Measuring the Cost of Aneuploidy by Counting Yeast Artificial Chromosomes

Evidence continues to mount that aneuploidy is a contributing factor in tumor development, and that aneuploidy may serve as a target for anti-cancer therapy. Aneuploidy is lethal in normal human cells. Little is known about the types of mutations that allow cancer cells to tolerate high levels of aneuploidy. However, one hypothesis is that damage to the mitotic spindle checkpoint may contribute to a cancer cell’s ability to develop and tolerate aneuploidy. To investigate this hypothesis, our goal is to determine the degree of aneuploidy arising in response to loss of MAD2 spindle checkpoint function in the well-defined model organism, the budding yeast. A direct measurement of the degree of aneuploidy in wild type and mad2 mutants, as determined by counting the average number of artificial chromosomes in a population of cells, has not been reported. Nor have the consequences on cellular health and viability been explored in wild type and mad2 mutant cells in response to a high degree of aneuploidy. I propose the follow three aims to address these questions. In Aim 1, our goal is to establish a budding yeast system to measure the amount of aneuploidy generated by using artificial chromosomes in a population of cells. In Aim 2, our goal is to determine how spindle checkpoint function contributes to the cost cells pay when they are forced to be aneuploid. In Aim 3, our goal is to select for mutations that allow cells to live when they should die when they are highly aneuploid, which are the kinds of mutations that may occur in cancer cells. To achieve Aim 1, we propose to employ a quantitative polymerase chain reaction (qPCR)-based system to count the average number of short-linear artificial chromosomes in a population of cells, using circular and long-linear chromosomes as controls. To achieve Aim 2, we propose analyze growth rates, retention rates, life-span, reproductive-span, and competitive fitness of wild type and mad2 mutant cells that are forced to be aneuploid. To achieve Aim 3, we propose to create a budding yeast aneuploid system that displays a strong lethal phenotype, and perform a genetic selection for mutations that allow cells to live in the presence of high levels of aneuploidy, when they should normally die. Working towards achieving these aims, we have already made an initial set of observations that indicate that MAD2 spindle checkpoint function is required to maintain high levels of aneuploidy, but that MAD2 also contributes to a decrease in chromosome retention rate in wild type cells when they were forced to have a high level of aneuploidy.

Project ID:PA10207-0700
External Project ID:NSC102-2311-B182-004