Wang Lili, Yang Jingang, Li Changling, Xing Sining, Yu Ying, Liu Shuo, Zhao Song, Ma Dongchu
Department of Experimental Medicine, Northern Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Department of Experimental Medicine, Northern Hospital, Shenyang Military Area Command, Shenyang 110016, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Oct;32(10):1336-1341.
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 cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, 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 cells. However, it only increased the phosphorylation of Thr389 in HEL cells 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 of 4E-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.
目的 探讨核糖体蛋白S6激酶1（S6K1）在不同分化阶段的不同巨核细胞白血病细胞系多倍体化中的调控作用。方法 用c-Jun氨基末端激酶（JNK）抑制剂SP600125诱导巨核细胞白血病细胞系（Dami、Meg-01和HEL细胞）向多倍体化发展。用环磷酸腺苷依赖性蛋白激酶（PKA）抑制剂H-89阻断SP600125的诱导过程。通过流式细胞术检测细胞表型（CD41a、CD42a和CD42b）和DNA倍性。通过蛋白质免疫印迹法检测S6K1及相关蛋白的表达和磷酸化情况。结果 SP600125诱导Dami、Meg-01和HEL细胞多倍体化，并增加真核起始因子4E结合蛋白1（4E-BP1）的磷酸化。然而，SP600125对这三种细胞系多倍体化的影响不同，对Dami细胞的影响最强，对Meg-01细胞的影响最弱。此外，SP600125增加Dami细胞中S6K1 Thr421/Ser424位点的磷酸化，降低Thr389位点的磷酸化。然而，它仅增加HEL细胞中Thr389位点的磷酸化，对Meg-01细胞中S6K1的磷酸化无影响。有趣的是，H-89仅部分阻断Dami细胞的多倍体化，尽管它降低了所有SP600125诱导的三种细胞系中4E-BP1的磷酸化。值得注意的是，H-89降低Dami细胞中S6K1 Thr421/Ser424位点的磷酸化，增加Thr389位点的磷酸化。然而，H-89对Thr421/Ser424位点的磷酸化无影响，尽管它增加了Meg-01和HEL细胞中Thr389位点的磷酸化。表型分析表明，这三种细胞系在巨核细胞谱系中的分化水平不同，Dami细胞的分化程度最高，Meg-01细胞的分化程度最低。结论 SP600125诱导的巨核细胞白血病细胞系多倍体化取决于SP600125在不同分化阶段对细胞系中S6K1磷酸化的影响。
