title: BubR1 is required for a sustained mitotic spindle checkpoint arrest in human cancer cells treated with tubulin-targeting pyrrolo-1,5-benzoxazepines.
abstract: Intrinsic or acquired resistance to chemotherapy is a major clinical problem that has evoked the need to develop innovative approaches to predict and ultimately reverse drug resistance. A prolonged G(2)M arrest has been associated with apoptotic resistance to various microtubule-targeting agents (MTAs). In this study, we describe the functional significance of the mitotic spindle checkpoint proteins, BubR1 and Bub3, in maintaining a mitotic arrest after microtubule disruption by nocodazole and a novel series of MTAs, the pyrrolo-1,5-benzoxazepines (PBOXs), in human cancer cells. Cells expressing high levels of BubR1 and Bub3 (K562, MDA-MB-231, and HeLa) display a prolonged G(2)M arrest after exposure to MTAs. On the other hand, cells with low endogenous levels of mitotic spindle checkpoint proteins (SK-BR-3 and HL-60) transiently arrest in mitosis and undergo increased apoptosis. The phosphorylation of BubR1 correlated with PBOX-induced G(2)M arrest in four cell lines tested, indicating an active mitotic spindle checkpoint. Gene silencing of BubR1 by small interfering RNA interference reduced PBOX-induced G(2)M arrest without enhancing apoptotic efficacy. Further analysis demonstrated that PBOX-treated BubR1-depleted cells were both mononucleated and multinucleated with a polyploid DNA content, suggesting a requirement for BubR1 in cytokinesis. Taken together, these results suggest that BubR1 contributes to the mitotic checkpoint induced by the PBOXs.
authors.affiliation: School of Biochemistry and Immunology, Trinity College, Dublin 2, United Kingdom. greeneli@tcd.ie
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Greene
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Campiani
authors.forename: Mark
authors.initials: M
authors.lastname: Lawler
authors.forename: D Clive
authors.initials: DC
authors.lastname: Williams
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Zisterer
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D010077:Oxazepines / Q000494:pharmacology* / Q000627:therapeutic use; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011758:Pyrroles / Q000494:pharmacology* / Q000627:therapeutic use; D008941:Spindle Apparatus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C514160:4-acetoxy-5-(1-(naphthyl))naphtho(2,3-b)pyrrolo(2,1-d)(1,4)oxazepine; D010077:Oxazepines; D011758:Pyrroles; D014404:Tubulin; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; C418868:PBOX-6