Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis

Abstract

A subset of mutant cell lines selected for resistance to the antitumor drug paclitaxel are unable to progress normally through mitosis unless the drug is present in the growth medium. Without paclitaxel the cells form defective spindles, undergo aberrant mitoses, fail to complete cell division and eventually die. Analysis of these drug-dependent cells revealed a low amount of microtubule polymer and less tubulin production than wild-type cells. Ribonuclease protection experiments indicated that the decreased tubulin protein was due to decreased tubulin mRNA. Enhancing microtubule assembly by treating the cells with paclitaxel, restored tubulin to levels comparable with those of paclitaxel-treated wild-type cells, which demonstrated that the drug-dependent cells do not have a permanent impairment in their capacity to synthesize tubulin. Paclitaxel-resistant (but not dependent) cells have a smaller reduction in microtubule polymer with little or no decrease in tubulin production, whereas colcemidresistant cells have increased microtubule assembly but also exhibit little or no change in tubulin production. Finally,a mutant cell line producing an unstable β-tubulin protein has normal growth as well as normal synthesis and polymerization of tubulin, despite an approximately 30% decrease in steady state tubulin content. These studies establish a lower limit of tubulin assembly needed for cell survival and indicate that tubulin assembly must fall below this point to trigger a significant decrease in tubulin synthesis.