title: Cellular uptake mechanism for oligonucleotides: involvement of endocytosis in the uptake of phosphodiester oligonucleotides by a human colorectal adenocarcinoma cell line, HCT-15.
abstract: 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. Doligo up-take consisted of a saturable component (Michaelis constant K(m) = 0.16 microM and maximal uptake rate Vmax = 4.8 pmol/min/mg for HCT-15 cells and K(m) = 0.21 microM and Vmax = 35.6 pmol/min/mg for HeLa cells), i.e., the difference between the cell types was mainly in Vmax. The uptake of labeled D-oligo was inhibited by unlabeled S-oligo at a much lower concentration (Ki = 0.01 microM) than by unlabeled D-oligo (K(m) = 0.16 microM), 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.
authors.affiliation: Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Nakai
authors.forename: T
authors.initials: T
authors.lastname: Seita
authors.forename: T
authors.initials: T
authors.lastname: Terasaki
authors.forename: S
authors.initials: S
authors.lastname: Iwasa
authors.forename: Y
authors.initials: Y
authors.lastname: Shoji
authors.forename: Y
authors.initials: Y
authors.lastname: Mizushima
authors.forename: Y
authors.initials: Y
authors.lastname: Sugiyama
mesh_terms: D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000230:Adenocarcinoma / Q000378:metabolism; D000838:Anions; D002451:Cell Compartmentation; D015179:Colorectal Neoplasms / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D019450:Genes, MDR; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000838:Anions; D007473:Ion Channels; D016376:Oligonucleotides, Antisense; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid