Objective: Polyploidy is an important mechanism of speciation, which determines the cell size and function of some critical organs. Polyploidization also accompanies some pathological conditions such as oncogenesis. By using a relatively synchronized polyploid cell model, we have confirmed that Mt0r/S6Kl pathway may play an important role in polyploidization of megakaryocytes. This study focused on whether 4E-BP1, as another important downstream signals of Mtor, also involved in polyploidization of megakaryocytes. Methods: Nocodazole-induced Dami cell model was a relatively synchronized polyploid cell model. The expression and phosphorylation of Mtor/4E-BPl pathway proteins was detected by western-blot. Double-labeling techniques were used to investigate in which of the phase of the polyploid cell cycle 4E-BP1 at Thr37/46 and Ser65 are phosphorylated. Results: Nocodazole induced a relatively synchronized polyploidization in Dami cells. In diploid cells and polyploid megakaryocytes, the expression of Mtor and the phosphorylation of Mtor at Ser2448 increased when Dami cells begin to progress from Gl to S-phase in cell cycle. Analysis of flow cytometry showed that phosphorylation of 4E-BP1 at Thr37/46 and Ser65 increased at G2/M-phase. Conclusion: Mtor/4E-BPl pathway is involved in polyploid cell cycle control.%目的:多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程(如恶性肿瘤)的发生有密切关系.我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR/S6K1参与多倍体细胞周期的调控.本课题主要研究mTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控.方法:诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR/4E-BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E-BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系.结果:诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E-BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2/M期.结论:mTOR/4E-BP1通路参与多倍体细胞周期的调控.
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