NF-κB in the Mammalian Cellular Response to Exposure with Ionising Radiation

Kurzfassung

During space missions, astronauts are exposed not only to greater amounts of radiation than on Earth but also to different radiation qualities, which can result in immediate and long-term risks. Activation of the Nuclear Factor κB (NF-κB) pathway resulting in increased survival of irradiated cells might contribute to cancer risk after space radiation exposure. In this work, the effect of ionising radiation and/or of inhibition or knock out of several pathway components on the NF-κB signalling was investigated. Activation of NF-κB dependent gene expression was detected by means of stably transfected human embryonic kidney cells (HEK/293) which express the reporter gene destabilized Enhanced Green Fluorescent Protein (d2EGFP) under control of a NF-κB responsive promoter (HEK-pNF-κB-d2EGFP/Neo L2). d2EGFP fluorescence was determined by means of FACS analysis after diffuse or cell nucleus-targeted irradiation with α-particles performed at the PTB, Braunschweig. Both irradiation conditions were capable of activating the NF-κB pathway with a peak at 5x10⁶ particles per cm² (1.26 Gy) for diffuse irradiation and after seven nuclear particle traversals (1.2 Gy) for targeted irradiation. Gene expression of NF-κB-regulated genes was analysed using quantitative real time qRT-PCR. For diffuse irradiation, a time-dependent expression pattern was observed for gadd45β, nfkbia, and bcl-2. For targeted irradiation the gene expression patterns were less pronounced. As NF-κB activation is discussed to result in a survival advantage of irradiated cells, the influence of NF-κB essential modulator (NEMO) on cellular survival (colony forming ability assay) after X-irradiation was analysed using wild-type and NEMOdeficient mouse embryonic fibroblasts (MEF). Results clearly show a better survival for the NF-κB proficient cell line, supporting this hypothesis. Analysing the upstream events in the NF-κB pathway it was shown that the proteasome inhibitor MG-132 abolished EGFP expression after treatment with TNF-α or with X-rays, pointing out that proteasomal degradation of IκB is involved after both treatments. Inhibition of the ataxia telangiectasia mutated (ATM) protein kinase by KU-55933 clearly demonstrated that NF-κB activation upon DNA double strand break processing depends on functional ATM after X-irradiation but not after TNF-α treatment of HEK cells. In conclusion, NF-κB activation via ATM kinase seems to be a relevant regulator of the cellular response to ionising radiation.