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Cloning of promoter of squamous cell carcinoma antigen and tissue secific gene therapy for cervical cancer

鳞状细胞癌抗原启动子的克隆及宫颈癌组织特异性基因治疗

基本信息

批准号：
09671688
负责人：
HAMADA Katsuyuki
金额：
$ 1.98万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671688/
关键词：
cervical cancer squamous cell carcinoma promoter gene therapy tissue specific squamous cell carcinoma antigen 1 squamous cell carcinoma antigen 2 transcription regulation 子宮頚癌

项目摘要

To select the suitable cells for promoter assay, expression of RNA arid protein of squamous cell carcinoma antigen (SCCA) 2 was studied in 40 human cell lines including cervical, skin and other squamous cell carcinoma cell lines, and endometrial, ovarian and other adenocarcinoma cell lines, and normal keratiocytes. ELIZA was used to detect SCCA protein expression in supernatant of each cell. This ELIZA did not differentiate SCCA1 and SCCA2 because of high homology of these 2 proteins. RT-PCR was used to detect SCCA2 RNA expression in each cell. Northern blot analysis was not used because of high homology (98%) of DNAs between SCCA1 and SCCA2. SCCA protein was detested in the supernatant of each squamous cell carcinoma cell line ; 18 ng/ml in SKG llla cells, 1 ng/ml in normal human keratinocyte, NHK, and <0.01 ng/ml in all adenocarcinoma cells. DNA-PCR analysis showed the clear bands of cDNAs of SCCA 1 and SCCA2 with high specificity from 1O^2 to 10^9 copies. RT-PCR analysis demonstrated the results which was well correlated with those of the ELIZA and adenocarcinoma cells did not show any bands. Transcription start site was 9bp upstream from the first codon, which was determined by SMART system. Deletion assay demonstrated the high promoter activity in the 250-500 bp upstream from the first codon. This promoter activity is the most in the SKG llla cells, 10% of that of SKGllla in the NHK, and absent in the SKOV3 cells. Promoter activity of SCCA2 is highly specific in the squamous cell carcinoma and promissing for the tissue specific gene therapy for squamous cell carcinoma.

为了筛选适合于启动子检测的细胞，我们研究了40个人细胞系中鳞状细胞癌抗原(SCCA)2的RNA和蛋白的表达，其中包括宫颈、皮肤等鳞状细胞癌细胞系，子宫内膜、卵巢等腺癌细胞系和正常角质细胞。ELIZA法检测细胞培养上清液中SCCA蛋白的表达。由于SCCA1和SCCA2蛋白具有很高的同源性，所以该ELIZA没有区分这两种蛋白。逆转录-聚合酶链式反应(RT-PCR)检测细胞内SCCA2RNA的表达。因为SCCA1和SCCA2的DNA同源性很高(98%)，所以没有使用Northern杂交分析。SCCA蛋白在各鳞癌细胞系培养上清液中均有表达，SKG111a细胞为18 ng/ml，正常角质形成细胞NHK为1 ng/ml，腺癌细胞均为0.01 ng/ml。DNA-PCR分析显示，SCCA1和SCCA2的cDNAs条带清晰，特异性强，拷贝数在10~109个拷贝之间。RT-PCR分析表明，结果与Eliza的结果有很好的相关性，腺癌细胞没有显示任何条带。经SMART系统测定，转录起始点位于第一个密码子上游9bp处。缺失分析表明，在第一密码子上游250-500bp处有较高的启动子活性。这种启动子活性在SKG111a细胞中最高，是SKG111a在NHK中的10%，在SKOV3细胞中不存在。SCCA2启动子活性在鳞状细胞癌中具有高度的特异性，有望用于组织特异性的鳞状细胞癌的基因治疗。