Visit our Expo - Redox and Inflammation signaling 2012

Session XIV : Transcriptional and translational control Poster XIV, 28
Activation of NF-kappaB by different agents – influence of culture conditions.
Christine E. Hellweg, Andrea Arenz, Susanne Bogner, Claudia Schmitz and Christa
Baumstark-Khan.
Cellular Biodiagnostics, Department of Radiobiology, Institute of Aerospace Medicine,
German Aerospace Center, 51170 Köln, Germany. E-mail : christine.hellweg@dlr.de
The transcription factor 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 the response to different stressors,
a cell-based screening assay for activation of NF-kappaB dependent gene transcription in
human embryonic kidney cells (HEK/293) was developed (J. Biomol. Screen. 2003, 8, 511-
521). This assay allows detection of NF-kappaB activation by measurement of the
fluorescence of the reporter protein destabilized Enhanced Green Fluorescent Protein
(d2EGFP). HEK/293 cells are stably transfected with a vector carrying the d2EGFP gene
under control of a synthetic promoter consisting of four kappa B elements and the minimal
thymidine kinase promoter. Treatment of the recombinant HEK-pNF-kB-d2EGFP/Neo cells
with the known NF-kappaB activator tumour necrosis factor alpha (TNF-alpha) results in
strong induction of NF-kappa B dependent gene expression in up to 90 % of the cells.
Involvement of the p65 subunit in this response was shown using an oligonucleotide-assay.
For a better characterisation of the cell-based assay, activation of the pathway by other agents,
e.g. interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin, and phorbol ester
(PMA), the influence of the culture conditions on NF-kappa activation by TNF-alpha, and the
expression of possible target genes were examined. NF-kappa B was activated by TNF-alpha,
IL-1beta and PMA in a dose-dependent manner, but not by camptothecin or LPS. TNF-alpha
results in the strongest induction of NF-kappa B dependent gene expression. However, this
response fluctuated from 30 to 90 %. Activation of NF-kappaB by TNF-alpha was maximal
within 48 hours after seeding of cells. With increasing confluence of the cells, the activation
potential decreased. In a confluent cell layer, only 30-50 % of the cell population showed
d2EGFP expression. This effect was also observed when cells were seeded in different cell
densities and treated at the same time point; the higher the cell density, the lower the d2EGFP
expression was after 20 hours incubation with TNF-alpha. The underlying mechanism to this
phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB
activation or the direct communication via gap junctions in the cell layer diminishing the
TNF-alpha response. The quantification of the transcripts of several possible NF-kappaB
target genes by quantitative PCR after treatment of cells with TNF-alpha revealed a strong
induction of IkappaB alpha and IL-6. In conclusion, the sequence of events from liberation of
NF-kappa B subunit p65 and its binding to DNA, the initiation of transcription and translation
in response to the NF-kappaB binding have been shown for the cell-based NF-kappaB assay.
The experiment conditions of this assay have to be very well controlled since cell density and
growth duration influence the NF-kappa B activation.
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