Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites.
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.
Hewett
Peter W
PW
Department of Vascular and Reproductive Biology, Institute for Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK. p.w.hewett.@bham.ac.uk
Daft
Emma L
EL
Laughton
Charles A
CA
Ahmad
Shakil
S
Ahmed
Asif
A
Murray
J Clifford
JC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects*; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D050783:Proto-Oncogene Proteins c-ets / Q000378:metabolism*; D042810:Receptor, TIE-1 / Q000235:genetics*
D009841:Oligonucleotides; D050783:Proto-Oncogene Proteins c-ets; C051802:triplex DNA; D004247:DNA; D008156:Luciferases; D042810:Receptor, TIE-1