Hartmann G, Krug A, Waller-Fontaine K, Endres S
Medizinische Klinik, Ludwig Maximilians-University, Munich, Germany.
Mol Med. 1996 Jul;2(4):429-38.
Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects.
We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied.
The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis.
The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.
反义寡脱氧核苷酸对靶蛋白的特异性抑制是一种经过广泛研究的实验方法。目前，该技术正在临床试验中进行测试，使用硫代磷酸酯修饰的寡核苷酸作为治疗药物。然而，这些聚阴离子分子也可能产生非反义介导的效应。
我们研究了寡核苷酸对新鲜分离的人外周血单核细胞中脂多糖（LPS）刺激的肿瘤坏死因子α（TNFα）合成的影响。研究了不同程度硫代磷酸酯修饰的寡核苷酸（18聚体）。
与仅用LPS的对照组相比，添加硫代磷酸酯寡核苷酸（5μM）可使TNF合成放大高达410%。在没有LPS刺激的情况下，硫代磷酸酯寡核苷酸不会诱导TNF产生。我们证明，硫代磷酸酯寡核苷酸对LPS刺激的TNF产生的增强不依赖于寡核苷酸在细胞内的存在，也不是由LPS污染介导的。部分硫代磷酸酯修饰的寡核苷酸和未修饰的寡核苷酸不会增加TNF合成。高浓度的聚阴离子肝素可逆转寡核苷酸诱导的TNF合成增强。
数据表明，TNF合成的放大可能是由于聚阴离子硫代磷酸酯寡核苷酸与细胞表面阳离子位点的结合所致。细胞外基质的聚阴离子糖胺聚糖也被认为存在这样的结合位点，并且也被描述为可增强LPS刺激的TNF合成。目前的结果与所有试图通过使用硫代磷酸酯寡核苷酸影响单核细胞中蛋白质合成的体外研究相关。对于硫代磷酸酯在体内应用（如在脓毒症中有TNF合成刺激的情况下）的意义，应予以阐明。
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