Validation of FLT uptake as a measure of thymidine kinase-1 activity in A549 carcinoma cells.
The thymidine analog (18)F-3'-deoxy-3' -fluorothymidine (FLT) is being used clinically for PET imaging of tumor proliferation. Appropriate use of this tracer requires validating the mechanisms by which it accumulates in dividing cells. We tested the accuracy with which FLT uptake predicted the activity of cytosolic thymidine kinase-1 (TK(1)), an enzyme that is upregulated before and during DNA synthesis.


METHODS
Cultured A549 human lung carcinoma cells were manipulated to a range of proliferation rates from actively dividing to growth arrested. Uptake of radiolabeled FLT was compared with cell cycle activity, which was expressed as the percentage of cells in S phase, and with activity of cytosolic TK(1). We also compared uptake of FLT and deoxyglucose. We genetically manipulated A549 cells by transfecting them with human papillomavirus type 16 E6 (designated A549-E6) to abrogate function of the tumor suppressor gene, p53. Although radiation typically inhibits progression of mammalian cells through the cell cycle, abrogation of p53 function eliminates this inhibition. We then compared FLT uptake with the percentage of cells in S phase and TK(1) activity in irradiated A549-E6 cells and in irradiated control cells having normal p53 function and the expected radiation-induced growth delay.


RESULTS
A549 cells with only 3%-5% cells in S phase took up little FLT and had low levels of TK(1) activity. When cells were stimulated to grow by being placed into fresh medium, we observed a strong correlation between increased FLT uptake and increased TK(1) activity. As expected, FLT uptake varied much more as a function of growth than did uptake of deoxyglucose. Nonproliferating A549 cells did not enter the cell cycle if they were irradiated before being placed into fresh medium, and they did not accumulate FLT or show elevated TK(1) activity. In contrast, radiation did not inhibit the cell cycle progression of A549-E6 cells. When subcultured, they began to grow and showed increased uptake of FLT commensurate with greater TK(1) activity.


CONCLUSIONS
In cultured A549 cells FLT uptake is positively correlated with cell growth and TK(1) activity. Inhibition of cell cycle progression prevents FLT uptake and increased TK(1) activity. These results suggest that FLT images reflect TK(1) activity and the percentage of cells in S phase.
Rasey
Janet S
JS
Department of Radiation Oncology, University of Washington School of Medicine, Seattle, Washington 98195, USA. rasey@u.washington.edu
Grierson
John R
JR
Wiens
Linda W
LW
Kolb
Pamella D
PD
Schwartz
Jeffrey L
JL
D000230:Adenocarcinoma / Q000473:pathology; D002453:Cell Cycle; D002455:Cell Division; D003470:Culture Media; D015224:Dideoxynucleosides / Q000493:pharmacokinetics; D005462:Fluorine Radioisotopes; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D013937:Thymidine Kinase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003470:Culture Media; D015224:Dideoxynucleosides; D005462:Fluorine Radioisotopes; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C002854:alovudine