Kathy Kamath

Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics

Microtubule dynamics are crucial for mitotic spindle assembly and chromosome movement. Suppression of dynamics by Taxol appears responsible for the drugs potent ability to inhibit mitosis and cell proliferation. Although Taxol is an important chemotherapeutic agent, development of resistance limits its efficacy. To examine the role of microtubule dynamics in Taxol resistance, we measured the dynamic instability of individual rhodamine-labeled microtubules in Taxol-sensitive and -resistant living human cancer cells. Taxol-resistant A549-T12 and -T24 cell lines were selected from a human lung carcinoma cell line, A549. They are, respectively, 9- and 17-fold resistant to Taxol and require low concentrations of Taxol for proliferation. We found that microtubule dynamic instability was significantly increased in the Taxol-resistant cells. For example, with A549-T12 cells in the absence of added Taxol, microtubule dynamicity increased 57% as compared with A549 cells. The length and rate of shortening excursions increased 75 and 59%, respectively. These parameters were further increased in A549-T24 cells, with overall dynamicity increasing by 167% compared with parental cells. Thus, the decreased Taxol-sensitivity of these cells can be explained by their increased microtubule dynamics. When grown without Taxol, A549-T12 cells were blocked at the metaphase/anaphase transition and displayed abnormal mitotic spindles with uncongressed chromosomes. In the presence of 2–12 nM Taxol, the cells grew normally, suggesting that mitotic block resulted from excessive microtubule dynamics. These results indicate that microtubule dynamics play an important role in Taxol resistance, and that both excessively rapid dynamics and suppressed dynamics impair mitotic spindle function and inhibit proliferation.

βIII-Tubulin Induces Paclitaxel Resistance in Association with Reduced Effects on Microtubule Dynamic Instability

The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the βIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the β-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that βIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either βI- or βIII-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two β-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing βIII-tubulin than in cells overexpressing similar levels of βI-tubulin (suppressed 47%). The results suggest that overexpression of βIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.

How do microtubule-targeted drugs work? An overview.

The importance of microtubules in mitosis makes them a superb target for a group of highly successful, chemically diverse anticancer drugs. Knowledge of the mechanistic differences among the many drugs of this class is vital to understanding their tissue and cell specificity, the development of resistance, the design of novel improved drugs, optimal scheduling of treatment, and potential synergistic combinations. This overview covers microtubule assembly dynamics, the exquisite regulation of microtubule dynamics in cells by endogenous regulators, the important role of microtubule dynamics in mitosis, the diversity and number of microtubule-targeted drugs undergoing clinical development, the antimitotic mechanisms of microtubule-targeted drugs with emphasis on suppression of microtubule dynamics by vinblastine and taxol, the role of drug uptake and retention in the efficacy of microtubule-targeted drugs, and the anti-angiogenic and vascular-disrupting mechanisms of microtubule targeted drugs. In view of the success of this class of drugs, it has been argued that microtubules represent the single best cancer target identified to date, and it seems likely that drugs in this class will continue to remain an important chemotherapeutic class of drugs even as more selective chemotherapeutic approaches are developed.

Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978–1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC50s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G2-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.

FTDP-17 Mutations Compromise the Ability of Tau to Regulate Microtubule Dynamics in Cells

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), ΔK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.

Microtubule Dynamics, Mitotic Arrest, and Apoptosis: Drug-Induced Differential Effects of βIII-Tubulin

Overexpression of βIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of βIII-tubulin expression hypersensitized non–small cell lung cancer cells to TBAs. To determine whether βIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of βIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non–small cell lung cancer cells stably expressing green fluorescent protein-βI-tubulin. Interphase cells were examined at three vincristine and paclitaxel concentrations that (a) inhibited cell proliferation, (b) induced 5% to 10% mitotic arrest, and (c) induced 30% to 40% mitotic arrest. In the absence of either drug, βIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC50), overall microtubule dynamicity was significantly suppressed in βIII-tubulin knockdowns (−31.2%) compared with controls (−6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of βIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (≥40 nmol/L vincristine; ≥20 nmol/L paclitaxel), βIII-tubulin knockdown resulted in significantly reduced suppressive effects on microtubule dynamicity with little or no further increase in mitotic arrest, compared with control cells. Importantly, apoptosis was markedly increased by βIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that βIII-tubulin knockdown enhances the effectiveness of TBAs through two mechanisms: suppression of microtubule dynamics at low drug concentrations and a mitosis-independent mechanism of cell death at higher drug concentrations. Mol Cancer Ther; 9(5); 1339–48. ©2010 AACR.

Drug resistance associated with loss of p53 involves extensive alterations in microtubule composition and dynamics.

In the present study, we compared the dynamics and composition of microtubules in cell lines derived from the human breast adenocarcinoma MCF-7 containing either the wild-type p53 (wt-p53; MN1) or a dominant-negative variant of p53 gene (mut-p53; MDD2). Mut-p53 cells were significantly resistant to the cytotoxicity of the microtubule-targeted drugs (vinca alkaloids and taxanes), as compared with wt-p53 cells. Studies by high-resolution time-lapse fluorescence microscopy in living cells indicated that the dynamics of microtubules of mut-p53 cells were altered in complex ways and were significantly increased as compared with microtubules in wt-p53 cells. The percentage of time microtubules spent in growing and shortening phases increased significantly, their catastrophe frequency increased, and their overall dynamicity increased by 33%. In contrast, their shortening rate and the mean length shortened decreased. Cells containing mut-p53 displayed increased polymerisation of tubulin, increased protein levels of the class IV β-tubulin isotype, STOP and survivin, and reduced protein levels of class II β-tubulin isotype, MAP4 and FHIT. We conclude that p53 protein may contribute to the regulation of microtubule composition and function, and that alterations in p53 function may generate complex microtubule-associated mechanisms of resistance to tubulin-binding agents.

2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules

2-Methoxyestradiol (2ME2), a metabolite of estradiol-17β, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5–100 μmol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 μmol/L 2ME2. However, with microtubule-associated protein–containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 μmol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 μmol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC50 for mitotic arrest (1.2 μmol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro, and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se. [Mol Cancer Ther 2006;5(9):2225–33]

Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

Determination of microtubule dynamic instability in living cells.

The precise regulation of microtubules and their dynamics is critical for cell cycle progression, cell signaling, intracellular transport, cell polarization, and organismal development. For example, mitosis, cell migration, and axonal outgrowth all involve rapid and dramatic changes in microtubule organization and dynamics. Microtubule-associated proteins (MAPs) such as MAP2 and tau (Bunker et al., 2004; Dhamodharan and Wadsworth, 1995) and microtubule-interacting proteins such as stathmin, the kinesin MCAK, and EB1 (Cassimeris, 1999; Moore and Wordeman, 2004; Ringhoff and Cassimeris, 2009; Rusan et al., 2001) as well as numerous clinically approved or experimental anti-mitotic drugs including the taxanes, vinca alkaloids, and colchicine-like compounds modulate microtubule dynamic in cells (Jordan, 2002; Jordan and Kamath, 2007). In this chapter, we describe methods to analyze the dynamic instability of microtubules in living cells by microscopy of microinjected or expressed fluorescent tubulin, time-lapse microscopy, and analysis of time-dependent microtubule length changes.