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

Chloe C Lepage,Bradley Larson,Kirk J Mcmanus,Laura L Thompson

doi:10.3390/cells9020344

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.

Highlights

Micronuclei are small, membrane bound, DNA-containing bodies located outside the primary cell nucleus and whose presence is synonymous with genome instability
Previous studies involving micronuclei were limited in that they typically only focus on micronucleus detection and rarely determine the frequency of micronuclei within a given experimental or clinical context [2,3,33,34,35]
Those studies are inherently limited in their capacity to rank the severity of a specific experimental condition, or to correlate the frequency of micronucleus formation with disease development, progression, and outcomes

Summary

Introduction

Micronuclei are small, membrane bound, DNA-containing bodies located outside the primary cell nucleus and whose presence is synonymous with genome instability. Many genotoxic studies have sought to determine the impact compounds have on genome stability by manually assessing the presence of micronuclei following a treatment [4,5], while more recent studies have employed micronucleus formation assays to uncover genes with pathogenic implications in cancer [6,7,8]. In this regard, numerous cytological studies have identified micronuclei within various cancer contexts including head and neck, ovarian, and breast cancers [9,10,11]. The ability to accurately identify and enumerate micronuclei is relevant in both laboratory and clinical settings

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Results

Discussion

Conclusion