24 h. In 10 mice treated with saline alone, no hyperdiploid cells were observed. Unlike cell cultures where vinblastine and colcemid had been shown to be equally effective in producing aneuploidy, vinblastine was found in this study to be a much more potent aneuploidy inducer than colcemid in mice.     

Slow infusions of vinblastine in the treatment of adult idiopathic thrombocytopenic purpura: a report on 43 cases

Forty-three adult patients with idiopathic thrombocytopenic purpura (ITP) were treated by slow intravenous infusions of vinblastine. Nineteen had ITP of recent onset (i.e. of less than 6 months duration) and had contraindication to steroids (3 patients), refractoriness to steroids (6 patients) or to steroids and high dose intravenous immunoglobulins (IVIg, 10 patients). Of the 19 patients, 10 achieved complete response (CR), 2 achieved partial response (PR), 2 had minor response (MR) and the remaining 5 patients had no response (NR). Six of the complete responders remained in CR after 12 to 48 months, whereas all other responders relapsed within 3 months, in spite of maintenance therapy. Twenty-four patients had chronic ITP (i.e. of 6 months duration or more) and had showed no or only transient response to steroids and/or splenectomy, and in many of them, to other therapeutic approaches. Four achieved CR, 4 PR, 6 MR and 10 NR. All but 3 responses were shorter than 3 months, in spite of maintenance therapy. Most responses to slow infusions of vinblastine began after the first infusion. Main side effects included leukopenia in 9 patients (but with absolute neutropenia in only one) and peripheral neuropathy in 2 patients. Interval from diagnosis was the only prognostic factor of response to treatment. We conclude that slow infusions of vinblastine may be a useful approach in ITP of recent onset, when contraindication or refractoriness to steroids and/or IVIg exists. In our experience, this treatment has limited benefit in chronic ITP. In addition, it remains to be demonstrated that slow infusions of vinca alkaloids have any superiority over intravenous bolus injections of the same drugs.     

The effect of inhibitors of microtubule and microfilament function on the cellular slime mould Dictyostelium discoideum

Colchicine, vinblastine, griseofulvin and isopropyl N-phenyl carbamate (IPC) (agents which inhibit microtubule function) inhibit division of the amoebae of the cellular slime mould Dictyostelium discoideum. These inhibitors also either inhibit or delay the aggregation process characteristic of these amoebae as does cytochalasin B (CB) (an inhibitor of microfilament function). Even when apparently normal fruiting bodies are formed in the presence of these inhibitors the spores may have a diminished heat resistance (CB and IPC) and a volume which is either larger (CB), very much larger (IPC) or smaller (colchicine, vinblastine) than normal. Some of the spores that are formed in the presence of IPC resemble in shape, as well as size, those typical of diploid strains.     

Effect of depletion of L3T4+ cells on resistance to early primary infection of mice with Taenia taeniaeformis

The role of L3T4+ T lymphocytes in early primary infection with the metacestode of T. taeniaeformis was investigated by selective removal of these cells in vivo by parenteral injections with the rat monoclonal antibody (MAb) GK1.5 directed against the L3T4 molecule. Comparisons between treated and non-treated BALB/cByJ mice, normally resistant to infection with T. taeniaeformis, demonstrated that the treated mice had a greater percentage of viable parasites in the livers. Eosinophils were prominent in the region immediately surrounding parasite larvae in control mice, whereas treated mice showed virtually no eosinophil infiltration. Additionally, fewer tissue macrophages were evident near parasite larvae in the treatment group when compared to controls. The more susceptible C3H/HeDub strain mice demonstrated similar responses following treatment with the MAb, including diminished parasite killing and limited inflammatory cell infiltration. When C3H/HeDub mice were injected with the cytotoxic agent vinblastine sulfate, which has been shown to diminish Lyt-2+ suppressor cell activity, these mice remained unable to mount a strong local cellular response to the larval parasite. It is suggested that L3T4+ T lymphocytes play a crucial role in the innate resistance to T. taeniaeformis infection during the first 6 days post-infection. Effects seen following vinblastine treatment may be a result of drug-induced alterations in leukocyte chemotaxis, toxicity to other effector T cell populations, or a specific depletion of a functional Lyt-2+ T cell population that is required in addition to L3T4+ T cells for the expression of resistance to primary infection with T. taeniaeformis.     

Studies on cellular autophagocytosis : Vinblastine-induced autophagy in the rat liver

Administration of vinblastine (5 mg/kg) in two doses intraperitoneally to rats induced a prominent formation of autophagic vacuoles in rat liver parenchymal cells. Four hours after the first injection of vinblastine, clearly recognizable organelles were seen inside these vacuoles, and by 12 h the cells were filled with residual body type of lysosomes. As compared with glucagon administration, vinblastine was followed by a far greater degree of autophagy, and therefore, offers an excellent model for future studies on autophagy.     

Stimulation of Tubulin-Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Vinblastin

Vinblastine, a plant alkaloid which inhibits tubulin polymerization, stimulated an ATPase activity in microtubules. When microtubule proteins were separated into microtubule-associated proteins (MAPs) and tubulin by phosphocellulose column chromatography, vinblastine did not stimulate an ATPase activity recovered in the MAPs fraction unless tubulin was present. Therefore, vinblastine is considered to act through its binding to the tubulin molecule on MAPs ATPase. Divalent cations that activate tubulin-dependent MAPs ATPase activity were also required for the stimulation by vinblastine. In the presence of Ca2+ and vinblastine the ATPase activity was most active and the extent of stimulation reached about 200% of the original level in the absence of vinblastine. Half-maximal stimulation was attained when the molar ratio of vinblastine to tubulin was 0.5. The concentration of tubulin for half-maximal stimulation was increased in the presence of vinblastine, while divalent cation requirements were decreased. Several factors such as KCl (100 mM), alkaline pH (pH 7.5), and low temperature (10 degrees C) were not responsible for the disappearance of the stimulation. Vincristine stimulated tubulin-dependent MAPs ATPases activity as vinblastine did, whereas the activity was scarcely affected by colchicine, podophyllotoxin, strychnine, and chlorpromazine. Actin had no effect on MAPs ATPase activity in the absence and presence of vinblastine when it was used in place of tubulin.     

Vinblastine-induced disruption of microtubules in cockroach hemocytes

Intrahemocoelic injections of vinblastine (10(-3), 10(-4)M) resulted in the disappearance of microtubule marginal bundles in the hemocytes of Periplaneta americana L. concomitant with a change in lenticular cell shape as determined by electron microscopy. In contrast to control cells, which in section were elliptical in shape with smooth edges, treated cells displayed large vacuoles apparently resulting from folds in the cytoplasm and an irregular shape. Vinblastine concentrations lethal to insects (25mu1/insect, 10(-3)M) resulted in marginal bundle loss, and the formation of cytoplasmic linear material with associated ribosomes, although microtubule crystals were not found.     

Interaction of Vinblastine with Calf Brain Microtubule protein.

The interaction of vinblastine with calf brain tubulin has been studied by velocity sedimentation, gel filtration, and fluorescence. It has been established that vinblastine induces the stable tubulin dimers to dimerize further to tetramers. The sedimentation patterns at low vinblastine concentration were analyzed by the ligand-induced dimerization theory of Cann and Goad ((1972) Arch. Biochem. Biophys. 153, 603-609). The association constant and stoichiometry for the binding of vinblastine to tubulin, determined by gel filtration and spectrofluorometry, were (2.3 +/- 0.1) X 10(4) liters/mol at 25 degrees and two vinblastine binding sites per tubulin dimer of molecular weight 110,000. The binding of vinblastine to tubulin is characterized by an enthalpy change of 5.8 kcal/mol and a positive unitary entropy change. Binding of vinblastine did not induce any significant conformational changes in tubulin as monitored by circular dichroism. However, the vinblastine-tubulin complex displayed an ultraviolet difference spectrum, which appears to reflect mostly the transfer of vinblastine to a less polar environment. Besides binding vinblastine, tubulin was shown to bind vincristine with identical free energy and stoichiometry and to have a single binding site for 8-anilino-1-naphthalene sulfonic acid per tubulin dimer, which is independent of those for vinblastine.     

Effect of tau on the vinblastine-induced aggregation of tubulin

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.     

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

The effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-D-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes. Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-D-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AV's in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-D-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AV's. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AV's resembled endoplasmic reticulum and still had attached ribosomes. It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.     

Studies on vinblastine-induced autophagocytosis in mouse liver. III. A quantitative study

The microtubule inhibitor vinblastine (25 mg/kg, i.p.) induces autophagocytosis in mouse hepatocytes. The formation of autophagic vacuoles, their contents, and other cellular changes after vinblastine injection in hepatocytes, were studied by light and electron microscopic morphometric analysis. The volume density of autophagic vacuoles increased significantly during the experimental period (24 h). This increase was due to the significant increase in their number, which was approximately 5-fold 4 h, 12 h and 24 h after vinblastine injection. The mean volume of the autophagic vacuoles increased significantly 1 h after vinblastine injection, at which time the formation of new autophagic vacuoles was at its greatest. There was an accumulation of single membrane-limited, obviously older autophagic vacuoles in the cytoplasm. Their volume density was at its maximum 12 h after injection, suggesting a retarded turnover of autophagic vacuoles. The segregation of cytoplasmic components into autophagic vacuoles may not be selective after vinblastine injection. The injurious effects of vinblastine were evident both in light and electron microscopic studies. In the parenchymal cells the Golgi cisternae were dilated and disorganized and the volume density of the Golgi apparatus was significantly decreased 12 h after vinblastine injection. The volume density of lysosomes was increased during the 12 h after vinblastine injection. Vesicles containing very low density lipoprotein particles accumulated in the cytoplasm so that their volume density was significantly increased during the entire experimental period. Vinblastine apparently interfered with the transport and secretion of the very low density lipoproteins from the parenchymal cells.     

The induction of immunological unresponsiveness in previously immunized mice.

HGG unresponsiveness can be induced in primed A/J mice; however, such induction is difficult and requires multiple injections of large doses of soluble HGG (SHGG). Although single injections (1.5–25 mg) of deaggregated HGG (DHGG) did not result in a significant secondary immune response, an unresponsive state to a subsequent injection of aggregated HGG (AHGG) was not induced. When the dose of DHGG was even smaller (0.5 mg), a normal secondary response was obtained similar to that observed following injection of AHGG. Evidence is presented here which suggests that the difficulty encountered in inducing unresponsiveness may be in part due to partial aggregation of DHGG by persisting antibody in the circulation. The PFC to HGG produced after injection of either AHGG or DHGG or during induction of unresponsiveness to SHGG apparently involved cell division, since all three responses were inhibited by vinblastine. The reduction in PFC in primed mice injected with DHGG or SHGG was not due to selective inhibition of PFC secreting certain classes or subclasses of immunoglobulins.     

The effects of vinblastine in assessment of the influence of age on proliferative activity of murine palate and footpad epithelium

Abstract
Data concerning changes in the rate of cell proliferation of stratified epithelia with increasing age are conflicting. In the present study young (3-month-old) and old (22-month-old) C57B1/6NNia male mice were injected intraperitoneally with 2, 3, 4 or 8 mg vinblastine sulfate/kg body weight and killed after 1.5, 3, 4.5 or 6 h. The number of arrested metaphase figures per 1000 basal cells was counted in histological sections. Data were analysed using a multivariate analysis of variance. There was a significant difference between the accumulation of mitotic figures in footpad epidermis and palate epithelium and both tissues contained an increased number of mitotic figures with increasing periods of accumulation at all dose levels. In the footpad epidermis neither the age of the animal nor the dose of vinblastine had a significant effect on the number of mitotic figures. In contrast, for palate epithelium the accumulation of mitotic figures was significantly less in the old mice compared with the young mice and at a dose of vinblastine of 2mg/kg compared with the higher doses. There was a statistically signifycant interaction between the dose of vinblastine and its period of action. It was concluded that the different tissues manifest a differential sensitivity to vinblastine and that only palate epithelium showed a significant reduction in proliferative activity with age.     

A cytochemical study on the effects of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells

Summary The effect of energy deprivation on autophagocytosis in Ehrlich ascites tumor cells was studied using cytochemical techniques. Autophagocytosis was induced with vinblastine incubation (0.1 mM) and the cellular ATP-level was lowered with 2-deoxy-d-glucose (0.35 mM). Acid phosphatase was used as a marker for lysosomal enzymes and imidazole-buffered osmium tetroxide impregnation in order to study the effects of energy deprivation on the maturation of autophagic vacuole (AV) membranes.Control and vinblastine treated cells maintained their ATP-levels throughout the incubation period tested (120 min). 2-Deoxy-d-glucose alone and with vinblastine decreased the intracellular ATP-level significantly after only 3 min incubation. Most of the AV's in control and vinblastine treated cells contained degraded material and acid phosphatase activity. Their membranes were stained only slightly or not at all with imidazole-buffered osmium tetroxide. 2-Deoxy-d-glucose alone as well as with vinblastine induced in particular an accumulation of early stages of AV's. These vacuoles contained undegraded cytoplasmic material and no acid phosphatase activity and their membranes were stained usually partly with imidazole-buffered osmium tetroxide. The membranes of some early AV's resembled endoplasmic reticulum and still had attached ribosomes.It was concluded that the inhibition of cellular energy production used in the present study did not inhibit autophagic sequestration but retarded the maturation of AV membranes and impaired the functioning of lysosomal hydrolases.     

Identification of a distinct class of vinblastine binding sites on microtubules

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Harnessing WYE-132 as an inhibitor of the mTOR signaling enriches the cytotoxicity effect of vinblastine in B16F10 melanoma cancer cells

This study aimed to evaluate the synergistic anticancer effect of vinblastine and WYE-132, a specific inhibitor of the mammalian target of rapamycin (mTOR), on B16F10 melanoma cancer cells. MTT assay, Annexin V, and DAPI staining along with Real-Time PCR were conducted to understand the mechanistic roles of mTOR pathway in melanoma cancer cells. The IC50 values for vinblastine and WYE-132 were 39.4 ± 1.8 nM and 145.2 ± 4.5 nM, respectively. The co-administration of WYE-132 and vinblastine in B16F10 cells offered a significant increase in the growth inhibitory effect of vinblastine along with a two-fold escalation in the percentage of apoptotic cells. The calculation of gene expression levels confirmed a visible fall in anti-apoptotic Bcl-2, Ki-67 and Mcl-1 accompanied by a surge in pro-apoptotic Bax mRNA levels (p < 0.05). Thus, we showed that the combination of WYE-132 and vinblastine could be a promising approach for dealing with patients with melanoma cancers.     

Alternate overexpression of two P-glycoprotein [corrected] genes is associated with changes in multidrug resistance in a J774.2 cell line

In multidrug-resistant murine J774.2 cells, the mdr1a and mdr1b genes encode the 120- and 125-kDa P-glycoprotein precursors, respectively (Hsu, S. I., Lothstein, L., and Horwitz, S.B. (1989) J. Biol. Chem. 264, 12053-12062). It is shown here that a J774.2 cell line selected for vinblastine resistance (J7.V3) switched from the 125- to 120-kDa precursor when cells that were maintained in 20 nM vinblastine were grown in 40 nM vinblastine for 20 months. The rate of switching was accelerated by growing cells in higher levels of vinblastine. These findings suggest that cells which express mdr1a have a selective growth advantage compared to cells which express mdr1b. Consistent with this hypothesis, the switching event that occurs in cells maintained at 40 nM vinblastine was correlated with 3.5-5-fold higher levels of resistance to vinblastine, taxol, and doxorubicin in the absence of any detectable increase in the amount of immunoreactive P-glycoprotein. These findings suggest that P-glycoproteins derived from mdr1a and mdr1b are functionally distinct.