Evaluation of 5'-deoxy-5'-[F-18]fluorothymidine as a tracer of intracellular thymidine phosphorylase activity.

Two human cell lines (A549 and U937) with cytosolic thymidine phosphorylase (TP) activity were used to evaluate the potential of 5'-deoxy-5'-[F-18]fluorothymidine ([F-18]DFT) as a tracer of intracellular TP expression. Cellular metabolism of DFT led to the production of 5-[F-18]fluoro-2,5-dideoxy-D-ribose-1alpha-phosphate ([F-18]FddR-1P), in analogy to the metabolism of thymidine, which produces 2-deoxy-D-ribose-1alpha-phosphate (dR-1P). A549 cells showed the highest production rate of FddR-1P. After A549 cells were exposed to [F-18]DFT for 40 min, the relative intracellular concentration of [F-18]FddR-1P was more than sevenfold higher in cells than its precursor in the incubating medium. For the same amount of time, a twofold concentration was seen in U937 cells. However, uptake ratios did not rank with the corresponding TP activities found in cell extracts [TP activity ratio (U937:A549)=1.6] that were independently determined with a labeled thymidine/thymine cleavage assay. The discrepancy of TP activity ratios was traced to the instability of FddR-1P in cells. This was evident from the fact that cells accumulated radioactivity by producing FddR-1P, but activity also effluxed from cells over 1 h when the medium was subsequently made tracer free. The dominant labeled molecule released by cells was characterized as a neutral and lipophilic molecule, which was presumed to be a deoxynucleoside. 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.

Metabolism of 3'-deoxy-3'-[F-18]fluorothymidine in proliferating A549 cells: validations for positron emission tomography.

3'-Deoxy-3'-[F-18]fluorothymidine (FLT) is under clinical evaluation as a metabolic probe for imaging cell proliferation in vivo using positron emission tomography (PET). As part of our validation effort, we followed the short-term metabolism of FLT in exponentially growing tumor cells to demonstrate the enzyme activities within the DNA salvage pathway that influence retention of radioactivity. In A549 cells, thymidine kinase-1 (TK1) activity produced FLTMP, which dominated the labeled nucleotide pool. Subsequent nucleotide phosphorylations by thymidylate kinase (TMPK) and nucleotide diphosphate kinase (NDPK) led to FLTTP. After 1h, the cellular metabolic pool contained approximately 30% FLTTP. A putative deoxynucleotidase (dNT), which degrades FLTMP to FLT, provided the primary mechanism for tracer efflux from cells. In contrast, FLTTP was resistant to degradation and highly retained. The uptake and retention characteristics of FLT were also compared to those of thymidine, FMAU (2'-arabino-fluoro-TdR) and FIAU (2'-arabino-fluoro-5-iodo-2'-dexoyuridine). Despite the fact that FLT lacks the 3'-hydroxy necessary for its incorporation into DNA it out performed both FMAU and FIAU in terms of uptake and retention.