The mechanisms of the spindle assembly checkpoint and the mitotic cell death

Abstract

The spindle assembly checkpoint (SAC) monitors the chromosomes and kinetochore– microtubule attachment to prevent premature anaphase onset (Lara-Gonzalez et al., 2012), and this ensures the fidelity of cell division. The mitotic checkpoint complex (MCC), the core SAC effector, contains two sub-complexes, CDC20-MAD2 and BUBR1-BUB3 (Sudakin et al., 2001). However, the exact mechanism underlying the assembly of the MCC regarding when, where and how still is not fully addressed. It is believed that the formation of the CDC20-MAD2 sub-complex is an initial and essential step in MCC assembly (Sudakin et al., 2001), thus the assembly of the MCC can be depicted by the observation of the formation of the CDC20-MAD2 complex (Fraschini et al., 2001, Meraldi et al., 2004, Poddar et al., 2005). Using the Duolink based in situ proximity ligation assay (PLA), the lab has previously used individual cell analysis to show the temporal and spatial in vivo formation of the CDC20-MAD2 complex throughout the cell cycle in HeLa cells and existence of a specific prophase form of the CDC20-MAD2 complex (Li et al., 2017). In this study, we provide evidences showing that the profile of the assembly of the CDC20-MAD2 complex revealed by using the PLA can genuinely reflect the dynamic in vivo interaction of these two proteins in individual cells. We also provide evidences to support the idea that the prophase specific CDC20-MAD2 complex is functional in preventing the premature degradation of cyclin B1 in prophase which ensures the proper G2/M transition. p31comet (MAD2L1BP), as an important SAC silencer (Habu et al., 2002, Yang et al., 2007), which, in conjunction with TRIP13 (Thyroid hormone receptor interacting protein 13, or PCH-2 in C. elegans) promotes the disassembly of the MCC and the conversion of the ii C-MAD2 back to O-MAD2 for recycling (Xia et al., 2004). It has been suggested that expression of p31comet is cell cycle regulated, that there are two putative destruction motifs of D-box and KEN-box (Habu et al., 2002), and that it is an ubiquitin substrate (Udeshi et al., 2013, Wagner et al., 2011)，which implying that it is a substrate of the APC/C. However, the potential turnover property of p31comet is yet to be characterized. In this study, we have examined the expression profile of p31comet throughout the cell cycle using a specific anti-p31comet antibody fluorescent staining and have confirmed that it is cell cycle regulated. It starts to accumulate from prophase and peaks at prometaphase and is sustained until telophase and then it declined. This is supported by the observation that the level of p31comet is reduced at three hours after cells released from a mitotic block caused by nocodazole treatment and re-enters the cell cycle. The accumulation of p31comet before the prometaphase and its accumulation in response to the activation of the SAC by nocodazole treatment are the results of protein synthesis. However, the potential accumulation of p31comet levels in cell samples after siRNA with the components of the APC/C, especially the APC3, examined by western blot produced ambiguous inconclusive results. The preliminary data produced by depletion of Cullin-1, another E3 ligase responsible for the degradation of cell cycle substrates in G1/S, using siRNA resulted in an increase in p31comet, which implies that Cullin-1 is potentially response to p31comet degradation though this would require further confirmation. The various kinds of cell death occurred during mitosis are collectively termed ‘Mitotic Catastrophe’(Vitale et al., 2011). Targeting the mitotic catastrophe signaling pathway in cancer offers a significant therapeutic advantage, and the anti-mitotic drugs such as taxanes, epothilones, and vinca alkaloids are widely used in the cancer clinic. M2I-1 (MAD2 inhibitor-1), a small molecule, has been shown to disrupt the CDC20-MAD2 interaction in vitro and weaken the SAC in vivo(Kastl et al., 2015). We report here that M2I-1 can disrupt the in vivo interaction of CDC20 and MAD2, and significantly increases the sensitivity of several cancer cell lines to anti-mitotic drugs, with cell death occurring after a prolonged mitotic arrest, when cell death is triggered by the premature degradation of cyclin B1 and the perturbation of the microtubule network by nocodazole or Taxol. Interestingly, the level of iii MCL-1, a pro-survival protein of the Bcl-2 family is significantly elevated, but higher levels of the pro-apoptotic proteins MCL-1s, a short form of the MCL-1, act antagonistically. Taken together, our results demonstrate that M2I-1 exhibits antitumor activity in the presence of current anti-mitotic drugs such as Taxol and nocodazole and has the potential to be developed as an anticancer agent.