A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

Abstract

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. 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. 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. 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.