Periyasamy S, Ammanamanchi S, Tillekeratne M P, Brattain M G
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43614, USA.
Oncogene. 2000 Sep 21;19(40):4660-7. doi: 10.1038/sj.onc.1203822.
In this report, we describe the mechanism of TGF-beta receptor type I (RI) repression in the GEO human colon carcinoma cells. Treatment of GEO cells with the DNA methyltransferase inhibitor, 5 azacytidine induced RI expression and restored TGF-beta response. A stably transfected RI promoter-reporter construct (RI-Luc) expressed higher activity in the 5 aza C treated GEO cells, suggesting the activation of a transactivator for RI transcription. Gel shift analysis indicated enhanced binding of proteins from the 5 aza C treated nuclear extracts to radiolabeled Sp1 oligonucleotides specifically contained in the RI promoter. Protein stability studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5 aza C treated GEO cells. Further, transfection of Sp1 cDNA into untreated GEO control cells increased RI promoter activity and thus induced RI expression. 5 aza C mediated Sp1 expression in Sp1 deficient GEO colon and MCF-7 breast cancer cells also enhanced the activity of several other Sp1 dependent promoters such as TGF-beta receptor type II (RII), Cyclin A and p21/waf1/cip1. These results indicate that restoration of Sp1 in several different types of Sp1 deficient cells leads to enhanced activation of a wide range of Sp1 dependent promoters.
在本报告中，我们描述了GEO人结肠癌细胞中转化生长因子βⅠ型受体（RI）抑制的机制。用DNA甲基转移酶抑制剂5-氮杂胞苷处理GEO细胞可诱导RI表达并恢复转化生长因子β反应。一个稳定转染的RI启动子-报告基因构建体（RI-Luc）在经5-氮杂胞苷处理的GEO细胞中表现出更高的活性，提示存在一种RI转录反式激活因子被激活。凝胶迁移分析表明，来自经5-氮杂胞苷处理的细胞核提取物中的蛋白质与RI启动子中特有的放射性标记的Sp1寡核苷酸的结合增强。环己酰亚胺处理后的蛋白质稳定性研究表明，经5-氮杂胞苷处理的GEO细胞中Sp1蛋白的稳定性增加。此外，将Sp1 cDNA转染入未处理的GEO对照细胞可增加RI启动子活性，从而诱导RI表达。5-氮杂胞苷在Sp1缺陷的GEO结肠癌细胞和MCF-7乳腺癌细胞中介导的Sp1表达还增强了其他几种Sp1依赖性启动子的活性，如转化生长因子βⅡ型受体（RII）、细胞周期蛋白A和p21/waf1/cip1。这些结果表明，在几种不同类型的Sp1缺陷细胞中恢复Sp1可导致多种Sp1依赖性启动子的激活增强。
