Random mutagenesis of ß-tubulin defines a set of dispersed mutations that confer paclitaxel resistance.

PURPOSE: Previous research showed that mutations in ß1-tubulin are frequently involved in paclitaxel resistance but the question of whether the mutations are restricted by cell-type specific differences remains obscure. METHODS: To circumvent cellular constraints, we randomly mutagenized ß-tubulin cDNA, transfected it into CHO cells, and selected for paclitaxel resistance. RESULTS: A total of 26 ß1-tubulin mutations scattered throughout the sequence were identified and a randomly chosen subset were confirmed to confer paclitaxel resistance using site-directed mutagenesis of ß-tubulin cDNA and transfection into wild-type cells. Immunofluorescence microscopy and biochemical fractionation studies indicated that cells expressing mutant tubulin had decreased microtubule polymer and frequently suffered mitotic defects that led to the formation of large multinucleated cells, suggesting a resistance mechanism that involves destabilization of the microtubule network. Consistent with this conclusion, the mutations were predominantly located in regions that are likely to be involved in lateral or longitudinal subunit interactions. Notably, fourteen of the new mutations overlapped previously reported mutations in drug resistant cells or in patients with developmental brain abnormalities. CONCLUSIONS: A random mutagenesis approach allowed isolation of a wider array of drug resistance mutations and demonstrated that similar mutations can cause paclitaxel resistance and human neuronal abnormalities.

Preclinical pharmacologic evaluation of MST-997, an orally active taxane with superior in vitro and in vivo efficacy in paclitaxel- and docetaxel-resistant tumor models.

PURPOSE: Because resistance to paclitaxel and docetaxel is frequently observed in the clinic, new anti-microtubule agents have been sought. The aim of this study was to evaluate the efficacy and oral activity of a novel taxane (MST-997) in paclitaxel- and docetaxel-resistant tumor models in vitro and in vivo. EXPERIMENTAL DESIGN: Tubulin polymerization assays, immunohistochemistry, and cell cycle analysis was used to evaluate mechanism of action of MST-997. The effect of MST-997 on growth inhibition in a panel of paclitaxel- and docetaxel-resistant cell lines that overexpressed P-glycoprotein (MDR1) or harbored beta-tubulin mutations were assayed in vitro and in murine xenografts. RESULTS: MST-997 induced microtubule polymerization (EC50 = 0.9 micromol/L) and bundling, resulting in G2-M arrest and apoptosis. In addition, MST-997 was a potent inhibitor of paclitaxel- and docetaxel-sensitive tumor cell lines that did not have detectable P-glycoprotein (IC50 = 1.8 +/- 1.5 nmol/L). Minimal resistance (1- to 8-fold) to MST-997 was found in cell lines that either overexpressed MDR1 or harbored point mutations in beta-tubulin. Most notable, MST-997 displayed superior in vivo efficacy as a single i.v. or p.o. dose either partially or completely inhibited tumor growth in paclitaxel- and docetaxel-resistant xenografts. CONCLUSIONS: MST-997 represents a potent and orally active microtubule-stabilizing agent that has greater pharmacologic efficacy in vitro and in vivo than the currently approved taxanes. Our findings suggest that MST-997, which has entered phase I clinical trials, may have broad therapeutic value.

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules.

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.

HTI-286, a synthetic analogue of the tripeptide hemiasterlin, is a potent antimicrotubule agent that circumvents P-glycoprotein-mediated resistance in vitro and in vivo.

Hemiasterlin is a natural product derived from marine sponges that, like other structurally diverse peptide-like molecules, binds to the Vinca-peptide site in tubulin, disrupts normal microtubule dynamics, and, at stoichiometric amounts, depolymerizes microtubules. Total synthesis of hemiasterlin and its analogues has been accomplished, and optimal pharmacological features of the series have been explored. The biological profile of one analogue, HTI-286, was studied here. HTI-286 inhibited the polymerization of purified tubulin, disrupted microtubule organization in cells, and induced mitotic arrest, as well as apoptosis. HTI-286 was a potent inhibitor of proliferation (mean IC(50) = 2.5 +/- 2.1 nM in 18 human tumor cell lines) and had substantially less interaction with multidrug resistance protein (P-glycoprotein) than currently used antimicrotubule agents, including paclitaxel, docetaxel, vinorelbine, or vinblastine. Resistance to HTI-286 was not detected in cells overexpressing the drug transporters MRP1 or MXR. In athymic mice implanted with human tumor xenografts, HTI-286 administered i.v. in saline inhibited the growth of numerous human tumors derived from carcinoma of the skin, breast, prostate, brain, and colon. Marked tumor regression was observed when used on established tumors that were >1 gram in size. Moreover, HTI-286 inhibited the growth of human tumor xenografts (e.g., HCT-15, DLD-1, MX-1W, and KB-8-5) where paclitaxel and vincristine were ineffective because of inherent or acquired resistance associated with P-glycoprotein. Efficacy was also achieved with p.o. administration of HTI-286. These data suggest that HTI-286 has excellent preclinical properties that may translate into superior clinical activity, as well as provide a useful synthetic reagent to probe the drug contact sites of peptide-like molecules that interact with tubulin.

Mutations in alpha- and beta-tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine.

Single-step selections were used to obtain Chinese hamster ovary cell lines resistant to Colcemid and vinblastine. Verapamil was included in the selections to circumvent the isolation of cells with P-glycoprotein-mediated multidrug resistance and thereby enrich for cells with tubulin alterations. The isolated cell lines were 2-fold resistant to the selecting drug, exhibited cross-resistance to other drugs that inhibit microtubule assembly, and had enhanced sensitivity to the microtubule-stabilizing drug paclitaxel. The concomitant resistance to microtubule-destabilizing drugs and enhanced sensitivity to paclitaxel suggested that these cell lines have changes in microtubule assembly. Consistent with this interpretation, drug-resistant cell lines exhibited altered alpha- or beta-tubulin mobility on two-dimensional gels and higher levels (47-54%) of assembled tubulin compared with wild-type (39%) or paclitaxel-resistant cells (25%). Some drug-resistant cells also had bundled microtubules as judged by immunofluorescence. Genomic sequencing of 11 drug-resistant cell lines predicted five different alterations (D45Y, C211F, D224N, S234N, and K350N) in beta-tubulin and four different alterations (H283Y, E55K, A383V, and R390C) in alpha-tubulin. The amino acid substitutions are dispersed on the primary and tertiary structures of tubulin and, together with the other mutant properties, argue against a mechanism involving changes in drug binding. Rather, we propose that the alterations in alpha- and beta-tubulin increase microtubule stability by promoting longitudinal interdimer and intradimer interactions and/or lateral interactions between protofilaments. This enhanced stability of microtubules increases their resistance to drugs that inhibit assembly.

Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin.

HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [(3)H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [(14)C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major beta-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in alpha-tubulin that substitutes Ser for Ala(12) near the nonexchangeable GTP-binding site of alpha-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala(12) mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of alpha-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.

Paclitaxel resistance by random mutagenesis of α-tubulin.

Many mammalian ß-tubulin mutations that confer paclitaxel resistance have been characterized, but little is currently known about the role of α-tubulin mutations in drug resistance. Previous studies using two-dimensional gel electrophoresis showed that α-tubulin mutations occur with a frequency equal to ß-tubulin mutations among CHO cells selected for resistance to paclitaxel but the identities of those mutations are largely unknown. We have now sequenced the major α-tubulin gene in several paclitaxel resistant CHO cell lines with lesions in genomic DNA and identified five mutations that predominately affect the amino terminal part of the protein. We also used random mutagenesis and transfection of α-tubulin cDNA to select further paclitaxel resistant mutants in an effort to remove genomic constraints that may limit the diversity of mutations. This approach led to the identification of 16 additional mutations that were distributed throughout the α-tubulin sequence. The mutations were confirmed as sufficient to confer resistance by site-directed mutagenesis, and they acted by a mechanism that involved reductions in microtubule assembly. One mutation prevented the acetylation of α-tubulin but otherwise produced a phenotype similar to the other mutations. A scan of the literature revealed that a significant number of drug resistance mutations overlap or lie close to lesions that have been reported in patients with brain disorders suggesting that alterations in microtubule assembly underlie both cellular resistance and developmental defects.

Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin.

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.

Expression of class III beta-tubulin reduces microtubule assembly and confers resistance to paclitaxel.

Human brain and testis specific betaIII-tubulin was amplified from a cDNA library, modified to encode a C-terminal hemagglutinin antigen epitope tag, and cloned into a vector that allows tetracycline regulated expression in mammalian cells. Immunofluorescence analysis of transfected Chinese hamster ovary cells demonstrated that expressed HA-tagged betaIII-tubulin is able to assemble with endogenous tubulin into microtubules even though betaIII-tubulin is not a normal constituent of these cells. A stable G418-resistant clone with moderate HAbetaIII-tubulin expression displayed weak (1.5-2-fold) resistance to paclitaxel. A second clone with higher HAbetaIII-tubulin expression could not grow unless tetracycline was present to repress transcription of the transfected cDNA. Analysis of cellular microtubules in each of these clones indicated that incorporation of HAbetaIII-tubulin led to a significant expression-dependent decrease in assembled tubulin. Paclitaxel resistant cells were also directly selected from the transfected cell population using a paclitaxel concentration 4 times higher than the minimum toxic dose. Few cells were able to survive the selection and they grew very slowly. Western blot analysis of these resistant cells revealed very high HAbetaIII-tubulin expression that led to almost complete replacement of endogenous beta-tubulin at steady state. Transfected betaIII-tubulin with no epitope tag behaved in a very similar fashion indicating that presence of the HA tag had no discernible functional effect. The results demonstrate that betaIII-tubulin diminishes microtubule assembly, is toxic when present at high levels, but is able to confer weak resistance to paclitaxel when expressed at moderate levels in mammalian cells.