Constitutive activation of c-Met is correlated with c-Met overexpression and dependent on cell-matrix adhesion in lung adenocarcinoma cell lines
Constitutive activation of c-Met is correlated with c-Met overexpression and dependent on cell-matrix adhesion in lung adenocarcinoma cell lines
复制标题
DOI：
10.1111/j.1349-7006.2007.00640.x
复制
发表时间：
2008-01-01
期刊：
影响因子：
5.7
通讯作者：
Niki, Toshiro
中科院分区：
文献类型：
--
作者：
Nakamura, Yu;Matsubara, Daisuke;Niki, Toshiro
本研究探讨了c-Met在肺腺癌细胞系中的组成性激活机制。首先，我们通过Western印迹分析检测了一组肺腺癌细胞系中c-Met和磷酸化c-Met（Y1234/Y1235）的水平。在14个细胞系中的12个中发现c-Met表达，并且注意到c-Met和磷酸化c-Met的表达之间的总体相关性。c-Met在五种细胞系（PC 3、LC-2/ad、L27、H1648和H2009）中被组成性激活，尤其是在高水平下。通过单核苷酸多态性阵列分析，在L27和H1648中鉴定出c-Met扩增，但在Sema结构域或c-Met胞质结构域的任何部分均未鉴定出突变。用中和抗肝细胞生长因子（HGF）抗体、Madin-Darby犬肾细胞散射试验和细胞系条件培养基上的Western印迹实验表明，c-Met的组成性磷酸化在很大程度上不依赖于配体。细胞-基质粘附的抑制诱导c-Met的去磷酸化在测试的五个细胞系。这是伴随着下调c-Met在三个五个细胞系。相反，通过中和E-钙粘蛋白抗体抑制细胞-细胞粘附对c-Met的表达和磷酸化的影响最小。这些结果揭示了我们的肺腺癌细胞系组中c-Met组成性激活的三个特征：（i）它与c-Met过表达相关，无论是否有基因扩增;（ii）它在很大程度上不依赖于配体;（iii）它依赖于细胞-基质粘附。
In this study we explored the mechanisms of constitutive activation of c-Met in lung adenocarcinoma cell lines. First, we examined levels of c-Met and phospho-c-Met (Y1234/Y1235) in a panel of lung adenocarcinoma cell lines by Western blot analysis. c-Met expression was found in 12 of 14 cell lines and an overall correlation between the expressions of c-Met and phospho-c-Met was noted. c-Met was constitutively activated particularly at high levels in five cell lines (PC3, LC-2/ad, L27, H1648, and H2009). c-Met amplification was identified in L27 and H1648 by single nucleotide polymorphism array analysis, but no mutations were identified in the Sema domain or in any part of the cytoplasmic domain of c-Met. Experiments with neutralizing anti-hepatocyte growth factor (HGF) antibody, scatter assay using Madin-Darby canine kidney cells, and Western blotting on conditioned media of the cell lines revealed that the constitutive phosphorylation of c-Met was largely ligand-independent. The inhibition of cell-matrix adhesion induced the dephosphorylation of c-Met in the five cell lines tested. This was accompanied by downregulation of c-Met in three of the five cell lines. In contrast, the inhibition of cell-cell adhesion by neutralizing E-cadherin antibody had a minimal effect on the expression and phosphorylation of c-Met. These results reveal three features of the constitutive activation of c-Met in our panel of lung adenocarcinoma cell lines: (i) it correlates with c-Met overexpression, either with or without gene amplification; (ii) it is largely ligand-independent; and (iii) it depends on cell-matrix adhesion.