An Easy-to-use Genotoxicity Assay Using EGFP-MDC1-expressing Human Cells

Genes and Environment

Masae Ikura¹  Shun Matsuda²  Tsuyoshi Ikura¹  Tomonari Matsuda²  Shin-ya Yanagisawa²  Yoko Matsuda²  Ryo Matsuda¹ 

[1] Radiation Biology Center, Kyoto University;Research Center for Environmental Quality Management, Kyoto University

关键词: MDC1;    γ-H2AX;    fluorescence microscopy;    image-processing;    R;   

DOI  :  10.3123/jemsge.2014.001

学科分类：分子生物学，细胞生物学和基因

来源: Japanese Environmental Mutagen Society / Nihon Kankyo Hen igen Gakkai

【 摘 要 】

References(92)Cited-By(3)Supplementary materials(1)Histone H2AX phosphorylated at Ser139 (γ-H2AX) is a useful biomarker for DNA double-strand breaks. However, γ-H2AX detection has methodological disadvantages such as the requirement of expensive anti-γ-H2AX antibody and time-consuming handling for its staining. Mediator of DNA damage checkpoint 1 (MDC1) is a central adaptor protein which recruits various DNA damage response proteins to γ-H2AX and thus forms nuclear foci in the same location as γ-H2AX in response to DNA damage. Here, we describe an easy-to-use genotoxicity assay which combines enhanced green fluorescence protein (EGFP)-fused MDC1-expressing cells with a free R program for image-processing and quantification of foci area/nucleus. The workflow of this assay is simple: mutagen treatment, imaging, and R-processing. This assay does not need antibodies or staining handling and it detected the genotoxicity of a range of mutagens, including camptothecin (topoisomerase I inhibitior), cisplatin (crosslinker), and 4-nitroquinoline 1-oxide and benzo[a]pyrene (bulky DNA-adduct forming compounds), as increased fluorescence of EGFP-MDC1 foci. Furthermore, cotreatment with arabinofuranosyl cytosine/hydroxyurea and mutagens sensitized EGFP-MDC1 foci formation to bulky DNA adduct-type mutagens. Additionally, the established cells can be monitored in real-time using live cell imaging to obtain detailed dynamics of MDC1 in response to mutagens. The simple handling of this assay is expected to enable its full automation, thus making it useful for high-throughput genotoxicity screening of chemicals and monitoring of environmental mutagens.

【 授权许可】

Unknown