Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression

ABSTRACT

Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoid^tetO-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption.

KEYWORDS:

• aneuploidy
• assay development
• chromosome instability (CIN)
• chromosome segregation
• human artificial chromosome (HAC)

This article is referred to by:

Visualizing chromosome segregation in live cells

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank all members of the Dasso and Larionov laboratories for helpful discussions. We thank Artem Kononenko for providing the p#264-GFP knot linker CAGpr vector and help with HAC reengineering techniques, Alexander Samoshkin for providing the TetR-f-GFP_HyTK vector and technical help, and Hee-Sheung Lee for help with MMCT. We thank Dr. Malte Renz (Albert Einstein College of Medicine) for providing the tandem GFP plasmids. We thank Dr. Talar Markossian (Loyola University Medical Center) for helpful discussions regarding statistical analysis and for assistance in running Stata analysis. This work was supported through NICHD Intramural Project HD008954.

Author contributions

S.M., A.A., and M.D. designed and analyzed the experiments. V.L. provided the CHO cells carrying the alphoid ^tetO-HAC and had some input in experimental analysis. S.M. and N.S. executed and analyzed the flow cytometry experiment. S.M. executed all remaining experiments. S.M. and MD wrote the manuscript with input from all authors.