Zhang Zhiren, Weinschenk Toni, Schluesener Hermann J
Institute of Brain Research, University of Tuebingen, Tuebingen, Germany.
Mol Cell Biochem. 2005 Apr;272(1-2):35-46. doi: 10.1007/s11010-005-6605-0.
Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.
胶质母细胞瘤是最恶性且最常见的脑肿瘤，也是基因治疗和免疫治疗令人瞩目的靶点。尽管实验性治疗发展迅速，但对于治疗性寡脱氧核苷酸（ODN）的细胞行为却知之甚少。在此，我们通过流式细胞术、荧光显微镜和质谱分析，设计研究了免疫刺激性CpG-ODN在胶质母细胞瘤细胞中的摄取、细胞分布及细胞结合蛋白。我们的数据表明，硫代磷酸酯（PS）CpG-ODN在T98G和C6细胞中的摄取具有剂量、时间和温度依赖性，且与CpG二核苷酸无关。叠氮化钠、聚阴离子可抑制摄取，但氯喹无此作用。内化后，异硫氰酸荧光素（FITC）标记的CpG-ODN在细胞质中呈斑点状分布。通过质谱分析鉴定出了数十种细胞结合蛋白。ODN与蛋白质的结合取决于修饰和序列，但与CpG基序无关。ODN与对RNA加工和转运至关重要的细胞蛋白结合。此外，还鉴定出了三种新型膜蛋白，它们可能有助于ODN的摄取。ODN与这些蛋白的结合可能会干扰其生理功能，从而导致不良影响。这种结合也可能会影响治疗性ODN的摄取效率或细胞分布。
