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The clinical application of gene directed enzyme prodrug therapy (GDEPT) is severely limited by the lack of strong cancer specific promoters. Carcinoembryonic antigen (CEA) promoter is a cancer specific promoter but the transcription activity of CEA in most cancer cell lines is 10- to 300-fold lower than that of CMV and RSV viral promoters. The low transcription activity is the major limitation for using CEA promoter-based GDEPT in vivo and in clinical trials.

In this study, we developed a novel cancer specific enhancer system and tested the enhancing activity and specificity of the novel enhancer on the CEA promoter using a panel of 9 colon cancer cell lines and 8 normal primary cells and cell lines (4 fibroblast, 2 endothelial, 2 epithelial). High CEA protein and mRNA expression was only detected in 30% cancer cell lines. Luciferase assay showed that CEA promoter has low transcriptional activity in most cancer cell lines. The transcriptional activity of CEA promoter was significantly enhanced by the combination of the novel enhancer (3 - 8-fold) in cancer cell lines but not in normal cells. In 90% cancer cell lines, the transcription activity of the novel enhancer-CEA promoter cassette was comparable to those of SV40 promoter. Thymidine phosphorylase (TP) is an enzyme which converts a prodrug 5’-deoxy-5-fluorouradine (5’-DFUR) into an active anticancer drug, 5-fluorouracil (5-FU). Furthermore, we constructed the vectors in which the TP cDNA was under the control of CEA, novel enhancer, novel enhancer-CEA and CMV promoters respectively. The novel enhancer-CEA-TP transiently transfected cancer cell lines (H630 and RKO) expressed high levels of TP protein and enzyme activity which are comparable to those in the pCMV-TP transfected cells. The novel enhancer-TP and pCEA-TP transfected cell lines respectively only demonstrated negligible and low levels of TP protein and enzyme activity. Both pCMV-TP and the novel enhancer-CEA-TP transfected cells were 8 - 10-fold sensitized to 5’-DFUR. The sensitivity of the novel enhancer-TP and pCEA-TP transfected H630 and RKO cells to 5’-DFUR was only 1.5 - 2-fold enhanced. To test the bystander effect, H630 and RKO cell lines were also stably transfected. The novel enhancer-CEA-TP and pCMV-TP transfected H630 and RKO cells were mixed with the mock transfected cells at different ratios (0, 5, 10, 20, 40, 60 and 100%) and the mixtures were subjected to cytotoxicity analysis. The MTT assay showed that only 5% of the novel enhancer-CEA-TP transfected cells were required to efficiently convert 5’-DFUR into 5-FU and demonstrated strong cytotoxic effect on the neighbor cells. The bystander effect of the novel enhancer-CEA-TP transfection in both cell lines was also comparable to that of pCMV-TP transfected cells. Our work indicates that the novel cancer specific enhancer system can be used to improve cancer specific promoter activity for GDEPT.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA