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

Ionizing radiation modulates several signaling pathways resulting in transcription factor activation. Nuclear factor kappa B (NF-kappa B) is one of the most important transcription factors that respond to changes in the environment of a mammalian cell. NF-kappa B plays a key role not only in inflammation and immune regulation but also in cellular radiation response. In response to DNA damage, NF-kappa B 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-kappa B signaling cascade. The current study aims to improve existing mammalian cell-based reporter assays for NF-kappa B 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-kappa B binding sites in its promoter) to ionizing radiation induces NF-kappa B-dependent DD-tdTomato expression. Using this reporter assays, NF-kappa B signaling in mammalian cells was monitored by flow cytometry and fluorescence microscopy. Activation of NF-kappa B by the canonical pathway was found to be quicker than by the genotoxin- and stress-induced pathway. X-rays activate NF-kappa B in HEK cells in a dose-dependent manner, and the extent of NF-kappa B activation is higher as compared to camptothecin.