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BACKGROUND To evaluate their relative activity and specificity for prostate cells promoter and regulatory regions from three prostate-expressed genes–prostate-specific antigen (PSA), probasin, and relaxin H2–have been compared in prostate cell lines and in lines of breast, bladder, liver, kidney, lung, and ovarian origin. METHODS After transfection into different cell types, the activity of promoters was assayed using linked reporter genes and normalized against that of the Rous sarcoma virus. Activity was measured both in the presence and in the absence of co-transfected androgen receptor (AR). RESULTS PSA and probasin regulatory regions showed strong responsiveness to co-transfection of the AR in most cell types. The core PSA promoter region showed low activity and specificity, but the specificity and level of expression were substantially increased by inclusion of upstream sequences, particularly the enhancer region. Probasin promoter fragments showed specificity of expression for prostate cell lines but required AR for significant levels of expression. Relaxin promoter fragments directed significant AR-inducible expression in prostate cells but showed little specificity and variable AR responsiveness in other cell types. CONCLUSIONS Of regulatory regions tested, a 430-base pair probasin promoter and PSA enhancer/core promoter showed the best combination of AR-stimulated prostate cell expression with limited expression in other cell types. Prostate 35:18–26, 1998. © 1998 Wiley-Liss, Inc.

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.

Prostate-specific membrane antigen (PSMA) is an integral membrane protein that is highly expressed on the surface of prostate epithelial cells. It is also expressed on the vascular endothelium of a number of tumor types. We have used an enhancer trap approach with randomly cleaved overlapping DNA fragments from an approximately 55-kb P1 cosmid insert encompassing the 5' half and upstream sequences of the PSMA gene (FOLH1) to isolate an enhancer that strongly activates the FOLH1 core promoter region. The enhancer (PSME) is located in the third intron about 12 kb downstream from the start site of transcription and is characterized by a 72-bp direct repeat within a 331-bp core region. The PSME activates transcription from its own and heterologous promoters in prostate cell lines; enhancement is greatest in the PSMA-expressing cell line LNCaP (>250-fold). The PSME shows essentially no activity in five nonprostate cell lines. PSME-enhanced expression is repressed in the presence of androgen, mimicking the repression of the endogenous FOLH1 gene. The data demonstrate that both cell-type specificity and androgen regulation are intrinsic properties of the enhancer. These properties make the PSME an excellent candidate for regulation of gene expression in gene therapy approaches to prostate cancer.