CELLULAR UPTAKE MECHANISM FOR OLIGONUCLEOTIDES - INVOLVEMENT OF ENDOCYTOSIS IN THE UPTAKE OF PHOSPHODIESTER OLIGONUCLEOTIDES BY A HUMAN COLORECTAL ADENOCARCINOMA CELL-LINE, HCT-15
D. Nakai et al., CELLULAR UPTAKE MECHANISM FOR OLIGONUCLEOTIDES - INVOLVEMENT OF ENDOCYTOSIS IN THE UPTAKE OF PHOSPHODIESTER OLIGONUCLEOTIDES BY A HUMAN COLORECTAL ADENOCARCINOMA CELL-LINE, HCT-15, The Journal of pharmacology and experimental therapeutics, 278(3), 1996, pp. 1362-1372
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.