A novel in vitro assay to assess phosphorylation of 3′-[ ¹⁸F]fluoro-3′-deoxythymidine

Purpose: 3′-[ ¹⁸F]fluoro-3′-deoxythymidine ([ ¹⁸F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [ ¹⁸F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [ ¹⁸F]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 [ ¹⁸F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured. Results: The apparent Michaelis-Menten kinetic parameters for [ ¹⁸F]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 [ ¹⁸F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [ ¹⁸F]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.