Kinetics of endomitosis in primary murine megakaryocytes

Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.