Wilhide C C, Van Dang C, Dipersio J, Kenedy A A, Bray P F
Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Blood. 1995 Jul 1;86(1):294-304.
The maturation of megakaryocytes in vivo requires polyploidization or repeated duplication of DNA without cytokinesis. As DNA replication and cytokinesis are tightly regulated in somatic cells by cyclins and cyclin-dependent kinases, we sought to determine the pattern of cyclin gene expression in cells that undergo megakaryocytic differentiation and polyploidization. The Dami megakaryocytic cell line differentiates and increases ploidy in response to phorbol 12-myristate 13-acetate (PMA) stimulation in vitro. We used Northern blotting to analyze mRNA levels of cyclins A, B, C, D1, and E in PMA-induced Dami cells and found that cyclin D1 mRNA levels increased dramatically (18-fold). Similar increases in cyclin D1 mRNA were obtained for other cell lines (HEL and K562) with megakaryocytic properties, but not in HeLa cells. The increase in cyclin D1 was confirmed by Western immunoblotting of PMA-treated Dami cells. This finding suggested that cyclin D1 might participate in megakaryocyte differentiation by promoting endomitosis and/or inhibiting cell division. To address these possibilities, we constructed two stable Zn+2-inducible, cyclin D1-overexpressing Dami cell lines. Cyclin D1 expression alone was not sufficient to induce polyploidy, but in conjunction with PMA-induced differentiation, polyploidization was slightly enhanced. However, unlike other cell systems, cyclin D1 overexpression caused cessation of cell growth. Although the mechanism by which cyclin D1 may affect megakaryocyte differentiation is not clear, these data demonstrate that cyclin D1 is upregulated in differentiating megakaryocytic cells and may contribute to differentiation by arresting cell proliferation.
巨核细胞在体内的成熟需要多倍体化，即DNA在没有胞质分裂的情况下重复复制。由于体细胞中DNA复制和胞质分裂受细胞周期蛋白和细胞周期蛋白依赖性激酶的严格调控，我们试图确定经历巨核细胞分化和多倍体化的细胞中细胞周期蛋白基因的表达模式。Dami巨核细胞系在体外受佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）刺激后会发生分化并增加倍性。我们用Northern印迹法分析了PMA诱导的Dami细胞中细胞周期蛋白A、B、C、D1和E的mRNA水平，发现细胞周期蛋白D1的mRNA水平显著增加（18倍）。具有巨核细胞特性的其他细胞系（HEL和K562）也有类似的细胞周期蛋白D1 mRNA增加情况，但HeLa细胞中没有。通过对PMA处理的Dami细胞进行Western免疫印迹证实了细胞周期蛋白D1的增加。这一发现表明，细胞周期蛋白D1可能通过促进核内有丝分裂和/或抑制细胞分裂参与巨核细胞分化。为了研究这些可能性，我们构建了两个稳定的锌离子诱导型、过表达细胞周期蛋白D1的Dami细胞系。单独的细胞周期蛋白D1表达不足以诱导多倍体化，但与PMA诱导的分化相结合时，多倍体化略有增强。然而，与其他细胞系统不同，细胞周期蛋白D1过表达导致细胞生长停止。尽管细胞周期蛋白D1影响巨核细胞分化的机制尚不清楚，但这些数据表明，细胞周期蛋白D1在分化的巨核细胞中上调，可能通过阻止细胞增殖促进分化。
