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

Hewett, Peter W., Daft, Emma L., Laughton, Charles A., Ahmad, Shakil, Ahmed, Asif and Murray, J. Clifford (2006). Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites. Molecular Medicine, 12 (1-3), pp. 8-16.

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: Life & Health Sciences > Biosciences
Life & Health Sciences
Aston Medical School
Additional Information: Creative Commons attribution
Full Text Link: http://molmed.o ... /articles/6/250
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Published Date: 2006-01
Authors: Hewett, Peter W.
Daft, Emma L.
Laughton, Charles A.
Ahmad, Shakil ( 0000-0002-9294-0475)
Ahmed, Asif
Murray, J. Clifford

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