(111a) Quantifying Differential Activation Of Nf-êb-Response Genes By Lps In Macrophages Using 3,4-Dcpa As A Molecular Probe

Nuclear Factor kappa B (NF-κB) is an important regulator of inflammation and adaptive immunity. Fibroblasts persistently stimulated with tumor necrosis factor-alpha (TNF-α) show oscillations in DNA-binding activity of NF-κB resulting from the coordinated regulation of IκB isoforms. We have previously described that dichloropropionaniline (DCPA) treatment of LPS-stimulated macrophages reduced TNF-α production that correlated with reduced NF-?ÛB DNA binding. Yet, reduced TNF-α did not correlate with reduced IκBα degradation. To test the hypothesis that reduced DNA binding levels were due to a change in the IκB isoforms, we undertook an iterative computational and experimental investigation to quantify the effects of DCPA on dynamic properties of lipopolysaccharide (LPS)-mediated activation of the transcription factor NF-κB. We found that oscillations in active NF-κB could be reproduced in a macrophage cell line, but exhibited significant variability between replicates. As biological variability has a confounding effect on quantifying the effect of DCPA on oscillations, we focused on the early activation of NF-κB. In contrast to our previous studies using a different DNA binding site, our kinetic analysis suggests that DCPA induced a dose-dependent increase in active NF-κB following LPS stimulation. We also found that transcription factor assay selection significantly impacts the ability to observe significant changes in NF-κB activation. This iterative approach is an important step towards improving our understanding of the quantitative role of NF-κB in adaptive immunity.