Hewett Peter W, Daft Emma L, Laughton Charles A, Ahmad Shakil, Ahmed Asif, Murray J Clifford
Department of Vascular and Reproductive Biology, Institute for Biomedical Research, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK.
Mol Med. 2006 Jan-Mar;12(1-3):8-16. doi: 10.2119/2005-00046.Hewett.
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.
Tie受体(Tie-1和Tie-2/Tek)对血管生成以及血管重塑/完整性至关重要。Tie受体在肿瘤相关内皮细胞中上调,抑制它们会破坏血管生成,并因此阻止肿瘤生长。为了研究反基因方法抑制tie基因表达用于抗血管生成治疗的潜力,我们检测了人tie-1启动子中2个串联的Ets转录因子结合基序(命名为E-1和E-2)处三链DNA的形成。构建了各种tie-1启动子缺失/突变荧光素酶报告基因构建体,并转染到内皮细胞中以检测E-1和E-2的相对活性。通过质粒DNA片段结合和电泳迁移率变动分析评估靶向E-1和E-2的反平行和平行(对照)嘌呤基序寡核苷酸(21 - 22 bp)的结合情况。将形成三链的寡核苷酸与tie-1报告基因构建体一起孵育并转染到内皮细胞中以确定它们的活性。E-1序列中的Ets结合基序对内皮细胞中人tie-1启动子活性至关重要,而E-2的缺失没有影响。靶向E-1或E-2的反平行嘌呤基序寡核苷酸在37℃时选择性地形成强三链DNA(解离常数K(d)约为10^(-7) M)。与对照寡核苷酸相比,在内皮细胞中用E-1处的三链DNA转染tie-1报告基因构建体,而非E-2处的,可特异性抑制tie-1启动子活性高达75%。由于类似的多个Ets结合位点对几种内皮细胞特异性基因的调控很重要,这种方法可能对癌症和其他涉及内皮细胞增殖/功能障碍的疾病具有广泛的治疗潜力。