Inhibition of centromere dynamics by eribulin (E7389) during mitotic metaphase.
Eribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase of microtubule dynamic instability in interphase cells, arrests mitosis, and induces apoptosis, suggesting that suppression of spindle microtubule dynamics induces mitotic arrest. To further test this hypothesis, we measured the effects of eribulin on dynamics of centromeres and their attached kinetochore microtubules by time-lapse confocal microscopy in living mitotic U-2 OS human osteosarcoma cells. Green fluorescent protein-labeled centromere-binding protein B marked centromeres and kinetochore-microtubule plus-ends. In control cells, sister chromatid centromere pairs alternated under tension between increasing and decreasing separation (stretching and relaxing). Eribulin suppressed centromere dynamics at concentrations that arrest mitosis. At 60 nmol/L eribulin (2 x mitotic IC(50)), the relaxation rate was suppressed 21%, the time spent paused increased 67%, and dynamicity decreased 35% (but without reduction in mean centromere separation), indicating that eribulin decreased normal microtubule-dependent spindle tension at the kinetochores, preventing the signal for mitotic checkpoint passage. We also examined a more potent, but in tumors less efficacious antiproliferative halichondrin derivative, ER-076349. At 2 x IC(50) (4 nmol/L), mitotic arrest also occurred in concert with suppressed centromere dynamics. Although media IC(50) values differed 15-fold between the two compounds, the intracellular concentrations were similar, indicating more extensive relative uptake of ER-076349 into cells compared with eribulin. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic arrest indicates that the primary mechanism by which eribulin blocks mitosis is suppression of spindle microtubule dynamics.
Okouneva
Tatiana
T
Department of Molecular, Cellular, and Developmental Biology and Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, CA 93106, USA.
Azarenko
Olga
O
Wilson
Leslie
L
Littlefield
Bruce A
BA
Jordan
Mary Ann
MA
D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002503:Centromere / Q000187:drug effects* / Q000378:metabolism*; D051936:Centromere Protein B / Q000378:metabolism; D005663:Furans / Q000737:chemistry / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000494:pharmacology*; D008677:Metaphase / Q000187:drug effects*; D008870:Microtubules / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D008941:Spindle Apparatus / Q000187:drug effects; D013997:Time Factors; D000095662:Polyether Polyketides
D000970:Antineoplastic Agents; D051936:Centromere Protein B; D005663:Furans; D049452:Green Fluorescent Proteins; D006576:Heterocyclic Compounds, 4 or More Rings; D007659:Ketones; C421616:ER 076349; C490954:eribulin; D000095662:Polyether Polyketides