Differential responses of mitotic spindle pole formation to microtubule-stabilizing agents epothilones A and B at low concentrations

Abstract

We previously reported that the microtubule-stabilizing agent docetaxel induced formation of fragile acentrosomal spindle poles but that structurally related paclitaxel did not. In the present study, we examined whether the microtubule-stabilizing agents epothilones A and B, which are structurally similar, affect the centrosome/spindle pole architecture.We investigated mitotic processes in epothilone A or B-treated human MDA-MB-435 cells, in which the centrosomes, spindle poles and mitotic micro-tubules were simultaneously visualized by GFP-Aurora A kinase. Fluorescence microscopy of metaphase cells indicated that several chromosomes were misaligned away from the metaphase plate when treated with IC(50) concentrations of epothilone A or B (4.5 or 2 nM, respectively), suggesting that microtubule dynamics was impaired. Interestingly, epothilone B induced formation of acentro-somal spindle poles, but this effect was not observed for epothilone A. Live-cell imaging showed that aster-like structures ectopically arose around the nuclear envelope at the onset of mitosis in epothilone B-treated cells and that one of these asters became an acentrosomal spindle pole. Aster-like structures also arose in the presence of epothilone A, but they were merged into centro-some-derived spindle poles during prometaphase and completely disappeared until metaphase. These results indicate that the centro-some/spindle pole integrity is strongly affected by epothilone B but is not greatly affected by epothilone A. Our findings show that the two epothilones cause different cellular responses at equipotent concentrations and suggest that they have different mechanisms of activity in cells.

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

“…Thus, the mechanism of plate disintegration for the two MT stabilizers are quite different. Pole The formation of supernumerary poles may be viewed as a competitive element in the establishment of kinetochore-MT interactions, which is a model recently proposed by Sakaushi et al 37 Centrosomal spindle poles are still present but they are not able to restrict aberrant poles from attempts to organize the chromosomal mass. Although this effect is similar with the two stabilizers, the frequency of the multipolar phenotype is quite different.…”

“…19,33 The outcome of premature aster formation ranges from mild effects on the spindle as seen with epothilone A, 37 where the bipolar spindle is retained, to severe as seen with paclitaxel, 12 epothilone B 12,37 and docetaxel, 19 where multipolar spindles are formed with inhibition of pole separation at higher doses. When compared with these other MT-stabilizing agents, laulimalide favors an intermediate spindle phenotype, as long as the drug is present before nuclear envelope breakdown.…”

Visualization of Aberrant Perinuclear Microtubule Aster Organization by Microtubule-Destabilizing Agents

“…We have constructed a multicolor cell line in which the centrosomes and chromosomes could be simultaneously visualized, 7) and using this cell line, we recently characterized the in vivo effect of the microtubule-stabilizing agents taxanes and epothilones on centrosomes at low concentrations that do not cause mitotic arrest. 8,9) Since centrosomes are the dominant microtubule organizing centers (MTOCs) for spindle formation, it is thought that microtubule-targeting agents disturb the spindle architecture by inhibiting microtubule organization. In the present study, to characterize in detail the involvement of microtubule organization in spindle assembly disturbance, we constructed another multicolor MDA-MB-435 cell line in which the centrosomes, spindle microtubules, and chromosomes can be simultaneously visualized with EGFP-Aurora A, mCherry--tubulin, and ECFP-histone H3.…”

“…7) Live cell imaging and fluorescence microscopy were performed as described previously. 7,9) Time-lapse images of mitosis of this cell line are shown in Fig. 1.…”

Novel microtubule-targeting agents – the epothilones

“…Using human MDA-MB-435 cells and fluorescence microscopy, investigators have noted a biologic difference in the spindle pole effects of epothilone A and of patupilone (Sakaushi et al 2008). These results suggest that small changes in the epothilone structure may have major differences in biological effect.…”