A Jurkat transcriptional reporter cell line for high-throughput analysis of the nuclear factor-kappaB signaling pathway.
The transcription factor, Nuclear Factor-kappaB (NF-kappaB), regulates many genes involved in host immunity and cell survival. Unregulated NF-kappaB activity has been linked to many chronic inflammatory diseases and is an important target for the identification of inhibitors to better manage these disorders. We present a novel screening system to identify NF-kappaB inhibitors that combines sensitive fluorescence detection with medium- to high-throughput flow cytometry (HyperCyt). To validate this approach, we quantified the activation of NF-kappaB by standard flow cytometry and the HyperCyt platform. Results were comparable with regard to EC(50) values for TNFalpha-mediated activation; however, the HyperCyt platform provided more sensitive signal detection and a greater linear range for detection. To demonstrate the usefulness of this screening tool, we identified a novel inhibitor of NF-kappaB activation from a resveratrol-based chemical library. The inhibition of NF-kappaB activation by analog 6q (IC(50) = 19 microm) showed a 3.7-fold improvement over that of resveratrol (IC(50) approximately 70 microm).
Gonzales
Amanda M
AM
Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, Albuquerque, NM 87131, United States.
Orlando
Robert A
RA
D016923:Cell Death / Q000187:drug effects; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D047311:Luteolin / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D047311:Luteolin