Ma Dongchu, Yu Huiying, Lin Di, Sun Yinghui, Liu Liping, Liu Yage, Dai Bing, Chen Wei, Cao Jianping
Department of Experimental Medicine, Northern Hospital, Shenyang, Liaoning, China.
J Cell Physiol. 2009 Apr;219(1):31-44. doi: 10.1002/jcp.21647.
Studies on polyploidization of megakaryocytes have been hampered by the lack of synchronized polyploid megakaryocytes. In this study, a relatively synchronized polyploid cell model was successfully established by employing Dami cells treated with nocodazole. In nocodazole-induced cells, cyclin B expression oscillated normally as in diploid cells and polyploid megakaryocytes. By using the nocodazole-induced Dami cell model, we found that 4E-BP1 and Thr421/Ser424 of ribosomal S6 kinase 1(S6K1) were phosphorylated mostly at M-phase in cytoplasm and oscillated in nocodazole-induced polyploid Dami cells, concomitant with increased expression of p27 and cyclin D3. However, phosphorylation of 4E-BP1 and S6K1 on Thr421/Ser424 was significantly decreased in differentiated Dami cells induced by phorbol 12-myristate 13-acetate (PMA), concomitant with increased expression of cyclin D1 and p21 and cyclin D3. Overexpression of the kinase dead form of S6K1 containing the mutation Lys 100 --> Gln in PMA-induced Dami cells increased ploidy whereas overexpression of rapamycin-resistant form of S6K1 containing the mutations Thr421 --> Glu and Ser424 --> Asp significantly dephosphorylated 4E-BP1 and reduced expression of cyclin D1, cyclin D3, p21 and p27, and slightly decreased the ploidy of PMA-induced Dami cells, compared with treatment with PMA alone. Moreover, overexpression of rapamycin-resistant form of S6K1 significantly reversed polyploidization of nocodazole-induced Dami cells. Furthermore, MAP (a novel compound synthesized recently) partly blocked the phosphorylation of S6K1 on Thr421/Ser424 and decreased the expression of p27 and polyploidization in nocodazole-induced Dami cells. Taken together, these data suggested that S6K1/4E-BP1 pathway may play an important role in polyploidization of megakaryocytes.
由于缺乏同步化的多倍体巨核细胞，巨核细胞多倍体化的研究受到了阻碍。在本研究中，通过使用经诺考达唑处理的达米细胞成功建立了相对同步化的多倍体细胞模型。在诺考达唑诱导的细胞中，细胞周期蛋白B的表达与二倍体细胞和多倍体巨核细胞一样正常振荡。通过使用诺考达唑诱导的达米细胞模型，我们发现核糖体S6激酶1（S6K1）的4E-BP1和Thr421/Ser424主要在细胞质的M期磷酸化，并且在诺考达唑诱导的多倍体达米细胞中振荡，同时p27和细胞周期蛋白D3的表达增加。然而，在佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的分化达米细胞中，4E-BP1和S6K1在Thr421/Ser424上的磷酸化显著降低，同时细胞周期蛋白D1、p21和细胞周期蛋白D3的表达增加。在PMA诱导的达米细胞中过表达含有K100→Q突变的S6K1激酶失活形式增加了倍性，而在PMA诱导的达米细胞中过表达含有Thr421→Glu和Ser424→Asp突变的雷帕霉素抗性形式的S6K1显著使4E-BP1去磷酸化，并降低了细胞周期蛋白D1、细胞周期蛋白D3、p21和p27的表达，与单独用PMA处理相比，略微降低了PMA诱导的达米细胞的倍性。此外，过表达雷帕霉素抗性形式的S6K1显著逆转了诺考达唑诱导的达米细胞多倍体化。此外，MAP（一种最近合成的新型化合物）部分阻断了S6K1在Thr421/Ser424上的磷酸化，并降低了诺考达唑诱导的达米细胞中p27的表达和多倍体化。综上所述，这些数据表明S6K1/4E-BP1通路可能在巨核细胞多倍体化中起重要作用。
