Received for publication May 19, 2008.
Revised July 29, 2008.
Accepted for publication July 30, 2008.

The Role of Human Nucleoside Transporters in Uptake of 3'-Deoxy-3'-Fluorothymidine

Abstract

3'-Deoxy-3'-fluorothymidine (FLT) is a positron emission tomography (PET) tracer used to identify proliferating tumor cells. The purpose of this study was to characterize FLT transport by each human nucleoside transporter (hNT) and to determine the role of hNTs for FLT uptake in various human cancer cell lines. FLT binding to hNTs was monitored by the inhibitory effects of FLT on ³H-uridine uptake in yeast cells producing recombinant hNT proteins. hCNT1 displayed the lowest FLT K_i value for inhibition of ³H-uridine uptake, followed by hCNT3, hENT2, hENT1, and hCNT2. ³H-FLT was efficiently transported in Xenopus laevis oocytes individually producing hENT1, hENT2, hCNT1 or hCNT3. ³H-FLT uptake in MCF-7, A549, U251, A498, MIA PaCa-2, and Capan-2 cells was inhibited at least 50% by the hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR). Using real-time PCR, hENT1 and hENT2 had the most abundant hNT transcripts in all cell lines. Cell lines also underwent (i) ³H-NBMPR equilibrium binding assays with or without 5-S-{2-(1-[(fluorescein-5-yl)thioureido]hexanamido)ethyl}-6-N-(4-nitrobenzyl)-5-thioadenosine (FTH-SAENTA), a membrane impermeable NBMPR analog, to determine plama membrane hENT1 levels, and (ii) dose-response NBMPR inhibition of ³H-FLT uptake. MCF-7, A549, and Capan-2 cells displayed NBMPR IC₅₀ values that were smaller or equal to NBMPR K_d values, suggesting that 50% inhibition of hENT1 reduced ³H-FLT uptake by at least 50%. A strong correlation between extracellular NBMPR binding sites/cell and ³H-FLT uptake was observed for all cell lines except MIA PaCa-2. These data suggest that plasma membrane hNTs (especially hENT1) are important determinants of cellular FLT uptake.

Key words: Nucleoside/Nucleotide, Nucleoside/Nucleotide derivatives