MULTITRACER STUDIES DURING GENE-THERAPY OF HEPATOMA-CELLS WITH HERPES-SIMPLEX VIRUS THYMIDINE KINASE AND GANCICLOVIR

Using different tracers of tumor metabolism, the application of PET fo r monitoring gene therapy with the suicide gene herpes simplex virus t hymidine kinase (HSVtk) is investigated in this in vitro study. Method s: Morris hepatoma cells were transfected with a retroviral vector bea ring the HSVtk gene, and different clones were established by selectio n with the neomycin analog G418. Thereafter, uptake measurements using fluorodeoxyglucose (FDG), 3-O-methylglucose, aminoisobutyric acid and methionine were performed in a thymidine kinase (TK)-expressing cell line and in control cells bearing the empty vector in the presence of different concentrations of ganciclovir (GCV). These experiments were done up to 48 hr after the onset of therapy. The values were expressed as Bq/well or as Bq/10(5) cells. Results: During GCV treatment therap y, a decrease of the uptake/well was measured for all tracers in the T K-expressing cell line. After normalization to the viable cell number, the uptake for FDG and 3-O-methylglucose increases up to 195% after 2 4 hr incubation with GCV. A high-pressure liquid chromatography analys is revealed a decline of the FDG-6-phosphate fraction after 48 hr incu bation with GCV. Consequently, a normalization of FDG uptake was obser ved after this incubation period, whereas the 3-O-methylglucose uptake was still increased. Experiments performed with different amounts of TK-expressing cells and control cells showed that these effects are de pendent on the percentage of TK-expressing cells. The aminoisobutyric acid uptake decreases to 47%, while the methionine uptake decreases in the acid-insoluble fraction (to 17%) and increases in the acid-solubl e fraction (to 150%). Conclusion: These data indicate that combination s of the PET tracers used in these experiments may be applied for moni toring gene therapy with HSVtk. The increase in FDG and 3-O-methylgluc ose uptake in vitro is interpreted as stress reaction of the tumor cel ls. However, an uncoupling of transport and phosphorylation was observ ed after 48 hr incubation. The amino acid uptake experiments point to an inhibition of protein synthesis as well as of the neutral amino aci d transport.