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Cloning of promoter of CA125 gene and tissue specific gene therapy for ovarian cancer

CA125基因启动子克隆及卵巢癌组织特异性基因治疗

基本信息

批准号：
11671624
负责人：
HAMADA Katsuyuki
金额：
$ 2.3万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671624/
关键词：
ovarian cancer promoter gene therapy tissue specificity CA125 transcription regulation IAI3B tumor marker 転写因子腺癌エンハンサー

项目摘要

Genomic DNA was cloned from EMBL SP6/T7 phage library using 500 bp of 5' end of CA125 gene and sequenced 5 kb upstream of exon 1 of CA125 genomic DNA.Transcriptional start site was determined by SMART PCR cDNA Synthesis kit. Exon 1 is devided into exon 1A and exon 1B.It is reported that transcriptional start site of normal lymphocite is exon 1A.However, this study demonstrated that transcriptional start site of ovarian cancer cell line was exon 1B.Deletion mutant was constructed by using PCR, in which antisense primer was contructed including transcriptional start site of exon 1B.Sense primer was setted in every 250-500 bp size 5' upstream of promoter region. Luciferase assay was determined by dual luciferase assay kit (Promega). Significant transcriptional activity was demonstrated in 500 bp upstream of transctiprional start site. Enhancer activity was shown in 1825 bp uptream of transctiprional start site. Ovarian carcinoma HEY cell line, cervical carcinoma SKGIIIa cell lilne and normal human keratinocyte K42 cell line were used to determine the tissue specificity in the transcriptional activity of CA125 gene. The highest transcriptional activity of CA125 gene was shown in 1825 bp upstream of transcriptional start site. The transcriptional activity of CA125 gene was 7 times that of SV40 gene in HEY cells, 1.9 times that of SV40 gene in SKGIIIa and 1.8 times that of SV40 gene in K42 cells. These results shows the tissue specific activity of CA125 gene in ovarian carcinoma cells. Then we inserted the 1875 bp of CA125 gene into E1A promoter region of adenovirus to make a conditioned replicative adenovirus. This vector showed the high tissue specific anti-proliferative activity in ovarican carcinoma cells, because IC50 of this vector was 0.01 MOI in HEY cells and 100 MOI in SKGIIIa cells. From these results, gene therapy by using CA125 gene promoter may be tissue specific for ovarian carcinoma and promissing in the clinical trial of gene therapy of ovarian cancer.

从EMBL SP6/T7噬菌体文库中，利用CA125基因5'端500 bp克隆基因组DNA，在CA125基因组DNA 1外显子上游5 kb处测序。采用SMART PCR cDNA合成试剂盒测定转录起始位点。外显子1分为外显子1A和外显子1B。据报道，正常淋巴细胞的转录起始位点在1A外显子。然而，本研究表明卵巢癌细胞系的转录起始位点为外显子1B。利用PCR构建缺失突变体，构建包含1B外显子转录起始位点的反义引物。在启动子区上游5'处每隔250-500 bp大小设置Sense引物。采用双荧光素酶测定试剂盒（Promega）测定荧光素酶含量。在转录起始位点上游500bp处有显著的转录活性。增强子在转录起始点上游1825bp处有活性。采用卵巢癌HEY细胞系、宫颈癌SKGIIIa细胞系和正常人角质形成细胞K42细胞系测定CA125基因转录活性的组织特异性。CA125基因在转录起始位点上游1825bp处的转录活性最高。CA125基因在HEY细胞中的转录活性是SV40基因的7倍，在SKGIIIa细胞中是SV40基因的1.9倍，在K42细胞中是SV40基因的1.8倍。这些结果表明CA125基因在卵巢癌细胞中的组织特异性活性。将CA125基因1875 bp插入腺病毒E1A启动子区，制备条件复制腺病毒。该载体对卵巢癌细胞具有较高的组织特异性抗增殖活性，其在HEY细胞中的IC50为0.01 MOI，在SKGIIIa细胞中的IC50为100 MOI。由此可见，利用CA125基因启动子进行卵巢癌基因治疗可能具有组织特异性，在卵巢癌基因治疗的临床试验中具有广阔的应用前景。