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.
Department of Radiology and Radiological Sciences, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232, USA. ning.guo@vanderbilt.edu
Ning
N
Guo
Jingping
J
Xie
H Charles
HC
Manning
Natasha G
NG
Deane
M Sib
MS
Ansari
Robert J
RJ
Coffey
John
J
Gore
Ronald R
RR
Price
Ronald M
RM
Baldwin
J Oliver
JO
McIntyre
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