THE CELL-CYCLE IN POLYPLOID MEGAKARYOCYTES IS ASSOCIATED WITH REDUCEDACTIVITY OF CYCLIN B1-DEPENDENT CDC2 KINASE

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 o ur megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocyt es 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 cel l cycle to an endomitotic cell cycle consisting of S/Gap phases. The l evel of the G(1)/S cyclin, cyclin A, as well as of the G, phase cyclin , cyclin D3, were elevated at the onset of DNA synthesis, either in Me gT cells undergoing a mitotic cell cycle or during endomitosis. In con trast, the level of the mitotic cyclin, cyclin B1, cycled in cells dis playing a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected d uring 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 tempera ture nor do they become polyploid, indicating that reduced levels of c yclin B1 is a property of megakaryocytes and not of the T-antigen muta nt. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1.