Published in:
01-04-2011 | Research Article
A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine
Published in: Molecular Imaging and Biology | Issue 2/2011
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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 \( {K_{\rm{m}}} = {4}.{8}\pm 0.{3}\;{{\mu M}} \) and V
max = 7.4 pmol min−1 per 1 × 106 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.