McKillop I H, Vyas N, Schmidt C M, Cahill P A, Sitzmann J V
Department of Surgery, Georgetown University Medical Center, Washington, DC, USA.
Hepatology. 1999 Feb;29(2):412-20. doi: 10.1002/hep.510290218.
Hepatocellular carcinoma (HCC) is associated with increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the effects of chronic ethanol exposure on the expression and function of adenylyl cyclase (AC)-linked G-proteins (Gs and Gi) and growth in experimental HCC. G-protein expression and function was determined by immunoblot in the hepatic tumorigenic H4IIE cell line and isolated cultured hepatocytes in the absence or presence of ethanol (5-100 mmol/L). Chronic exposure (24 hours) to ethanol dose-dependently increased Gialpha1/2 expression in the H4IIE cell line, but not in cultured hepatocytes. Gsalpha-protein expression remained unchanged in both H4IIE cells and cultured hepatocytes following ethanol treatment. In addition, ethanol directly activated a Gi-protein, because pertussis toxin (PTx)-catalyzed, adenosine diphosphate (ADP)-dependent ribosylation of Gialpha substrates decreased following ethanol treatment. The increased functional activity of Gialpha1/2-protein expression was confirmed by demonstrating that ethanol dose-dependently inhibited basal and stimulated AC activity in H4IIE cells, while not significantly altering basal AC activity in isolated cultured hepatocytes. Furthermore, while ethanol had no significant effect on basal mitogenesis in H4IIE cells or hepatocytes, increased mitogenesis caused by direct Gialpha-protein stimulation (mastoparan M7; 10-5,000 nmol/L) was further enhanced in the presence of ethanol, an effect that was completely blocked following Gi-protein inhibition (PTx; 100 ng/mL). In contrast, activation of Gi-proteins using M7 failed to alter cellular mitogenesis in isolated cultured hepatocytes, whether in the absence or presence of ethanol. Finally, analysis of mitogen-activated protein kinase (MAPK) activity demonstrated that chronic ethanol treatment further enhanced Gi-protein-stimulated MAPK activity in hepatic tumorigenic cells. In conclusion, these data demonstrate that ethanol enhances cellular mitogenesis in experimental HCC as a result of, at least in part, a Gi-MAPK-dependent pathway. Furthermore, this effect may be caused by ethanol's direct up-regulation of the expression and activity of Gi-proteins in HCC.
肝细胞癌（HCC）与抑制性鸟嘌呤核苷酸调节蛋白（Gi蛋白）的表达增加及功能增强有关。本研究探讨慢性乙醇暴露对腺苷酸环化酶（AC）相关G蛋白（Gs和Gi）的表达及功能以及实验性HCC生长的影响。通过免疫印迹法在肝致瘤性H4IIE细胞系和分离培养的肝细胞中（有无乙醇（5 - 100 mmol/L）存在的情况下）测定G蛋白的表达及功能。乙醇慢性暴露（24小时）剂量依赖性地增加H4IIE细胞系中Gialpha1/2的表达，但在培养的肝细胞中未增加。乙醇处理后，H4IIE细胞和培养的肝细胞中Gsalpha蛋白表达均保持不变。此外，乙醇直接激活了Gi蛋白，因为乙醇处理后百日咳毒素（PTx）催化的Gialpha底物的腺苷二磷酸（ADP）依赖性核糖基化减少。通过证明乙醇剂量依赖性地抑制H4IIE细胞中的基础AC活性并刺激其活性，同时未显著改变分离培养的肝细胞中的基础AC活性，证实了Gialpha1/2蛋白表达的功能活性增加。此外，虽然乙醇对H4IIE细胞或肝细胞中的基础有丝分裂没有显著影响，但在乙醇存在的情况下，由直接Gialpha蛋白刺激（马斯托帕兰M7；10 - 5000 nmol/L）引起的有丝分裂增加进一步增强，该效应在Gi蛋白抑制（PTx；100 ng/mL）后完全被阻断。相反，无论有无乙醇存在，使用M7激活Gi蛋白均未改变分离培养的肝细胞中的细胞有丝分裂。最后，对丝裂原活化蛋白激酶（MAPK）活性的分析表明，慢性乙醇处理进一步增强了肝致瘤性细胞中Gi蛋白刺激的MAPK活性。总之，这些数据表明，乙醇至少部分通过Gi - MAPK依赖性途径增强实验性HCC中的细胞有丝分裂。此外，这种效应可能是由于乙醇直接上调HCC中Gi蛋白的表达和活性所致。
