A β-Tubulin Leucine Cluster Involved in Microtubule Assembly and Paclitaxel Resistance

Abstract

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...

Amino acid substitutions at proline 220 of β-tubulin confer resistance to paclitaxel and colcemid

“…Cells transfected with WT HAh1-tubulin cDNA (Fig. 3A) had normal cytoplasmic microtubule networks and mitotic spindles, consistent with previous studies (25,30,31). In the case of P220L and P220V (Fig.…”

“…Cell Lines, Plasmids, and Mutagenesis The CHO tTApuro 6.6a cells used for transfection express a tetracycline-regulated transactivator and were maintained as described previously (25). CHO Cb1 cDNA (Genbank accession no.…”

“…CHO Cb1 cDNA (Genbank accession no. U08342) was modified to encode a COOH-terminal hemagglutinin antigen (HA) epitope tag and cloned into a pTOPneo vector to create pTOP/HAb1 that allows tetracycline-regulated transcription of the HAb1 cDNA (25).…”

“…The high frequency of mutations affecting leucine residues in the H6/H7 loop of h-tubulin in paclitaxel-resistant CHO cells (25) suggested that these leucines might play a unique role in the acquisition of resistance to paclitaxel, a microtubule-stabilizing drug. Moreover, no mutations in this region have yet been shown to confer resistance to drugs such as colcemid that destabilize microtubules.…”

“…Among resistant cell lines with mutations in tubulin, alterations in L215, L217, and L228 of h-tubulin occur at very high frequency (25). These altered residues are located in a loop that connects helix 6 with helix 7 (the H6/H7 loop) as well as in helix 7 itself.…”