Susan Band Horwitz

Promotion of microtubule assembly in vitro by taxol.

TAXOL (Fig. 1) was isolated from the plant Taxus brevifolia (western yew) by Wani et al., who reported that the molecule has antitumour activity in several experimental systems1. In our laboratory we have found that taxol, a low molecular weight neutral compound, completely inhibits division of exponentially growing HeLa cells at low concentrations of drug (0.25 µM) that have no significant effects on DNA, RNA or protein synthesis during a 4-h incubation with the cells. HeLa cells incubated with taxol for 20 h are blocked in late G2 and/or M (ref. 2). We report here that taxol acts as a promoter of calf brain microtubule assembly in vitro, in contrast to plant products such as colchicine and podophyllotoxin, which inhibit assembly. Taxol decreases the lag time for microtubule assembly and shifts the equilibrium for assembly in favour of the microtubule, thereby decreasing the critical concentration of tubulin required for assembly. Microtubules polymerised in the presence of taxol are resistant to depolymerisation by cold (4 °C) and CaCl2 (4 mM).

Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes.

The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the beta subunit of tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six beta-tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa beta-tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific beta-tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.

Mechanisms of Taxol resistance related to microtubules

Since its approval by the FDA in 1992 for the treatment of ovarian cancer, the use of Taxol has dramatically increased. Although treatment with Taxol has led to improvement in the duration and quality of life for some cancer patients, the majority eventually develop progressive disease after initially responding to Taxol treatment. Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients. This review focuses on mechanisms of Taxol resistance that occur directly at the microtubule, such as mutations, tubulin isotype selection and post-translational modifications, and also at the level of regulatory proteins. A review of tubulin structure, microtubule dynamics, the mechanism of action of Taxol and its binding site on the microtubule are included, so that the reader can evaluate Taxol resistance in context.

A role for ferrous ion and oxygen in the degradation of DNA by bleomycin

Abstract An interaction between bleomycin and low concentrations of Fe(II) in the degradation of DNA is reported. Complete conversion of simian virus 40 DNA to acid-soluble products occurs at approximately equimolar levels of Fe(II), bleomycin, and DNA; Fe(III) does not substitute for Fe(II) in this reaction. Anaerobiosis inhibits the observed DNA degradation by bleomycin and Fe(II). Optical spectral studies reveal that an oxygen-labile complex is formed between bleomycin and Fe(II).

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.

Taxol: Mechanisms of Action and Resistance

Information on the mechanisms of action and of resistance to Taxol, as well as new data from our laboratory on the promoter regions of the genes that encode P-glycoprotein in a murine Taxol-resistant cell line, is discussed. Taxol induces the formation of stable bundles of microtubules, thereby interfering with the normal function of cellular microtubules. The drug can induce the multidrug-resistance (MDR) phenotype that includes the overproduction of P-glycoprotein, a membrane glycoprotein that acts as a drug efflux pump. In human tumors resistant to Taxol, P-glycoprotein could be responsible for maintaining the drug below cytotoxic levels. Analyses of the MDR promoters that are involved in P-glycoprotein expression and overproduction revealed an interesting recombination event in a Taxol-resistant cell line. As an important new clinical agent for the treatment of malignancies, Taxol requires further mechanistic investigations at the preclinical level.

Insights into the mechanism of microtubule stabilization by Taxol

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the α/β-tubulin heterodimer, GTP bound to β-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to β-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both β- and α-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography–electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both β-and α-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer–dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography–electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers.

Phase I Clinical and Pharmacokinetic Study of BMS-247550, a Novel Derivative of Epothilone B, in Solid Tumors

Purpose: The purpose of this study was to determine the maximum tolerated dose, toxicity, and pharmacokinetics of BMS-247550 administered as a 1-h i.v. infusion every 3 weeks. Experimental Design: Patients with advanced solid malignancies were premedicated and treated with escalating doses of BMS-247550. Blood sampling was performed to characterize the pharmacodynamics and pharmacokinetics of BMS-247550. Results: Twenty-five patients were treated at six dose levels ranging from 7.4 to 59.2 mg/m2. At 50 mg/m2, 4 of 9 patients (44.4%) had dose-limiting toxicity (neutropenia, abdominal pain/nausea). At 40 mg/m2 (the recommended Phase II dose), 2 of 12 patients (16.7%) had dose-limiting neutropenia. Overall, the most common nonhematological toxicity was fatigue/generalized weakness (grade 3–4 seen in 9.0% of patients), followed by neurosensory deficits manifested as peripheral neuropathy and by gastrointestinal discomfort. At 40 mg/m2, the incidence of grade 3 fatigue, abdominal pain, diarrhea, and neuropathy was 7.7%. Grade 1–2 neuropathy was observed in all patients enrolled and treated at 40 mg/m2. Two patients with paclitaxel-refractory ovarian cancer, one patient with taxane-naïve breast cancer, and another patient with docetaxel-refractory breast cancer had objective partial responses (lasting 6.0, 5.3, 3.0, and 4.5 months, respectively). The mean pharmacokinetic parameter values during course 1 for clearance, volume of distribution, and apparent terminal elimination half-life at the 40 mg/m2 (recommended Phase II dose) dose level were 21 liters/h/m2, 826 liters/m2, and 35 h (excluding one outlier of 516 h), respectively. Values during course 1 and course 2 were similar. Conclusions: The recommended dose for Phase II evaluation of BMS-247550 is 40 mg/m2, although more long-term observations are needed. BMS-247550 has advantages over taxanes in relation to drug resistance and warrants further study.

Antisense oligonucleotides to class III β-tubulin sensitize drug-resistant cells to Taxol

SummaryA major impediment to the successful use of Taxol in the treatment of cancer is the development of drug resistance. The major cellular target of Taxol is the microtubule that is comprised of α- and β-tubulin heterodimers. Binding sites for Taxol have been delineated on the β-tubulin subunit that has six isotypes. We have recently described increased expression of the brain-specific human class III β-tubulin isotype, encoded by the Hβ4 gene, in both Taxol-resistant ovarian tumours and non-small-cell lung cancer cell lines. To evaluate directly the role of the class III β-tubulin isotype in mediating Taxol resistance, antisense phosphorothioate oligodeoxynucleotides (ODN) targeted against various regions of the Hβ4 gene have been designed and examined for their efficacy in reducing Hβ4 gene and protein expression. Taxol-resistant lung cancer cells, A549-T24, which are 17-fold resistant to Taxol and display a fourfold increase in Hβ4 expression compared to the parental A549 cells, were treated with 1 μM antisense ODNs. Two ODNs, AS1 and AS3, were found to reduce mRNA expression by 40–50%, as determined by reverse transcription polymerase chain reaction. A concentration-dependent reduction in Hβ4 mRNA expression was demonstrated with AS1 ODN. Immunofluorescence staining of cells treated with AS1 ODN revealed a decrease in class III protein expression which corresponded to a 39% increase in sensitivity to Taxol (P < 0.005). These findings support an important role for Hβ4 (class III) β-tubulin expression in Taxol resistance and have potential implications for the treatment of Taxol-resistant tumours.

Upregulation of caveolin‐1 and caveolae organelles in Taxol‐resistant A549 cells

Caveolin is a principal component of caveolae membranes. It has been demonstrated that the interaction of the caveolin scaffolding domain with signaling molecules can functionally inhibit the activity of these molecules. Taxol is an antitumor agent that suppresses microtubule dynamics and binds to microtubules thereby stabilizing them against depolymerization. The drug also has been implicated in the induction of apoptosis through activation of components in signal transduction cascades. Here we have investigated the role of caveolin in the development of drug resistance by examining the expression of caveolins in low‐ and high‐level drug‐resistant cell lines. Caveolin‐1, but not caveolin‐2, was upregulated in highly multidrug resistant SKVLB1 cells that express high levels of P‐glycoprotein, and in low‐level Taxol‐resistant A549 cell lines that express low amounts of P‐glycoprotein. Two drug‐resistant A549 cell lines (one 9‐fold resistant to Taxol and the other 1.5‐fold resistant to epothilone B), both of which express no P‐glycoprotein, demonstrate a significant increase in the expression of caveolin‐1. These results indicate that in low‐level epothilone B‐ or Taxol‐resistant A549 cells, increased caveolin‐1 expression occurs independently of P‐glycoprotein expression. Electron microscopic studies clearly demonstrate the upregulation of caveolae organelles in Taxol‐resistant A549 cells. Upregulation of caveolin‐1 expression in drug‐sensitive A549 cells was observed acutely beginning 48 h after incubation with 10 nM Taxol. Thus, caveolin‐1 may play a role in the development of Taxol resistance in A549 cells.