Cell-specific expression of the diphtheria toxin A-chain coding sequence under the control of the upstream region of the human alpha-fetoprotein gene.
OBJECTIVE
Development of the system to express a suicide gene selectively in tumor cells is essential for gene therapy. We constructed a plasmid containing the diphtheria toxin A (DTA) fragment linked to human alpha-fetoprotein (AFP) promoter and enhancer, and tested whether it can exert its cytocidal effect selectively on AFP-producing cells.


METHODS
The chloramphenical acetyltransferase (CAT) reporter gene or DTA gene was linked to the 5' upstream region of the AFP gene. The plasmids were transfected into AFP-producing or non-producing cells by the lipopolyamine-coated DNA method. Expression of CAT activity and effects on cell growth of transfected cells were assessed.


RESULTS
When the AFP-producing cells HuH-7 or HepG2 were cotransfected with CAT reporter plasmid and pAF5.1DTA plasmid, the CAT activity was greatly suppressed. In contrast, cotransfection with pAF5.1DTA-R, the inversely inserted DTA gene, did not inhibit CAT activity. Furthermore, cell growth of HuH-7 cells transfected with pAF5.1DTA plasmid was significantly inhibited compared with HuH-7 cells transfected with DTA-R plasmid.


CONCLUSIONS
Our results indicate that selective killing of AFP-producing cells will be attained by introducing the DTA gene linked to the promoter and enhancer region of AFP.
Murayama
Y
Y
Department of Surgery, Keio University School of Medicine, Tokyo, Japan.
Tadakuma
T
T
Kunitomi
M
M
Kumai
K
K
Tsutsui
K
K
Yasuda
T
T
Kitajima
M
M
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004167:Diphtheria Toxin / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D015316:Genetic Therapy; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000235:genetics*
D004167:Diphtheria Toxin; D010446:Peptide Fragments; D000509:alpha-Fetoproteins; C029795:diphtheria toxin fragment A; D015500:Chloramphenicol O-Acetyltransferase