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Endomitosis of human megakaryocytes are due to abortive mitosis
N Vitrat1, K Cohen-Solal, C Pique
1INSERM U 362, CNRS URA 1156, and CNRS URA 147, Institut Gustave Roussy, Villejuif, France.
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.
Insights
Human megakaryocyte (MK) endomitosis, a process increasing cell ploidy, results from abortive mitosis. This study reveals complex spindle structures and altered cell cycle regulation, not cyclin B1 absence, drives this unique cell division. Keywords: megakaryocyte, endomitosis, abortive mitosis, cell ploidy, cyclin B1.
Area of Science:
- Cell Biology
- Hematopoiesis
Background:
- Megakaryocyte (MK) differentiation involves endomitosis, a poorly understood process where cells increase DNA content without dividing.
- This process leads to the formation of large MKs that produce platelets.
Purpose of the Study:
- To elucidate the mechanism of human MK endomitosis.
- To investigate the roles of cell cycle regulators, specifically cyclin B1 and CDK1, in this process.
Main Methods:
- Human MKs were generated from CD34(+) precursors.
- Microscopy, nocodazole treatment, flow cytometry, and Western blot analysis were employed.
- MKs were sorted based on ploidy for further analysis.
Main Results:
- Human MK endomitosis is characterized by abortive mitosis with multiple centrosomes and complex, multi-aster spindle structures.
- Cells exhibit breakdown of the nuclear envelope and limited chromosome segregation before nuclear reformation without cytokinesis.
- Cyclin B1 is present and associated with the mitotic spindle, and CDK1 activity is detected, refuting absence of these as the cause.
Conclusions:
- Human MK endomitosis is a consequence of abortive mitosis, not a failure of cell cycle regulators like cyclin B1.
- Alterations in the regulation of mitotic exit are likely responsible for endomitosis in MKs.