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The functional role of Mcl-1 in the dynamics of mitotic cell fate.
[Thesis]. Manchester, UK: The University of Manchester; 2015.
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Abstract
Drugs that alter microtubule dynamics are often used in chemotherapy regimes in combination with other agents in order to treat various cancers. Despite the success over many years, there remain problems in toxicity, resistance and predictability to the drugs. In order to overcome these problems, there is a need to gain an understanding of how these drugs kill cancer cells in cell culture. As microtubule function is particularly important for chromosome movement in mitosis, cells treated with these agents cause a mitotic arrest through activation of the spindle assembly checkpoint. Following induction of a mitotic arrest, cells can escape this arrest (mitotic slippage) or undergo mitotic death, determined in part by the response of the apoptotic network. Levels of an anti-apoptotic protein, Mcl-1, are often lost over time in mitosis. Using time-lapse analysis on a cell line unable to escape the mitotic arrest, this thesis shows that Mcl-1 protein contributes to cell death both in mitosis and the subsequent interphase in response to microtubule toxin, taxol. This suggests that inhibiting Mcl-1 may increase the efficacy of anti-mitotic agents. In addition, mitotic cell lines prone to mitotic slippage were found to have higher levels anti-apoptotic protein, Bcl-xL, in comparison to Mcl-1, indicating one way in which these cells can cope with loss of Mcl-1 during mitosis. Secondly, an evaluation of the contribution of the previously identified interaction between Mcl-1 and mitotic E3 ligase complex, the APC/C-Cdc20, to the rate of mitotic death and mitotic slippage was assessed. Inhibition of APC/C-Cdc20 activity or mutation of a Mcl-1 motif (RxxL) thought to engage with the APC/C-Cdc20 complex did not have a substantial effect on Mcl-1 degradation or mitotic death, thereby questioning the functional significance of this interaction. However, it appears that Mcl-1 protein levels can influence the rate of mitotic slippage and this influence was affected via Mcl-1’s RxxL motif within Mcl-1. This suggests that Mcl-1 protein may delay mitotic slippage via substrate competition for the APC/C-Cdc20 complex with Cyclin B1, whose degradation is required for mitotic exit. Further analysis of this effect showed that this interaction may not be a universal effect. This together with the specific functional effect on mitotic slippage rather than mitotic death, suggests that this is an indirect effect caused by network interference between the components of the death and slippage pathways.
Layman's Abstract
Spindle toxins such as taxol are used in the clinic to kill cancer cells in patients. These drugs work by inhibiting cell and genome division in mitosis, causing what is known as a mitotic arrest. A cell cannot remain in this mitotic arrest forever and can either be forced to undertake cell death or escape the mitotic arrest. By understanding the contributing factors to both mitotic death and escape from this mitotic arrest we may be able to find ways to enhance mitotic death of cancer cells to these drugs.This thesis shows that Mcl-1, a protein that is normally required for cell survival, is lost during a mitotic arrest and this helps drive cell death in response to a spindle toxin drug both during the mitotic arrest and after the cells have escaped the arrest. We investigated further ways in which Mcl-1 is lost in a mitotic arrest, specifically concentrating on enzyme complexes SCF, MULE and APC/C. However, inhibition of these enzyme complexes did not stabilise Mcl-1 in mitosis and therefore had no effect on mitotic death. This suggests that Mcl-1 protein destruction in a drug-induced mitotic arrest may be independent of the activity of enzyme complexes SCF, MULE and APC/C. Moreover, cell lines that are more likely to escape mitotic arrest instead of mitotic death are highly dependent on another pro-survival protein, Bcl-xL. Importantly, Bcl-xL does not get degraded in mitosis like Mcl-1 and is therefore able to counteract mitotic death in certain cancer cell types when Mcl-1 is lost. This suggests that targeting Bcl-xL in combination with the spindle toxin drugs may enhance mitotic death.