Evaluation of pyrimidine metabolising enzymes and in vitro uptake of 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) in pancreatic cancer cell lines

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Abstract
Here we report the expression of major pyrimidine metabolising enzymes in pancreatic cancer cell lines, chronic pancreatitis tissue and human pancreatic cancer and the in vitro uptake of 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT). The expression of pyrimidine metabolising enzymes was evaluated with real-time PCR, Western blot and immunostaining. Thymidine kinase 1 (TK-1) activity was measured with a fluorocytometric assay. The cellular uptake and intracellular metabolism of [18F]FLT were evaluated in pancreatic lobules and in transformed cancer cell lines. TK-1 and thymidine synthetase mRNA were increased in six pancreatic cancer cell lines, while mRNA levels of thymidine kinase 2 and deoxycytidine kinase were down-regulated. High TK-1 activity was confirmed in all cell lines. Furthermore, TK-1 was overexpressed in human pancreatic cancer as compared with normal pancreatic tissue and samples from patients with chronic pancreatitis. The cellular uptake of [18F]FLT was 18.4%±3.6% and 5.2%±1.4% of the applied radioactivity after 240 min in SW-979 and BxPc-3 cells, respectively, while uptake of [18F]fluorodeoxyglucose ([18F]FDG) was only 0.6%±0.04% (SW-979) and 0.3%±0.13% (BxPc-3) after 240 min of incubation. In contrast, cellular uptake of [18F]FLT in isolated pancreatic lobules and growth-arrested HT1080 cells was lower as compared with the uptake of [18F]FDG and with the malignant pancreatic cancer cell lines. HPLC analysis of the perchloric acid-soluble cell fraction demonstrated the phosphorylation of [18F]FLT to the respective monophosphate in both cell lines. Furthermore, 0.8%±0.12% (BxPc-3) and 1.3%±0.38% (SW-979) of the applied radioactivity was detected in the perchloric acid-insoluble cell fraction, indicating the incorporation of [18F]FLT into the DNA. Our results demonstrate the cellular uptake, intracellular trapping and incorporation into the DNA of [18F]FLT in pancreatic cancer cells in vitro. TK-1, as the rate-limiting enzyme of [18F]FLT metabolism, is overexpressed in pancreatic cancer cell lines and in human pancreatic cancer. Thus, we propose [18F]FLT as a promising tracer for positron emission tomography that might overcome current limitations in the diagnosis of pancreatic cancer.

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