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Endomitosis of Human Megakaryocytes Are Due to Abortive Mitosis
Natacha Vitrat,
Karine Cohen-Solal,
Claudine Pique,
Jean Pierre LeCouedic,
Françoise Norol,
Annette K. Larsen,
André Katz,
William Vainchenker, and
Najet Debili
From INSERM U 362, CNRS URA 1156, and CNRS URA 147, Institut Gustave
Roussy, Villejuif, France.
During megakaryocyte differentiation, the promegakaryoblast
(immature megakaryocyte) increases its ploidy to a 2x 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
endomitotis 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. Microcoscopic 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 chromatides 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.
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3711-3723
© 1998 by The American Society of Hematology.
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