Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
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Jung-Hyun Kim1, Hee-Sheung Lee1, Nicholas C. O. Lee1, Nikolay V. Goncharov1,2, Vadim Kumeiko2, Hiroshi Masumoto3, William C. Earnshaw4, Natalay Kouprina1 and Vladimir Larionov1
1 Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD, USA
2 School of Biomedicine, Far Eastern Federal University, A. V. Zhirmunsky Institute of Marine Biology, FEB RAS, Vladivostok, Russia
3 Laboratory of Cell Engineering, Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Japan
4 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland
Vladimir Larionov, email:
Keywords: chromosome instability, CIN, human artificial chromosome, HAC, anticancer drugs
Received: November 04, 2015 Accepted: January 29, 2016 Published: March 02, 2016
Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene (“loss of signal” assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this “loss of signal” assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this “gain of signal” assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The “gain of signal” assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level.
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