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

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.

• Authors: Peter W Hewett, Emma L Daft, Charles A Laughton, Shakil Ahmad, Asif Ahmed, J Clifford Murray
• Journal: Molecular medicine (Cambridge, Mass.) (Mol Med) 2006 Jan-Mar Vol. 12 Issue 1-3 Pg. 8-16 ISSN: 1076-1551 [Print] England
• PMID: 16838069 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Oligonucleotides
• Proto-Oncogene Proteins c-ets
• triplex DNA
• DNA
• Luciferases
• Receptor, TIE-1

Topics

• Animals
• Base Sequence
• Binding Sites
• Cattle
• DNA (genetics)
• Down-Regulation (drug effects)
• Electrophoretic Mobility Shift Assay
• Endothelial Cells (drug effects)
• Genes, Reporter (genetics)
• Humans
• Luciferases (metabolism)
• Molecular Sequence Data
• Nucleic Acid Conformation (drug effects)
• Oligonucleotides (genetics, metabolism, pharmacology)
• Promoter Regions, Genetic (genetics)
• Proto-Oncogene Proteins c-ets (metabolism)
• Receptor, TIE-1 (genetics)