Prostate Tissue Specificity of the Prostate-Specific Antigen Promoter Isolated from a Patient with Prostate Cancer

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Abstract
We have cloned and characterized a 620-bp fragment of DNA that flanks 5′ of the prostate-specific antigen (PSA) gene from a prostate cancer patient. Using DNA transfection, the efficacy of this putative promoter in regulating gene expression was quantitated in several prostate and nonprostate tissue cell lines. Our results demonstrated that the 620-dp DNA fragment actively drives gene expression in LNCaP, a PSA-producing prostate tumor cell line. No promoter activity was detected in the non-PSA-producing prostate tumor lines, DU145 and PC-3, nor in a renal (R11) or breast (MCF-7) cancer cell line. Furthermore, the promoter activity could be regulated in vitro by androgen stimulation. Dihydrotestosterone (DHT) concentrations between 3 and 30 nM induced the highest promoter activity in the transfected LNCaP cells, which parallels the expression profile of the androgen receptor in LNCaP cells. In addition, our PSA promoter exhibited competitive inhibition of the endogenous genomic PSA promoter in transfected LNCaP cells, suggesting that prostate cell-specific DNA-binding proteins are required to activate the PSA promoter. A cytomegalovirus IE1 promoter (CMV promoter) attached to the 5′-flanking region of the PSA promoter increased its potency four-to five-fold while retaining tissue specificity. Our data suggest that a strong tissue-specific negative regulatory element capable of overriding the nonspecific CMV promoter is present in the PSA promoter and confers its tissue specificity. The use of a highly specific promoter-driven gene vector will allow selective expression of therapeutic genes within PSA-producing prostate cancer cells, providing a unique strategy for prostate cancer gene therapy. High levels of prostate-specific antigen (PSA) expression are detected in most patients with metastatic prostate cancer. We have cloned the PSA promoter from such a patient with very high levels of serum PSA. Using the luciferase gene as a reporter gene, the cloned PSA promoter (which has been designated PCPSA promoter) demonstrated strong prostate tissue specificity. The PCPSA promoter was modified with a cytomegalovirus (CMV) promoter sequence. A four-fold increase in activity was observed after the modification, without significant changes in tissue specificity. Models to explain these results are proposed. The results suggest that the PCPSA promoter has the potential for use in constructing therapeutic gene vectors for prostate cancer gene therapy.

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