TRANSCRIPTIONAL REGULATORY SEQUENCES OF CARCINOEMBRYONIC ANTIGEN - IDENTIFICATION AND USE WITH CYTOSINE DEAMINASE FOR TUMOR-SPECIFIC GENE-THERAPY

The 5' sequences from the human carcinoembryonic antigen gene (CEA) we re analyzed using luciferase reporter gene assays. This analysis ident ified important cis-acting sequences needed for selective expression i n CEA-positive cells. Over 50 CEA/luciferase reporter clones were cons tructed and analyzed in two CEA-positive and two CEA-negative cell lin es. The CEA sequences analyzed extended from the translational start t o 14.5 kb 5' of the CEA gene. A 408-bp region from the CEA 3' untransl ated region was also examined for its effect on reporter gene activity . The CEA promoter was located between bases -90 and +69 of the transc riptional start site. Sequences between -41 and -18 were essential for expression from the CEA promoter. Multimerization of sequences betwee n -89 and -40 resulted in copy number-related increases in both expres sion level and selectivity for CEA-positive cells. Two upstream region s of CEA, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the mul timerized promoter led to high-level, selective expression in CEA-posi tive cell lines. Several CEA/luciferase constructs demonstrated 80- to 120-fold higher expression in CEA-positive cell lines compared to exp ression in CEA-negative Hep3B cells. The expression from these constru cts was quite strong in CEA-positive cells, being two- to four-fold hi gher than an SV40 enhancer/promoter construct. The most promising CEA transcriptional regulatory sequences were used to regulate the express ion of cytosine deaminase (CD) in stable cell lines. The expression of CD was assessed directly by an enzymatic assay and indirectly by dete rmining the in vitro IC50 to 5-fluorocytosine (5FC). The chimeric gene pCEA/CD-145 displayed the desired expression spectrum-high-level expr ession in the CEA-positive cells and low-level expression in CEA-negat ive cells. CD expression from this chimera correlated well with the ex pression of the endogenous CEA gene. Treatment of mice bearing NCI H50 8 pCEA/CD-145 tumor xenografts with 5FC lead to significant antitumor effects in vivo. The CEA/CD chimeric gene should be useful for tumor-s pecific suicide gene therapy of CEA-positive tumors.