Evaluation of pyrimidine metabolising enzymes and in vitro uptake of 3'-[(18)F]fluoro-3'-deoxythymidine ([(18)F]FLT) in pancreatic cancer cell lines.
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'-[(18)F]fluoro-3'-deoxythymidine ([(18)F]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 [(18)F]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 [(18)F]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 [(18)F]fluorodeoxyglucose ([(18)F]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 [(18)F]FLT in isolated pancreatic lobules and growth-arrested HT1080 cells was lower as compared with the uptake of [(18)F]FDG and with the malignant pancreatic cancer cell lines. HPLC analysis of the perchloric acid-soluble cell fraction demonstrated the phosphorylation of [(18)F]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 [(18)F]FLT into the DNA. Our results demonstrate the cellular uptake, intracellular trapping and incorporation into the DNA of [(18)F]FLT in pancreatic cancer cells in vitro. TK-1, as the rate-limiting enzyme of [(18)F]FLT metabolism, is overexpressed in pancreatic cancer cell lines and in human pancreatic cancer. Thus, we propose [(18)F]FLT as a promising tracer for positron emission tomography that might overcome current limitations in the diagnosis of pancreatic cancer.
Seitz
Ulrike
U
Department of Nuclear Medicine, University of Ulm, Robert-Koch-Strasse 8, 89081 Ulm, Germany. ulrike.seitz@medizin.uni-ulm.de
Wagner
Martin
M
Neumaier
Bernd
B
Wawra
Edgar
E
Glatting
Gerhard
G
Leder
Gerhard
G
Schmid
Roland M
RM
Reske
Sven N
SN
D001483:Base Sequence; D001692:Biological Transport; D002908:Chronic Disease; D015224:Dideoxynucleosides / Q000493:pharmacokinetics*; D005462:Fluorine Radioisotopes / Q000493:pharmacokinetics*; D006801:Humans; D010190:Pancreatic Neoplasms / Q000000981:diagnostic imaging / Q000201:enzymology*; D010195:Pancreatitis / Q000000981:diagnostic imaging / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011743:Pyrimidines / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured
D015224:Dideoxynucleosides; D005462:Fluorine Radioisotopes; D011743:Pyrimidines; D012334:RNA, Neoplasm; D019275:Radiopharmaceuticals; C002854:alovudine