Abstract
This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.
Highlights
Interest is growing in high-content assays that can reliably measure chromosomal instability (CIN)because of its important role in causing developmental defects, cancer, and accelerated aging [1,2,3,4].Chromosomal aberrations can be measured directly using metaphase analysis of chromosomes or indirectly by interphase cytogenetic techniques that analyse the DNA content of nuclei and abnormalities of their morphology
Blocking of cells in the binucleated cell stage allowed the efficient measurement of the cytokinesis-block method: formation of nucleoplasmic bridges (NPBs) that originate from dicentric chromosomes caused by
We have identified additional nuclear anomalies formed under folate-deficient conditions, defined as fused (FUS), circular (CIR), and horseshoe (HS) nuclei, and investigated their suitability for inclusion as additional CIN biomarkers in the lymphocyte cytokinesis-block micronucleus cytome (CBMN-Cyt) assay (Figure 6) [31,44]
Summary
Interest is growing in high-content assays that can reliably measure chromosomal instability (CIN). Because of its important role in causing developmental defects, cancer, and accelerated aging [1,2,3,4]. Chromosomal aberrations can be measured directly using metaphase analysis of chromosomes or indirectly by interphase cytogenetic techniques that analyse the DNA content of nuclei and abnormalities of their morphology. One of the most prominent of these nuclear aberrations are micronuclei (MNi), which were discovered more than 100 years ago. This brief review explains the origin of MNi and the evolution of their measurement, together with other nuclear anomalies, into a multi-endpoint assay of CIN
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