Zhang Y, Wang Z, Ravid K
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 1996 Feb 23;271(8):4266-72. doi: 10.1074/jbc.271.8.4266.
The platelet precursor, the megakaryocyte, matures to a polyploid cell as a result of DNA replication in the absence of mitosis (endomitosis). The factors controlling endomitosis are accessible to analysis in our megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocytes of transgenic mice. We aimed to define whether endomitosis consists of a continuous phase of DNA synthesis (S) or of S phases interrupted by gaps. Analysis of the cell cycle in MegT cells revealed that, upon inactivation of large T antigen, the cells shifted from a mitotic cell cycle to an endomitotic cell cycle consisting of S/Gap phases. The level of the G1/S cyclin, cyclin A, as well as of the G1 phase cyclin, cyclin D3, were elevated at the onset of DNA synthesis, either in MegT cells undergoing a mitotic cell cycle or during endomitosis. In contrast, the level of the mitotic cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected during the mitotic cell cycle or during endomitosis; however, cyclin B1-dependent Cdc2 kinase activity was largely abolished in the polyploid cells. Fibroblasts immortalized with the same heat-labile oncogene do not display reduced levels of cyclin B1 upon shifting to high temperature nor do they become polyploid, indicating that reduced levels of cyclin B1 is a property of megakaryocytes and not of the T-antigen mutant. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1.
血小板前体巨核细胞由于在无有丝分裂情况下进行DNA复制（核内有丝分裂）而成熟为多倍体细胞。通过将温度敏感型猿猴病毒40大T抗原靶向表达于转基因小鼠的巨核细胞而产生的巨核细胞系MegT，为分析控制核内有丝分裂的因素提供了便利。我们旨在确定核内有丝分裂是由连续的DNA合成（S）期组成，还是由被间隙打断的S期组成。对MegT细胞的细胞周期分析表明，大T抗原失活后，细胞从有丝分裂细胞周期转变为由S/间隙期组成的核内有丝分裂细胞周期。无论是处于有丝分裂细胞周期的MegT细胞，还是在核内有丝分裂期间，G1/S期细胞周期蛋白cyclin A以及G1期细胞周期蛋白cyclin D3的水平在DNA合成开始时均升高。相比之下，有丝分裂细胞周期蛋白cyclin B1的水平在显示有丝分裂细胞周期的细胞中呈周期性变化，而在核内有丝分裂期间无法检测到。在有丝分裂细胞周期或核内有丝分裂期间检测到相当水平的有丝分裂激酶蛋白Cdc2；然而，在多倍体细胞中，cyclin B1依赖性Cdc2激酶活性基本被消除。用相同热不稳定癌基因永生化的成纤维细胞在转移到高温时，cyclin B1水平不会降低，也不会变成多倍体，这表明cyclin B1水平降低是巨核细胞的特性，而非T抗原突变体的特性。我们得出结论，巨核细胞核内复制过程中的细胞编程与cyclin B1水平降低有关。
