Received for publication June 21, 2007.
Revised October 16, 2007.
Accepted for publication November 8, 2007.

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 which has evoked the need to develop innovative approaches to predict and ultimately reverse drug resistance. A prolonged G₂M 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 following 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 G₂M arrest following 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 G₂M arrest in four cell lines tested indicating an active mitotic spindle checkpoint. Gene silencing of BubR1 by siRNA interference reduced PBOX-induced G₂M 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.

Key words: Fluorescence techniques, RNA/siRNA, Mechanisms of cell killing/apoptosis, Cytoskeletal targets