甾醇类新药NSC67657诱导的HL60细胞单核系分化中β-catenin/ICAT蛋白差异表达及相互作用研究

Differential expression and interaction of β-catenin/ICAT proteins in NSC67657 induced monocytic differentiation of HL60 cells

目的分析甾醇类新药NSC67657诱导HL60细胞单核系分化中,β-catenin/ICAT蛋白的差异表达及相互作用,探讨NSC67657诱导细胞分化的作用机制。方法应用10μmol·L^(-1)NSC67657诱导HL60细胞分化,采用细胞化学染色和流式细胞技术评估细胞的分化方向和分化程度;通过RT-PCR和Western blot方法验证药物作用细胞前后β-catenin和ICAT基因和蛋白的表达差异;采用免疫共沉淀技术分析β-catenin与ICAT蛋白的相互作用情况;采用激光共聚焦技术协同分析目的蛋白的差异表达及细胞内定位。结果NSC67657可诱导HL60细胞向单核系分化。在10μmol·L^(-1)NSC67657作用5d后,CD14的表达可达到90%以上,细胞化学染色支持细胞单核系分化结论。分化后ICAT基因和蛋白表达明显升高(P<0.01),而β-catenin基因和蛋白表达明显下降(P<0.05)。免疫共沉淀结果显示,ICAT与β-catenin蛋白在细胞分化前后都存在相互作用,药物诱导细胞分化后两者蛋白相互作用条带吸光度明显增加。激光共聚焦结果显示,ICAT蛋白和β-catenin蛋白在胞质和胞核均有荧光,药物诱导HL60细胞分化后,ICAT蛋白胞质和胞核荧光均明显增加,β-catenin蛋白在核内荧光明显减弱,但胞质荧光强度增加,有蛋白胞质转位现象。结论 NSC67657能够诱导HL60细胞向单核系分化,且引起ICAT蛋白和β-catenin蛋白的表达差异;ICAT蛋白与β-catenin蛋白的相互作用增强及β-catenin蛋白从胞核向胞质的转位现象,提示NSC67657可能通过下调并阻止β-catenin蛋白入核,从而导致HL60细胞单核系分化。

Aim To analyze differential expression and interaction of β-catenin/ICAT proteins in HL60 cells when they were induced into monocytic differentiation, and to figure out the mechanism of NSC67657 in cellu-lar induction. Methods HL60 cells were treated by 10 μmol · L-1 NSC67657 , and cellular differentiation could be observed by cytochemical staining and flow cytometry. Then, RT-PCR and Western blot were em-ployed to determine the differential expression of β-catenin/ICAT genes and proteins. Co-immunoprecipi-tation assay was used to confirm the interaction of β-catenin/ICAT proteins, and laser co-focus light mi-croscopy technology was used to co-indentify proteins differential expression and intracellular location. Re-sults HL60 cells could be induced into monocytic dif-ferentiation after 5 days treatment using 10μM NSC67657 . The CD14 ( +)% cells could be up to o-ver 90%, and cytochemical staining reports were con-sistent with this result. The expressions of ICAT gene and protein were up-regulated significantly ( P <0. 01 ) , but the expressions ofβ-catenin gene and pro-tein, on the contrary, were down-regulated(P<0. 05) when HL60 cells were induced into monocytic differen-tiation. From co-immunoprecipitation assay findings, ICAT protein interacted with β-catenin protein, and the absorbance of protein electrophoresis bands in-creased in differentiated cells. From laser co-focus light microscopy assay findings, the fluorescence of ICAT and β-catenin protein could be both observed in cytoplasm and nucleus. In drug treated HL60 cells, the fluorescence of ICAT protein was enhanced both in cytoplasm and nucleus, however, the fluorescence ofβ-catenin protein, which looked like transferring into different organelles, decreased significantly in nucleus, but increased in cytoplasm. Conclusions HL60 cells could be induced into monocytic differentiation by NSC67657 and β-catenin/ICAT proteins differentially expressed during cellular differentiation. The enhanced interaction of β-catenin/ICAT proteins and β-catenin protein transferring from nucleus into cytoplasm indi-cates that NSC67657 probably induces HL60 cells into monocytic differentiation through down-regulating β-catenin protein and blockingβ-catenin protein from nu-cleus.