Abstract
Chromosomal instability (CIN) is a hallmark of cancer, which is characterized by the gain or loss of chromosomes as well as the rearrangement of the genetic material during cell division. Detection of mitotic errors such as misaligned chromosomes or chromosomal bridges (also known as lagging chromosomes) is challenging as it requires the analysis and manual discrimination of chromosomal aberrations in mitotic cells by molecular techniques. In interphase cells, more frequent in the cell population than mitotic cells, two distinct nuclear phenotypes are associated with CIN: the micronucleus and the toroidal nucleus. Several methods are available for the detection of micronuclei, but none for toroidal nuclei. Here, we provide a method to quantify the presence of both nuclear biomarkers for the evaluation of CIN status in non-mitotic cells particularly suited for genotoxicity screens.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
Abbreviations
- BSA:
-
Bovine serum albumin
- CIN:
-
Chromosomal instability
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DMEM:
-
Dulbecco’s modified eagle medium
- DNA:
-
Deoxyribonucleic acid
- LAMP2:
-
Lysosome-associated membrane protein 2
- LC3:
-
Microtubule-associated proteins 1A/1B light chain 3
- PBS:
-
Phosphate-buffered saline
- PFA:
-
Paraformaldehyde
- UPS:
-
Ubiquitin proteasome system
- UV:
-
Ultraviolet
References
McClelland SE (2017) Role of chromosomal instability in cancer progression. Endocr Relat Cancer 24:T23–T31
Watkins TBK, Lim EL, Petkovic M et al (2020) Pervasive chromosomal instability and karyotype order in tumour evolution. Nature 587:126–132
Hoevenaar WHM, Janssen A, Quirindongo AI et al (2020) Degree and site of chromosomal instability define its oncogenic potential. Nat Commun 11:1501
Vargas-Rondón N, Pérez-Mora E, Villegas VE et al (2020) Role of chromosomal instability and clonal heterogeneity in the therapy response of breast cancer cell lines. Cancer Biol Med 17:970–985
Bakhoum SF, Landau DA (2017) Chromosomal instability as a driver of tumor heterogeneity and evolution. Cold Spring Harb Perspect Med 7(6):a029611
Almacellas E, Pelletier J, Day C et al (2021) Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability. Autophagy 17:796–813
Hämälistö S, Stahl JL, Favaro E et al (2020) Spatially and temporally defined lysosomal leakage facilitates mitotic chromosome segregation. Nat Commun 11:229
Holdgaard SG, Cianfanelli V, Pupo E et al (2019) Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites. Nat Commun 10:4176
Gisselsson D, Björk J, Höglund M et al (2001) Abnormal nuclear shape in solid tumors reflects mitotic instability. Am J Pathol 158:199–206
Sommer S, Buraczewska I, Kruszewski M (2020) Micronucleus assay: the state of art, and future directions. Int J Mol Sci 21(4):1534
Krupina K, Goginashvili A, Cleveland DW (2021) Causes and consequences of micronuclei. Curr Opin Cell Biol 70:91–99
Hintzsche H, Hemmann U, Poth A et al (2017) Fate of micronuclei and micronucleated cells. Mutat Res Rev Mutat Res 771:85–98
Lepage CC, Thompson LL, Larson B et al (2020) An automated, single cell quantitative imaging microscopy approach to assess micronucleus formation, genotoxicity chromosome instability. Cells 9:344
Mosesso P, Cinelli S (2019) In vitro cytogenetic assays: chromosomal aberrations and micronucleus tests. Methods Mol Biol 2031:79–104
Beliën JA, Copper MP, Braakhuis BJ et al (1995) Standardization of counting micronuclei: definition of a protocol to measure genotoxic damage in human exfoliated cells. Carcinogenesis 16:2395–2400
Ramadhani D, Purnami S (2013) Automated detection of binucleated cell and micronuclei using CellProfiler 2.0 software. HAYATI J Biosci 20:151–156
Naso FD, Sterbini V, Crecca E et al (2020) Excess TPX2 interferes with microtubule disassembly and nuclei reformation at mitotic exit. Cells 9:374
Verstraeten VLRM, Peckham LA, Olive M et al (2011) Protein farnesylation inhibitors cause donut-shaped cell nuclei attributable to a centrosome separation defect. Proc Natl Acad Sci U S A 108:4997–5002
Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Acknowledgments
C.P. was supported by a Ramon y Cajal fellowship (RYC-2017-22959). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no 799000 to C.M. We thank CERCA Program/Generalitat de Catalunya for institutional support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Pons, C., Almacellas, E., Tauler, A., Mauvezin, C. (2022). Detection of Nuclear Biomarkers for Chromosomal Instability. In: Norberg, H., Norberg, E. (eds) Autophagy and Cancer. Methods in Molecular Biology, vol 2445. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2071-7_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2071-7_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2070-0
Online ISBN: 978-1-0716-2071-7
eBook Packages: Springer Protocols