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 journalArticle

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
Number of pages9
JournalMolecular Medicine
Volume12
Issue number1-3
DOIs
Publication statusPublished - Jan 2006

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Oligonucleotides
Endothelial Cells
Binding Sites
Proto-Oncogene Proteins c-ets
Neoplasms
Sequence Deletion
Electrophoretic Mobility Shift Assay
Luciferases
Genes
Endothelium
Transfection
Plasmids
Pathology
Gene Expression
DNA
Therapeutics
Growth
triplex DNA
purine

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Hewett, Peter W. ; Daft, Emma L. ; Laughton, Charles A. ; Ahmad, Shakil ; Ahmed, Asif ; Murray, J. Clifford. / Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites. In: Molecular Medicine. 2006 ; Vol. 12, No. 1-3. pp. 8-16.
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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; Murray, J. Clifford.

In: Molecular Medicine, Vol. 12, No. 1-3, 01.2006, p. 8-16.

Research output: Contribution to journalArticle

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AU - Daft, Emma L.

AU - Laughton, Charles A.

AU - Ahmad, Shakil

AU - Ahmed, Asif

AU - Murray, J. Clifford

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AB - 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.

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