McKillop I H, Schmidt C M, Cahill P A, Sitzmann J V
Department of Surgery, Georgetown University Medical Center, Washington, DC 20007, USA.
Eur J Gastroenterol Hepatol. 1999 Jul;11(7):761-8. doi: 10.1097/00042737-199907000-00014.
Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC.
Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC.
These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling.
Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx.
These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.
肝细胞癌（HCC）与抑制性鸟嘌呤核苷酸调节蛋白（Gi蛋白）的表达和功能改变有关。本研究探讨在HCC实验模型中，Gi蛋白与丝裂原活化蛋白激酶（MAPK）级联反应的细胞外调节激酶（ERK）组分之间在调节有丝分裂发生过程中的相互作用。
最近已获得能通过Gi-MAPK途径与信号转导中特定靶蛋白选择性相互作用的药物制剂。这些药物制剂与科学检测方法相结合，使我们能够研究该级联反应各个组分在HCC有丝分裂发生调节中的作用。
这些研究使用大鼠肝癌细胞（H4IIE）和体外分离培养的肝细胞，并结合与细胞内信号传导的Gi蛋白或MAPK组分相互作用的药物制剂进行。
用马斯托帕兰M7（M7）直接激活Gi蛋白，以剂量依赖方式（10 - 1000 nM，n = 4，P < 0.05）显著增加肝癌性H4IIE细胞中的核胸苷掺入，百日咳毒素（PTx）或选择性丝裂原活化ERK调节激酶（MEK）抑制剂PD098059处理可消除该效应。相反，M7抑制血清刺激的分离肝细胞中的核胸苷掺入。然后将ERK2活性确定为免疫沉淀的ERK2蛋白磷酸化ERK底物髓鞘碱性蛋白的能力。这些研究表明，用M7激活Gi蛋白后，H4IIE细胞中ERK2活性呈时间和剂量依赖性增加，在20分钟时达到最大反应。相反，在所检测的任何剂量或时间点（10 - 5000 nM，10 - 120分钟），M7均未显著改变分离培养的肝细胞中的ERK2活性。用PTx处理后，Gi刺激的ERK激活在致瘤细胞中被完全阻断。
这些数据首次证明实验性HCC中存在Gi连接的MAPK级联反应，其激活可刺激细胞有丝分裂发生。
