Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter.
Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter.
复制标题
使用由大鼠胰岛素启动子指导的胞嘧啶脱氨酶基因进行胰腺癌的特异性基因表达和治疗。
DOI：
10.1016/j.gassur.2003.10.008
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Brunicardi,FCharles
中科院分区：
文献类型：
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作者：
Wang,Xiao-Ping;Yazawa,Kazuyuki;Yang,Jie;Kohn,Deborah;Fisher,WilliamE;Brunicardi,FCharles
自杀基因治疗已被证明是摧毁胰腺癌细胞的有效手段，但需要细胞特异性的基因传递来限制宿主的毒性。本研究的目的是确定大鼠胰岛素启动子(RIP)是否允许细胞特异性基因在人胰腺癌细胞中传递和随后的细胞死亡。用聚合酶链式反应(PCR)扩增RIP DNA，将纯化的片段克隆到含有酵母胞嘧啶脱氨酶(CD)编码序列的pCR-BLUNT II-TOPO载体的SPI位点上。用RIP-LacZ和RIP-CD DNA构建物在两种人胰腺癌细胞系PANC-1和MIA Paca-2中进行了转染实验。用X-Gal染色进行报告分析，并用5-.ucytosine处理RIP-CD细胞5天，检测其体外细胞毒作用。2天后用Western印迹法检测细胞CD蛋白的表达水平。用原位免疫组织化学方法检测胰岛素促进因子(IPF-1/PDX-1)在这些人胰腺癌细胞系以及新鲜分离的人胰腺癌标本中的表达水平。转染RIP-LacZ后，只有PANC-1细胞表达RIP-LacZ，而MIA Paca-2细胞未检测到RIP-LacZ表达，表明RIP介导的报告基因仅在PANC-1细胞中表达。经RIP-CD基因转染和5-氟胞嘧啶处理后，PANC-1细胞的细胞死亡率明显高于MIA-PACA-2细胞，提示RIP介导的自杀基因表达仅在PANC-1细胞中发生。Western印迹分析表明，只有PANC-1细胞能够表达CD蛋白，PANC-1细胞中PDX-1的表达水平显著增加，而Mia Paca-2细胞则不表达。原位免疫组织化学分析显示，PDX-1仅在PANC-1细胞的胞核中表达，在MIA-Paca-2细胞中不表达。此外，两个新分离的人类胰腺癌样本的PDX-1水平显著增加。RIP在PANC-1细胞中被激活，但在Mia Paca-2细胞中不被激活，其激活机制是通过PDX-1。体外应用RIP-CD和5-氟胞嘧啶可实现胰腺癌特异性细胞毒作用。在人类胰腺癌标本中发现PDX-1水平显著增加。这些结果表明，RIP可用于针对人胰腺肿瘤的细胞特异性自杀基因治疗。
Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-.ucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors.