El-Tanani M, Barraclough R, Wilkinson M C, Rudland P S
Cancer and Polio Research Fund Laboratories, Life Sciences Building, School of Biological Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom.
Cancer Res. 2001 Jul 15;61(14):5619-29. doi: 10.1100/tsw.2002.238.
Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.
当从人类恶性乳腺癌细胞系中获得的1000个碱基对的小基因组DNA片段转染到良性大鼠乳腺细胞系中时，会增强骨桥蛋白基因的转录，从而导致细胞在同基因大鼠中发生转移。为了确定这一过程背后的分子事件，骨桥蛋白启动子-报告基因构建体与一种转移诱导DNA（Met-DNA）片段的瞬时共转染已确定Met-DNA中的活性成分是转录因子T细胞因子（Tcf）家族的结合位点。用含有序列CAAAG的活性DNA片段孵育细胞提取物会导致其在聚丙烯酰胺凝胶上的迁移率减慢，蛋白质印迹法在体外相关DNA复合物中鉴定出了Tcf-4、β-连环蛋白和E-钙黏蛋白。转染Tcf-4的表达载体可抑制由Met-DNA的瞬时转染活性片段或永久转染的Met-DNA引起的骨桥蛋白启动子-报告基因构建体的刺激活性。骨桥蛋白启动子-报告基因构建体的这种刺激活性伴随着转染细胞中内源性骨桥蛋白mRNA的增加，但fos或肌动蛋白mRNA没有增加。用来自Met-DNA的含有Tcf识别序列CAAAG的20个碱基对片段永久转染良性大鼠乳腺细胞系，在体外导致内源性骨桥蛋白的永久产生增加，并在体内诱导细胞在同基因大鼠中转移。没有CAAAG序列的相应片段则没有任何作用。因此，C9-Met-DNA的调节作用至少部分是由一个CAAAG序列发挥的，该序列可以隔离内源性抑制性Tcf-4，从而促进骨桥蛋白的转录，骨桥蛋白是该系统中转移的直接效应物。
