In vitro evaluation of cancer-specific NF-kappaB-CEA enhancer-promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines.
Nuclear factor-kappa B (NF-kappaB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer-promoter system, kappaB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kappaB DNA-binding sites (kappaB4). In combination with a kappaB4 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 kappaB4-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, kappaB4, kappaB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kappaB4-CEA205- and CMV-driven TP cDNA-transfected cancer cell lines (H630 and RKO). The kappaB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV- and kappaB4-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 kappaB4- and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV- and kappaB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kappaB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.
Guo
X
X
Oncology Group, Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton, Wolverhampton, UK.
Evans
T R J
TR
Somanath
S
S
Armesilla
A L
AL
Darling
J L
JL
Schatzlein
A
A
Cassidy
J
J
Wang
W
W
D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000235:genetics*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000628:therapy*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000628:therapy; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary; D005472:Fluorouracil / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016328:NF-kappa B / Q000235:genetics*; D011355:Prodrugs / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013939:Thymidine Phosphorylase / Q000235:genetics; D014162:Transfection
D002272:Carcinoembryonic Antigen; D018076:DNA, Complementary; D016328:NF-kappa B; D011355:Prodrugs; D013939:Thymidine Phosphorylase; D005472:Fluorouracil