Abstract
1457
Objectives: Bioactivation of the chemotherapy agent 5-fluorouracil (5-FU) depends on thymidine phosphorylase (TP) enzyme activity. A PET-based metabolic tracer for imaging TP activity might help select tumors that will respond to 5-FU. 5'-Deoxy-5'-fluorothymidine (DFT) is a substrate for TP, with 5-fluoro-2,5-dideoxy-D-ribose-1(alpha)-phosphate (F-ddR-1P) as a product. We evaluated the uptake and retention of [F-18]DFT in cells known to express TP. We proposed that the production and retention of [F-18]F-ddR-1P in cells would reflect TP activity. This requires: (1) cells concentrate radioactivity from their environment; (2) cellular activity is specific to [F-18]F-ddR-1P production; (3) [F-18]F-ddR-1P remains trapped in cells. Methods: Uptake and washout experiments were performed with [F-18]DFT using A549 and U937 human cell lines with known TP activity. Cells were incubated for 1 hour in medium containing [F-18]DFT, then the medium was made tracer-free and washout of cellular activity was monitored. Cell lysates and efflux medium samples were obtained. Activity in samples was identified by chromatography using tandem anion exchange and C18 SepPaks. Results: Accummulation of radioactivity was due to production of cellular 5-FddR-1P. A549 cells showed the highest production rate of 5-FddR-1P. After 40 minutes of DFT exposure, the relative intracellular concentration of this metabolite was >7-fold that of its precursor in the medium. A much smaller, 2-fold, concentration was seen with U937 cells. The uptake ratios observed did not rank with the measured relative activity of TP in cells (U937:A549-cell extract TP activity ratio = 1.6). Futhermore, accumulated cell activity subsequently effluxed from cells into tracer-free medium within 1h. Radioactivity efflux was predominantly in the form of a neutral lipophilic species, presumed to be a deoxynucleoside and was not the dephosphorylated fluoro-sugar. Conclusions: Our results indicate that [F 18]DFT would not be effective for imaging TP expression because its initial metabolite undergoes further conversion to a diffusible secondary metabolite, allowing activity loss from cells.
Research Support (if any): This work was supported by NIH grant RO1 CA 115559.
- Society of Nuclear Medicine, Inc.