Monitoring gene therapy with herpes simplex virus thymidine kinase in hepatoma cells: uptake of specific substrates.
This study investigates the application of PET with specific substrates for the assessment of enzyme activity after transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene.


METHODS
After transfection of a rat hepatoma cell line with a retroviral vector containing the HSV-tk gene, different clones were established by G418 selection. Uptake measurements were performed up to 48 hr in a TK-expressing cell line and in a control cell line using thymidine (TdR; measured under therapy conditions), fluorodeoxycytidine (FdCyt) and ganciclovir (GCV). Additionally, bystander experiments and inhibition/competition studies were done.


RESULTS
In TK-expressing cells GCV treatment caused an increased (up to 250%) TdR uptake in the acid-soluble fraction and a decrease to 5.5% in the acid-insoluble fraction. The FdCyt uptake was higher in the TK-expressing cells than in controls with a maximum after 4 hr (12-fold and 3-fold higher in the acid-insoluble and acid-soluble fraction). GCV accumulated up to 180-fold more in the acid-insoluble and 26-fold more in the acid-soluble fraction. GCV uptake occurred mainly by the nucleoside transport systems. Bystander experiments revealed a relation between growth inhibition or GCV uptake and the amount of TK-expressing cells. GCV uptake and growth inhibition were correlated with r = 0.96.


CONCLUSIONS
Assessment of GCV accumulation may serve as an indicator of the enzyme activity and of therapy outcome. TdR may be useful to measure therapy effects on DNA synthesis, whereas the potential of FdCyt has to be investigated in further studies.
Haberkorn
U
U
Department of Oncological Diagnostics and Therapy, German Cancer Research Center, Heidelberg, FRG.
Altmann
A
A
Morr
I
I
Knopf
K W
KW
Germann
C
C
Haeckel
R
R
Oberdorfer
F
F
van Kaick
G
G
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002470:Cell Survival; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015774:Ganciclovir / Q000494:pharmacology; D005814:Genes, Viral; D015316:Genetic Therapy; D008114:Liver Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000201:enzymology / Q000628:therapy*; D012588:Scintillation Counting; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000235:genetics*; D014055:Tomography, Emission-Computed; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000000981:diagnostic imaging / Q000187:drug effects / Q000201:enzymology; D014764:Viral Proteins
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D014764:Viral Proteins; D014316:Tritium; D003843:Deoxycytidine Monophosphate; C067000:N(4)-hydroxy-5-fluorodeoxycytidine monophosphate; D004247:DNA; D013937:Thymidine Kinase; D015774:Ganciclovir