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

Abstract

Nuclear factor-kappa B (NF-kB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer -promoter system, kB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kB DNA-binding sites (kB4). 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 kB4-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, kB4, kB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kB4-CEA205-and CMV-driven TP cDNAtransfected cancer cell lines (H630 and RKO). The kB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV-and kB4-CEA205-driven TP cDNA transiently transfected cells were 8-to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5 0 -deoxy-5-fluorouradine (5 0 -DFUR), in contrast to only 1.5-to 2-fold sensitised by the kB4-and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV-and kB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy. Lack of selectivity for malignant cells leading to dose-limiting toxicity is one of the major obstacles for the success of cancer chemotherapy. Gene-directed enzyme prodrug therapy (GDEPT) is potentially an elegant way to improve the therapeutic index of active anticancer drugs, but one of the major limitations for the clinical application of this approach is the lack of strong cancerspecific promoters (Aghi et al, 2000;Rooseboom et al, 2004).Carcinoembryonic antigen (CEA) is not expressed in normal and overexpressed in epithelial cancer cells (Shively and Beatty, 1985). Four cis-acting DNA-binding elements mapped on the CEA promoter region within 300 bp upstream of CEA translation starting point, especially, the first three elements are essential for specific CEA transcription (Hauck and Stanners, 1995;Richards et al, 1995a). Utilisation of the CEA promoter in an adenovirus vector for cytosine deaminase (CD) gene expression improves the selectivity of 5 0 -deoxy-5-fluorocytidine (5-DFCR)/5-fluorouracil (5-FU) conversion in CEA-expressing tumours (Richards et al, 1995a;Lan et al, 1996a). However, the transcriptional activity of CEA in most cancer cell lines is 10-to 300-fold lower than that of CMV and RSV viral promoters (Lan et al, 1996b;Ueda et al, 2001).Even when using adenovirus to deliver CEA promoter-driven suicide gene in vivo, few reports have demonstrated sufficient antitumou...

Specific Colon Cancer Cell Cytotoxicity Induced by Bacteriophage E Gene Expression under Transcriptional Control of Carcinoembryonic Antigen Promoter

“…We found the highest expression among all colon cancer cells in HTC-116 cell lines (33.4%) and the lowest in T-84 cells (3.49%); whereas normal CCD18co cells revealed practically undetectable levels of CEA. These results for CEA promoter activity are similar to those described by Gou et al [ 31 ] and Dąbrowska et al [ 32 ], who also observed a greater CEA promoter activity in colon cancer cell lines than in non-tumor cancer cell lines using a luciferase assay.…”

Therapeutic Improvement of a Stroma-Targeted CRAd by Incorporating Motives Responsive to the Melanoma Microenvironment

“…Few reports described the incorporation of genetic responsive elements to promoters for enhanced transcriptional regulation of therapeutic genes (Yew et al , 2001; Greco et al , 2002; Lee et al , 2002; Liu et al , 2004; Lee et al , 2006; Guo et al , 2007; Poulsen et al , 2008), but only one study attempted to combine NF-κB and HRE elements in a plasmid that conferred only a slight enhanced activity to the KDR promoter when assessed in primary murine endothelial cells (Modlich et al , 2000). Here we observed that both in a plasmid and in an adenoviral context the triple chimeric promoter increased its transcriptional activity greatly, leading to enhanced viral efficacy under hypoxia and upon TNF-α addition in human melanoma cells and fetal fibroblasts.…”

Viral-based gene delivery and regulated gene expression for targeted cancer therapy

“…Carcinoembryonic antigen (CEA) promoter is active only in CEA-positive cells, an exclusive characteristic of vast tumor cells; the CEA promoter-controlled CD strategy confers toxic specificity for CEA-producing colorectal cancer cells while preserving activity [54]. Furthermore, by taking advantage of the knowledge that nuclear transcriptional factor NFkB is often highly transcribed in cancer cells, the addition of NF-kB enhancer to the CEA promoter significantly enhanced the efficacy of its driven CD/5-FC system in colon cancer cells [65]. In a study to select the optimal promoter to drive CD in a GDEPT strategy against gastric cancer, promoters of SEL1L, mucin-1 and KRT19 displayed the highest activity levels among several genes that are significantly highly expressed in gastric tumors [66].…”