CELLULAR UPTAKE MECHANISM FOR OLIGONUCLEOTIDES - INVOLVEMENT OF ENDOCYTOSIS IN THE UPTAKE OF PHOSPHODIESTER OLIGONUCLEOTIDES BY A HUMAN COLORECTAL ADENOCARCINOMA CELL-LINE, HCT-15

We used a 20-mer antisense oligonucleotide against human MDR1 mRNA to study the mechanism of uptake of oligonucleolides by a human colorecta l adenocarcinoma cell line, HCT-15. 5'-end-P-32-labeled oligonucleotid es were mainly used. The uptake of phosphodiester oligonucleotides (D- oligo) by HCT-15 cells increased with time, but the uptake of phosphor othioate oligonucleotides (S-oligo) showed minimal increases with time . HeLa cells showed a 4-fold greater D-oligo uptake than did HCT-15 ce lls, and other cell lines exhibited a 25-fold lower uptake than did HC T-15 cells. On the other hand, S-oligo uptake varied only severalfold among the cell lines used. D-oligo uptake consisted of a saturable com ponent (Michaelis constant K-m = 0.16 mu M and maximal uptake rate V-m ax = 4.8 pmol/min/mg for HCT-15 cells and K-m = 0.21 mu M and Vmax = 3 5.6 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 (K-i = 0.01 mu M) than by unlabeled D-oligo (K-m = 0.16 mu M), although the uptake rate of D-oligo was greater than that of S-oligo. The D-oligo uptake was hi ghly temperature dependent. Of the sulfhydryl-modifying reagents (p-ch loromercuriphenyl-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 know n to alter the intracellular fate of ligands and receptors, did not af fect D-oligo uptake, but phenylarsine oxide, an inhibitor of internali zation processes, inhibited uptake significantly. Down-regulation and recovery of D-oligo uptake induced by excess unlabeled D-oligo pretrea tment occurred, but this was only partial. Studies with a digitonin eq uilibrium method and confocal microscopy indicated that intracellular localization of D-oligo was mainly in a vesicular compartment. More st able 3'-end-P-32-labeled oligonucleotides were also used, to compare t heir uptake characteristics with those of the 5'-end-labeled oligonucl eotides. 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, th e 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 e ndocytosis involving binding sites on the plasma membrane.