The identification of the transport proteins responsible for the uptake and the 
efflux of nucleosides and their metabolites enables the characterization of 
their vectorial transport and a better understanding of their absorption, 
distribution, and elimination. Human concentrative nucleoside transporters 
(hCNTs/SLC28A) are known to mediate the transport of natural nucleosides and 
some nucleoside analogs into cells in a sodium-dependent and unidirectional 
manner. On the other hand, several human multidrug resistance proteins [human 
ATP-binding cassette transporter, subfamily C (ABCC)] cause resistance against 
nucleoside analogs and mediate transport of phosphorylated nucleoside 
derivatives out of the cells in an ATP-dependent manner. For the integrated 
analysis of uptake and efflux of these compounds, we established a 
double-transfected Madin-Darby canine kidney (MDCK) II cell line stably 
expressing the human uptake transporter hCNT3 in the apical membrane and the 
human efflux pump ABCC4 in the basolateral membrane. The direction of transport 
was from the apical to the basolateral compartment, which is in line with the 
unidirectional transport and the localization of both recombinant proteins in 
the MDCKII cells. Recombinant hCNT3 mediated the transport of several known 
nucleoside substrates, and we identified 5-azacytidine as a new substrate for 
hCNT3. It is of interest that coexpression of both transporters was confirmed in 
pancreatic adenocarcinomas, which represent an important clinical indication for 
the therapeutic use of nucleoside analogs. Thus, our results establish a novel 
cell system for studies on the vectorial transport of nucleosides and their 
analogs from the apical to the basolateral compartment. The results contribute 
to a better understanding of the cellular transport characteristics of 
nucleoside drugs.