title: Suppression of centromere dynamics by Taxol in living osteosarcoma cells.
abstract: Taxol potently blocks mitosis at the transition from metaphase to anaphase, leading to apoptosis in many types of tumor cells. However, the precise mechanism of action of Taxol is not understood. Here we have tested the hypothesis that a primary mechanism of action of Taxol involves suppression of spindle microtubule dynamics. We have used centromere-binding protein B coupled to green fluorescent protein as a marker for the kinetochores and centromeres of chromosomes and analyzed the effects of low Taxol concentrations on the dynamics of centromeres during metaphase of mitosis in living human osteosarcoma (U2OS) cells by quantitative time-lapse confocal microscopy. In the absence of Taxol, the centromere pairs on attached sister chromatids alternately stretch apart and relax back together approximately 1.2 times/min due to tension on the kinetochores produced by the spindle microtubules (referred to here as centromere dynamics). We found that 50-100 nM Taxol significantly suppressed centromere dynamics. For example, Taxol reduced the mean separation distance between the sister centromeres from 0.73 to 0.65 microm, a distance equivalent to that observed in the complete absence of microtubules. The frequency of transitions between stretching and relaxing was also significantly diminished by Taxol (by 27%-35%). The suppressive effects of Taxol on centromere dynamics were associated with maximal accumulation of cells at mitosis (63%), a >90% block of the metaphase/anaphase transition, and complete inhibition of cell proliferation. The data strongly support the idea that the inhibition of centromere dynamics by Taxol prevents silencing of the mitotic spindle surveillance (checkpoint) mechanism. Because Taxol strongly suppresses microtubule dynamics, the data also indicate that centromere dynamics can be accounted for by microtubule dynamics and may not require significant energetic contributions from microtubule motors. The strict correlation between the degree of suppression of centromere dynamics by Taxol and the degree of mitotic block strongly indicates that the primary mechanism responsible for the mitotic block by Taxol in U2OS cells involves suppression of the polymerization dynamics of kinetochore microtubules.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California 93106, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kelling
authors.forename: Kevin
authors.initials: K
authors.lastname: Sullivan
authors.forename: Leslie
authors.initials: L
authors.lastname: Wilson
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Jordan
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001324:Autoantigens; D001859:Bone Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002503:Centromere / Q000187:drug effects* / Q000378:metabolism; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects; D008938:Mitosis / Q000187:drug effects; D012516:Osteosarcoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017239:Paclitaxel