1033. A Novel Mechanism of Synergistic Cytoxicity between Prodrugs, 5-FC and GCV in Double Suicide Gene Therapy

Abstract

The simultaneous use of CD/5-FC and HSV-TK/GCV suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cell lines as well as in mouse models. It has been suggested that the CD/5-FC-mediated decrease in dTTP leads to lower thymidine levels and decreased competition for GCV phosphorylation by HSV-TK. In this study, we demonstrate that concurrent addition of prodrugs, 5-FC and GCV, was less efficacious than sequential addition of 5-FC followed by GCV treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. The level of radiolabeled GCV-TP in cells treated simultaneously with 200 μg/ml 5-FC and 1.0μM GCV for 24 hr was 188 pmol/106 cells compared to 143 pmol/106 cells in cells treated with GCV alone. However, if the cells were pretreated 24 hr with 5-FC and then incubated 24 hr with GCV, GCV-TP levels were 2.6-fold higher compared to GCV treatment alone. [H3]GCV incorporation into DNA was also elevated more than 2-fold in pretreated cells compared to simultaneous addition. In addition, compared to GCV treatment alone, sequential addition of 5-FC and GCV resulted in an additional 60% growth inhibition, whereas only 15% inhibition was observed when both drugs were given together. We measured the effect of 5-FC pre-incubation on endogenous deoxynucleotide concentrations by incubating DU145 cells transduced with the CD/HSV-TK adenovirus with 200 μg/ml 5-FC over a 24 hr period. As expected, cellular dTTP levels were gradually depleted over the 24 hr incubation (from 82 to 11 pmol/106 cells). However, the level of dGTP also decreased (from 9 to 1 pmol/106 cells) concurrently with the dTTP pool decrease over this time period. This reduction of dTTP and dGTP concentrations was not observed when CD/HSV-TK-expressing DU145 cells were treated over 24 hr with both 5-FC and GCV simultaneously. The results from these experiments allowed us to propose and test a novel hypothesis for the synergistic interaction between CD/5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC-mediated reduction of dTTP concentrations results in concurrent decrease of dGTP levels due to allosteric regulation of ribonucleotide reductase. Since dGTP is the endogenous competitor of GCV-TP, a depleted dGTP pool at the time of GCV addition can result in increased GCV incorporation into DNA and cell kill. In fact, including increasing amounts of deoxyguanosine during the 5-FC pre-incubation reverses the depletion of endogenous dGTP, reduces the amount of GCV incorporated into DNA and negates the GCV-mediated cytotoxicity associated with 5-FC pre-incubation in DU145 cells expressing CD and HSV-TK. Understanding this mechanistic interaction between these two enzyme/prodrug systems may help recognize better strategies for creating more efficacious clinical protocols.