Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase.
Assessment of suicide enzyme activity would have considerable impact on the planning and the individualization of suicide gene therapy of malignant tumors. This may be done by determining the pharmacokinetics of specific substrates. We generated ganciclovir (GCV)-sensitive human mammary carcinoma cell lines after transfection with a retroviral vector bearing the herpes simplex virus thymidine kinase (HSV-tk) gene. Thereafter, uptake measurements and HPLC analyses were performed up to 48 h in an HSV-tk-expressing cell line and in a wild-type cell line using tritiated GCV. HSV-tk-expressing cells showed higher GCV uptake and phosphorylation than control cells, whereas in wild-type MCF7 cells no phosphorylated GCV was detected. In bystander experiments the total GCV uptake was related to the amount of HSV-tk-expressing cells. Furthermore, the uptake of GCV correlated closely with the growth inhibition (r = 0.92). Therefore, the accumulation of specific substrates may serve as an indicator of the HSV-tk activity and of therapy outcome. Inhibition and competition experiments demonstrated slow transport of GCV by the nucleoside carriers. The slow uptake and low affinity to HSV-tk indicate that GCV is not an ideal substrate for the nucleoside transport systems or for HSV-tk. This may be the limiting factor for therapy success, necessitating the search for better substrates of HSV-tk.
Haberkorn
U
U
Department of Oncological Diagnostics and Therapy, German Cancer Research Center, Heidelberg, Federal Republic of Germany. u.haberkorn@dkfz-heidelberg.de
Khazaie
K
K
Morr
I
I
Altmann
A
A
Müller
M
M
van Kaick
G
G
D000998:Antiviral Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015774:Ganciclovir / Q000378:metabolism / Q000493:pharmacokinetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D009369:Neoplasms / Q000628:therapy; D010766:Phosphorylation; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics*; D014055:Tomography, Emission-Computed; D014162:Transfection; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D013937:Thymidine Kinase; D015774:Ganciclovir