Rhee K, Ma T, Thompson E A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
J Biol Chem. 1994 Jun 24;269(25):17035-42.
Glucocorticoids inhibit transcription of the proto-oncogene c-myc in lymphoid cells of thymic origin. To determine if this effect is associated with changes in the properties of the transcription factor E2F, extracts were prepared from control and glucocorticoid-treated P1798 murine T lymphoma cells, and the macromolecular state of E2F was assessed by gel-mobility shift. Control extracts exhibit two predominant gel-mobility shift entities of which one corresponds to "free" E2F. A second entity, complex C, has properties similar to those described for the complex containing E2F, p107, cyclin A, and Cdk2. Complex C disappears after addition of dexamethasone and is replaced by complex D. The mobility of this complex and its sensitivity to SV40 T antigen suggest that complex D corresponds to an E2F-p105Rb-1 complex. Extracts from control and glucocorticoid-treated cells yield identical DNase I protection patterns on the c-myc P2 promoter. Furthermore, such extracts transcribe the c-myc P2 promoter in vitro with equal activity. The relative abundance of the E2F complexes was measured after addition of dexamethasone. Complex C disappears as cells withdraw from S phase, and complex D appears at this time. The genes encoding thymidine kinase (Tk-1) and p34cdc2 (cdc2) are regulated with kinetics similar to those observed for changes in the macromolecular state of E2F. However, regulation of c-myc expression occurs long before any change in E2F. The macromolecular state of E2F may regulate expression of genes at the G1/S boundary. However, the data are not consistent with the hypothesis that association of E2F with tumor suppressor gene products such as p107 or p105Rb-1 is relevant to glucocorticoid regulation of c-myc transcription.
糖皮质激素可抑制源自胸腺的淋巴细胞中原癌基因c-myc的转录。为确定这种效应是否与转录因子E2F特性的改变相关，我们从对照及经糖皮质激素处理的P1798小鼠T淋巴瘤细胞中制备了提取物，并通过凝胶迁移率变动分析评估E2F的大分子状态。对照提取物呈现出两种主要的凝胶迁移率变动条带，其中一个对应“游离”E2F。另一个条带，即复合物C，其特性与含有E2F、p107、细胞周期蛋白A和Cdk2的复合物相似。加入地塞米松后复合物C消失，并被复合物D取代。该复合物的迁移率及其对SV40 T抗原的敏感性表明复合物D对应于E2F-p105Rb-1复合物。对照细胞和经糖皮质激素处理的细胞提取物在c-myc P2启动子上产生相同的DNA酶I保护模式。此外，这些提取物在体外以相同活性转录c-myc P2启动子。加入地塞米松后测定了E2F复合物的相对丰度。随着细胞退出S期，复合物C消失，此时复合物D出现。编码胸苷激酶（Tk-1）和p34cdc2（cdc2）的基因的调控动力学与E2F大分子状态变化所观察到的相似。然而，c-myc表达的调控发生在E2F任何变化之前很久。E2F的大分子状态可能在G1/S边界调控基因表达。然而，这些数据与E2F与肿瘤抑制基因产物如p107或p105Rb-1的结合与糖皮质激素对c-myc转录的调控相关这一假说不一致。
