title: Endomitosis of human megakaryocytes are due to abortive mitosis.
abstract: During megakaryocyte differentiation, the promegakaryoblast (immature megakaryocyte) increases its ploidy to a 2(x) DNA content by a poorly understood process called endomitosis. This leads to the formation of a giant cell, the megakaryocyte (MK), which subsequently gives rise to platelets. In this report, we show that endomitosis of human MKs is due to abortive mitosis. Human MKs were obtained by a two-step purification of CD34(+) blood or marrow precursors followed by in vitro culture in the presence of MK growth factors. Microscopic examination shows that a large number of centrosomes (up to 32) and centrioles are present in polyploid MKs. After nocodazole treatment, more than 20% of the MK are blocked in a typical pseudo-metaphase. Both spontaneous and nocodazole-induced endomitosis are associated with a breakdown of the nuclear envelope and possess a complex mitotic spindle composed of several asters. Spindle microtubules radiate from each aster, creating a spherical structure. At metaphase, expression of the kinetochore phosphoepitope recognized by the 3F3/2 antibody is lost, and the sister chromatids segregate moving toward the spindle poles. After limited segregation, the chromosomes decondense and the nuclear envelope reforms in the absence of cytokinesis, isolating all chromosomes in a single nucleus. It has been proposed that endomitosis could be due to an abnormal CDK1 activity or an absence of cyclin B1. Our results show that cyclin B1 can be detected in all MKs, including those with a ploidy of 8N or more. The cyclin B1 staining colocalizes with the mitotic spindle. Using flow cytometry, the level of cyclin B1 increased until 8N, but remained identical in 16N and 32N MKs. Cell sorting was used to separate the MKs into a 2N/4N and >4N population. Both cyclin B1 and CDK1 could be detected in the endomitotic polyploid MKs using Western blot analysis, and a histone H1 kinase activity was associated with immunoprecipitated cyclin B1. We conclude that endomitosis of human MKs is due to abortive mitosis, possibly due to alterations in the regulation of mitotic exit.
authors.affiliation: INSERM U 362, CNRS URA 1156, and CNRS URA 147, Institut Gustave Roussy, Villejuif, France.
authors.forename: N
authors.initials: N
authors.lastname: Vitrat
authors.forename: K
authors.initials: K
authors.lastname: Cohen-Solal
authors.forename: C
authors.initials: C
authors.lastname: Pique
authors.forename: J P
authors.initials: JP
authors.lastname: Le Couedic
authors.forename: F
authors.initials: F
authors.lastname: Norol
authors.forename: A K
authors.initials: AK
authors.lastname: Larsen
authors.forename: A
authors.initials: A
authors.lastname: Katz
authors.forename: W
authors.initials: W
authors.lastname: Vainchenker
authors.forename: N
authors.initials: N
authors.lastname: Debili
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D016203:CDC2 Protein Kinase / Q000502:physiology; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D002502:Centrioles / Q000648:ultrastructure; D018385:Centrosome / Q000648:ultrastructure; D019926:Cyclin B / Q000502:physiology; D056744:Cyclin B1; D005434:Flow Cytometry; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D011003:Ploidies; D011092:Polyethylene Glycols / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D008941:Spindle Apparatus / Q000187:drug effects / Q000648:ultrastructure; D013926:Thrombopoietin / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D011994:Recombinant Proteins; C103117:polyethylene glycol-recombinant human megakaryocyte growth and development factor; D011092:Polyethylene Glycols; D013926:Thrombopoietin; D016203:CDC2 Protein Kinase; D015739:Nocodazole