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


CONCLUSIONS
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.
Paproski
Robert J
RJ
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada.
Wuest
Melinda
M
Jans
Hans-Sonke
HS
Graham
Kathryn
K
Gati
Wendy P
WP
McQuarrie
Steve
S
McEwan
Alexander
A
Mercer
John
J
Young
James D
JD
Cass
Carol E
CE
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D015224:Dideoxynucleosides / Q000378:metabolism* / Q000493:pharmacokinetics*; D033721:Equilibrative Nucleoside Transporter 1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D005260:Female; D055785:Gene Knockdown Techniques; D055786:Gene Knockout Techniques; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D049268:Positron-Emission Tomography; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D013154:Spleen / Q000378:metabolism; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013936:Thymidine / Q000097:blood; D013937:Thymidine Kinase / Q000378:metabolism; D014162:Transfection
D015224:Dideoxynucleosides; D033721:Equilibrative Nucleoside Transporter 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C438649:SLC29A1 protein, human; D013868:Thioinosine; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C001789:4-nitrobenzylthioinosine; C002854:alovudine; D013936:Thymidine