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 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.
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 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.
Authors: Vijaya L Damaraju; Sambasivarao Damaraju; James D Young; Stephen A Baldwin; John Mackey; Michael B Sawyer; Carol E Cass Journal: Oncogene Date: 2003-10-20 Impact factor: 9.867
Authors: Ulrike Seitz; Martin Wagner; Bernd Neumaier; Edgar Wawra; Gerhard Glatting; Gerhard Leder; Roland M Schmid; Sven N Reske Journal: Eur J Nucl Med Mol Imaging Date: 2002-06-04 Impact factor: 9.236
Authors: Meg Perumal; Radhakrishna G Pillai; Henryk Barthel; Julius Leyton; John R Latigo; Martin Forster; Fraser Mitchell; Ann L Jackman; Eric O Aboagye Journal: Cancer Res Date: 2006-09-01 Impact factor: 12.701