IDENTIFICATION OF A POSITIVE REGULATORY ELEMENT RESPONSIBLE FOR TISSUE-SPECIFIC EXPRESSION OF PROSTATE-SPECIFIC ANTIGEN

The prostate-specific antigen (PSA) promoter (PSA-P) has been identifi ed, characterized, and determined to be tissue specific. Compared with high expression of the genomic PSA gene in prostate cells, expression of the transgene driven by the putative PSA promoter is low, This sug gests that the identified promoter may be incomplete or may function o ptimally with additional regulatory elements, To identify the presence of additional regulatory elements, we screened sequences upstream of the PSA promoter and identified a DNA fragment of 822 bp, which enhanc es PSA gene expression, Combining the newly identified PSA gene regula tory sequence (PSAR) with our previously identified PSA promoter (PCPS A-P) exhibited enhanced expressional activity in the PSA-producing LNC aP cell line, With the addition of 10 to 100 nM dihydrotestosterone, a more than 1000-fold increase in expression was observed as compared t o androgen-negative controls, Furthermore, although the combined regul atory element (PSAR)-PSA promoter (PCPSA-P) sequence resulted in high transgene expression in LNCaP cell lines, the combined regulatory elem ent-promoter sequence resulted in minimal expression in the non-PSA-pr oducing prostate cell line PC-3, renal tumor cell line R11, and cervic al adenocarcinoma cell line HeLa. The newly identified 822 bp alone co uld also function as a promoter, Compared with the combined promoter, however, the 822-bp fragment alone demonstrated lower activity and low er responsiveness to androgen stimulation, Our results suggest that co upling the PSA promoter with an upstream regulatory element results in a marked increase in PSA expression, suggesting that the complete PSA promoter contains two functional domains: a proximal promoter and a d istal promoter, which can also function as an enhancer, The enhanced g ene expression of the new construct, combined with its tissue specific ity and androgen responsiveness, in turn provides a foundation for the development of tissue-specific vectors for prostate cancer gene thera py.