Vinca alkaloids are antimitotic drugs that inhibit microtubule assembly and induce tubulin self-association into coiled spiral aggregates. Previous sedimentation velocity results with vinblastine have been interpreted by a mechanism involving isodesmic ligand-mediated or ligand-mediated plus ligand-facilitated self-association [Lobert et al. (1995) Biochemistry 34, 8050-8060]. In this study, we compare the vincristine- or vinorelbine-induced self-association of porcine brain tubulin with our prior vinblastine studies in the presence of 50 microM GDP or 50 microM GTP. Vincristine demonstrates the highest overall affinity for tubulin, K1K2, and vinorelbine the lowest (vincristine > vinblastine > vinorelbine). These and the first quantitative studies comparing the interaction of a new vinca alkaloid derivative, vinorelbine (Navelbine), with other vinca alkaloids. The relative binding affinities reported here correlate with the weekly drug doses used clinically in cancer chemotherapy, where vincristine is used at the lowest dosages and vinorelbine at the highest. Surprisingly, K1, the affinity of drug for tubulin heterodimers, is identical for all three drugs. When data are fit with the ligand-mediated model, the differences in overall affinity are due to effects on K2, the affinity of liganded heterodimers for spiral polymers. When data are fit with the ligand-mediated plus-facilitated model, affinity differences are also reflected in K3, the binding of the drug to unliganded polymers. We find that GDP enhances self-association in the presence of all three drugs 3-5-fold over GTP. The enhancement is manifested in K2 and K3 and amounts to an average of 0.90 +/= 0.17 kcal/mol. Thus, nucleotide enhancement is linked to the self-association step. Data collected at 5, 25, and 36 degrees C for all three drugs show increased maximum s-20,w values with increasing temperature and are consistent with an entropically driven reaction for the overall process. To investigate these results further, stopped-flow light scattering experiments have been conducted. Relaxation times are longest for the largest vincristine polymers and shortest for the smallest vinorelbine polymers, consistent with a cascade of events corresponding to successive dissociation events from spiral polymers, the larger the polymers the longer the relaxation time. Relaxation times for any single drug decrease with increasing tubulin concentration, consistent with the occurrence of oligomer annealing in addition to the association of liganded heterodimers to the ends of the growing spirals. Relaxation times were used to estimate on and off rates for liganded heterodimer association with spirals, and their ratio gives affinity constants (Kapp) that are independently consistent with K2 estimates from sedimentation velocity results for vinblastine and vinorelbine. For vincristine-induced tubulin polymers, a two-step process is observed with a second relaxation time more than 20-fold longer than times observed for vinblastine or vinorelbine. Sedimentation velocity ...
Vinblastine is an antimitotic drug that inhibits microtubule assembly and induces the self-association of tubulin into coiled spiral aggregates. Previous quantitative binding and sedimentation velocity results have been interpreted by a mechanism involving isodesmic ligand-mediated plus ligand-facilitated self-association [Na, G., & Timasheff, S. N. (1986) Biochemistry 25, 6214-6222, 6222-6228]. In this study, the vinblastine-induced self-association of porcine brain tubulin has been compared in the presence of 50 microM GDP or 50 microM GTP to investigate the role of nucleotides. Experiments at 1-4 microM tubulin in 10 mM Pipes, 1 mM MgSO4, 2 mM EGTA (pH 6.9), and varying concentrations of vinblastine (0.05-70 microM) demonstrate that GDP enhances self-association by 2-4-fold over GTP. In the presence of GDP or GTP, sedimentation velocity data can be best fit by either an indefinite ligand-mediated model or an indefinite ligand-mediated plus ligand-facilitated model. The association constant, K2, for the vinblastine-tubulin complex binding to a polymer is larger when GDP is present, while the association constant, K1, for the binding of vinblastine to tubulin heterodimers is identical in the presence of either nucleotide. The enhancement of K2 by GDP is confirmed by micropartition binding experiments with [3H]vinblastine. The fitting of sedimentation velocity and binding studies gives parameters for the interaction of vinblastine with GTP-tubulin that are identical, within error, to the previous results of Na and Timasheff. van't Hoff analysis of multiple temperature data reveals that this enhancement in the presence of GDP is due to a change in the enthalpy of self-association. Additional results suggest that the interaction of vinblastine with tubulin is identical for all beta-isotypes. Sedimentation velocity experiments in the presence of GDP or GTP show that the vinblastine-induced association of affinity-purified alpha,beta-class III tubulin is identical to that of unfractionated tubulin, although there is a difference in the abilities of unfractionated tubulin and alpha,beta III-tubulin to associate into taxol-stabilized microtubules.
Antimitotic drugs are chemotherapeutic agents that bind tubulin and microtubules. Resistance to these drugs is a major clinical problem. One hypothesis is that the cellular composition of tubulin isotypes may predict the sensitivity of a tumor to antimitotics. Reliable and sensitive methods for measuring tubulin isotype levels in cells and tissues are needed to address this hypothesis. Quantitative measurements of tubulin isotypes have frequently relied upon inferring protein amounts from mRNA levels. To determine whether this approach is justified, protein and mRNA levels of beta-tubulin isotypes from 12 human cancer cell lines were measured. This work focused on only beta-tubulin isotypes because we had readily available monoclonal antibodies for quantitative immunoblots. The percentage of beta-tubulin isotype classes I, II, III, and IVa + IVb mRNA and protein were compared. For beta-tubulin class I that comprises >50% of the beta-tubulin protein in 10 of the 12 cell lines, there was good agreement between mRNA and protein percentages. Agreement between mRNA and protein was also found for beta-tubulin class III. For beta-tubulin classes IVa + IVb, we observed higher protein levels compared to mRNA levels.Beta-tubulin class II protein was found in only four cell lines and in very low abundance. We conclude that quantitative Western blotting is a reliable method for measuring tubulin isotype levels in human cancer cell lines. Inferring protein amounts from mRNA levels should be done with caution, since the correspondence is not one-to-one for all tubulin isotypes.
A diverse group of natural biological compounds bind to microtubules and suppress microtubule dynamics. Here we review the mechanism of microtubule assembly and dynamics as well as structural features that are important for nucleotide binding, GTP hydrolysis and stabilization of longitudinal and lateral protofilament contacts. Specific emphasis is placed upon the polar structure of the microtubule, the exposure of the nucleotide hydrolysis site at the + end and the conformational and configurational plasticity of the microtubule lattice. These features have important implications for the mechanism of dynamic instability and the disruptive action of antimitotic drugs. We then discuss the various classes of tubulin binding drugs emphasizing their site and mode of binding as well as the structural and energetic basis for their effects on microtubule assembly and dynamics. A common feature of tubulin-interacting compounds is a linkage to assembly, either the stabilization of a microtubule lattice by compounds like taxol or epothilone A, or the preferential formation of alternate lattice contacts and polymers at microtubule ends by compounds like colchicine, vinca alkaloids and cryptophycin-52. Finally, we explore the likely possibility that these drugs also disrupt the regulation of microtubule dynamics. Future generations of these compounds may be selectively developed to directly target the proteins that regulate mitotic spindle dynamics.
Common agents currently used in treating metastatic breast cancer are the antimitotics paclitaxel and docetaxel [1,2]. These drugs bind to β-tubulin, a major protein in mitotic spindles, and halt cell division at metaphase. Their effectiveness in cancer chemotherapy is thought to be due to their ability to reduce the dynamics of microtubules in mitotic spindles, thus preventing spindle assembly and interrupting the normal movement of sister chromatids toward the spindle poles [3][4][5][6][7][8][9]. Tubulin, a 100 kDa αβ heterodimer, is structurally heterogeneous, with seven genes encoding α-tubulin and β-tubulin isotypes. The major differences between isotype classes reside in the last 15-20 amino acids of the carboxy-termini et al., licensee BioMed Central Ltd (Print ISSN 1465-5411; Online ISSN 1465-542X). This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. AbstractBackground: Antimitotic chemotherapeutic agents target tubulin, the major protein in mitotic spindles. Tubulin isotype composition is thought to be both diagnostic of tumor progression and a determinant of the cellular response to chemotherapy. This implies that there is a difference in isotype composition between normal and tumor tissues.
The ability of a class of C-20' modified vinca alkaloid congeners to induce tubulin spiral formation was investigated relative to their ability to inhibit microtubule assembly, their cytotoxicity against a leukemic cell line, L1210, and their measured and calculated partition coefficients. These studies were prompted by the observation that the energetics of vinca alkaloid-induced tubulin spiral polymers, or spiraling potential, is inversely related to their clinical dosage and are aimed at the long-term goal of developing the ability to predict the cytotoxic and antineoplastic properties of antimitotic drugs. We demonstrate here that vinca-induced tubulin-spiraling potential is significantly correlated with cytotoxicity against L1210 cells. This is consistent with the size of spirals formed being proportional to the relaxation time for polymer redistribution, the lifetime of cell retention, and effects on microtubule ends and dynamics. Spiraling potential also correlates with calculated but not measured partition coefficients. Surprisingly, spiraling potential does not correlate with the ability to inhibit microtubule formation with purified tubulin or microtubule protein. For the set of C-20' modified compounds studied, the largest inhibitory effects on spiraling potential and cytotoxicity are caused by multiple sites of halogen (-F, -Cl) substitution with the introduction of increased rigidity in the ring. This suggests the C-20' position interacts with a hydrogen bond acceptor or an electrophilic region on the protein that electrostatically disfavors halogen substitutions. These studies are discussed in terms of the cellular mode of action of antimitotic drugs, particularly the importance of microtubule dynamics during mitosis and the factors that regulate those dynamics.
Antimitotic drugs are key components of combination chemotherapy protocols for hematological and solid tumors. The taxanes (e.g., paclitaxel) bind to the β subunit of the tubulin heterodimer and reduce microtubule dynamics, leading to cell cycle arrest in G2/M. The effectiveness of combination chemotherapy is limited by tumor resistance to drugs initially or as a cumulative effect after several cycles of treatment. Because changes in the drug receptor may be linked to drug resistance, we investigated changes in β-tubulin isotypes in response to paclitaxel treatment in MCF7 breast cancer cells. We found that paclitaxel induced a 2-3 fold increase in mRNA for β-tubulin IIA and III genes, TUBB2A, and TUBB3. β-Tubulin class III protein increased; however, β-tubulin class II protein was not detected in these cells. Paclitaxel treatment following pretreatment with actinomycin D showed that the change in β-tubulin class III was due to increased transcription and linked to G2/M arrest. The increase in β-tubulin IIA mRNA was due to both enhanced stability and increased transcription, unassociated with G2/M arrest. We used micro-RNA superarrays to look for changes in families of micro-RNAs that might be linked to drug-induced changes in β-tubulin isotype mRNA and/or protein. We found a significant decrease in the tumor suppressor, miR-100, in MCF7 cells in response to paclitaxel treatment. Transfection of MCF7 cells with miR-100 significantly reduced β-tubulin I, IIA, IIB and V mRNA and prevented paclitaxel-induced increases in β-tubulin isotypes. This is the first report of a micro-RNA that regulates these specific β-tubulin isotype mRNAs.
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