Pang S, Dannull J, Kaboo R, Xie Y, Tso C L, Michel K, deKernion J B, Belldegrun A S
Department of Urology, UCLA School of Medicine, Los Angeles, California 90095, USA.
Cancer Res. 1997 Feb 1;57(3):495-9.
The prostate-specific antigen (PSA) promoter (PSA-P) has been identified, 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 suggests that the identified promoter may be incomplete or may function optimally 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 enhances PSA gene expression. Combining the newly identified PSA gene regulatory sequence (PSAR) with our previously identified PSA promoter (PCPSA-P) exhibited enhanced expressional activity in the PSA-producing LNCaP cell line. With the addition of 10 to 100 nM dihydrotestosterone, a more than 1000-fold increase in expression was observed as compared to androgen-negative controls. Furthermore, although the combined regulatory element (PSAR)-PSA promoter (PCPSA-P) sequence resulted in high transgene expression in LNCaP cell lines, the combined regulatory element-promoter sequence resulted in minimal expression in the non-PSA-producing prostate cell line PC-3, renal tumor cell line R11, and cervical adenocarcinoma cell line HeLa. The newly identified 822 bp alone could also function as a promoter. Compared with the combined promoter, however, the 822-bp fragment alone demonstrated lower activity and lower responsiveness to androgen stimulation. Our results suggest that coupling 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 distal promoter, which can also function as an enhancer. The enhanced gene expression of the new construct, combined with its tissue specificity and androgen responsiveness, in turn provides a foundation for the development of tissue-specific vectors for prostate cancer gene therapy.
前列腺特异性抗原（PSA）启动子（PSA-P）已被识别、表征并确定具有组织特异性。与前列腺细胞中基因组PSA基因的高表达相比，由假定的PSA启动子驱动的转基因表达较低。这表明所识别的启动子可能不完整，或者可能需要额外的调控元件才能最佳发挥功能。为了确定是否存在其他调控元件，我们筛选了PSA启动子上游的序列，并鉴定出一个822 bp的DNA片段，该片段可增强PSA基因的表达。将新鉴定的PSA基因调控序列（PSAR）与我们之前鉴定的PSA启动子（PCPSA-P）相结合，在产生PSA的LNCaP细胞系中表现出增强的表达活性。添加10至100 nM双氢睾酮后，与雄激素阴性对照相比，表达增加了1000倍以上。此外，尽管组合的调控元件（PSAR）-PSA启动子（PCPSA-P）序列在LNCaP细胞系中导致了高转基因表达，但该组合的调控元件-启动子序列在不产生PSA的前列腺细胞系PC-3、肾肿瘤细胞系R11和宫颈腺癌细胞系HeLa中表达极少。单独的新鉴定的822 bp也可作为启动子发挥作用。然而，与组合启动子相比，单独的822 bp片段表现出较低的活性和对雄激素刺激的较低反应性。我们的结果表明，将PSA启动子与上游调控元件偶联会导致PSA表达显著增加，这表明完整的PSA启动子包含两个功能域：近端启动子和远端启动子，其也可作为增强子发挥作用。新构建体增强的基因表达，连同其组织特异性和雄激素反应性，反过来为开发用于前列腺癌基因治疗的组织特异性载体提供了基础。
