Leslie Wilson

Microtubules as a target for anticancer drugs

Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy. Microtubule-targeted drugs, including paclitaxel and Vinca alkaloids, were previously considered to work primarily by increasing or decreasing the cellular microtubule mass. Although these effects might have a role in their chemotherapeutic actions, we now know that at lower concentrations, microtubule-targeted drugs can suppress microtubule dynamics without changing microtubule mass; this action leads to mitotic block and apoptosis. In addition to the expanding array of chemically diverse antimitotic agents, some microtubule-targeted drugs can act as vascular-targeting agents, rapidly depolymerizing microtubules of newly formed vasculature to shut down the blood supply to tumours.

Microtubule dynamics: taking aim at a moving target

Antitumor drugs of the vinca alkaloid and taxane classes function by suppressing the dynamics of microtubules in spindles, blocking cell division at metaphase. The drugs bind to various sites on the tubulin dimer and at different positions within the microtubule, suggesting that there are many unexplored targets for the design of novel drugs of this type.

MICROTUBULE TREADMILLING : WHAT GOES AROUND COMES AROUND

“Ill see it when I believe it”u2003Daniel Mazia

Maytansinoid-Antibody Conjugates Induce Mitotic Arrest by Suppressing Microtubule Dynamic Instability

Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability. Mol Cancer Ther; 9(10); 2700–13. ©2010 AACR.

THE USE AND ACTION OF DRUGS IN ANALYZING MITOSIS

Publisher Summary This chapter describes the use of drugs in studies of mitosis and their mode of action at a biochemical/mechanistic level and present general guidelines for using the drugs as tools. The chapter presents a brief overview of microtubule assembly dynamics and summarizes the current understanding of (i) the binding of vinblastine, colchicine, nocodazole, and taxol to tubulin and microtubules; (ii) the effects of the drugs on microtubule polymerization and dynamics in vitro and in cells; (iii) the levels and kinetics of cellular accumulation and loss of the compounds; (iv) the drug concentration dependence for effects on mitotic spindle organization; and (v) the advantages and disadvantages of using a particular drug for the study of microtubule assembly and dynamics. The chapter also describes the methods for measuring drug uptake into cultured cells, and summarizes some practical guidelines for drug use. First of all, one has to determine the effects of the drug on spindle microtubule organization and mitosis over a range of concentrations in the cells of choice. One should also ensure that the drug is completely solubilized and should determine the effects of varying the duration of drug incubation.

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser16, Ser25, Ser38, and Ser63) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser16 or Ser63, and doubly phosphorylated at Ser25 and Ser38, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser16 or Ser63 strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser25 and Ser38 did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser16 and Ser63 and support the hypothesis that selective targeting by Ser16-specific or Ser63-specific kinases provides complimentary mechanisms for regulating microtubule function.

Mechanism of action of the microtubule-targeted antimitotic depsipeptide tasidotin (formerly ILX651) and its major metabolite tasidotin C-carboxylate.

Tasidotin (ILX-651), an orally active synthetic microtubule-targeted derivative of the marine depsipeptide dolastatin-15, is currently undergoing clinical evaluation for cancer treatment. Tasidotin inhibited proliferation of MCF7/GFP breast cancer cells with an IC(50) of 63 nmol/L and inhibited mitosis with an IC(50) of 72 nmol/L in the absence of detectable effects on spindle microtubule polymer mass. Tasidotin inhibited the polymerization of purified tubulin into microtubules weakly (IC(50) approximately 30 micromol/L). However, it strongly suppressed the dynamic instability behavior of the microtubules at their plus ends at concentrations approximately 5 to 10 times below those required to inhibit polymerization. Its major actions were to reduce the shortening rate, the switching frequency from growth to shortening (catastrophe frequency), and the fraction of time the microtubules grew. In contrast with all other microtubule-targeted drugs thus far examined that can inhibit polymerization, tasidotin did not inhibit the growth rate. In contrast to stabilizing plus ends, tasidotin enhanced microtubule dynamic instability at minus ends, increasing the shortening length, the fraction of time the microtubules shortened, and the catastrophe frequency and reducing the rescue frequency. Tasidotin C-carboxylate, the major intracellular metabolite of tasidotin, altered dynamic instability of purified microtubules in a qualitatively similar manner to tasidotin but was 10 to 30 times more potent. The results suggest that the principal mechanism by which tasidotin inhibits cell proliferation is by suppressing spindle microtubule dynamics. Tasidotin may be a relatively weak prodrug for the functionally active tasidotin C-carboxylate.

Tau isoform-specific modulation of kinesin-driven microtubule gliding rates and trajectories as determined with tau-stabilized microtubules.

We have utilized tau‐assembled and tau‐stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin‐driven MT gliding. MTs were assembled in the presence of either 3‐repeat tau (3R tau) or 4‐repeat tau (4R tau) at tau:tubulin dimer molar ratios that approximate those found in neurons. MTs assembled with 3R tau glided at 31.1 μm/min versus 25.8 μm/min for 4R tau, a statistically significant 17% difference. Importantly, the gliding rates for either isoform did not change over a fourfold range of tau concentrations. Further, tau‐assembled MTs underwent minimal dynamic instability behavior while gliding and moved with linear trajectories. In contrast, MTs assembled with taxol in the absence of tau displayed curved gliding trajectories. Interestingly, addition of 4R tau to taxol‐stabilized MTs restored linear gliding, while addition of 3R tau did not. The data are consistent with the ideas that (i) 3R and 4R tau‐assembled MTs possess at least some isoform‐specific features that impact upon kinesin translocation, (ii) tau‐assembled MTs possess different structural features than do taxol‐assembled MTs, and (iii) some features of tau‐assembled MTs can be masked by prior assembly by taxol. The differences in kinesin‐driven gliding between 3R and 4R tau suggest important features of tau function related to the normal shift in tau isoform composition that occurs during neural development as well as in neurodegeneration caused by altered expression ratios of otherwise normal tau isoforms.

A Molecular and Structural Mechanism for G Protein-mediated Microtubule Destabilization

The heterotrimeric, G protein-coupled receptor-associated G protein, Gαs, binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activated form of Gαs binds tubulin with a KD of 100 nm, stimulates tubulin GTPase, and promotes microtubule dynamic instability. Moreover, the data reveal that the α3–β5 region of Gαs is a functionally important motif in the Gαs-mediated microtubule destabilization. Indeed, peptides corresponding to that region of Gαs mimic Gαs protein in activating tubulin GTPase and increase microtubule dynamic instability. We have identified specific mutations in peptides or proteins that interfere with this process. The data allow for a model of the Gαs/tubulin interface in which Gαs binds to the microtubule plus-end and activates the intrinsic tubulin GTPase. This model illuminates both the role of tubulin as an “effector” (e.g. adenylyl cyclase) for Gαs and the role of Gαs as a GTPase activator for tubulin. Given the ability of Gαs to translocate intracellularly in response to agonist activation, Gαs may play a role in hormone- or neurotransmitter-induced regulation of cellular morphology.

Nanoscale Assembly in Biological Systems: From Neuronal Cytoskeletal Proteins to Curvature Stabilizing Lipids

The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of the interactions, structures, and structure-function correlations remain poorly understood. We describe how three-dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter-ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT-associated-protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio- nanotechnology. In another set of experiments we describe lipid-protein-nanotubes, and lipid nano-tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio-nanotechnology.