940. Chemotherapeutic Agent DFMO as an Intensifier of Suicide Gene Therapy

Even though suicide gene therapy with herpes simplex virus thymidine kinase have recently demonstrated to be an effective way to treat cancer patients, there is still need for improvement before a true clinical success. We have earlier demonstrated the enhancement of the HSV-TK/GCV gene therapy with ornithine decarboxylase (ODC) inhibitor DFMO in vitro. ODC is a rate-limiting enzyme in polyamine biosynthetic pathway and is frequently up-regulated in preneoplastic cells. By inhibiting the ODC activity, DFMO causes polyamine deprivation that inhibits cell growth and division. Here we show the utility of combination of polyamine biosynthesis inhibition and HSV-TK/GCV gene therapy in nude mouse model.Subcutaneous rat glioma tumors bearing mice received 2% DFMO in drinking water for 7 days causing almost complete suppression of ODC activity. Consequently, levels of all polyamines; putrescine, spermidine and spermine were down-regulated and started to rise four days post-DFMO removal. Western blot analyses of proliferating cell nuclear antigen (PCNA) revealed respective suppression and recovery of tumor cell proliferation activity. For the combination treatment, 9L tumors with 30% of TK-GFP positive cells were induced and the animals were subjected to two different combinations of DFMO and GCV (the treatments overlapped 2 or 5 days). The analysis of tumor sizes at the end of the treatment revealed enhanced HSV-TK/GCV cytotoxicity in both combination treatment groups. A significant (p < 0.05, ANOVA) enhancement was achieved when the overlap between DFMO and GCV treatments was two days. Flow cytometric analyses of the tumor samples showed also that there were still 7% of TK-GFP positive cells in treated tumors, indicating the anti-apoptotic features of DFMO. Further analysis of the anti-apoptotic features of DFMO will reveal whether this mechanism also contributes to the enhancement of HSV-TK cytotoxicity. In conclusion, our data confirms that the combination of two clinically relevant treatment modalities, polyamine deprivation and HSV-TK/GCV gene therapy, can be used synergistically to enhance the tumor cell killing in vivo.