Resumen de Unconventional cell cycles in polyploidization: the megakaryocyte paradigm
Polyploidization is a natural process by which specific cell types increase ploidy by replicating DNA in the absence of chromosome segregation. This process is part of the normal development of specific tissues in plants and animals, and altered ploidy is also observed as an accompanying feature of malignant transformation in cancer. Megakaryocytes normally poplyploidize to become mature giant cells able to generate platelets However, the molecular basis of polyploidization in these cells is mostly unknown. In the present study we have analyzed the relevance of well-established cell cycle regulators in the process of megakaryopoiesis and more specific in the particular polyploidization events that take place during the development and maturation of these platelet-producing cells. Mastl, a kinase regulating phosphatase complexes during mitosis, was initially found in humans as a gene mutated in human thrombocytopenia. By generating knockin mice carrying the corresponding mutation, we describe here that this protein is required to attenuate PP2A-B55 activity and to modulate MAPK signaling and proplatelet formation during the development of megakaryocytes. To further understand the control of megakaryocyte maturation and platelet formation by cell cycle regulators, we specifically ablated several cell cycle regulators involved in mitotic entry, progression and exit, using a megakaryocyte-specific Cre model in the mouse. Using these models, we demonstrate here that unperturbed megakaryocytes undergo endomitosis in vivo, a process in which cells replicate the DNA but chromosomes are not segregated due to incomplete mitosis. The Anaphase-promoting Complex (APC/C)- Cdc20 is necessary for endomitotic exit since Cdc20-null megakaryocytes were arrested in mitosis leading to severe thrombocytopenia. However, APC/C is dispensable for platelet production when activated by the Cdc20-related cofactor Cdh1, a protein required for endoreplication in other systems. Strikingly, ablation of Cdk1, a cell cycle kinase essential for mitosis in all cells analyzed, does not lead to thrombocytopenia development. The reason behind this observation is the ability of Cdk1-null megakaryocytes to endoreplicate as an alternative cell cycle. Mature megakaryocytes arising from endoreplicative cell cycles were able to shed platelets upon TPO stimulation in vitro, even though this procedure was carried out less efficiently. Further ablation of Cdk2 kinase in Cdk1-null megakaryocytes resulted in re-replication events. Unexpectedly, megakaryocytes were able to undergo functional polyploidization using re-replication and Cdk1; Cdk2 double mutant mice displayed normal differentiation of megakaryocytes in the absence of thrombocytopenia. Interestingly, genetic ablation of Cdk1 and Cdk2 rescued platelet defects in Cdc20-deficient mice indicating that endoreplication and re-replication can functional replace endomitosis in vivo. Altogether, this analysis suggests the presence of multiple endocycles that can be alternatively used during functional polyploidization. These data not only shed light on basic cellular biology questions but may also be of relevance for possible therapeutic approaches against cancer mitotic cell cycles.
