Schmidt C M, McKillop I H, Cahill P A, Sitzmann J V
Department of Surgery, Georgetown University Medical Center, Washington, DC, USA.
Eur J Gastroenterol Hepatol. 1999 Dec;11(12):1393-9. doi: 10.1097/00042737-199912000-00009.
We have previously identified that primary human hepatocellular carcinoma (HCC) is associated with altered guanine nucleotide regulatory protein (G-protein) expression concomitant with decreased adenylyl cyclase (AC) and increased mitogen activated protein kinase (MAPK) activity in vivo. This study aims to address the potential link between Gs protein regulation of AC activity/ cyclic adenosine monophosphate (cAMP) production and the subsequent downstream regulation of MAPK activity and mitogenesis.
Pharmacological agents which selectively interact with specific target proteins involved in signal transduction via the Gs-AC-cAMP-MAPK signalling pathway were employed in cultured human HCC cell lines in these studies. These agents allow us to address the role of individual components of these pathways in the regulation of mitogenesis in HCC.
These studies utilized three distinct human HCC cell lines (HepG2, Hep3B and SKHep) in the absence and presence of agents that alter AC-cAMP dependent signalling. De novo DNA synthesis was determined as a marker of altered cellular proliferation, and MAPK activity was determined as the ability to catalyse myelin basic protein (MBP) phosphorylation.
8-Bromo-cAMP (8-Br-cAMP; a cell-permeable cAMP analogue) and forskolin (AC activator) dose-dependently decreased thymidine incorporation in all three cell lines. In addition, serum-stimulated [3H] thymidine incorporation was significantly decreased in HepG2, Hep3B and SKHep cell lines following treatment with either 8-Br-cAMP or forskolin. By contrast, MDL12330A (MDL; irreversible AC inhibitor) enhanced thymidine incorporation in all three cell lines. Treatment with either 8-Br-cAMP or forskolin significantly decreased serum-stimulated MAPK activity.
These data suggest that cAMP acts as an anti-mitogenic agent in these hepatic tumorigenic cell lines in vitro such that inhibition of AC activity promotes MAPK activity and cellular mitogenesis in HCC.
我们之前已经确定，原发性人类肝细胞癌（HCC）与鸟嘌呤核苷酸调节蛋白（G蛋白）表达改变相关，同时体内腺苷酸环化酶（AC）活性降低，丝裂原活化蛋白激酶（MAPK）活性增加。本研究旨在探讨Gs蛋白对AC活性/环磷酸腺苷（cAMP）生成的调节与随后MAPK活性及有丝分裂的下游调节之间的潜在联系。
在这些研究中，使用了与通过Gs-AC-cAMP-MAPK信号通路参与信号转导的特定靶蛋白选择性相互作用的药理试剂，作用于培养的人类肝癌细胞系。这些试剂使我们能够研究这些信号通路中各个组分在肝癌细胞有丝分裂调节中的作用。
这些研究使用了三种不同的人类肝癌细胞系（HepG2、Hep3B和SKHep），在有无改变AC-cAMP依赖性信号转导的试剂存在的情况下进行实验。将从头DNA合成作为细胞增殖改变的标志物进行测定，将MAPK活性作为催化髓鞘碱性蛋白（MBP）磷酸化的能力进行测定。
8-溴-cAMP（8-Br-cAMP；一种可透过细胞的cAMP类似物）和福斯可林（AC激活剂）在所有三种细胞系中均呈剂量依赖性地降低胸苷掺入。此外，用8-Br-cAMP或福斯可林处理后，HepG2、Hep3B和SKHep细胞系中血清刺激的[3H]胸苷掺入显著降低。相比之下，MDL12330A（MDL；不可逆AC抑制剂）增强了所有三种细胞系中的胸苷掺入。用8-Br-cAMP或福斯可林处理显著降低了血清刺激的MAPK活性。
这些数据表明，cAMP在这些体外肝癌细胞系中作为一种抗有丝分裂剂起作用，即抑制AC活性可促进肝癌细胞中的MAPK活性和细胞有丝分裂。
