Bigey P, Knox J D, Croteau S, Bhattacharya S K, Théberge J, Szyf M
Department of Pharmacology, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada.
J Biol Chem. 1999 Feb 19;274(8):4594-606. doi: 10.1074/jbc.274.8.4594.
The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.
由于缺乏直接且特异的抑制剂，DNA甲基转移酶（DNA MeTase）生物学作用的研究受到了阻碍。本报告描述了基于DNA的高效DNA MeTase拮抗剂的设计，以及利用它们来确定DNA MeTase与其底物的相互作用并研究其生物学作用。我们证明，大小、二级结构、半甲基化和硫代磷酸酯修饰强烈影响拮抗剂与DNA MeTase的相互作用，而碱基替换则没有显著影响。为了研究DNA MeTase对细胞转化是否至关重要，用人肺非小细胞癌细胞进行了DNA MeTase拮抗剂处理。在体外，DNA MeTase的发夹抑制剂定位于肺癌细胞的细胞核中。它们以剂量依赖的方式抑制DNA MeTase、细胞生长和非锚定依赖性生长（细胞培养中肿瘤发生的一个指标）。本研究中开发的抑制剂是活细胞中DNA MeTase直接抑制剂以及修饰寡核苷酸作为关键细胞蛋白的真正拮抗剂的首个有记录的实例。
