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

为了选择合适的启动子细胞，我们研究了40种人类细胞系（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化细胞）中SCCA抗原（SCCA） 2 RNA和蛋白的表达。elisa法检测各组细胞上清液中SCCA蛋白的表达。由于SCCA1和SCCA2的高度同源性，该elisa不能区分SCCA1和SCCA2。RT-PCR检测SCCA2 RNA在每个细胞中的表达。由于SCCA1和SCCA2之间的dna同源性很高（98%），因此没有使用Northern blot分析。在各鳞状细胞癌细胞系的上清液中检测SCCA蛋白；在SKG细胞中为18 ng/ml，在正常人角质细胞，NHK中为1 ng/ml，在所有腺癌细胞中均<0.01 ng/ml。DNA-PCR分析显示scca1和SCCA2在10^ 2 ~ 10^9拷贝间有清晰的条带，特异性高。RT-PCR分析结果与elisa检测结果有较好的相关性，腺癌细胞未显示任何条带。转录起始位点在第一个密码子上游9bp处，由SMART系统确定。缺失实验表明，在第一个密码子上游250-500 bp处有较高的启动子活性。该启动子活性在SKGllla细胞中最高，为NHK中SKGllla的10%，而在SKOV3细胞中不存在。SCCA2启动子活性在鳞状细胞癌中具有高度特异性，有望用于鳞状细胞癌的组织特异性基因治疗。

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.