Monitoring gene therapy with cytosine deaminase: in vitro studies using tritiated-5-fluorocytosine.
Genetically modified mammalian cells that express the cytosine deaminase (CD) gene are able to convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). PET with 18F-5-FC may be used for in vivo measurement of CD activity in genetically modified tumors.


METHODS
A human glioblastoma cell line was stably transfected with the Escherichia coli CD gene. After incubation of lysates of CD-expressing cells and control cells with 3H-5-FC high-performance liquid chromatography (HPLC) was performed. The uptake of 5-FC was measured after various incubation times using therapeutic amounts of 5-FC. In addition, saturation and competition experiments with 5-FC and 5-FU were performed. Finally, the efflux was measured.


RESULTS
We found that 3H-5-FU was produced in CD-expressing cells, whereas in the control cells only 3H-5-FC was detected. Moreover, significant amounts of 5-FU were found in the medium of cultured cells, which may account for the bystander effect observed in previous experiments. However, uptake studies revealed a moderate and nonsaturable accumulation of radioactivity in the tumor cells, suggesting that 5-FC enters the cells only through diffusion. Although a significant difference in 5-FC uptake was seen between CD-positive and control cells after 48 hr of incubation, no difference was observed after 2 hr of incubation. Furthermore, a rapid efflux could be demonstrated.


CONCLUSIONS
5-Fluorocytosine transport may be a limiting factor for this therapeutic procedure. Quantitation with PET has to rely more on dynamic studies and modeling, including HPLC analysis of the plasma, than on nonmodeling approaches.
Haberkorn
U
U
Department of Oncological Diagnostics, German Cancer Research Center, Heidelberg, Germany.
Oberdorfer
F
F
Gebert
J
J
Morr
I
I
Haack
K
K
Weber
K
K
Lindauer
M
M
van Kaick
G
G
Schackert
H K
HK
D002851:Chromatography, High Pressure Liquid; D043525:Cytosine Deaminase; D005437:Flucytosine / Q000493:pharmacokinetics / Q000627:therapeutic use*; D005472:Fluorouracil / Q000378:metabolism / Q000627:therapeutic use*; D015316:Genetic Therapy / Q000379:methods*; D005909:Glioblastoma / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D066298:In Vitro Techniques; D009700:Nucleoside Deaminases / Q000235:genetics / Q000378:metabolism*; D011355:Prodrugs / Q000493:pharmacokinetics / Q000627:therapeutic use*; D014055:Tomography, Emission-Computed; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured
D011355:Prodrugs; D014316:Tritium; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil