Paclitaxel resistance by random mutagenesis of α-tubulin.

Many mammalian β-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 β-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.