Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer

BACKGROUND. Prostate-specific membrane antigen (PSMA) is abundantly express ed in virtually 100% of prostate cancers and metastases. in addition, unlik e prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target f or advanced prostate cancer. Recently, both the promoter and the enhancer d riving prostate-specific expression of the PSMA gene were cloned. We descri be 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 cytosi ne deaminase gene for suicide-driven gene therapy that converts the nontoxi c 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 subclone d into a luciferase reporter vector containing either the PSMA or SV-40 pro moter. The most active portion of the enhancer was then determined via luci ferase 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 m ost 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 app roximately 2 kb. Deletion constructs revealed that at least two distinct re gions seem to contribute to expression of the gene in prostate cancer cells , and therefore the best construct for prostate-specific expression was det ermined to be 1,648 bp long. The IC50 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 expres s 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. Prostate 45:149-157, 2000. (C) 2000 Wiley-Liss, Inc.