Interaction of alpha adrenergic antagonists with calmodulin

Several alpha-adrenergic antagonists inhibited the activation of calmodulin-stimulated phosphodiesterase at concentrations that had little or no effect on basal phosphodiesterase activity. The most potent of these compounds were phenoxybenzamine and dibenamine (IC50 values of about 1 microM); the amino acid ergot alkaloids ergocryptine, ergocristine, ergotamine and their dihydrogenated derivatives were less potent calmodulin-inhibitors (IC50 values of 35-80 microM). The amino ergot alkaloids ergonovine and methysergide were essentially devoid of inhibitory activity. A variety of other alpha 1-antagonists (phentolamine, tolazoline and prazosin), an alpha 2-antagonist (yohimbine), alpha-agonists (norepinephrine, phenylephrine and clonidine), beta-adrenergic antagonists (propranolol and practolol) and the beta-adrenergic agonist methoxyphenamine displayed little or no anti-calmodulin activity (IC50 values greater than 300 microM). Similarly, the alkylating agents chlorambucil and mechlorethamine also failed to inhibit calmodulin activity. Phenoxybenzamine and dibenamine inhibited calmodulin activity irreversibly, whereas the inhibition caused by other alpha adrenergic blocking agents was reversible. Phenoxybenzamine inhibited calmodulin activity by binding directly to it. This binding was calcium-dependent and irreversible. The irreversible binding and inhibition of calmodulin activity by phenoxybenzamine (or dibenamine) may serve as a useful tool for studying the sites at which drugs bind to calmodulin and may also be useful for studying the distribution and turnover of calmodulin.     

Vinflunine, a novel microtubule inhibitor, suppresses calmodulin interaction with the microtubule-associated protein STOP

Vinca alkaloids vinblastine and vincristine and some of their derivatives such as vinorelbine are widely used in therapy of leukemia and several solid tumors. Their action is associated with alterations of the mitotic spindle functions that prevent the cell cycle progression and lead to mitotic block. A number of studies show that some Vinca alkaloids inhibit CaM-target interaction. The newest microtubule inhibitor, vinflunine (Javlor), currently in clinical trials, is remarkably more active than vinblastine against a number of tumors. Moreover, vinflunine is significantly less toxic than other Vinca alkaloids. The high antitumor activity of this molecule is not well understood since it binds to tubulin with an overall affinity several-fold lower than that of vinblastine or vincristine. In this study, we examined the interaction of Ca2+-CaM with vinflunine, vinblastine, and stable tubule only polypeptide (STOP) by using a combination of thermodynamic and mass spectrometric approaches. We characterized the influence of Vinca alkaloids on Ca2+-CaM-STOP complex formation. Our results revealed different binding modes to Ca2+-CaM for vinflunine and vinblastine, highlighting that adding fluorine atoms on the cleavamine moiety of the Vinca alkaloid molecule is critical for the localization of the drug on calmodulin. We demonstrate that vinflunine is a better inhibitor for STOP binding to calmodulin than vinblastine. We suggest that vinflunine action on calmodulin can have an effect on microtubule dynamics. These data may contribute to a better understanding of the superior antitumor efficiency and lower toxicity of vinflunine.     

Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.     

Mechanism of interaction of vinca alkaloids with tubulin: catharanthine and vindoline

The interactions of the vinca alkaloid drugs catharanthine and vindoline with tubulin have been investigated and compared with those of vinblastine and vincristine. Both drugs were found to be less effective in bringing about the inhibition of tubulin self-assembly into microtubules than vincristine and vinblastine, the drug to protein molar ratio required being 3 orders of magnitude greater. An analytical ultracentrifuge study has shown that catharanthine can induce the self-association of tubulin into linear indefinite polymers with an efficacy that is 75% that of vinblastine or vincristine, the intrinsic dimerization constant for the liganded protein being K2 congruent to 1 x 10(5) M-1. The effect of vindoline was marginally detectable. Binding studies of catharanthine using the gel batch and fluorescence perturbation techniques showed a polymerization-linked binding of one catharanthine molecule per tubulin alpha-beta dimer with a binding constant of (2.8 +/- 0.4) x 10(3) M-1. For vindoline, binding to tubulin was marginally detectable by fluorescence spectroscopy, although addition of vindoline to tubulin did generate a difference spectrum. It was concluded that the binding of vinblastine/vincristine to tubulin and its consequences are determined by the interaction of the indole part of catharanthine with tubulin, the role of vindoline being that of an anchor.     

Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of tubulin-based mechanism of action by analysis of differential cytotoxicity data

Data generated in the new National Cancer Institute drug evaluation program, which is based on inhibition of cell growth in 60 human tumor cell lines, were used to compare new compounds with agents of known mechanism of action in terms of their differential cytotoxicity. Two marine natural products, halichondrin B and homohalichondrin B, appeared repeatedly when the data base was probed with known antimitotic agents. We confirmed that both compounds were highly cytotoxic (IC50 values for L1210 murine leukemia cells of 0.3 and 1 nM, respectively), with accumulation of cells arrested in mitosis at toxic concentrations, that both inhibited the polymerization of purified tubulin, and that both inhibited microtubule assembly dependent on microtubule-associated proteins. Limited amounts of homohalichondrin B, the less active agent, were available, so only halichondrin B was studied in detail. Halichondrin B did not interfere with colchicine binding to tubulin, but it was a noncompetitive inhibitor of the binding of vinblastine to tubulin (apparent Ki, 5.0 microM). Halichondrin B was therefore compared with other agents which interfere with the binding of vinca alkaloids to tubulin (vinblastine, maytansine, dolastatin 10, phomopsin A, rhizoxin) in terms of its effects on tubulin polymerization, inhibition of GTP hydrolysis, inhibition of nucleotide exchange, and stabilization of tubulin, as well as the quantitative assessment of its effects on vinca alkaloid binding and inhibition of cell growth. Since halichondrin B was originally isolated from the same organism as the phosphatase inhibitor okadaic acid, and since it is about 50-fold more effective than okadaic acid as an inhibitor of L1210 cell growth, perturbations of cellular microtubules observed following treatment with okadaic acid should be interpreted cautiously.     

Aging of tubulin at neutral pH: the destabilizing effect of vinca alkaloids.

The effect of the vinca alkaloid drugs, vincristine, vinblastine, catharanthine, and vindoline, on the aging process of tubulin has been examined. It was found that addition of vincristine or vinblastine accelerated by a factor of 3-3.5 the transformation of tubulin from the 5.8 S alpha-beta-tubulin dimer to paucidisperse polymers, with an average sedimentation coefficient of 9 S, previously observed in the absence of drugs (V. Prakash and S. N. Timasheff, 1982, J. Mol. Biol. 160, 499-515). This transformation of tubulin from 5.8 S to "9 S" followed pseudo-first-order kinetics whether the starting protein was predominantly dimeric (i.e., at low drug concentration) or self-associated into the reversible linear polymers induced by the vinca alkaloid drugs at high drug concentration (G. C. Na and S. N. Timasheff, 1980, Biochemistry 19, 1355-1365; V. Prakash and S. N. Timasheff, 1985, Biochemistry 24, 5004-5010). Identical kinetics were found in a fluorescence examination of the loss by tubulin of its ability to bind colchicine specifically, indicating that the rate determining step is a protein conformational change that induces a major change in the far uv circular dichroism spectrum of tubulin. The found lack of an effect of dithiothreitol on the aging and aggregation processes is consistent with the irreversible aggregation being due to the intermolecular coalescence of nonpolar patches on the protein. The observations that vincristine binds to aged tubulin and that the aging of tubulin is accompanied by quenching of the tryptophan fluorescence similar to that which occurs on the binding of the vinca drugs has led to the proposal that the vinca alkaloids stabilize the aged conformation of the protein by interacting with nonpolar regions that may be related to the aggregation sites.     

P-glycoprotein possesses a 1,4-dihydropyridine-selective drug acceptor site which is alloserically coupled to a vinca-alkaloid-selective binding site.

[3H]Vinblastine bound with high affinity to surface membranes prepared from H69/LX4 cells which express P-glycoprotein (P-gp) and as a consequence are multidrug resistant (MDR). The KD was 9.8 +/- 1.5 nM and density of sites 31.2 +/- 8.6 pmol/mg of protein. [3H]Vinblastine binding was inhibited by cytotoxics and agents known to reverse MDR. 1,4-Dihydropyridine MDR reversing agents including nicardipine and nifedipine accelerated the dissociation of [3H]vinblastine from P-gp indicating a negative heterotropic allosteric effect. Cyclosporin A, vincristine and actinomycin D did not alter [3H]vinblastine dissociation kinetics. It is concluded that P-gp possesses at least two allosterically coupled drug acceptor sites, receptor site-1 that is selective for vinca alkaloids and cyclosporin A, and receptor site-2 that is selective for 1,4-dihydropyridines.     

In situ response to vinka alkaloids by microtubules in cultured post-implanted mouse embryos

The response of microtubules to treatment with vinca alkaloids was investigated in vivo and in situ in the embryonic nervous system of mice. For this purpose we used rotatory cultures of post-implanted embryos in a serum medium containing the alkaloid combined with immunofluorescence using a tubulin-specific polyclonal antibody on high molecular weight polyethylene glycol embedded semithin sections. In mitotic cells, kinetochore microtubules were seen to be more resistant to the action of vinca alkaloids than interpolar microtubules. Increasing drug concentrations induced an increasing rate of mitosis together with an increasing rate of disassembly of the cytoplasmic microtubule complex, suggesting a probable relation between these events. In bipolar neuroepithelial cells at interphase, a small pool of microtubules was resistant to the vinca alkaloids. These microtubules were located near the centriolar apparatus associated with the primary cilium; they were short, curly and bent. Disruption of the cytoplasmic microtubule complex did not alter the shape of the bipolar neuroepithelial cells. In the axonal profiles, a drug-stable pool of microtubules were not disrupted by the alkaloids and were also short. They seem to act as microtubule organizing centres. These observations suggest vinca alkaloids seem to act in vivo much more by inducing, at a given concentration, the disruption of a particular group of microtubules without altering the others. The fact that these drugs affect the number, but not the length, of the microtubules raises the hypothesis that these drugs act on microtubules by a mechanism similar to that described as "dynamic instability".     

Comparative genotoxic effect of vincristine,vinblastine, and vinorelbine in somatic cells of Drosophila melanogaster

In this study, the vinca alkaloids vincristine (VCR), vinblastine (VBL) and vinorelbine (VNR) were investigated for genotoxicity in the wing Somatic Mutation and Recombination Test (SMART) of Drosophila. Our in vivo experiments demonstrated that all drugs assessed induced genetic toxicity, causing increments in the incidence of mutational events, as well as in somatic recombination. Another point to be considered is the fact that VNR was able to induce, respectively, approximately 13.0 and 1.7 times more mutant clones per millimolar exposure unit as their analogues VCR and VBL. The replacement of a CH(3) attached to vindoline group in VBL by a CHO in VCR seems to be responsible for the approximately seven times higher potency of the former. In contrast, the structural modifications on VNR's catharantine group could be related to its higher genotoxic potency, as well as its similar mutagenic and recombinagenic action.     

The mechanism of action of vinblastine. Binding of [acetyl-3H]vinblastine to embryonic chick brain tubulin and tubulin from sea urchin sperm tail outer doublet microtubules.

Tritium-labeled viblastine, specific activity 107 Ci/mol, was prepared by acetylation of desacetylvinblastine with [3H]acetic anhydride, and has been employed in a study of vinblastine binding to tubulin. There are two high affinity vinblastine-binding sites per mole of embryonic chick brain tubulin (KA = 3-5 X 10(5) l./mol). Binding to these sites was rapid, and relatively independent of temperature between 37 and 0degreeC. Vincristin sulfate and desacetylvinblastine sulfate, two other active vinca alkaloid derivatives, competitively inhibited the binding of vinblastine. The inhibition constant for vincristine was 1.7 X 10(-5) M; and for desacetylvinblastine, 2 X 10(-5) M. The vinblastine binding activity of tubulin decayed upon aging, but this property was not studied in detail. Vinblastine did not depolymerize stable sea urchin sperm tail outer doublet microtubules, nor did it bind to these microtubules. However, tubulin solubilized from the B subfiber of the outer doublet microtubules possessed the two high affinity binding sites (KA = 1-3 X 105 l./mol). These data suggest that vinblastine destroys microtubules in cells primarily by inhibition of microtubule polymerization, and does not directly destroy preformed microtubules.     

Interplay of cyclic AMP and microtubules in modulating the initiation of DNA synthesis in 3T3 cells

The results presented here show that disruption of the microtubule network acts synergistically with cAMP-elevating agents to stimulate the entry into DNA synthesis of 3T3 cells. Antimicrotubule agents and increased cAMP levels require an additional growth-promoting factor for inducing initiation of DNA synthesis; such requirement can be furnished by insulin, vasopressin, epidermal growth factor, platelet-derived growth factor, or fibroblast-derived growth factor. The involvement of the microtubules is indicated by the fact that enhancement of the DNA synthetic response was demonstrated with the chemically diverse agents colchicine, nocodazole, vinblastine, or demecolcine, all of which elicited the response in a dose-dependent manner. We verified that colchicine and nocodazole, at the doses used in this study, induced microtubule disassembly in the absence as well as in the presence of cAMP-elevating agents as judged by measurement of [3H]colchicine binding of total and pelletable tubulin. The involvement of cAMP was revealed by increasing its endogenous production by cholera toxin or by treatment with 8BrcAMP. The enhancing effects of antimicrotubule drugs and cAMP-elevating agents could be demonstrated by incorporation of [3H]thymidine into acid-insoluble material, autoradiography of labeled nuclei, or flow cytofluorometric analysis. The addition of antimicrotubule drugs does not increase the intracellular level of cAMP nor does addition of cAMP-elevating agents promote disassembly of microtubules (as judged by measuring [3H]colchicine binding of total and pelletable tubulin) in 3T3 cells. In view of these findings and the striking synergistic effects between these agents in stimulating DNA synthesis in the presence of a peptide growth factor, we conclude that increased cAMP levels and a disrupted microtubule network regulate independent pathways involved in proliferative response.     

Block of axoplasmic transport in vitro by vinca alkaloids

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1–100μM was examined. The relative order of potency in blocking axoplasmic transport was VCR > VLB > VDS at a concentration of 25μM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block ot the transport mechanism is discussed.     

Structural insight into the inhibition of tubulin by vinca domain peptide ligands

The tubulin vinca domain is the target of widely different microtubule inhibitors that interfere with the binding of vinblastine. Although all these ligands inhibit the hydrolysis of GTP, they affect nucleotide exchange to variable extents. The structures of two vinca domain antimitotic peptides--phomopsin A and soblidotin (a dolastatin 10 analogue)--bound to tubulin in a complex with a stathmin-like domain show that their sites partly overlap with that of vinblastine and extend the definition of the vinca domain. The structural data, together with the biochemical results from the ligands we studied, highlight two main contributors in nucleotide exchange: the flexibility of the tubulin subunits' arrangement at their interfaces and the residues in the carboxy-terminal part of the beta-tubulin H6-H7 loop. The structures also highlight common features of the mechanisms by which vinca domain ligands favour curved tubulin assemblies and destabilize microtubules.     

Cytoplasmic microtubules are essential for the formation of membrane-bound polyribosomes

Colchicine, at low intracellular concentrations, causes a rapid depolymerization of membrane-associated polyribosomes. Poly(A+) mRNA is rapidly lost from these polysomes, and 80 S monomers are left attached to the membranes of the endoplasmic reticulum. Binding studies and measurements of intracellular colchicine concentrations indicate that the drug is acting via depolymerization of cytoplasmic microtubules. The vinca alkaloids, vincristine and vinblastine, have the same effect on polyribosomes, whereas lumicolchicine is ineffective. Furthermore, cordycepin and actinomycin D are without effect on polyribosomes indicating that colchicine is not simply inhibiting the production or transport of new mRNA. It appears that disruption of the cytoplasmic microtubule network prevents membrane-associated ribosomes from reinitiating protein synthesis resulting in the rapid loss of mRNA.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

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.     

Microtubules and axoplasmic transport. Inhibition of transport by podophyllotoxin: an interaction with microtubule protein

Pharmacological evidence is presented for the involvement of microtubules in the process of fast axoplasmic transport. A quantitative measure of the inhibition of axoplasmic transport in an in vitro preparation of rat sciatic nerve is described. The alkaloids colchicine, podophyllotoxin, and vinblastine, which are known both to disrupt microtubules and to bind to the protein subunit of microtubules, are inhibitors of axoplasmic transport. Lumicolchine and picropodophyllin, unlike their respective isomers colchicine and podophyllotoxin, are poor inhibitors of axoplasmic transport. The dissociation constants for the binding of colchicine, lumicolchicine, podophyllotoxin, and picropodophyllin to purified microtubule protein from rat brain have been measured. Inhibition of axoplasmic transport by these drugs correlates favorably with their affinities of microtubule protein.     

Vinca alkaloids inhibit conversion of arachidonic acid to thromboxane by human platelet microsomes: comparison with other microtubule-active drugs

The Vinca alkaloid vinblastine causes dose-dependent inhibition of malondialdehyde formation and aggregation in activated human platelets as a result of inhibition of arachidonic acid metabolism via the thromboxane pathway (Brammer, J.P., Kerecsen, L. and Maguire, M.H. (1982) Eur. J. Pharmacol. 81, 577). The nature of the inhibition by vinblastine has been investigated with human platelet microsomes, measuring conversion of arachidonic acid to malondialdehyde and thromboxane B2 via spectrophotometric assay and RIA, respectively, determining arachidonate oxygenation by monitoring oxygen consumption, and identifying metabolites formed from [1-14C]arachidonic acid. Vinblastine was compared with other Vinca alkaloids and with structurally unrelated microtubule-active drugs. Vinca alkaloids were unique in causing dose-dependent inhibition of both malondialdehyde and thromboxane B2. Order of potency was vinblastine = vincristine = vindesine greater than leurosine greater than vinepidine. Inhibition of malondialdehyde and thromboxane B2 by 50 microM vinblastine was at least 60%. Microsomal cyclooxygenase was not inhibited by 200 microM vinblastine. Inhibition by vinblastine of [1-14C]arachidonic acid conversion to thromboxane B2 was associated with a 4-fold increase in prostaglandin E2 formation. Thromboxane B2, but not malondialdehyde, formation was inhibited by colchicine less than nocodazole much less than vinblastine. Results indicate that microsomal thromboxane synthetase is inhibited by Vinca alkaloids and other tubulin-binding drugs, and suggest that the action of vinblastine in inhibiting thromboxane synthesis, aggregation and release in intact platelets is not dependent upon its antimicrotubular actions.     

Axotomy-induced ornithine decarboxylase activity in the mouse dorsal root ganglion is inhibited by the vinca alkaloids

Vinca alkaloids were used to study the role of retrograde axon transport (RT) in activating neuron perikaryal repair response to nerve transection. Mouse lumbar dorsal root ganglia (DRG) (L₄-L₆) were excised 48 hours after unilateral transection of the sciatic nerve and ornithine decarboxylase (ODC) activity determined. ODC activity in DRG ipsilateral to nerve transection was increased 10–20 fold over contralateral values. Typical ODC activities in ipsilateral and contralateral DRG samples were 6.18±1.4 and 0.31±0.09 pmol¹⁴CO₂ released/h/3DRG, respectively. Systemic administration of single doses of either vincristine (1 mg/kg) or vinblastine (5 mg/kg) immediately prior to axotomy attenuated ODC induction in ipsilateral DRG by 39% and 47%, respectively. A direct inhibition of ODC activity in the DRG appears unlikely since only high concentrations of vinblastine (0.5–1.0 mM) were able to inhibit ODC activity in vitro. We suggest vinca alkaloids inhibit ODC induction as a consequence of distupting retrograde axonal transport. Interruption of this intracellular communication mechanism may be etiologically linked to the distal axon degeneration which follows repetitive exposure to vinca alkaloids and other agents that induce toxic axonal neuropathy.     

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.