肝癌细胞生物学行为改变涉及p120ctn酪氨酸磷酸化的研究

目的 以表皮生长因子刺激肝癌细胞内p120^ctn酪氨酸磷酸化,观察p120^ctn酪氨酸磷酸化与p120^ctn在细胞内转位及肝癌细胞生物学行为的关系,探讨p120^ctn在肝癌细胞粘附和信号转导中的作用。方法 使用免疫沉淀和免疫印迹法检测表皮生长因子引起的p120^ctn酪氨酸磷酸化改变情况,用间接免疫荧光法观察了表皮生长因子引起的p120^ctn在BEL—7404人肝癌细胞内的分布、转位。用相应的方法检测表皮生长因子促使BEL—7404人肝癌细胞的细胞形态和细胞粘附能力的改变情况。人全长p120^ctn反义核苷酸转染细胞,观察表皮生长因子促使的p120^ctn磷酸化改变情况是否受到明显抑制。结果表皮生长因子可使BEL—7404人肝癌细胞内的p120^ctn酪氨酸发生磷酸化,以表皮生长因子作用20min时最明显;在细胞内转染人全长p120^ctn反义核苷酸后,表皮生长因子对p120^ctn酪氨酸磷酸化的上调作用明显减弱。表皮生长因子刺激下,磷酸化的p120^ctn发生了细胞内转位,表现为p120^ctn细胞膜的表达明显减少,核表达增加;同时β—连环蛋白也发生相似的改变;表皮生长因子刺激下,BEL—7404人肝癌细胞的粘附能力明显下降;细胞形态变为细长和不规则,伪足增多。结论 在表皮生长因子刺激下BEL—7404中p120^ctn酪氨酸磷酸化增加,p120^ctn发生从细胞膜到核内的转位,并同表皮生长因子引起的细胞形态和细胞粘附能力的改变密切相关。结果 提示p120^ctn酪氨酸磷酸化与p120^ctn在细胞内的转位和细胞的生物学行为密切相关。

p120(ctn) tyrosine phosphorylation are involved in the biologic behavior changes of hepatocellular carcinoma cells

OBJECTIVE: In hepatocellular carcinoma cells, the tyrosine phosphorylation of p120(ctn) was stimulated by epidermal growth factor (EGF) to investigate the relationship between the tyrosine phosphorylation of p120(ctn) and the translocation of p120(ctn), also the relationship between the tyrosine phosphorylation of p120(ctn) and the biological behaviour of hepatocellular carcinoma cells. The role of p120(ctn) in the cell adhesion and signaling of hepatocellular carcinoma is to be investigated. METHODS: In BEL-7404 human hepatocellular carcinoma cells, the tyrosine phosphotyrosine of p120(ctn) stimulated by EGF were detected by immunoprecipitation (IP) and Immunoblotting (IB). The cellular distribution and translocation of p120(ctn) and beta-catenin were detected and examined by indirect intracellular immunofluorescence. Cell morphology and cell adhesion potential were also detected using correspondent methods. Antisense nucleotide of p120(ctn) was transfected into BEL-7404 cells. RESULTS: The tyrosine phosphorylation of p120(ctn) was enhanced after EGF treatment than control, especially at 20min after EGF treatment; When BEL-7404 cells were transfected with antiseuse nucleotide of p120(ctn) before EGF treatment, the tyrosine phosphorylation of p120(ctn) stimulated by EGF was obviously lowered. We also observed that the tyrosine phosphorylation of p120(ctn) stimulated by EGF was accompanied by the nuclear translocation of p120(ctn); the similar translocation was also observed in beta-catenin after EGF stimulation. At the meantime, cell adhesion potential was reduced after EGF treatment and cell morphology became thin, elongated and irregular, speudopods increased. CONCLUSIONS: In BEL-7404 cells,the tyrosine phosphorylation of p120(ctn) could be stimulated by EGF, which was accompanied by the nuclear accumulation of p120(ctn). The tyrosine phosphorylation of p120(ctn) stimulated by EGF was also in correlation with the changes of cell adhesion and cell morphology. The results indicated that the tyrosine phosphorylation of p120(ctn) cell correlated with the translocation of p120(ctn) and the biological behavior of cells.