INTERACTION OF OLIGODEOXYNUCLEOTIDES WITH MAMMALIAN-CELLS

Many previous studies have demonstrated that antisense oligodeoxynucle otides (ODNs) bind to surface proteins in a manner compatible with rec eptor-mediated endocytosis and, unless specifically modified, are inte rnalized into endosomes with little access to the cytoplasmic structur es or to the nucleus, Reports vary as to the specific proteins involve d in the mechanism, and this study examines the conditions of binding, some proteins that might contribute to the process, and whether chang es in binding patterns occur during differentiation. Native gel electr ophoresis was used to optimize the surface binding of a phosphorothioa te end-capped 16-mer to T15 mouse fibroblast cells, and comparisons ar e made with some human epithelial tumor cell lines, Binding to individ ual proteins was visualized using SDS-PAGE and autoradiography, Bindin g at 4 degrees C was almost exclusively to a 46 kDa protein and decrea sed 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 bin ding to the total cytoplasmic and membrane proteins immobilized on a m embrane showed a greater number of binding proteins, the most prominen t 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 increase d total binding seen on native gels coinciding with increased binding to a 46 kDa protein, Demonstration that changes in binding proteins oc cur when cells differentiate was made using the premacrophage cell lin e THP-1. Differentiation of these cells increased the total ODN bindin g and appeared to initiate the synthesis of some new binding proteins, although binding to a 46 kDa protein was reduced.