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Detection of UV-induced activation of NF-κB in a recombinant human cell line by means of Enhanced Green Fluorescent Protein (EGFP)

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Abstract

The cellular protection reaction known as ultraviolet (UV) response leads to increased transcription of several genes. Parts of this transcriptional response are transmitted via activation of the Nuclear factor κB (NF-κB). The contribution of different UV radiation qualities to this process is not yet known. In a previous work, a stably transfected human cell line was developed which indicates activation of the NF-κB pathway by fluorescence of the reporters Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) thereby allowing a fast and reliable monitoring of UV effects on the NF-κB pathway. Cells were exposed to a mercury low-pressure lamp or to simulated sunlight of different wavelength ranges and subjected to flow cytometric analysis after different post-irradiation periods. Growth capacity of cells after UV irradiation was quantified using a luminance measurement of crystal violet stained cell layers. In contrast to UVC and UVB, UVA radiation induced d2EGFP expression and NF-κB activation in a non-cytotoxic dose range. These results show that NF-κB plays a role in the UVA-induced gene activation in a non-cytotoxic dose range in a human epithelial cell line.

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Abbreviations

AP-1:

Activated protein 1

cIAP:

Cellular inhibitor of apoptosis protein

CK2:

Casein kinase II

d2EGFP:

Destabilized Enhanced Green Fluorescent Protein

EGFP:

Enhanced Green Fluorescent Protein

Egr-1:

Early growth response gene

EMSA:

Electrophoretic mobility shift assay

FACS:

Fluorescence activated cell scanner

FBS:

Fetal bovine serum

Gadd45β:

Growth arrest and DNA damage inducible protein 45β

HEK:

Human embryonic kidney

IκB:

Inhibitor of NF-κB

IL:

Interleukin

NF-κB:

Nuclear factor κB

NRE:

NF-κB response element

PBS:

Phosphate-buffered saline

PTFE:

Polytetrafluoroethylene

Rel:

v-rel reticuloendotheliosis viral oncogene homolog

ROS:

Reactive oxygen species

SSL:

Simulated sunlight

TNF-α:

Tumor necrosis factor α

TRAF2:

TNF-receptor associated factor-2

UV:

Ultraviolet

XIAP:

X-linked inhibitor of apoptosis protein

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Acknowledgments

This work has been supported in part by the German Academy of Aviation and Travel Medicine (Deutsche Akademie für Flug- und Reisemedizin). The authors would like to thank Anna Brozyna, Patrick Lau, Susanne Bogner, and Claudia Schmitz for excellent technical assistance and Dr. Rainer Facius for recording the spectrum of the sun simulator.

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  1. Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, 51147, Köln, Germany

    Christine E. Hellweg & Christa Baumstark-Khan

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  1. Christine E. Hellweg
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  2. Christa Baumstark-Khan
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Correspondence to Christine E. Hellweg.

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Hellweg, C.E., Baumstark-Khan, C. Detection of UV-induced activation of NF-κB in a recombinant human cell line by means of Enhanced Green Fluorescent Protein (EGFP). Radiat Environ Biophys 46, 269–279 (2007). https://doi.org/10.1007/s00411-007-0104-5

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