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.
Department of Oncological Diagnostics, German Cancer Research Center, Heidelberg, Germany.
U
U
Haberkorn
F
F
Oberdorfer
J
J
Gebert
I
I
Morr
K
K
Haack
K
K
Weber
M
M
Lindauer
G
G
van Kaick
H K
HK
Schackert
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