Kim D H, Chang J H, Lee K H, Lee H Y, Kim S J
Laboratory of Chemoprevention, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.
J Biol Chem. 1997 Jan 3;272(1):688-94. doi: 10.1074/jbc.272.1.688.
Cellular transformation driven by the E1A oncogene is associated with the development of cellular resistance to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). We demonstrate that development of resistance occurs simultaneously with decreased expression of TGF-beta type II receptor (TGF-beta RII) mRNA and protein. To determine whether changes in transcriptional regulation are responsible for the decreased receptor expression in E1A-transformed cells, a series of mobility shift assays was performed utilizing nuclear extracts from E1A-transformed and untransformed murine keratinocytes using radiolabeled positive regulatory elements (PRE1 and PRE2) of the TGF-beta RII promoter. The results from these assays suggest that E1A-transformed cells express markedly lower levels of nuclear proteins that bind specifically to PRE1 and PRE2. Transfection of both E1A-transformed and untransformed cell lines with a series of mutant promoter constructs confirmed that both PREs contribute significantly to basal expression of TGF-beta RII and that inactivation of either element leads to markedly reduced promoter activity. We conclude that development of TGF-beta resistance in E1A-transformed cells is achieved in part through transcriptional down-regulation of the TGF-beta RII gene and that this down-regulation is the result of decreased expression of unidentified transcription factor complexes that interact with PRE1 and PRE2.
由E1A癌基因驱动的细胞转化与细胞对转化生长因子-β（TGF-β）生长抑制作用产生抗性相关。我们证明，抗性的产生与TGF-βⅡ型受体（TGF-βRII）mRNA和蛋白表达的降低同时发生。为了确定转录调控的变化是否是E1A转化细胞中受体表达降低的原因，我们使用来自E1A转化和未转化的小鼠角质形成细胞的核提取物，利用TGF-βRII启动子的放射性标记正调控元件（PRE1和PRE2）进行了一系列凝胶迁移实验。这些实验结果表明，E1A转化细胞中与PRE1和PRE2特异性结合的核蛋白水平明显较低。用一系列突变启动子构建体转染E1A转化和未转化细胞系证实，两个PRE对TGF-βRII的基础表达均有显著贡献，且任一元件失活都会导致启动子活性显著降低。我们得出结论，E1A转化细胞中TGF-β抗性的产生部分是通过TGF-βRII基因的转录下调实现的，且这种下调是与PRE1和PRE2相互作用的未鉴定转录因子复合物表达降低的结果。
