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5-Fluoro-1-(2'-Deoxy-2'-Fluoro-ß-D-Ribofuranosyl) Uracil Trapping in Morris Hepatoma Cells Expressing the Herpes Simplex Virus Thymidine Kinase Gene

Department of Oncological Diagnostics and Therapy, German Cancer Research Center, Heidelberg, Germany; Karmanos Cancer Institute, Detroit Medical Center, Wayne State University, Detroit, Michigan; Department of Radiology, University of Washington, Seattle, Washington

Correspondence: For correspondence or reprints contact: Uwe Haberkorn, MD, Department of Oncological Diagnostics and Therapy, German Cancer Research Center, Im Neuenhelmer Feld 280, D-69120 Heidelberg, Germany.

ABSTRACT

The planning and individualization of gene therapy with suicide genes such as herpes simplex virus thymidine kinase (HSV-tk) necessitates the assessment of the enzyme activity expressed in the tumor. This can be done by uptake measurements of specific substrates for HSV-tk. Due to the molecular structure of 5-fluoro-1-(2'-deoxy-fluoro-ß-D-ribofuranosyl)uracil (FFUdR), it may be a substrate for both the mammalian thymidine kinase and HSV-tk. Methods: Using a HSV-tk-expressing rat hepatoma cell line and a control cell line (bearing the empty vector) the uptake of ³H-FFUdR was determined with increasing incubation periods. Furthermore, measurements with graded mixtures of HSV-tk-expressing cells and control cells were made. To elucidate the mechanism of FFUdR transport into cells, a series of inhibition/competition experiments was performed with challenge inhibitors of the nucleoside and the nucleobase transport systems. Results: The uptake studies with tritiated FFUdR revealed a 14- to 19-fold higher accumulation in the HSV-tk-expressing cell line compared to the control cell line. While the ³H-FFUdR uptake was 3- to 4-fold higher than the ³H-ganciclovir uptake in the HSV-tk-expressing cells, it was also higher in control cells (5-fold). Furthermore, FFUdR accumulation was linearly correlated with the amount of HSV-tk-expressing cells. FFUdR uptake and growth inhibition by therapeutic doses of ganciclovir were highly correlated, with r = 0.96. Inhibition/competition experiments showed that FFUdR is transported mainly by the equilibrative and the concentrative nucleoside transporter but not by the nucleobase transport systems. Conclusion: The FFUdR uptake is an indicator of the HSV-tk activity in tumor cells and can be used as a prognostic marker during gene therapy with HSV-tk. The relative merits of ganciclovir and FFUdR as specific substrates for HSV-tk will need to be further explored in vivo.

Key Words: gene therapy • specific substrate • hepatoma • PET