细胞周期蛋白依赖性激酶抑制蛋白2A(CDKN2A/p16^(INK4a))在MCF7细胞中的可诱导表达并引起F-actin的重分布

Subcellular re-distribution of F-actin in MCF7 cells upon inducible expression of CDKN2A/p16^(INK4a)

目的使用四环素操纵子(Tet-on)可控表达系统研究细胞周期蛋白依赖性激酶抑制蛋白2A(CDKN2A/p16^(INK4a))对MCF7乳腺癌细胞形态及细胞骨架的影响。方法以p16^(INK4a)表达缺失的MCF7乳腺癌细胞为宿主细胞,用慢病毒通过"两步感染法"建立MCF7-p Tet-on-p16^(INK4a)可诱导表达细胞系,加入诱导剂强力霉素(Dox)诱导目的基因表达;细胞计数检测细胞生长变化;采用免疫细胞化学染色检测上皮钙黏素(E-cadherin)表达,采用鬼笔环肽(phalloidin)标记纤维型肌动蛋白(F-actin),观察p16^(INK4a)对细胞间黏附连接、细胞骨架的影响;用显微镜对细胞拍照后,用Image J软件进行形态分析。结果成功建立了可诱导表达p16^(INK4a)的MCF7稳定细胞系,Dox可以有效诱导p16^(INK4a)的表达;诱导条件下稳定表达p16^(INK4a)的MCF7细胞增殖受抑制,细胞体积增大,轮廓线延长;而对照MCF7细胞F-actin主要定位于胞质,p16^(INK4a)表达的细胞内F-actin向细胞边缘分布增加。结论 p16^(INK4a)在MCF7细胞中的表达可以导致细胞体积增大、细胞间黏附连接弱化、F-actin向细胞外周重新分布。

Objective To investigate the effect of CDKN2A/p16INK4a ,a cyclin-dependent kinase inhibitor, on cell morphology and cytoskeleton in MCF7 breast cancer cells by tetracycline operon （Tet-on） inducible gene expression system. Methods MCF7 cells, genetically null for p16INK4a expression, were sequentially infected with two viruses of Tet-on systems to make MCF7-pTet-on-p16INK4a cells, which were induced to express p16INK4a by doxycycline （Dox） treatment. Cell growth was evaluated by cell counting. To observe the effect of p16INK4a on cell adhesion and cytoskeleton, intercellular adhesion and intracellular F-actin were examined by fluorescent staining with anti-E-cadherin antibody and phalloidin, respectively. Changes in cell morphology were analyzed by ImageJ software after microscopic imaging. Results Cell line expressing p16INK4a upon Dox induction was successfully made in MCF7 breast cancer cells. The inducible expression of p16INK4ainhibited cell proliferation while significantly increasing cell size as marked by prolonged contour lines. Furthermore, intracellular F-actin were endched at the cortical peripheries of p16INK4a-expressing cells, which was different from those in control MCF7 cells where F-actin were enriched in cytoplasmic regions. Conclusion p16INK4a expression in MCF7 breast cancer cells leads to increased cell size, weakened intercellular adhesion and cortical re-distribution of cytoskeletal F-actin.