A novel in vitro assay to assess phosphorylation of 3'-[(18)F]fluoro-3'-deoxythymidine.
OBJECTIVE
3'-[(18)F]fluoro-3'-deoxythymidine ([(18)F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [(18)F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [(18)F]FLT both in tumor cell lysates and tumor cells.


METHODS
The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [(18)F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured.


RESULTS
The apparent Michaelis-Menten kinetic parameters for [(18)F]FLT are K(m) = 4.8 ± 0.3 μM and V(max) = 7.4 pmol min(-1) per 1 × 10(6) cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates.


CONCLUSIONS
The apparent K (m) of [(18)F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [(18)F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells.
Guo
Ning
N
Department of Radiology and Radiological Sciences, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232, USA. ning.guo@vanderbilt.edu
Xie
Jingping
J
Manning
H Charles
HC
Deane
Natasha G
NG
Ansari
M Sib
MS
Coffey
Robert J
RJ
Gore
John
J
Price
Ronald R
RR
Baldwin
Ronald M
RM
McIntyre
J Oliver
JO
D001692:Biological Transport / Q000187:drug effects; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D015224:Dideoxynucleosides / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009705:Nucleosides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013997:Time Factors
D002457:Cell Extracts; D015224:Dideoxynucleosides; D004791:Enzyme Inhibitors; D009705:Nucleosides; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C002854:alovudine