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Imaging of nuclear factor κB activation induced by ionizing radiation in human embryonic kidney (HEK) cells

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Abstract

Ionizing radiation modulates several signaling pathways resulting in transcription factor activation. Nuclear factor kappa B (NF-κB) is one of the most important transcription factors that respond to changes in the environment of a mammalian cell. NF-κB plays a key role not only in inflammation and immune regulation but also in cellular radiation response. In response to DNA damage, NF-κB might inhibit apoptosis and promote carcinogenesis. Our previous studies showed that ionizing radiation is very effective in inducing biological damages. Therefore, it is important to understand the radiation-induced NF-κB signaling cascade. The current study aims to improve existing mammalian cell-based reporter assays for NF-κB activation by the use of DD-tdTomato which is a destabilized variant of red fluorescent protein tdTomato. It is demonstrated that exposure of recombinant human embryonic kidney cells (HEK/293 transfected with a reporter constructs containing NF-κB binding sites in its promoter) to ionizing radiation induces NF-κB-dependent DD-tdTomato expression. Using this reporter assays, NF-κB signaling in mammalian cells was monitored by flow cytometry and fluorescence microscopy. Activation of NF-κB by the canonical pathway was found to be quicker than by the genotoxin- and stress-induced pathway. X-rays activate NF-κB in HEK cells in a dose-dependent manner, and the extent of NF-κB activation is higher as compared to camptothecin.

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Acknowledgments

This work was supported by the Deutsches Zentrum für Luft-und Raumfahrt e.V. (DLR), Helmholtz SpaceLife program for accomplishment of Ph.D degree at the University of Bonn. The authors would like to thank all the members of Biodiagnostics group for their timely help.

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The authors declare that they have no conflict of interest.

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The experiments shown in this manuscript comply with the current laws of Germany where they were performed.

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  1. Radiation Biology, Institute of Aerospace Medicine, German Aerospace Centre (DLR), Linder Höhe, 51147, Köln, Germany

    Arif Ali Chishti, Christa Baumstark-Khan, Christine E. Hellweg & Günther Reitz

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  1. Arif Ali Chishti
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  2. Christa Baumstark-Khan
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  3. Christine E. Hellweg
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Correspondence to Christine E. Hellweg.

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Chishti, A.A., Baumstark-Khan, C., Hellweg, C.E. et al. Imaging of nuclear factor κB activation induced by ionizing radiation in human embryonic kidney (HEK) cells. Radiat Environ Biophys 53, 599–610 (2014). https://doi.org/10.1007/s00411-014-0541-x

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