Generation of stable reporter breast and lung cancer cell lines for NF-κB activation studies

Abstract

Nuclear Factor kappa B (NF-κB) is a conserved transcription factor involved in the expression of genes that are critical to inflammation and cell survival. Exposure to particular signals results in phosphorylation of NF-κB inhibitor proteins, which in turn allows NF-κB dimers to translocate to the nucleus and induce gene expression. Pathologic consequences of NF-κB activation are vast, mainly because of the pleiotropic roles that NF-κB-induced genes have on inflammation, cell proliferation and apoptosis. Thus, experimental models assessing NF-κB activation have direct screening applications for drug discovery. In this scenario, pathway-specific reporter cell systems become valuable tools to identify and elucidate the mechanism of action of novel compounds. Here, we describe the generation, characterization, and validation of human cancer epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory mediators. Human lung (H460) and breast (T-47D) cancer cell lines were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. The pro-inflammatory cytokine TNF-α was able to activate the reporter systems in a concentration-response manner, correlating to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using dexamethasone, a potent anti-inflammatory drug, a synthetic inhibitor of NF-κB (BAY 11-7082) and a new anti-cancer peptide (CIGB-552). We have established robust H460-NF-κB-hrGFP and T-47D-NF-κB-hrGFP reporter cell lines which represent a useful cancer model for primary screening and identification of compounds with anti-inflammatory activity.

Keywords: In vitro; Inflammation; NF-κB; Reporter cell lines; Stable cell lines; assays.