Shevelev A, Burfeind P, Schulze E, Rininsland F, Johnson T R, Trojan J, Chernicky C L, Hélène C, Ilan J, Ilan J
Department of Pathology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106-4943, USA.
Cancer Gene Ther. 1997 Mar-Apr;4(2):105-12.
Oligonucleotide-directed triple helix formation is a powerful approach to block transcription of specific genes. Although the oligonucleotide triplex approach is efficient for inhibiting gene expression in cultured cells, suppression is transient. We developed an approach which inhibits insulin-like growth factor-I (IGF-I) expression following stable transfection of C6 rat glioblastoma cells with a plasmid from which an RNA is transcribed that codes for the third strand of a potential triple helix. We tested the ability of this expression vector to inhibit IGF-I gene expression in vitro as well as tumorigenesis in an animal. A dramatic reduction of IGF-I RNA and protein levels in cultured cells occurred following transfection of rat C6 cells with a eukaryotic expression plasmid encoding the oligopurine variant of the triple helix but not the oligopyrimidine or a control sequence. The cells transfected with the oligopurine variant displayed morphological changes, upregulation of major histocompatibility complex I, and increased expression of protease nexin I. Dramatic inhibition of tumor growth occurred in nude mice following injection of transfected C6 cells. To our knowledge, this is the first example of tumor growth inhibition in an animal model employing a triple helix approach.
寡核苷酸定向三链螺旋形成是一种阻断特定基因转录的有效方法。尽管寡核苷酸三链体方法在抑制培养细胞中的基因表达方面很有效，但抑制作用是短暂的。我们开发了一种方法，在用一种质粒稳定转染C6大鼠胶质母细胞瘤细胞后抑制胰岛素样生长因子-I（IGF-I）的表达，该质粒转录出一种RNA，该RNA编码潜在三链螺旋的第三条链。我们测试了这种表达载体在体外抑制IGF-I基因表达以及在动物体内抑制肿瘤发生的能力。在用编码三链螺旋的寡嘌呤变体而非寡嘧啶或对照序列的真核表达质粒转染大鼠C6细胞后，培养细胞中IGF-I RNA和蛋白质水平显著降低。用寡嘌呤变体转染的细胞表现出形态变化、主要组织相容性复合体I上调以及蛋白酶nexin I表达增加。在注射转染的C6细胞后，裸鼠体内的肿瘤生长受到显著抑制。据我们所知，这是在动物模型中采用三链螺旋方法抑制肿瘤生长的首个实例。
