核糖体蛋白S6激酶1(S6K1)介导SP600125诱导的巨核细胞白血病细胞系多倍体化依赖于细胞分化程度

SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation

目的探讨核糖体蛋白S6激酶1(S6K1)在不同分化程度巨核细胞白血病细胞系多倍体化中的调控作用。方法采用c-Jun氨基末端激酶(JNK)抑制剂SP600125诱导Dami、Meg-01和HEL细胞多倍体化,使用蛋白激酶A抑制剂H-89阻断SP600125药物作用,流式细胞术分析表型(CD41a、CD42a和CD42b)和DNA倍性,Western blot法检测S6K1相关蛋白的表达及磷酸化修饰位点的变化。结果 SP600125诱导多倍体化和真核翻译起始因子4E结合蛋白1(4E-BP1)磷酸化。然而,SP600125诱导多倍体化的程度不同,对Dami细胞作用最强、HEL细胞次之、Meg-01细胞最弱。而且SP600125上调Dami细胞的S6K1Thr421/Ser424磷酸化并下调Thr389磷酸化,仅上调HEL细胞的Thr389磷酸化,对Meg-01细胞的S6K1无影响。虽然H-89下调SP600125诱导的3种细胞系中4E-BP1磷酸化,但只部分阻断Dami细胞多倍体化及下调S6K1 Thr421/Ser424磷酸化和上调Thr389磷酸化。尽管H-89上调Meg-01细胞和HEL细胞的S6K1 Thr389磷酸化,但其对Thr421/Ser424磷酸化无影响,而且也不能阻断两者的多倍体化。表型分析显示3种细胞系处于不同的分化水平,即Dami细胞最高、HEL细胞次之、Meg-01细胞最低。结论SP600125诱导巨核细胞白血病细胞系的多倍体化依赖于不同分化程度的细胞系内S6K1对SP600125诱导磷酸化修饰的应答。

Objective To investigate regulatory role of ribosomal protein S6 kinase 1（ S6K1） in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines（ Dami,Meg-01 and HEL cells） were induced towards polyploidization by SP600125,a c-Jun N-terminal kinase（ JNK） inhibitor. The SP600125-inducing process was blocked by H-89,a c AMP-dependent protein kinase（ PKA） inhibitor. The phenotype（ CD41 a,CD42a and CD42b） and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1（ 4E-BP1） in Dami,Meg-01 and HEL cells. However,the effect of SP600125 on polyploidization of the three cell lines was different,with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover,SP600125 increased the phosphorylation of S6K1 Thr421/ Ser424 and decreased the phosphorylation of Thr389 in Dami cel s. However,it only increased the phosphorylation of Thr389 in HEL cel s and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly,H-89 only partially blocked the polyploidization of Dami cells,although it decreased the phosphorylation of4E-BP1 in all SP600125-induced three cell lines. Noticeably,H-89 decreased the phosphorylation of S6K1 Thr421 / Ser424 and increased the phosphorylation of Thr389 in Dami cells. However,H-89 had no effect on the phosphorylation of Thr421 /Ser424,although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage,with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect of SP600125 on phosphorylation of S6K1 in cell lines at the different differentiation stages.