We are evaluating the therapeutic potential of gene directed enzyme prodrug therapy (GDEPT) using the fusion gene,cytosine deaminase uracil phosphoribosyl transferase (CDUPRT) for treating prostate cancer (PCa). Objective: To test the efficacy of CDUPRT-GDEPT against RM1 mouse androgen-refractory PCa grown in C57BL/6 mice: RM1 cells were stably transfected with green fluorescence protein (GFP) and the fusion gene, CDUPRT, derived from E coli (RM1-GFP/CDUPRT). CD/UPRT converts 5 fluoro-cytosine (5FC) to freely diffusible metabolites including 5-fluorouracil (5FU), that disrupt the metabolic pathways for both DNA and RNA synthesis, thus killing both dividing and non-dividing cells. This is especially relevant to PCa, which is characterized by a low proportion of dividing cells. Experimental Design: RM1 cells were stably transfected with plasmids containing GFP/CDUPRT, GFP or GFP/LacZ (controls) using lipofectamine. Cells that highly expressed GFP were selected by flow cytometry and used for further study. Transgene CDUPRT expression in cell lysates from cells grown in vitro or after in vivo implantation of RM1-GFP/CDUPRT was assessed by enzymic conversion of its substrate using HPLC. To assess the local bystander effect of CDUPRT-GDEPT, C57BL/6 mice were implanted directly into the prostate with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions; 4 days later, 5FC was given intraperitoneally (ip) for 13 days at 500mg/kg/mouse/day. Pseudo-metastases in the lungs were established by a tail vein injection (iv) of untransfected RM1 cells 4 days post intraprostatic implantation. Mice were euthanased on day 19, and prostate weight and volume, and lung weight and colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. Results: Intraprostatic RM1-GFP/CDUPRT tumors on treatment with 5FC for 13 days resulted in complete regression of the tumors. Injection of cell mixtures (RM1-GFP/CDUPRT + RM1-GFP) resulted in a local bystander effect when only 20% of the cells were expressing the CDUPRT transgene. Interestingly, the lung colony counts indicated the presence of a distant bystander effect. The pseudo-metastases were absent in ∼50% of mice in the RM1-GFP/CDUPRT+5FC group compared with the control groups. This is the first demonstration of a distant bystander effect using CDUPRT-GDEPT. Conclusions and future work: The CDUPRT GDEPT leads to a significant local and a distant bystander effect when used to treat androgen refractory RM1 tumors in mice. The role of the immune system in this distant bystander effect is currently under investigation.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]