Mechanism of oligonucleotide uptake by cells: involvement of specific receptors?

Abstract

We have investigated the interaction of ohgonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (<1 jAM), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single-and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.Antisense oligodeoxynucleotides and their derivatives have been shown to be specific inhibitors ofgene expression. They have been considered as a potential new generation of drugs, capable perhaps of inhibiting various pathogens and of regulating specific gene expression by inhibiting the translation of mRNA molecules in a highly specific manner (1-4). However, a prevailing view is that cells are not very permeable to oligonucleotides. Considerable efforts have been made to design nonionic membrane-permeable analogs (5) and to develop special delivery techniques (6, 7). Nevertheless, it has also been shown that normal unsubstituted oligonucleotides can cause hybridization arrest of specific mRNAs and viruses in cell culture (8-11). These observations provide evidence that oligodeoxynuicleotides do indeed enter cells. Recently, the ability of oligonucleotides to enter mammalian cells has been proved experimentally (9,(11)(12)(13)(14).The present investigation studied the interaction of oligodeoxynucleotide derivatives with mammalian cells. We examined the efficiency of oligomer binding and the time course of oligomer internalization under various conditions. We also investigated the stability of the internalized oligomers. Experiments involving the reactive 4-[N-(2-chloroethyl)-N-methyl]aminobenzyl phosphamide derivative of oligodeoxynucleotides [general formula ClRCH2NHpTTFr..., where R = -CH2CH2N(CH3)C6Hr---(1)]t were also undertaken: reagents of this type have recently been used by us as inhibitors of the influenza and tick-borne encephalitis viruses (4,15). In the present study, these reagents were used to chemically modify the putative cellular receptors binding oligonucleotides. MATERIALS AND METHODSOligodeoxynucleotides and Their Derivatives. The oligodeoxyribonucleotides pT, (n = 8, 9, 10, 16)

Inhibition of LNCaP prostate cancer cells by means of androgen receptor antisense oligonucleotides

“…The gradual abatement of the antisense effect may be explained by inactivation of ODNs by enzymatic degradation or by the release of ODNs by exocytosis. 39 Proliferation of antisense-treated LNCaP cells was completely inhibited compared with mock-treated controls after a 24-to 48-hour lag-phase.…”

Spontaneous transgenesis of human B lymphocytes

“…Plasmid g1NANP carries an insertion in the third complementarity-determining region (CDR3) of the variable domain coding for three repeats of the tetrapeptide NANP. 22 Plasmid g1MART (used in experiments shown in Figure 6) codes for the amino-acid sequence AAGIGILTV (position [27][28][29][30][31][32][33][34][35] from the MART-1 melanoma-associated antigen 23 in lieu of the three NANP repeats. Both plasmids are under the control of a B-cell-specific promoter.…”

“…In spite of the ease of reproducibility, the exact mechanism of DNA internalization can only be speculated. Studies on spontaneous DNA internalization in vitro (using viral DNA or oligonucleotides) 25,32,33 or in vivo (using plasmid DNA) 34 exclude the fact that DNA molecules penetrate through pores or areas of partial disruption of the membrane. Thus, two possibilities are considered.…”

“…No lysis was observed at any of the E:T ratios used (Figure 8). Therefore, lysis of transgenic cells requires expression, processing, and presentation of the MART-1 [27][28][29][30][31][32][33][34][35] peptide.…”

Suppression of ischemia‐induced fos expression and AP‐1 activity by an antisense oligodeoxynucleotide to c‐fos mRNA

“…In special circumstances, similar to the delivery of cloned genes in plasmid, anti-sense oligodeoxynucleotides longer than 30 nucleotides can be internalized with the aid of lipofectin [36][37][38]. Although the cellular uptake of antisense oligodeoxynucleotides can be mediated via carrier-mediated endocytosis [39,40], phosphorothioated oligodeoxynucleotide-lipofectin uptake by cells does not appear to involve endosomes or lysosomes [41,42]. The present study of an in vivo antisense strategy utilizes a focal cerebral ischemia model in rats, in which the expression of c-fos and Fos protein and the enhanced AP-1 binding activity have been established and characterized [6,8,9].…”