Interaction of oligodeoxynucleotides with mammalian cells.
Many previous studies have demonstrated that antisense oligodeoxynucleotides (ODNs) bind to surface proteins in a manner compatible with receptor-mediated endocytosis and, unless specifically modified, are internalized into endosomes with little access to the cytoplasmic structures or to the nucleus. Reports vary as to the specific proteins involved in the mechanism, and this study examines the conditions of binding, some proteins that might contribute to the process, and whether changes in binding patterns occur during differentiation. Native gel electrophoresis was used to optimize the surface binding of a phosphorothioate end-capped 16-mer to T15 mouse fibroblast cells, and comparisons are made with some human epithelial tumor cell lines. Binding to individual proteins was visualized using SDS-PAGE and autoradiography. Binding at 4 degrees C was almost exclusively to a 46 kDa protein and decreased in the presence of an excess of unlabeled ODN and heparin but not ATP. Increasing the temperature of ODN binding from 4 degrees C to 37 degrees C for 10 minutes changed the binding pattern observed. ODN binding to the total cytoplasmic and membrane proteins immobilized on a membrane showed a greater number of binding proteins, the most prominent being one of 30 kDa. Examination of the effects of serum on binding were made using the human lung carcinoma cell line COR-L23, which can be grown in serum-free conditions. Serum starvation led to an increased total binding seen on native gels coinciding with increased binding to a 46 kDa protein. Demonstration that changes in binding proteins occur when cells differentiate was made using the premacrophage cell line THP-1. Differentiation of these cells increased the total ODN binding and appeared to initiate the synthesis of some new binding proteins, although binding to a 46 kDa protein was reduced.
School of Biological Sciences, Norwich, UK.
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P
Hawley
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Gibson
D000818:Animals; D001667:Binding, Competitive; D002277:Carcinoma / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000502:physiology*; D004734:Energy Metabolism / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense