Chiang M Y, Chan H, Zounes M A, Freier S M, Lima W F, Bennett C F
Department of Molecular and Cellular Biology and Medicinal Chemistry, ISIS Pharmaceuticals, Carlsbad, California 92008.
J Biol Chem. 1991 Sep 25;266(27):18162-71.
Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.
细胞间黏附分子1（ICAM-1）是一种糖蛋白，在造血细胞和非造血细胞表面均有表达，它在一定程度上介导白细胞从脉管系统中移出。白细胞介素-1β、肿瘤坏死因子α和干扰素γ可使人类脐静脉内皮细胞和一种人肺癌细胞系（A549）表面的ICAM-1表达呈浓度依赖性增加。设计用于与人ICAM-1 mRNA上10个靶位点杂交的硫代磷酸酯反义寡核苷酸，通过ICAM-1酶联免疫吸附测定法检测其对两种细胞系中ICAM-1表达的抑制作用。基于效力和独特的mRNA靶位点，对两种寡核苷酸进行了更详细的研究：ISIS 1570靶向AUG翻译起始密码子，ISIS 1939靶向mRNA 3'非翻译区的特定序列。通过对35S标记蛋白的免疫沉淀分析，两种寡核苷酸均能特异性抑制ICAM-1的表达。用ISIS 1939处理细胞可使ICAM-1 mRNA减少，而ISIS 1570未改变ICAM-1 mRNA水平，这表明两种寡核苷酸可能通过两种不同机制抑制ICAM-1表达。在加入细胞之前，寡核苷酸与其互补的有义链杂交可阻断两种寡核苷酸的活性。ISIS 1570和ISIS 1939均未改变ICAM-1基因的转录速率，表明两种寡核苷酸均通过转录后机制发挥作用。使用不支持RNase H介导的靶mRNA切割的2'-O-甲基硫代磷酸酯类似物来确定活性反义寡核苷酸是否通过依赖RNase H的机制抑制ICAM-1表达。ISIS 1939的2'-O-甲基硫代磷酸酯类似物未显著降低白细胞介素-1β诱导的ICAM-1表达，而ISIS 1570的2'-O-甲基硫代磷酸酯类似物确实抑制了ICAM-1表达，这表明用ISIS 1939处理后ICAM-1 mRNA的减少部分归因于RNase H介导的水解。ISIS 1570和ISIS 1939均可抑制HL-60细胞与人脐静脉细胞单层的黏附，表明ICAM-1水平的降低影响了与ICAM-1相关的功能。
