Resistance to Microtubule-Stabilizing Drugs Involves Two Eventse: β-Tubulin Mutation in One Allele Followed by Loss of the Second Allele

Resistance to paclitaxel (PTX) or the epothilones (Epo) occurs via the acquisition of point mutations in β-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with PTX or Epo A which harbor distinct β-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant β-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant β-tubulin sequence exhibited 30 to 50-fold drug resistance. The drug-sensitive parental 1A9 ovarian carcinoma cell line and the drug resistant clones (1A9-A8, 1A9-PTX10 and 1A9-PTX22) were evaluated for loss of heterozygosity (LOH) for β-tubulin (6p25) by single nucleotide polymorphism (SNP) and fluorescent in situ hybridization (FISH) analyses. Functional assays such as drug-induced tubulin polymerization, cell cycle analysis by FACS, DNA sequencing for β-tubulin and mitotic index by immunofluorescence were performed to correlate the β-tubulin LOH status with drug response in the early- and late-step drug-resistant clones. Late-step drug resistant clones revealed LOH in one allele for wt b-tubulin in addition to a β-tubulin mutation in the other allele leading to increased levels of drug resistance, while the early-step clones that contained both a wt and a mutant b-tubulin allele were considerably less drug resistant. The LOH and functional assays revealed cell response that was proportional to the tubulin gene and heterozygosity status. Acquired tubulin mutations in conjunction with LOH for the wt tubulin resulted in a highly resistant phenotype, revealing a new mechanism for taxane resistance.