Zhang Peng, Zeng Hao, Wei Qiang, Lu Yiping, Li Xiang, Wang Jia, Zhao Fujun, Li Hong
Department of Urology, West China Hospital, Sichuan University, Sichuan, China.
Int J Urol. 2008 May;15(5):442-8. doi: 10.1111/j.1442-2042.2008.02034.x.
To explore the specific killing effect on prostate cancer cells of a dual cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) expression plasmid system controlled by the prostate-specific membrane antigen (PSMA) promoter and enhancer.
The CD gene was used to construct the recombinant plasmid prostate-specific membrane antigen(promoter/enhancer)-CD (pPSMA(E/P)-CD). The specific regulatory function of the pPSMA(E/P) promoter was demonstrated by detection of enhanced green fluorescent protein (EGFP) expression in the LNCaP cell line. Survival of cells transfected with different plasmids and treated with 5-fluorocytosine (5-FC) was measured by microculture tetrazolium assay. Cell cycle changes were measured by flow cytometry.
Target-specific expression of PSMA(E/P) was observed in the prostate cancer cell line. Cytotoxicity of 5-FC was greater against LNCaP cells transfected with pPSMA(E/P)-CD and UPRT and pPSMA(E/P)-CD than control groups. Percentages of cells in S phase were 37.5% (LNCaP) and 30.6% (5-FC treatment) in the un-transfected groups, whereas they were 23.9% and 12.4% in the double and single suicide gene groups, respectively.
Our findings confirm the cytotoxic efficacy of the pPSMA(E/P)-CD + 5-FC and pPSMA(E/P)-CD and UPRT + 5-FC suicide gene systems. The CD and UPRT gene system quickly and directly converted 5-FC into 5-FU, and then into toxic metabolites. The CD and UPRT double suicide gene system was more effective in inducing tumor cell apoptosis with 5-FC than the single suicide gene system. Thus, this construct can specifically target prostate cancer cells and might have a role in gene therapy against prostate cancer.
探讨由前列腺特异性膜抗原（PSMA）启动子和增强子控制的双胞嘧啶脱氨酶（CD）和尿嘧啶磷酸核糖转移酶（UPRT）表达质粒系统对前列腺癌细胞的特异性杀伤作用。
利用CD基因构建重组质粒前列腺特异性膜抗原（启动子/增强子）-CD（pPSMA(E/P)-CD）。通过检测LNCaP细胞系中增强型绿色荧光蛋白（EGFP）的表达，证实pPSMA(E/P)启动子的特异性调控功能。采用微量培养四氮唑蓝法测定转染不同质粒并经5-氟胞嘧啶（5-FC）处理的细胞存活率。通过流式细胞术检测细胞周期变化。
在前列腺癌细胞系中观察到PSMA(E/P)的靶向特异性表达。与对照组相比，5-FC对转染pPSMA(E/P)-CD和UPRT以及pPSMA(E/P)-CD的LNCaP细胞的细胞毒性更大。未转染组S期细胞百分比分别为37.5%（LNCaP）和30.6%（5-FC处理），而在双自杀基因组和单自杀基因组中分别为23.9%和12.4%。
我们的研究结果证实了pPSMA(E/P)-CD + 5-FC和pPSMA(E/P)-CD及UPRT + 5-FC自杀基因系统的细胞毒性疗效。CD和UPRT基因系统迅速将5-FC直接转化为5-氟尿嘧啶（5-FU），然后转化为有毒代谢产物。与单自杀基因系统相比，CD和UPRT双自杀基因系统在5-FC诱导肿瘤细胞凋亡方面更有效。因此，该构建体可特异性靶向前列腺癌细胞，可能在前列腺癌基因治疗中发挥作用。
