O'Keefe D S, Uchida A, Bacich D J, Watt F B, Martorana A, Molloy P L, Heston W D
George M. O'Brien Urology Research Center, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Prostate. 2000 Oct 1;45(2):149-57. doi: 10.1002/1097-0045(20001001)45:2<149::aid-pros9>3.0.co;2-o.
Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe 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 cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells.
Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase 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 most 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.
The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) 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 express the PSMA gene were not significantly sensitized by transfection.
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.
前列腺特异性膜抗原（PSMA）在几乎100%的前列腺癌及转移灶中大量表达。此外，与前列腺特异性抗原（PSA）不同，PSMA在雄激素剥夺条件下上调。因此，PSMA是晚期前列腺癌一个有吸引力的治疗靶点。最近，驱动PSMA基因前列腺特异性表达的启动子和增强子均已克隆。我们在此描述了对PSMA增强子最活跃区域的分析，并提出了一种将增强子与大肠杆菌胞嘧啶脱氨酶基因联合用于自杀驱动基因治疗的方法，该方法可在前列腺癌细胞中将无毒前药5-氟胞嘧啶（5-FC）转化为细胞毒性药物5-氟尿嘧啶（5-FU）。
将全长PSMA增强子的缺失构建体亚克隆到含有PSMA或SV-40启动子的荧光素酶报告载体中。然后通过C4-2细胞系中的荧光素酶活性确定增强子最活跃的部分。接着在显示最高荧光素酶活性的亚克隆中用大肠杆菌胞嘧啶脱氨酶基因替换荧光素酶基因。使用前列腺癌细胞系LNCaP、其雄激素非依赖性衍生物C4-2以及一些非前列腺细胞系在体外检测该技术的特异性。然后比较5-FC和5-FU对瞬时转染细胞系的毒性。
最初从PSMA基因分离的增强子区域约为2 kb。缺失构建体显示至少有两个不同区域似乎对前列腺癌细胞中该基因的表达有贡献，因此确定用于前列腺特异性表达的最佳构建体长度为1648 bp。在所有测试的细胞系中，5-FC的半数抑制浓度（IC50）相似（>10 mM）。然而，用1648 nt的PSMA增强子和PSMA启动子驱动胞嘧啶脱氨酶基因转染以剂量依赖性方式增强毒性超过50倍，而未表达PSMA基因的细胞未因转染而显著致敏。
使用PSMA增强子的自杀基因治疗可能对已接受雄激素剥夺治疗且疾病复发的患者有益。
