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Updated: Jun 12, 2026
Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates
Published on: May 8, 2020
A novel in vitro assay to assess phosphorylation of 3'-[(18)F]fluoro-3'-deoxythymidine
Ning Guo1, Jingping Xie, H Charles Manning
1Department of Radiology and Radiological Sciences, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232, USA. ning.guo@vanderbilt.edu
Purpose:
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.
Procedures:
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 microM 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.
