Abstract 1317: The metaphase checkpoint complex is controlled by events downstream to spindle assembly and microtubule attachment to the kinetochore

Abstract

Abstract Aneuploidy is a hallmark of cancer that frequently arises from cell cycle errors. The risks of such errors are minimized due to cell cycle checkpoints which lead to cell cycle arrests that are not overcome unless specific determinants of a normally functioning cell cycle machinery are verified. The metaphase checkpoint is especially important, as defects in mitosis can lead to severe aneuploidy due to abnormal chromosome segregation. Much of what is known about this checkpoint comes from work based on drug-induced defects to the mitotic spindle that artificially arrests cells in prometaphase or metaphase. This work led to the concept that cell cycle arrest at the metaphase checkpoint can be triggered by defects in microtubule assembly or attachment to the kinetochore. Treatment with nocodazole, which prevents microtubule assembly, induces this arrest. However, it is not clear whether or not spindle assembly and attachment are the main physiological stimuli of the metaphase checkpoint. We reported earlier that cell cultures derived from ovarian cystadenomas, which are the benign counterparts of ovarian carcinomas, when engineered to overcome senescence due to forced expression of SV40 large T antigen, undergo a mitotic arrest at the metaphase checkpoint as they age in culture. This arrest, which often leads to cell death, is the main determinant of in vitro crisis in this cell model. We sought to compare the mechanisms of this physiological arrest to those operating in cells treated with nocodazole. Here we show that formation of the metaphase checkpoint complex, which includes BUB3, BUBR1, CDC20, and MAD2, forms readily in our cell culture model similarly to what is observed following treatment with nocodazole. In contrast to cells treated with this inhibitor of spindle assembly, the checkpoint complex is formed and remains stable after spindle assembly has occurred in untreated cells undergoing the physiological arrest. Furthermore, examination of nocodazole untreated cells undergoing the arrest at the metaphase checkpoint showed that the checkpoint complex remained stable in cells despite showing localization of EB1, an indicator of microtubule anchoring to the kinetochore, to chromosomes. We conclude that neither complete mitotic spindle formation, nor attachment of the spindle to the kinetochore, is sufficient for disruption of the metaphase checkpoint complex and activation of the anaphase promoting complex. Progression to anaphase is therefore driven by events downstream to these steps. Citation Format: Theresa Austria, David Hinton, Louis Dubeau. The metaphase checkpoint complex is controlled by events downstream to spindle assembly and microtubule attachment to the kinetochore. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2014-1317