McGuffie E M, Pacheco D, Carbone G M, Catapano C V
Department of Experimental Oncology and Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, USA.
Cancer Res. 2000 Jul 15;60(14):3790-9.
The c-myc gene is frequently deregulated and overexpressed in human cancers, and strategies designed to inhibit c-myc expression in cancer cells may have considerable therapeutic value. The purpose of the present work was to characterize the antigene and antiproliferative activity of a triple helix-forming oligonucleotide (TFO) targeted to a homopurine-homopyrimidine sequence in the P2 promoter of the c-myc gene. The TFO was synthesized with phosphorothioate (PS) internucleotide linkages to confer resistance to intra- and extracellular nucleases. This property is required of oligonucleotides designed for in vivo testing and therapeutic applications. The PS-TFO was found to form triplex DNA with affinity and specificity comparable with that of the corresponding phosphodiester TFO, as shown by gel mobility shift and footprinting assays. Fluorescence microscopy and polyacrylamide gel analysis showed that the fluorescein-labeled PS-TFO accumulated in nuclei of CEM leukemia cells and remained intact for at least 72 h. Incubation of CEM cells with PS-TFO reduced c-myc RNA and protein levels. A single exposure of leukemia cells to the PS-TFO was sufficient to induce dose-dependent growth inhibitory effects. Growth inhibition correlated with accumulation of cells in S phase and with induction of cell death by apoptosis. The PS-TFO was also effective in other leukemia and lymphoma cell lines. Control oligonucleotides had minimal effects in all assays. These data indicate that the c-myc-targeted PS-TFO is an effective antigene and antiproliferative agent, with potential for testing in vivo as a novel approach to cancer therapy.
c-myc基因在人类癌症中经常发生失调并过度表达，旨在抑制癌细胞中c-myc表达的策略可能具有相当大的治疗价值。本研究的目的是表征一种靶向c-myc基因P2启动子中同型嘌呤-同型嘧啶序列的三链螺旋形成寡核苷酸（TFO）的反基因和抗增殖活性。TFO通过硫代磷酸酯（PS）核苷酸间连接合成，以赋予对细胞内和细胞外核酸酶的抗性。这一特性是为体内测试和治疗应用设计的寡核苷酸所必需的。凝胶迁移率变动分析和足迹分析表明，PS-TFO能与相应的磷酸二酯TFO一样，以相似的亲和力和特异性形成三链DNA。荧光显微镜和聚丙烯酰胺凝胶分析表明，荧光素标记的PS-TFO在CEM白血病细胞核中积累，并至少保持完整72小时。用PS-TFO孵育CEM细胞可降低c-myc RNA和蛋白质水平。白血病细胞单次暴露于PS-TFO足以诱导剂量依赖性生长抑制作用。生长抑制与细胞在S期的积累以及凋亡诱导的细胞死亡相关。PS-TFO在其他白血病和淋巴瘤细胞系中也有效。对照寡核苷酸在所有试验中作用极小。这些数据表明，靶向c-myc的PS-TFO是一种有效的反基因和抗增殖剂，有作为癌症治疗新方法进行体内测试的潜力。
