Abstract 2737: Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression

Abstract

Abstract Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). CIN is associated with poor prognosis in many cancer types, and targeting of CIN is an attractive strategy for anti-cancer therapeutics. The mechanisms causing CIN and its contributions to tumor initiation and growth are not well defined, partly because there is no straightforward, quantitative assays for CIN in human cells. To address this problem, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in mammalian cells, with which we can easily score the rates of CIN within one cell division under different experimental conditions. We have constructed a HAC encoding copies of enhanced green fluorescent protein (eGFP) fused to the destruction box (DB) of hSecurin, a substrate of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, which becomes active during anaphase to catalyze the proteolysis of critical mitotic target proteins. This HAC also contains tet operator (tetO) arrays and sequences encoding the tetracycline repressor fused to monomeric cherry fluorescent protein (tetR-mCherry). We have produced human U2OS cells (U2OS-Phoenix) carrying this HAC, in which we monitor HAC segregation in two ways: First, APC/C degrades the DB-eGFP fusion expressed from the HAC at anaphase onset, and DB-eGFP re-accumulates in the daughter cells after G1 phase, when APC/C becomes inactive. Daughter cells that do not obtain a copy of the HAC will thus be GFP negative in the subsequent interphase. Second, because tetR-mCherry binds to the tetO arrays, the HAC itself could be followed by live imaging. Following the HAC by live cell imaging experiments, we show that U2OS-Phoenix cells have low inherent levels of CIN, but HAC missegregation is markedly increased by treatment with Reversine, an inhibitor of Mps1, and microtubule agents Nocodazole and Paclitaxel. In summary, we have developed new assays to score CIN levels in human cells and have shown that CIN levels increase upon chemical disruption of mitotic progression, which makes our assays ideal for chemical screens. Citation Format: Sarine Markossian, Alexei Arnaoutov, Nakhle S. Saba, Vladimir Larionov, Mary Dasso. Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2737.