The influence of polarized epithelial (Caco-2) cell differentiation on the cellular binding of phosphodiester and phosphorothioate oligonucleotides.
Cell aging and the degree of cellular differentiation are thought to be important variables governing uptake of oligonucleotides but remain poorly understood. The Caco-2 colon carcinoma cell line has the ability 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 surface binding proteins are governed by the age and thus the degree of differentiation of Caco-2 epithelial cells in culture. Cellular association (normalized for cell number) of an all phosphodiester (PO), all phosphorothioate (PS), and a phosphodiester oligonucleotide containing two terminal phosphorothioate internucleotide linkages at the 3' end (EC-PO) gradually increased from day 3 to around day 17 of the culture, 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 temperature and pH dependent, but the magnitude of the effect was influenced by cell aging and the degree of differentiation. PS oligonucleotides exhibited greater binding (up to threefold) at the basolateral surface compared with the apical surface within the pH range 5-7. These findings could be directly correlated with the expression levels of cell surface oligonucleotide binding proteins during the aging study. A Caco-2 cell surface protein binding complex of around 46 kDa was identified as the major site of binding for both PO and PS oligonucleotides, although the latter also bound to several other proteins, especially at low pH.
Pharmaceutical Sciences Institute, Aston University, Birmingham, UK.
S
S
Akhtar
G F
GF
Beck
P
P
Hawley
W J
WJ
Irwin
I
I
Gibson
D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology*; D016922:Cellular Senescence / Q000502:physiology*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*; D011485:Protein Binding; D013873:Thionucleotides / Q000378:metabolism*
D002352:Carrier Proteins; D008565:Membrane Proteins; D009841:Oligonucleotides; D013873:Thionucleotides