Attenuation of NFKB binding activity using oligonucleotides as decoys

Academic Article

Abstract

  • Nuclear Factor Kappa B (NFKB) is a transcriptional regulatory protein involved in the activation of genes encoding a variety of proteins such as cytokines, immunoglobulin subunits and viral proteins. The gene for the cytokine IL-8, a powerful chemotactic agent, contains a binding site for NFKB in its promotor region. Previous work in this lab showed IL-8 production by monocytes can be attenuated by transfecting monocytes with various related oligonucleotides containing the consensus sequence for the IL-8 NFKB binding site. An oligonucleotide containing two repeats of the consensus sequence was more effective at suppressing IL-8 protein production than one containing one copy of the sequence. The purpose of this current study was to investigate whether NFKB binding activity could be reduced in monocytes by transfecting with similar oligonucleotides containing the consensus sequence for the IL-8 NFKB binding site. Monocytes obtained from healthy volunteers were transfected with either lipofectin (LP) alone, oligonucleotides containing a single repeat (NFKB1) or a double repeat (NFKB2) of the IL-8 consensus sequence. Cells were stimulated with IL-1 for twenty-four hours and then nuclear protein was extracted. NFKB binding activity of nuclear proteins was determined by gel shut assay and densitometry was used to quantity results. Results are shown in Table 1. Table 1 LP LP/IL1 LP/IL1/NFKB1 LP/IL1/NFKB2 21.5+/-5.5 30+/-4.0 35+/-6.0 18.2+/-3.4* Data represent density in random units+/-S.E.M. *p<0.05 vsLP/IL1 and LP/IL1/NFKB 1. NFKB binding activity was reduced when cells were transfected with the oligonucleotide containing two repeats of the consensus sequence but was not reduced in the cells containing the single repeat, hi conclusion, reduced IL-8 production by monocytes transfected with oligonucleotides containing the consensus sequence for the NFKB binding site is associated with a reduction in NFKB binding activity probably due to a decoy phenomenon exerted by the transfected oligonucleotides.
  • Author List

  • Parks JM; Cooper JAD
  • Volume

  • 44
  • Issue

  • 1