Biodistribution and uptake of 3'-deoxy-3'-fluorothymidine in ENT1-knockout mice and in an ENT1-knockdown tumor model.

Abstract

UNLABELLED:

(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 gamma-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.

• Authors: Robert J Paproski, Melinda Wuest, Hans-Sonke Jans, Kathryn Graham, Wendy P Gati, Steve McQuarrie, Alexander McEwan, John Mercer, James D Young, Carol E Cass
• Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (J Nucl Med) Vol. 51 Issue 9 Pg. 1447-55 (Sep 2010) ISSN: 1535-5667 [Electronic] United States
• PMID: 20720035 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Dideoxynucleosides
• Equilibrative Nucleoside Transporter 1
• RNA, Messenger
• RNA, Small Interfering
• SLC29A1 protein, human
• Thioinosine
• Thymidine Kinase
• thymidine kinase 1
• 4-nitrobenzylthioinosine
• alovudine
• Thymidine

Topics

• Animals
• Biological Transport (genetics)
• Cell Line, Tumor
• Cell Transformation, Neoplastic
• Dideoxynucleosides (metabolism, pharmacokinetics)
• Equilibrative Nucleoside Transporter 1 (antagonists & inhibitors, genetics)
• Female
• Gene Knockdown Techniques
• Gene Knockout Techniques
• Humans
• Mice
• Mice, Knockout
• Neoplasms (diagnostic imaging, genetics, metabolism, pathology)
• Positron-Emission Tomography
• RNA, Messenger (genetics, metabolism)
• RNA, Small Interfering (genetics)
• Spleen (metabolism)
• Thioinosine (analogs & derivatives, pharmacology)
• Thymidine (blood)
• Thymidine Kinase (metabolism)
• Transfection