Ca. Richards et al., TRANSCRIPTIONAL REGULATORY SEQUENCES OF CARCINOEMBRYONIC ANTIGEN - IDENTIFICATION AND USE WITH CYTOSINE DEAMINASE FOR TUMOR-SPECIFIC GENE-THERAPY, Human gene therapy, 6(7), 1995, pp. 881-893
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.