Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells

Abstract

We have examined the cellular association and internalization of phosphodiester (PO) oligodeoxynucleotides (oligos) with HL60 cells. At 4 degrees C, a 15-mer PO homopolymer of thymidine (FOdT15) exhibits apparent saturation binding (Km = 22 +/- 1 nM) that is competitive with the binding of phosphorothioate (PS) oligos. The value of Kc for SdC28, a PS 28-mer homopolymer of cytidine, is 5 +/- 2 nM. SdC28 was used to strip cell surface fluorescence: Internalized fluorescence accumulated in a (concentration)(time)-dependent fashion, consistent with a pinocytotic mechanism. PS, and to a lesser extent, PO oligos inhibited the rate of internalization of fluorescent albumin, also a marker of pinocytosis. This was correlated with direct in vitro inhibition of protein kinase C (PKC) beta 1 by the PS and PO oligos. Furthermore, other PKC inhibitors (H7, staurosporine, DMSO, PKC pseudosubstrate polypeptide) also inhibited intracellular accumulation of pinocytosed materials, perhaps by stimulating the exocytosis rate. In HL60 cells, the pinocytotic internalization of charged oligos appears to be dependent on intact PKC kinase activity, which is inhibited in vitro by PS and PO oligos.

Human cancer cell lines growth inhibition by GTn oligodeoxyribonucleotides recognizing single‐stranded DNA‐binding proteins

“…The reduction of the toxic effect of GT 3 since control CT 3 , GC 3 , AT 3 and GA 3 containing the same pyrimidine/purine values or the same number of T or G bases in PMA-treated cells is not due to a decrease of oligonucleotide uptake upon phorbol ester administration; in fact, administration with respect to toxic GT 3 do not result in significantly altered cellular growth. In addition, we verified that the administration of PMA can increase the oligonucleotide uptake during the first 24 h [8] and we found that 24 h after the administration of PMA of monophosphate or triphosphate-nucleotides at the same concentrations as those of toxic GT n do not alter the cellular growth or IL-2, the GT3 uptake does not differ from that of an untreated control culture (data not shown). In agreement with the decrease of the human cancer cell lines (data not shown).…”

Effect of unmodified triple helix‐forming oligodeoxyribonucleotide targeted to human multidrug‐resistance gene mdr1 in MDR cancer cells

“…Depending on the ODN extracellular concentration, they may enter the cells either by specific absorptive endocytosis or by pinocytosis [29][30][31]. Although natural phosphodiester ODNs undergo a rapid extra-intracellular degradation [32,33], they can partially persist in the cell and rapidly reach the nucleus [12,34,35].…”

Novel PDGFβR antisense encapsulated in polymeric nanospheres for the treatment of restenosis

“…32 Blocking the binding of these proteins to their natural receptor is a possible mechanism for our results. However, this is questionable since the affinity was shown to be length-dependent, rather than sequence-dependent, 33 and the observed antirestenotic effect was sequence-dependent (the SC sequence had no effect).…”