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

Peter W Hewett,Shakil Ahmad,J Clifford Murray,Asif Ahmed,Emma L Daft,Charles A Laughton

doi:10.2119/2005-00046.hewett

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.

Highlights

Tumor growth and the development of hematologic metastases depend on angiogenesis, and the inhibition of this process represents a target for therapy [1]
In addition to the disruption of transcriptional elongation [21,22,27,28,29,53], inhibition of gene expression has been achieved through triplex DNA formation at sequences encoding transcription factor binding sites including SP-1, SRF, CNBP, PuF, MAZ, Pax5, PU.1, STAT6, and NF-κB [21,22,24,25,26,31,32]
We demonstrate the ability of antiparallel purine motif triplex-forming oligonucleotide (TFO) to selectively form DNA triplex at two sequences encoding tandem Ets core DNA binding motifs in the tie-1 promoter and the triplex-mediated inhibition of promoter activity through TFO binding to one of these sequences (E-1)

Summary

Introduction

Tumor growth and the development of hematologic metastases depend on angiogenesis, and the inhibition of this process represents a target for therapy [1]. Several endothelial receptor tyrosine kinases (RTKs) play key roles in coordinating vascular proliferation, differentiation, and maintenance/integrity [2,3]. These include the vascular endothelial growth factor (VEGF) family of receptors (Flt-1, Flt-4, and KDR/Flk-1) and Tie (Tie-1 and Tie-2/ Tek) RTKs [2,3]. The Tie receptors are essential for embryonic development, and targeted disruption of the tie genes in mice highlights their importance in angiogenesis and vascular remodeling [4]. As with Tie-2, the activation of Tie-1 signaling has been reported to promote endothelial cell survival through the phosphoinositol-3kinase/Akt pathway [7]

Methods

Results

Conclusion