Resistance to microtubule-stabilizing drugs involves two events: beta-tubulin mutation in one allele followed by loss of the second allele.
Resistance to Taxol (paclitaxel) or the epothilones (Epo) occurs via the acquisition of point mutations in beta-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with Taxol or Epo A, which harbor distinct beta-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant beta-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant beta-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 beta-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 beta-tubulin and mitotic index by immunofluorescence were performed to correlate the beta-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 beta-tubulin in addition to a beta-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 beta-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.
Wang
Yuefang
Y
Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia, USA.
O'Brate
Aurora
A
Zhou
Wei
W
Giannakakou
Paraskevi
P
D000483:Alleles; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D002896:Chromosomes, Human, Pair 6 / Q000235:genetics; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D034261:Epothilones / Q000494:pharmacology; D005260:Female; D016195:G2 Phase / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019656:Loss of Heterozygosity / Q000187:drug effects / Q000235:genetics; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism*; D008940:Mitotic Index; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D011108:Polymers; D020033:Protein Isoforms / Q000378:metabolism; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism
D000970:Antineoplastic Agents; D034261:Epothilones; D011108:Polymers; D020033:Protein Isoforms; D014404:Tubulin; D017239:Paclitaxel