Activation of nuclear factor kappa B by different agents: influence of culture conditions in a cell-based assay.
The transcription factor nuclear factor kappaB (NF-kappaB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF-kappaB in epithelial cells in response to different stresses, a cell-based screening assay for activation of NF-kappaB-dependent gene transcription in human embryonic kidney cells (HEK/293) was developed. This assay allows detection of NF-kappaB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell-based assay, activation of the pathway by several agents, for example, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF-kappaB activation by TNF-alpha were examined. NF-kappaB was activated by TNF-alpha, IL-1beta, PMA, and camptothecin in a dose-dependent manner, but not by LPS. TNF-alpha results in the strongest induction of NF-kappaB-dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20-35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB activation or direct communication via gap junctions in the cell layer diminishing the TNF-alpha response.
Hellweg
Christine E
CE
Radiobiology Division, Institute of Aerospace Medicine, DLR, Linder Höhe, 51147 Köln, Germany. christine.hellweg@dlr.de
Arenz
Andrea
A
Bogner
Susanne
S
Schmitz
Claudia
C
Baumstark-Khan
Christa
C
D002166:Camptothecin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line / Q000187:drug effects / Q000502:physiology*; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D053583:Interleukin-1beta / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
D002273:Carcinogens; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D013755:Tetradecanoylphorbol Acetate; D002166:Camptothecin