A novel in vitro assay to assess phosphorylation of 3'-[(18)F]fluoro-3'-deoxythymidine.

Guo N, Xie J, Manning HC, Deane NG, Ansari MS, Coffey RJ, Gore J, Price RR, Baldwin RM, McIntyre JO
Mol Imaging Biol. 2011 13 (2): 257-64

PMID: 20532643 · PMCID: PMC4180216 · DOI:10.1007/s11307-010-0351-8

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.

MeSH Terms (13)

Biological Transport Cell Extracts Cell Line, Tumor Cell Membrane Dideoxynucleosides Enzyme Assays Enzyme Inhibitors Humans Kinetics Nucleosides Phosphorylation Thymidine Kinase Time Factors

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