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      An Automated, Single Cell Quantitative Imaging Microscopy Approach to Assess Micronucleus Formation, Genotoxicity and Chromosome Instability

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          Abstract

          Micronuclei are small, extranuclear bodies that are distinct from the primary cell nucleus. Micronucleus formation is an aberrant event that suggests a history of genotoxic stress or chromosome mis-segregation events. Accordingly, assays evaluating micronucleus formation serve as useful tools within the fields of toxicology and oncology. Here, we describe a novel micronucleus formation assay that utilizes a high-throughput imaging platform and automated image analysis software for accurate detection and rapid quantification of micronuclei at the single cell level. We show that our image analysis parameters are capable of identifying dose-dependent increases in micronucleus formation within three distinct cell lines following treatment with two established genotoxic agents, etoposide or bleomycin. We further show that this assay detects micronuclei induced through silencing of the established chromosome instability gene, SMC1A. Thus, the micronucleus formation assay described here is a versatile and efficient alternative to more laborious cytological approaches, and greatly increases throughput, which will be particularly beneficial for large-scale chemical or genetic screens.

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          Most cited references 59

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          Hallmarks of Cancer: The Next Generation

          The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the "tumor microenvironment." Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer. Copyright © 2011 Elsevier Inc. All rights reserved.
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            Genetic instabilities in human cancers.

            Whether and how human tumours are genetically unstable has been debated for decades. There is now evidence that most cancers may indeed be genetically unstable, but that the instability exists at two distinct levels. In a small subset of tumours, the instability is observed at the nucleotide level and results in base substitutions or deletions or insertions of a few nucleotides. In most other cancers, the instability is observed at the chromosome level, resulting in losses and gains of whole chromosomes or large portions thereof. Recognition and comparison of these instabilities are leading to new insights into tumour pathogenesis.
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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.
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                Author and article information

                Journal
                Cells
                Cells
                cells
                Cells
                MDPI
                2073-4409
                02 February 2020
                February 2020
                : 9
                : 2
                Affiliations
                [1 ]Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, MB R3E 0J9, Canada; Chloe.Lepage@ 123456umanitoba.ca (C.C.L.); Laura.Thompson@ 123456umanitoba.ca (L.L.T.)
                [2 ]Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, MB R3E 0V9, Canada
                [3 ]BioTek Instruments, Inc., Winooski, VT 05404, USA; larsonb@ 123456BioTek.com
                Author notes
                [* ]Correspondence: Kirk.McManus@ 123456umanitoba.ca ; Tel.: +1-204-787-2833
                Article
                cells-09-00344
                10.3390/cells9020344
                7072510
                32024251
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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