S. Akhtar et al., THE INFLUENCE OF POLARIZED EPITHELIAL (CACO-2) CELL-DIFFERENTIATION ON THE CELLULAR-BINDING OF PHOSPHODIESTER AND PHOSPHOROTHIOATE OLIGONUCLEOTIDES, ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT, 6(3), 1996, pp. 197-206
Citations number
29
Categorie Soggetti
Medicine, Research & Experimental","Biothechnology & Applied Migrobiology
Cell aging and the degree of cellular differentiation are thought to b
e important variables governing uptake of oligonucleotides but remain
poorly understood, The Caco-2 colon carcinoma cell line has the abilit
y to spontaneously differentiate into enterocytes in vitro and serves
as a useful model to further investigate the effect of differentiation
on oligonucleotide binding and uptake, In this study, we report that
the extent of oligonucleotide association and the expression of cell s
urface binding proteins are governed by the age and thus the degree of
differentiation of Caco-2 epithelial cells in culture. Cellular assoc
iation (normalized for cell number) of an all phosphodiester (PO), all
phosphorothioate (PS), and a phosphodiester oligonucleotide containin
g two terminal phosphorothioate internucleotide linkages at the 3' end
(PC-PO) gradually increased from day 3 to around day 17 of the cultur
e, followed by a plateau, or slight decrease, up to day 21 of the cell
aging study. Overall, a threefold to fourfold increase in binding was
observed from day 3 to day 17, Oligonucleotide binding was temperatur
e and pH dependent, but the magnitude of the effect was influenced by
cell aging and the degree of differentiation. PS oligonucleotides exhi
bited greater binding (up to threefold) at the basolateral surface com
pared with the apical surface within the pH range 5-7. These findings
could be directly correlated with the expression levels of cell surfac
e oligonucleotide binding proteins during the aging study. A Caco-2 ce
ll surface protein binding complex of around 46 kDa was identified as
the major site of binding for both PO and PS oligonucleotides, althoug
h the latter also bound to several other proteins, especially at low p
H.