In vitro evaluation of cancer-specific NF-κB-CEA enhancer-promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines

Nuclear factor-kappa B (NF-kappa B) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer-promoter system, kappa B4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kappa B DNA-binding sites (kappa B4). In combination with a kB4 enhancer, the transcriptional activity of the CEA promoter was significantly enhanced (three- to eight-fold) in cancer cell lines but not in normal cells. In cancer cell lines, the transcriptional activity of kappa B4-CEA205 was comparable with that of the SV40 promoter. We also constructed vectors in which the thymidine phosphorylase (TP) cDNA was under the control of CEA205, kappa B4, kappa B4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kappa B4-CEA205- and CMV-driven TP cDNA-transfected cancer cell lines (H630 and RKO). The kappa B4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV- and kappa B4-CEA205- driven TP cDNA transiently transfected cells were 8- to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5'-deoxy-5-fluorouradine (5'-DFUR), in contrast to only 1.5- to 2-fold sensitised by the kappa B4- and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV- and kappa B4- CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kappa B DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.