Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53

Using time-lapse confocal microscopy and enhanced green fluorescent protein-tubulin transfected MCF-7 cells, we found that a tubulin-targeted antimitotic agent, benomyl at its half-maximal proliferation inhibitory concentration (5 mu M) strongly suppressed the rate and extent of growing and shortening excursions of individual microtubules in MCF-7 cells without noticeably depolymerizing the microtubule network or decreasing the polymerized mass of tubulin. Further, benomyl treatment caused an increase in the acetylation level of microtubules suggesting that it stabilizes microtubules. Under the conditions that suppressed the dynamic instability, a sharp increase in the nuclear accumulation of p53 in MCF7 cells was observed in the presence of benomyl. Up regulation of bax and the increased nuclear accumulation of p21 upon benomyl treatment confirmed the activation of p53. Cisplatin caused an increase in the translocation of p53 into the nucleus in the presence of lower effective concentrations of benomyl while a decrease in the nuclear accumulation of p53 was observed in the presence of high concentrations of benomyl suggesting that the stabilized microtubules assist in the nuclear transportation of p53. Furthermore, increased localization of the light chain of the minus end directed motor protein dynein was detected on the microtubules in the benomyl-treated-cells indicating that the suppression of microtubule dynamics may influence the binding of dynein on the microtubules and dynein-mediated cargo transport. Together the data indicate that benomyl inhibits mitosis primarily by suppressing the dynamic instability of microtubules and support the hypothesis that the kinetic stabilization of microtubules enhances the microtubule-mediated transport of p53 into the nucleus. (c) 2008 Elsevier Inc. All rights reserved.