Han Shujie, Knoepp Stewart M, Hallman Mark A, Meier Kathryn E
Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164-6534, USA.
Mol Pharmacol. 2007 Jan;71(1):314-22. doi: 10.1124/mol.106.028639. Epub 2006 Oct 25.
The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.
小鼠EL4淋巴瘤细胞系存在对肿瘤启动子佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）敏感或耐药的变体。在敏感的EL4细胞中，PMA可导致强劲的细胞外信号调节激酶（Erk）丝裂原活化蛋白激酶激活，从而导致生长停滞。在耐药细胞中，PMA诱导的Erk激活极少，且不会导致生长停滞。PMA可刺激敏感细胞而非耐药细胞产生白细胞介素-2（IL-2）。研究了RasGRP1（一种PMA激活的Ras鸟嘌呤核苷酸交换因子）在EL4细胞表型中的作用。内源性RasGRP1蛋白在敏感细胞中的表达水平远高于耐药细胞。在敏感细胞中可观察到PMA诱导的Ras激活，但在缺乏Ras - GRP1的耐药细胞中则未观察到。PMA可诱导敏感细胞中RasGRP1蛋白的下调，但会使耐药细胞中的RasGRP1增加。将RasGRP1转染到耐药细胞中可增强PMA诱导的Erk激活。在反向实验中，引入针对RasGRP1的小干扰RNA（siRNA）可抑制敏感细胞中PMA诱导的Ras和Erk激活。用针对RasGRP1的siRNA孵育的敏感细胞表现出PMA耐药表型，即它们能够在PMA存在的情况下增殖，并且在受到PMA刺激时不分泌IL-2。这些研究表明，如先前为EL4细胞系所定义的PMA敏感表型是由RasGRP1蛋白的内源性表达赋予的。
