Monitoring NF-κB Transactivation Potential Via Real-Time PCR Quantification of IκB-α Gene Expression
作者:
Virginie Bottero,
Véronique Imbert,
Catherine Frelin,
Jean-Louis Formento,
Jean-François Peyron,
期刊:
Molecular Diagnosis
(ADIS Available online 2003)
卷期:
Volume 7,
issue 3
页码: 187-194
ISSN:1084-8592
年代: 2003
出版商: ADIS
关键词: Genetic polymorphism;Inflammation, diagnosis;Neurological disorders, diagnosis;Cancer, diagnosis
数据来源: ADIS
摘要:
BackgroundNuclear 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 resultsTermination 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-κB DNA binding activity and IκB-α mRNA levels. No accumulation or stabilization of IκB-α mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-κB activation in stimulated normal peripheral blood lymphocytes.ConclusionThe real-time PCR measure of IκB-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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