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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 references47

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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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            Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations.

            Various types of chromosomal aberrations, including numerical (aneuploidy) and structural (e.g., translocations, deletions), are commonly found in human tumors and are linked to tumorigenesis. Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, whereas structural aberrations are caused by improperly repaired DNA breaks. Here, we demonstrate that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM, Chk2, and p53. We show that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Our data show that segregation errors can cause translocations and provide insights into the role of whole-chromosome instability in tumorigenesis.
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              Mechanisms of chromosomal instability.

              Most solid tumors are aneuploid, having a chromosome number that is not a multiple of the haploid number, and many frequently mis-segregate whole chromosomes in a phenomenon called chromosomal instability (CIN). CIN positively correlates with poor patient prognosis, indicating that reduced mitotic fidelity contributes to cancer progression by increasing genetic diversity among tumor cells. Here, we review the mechanisms underlying CIN, which include defects in chromosome cohesion, mitotic checkpoint function, centrosome copy number, kinetochore-microtubule attachment dynamics, and cell-cycle regulation. Understanding these mechanisms provides insight into the cellular consequences of CIN and reveals the possibility of exploiting CIN in cancer therapy. 2010 Elsevier Ltd. All rights reserved.
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                Author and article information

                Journal
                Cells
                Cells
                cells
                Cells
                MDPI
                2073-4409
                02 February 2020
                February 2020
                : 9
                : 2
                : 344
                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
                Author information
                https://orcid.org/0000-0003-0081-8737
                Article
                cells-09-00344
                10.3390/cells9020344
                7072510
                32024251
                ebeb3cf2-90b4-421a-9e78-09e47510d0bc
                © 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/).

                History
                : 02 December 2019
                : 31 January 2020
                Categories
                Article

                micronucleus,micronuclei,genotoxicity,chromosome instability,single cell quantitative imaging microscopy (scquantim),cancer

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