Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression

Mol Diagn. 2003;7(3-4):187-94. doi: 10.1007/BF03260037.

Abstract

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.

Methods and 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Primers
  • Gene Expression Regulation, Neoplastic / genetics
  • HeLa Cells
  • Humans
  • I-kappa B Proteins / genetics*
  • Jurkat Cells
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / genetics*
  • NF-kappa B / metabolism
  • Polymerase Chain Reaction / methods*
  • RNA, Messenger / genetics
  • Transcription, Genetic
  • Transcriptional Activation / genetics*

Substances

  • DNA Primers
  • I-kappa B Proteins
  • NF-kappa B
  • NFKBIA protein, human
  • RNA, Messenger
  • NF-KappaB Inhibitor alpha