Background: Nuclear factor-kappa B (NF-κB) is an important transcription factor involved in the regulation of immune responses as well as in cell proliferation and survival. An abnormal and constitutive activation of NF-κB is observed in many pathological states as diverse as inflammation, neurological diseases, and cancer.
Methods and results: Termination of NF-κB transcription is mediated through the NF-κB-dependent synthesis of the IκB-α inhibitory subunit. To quantify NF-κB activation we measured by real-time PCR the expression of IκB-α mRNA. The PCR data perfectly matched the results obtained by Northern blot or gene reporter analysis when Jurkat leukemic T cells or HeLa carcinoma cells were stimulated with various activators of NF-κB, such as the cytokine tumor necrosis factor (TNF)-α or the phorbol ester PMA. Constitutive NF-κB activation in Hodgkin’s lymphoma cell line could also be evaluated by this approach. Kinetic experiments in HeLa cells show that TNF stimulation first induced NF-κB DNA binding within 30 minutes, followed by IκB-α gene transcription 30 minutes later. Removal of TNF after stimulation resulted in a faster decrease in both NF-αB DNA binding activity and IκB-α mRNA levels. No accumulation or stabilization of IαB-α mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-αB activation in stimulated normal peripheral blood lymphocytes.
Conclusion: The real-time PCR measure of IαB-α mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-κB. It can be easily used for clinical evaluation of NF-κB status.
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This work is supported by an institutional grant from INSERM and by a grant from La Fondation de France, Comité Leucémies.
We would like to express our thanks to Mrs Marie Noëlle Monthouel (INSERM U145, Nice) for her help and advice at the beginning of this study. We also thank Dr Gérard Milano (Centre Antoine Lacassagne, Nice, France) for his support.
The authors have no conflict of interest directly relevant to the content of this study.
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