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.

Publication DOI: https://doi.org/10.2119/2005-00046.Hewett
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Health & Life Sciences > Aston Medical School
College of Health & Life Sciences
Additional Information: Creative Commons attribution
Publication ISSN: 1528-3658
Last Modified: 04 Jan 2024 08:12
Date Deposited: 09 Jun 2014 13:55
Full Text Link:
Related URLs: http://molmed.o ... /articles/6/250 (Publisher URL)
PURE Output Type: Article
Published Date: 2006-01
Authors: Hewett, Peter W.
Daft, Emma L.
Laughton, Charles A.
Ahmad, Shakil (ORCID Profile 0000-0002-9294-0475)
Ahmed, Asif (ORCID Profile 0000-0002-8755-8546)
Murray, J. Clifford

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License: Creative Commons Attribution


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