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Biodistribution and Uptake of 3′-Deoxy-3′-Fluorothymidine in ENT1-Knockout Mice and in an ENT1-Knockdown Tumor Model

2010; Society of Nuclear Medicine and Molecular Imaging; Volume: 51; Issue: 9 Linguagem: Inglês

10.2967/jnumed.110.076356

1535-5667

Robert J. Paproski, Melinda Wuest, Hans-Sonke Jans, Kathryn Graham, Wendy P. Gati, S.A. McQuarrie, Alexander McEwan, John R. Mercer, James D. Young, Carol E. Cass,

Adenosine and Purinergic Signaling

18 F-3′-Deoxy-3′-fluorothymidine ( 18 F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for 18 F-FLT uptake in normal tissues and tumors. Methods: ENT1-knockout (ENT1 −/− ) mice were generated and compared with wild-type (ENT1 +/+ ) mice using small-animal 18 F-FLT PET. In addition, ENT1 +/+ mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by 18 F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography–mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and 18 F-FLT uptake using real-time polymerase chain reaction and 3 H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by 18 F-FLT small-animal PET. Results: Compared with noninjected ENT1 +/+ mice, ENT1 +/+ mice injected with NBMPR-P and ENT1 −/− mice displayed a reduced percentage injected dose per gram (%ID/g) for 18 F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for 18 F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1 −/− mice displayed 1.65-fold greater plasma thymidine levels than did ENT1 +/+ mice. Spleen tissue from ENT1 +/+ and ENT1 −/− mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold 3 H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo γ-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for 18 F-FLT uptake at 1 h after tracer injection. Conclusion: Loss of ENT1 activity significantly affected 18 F-FLT biodistribution in mice and 18 F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of 18 F-FLT uptake in normal and transformed cells.