Analysis of ␤-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged ␤-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin ␤-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.Paclitaxel is the prototype for a novel class of agents that inhibit cells in mitosis by promoting and stabilizing microtubule assembly. Early studies with this compound demonstrated that it binds to microtubules in a 1:1 stoichiometry with tubulin heterodimers (1) and inhibits microtubule disassembly. It is also able to induce microtubule assembly both in vitro and in vivo and induces microtubule bundle formation in treated cells (2, 3). Recent interest in this and related compounds has been fueled by clinical studies demonstrating remarkable activity of paclitaxel against a number of malignant diseases (reviewed in Ref. 4). Although still in clinical trials, the demonstrated activity of paclitaxel in phase II studies has led to FDA approval for its use in refractory cases of breast and ovarian cancer. As more patients are treated with this drug, clinical resistance is expected to become an increasingly significant problem.The mechanisms by which tumor cells acquire resistance to paclitaxel are not fully understood. Cell culture studies have shown that paclitaxel is a substrate for the multidrug resistance pump (gP170), 1 and cells selected for high levels of resistance to the drug have increased gP170 (reviewed in Ref. 5). Nevertheless, it has yet to be demonstrated that this mechanism is significant in paclitaxel refractory tumors. Indeed, the remarkable efficacy of paclitaxel in early clinical studies of patients who were pretreated with Adriamycin, a well known substrate for gP170, argues that the multidrug resistance (mdr) phenotype may not be as clinically prevalent as had initially been anticipated (4).Additional mechanisms of resistance to paclitaxel have been reported. For example, several laboratories have provided evidence that changes in the expression of sp...

.-In pressure-overload cardiac hypertrophy, microtubule network densification is one cause of contractile dysfunction. Cardiac transcriptional upregulation of ␤ 1-tubulin rather than the constitutive ␤ 4-tubulin and of microtubuleassociated protein (MAP)4 accompanies hypertrophy, with extensive microtubule decoration by MAP4. Because MAP4 stabilizes microtubules, and because the isoform-variable carboxy terminus of ␤-tubulin binds to MAP4, we wished to determine whether one or both of these proteins has etiologic significance for cardiac microtubule network densification. Recombinant adenoviruses encoding ␤ 1-tubulin, ␤4-tubulin, and MAP4 were used to infect isolated cardiocytes. Overexpressed MAP4 caused a shift of tubulin dimers to the polymerized fraction and formation of a dense, stable microtubule network. Overexpressed ␤ 1-or ␤4-tubulin had neither any independent effect on these variables nor any effect additive to that of simultaneously overexpressed MAP4. Results from transgenic mice with cardiac overexpression of ␤ 1-tubulin or MAP4 were confirmatory, but unlike the effects of brief adenovirus-mediated MAP4 overexpression in isolated cardiocytes, MAP4 transgenic hearts showed a marked increase in total ␣-and ␤-tubulin. Thus MAP4 overexpression caused increased tubulin expression, formation of stable microtubules, and altered microtubule network properties, such that MAP4 upregulation may be one cause for the dense, stable microtubule network characteristic of pressure-overloaded, hypertrophied cardiocytes. myocardium; hypertrophy; adenovirus ONE REASON for the contractile dysfunction characteristic of severe pressure-overload cardiac hypertrophy is increased density of the cellular microtubule network (41,42). This imposes a viscous load on active myofilaments, such that structural damping via intracellular frictional dissipation impedes sarcomere shortening (38). This is accompanied by persistent increases in ␣-and ␤-tubulin on both the mRNA and protein levels (37). Thus increased synthesis of ␣␤-tubulin heterodimers is one cause for the greater cardiac microtubule network density. However, given that the ␣␤-tubulin heterodimer-microtubule system is in dynamic equilibrium, enhanced microtubule stability is a potentially synergistic second mechanism for microtubule network densification. Indeed, we (34) found that the microtubules of pressure-hypertrophied cardiocytes demonstrate markedly enhanced stability, in which microtubule stabilization begins very shortly after cardiac pressure overloading and persists indefinitely thereafter.We then turned to two possible bases for this microtubule stabilization. The first of these was the role of microtubule-associated proteins (MAPs), which are known to regulate microtubule stability (11,23,26,28,44). Here, we focused on MAP4, the dominant structural MAP of the heart (29, 34), and found on both the mRNA and protein levels striking MAP4 upregulation in hypertrophied cardiocytes (34). This was associated with extensive microtubule decoration with MAP4, which wa...

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.

Increased microtubule density, through viscous loading of active myofilaments, causes contractile dysfunction of hypertrophied and failing pressure-overloaded myocardium, which is normalized by microtubule depolymerization. We have found this to be based on augmented tubulin synthesis and microtubule stability. We show here that increased tubulin synthesis is accounted for by marked transcriptional up-regulation of the ␤1-and ␤2-tubulin isoforms, that hypertrophic regulation of these genes recapitulates their developmental regulation, and that the greater proportion of ␤1-tubulin protein may have a causative role in the microtubule stabilization found in cardiac hypertrophy.When under pathological circumstances the heart is forced to eject blood against an increased impedance, the terminally differentiated cardiac muscle cell, or cardiocyte, responds by hypertrophic growth (1). The resultant increase in muscle mass constitutes the basic compensatory cardiac response to sustained hemodynamic overloading, but this initial compensation is frequently vitiated by a progressive decline in cardiocyte contractile function (2), so that congestive heart failure ensues.We have found that this cardiocyte contractile defect is caused by increased density of the cellular microtubule network (3), which imposes a viscous load on the shortening sarcomeres during contraction (4). Thus, microtubule depolymerization in hypertrophied cardiocytes restores normal cellular contractile function, and induced microtubule hyperpolymerization in normal cardiocytes causes these cells to exhibit the same contractile abnormality found in hypertrophied cells (3).The ␣␤-tubulin heterodimer-microtubule system is in a dynamic steady state. Therefore, in attempting to uncover the cause of increased microtubule density in hypertrophied cardiocytes, we focused on increased tubulin synthesis (5) and thus microtubule formation as well as on increased stability of the microtubules once formed. With respect to the latter, we have indeed found marked stabilization of the microtubule network in hypertrophied cardiocytes associated with a substantial increase in the predominant microtubule-associated protein of the heart, MAP4 1 (6). Although recent data (7, 8) put into question the previously accepted role of MAP4 in microtubule assembly and stability for some cell types, the muscle-specific variant of MAP4 appears to play a role in striated muscle (9). Given that MAPs and the expressed proteins of the ␤-tubulin multigene family exhibit coordinate developmental regulation (10) and that the latter may via their isoform-variable carboxyl-terminal domain confer differing MAP binding affinity and microtubule stability after assembly (11), the question of whether there is differential regulation of the members of the ␤-tubulin multigene family during cardiac hypertrophy assumed pivotal importance. EXPERIMENTAL PROCEDURESRight Ventricular Pressure Overloading-Pressure overload hypertrophy of the feline right ventricle (RV) was created (12) by placement of a 2.9-...

Recent studies have suggested a correlation between increased expression of specific beta-tubulin isotypes and paclitaxel resistance in drug-selected cell lines. In an attempt to establish a causal link, we have transfected Chinese hamster ovary cells with cDNAs encoding epitope-tagged class I, II, and IVb beta-tubulins, as well as a class I beta-tubulin with a mutation previously characterized in a paclitaxel resistant mutant. To eliminate possible toxicity that might be associated with overexpression of non-native tubulin, each of the cDNAs was placed under the control of a tetracycline regulated promoter. All transfected cDNAs produced assembly competent tubulin whose synthesis could be turned off or on by the presence or absence of tetracycline. Production of betaI, betaII, or betaIVb tubulin had no effect on the sensitivity of the cells to paclitaxel, but production of the mutant betaI-tubulin conferred clear resistance to the drug. We conclude from these experiments that simple overexpression of class I, II, or IVb isoforms of beta-tubulin is insufficient to confer resistance to paclitaxel.