Analysis of a Damaged Mitotic Spindle Checkpoint in Budding Yeast

A hallmark of a cancer cell is aneuploidy, an increase or loss in chromosomes along with chromosomal rearrangements or fragmentation. Aneuploidy has been proposed to contribute to tumor development by allowing cancer cells to find new genetic combinations that promote growth even, for example, in the presence of an anti-cancer chemical agent. One cellular pathway that prevents the development of aneuploidy is the mitotic spindle checkpoint, which is essential for cell viability in both wild type and cancer cells. This pathway is also essential to maintain the very high rate of accurate chromosome segregation that is observed in wild type cells. Thus, it has been hypothesized that cancer cells may contain a damaged spindle checkpoint which would allow for the development and maintenance of aneuploidy. During my post-doctoral training I have studied the spindle checkpoint in vivo and in vitro. In vivo I have found that a partial loss of Mad2 function, which damages the pathway, in cells that are forced to be aneuploid can be restored by a compensatory loss in Mad1 function. To build upon this work, in my first aim I propose to select budding yeast for mutations that allow cells to live when they are forced to carry extra artificial chromosomes, which would normally cause the cells to die. Such mutants might be informative with regard to the types of mutations cancer cells accumulate that allow them to live when they are aneuploid. In vitro I have developed a highly active and specific Cdc20-dependent APC (APCCdc20) enzyme assay using components from budding yeast. The APC is the master cell cycle regulator at metaphase, and is the target of spindle checkpoint regulation. I have used this assay to make detailed quantitative measurements of wild type checkpoint activity. One way that cancer cells might evolve a damaged spindle checkpoint is by accumulating mutations in checkpoint genes that ultimately affect the ability of the checkpoint to inhibit cell cycle progression. In my second aim I propose to use my in vitro system to perform titration analyses on aberrant forms of spindle checkpoint proteins. I hope to uncover mechanistic insight into the kinds of consequences that occur when spindle checkpoint components are mutated. Finally, I have identified phosphorylation sites within three highly conserved APC subunits that correlate with checkpoint activity by employing protein purification and mass spectroscopy. In my third aim I propose to perform this analysis on all APC subunits and then to investigate the functional consequence of these phosphorylation events both in vivo and in vitro. Checkpoint regulation of the APC by phosphorylation may play a contributing role in halting cell cycle progression, and this form of regulation may be susceptible to alteration in cancer cells. I suggest that the results from these studies will make a contribution to the mechanistic understanding of checkpoint function that shall be necessary for us to evaluate potential consequences on cell cycle regulation when the spindle checkpoint is damaged in cancer cells.

Project ID:PA10008-0314
External Project ID:NSC100-2311-B182-006