Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites

Peter W Hewett, Emma L Daft, Charles A Laughton, Shakil Ahmad, Asif Ahmed, J Clifford Murray

Research output: Contribution to journalArticlepeer-review

Abstract

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.
Original languageEnglish
Pages (from-to)8-16
JournalMolecular Medicine
Volume12
Issue number1-3
Publication statusPublished - 2006

Keywords

  • Animals
  • Base Sequence
  • Binding Sites
  • CATTLE
  • DNA
  • DOWN-REGULATION
  • Electrophoretic Mobility Shift Assay
  • Endothelial cell
  • Genes, Reporter
  • humans
  • Luciferases
  • molecular sequences data
  • Nucleic Acid Conformation
  • OLIGONUCLEOTIDES
  • Promoter Regions, Genetic
  • Proto-Oncogene Protein c-ets
  • receptor, tie-1

Fingerprint Dive into the research topics of 'Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites'. Together they form a unique fingerprint.

Cite this