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

为筛选适合启动子检测的细胞，我们研究了40种人细胞系（包括宫颈癌、皮肤癌等鳞状细胞癌细胞系、子宫内膜癌、卵巢癌等腺癌细胞系和正常角膜上皮细胞）中鳞状细胞癌抗原2（SCCA 2）的RNA和蛋白的表达。ELISA法检测细胞培养上清中SCCA蛋白的表达。由于SCCA 1和SCCA 2的高度同源性，该ELISA不能区分这两种蛋白。RT-PCR检测SCCA 2 RNA在各细胞中的表达。由于SCCA 1和SCCA 2之间的DNA具有高度同源性（98%），因此未使用北方印迹分析。SCCA蛋白在每个鳞状细胞癌细胞系的上清液中是不受欢迎的;在SKG 111 a细胞中为18 ng/ml，在正常人角质形成细胞、NHK中为1 ng/ml，并且在所有腺癌细胞中<0.01 ng/ml。DNA-PCR分析显示SCCA 1和SCCA 2的cDNA条带清晰，特异性高，拷贝数在10^2 ~ 10^9之间。RT-PCR结果与ELISA结果一致，腺癌细胞未出现条带。SMART系统确定转录起始位点为第一密码子上游9 bp处。缺失分析表明，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SKG IIIa细胞中最高，在NHK中为SKG IIIa的10%，而在SKOV 3细胞中不存在。SCCA 2启动子活性在鳞状细胞癌中具有高度特异性，有望成为鳞状细胞癌组织特异性基因治疗的靶点。

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.