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      A Novel Multiplexed, Image-Based Approach to Detect Phenotypes That Underlie Chromosome Instability in Human Cells

      1 , 2 , 1 , 2 , *

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          Chromosome instability (CIN) is characterized by a progressive change in chromosome numbers. It is a characteristic common to virtually all tumor types, and is commonly observed in highly aggressive and drug resistant tumors. Despite this information, the majority of human CIN genes have yet to be elucidated. In this study, we developed and validated a multiplexed, image-based screen capable of detecting three different phenotypes associated with CIN. Large-scale chromosome content changes were detected by quantifying changes in nuclear volumes following RNAi-based gene silencing. Using a DsRED-LacI reporter system to fluorescently label chromosome 11 within a human fibrosarcoma cell line, we were able to detect deviations from the expected number of two foci per nucleus (one focus/labelled chromosome) that occurred following CIN gene silencing. Finally, micronucleus enumeration was performed, as an increase in micronucleus formation is a classic hallmark of CIN. To validate the ability of each assay to detect phenotypes that underlie CIN, we silenced the established CIN gene, SMC1A. Following SMC1A silencing we detected an increase in nuclear volumes, a decrease in the number of nuclei harboring two DsRED-LacI foci, and an increase in micronucleus formation relative to controls (untreated and si GAPDH). Similar results were obtained in an unrelated human fibroblast cell line. The results of this study indicate that each assay is capable of detecting CIN-associated phenotypes, and can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer.

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          Most cited references 46

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          The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity

          The systematic translation of cancer genomic data into knowledge of tumor biology and therapeutic avenues remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacologic annotation is available 1 . Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number, and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacologic profiles for 24 anticancer drugs across 479 of the lines, this collection allowed identification of genetic, lineage, and gene expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Altogether, our results suggest that large, annotated cell line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of “personalized” therapeutic regimens 2 .
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            Molecular origins of cancer: Molecular basis of colorectal cancer.

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              Genetic instability in colorectal cancers.

              It has long been considered that genetic instability is an integral component of human neoplasia. In a small fraction of tumours, mismatch repair deficiency leads to a microsatellite instability at the nucleotide sequence level. In other tumours, an abnormal chromosome number (aneuploidy) has suggested an instability, but the nature and magnitude of the postulated instability is a matter of conjecture. We show here that colorectal tumours without microsatellite instability exhibit a striking defect in chromosome segregation, resulting in gains or losses in excess of 10(-2) per chromosome per generation. This form of chromosomal instability reflected a continuing cellular defect that persisted throughout the lifetime of the tumour cell and was not simply related to chromosome number. While microsatellite instability is a recessive trait, chromosomal instability appeared to be dominant. These data indicate that persistent genetic instability may be critical for the development of all colorectal cancers, and that such instability can arise through two distinct pathways.

                Author and article information

                Role: Academic Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                20 April 2015
                : 10
                : 4
                [1 ]Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada
                [2 ]Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada
                University of Science and Technology of China, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LLT KJM. Performed the experiments: LLT. Analyzed the data: LLT KJM. Contributed reagents/materials/analysis tools: LLT KJM. Wrote the paper: LLT KJM.


                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                Page count
                Figures: 3, Tables: 1, Pages: 17
                The authors acknowledge NSERC ( 401851-2012) for operational funds (KJM), and the Terry Fox Research Institute (, Research Manitoba ( and CancerCare Manitoba Foundation ( for studentships (LLT). The authors also acknowledge the strong support of the Manitoba Institute of Cell Biology, which is funded in part by the CancerCare Manitoba Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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