Selective Suicide Gene Therapy of Colon Cancer Cell Lines Exploiting Fibroblast Growth Factor 18 Promoter Ladan Teimoori-Toolabi, 1 Kayhan Azadmanesh, 2,3 and Sirous Zeinali 1 Abstract Fibroblast growth factor 18 (FGF18) is one of the genes downstream of Wnt, one of the most important signaling pathways activated in colon cancer. An FGF18 promoter containing a single T-cell factor=lymphocyte enhancing factor 1 (TCF=LEF1) binding site was inserted upstream of a thymidine kinase (TK) suicide gene module, while a bacterial b-Gal (LacZ) element served as the reporter gene. Following transient transfection with pUCFG- F18LacZ, b-Gal staining showed that 5% of SW480, 10% of HCT116, 0% of human umbilical vein endothelial cells (HUVECs) and 0% of normal colon cells (NCCs) had expressed LacZ. b-Gal enzyme-linked immunosorbent assay revealed that the ratio of pUCFGF18LacZ activity to that of positive control was 0.09 and 0.25 in SW480 and HCT116, respectively (significantly higher than mock plasmid), while there were no significant changes in the b-Gal expression in HUVEC and NCC cells transfected with pUCFGF18LacZ or mock plasmid. Following transfection with pUCFGF18TK and pUCCMVTK (positive control), cytotoxicity analysis of transfected cells showed that treatment with ganciclovir (GCV) significantly decreased SW480 and HCT116 cell survival at GCV concentrations above 20 mg=mL. An inverse correlation between GCV concentration and cell viability was evi- dent in both colon cancer cell lines following transfection with these suicide plasmids. pUCFGF18TK and pUCCMVTK induced apoptosis after the administration of GCV in HCT116, but not in SW480, as demonstrated by M30 cytodeath antibody. This discrepancy may stem from differences in the mechanisms of TK=GCV- induced apoptosis in p53-proficient (HCT116) and -deficient (SW480) cells. The specific activity of the FGF18 promoter in HCT116 and SW480 may reflect the advantage of this promoter over artificial promoters containing artificial TCF=LEF binding sites. Key words: genes, transgenic, suicide, neoplasm, colonic neoplasm, gene therapy Introduction C olon cancer is the third and fourth most prevalent cancer among Iranian women and men, respectively, 1 whereas in the United States, it is rated the third most common cancer for both men and women. 2 Conventional therapies cannot fully eradicate cancerous cells, and therefore, gene therapy has attracted much attention in recent years. 3 Efficacy of chemotherapy in complete eradication of a few types of can- cers, such as leukemia, has led to the hypothesis that all types of cancer can potentially be eradicated by higher doses of chemotherapy, 4 an assumption that is questionable given the side-effects associated with high-dose regimens of chemo- therapeutic agents. In suicide gene therapy, a gene construct that encodes an enzyme is introduced to the target tissues. The enzyme converts a nontoxic prodrug to a toxic drug. There- fore, suicide gene therapy opens a new horizon for the local administration of these toxic drugs. Thymidine kinase (TK) is the first, and the most studied, suicide gene, 5 which accounts for about 10% of all clinical trials evaluating cancer gene therapy. 6 The mechanism of its action exploits a unique characteristic of herpes simplex virus thy- midine kinase (HSV-TK), which, contrary to normal mamma- lian thymidine kinase, preferentially monophosphorylates gancyclovir (GCV), rendering it toxic to normal mammalian cells. Further phosphorylation of GCV-monophosphate by Departments of 1 Molecular Medicine, Biotechnology Research Center, 2 Hepatitis and AIDS, and 3 Virology, Pasteur Institute of Iran, Tehran, Iran. Address correspondence to: Sirous Zeinali; Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran; 69th Pasteur Avenue, Kargar Street, Tehran 13169-43551, Iran E-mail: siruszeinali@yahoo.com CANCER BIOTHERAPY AND RADIOPHARMACEUTICALS Volume 25, Number 1, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=cbr.2009.0643 105