FAK的活化促进悬浮肺肿瘤细胞的积聚、凋亡抑制与增殖

Activation of FAK Promotes Forming of Lung Carcinoma Cell Aggregation Which Inhibits Cell Apoptosis and Enhances Cell Proliferation in Suspension

【目的】研究在悬浮状态下细胞聚集与细胞凋亡和增殖的关系,以及参与细胞聚集的分子信号传导?【方法】 镜下观察不同肺上皮细胞悬浮状态聚集作用的改变;DNA琼脂糖凝胶电泳检测细胞凋亡;用MTT和台盼蓝细胞排斥实验检测细胞增殖;磷酸化蛋白的检测用免疫沉淀和Western blot; RNA interference沉默实验采用逆转录病毒载体感染的稳定表达细胞株?【结果】 肿瘤细胞在 polyHEMA悬浮状态下脱离细胞外基质不仅能够存活,而且不同细胞形成大小不同的聚集体:人类大细胞肺癌H460和人肺腺癌H1792细胞株的细胞聚集体大而紧密;人肺腺癌A549和SK-LU-1细胞聚集体小而松散;正常狗肾上皮细胞MDCK几乎不形成聚集体,这与它们的恶性程度来源有关?在悬浮状态下正常细胞MDCK发生凋亡? 细胞聚集体间的相互作用能够促进悬浮状态下肿瘤细胞的增殖?磷酸化FAK的水平和聚集体的大小有关,沉默FAK蛋白的表达能够部分地阻断肿瘤细胞聚集体的形成?【结论】肿瘤细胞脱离细胞外基质形成的聚集能力是判断肿瘤恶性程度和转移特性潜在的标志物?聚集体细胞间的相互作用能够抑制细胞凋亡和促进细胞增殖?聚集体的形成与FAK磷酸化有关?

Objective To investigate the role of cell aggregation in cell apoptosis and prolifera-tion on polyHEMA suspension culture, and the signal transduction involved in the cell aggregation. Methods Morphology changes of cells on polyHEMA culture were observed under microscope by aggrega-tion assay. Apoptosis was detected by DNA extraction and electrophoresis. Cell proliferation was mea-sured by MTT assay and trypan blue excluding experiment. Phosphorylated protein was detected by im-munoprecipitation and Western blot. Retrovirus vector mediated RNA interference stable cells were used in RNA interference experiments. Results We found that not only lung tumor cells can survive in polyHEMA suspension culture but also different kinds of lung tumor cells can form different size of aggregation: The aggregates of human lung adenocarcinoma NCI-1792 and human large cell lung can-cer H460 cells were large and compact spheroids; human lung adenocarcinoma A549 and SK-LU-1 cell aggregates were small and loose; Madin-Darby canine kidney epithelial (MDCK) cells formed no aggregates or very small and loose aggregates. This was consistent with their tumorigenicity. Disrup-tion of cell-extracellular matrix adhesion in suspension culture induced apoptosis only in normal epithe-lial cells but not in tumor cells. Level of phosphorylated FAK protein was related to sizes of cell aggrega-tion in polyHEMA dishes (regular suspension) and methylcellulose dishes (methyl-cellulose suspen-sion). Knockdown of FAK protein expression by RNA interference can partially block the forming of cell aggregation. Conclusions Cell aggregation in suspension culture can be a potential marker of tumori-genicity and it can prevent tumor cells from apoptosis and facilitate tumor cell proliferation. The form-ing of cell aggregation related with the phosphorylated FAK protein.