Beltinger C, Saragovi H U, Smith R M, LeSauteur L, Shah N, DeDionisio L, Christensen L, Raible A, Jarett L, Gewirtz A M
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
J Clin Invest. 1995 Apr;95(4):1814-23. doi: 10.1172/JCI117860.
An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.
对硫代磷酸酯修饰的寡脱氧核苷酸（P-ODN）摄取机制和细胞内运输的深入了解，可能会促进这些化合物在实验和治疗中的应用。我们通过鉴定与P-ODN特异性相互作用的细胞表面蛋白、研究P-ODN内化机制以及使用免疫化学和超微结构技术追踪细胞内的内化P-ODN来解决这些问题。用生物素标记的P-ODN（bP-ODN）进行的化学交联研究表明，存在五个主要的细胞表面P-ODN结合蛋白组，大小约为20-143 kD。未标记的P-ODN可竞争性抑制与这些蛋白的结合，但游离生物素则不能，这表明相互作用具有特异性。进一步的实验表明，结合蛋白可能以单链结构存在，并且碳水化合物部分可能在P-ODN结合中起作用。用35S标记的P-ODN进行的摄取研究表明，在P-ODN浓度<1 microM时，由受体样机制介导的内吞作用占主导，而在较高浓度时，液相内吞作用占优势。细胞分级分离和超微结构分析表明，网格蛋白包被小窝以及与内体和溶酶体一致的囊泡结构中存在ODN。在细胞核中也发现了大量标记的ODN，而没有一个与核糖体或与糙面内质网（ER）相关的核糖体有关。由于小麦胚凝集素或伴刀豆球蛋白A不能阻断核摄取，因此提示存在一种不依赖核孔蛋白、可能由扩散驱动的导入过程。这些数据表明反义DNA可能在细胞核中发挥作用。它们还提出了设计可能提高其效率的ODN的合理方法。
