Monitoring NF-κB Transactivation Potential Via Real-Time PCR Quantification of IκB-α Gene Expression

Abstract

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.