Ji L, Mochon E, Arcinas M, Boxer L M
Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center, Stanford, California 94305, USA.
J Biol Chem. 1996 Sep 13;271(37):22687-91. doi: 10.1074/jbc.271.37.22687.
The translocated and normal bcl-2 alleles in the DHL-4 cell line with the t(14;18) translocation were separated by pulsed field electrophoresis. An in vivo footprint over a cAMP response element (CRE) in the bcl-2 5'-flanking sequence was identified on the translocated allele. Electrophoretic mobility shift assays with the bcl-2 CRE demonstrated complexes with mobilities identical to those with a consensus CRE. UV cross-linking experiments revealed that proteins with molecular masses of 34, 43, and 67 kDa bound to the bcl-2 CRE site. Electrophoretic mobility shift assay with an antibody specific to the phosphorylated cAMP response-binding protein (CREB) demonstrated that phosphorylated CREB was present in DHL-4 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) led to an increase in both the amount of phosphorylated CREB and the bcl-2 promoter activity. The response to PMA was dependent on an intact CRE site. The activity of the bcl-2 promoter was increased 20-fold in a construct with the immunoglobulin heavy chain enhancers, and mutation of the CRE site abolished most of the induction. The addition of PMA increased the activity of the bcl-2-immunoglobulin enhancer construct by 3.5-fold. Access to the CRE site is blocked in the silent normal bcl-2 allele, while CREB proteins bind to the site on the translocated allele. We conclude that the CRE site functions as a positive regulatory site for the translocated bcl-2 allele in t(14;18) lymphomas.
通过脉冲场电泳分离了具有t(14;18)易位的DHL-4细胞系中的易位bcl-2等位基因和正常bcl-2等位基因。在易位等位基因上鉴定出bcl-2 5'侧翼序列中cAMP反应元件（CRE）上的体内足迹。用bcl-2 CRE进行的电泳迁移率变动分析表明，形成的复合物迁移率与共有CRE的复合物相同。紫外线交联实验显示，分子量为34、43和67 kDa的蛋白质与bcl-2 CRE位点结合。用磷酸化cAMP反应结合蛋白（CREB）特异性抗体进行的电泳迁移率变动分析表明，磷酸化CREB存在于DHL-4细胞中。用佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）处理导致磷酸化CREB的量和bcl-2启动子活性均增加。对PMA的反应依赖于完整的CRE位点。在带有免疫球蛋白重链增强子的构建体中，bcl-2启动子活性增加了20倍，CRE位点的突变消除了大部分诱导作用。添加PMA使bcl-2-免疫球蛋白增强子构建体的活性增加了3.5倍。在沉默的正常bcl-2等位基因中，CRE位点的可及性被阻断，而CREB蛋白则与易位等位基因上的该位点结合。我们得出结论，在t(14;18)淋巴瘤中，CRE位点作为易位bcl-2等位基因的正调控位点发挥作用。
