nm23-H1基因对人肺癌细胞中细胞外信号调节激酶活性的影响

目的探讨肿瘤转移抑制基因nm23-H1对人高转移大细胞肺癌细胞株L9981中细胞外信号调节激酶ERK1/2活性的影响.方法应用特异性识别总ERK1/2(p44/42 MAP kinase)和双磷酸化ERK1/2(phospho-p44/42 MAP kinase)的抗体及蛋白印迹法(Western blot),检测L9981(缺失nm23-H1基因的原代肺癌细胞株)、L9981-nm23-H1(转染了nm23-H1基因的L9981细胞株)、L9981-PLXSN(转染了空载体的L9981细胞株)中总ERK1/2和磷酸化ERK1/2的水平.磷酸化ERK1/2活性应用非放射性免疫沉淀和Western blot法以及p44/42 MAP kinase分析试剂盒予以检测.结果 L9981-nm23-H1细胞株中磷酸化ERK1/2的水平,以及ERK1/2活性均显著低于L9981细胞株和L9981-PLXSN细胞株(P<0.01),L9981和L9981-PLXSN细胞株间磷酸化ERK1/2水平和ERK1/2活性比较均无显著性差异(P＞0.05).三个细胞株间总ERK1/2水平比较均无显著性差异(P>0.05).结论 nm23-H1基因可明显靶向地抑制人高转移肺癌细胞株L9981中ERK1/2的转录表达和ERK1/2的活性.推测nm23-H1基因的作用机制可能与其抑制了MAPK/ERK信号传导通路有关.

Transfection of the tumor metastasis suppressor gene nm23-H1 can targetly suppress the activity of extracellular signal-regulated protein kinase (ERK) in human high-metastasis large cell lung cancer cell line L9981

Objective To investigate the influence of the tumor metastasis suppressor gene nm23 H1 on the activity of extracellular signal regulated protein kinase (ERK) in human high metastasis large cell lung cancer cell line L9981. Methods The levels of total ERK1/2 and phospho pERK1/2 were determined with p44/42 MAP kinase antibody and dually phosphospecific phospho 44/42 MAP kinase antibody in human high metastasis large cell lung cancer cell lines L9981 (cell line with nm23 H1 gene deletion), L9981 nm23 H1 (cell line with nm23 H1 transfected ) and L9981 PLXSN (cell line with vector transfected) by Western blot method, respectively. The activity of phospho ERK1/2 was determined with an ERK1/2 assay kit by immunopreciptation and Western blot analysis. Results The expression levels of phospho ERK1/2 kinase and the activity of phospho ERK1/2 in the lung cancer cell line L9981 nm23 H1 were remarkably higher than those of the L9981 cell line and L9981 PLXSN cell line ( P <0.01), but no significant difference in both the phospho ERK1/2 expression and phospho ERK1/2 activity was observed between the L9981 and L9981 PLXSN cell lines ( P > 0.05). There was no significant difference in the total ERK1/2 level among the three cell lines. Conclusion nm23 H1 gene can obviously targetly suppress the activity of ERK1/2 in human high metastasis large cell lung cancer cell line L9981. This suggest that the mechanisms of nm23 H1 gene as a tumor metastasis suppressor gene may be related to its suppression to the MAPK/ERK signal transduction pathway.