Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer.
BACKGROUND
Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells.


METHODS
Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line. We then replaced the luciferase gene with the E. coli cytosine deaminase gene in the subclone that showed the most luciferase activity. The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines. The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared.


RESULTS
The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) of 5-FC was similar in all cell lines tested (>10 mM). However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection.


CONCLUSIONS
Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease.
O'Keefe
D S
DS
George M. O'Brien Urology Research Center, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Uchida
A
A
Bacich
D J
DJ
Watt
F B
FB
Martorana
A
A
Molloy
P L
PL
Heston
W D
WD
D000954:Antigens, Surface; D001483:Base Sequence; D002268:Carboxypeptidases / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D043525:Cytosine Deaminase; D018076:DNA, Complementary; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005437:Flucytosine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D043425:Glutamate Carboxypeptidase II; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009700:Nucleoside Deaminases / Q000235:genetics* / Q000378:metabolism; D011355:Prodrugs / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000954:Antigens, Surface; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011355:Prodrugs; D049452:Green Fluorescent Proteins; D005437:Flucytosine; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil