SUICIDEAND IMMUNE GENE THERAPY USING TUMOR SPECIFIC PROMOTOR FOR BREAST CANCER

自杀或免疫基因(S)在肿瘤细胞中的选择性表达将对肿瘤产生优先的细胞毒作用，并为肿瘤的治疗提供一种有用的策略。C-erbB-2和Midkine基因在人类乳腺癌中经常过表达，是基因扩增和/或转录上调的结果。因此，我们研究了利用c-erbB-2和Midkine基因的启动子区域在乳腺癌细胞中优先表达自杀基因的可能性。利用c-erbB-2启动子区域的缺失突变进行报告基因分析表明，在乳腺癌细胞中，251个碱基(转录起始点-213/+38)而不是125个HP片段(-87/+38)能够更好地指导连接的荧光素酶基因的转录。此外，Midkine基因的1.0kb启动子区域可以激活融合报告基因和自杀基因在人乳腺癌细胞中的转录。相比之下，251bp的片段介导的Pro…在非乳腺癌细胞和正常成纤维细胞中更多的运动活性低于SV40启动子的活性。因此，126bp的片段(-213/-87)包含一个严格的元件(S)，该元件负责乳腺癌细胞的优先转录活性。电泳迁移率改变分析表明，转录因子的一个可能的修饰参与了肿瘤的特异性。用含有251-bp启动子(p256-TK)的单纯疱疹病毒胸苷激酶基因的DNA处理后，乳腺癌细胞对更昔洛韦(GCV)的敏感性增加，而非乳腺癌细胞对GCV的敏感性增加。对携带p256-TK DNA的人乳腺肿瘤裸鼠给予GCV可抑制随后移植瘤的生长。这些结果表明，携带与这些1.0kb中期因子或251bpc-erb B-2启动子连锁的自杀基因可能是一种可行的针对乳腺癌的治疗策略。较少

A selective expression of suicide or immune gene(s) in tumor cells should produce a preferential cytotoxic effect on tumors and a useful strategy for cancer treatment. The c-erbB-2 and midkine gene are frequently overexpressed in human breast cancers as a result of gene amplification and/or elevated transcription. We therefore examined a possible usage of promoter regions of the c-erbB-2 and midkine gene to express a suicide gene preferentially in breast cancer cells. The present reporter gene assays using deletion mutants of the c-erbB-2 promoter region demonstrated that the 251-bp (-213/+38 from the transcriplional start site) but not the 125-hp fragment (-87/+38) could direct transcription of the linked luciferase gene better than the SV40 immediate early promoter in breast cancer cells. Also the 1.0 kb promoter region of the midkine gene could activate transcription of a fused reporter gene and suicide gene in human breast cancer cells. In contrast, the 251-bp fragment-mediated pro … More moter activity in nonbreast cancer cells and in normal fibroblasts was lower than the activity by the SV40 promoter. The 126-bp fragment (-213/-87) thereby contains a exacting element(s) which is responsible for the preferential transcriptional activity in breast cancer cells. An electrophoretic mobility shift assay suggested that a possible modification of a transcriptional factor was involved in the tumor specificity. Transaction with the plasmid DNA containing the herpes simplex virus-thymidine kinase gene linked with the 251-bp promoter (p256-TK) resulted in increased sensitivity to ganciclovir (GCV) in breast cancer but not in nonbreast cancer cells. Administration of GCV into nude mice bearing human breast tumors that were transfecled with the p256-TK DNA suppressed subsequent growth of the transplanted tumors. These results suggest that delivery of a suicide gene linked with the these 1.0 kb midkine or 251 bp c-erbB-2 promoter can be a feasible therapeutic strategy specific to breast cancer. Less