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The role of endothelial cell on cancer cell metastasis

内皮细胞在癌细胞转移中的作用

基本信息

批准号：
10671096
负责人：
MINAMIYA Yoshihiro
金额：
$ 2.05万
依托单位：
AKITA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671096/
关键词：
Cancer metastasis endothelial cell myosin light chain kinase myosin light chain hepatic growth factor myosin light chain kinase HGF myosin light chair kinase myosin light chair

项目摘要

Chemotaxis is a key step in the process of cancer cell invasion and metastasis. We studied the role of nonmuscle myosin light chain kinase (nm-MLCK) in cancer cell chemotaxis toward hepatocyte growth factor (HGF) using A549 cells, a model cell line derived from lung adenocarcinoma. Our analysis entailed characterizing expression of nm-MLCK using Western blot ; examining chemotaxis toward HGF in Boyden chambers equipped with 8 μm pore polycarbonate membranes ; examining myosin II filament formation by immunolabeling cells with anti-myosin II antiserum ; and examining myosin light chain (MLC) phosphorylation by myosin II immunoprecipitation, followed by immunoblotting with anti-phosphoserine and anti-phosphotheonine antibodies. To assess the dependency on intracellular CaィイD12+ィエD1 and MLCK activity, experiments were performed in presence and absence of the CaィイD12+ィエD1 chelator, BAPTA, or the specific MLCK antagonist, ML-7. A549 cells were found to express a 214kDa nm-MLCK, which in the presence of HGF, phosphorylated MLC on both serine and threonine residues. HGF also elicited myosin II filament formation and chemotaxis which was maximal at 24 h in the presence of 30 ng/ml HGF. All of the aforementioned reactions were inhibited by both BAPTA and ML-7. Thus, it appears that CaィイD12+ィエD1-dependent nm-MLCK activity regulates HGF-induced A549 cell chemotaxis.

趋化性是癌细胞侵袭和转移过程中的关键步骤。我们研究了非肌肉肌球蛋白轻链激酶（nm-MLCK）在癌细胞对肝细胞生长因子（HGF）的趋化性中的作用，使用A549细胞，一种来自肺腺癌的模型细胞系。我们的分析需要使用蛋白质印迹表征nm-MLCK的表达;在配备8 μm孔聚碳酸酯膜的Boyden室中检查对HGF的趋化性;通过用抗肌球蛋白II抗血清免疫标记细胞检查肌球蛋白II细丝形成;通过肌球蛋白II免疫沉淀检查肌球蛋白轻链（MLC）磷酸化，然后用抗磷酸丝氨酸和抗磷酸苏氨酸抗体进行免疫印迹。为了评估对细胞内Ca ~（2+）D12+ Ca ~（2+）D1和MLCK活性的依赖性，在存在和不存在Ca ~（2+）D12+ Ca ~（2+）D1螯合剂BAPTA或特异性MLCK拮抗剂ML-7的情况下进行实验。发现A549细胞表达214 kDa的nm-MLCK，其在HGF存在下磷酸化MLC的丝氨酸和苏氨酸残基。HGF还引起肌球蛋白II丝的形成和趋化性，在30 ng/ml HGF存在下，在24 h时达到最大。所有上述反应均被BAPTA和ML-7抑制。因此，似乎Ca ~（2+）依赖性nm-MLCK活性调节HGF诱导的A549细胞趋化性。