Biodistribution and Uptake of 3′-Deoxy-3′-Fluorothymidine in ENT1-Knockout Mice and in an ENT1-Knockdown Tumor Model
| Autor: | John R. Mercer, Wendy P. Gati, Hans-Sonke Jans, Robert J. Paproski, S. A. McQuarrie, Alexander J.B. McEwan, Carol E. Cass, Melinda Wuest, James D. Young, Kathryn Graham |
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| Rok vydání: | 2010 |
| Předmět: |
Biodistribution
Transfection Equilibrative nucleoside transporter 1 Thymidine Kinase Equilibrative Nucleoside Transporter 1 Concentrative nucleoside transporter Gene Knockout Techniques Mice chemistry.chemical_compound Thioinosine Cell Line Tumor Neoplasms Animals Humans Radiology Nuclear Medicine and imaging RNA Messenger RNA Small Interfering Thymidine kinase 1 Mice Knockout A549 cell biology Biological Transport Molecular biology Dideoxynucleosides Cell Transformation Neoplastic chemistry Gene Knockdown Techniques Positron-Emission Tomography biology.protein Female Thymidine Nucleoside Spleen Ex vivo |
| Zdroj: | Journal of Nuclear Medicine. 51:1447-1455 |
| ISSN: | 2159-662X 0161-5505 |
| Popis: | (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.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.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 gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection.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. |
| Databáze: | OpenAIRE |
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