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Review
. 2022 Jun;16(2):159-177.
doi: 10.1007/s12079-021-00661-z. Epub 2021 Nov 29.

Tools used to assay genomic instability in cancers and cancer meiomitosis

Jennifer Gantchev  1 , Brandon Ramchatesingh  1 , Melissa Berman-Rosa  2 , Daniel Sikorski  3 , Keerthenan Raveendra  4 , Laetitia Amar  3 , Hong Hao Xu  5 , Amelia Martínez Villarreal  1 , Daniel Josue Guerra Ordaz  1 , Ivan V Litvinov  6   7
Affiliations
Review

Tools used to assay genomic instability in cancers and cancer meiomitosis

Jennifer Gantchev et al. J Cell Commun Signal. 2022 Jun.

Abstract

Genomic instability is a defining characteristic of cancer and the analysis of DNA damage at the chromosome level is a crucial part of the study of carcinogenesis and genotoxicity. Chromosomal instability (CIN), the most common level of genomic instability in cancers, is defined as the rate of loss or gain of chromosomes through successive divisions. As such, DNA in cancer cells is highly unstable. However, the underlying mechanisms remain elusive. There is a debate as to whether instability succeeds transformation, or if it is a by-product of cancer, and therefore, studying potential molecular and cellular contributors of genomic instability is of high importance. Recent work has suggested an important role for ectopic expression of meiosis genes in driving genomic instability via a process called meiomitosis. Improving understanding of these mechanisms can contribute to the development of targeted therapies that exploit DNA damage and repair mechanisms. Here, we discuss a workflow of novel and established techniques used to assess chromosomal instability as well as the nature of genomic instability such as double strand breaks, micronuclei, and chromatin bridges. For each technique, we discuss their advantages and limitations in a lab setting. Lastly, we provide detailed protocols for the discussed techniques.

Keywords: Chromosomal instability; Cytokinesis-block micronucleus assay; DNA damage; Genomic instability; H2B-GFP; Meiomitosis; Single-cell sequencing.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Immunofluorescence staining using indicators of DNA damage in the cutaneous squamous cell carcinoma cell line, A431. Examples of representative images for untreated and cells treated with 1 μm of etoposide are shown for A γ H2AX; B 53BP1; and C RAD51. Photos taken on a Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969). Scale bar 20 μm
Fig. 2
Fig. 2
Immunofluorescence staining patterns are indicative of levels of DNA damage in A431 cells. A Three types of γ H2AX staining patterns corresponding to the number of double strand DNA breaks are as follows: type 1 expression of less than 10 nuclear foci indicates low levels of DNA damage/DNA DSBs; type 2 expression of greater than 10 nuclear foci indicate a high level of DNA damage/DNA DSBs and type 3 pan-nuclear expression indicates pre-apoptotic state. B Four distinct types of 53BP1 expression patterns are as follows: type 1 stable expression of faint diffuse nuclear staining; type 2 expression of one or two discrete nuclear foci indicates low DNA damage response; type 3 expression of three or more discrete nuclear foci indicates high DDR; and type 4 expression of nuclear foci greater than 1.0 μM indicates abnormal/uncharacteristic expression for this protein. Photos taken on a Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969). Scale bar 10 μm
Fig. 3
Fig. 3
Immunofluorescence staining of human A431 cells with pericentrin antibody. A Normal metaphase A431 cell with two pericentrin foci indicative of two centrosomes at opposing poles of the cells compared to B–C abnormal number of pericentrin foci indicative of more than two centrosomes in a single dividing cell at metaphase. Arrows point to metaphase cells. D–E Pericentrin expression in interphase cells. (1) Demonstrates an interphase cell with normal level of pericentrin expression, magnified in F. (2) Demonstrates pericentrin overexpression, magnified in G. Photos taken on a Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969). Scale bar 20 μ m
Fig. 4
Fig. 4
Cytokinesis block micronucleus (CBMN) assay. A Mononucleated cell; B binuclear cell; C multinucleated cell; D binuclear cells with micronuclei (arrow); E nuclear bud (arrow); F late apoptotic cell. Photos taken on a Etaluma Lumascope LS720 microscope with a 60X objective (Meiji MA969). Scale bar 10 μ m
Fig. 5
Fig. 5
Double thymidine block synchronized A431 cells at anaphase showing events of lagging chromosomes (arrows). Cells were stained with DAPI and viewed under a fluorescence microscope. Photos taken on a Etaluma Lumascope LS720 microscope with a 40X objective (Olympus LUCPLFLN40X). Scale bar 10 μ m
Fig. 6
Fig. 6
Schematic representation of breakage-fusion-bridge (BFB) cycle. The BFB cycle is initiated when a double strand break occurs in an unreplicated chromosome leading to the loss of its telomere. 1 Following replication, both sister chromatids are telomere deficient. 2 The unprotected chromosomal ends of the sister chromatids fuse with one another, forming a dicentric chromosome. 3 The fused chromatids form a bridge during anaphase, where the two centromeres are pulled to opposing sides of the cell. 4 The bridge breaks at a random position, usually in a different site than the original fusion resulting in an inverted duplication in one of the daughter cells. The other daughter cell inherits a terminal deletion. Both chromosomes can then lead to more BFB cycles until the end acquires a telomere. Subsequent BFB cycles result in amplification of DNA sequences located near the telomere end and the progressive accumulation of terminal deletions. Adapted from Bailey and Murnane (Bailey and Murnane 2006)
Fig. 7
Fig. 7
Anaphase bridge formation in A431 cells synchronized with the double thymidine block method. Examples of DAPI-stained anaphase cells with chromatin bridge formation compared to normal anaphase cells, as indicated in the above images. Photos taken on a Etaluma Lumascope LS720 microscope with a 40X objective (Olympus LUCPLFLN40X). Scale bar 20 μm
Fig. 8
Fig. 8
Time-lapse live cells microscopy of cell cycle progression of an untreated A431-H2B-GFP cell. Representative microscopy images of fluorescent (GFP) A431cells expressing H2B-GFP. Phases of the cell cycle are indicated above sequential images of the same cell from interphase through mitosis to telophase. Time-lapse was taken on a Etaluma Lumascope LS720 microscope with a 40X objective (Olympus LUCPLFLN40X). Scale bar 20 μm
Fig. 9
Fig. 9
Display of the 24-colour spectral karyotype (SKY) hybridization of cutaneous T cell lymphoma cell line, Mac2A. A SKY reveals a diploid karyotype for Mac2A cells. The karyotype presented with the individual inverted 4′,6-diamidino-2-phenylindole (DAPI) (top left), the raw spectral image (top middle), and the SKY classified image (top right). The classified karyotype is presented with individual chromosome numbers indicated below. An example of a dicentric chromosome (der(15)t(2;15)(p11.2;p12)) indicated by the white arrow.
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References

    1. Aaij R, Abellan Beteta C, Adeva B, Adinolfi M, Aidala CA, Ajaltouni Z, Akar S, Albicocco P, Albrecht J, Alessio F, et al. Measurement of antiproton production in p-He collisions at sqrt[s_{NN}]=110 GeV. Phys Rev Lett. 2018;121:222001. - PubMed
    1. Adelaide J, Finetti P, Bekhouche I, Repellini L, Geneix J, Sircoulomb F, Charafe-Jauffret E, Cervera N, Desplans J, Parzy D, et al. Integrated profiling of basal and luminal breast cancers. Cancer Res. 2007;67:11565–11575. - PubMed
    1. Aganezov S, Goodwin S, Sherman RM, Sedlazeck FJ, Arun G, Bhatia S, Lee I, Kirsche M, Wappel R, Kramer M, et al. Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing. Genome Res. 2020;30:1258–1273. - PMC - PubMed
    1. Albertson DG, Pinkel D. Genomic microarrays in human genetic disease and cancer. Hum Mol Genet. 2003;12(2):R145–R152. - PubMed
    1. Amato A, Lentini L, Schillaci T, Iovino F, Di Leonardo A. RNAi mediated acute depletion of retinoblastoma protein (pRb) promotes aneuploidy in human primary cells via micronuclei formation. BMC Cell Biol. 2009;10:79. - PMC - PubMed