Abstract
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.
Footnotes
This work was supported in part by the German Cancer Research Center, Heidelberg, Germany; the Wilhelm Sander Foundation, Munich, Germany [Grant 2004.101.1]; the Deutsche Krebshilfe, Bonn, Germany [Grant 107150]; and the Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik (to M.R.).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.032664.
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ABBREVIATIONS:
- hCNT
- human concentrative nucleoside transporter
- hENT
- human equilibrative nucleoside transporter
- ABCC
- human ATP-binding cassette transporter, subfamily C
- MDCK
- Madin-Darby canine kidney
- 5-F-5′-dUrd
- 5-fluoro-5′-deoxyuridine
- 5-azaCyd
- 5-azacytidine
- 5-aza-2′-dCyd
- 5-aza-2′-deoxycytidine
- NBTI
- S-(4-nitrobenzyl)-6-thioinosine)
- NCBI
- National Center for Biotechnology Information
- PBS
- phosphate-buffered saline.
- Received February 9, 2010.
- Accepted April 1, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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