Cellular uptake mechanism for oligonucleotides: Involvement of endocytosis in the uptake of phosphodiester oligonucleotides by a human colorectal adenocarcinoma cell line, HCT-15

Daisuke Nakai, Takeshi Seita, Tetsuya Terasaki, Susumu Iwasa, Yoko Shoji, Yutaka Mizushima, Yuichi Sugiyama

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We used a 20-mer antisense oligonucleotide against human MDR1 mRNA to study the mechanism of uptake of oligonucleotides by a human colorectal adenocarcinoma cell line, HCT-15. 5'-end-32P-labeled oligonucleotides were mainly used. The uptake of phosphodiester oligonucleotides (D-oligo) by HCT- 15 cells increased with time, but the uptake of phosphorothioate oligonucleotides (S-oligo) showed minimal increases with time. HeLa cells showed a 4-fold greater D-oligo uptake than did HCT-15 cells, and other cell lines exhibited a 25-fold lower uptake than did HCT-15 cells. On the other hand, S-oligo uptake varied only severalfold among the cell lines used. D- oligo uptake consisted of a saturable component (Michaelis constant K(m) = 0.16 μM and maximal uptake rate V(max) = 4.8 pmol/min/mg for HCT-15 cells and K(m) = 0.21 μM and V(max) = 35.6 pmol/min/mg for HeLa cells), i.e., the difference between the cell types was mainly in V(max). The uptake of labeled D-oligo was inhibited by unlabeled S-oligo at a much lower concentration (K(i) = 0.01 μM) than by unlabeled D-oligo (K(m) = 0.16 μM), although the uptake rate of D-oligo was greater than that of S-oligo. The D-oligo uptake was highly temperature dependent. Of the sulfhydryl-modifying reagents (p- chloromercuriphenyl-sulfonic acid and p-chloromercuribenzoic acid), only p- chloromercuribenzoic acid inhibited D-oligo uptake, suggesting the involvement of a protein whose sulfhydryl residues inside the cell are critical for D-oligo uptake. Chloroquine and monensin, which are known to alter the intracellular fate of ligands and receptors, did not affect D- oligo uptake, but phenylarsine oxide, an inhibitor of internalization processes, inhibited uptake significantly. Down-regulation and recovery of D- oligo uptake induced by excess unlabeled D-oligo pretreatment occurred, but this was only partial. Studies with a digitonin equilibrium method and confocal microscopy indicated that intracellular localization of D-oligo was mainly in a vesicular compartment. More stable 3'-end-32P-labeled oligonucleotides were also used, to compare their uptake characteristics with those of the 5'-end-labeled oligonucleotides. The uptake characteristics for the two labels were similar in terms of saturability, greater D-oligo uptake, compared with S-oligo, and the inhibitory effect of p-chloromercuribenzoic acid; however, the stable 3'-end label showed greater uptake than the 5'-end label. Our findings suggest that D-oligo uptake by HCT-15 cells is mediated by endocytosis involving binding sites on the plasma membrane.

Original languageEnglish
Pages (from-to)1362-1372
Number of pages11
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
Publication statusPublished - 1996 Sep 1
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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