Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression.
BACKGROUND
Nuclear factor-kappa B (NF-kappa B) 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-kappa B is observed in many pathological states as diverse as inflammation, neurological diseases, and cancer.


RESULTS
Termination of NF-kappa B transcription is mediated through the NF-kappa B-dependent synthesis of the I kappa B-alpha inhibitory subunit. To quantify NF-kappa B activation we measured by real-time PCR the expression of I kappa B-alpha 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-kappa B, such as the cytokine tumor necrosis factor (TNF)-alpha or the phorbol ester PMA. Constitutive NF-kappa B 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-kappa B DNA binding within 30 minutes, followed by I kappa B-alpha gene transcription 30 minutes later. Removal of TNF after stimulation resulted in a faster decrease in both NF-kappa B DNA binding activity and I kappa B-alpha mRNA levels. No accumulation or stabilization of I kappa B-alpha mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-kappa B activation in stimulated normal peripheral blood lymphocytes.


CONCLUSIONS
The real-time PCR measure of I kappa B-alpha mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-kappa B. It can be easily used for clinical evaluation of NF-kappa B status.
Bottero
Virginie
V
INSERM U526, Hematopolatic Cell Activation, IFR 50, Faculty of Medicine Pasteur, Nice, France.
Imbert
Véronique
V
Frelin
Catherine
C
Formento
Jean-Louis
JL
Peyron
Jean-François
JF
D001483:Base Sequence; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D019169:Jurkat Cells; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics*
D017931:DNA Primers; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha