Stang Stacey L, Lopez-Campistrous Ana, Song Xiaohua, Dower Nancy A, Blumberg Peter M, Wender Paul A, Stone James C
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Exp Hematol. 2009 Jan;37(1):122-134. doi: 10.1016/j.exphem.2008.09.008.
Bryostatin-1 and related diacylglycerol (DAG) analogues activate RasGRPs in lymphocytes, thereby activating Ras and mimicking some aspects of immune receptor signaling. To define the role of RasGRPs in lymphocyte apoptosis and to identify potential therapeutic uses for DAG analogues in lymphocyte disorders, we characterized the response of lymphoma-derived cell lines to DAG analogues.
Human lymphoma-derived B cell lines and mouse primary B cells were treated with bryostatin-1 or its synthetic analogue "pico." Ras signaling partners and Bcl-2 family members were studied with biochemical assays. Cellular responses were monitored using growth and apoptosis assays.
Stimulation of B cells with DAG analogues results in activation of protein kinase C/RasGRP-Ras-Raf-Mek-Erk signaling and phosphorylation of the proapoptotic BH3-only protein Bim. In vitro, Bim is phosphorylated by Erk on sites previously associated with increased apoptotic activity. In Toledo B cells derived from a non-Hodgkin's lymphoma (B-NHL), DAG analogue stimulation leads to extensive apoptosis. Apoptosis can be suppressed by either downregulation of Bim or overexpression of Bcl-2. It is associated with the formation of Bak-Bax complexes and increased mitochondrial membrane permeability. Toledo B-NHL cell apoptosis shows a striking dependence on sustained signaling.
In B cells, Erk activation leads directly to phosphorylation of Bim on sites associated with activation of Bim. In Toledo B-NHL cells, the dependence of apoptosis on sustained signaling suggests that Bcl-2 family members could interpret signal duration, an important determinant of B cell receptor-mediated negative selection. Certain cases of B-NHL might respond to DAG analogue treatment by the mechanism outlined here.
苔藓抑素-1及相关二酰基甘油（DAG）类似物可激活淋巴细胞中的RasGRP，从而激活Ras并模拟免疫受体信号传导的某些方面。为了确定RasGRP在淋巴细胞凋亡中的作用，并确定DAG类似物在淋巴细胞疾病中的潜在治疗用途，我们对淋巴瘤来源的细胞系对DAG类似物的反应进行了表征。
用人淋巴瘤来源的B细胞系和小鼠原代B细胞用苔藓抑素-1或其合成类似物“皮可”处理。用生化分析研究Ras信号传导伙伴和Bcl-2家族成员。使用生长和凋亡分析监测细胞反应。
用DAG类似物刺激B细胞会导致蛋白激酶C/RasGRP-Ras-Raf-Mek-Erk信号传导激活以及仅含BH3结构域的促凋亡蛋白Bim磷酸化。在体外，Bim在先前与凋亡活性增加相关的位点被Erk磷酸化。在源自非霍奇金淋巴瘤（B-NHL）的托莱多B细胞中，DAG类似物刺激导致广泛凋亡。通过下调Bim或过表达Bcl-2可抑制凋亡。它与Bak-Bax复合物的形成和线粒体膜通透性增加有关。托莱多B-NHL细胞凋亡显示出对持续信号传导的显著依赖性。
在B细胞中，Erk激活直接导致Bim在与Bim激活相关的位点磷酸化。在托莱多B-NHL细胞中，凋亡对持续信号传导的依赖性表明Bcl-2家族成员可以解读信号持续时间，这是B细胞受体介导的阴性选择的重要决定因素。某些B-NHL病例可能通过此处概述的机制对DAG类似物治疗产生反应。
