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Endomitosis of Human Megakaryocytes Are Due to Abortive Mitosis
By Natacha Vitrat, Karine Cohen-Solal, Claudine Pique, Jean Pierre Le Couedic, Françoise Norol,
Annette K. Larsen, André Katz, William Vainchenker, and Najet Debili
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 CD341 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 G4N 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.
r 1998 by The American Society of Hematology.
P
phases (G11G2) and S phases up to the 2xN ploidy level.
Several recent reports on cell cycle regulation during polyploidization have favored this hypothesis by showing an alteration in
CDK1 kinase activity,19-23 which is necessary for entry into
mitosis.24,25 These changes in kinase activity could be due to the
absence or very low amounts of cyclin B1,19,22,23 an alteration of
the CDK1/cyclin B1 complex formation,20 or downregulation
of CDC25C phosphatase.21 However, except for the two intitial
reports,19,22 the precise description of the cell cycle abnormalities during polyploidization was established using murine and
human cell lines with an MK phenotype. The human cell lines
were derived from leukemia patients and treated with phorbol
esters to promote the MK phenotype.20,21,26 Third, it is possible
that endomitosis may correspond to incomplete mitosis with the
absence of karyokinesis and cytokinesis. The original nomenclature, endomitosis, implies that chromosome condensation occurs in the absence of spindle and nuclear membrane breakdown.27 However, the occurence of an incomplete mitosis with
breakdown of the nuclear envelope has been supported by
several morphologic observations of MKs undergoing endomitosis. Although preliminary, these early observations led to the
concept of a defective metaphase/anaphase transition in MK
endomitosis.28,29 The major difficulty in performing a detailed
LATELETS ARE ANUCLEATE blood cells formed by a
unique process of cytoplasmic fragmentation of their
polyploid bone marrow precursors, the megakaryocytes (MKs).
The number of platelets in the circulation is regulated by two
independent parameters1: (1) the total number of MKs produced
in the marrow and (2) the size of the MKs, which is a function of
their ploidy. Polyploidization begins in immature MK and leads
to a 2xN cell with a single polylobulated nucleus by a process
called endomitosis.2 This process is associated with an increase
in MK cytoplasmic volume and, thus, indirectly regulates
platelet production. Endomitosis only occurs during terminal
differentiation of the cell. At the MK progenitor level, hematopoietic growth factors induce proliferation regulating the number of MKs in the marrow.1,3-5 During differentiation, MK
progenitors begin to synthesize specific platelet proteins.6-9 This
acquisition of platelet markers is soon followed by a switch
from a mitotic to an endomitotic process,10 whereby the cell
begins to replicate its DNA in the absence of cytokinesis and
karyokinesis.11 The humoral regulator of platelet production has
recently been isolated12-15 and designated either Mpl-ligand
(Mpl-L),13,15 thrombopoietin (TPO),14 or megakaryocyte growth
and development factor (MGDF).12 Mpl-L acts at all stages of
MK differentiation, inducing proliferation of the progenitors,
polyploidization, and cytoplasmic maturation of more mature
cells.4,5,13,16,17
The process of MK polyploidization is poorly characterized.
To explain endomitosis, three main theories have been proposed. First, MK DNA replication may occur during a continuous S phase until the hyperploid stage is obtained. Thereafter,
DNA synthesis stops and MKs undergo cytoplasmic maturation
leading to platelet shedding. This abnormality has been described in Chinese hamster cells with the ts41 mutation.18
However, experiments showing that MKs undergo several
rounds of replication interrupted by short gaps2,19 and synthesize cyclin D3 have essentially invalidated this hypothesis.19
Second, it has been proposed that the MK cell cycle is abnormal
with the absence of mitosis and consists of alternating resting
Blood, Vol 91, No 10 (May 15), 1998: pp 3711-3723
From INSERM U 362, CNRS URA 1156, and CNRS URA 147, Institut
Gustave Roussy, Villejuif, France.
Submitted June 9, 1997; accepted January 6, 1998.
Supported by grants from the Institut National de la Santé et de la
Recherche Médicale, the Ligue Nationale contre le Cancer, and the
Institut Gustave Roussy.
Address reprint requests to William Vainchenker, MD, PhD, INSERM
U 362, Institut Gustave Roussy, Villejuif 94805 Cedex, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9110-0019$3.00/0
3711
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3712
VITRAT ET AL
study of endomitosis is related to the low frequency of MKs in
the marrow and to the rarity of MKs undergoing endomitosis.
However, due to the recent discovery of Mpl-L,12-15 it is now
possible to grow large numbers of MKs in vitro and to perform a
detailed analysis of the endomitotic process.
In this report, human MKs were obtained by a two-step
purification of CD341 blood or bone marrow precursors
followed by in vitro culture in the presence of pegylated MGDF
(PEG-rhuMGDF, a truncated form of Mpl-L). Microscopic
examination of endomitosis was performed in the presence or
absence of nocodozole, an agent that blocks the cell cycle in
prometaphase. The different components involved in mitosis
were studied, including centrosomes and centrioles, lamin B,
kinetochores, and tubulin. Our results clearly show a breakdown of the nuclear envelope during endomitosis, which is
accompanied by DNA condensation of normal appearing chromosomes, and the formation of a complex spherical mitotic
spindle. The metaphase was followed by chromatid separation
and movement of the chromosomes towards the spindle poles.
Finally, catalytically active cyclin B1 was demonstrated in
polyploid endomitotic MKs.
MATERIALS AND METHODS
Antibodies. Fluorescein isothiocyanate (FITC)-labeled anti-CD41b
(Tab; a generous gift from R. McEver [Oklahoma City, OK] and
conjugated by S. Burstein [Oklahoma City, OK]) and phycoerythrin
(R-PE)-conjugated anti-CD34 (HPCA-2; Becton Dickinson, Mountain
View, CA) were used for flow cytometric analysis. A monoclonal
antibody (MoAb) against cyclin B1 and its IgG1 isotype-specific
control were purchased from Pharmingen (San Diego, CA). MoAbs
against centrioles and centrosomes (CTR 210) were a generous gift
from M. Bornens (Institut Curie, Paris, France). MoAb 3F3/2 was
generously provided by G.J. Gorbsky (Charlottesville, VA). MoAbs
against a and b tubulins were purchased from Amersham (Buckinghamshire, UK). Antibodies against lamin B and kinetochores were of human
origin and were obtained from patients with a lupus-like syndrome.30
These sera were kindly provided by J.C. Brouet (Hôpital St Louis, Paris,
France). Rabbit polyclonal antibody against von Willebrand factor
(vWF) was obtained from Dako (Glostrup, Denmark). The rabbit
polyclonal antibodies raised against cyclin B1 used for analysis by
Western blotting and by fluorescence microcospy were provided from
Santa Cruz Biotechnology (Santa Cruz, CA) and from J. Pines
(Wellcome/CRC Institute, Cambridge, UK), respectively. The mouse
monoclonal and the rabbit polyclonal antibodies raised towards CDK1
were purchased respectively from Santa Cruz Biotechnology and from
GIBCO (Paisley, Scotland). FITC-conjugated sheep antimouse IgG
(Silenus, Hawthorn, Australia), tetramethylrhodamine isothiocyanate
(TRITC)-conjugated goat antimouse IgG (Jackson ImmunoResearch,
West Grove, CA), FITC-goat antirabbit IgG (Caltag, San Francisco,
CA), and TRITC-goat antirabbit or antihuman IgG (Jackson ImmunoResearch) were used for indirect immunofluorescence assays.
Purification of CD341 cells. CD341 cells were purified from
human blood or bone marrow using a two-step procedure. After
obtaining informed consent from the patients, precursor blood cells
were isolated either from normal bone marrow of patients undergoing
hip surgery or from the peripheral blood of patients after mobilization
by chemotherapy and granulocyte colony-stimulating factor. The precursor cells were separated over a Ficoll-metrizoate gradient (Lymphoprep;
Nycomed Pharma, Oslo, Norway) to obtain an enriched fraction of
mononucleated cells. CD341 cells were then isolated using the Miltenyi
immunomagnetic bead technique as previously reported.31 Cells were
incubated for 15 minutes at 4°C with a mouse MoAb directed against
CD34 (QBEND-10) and human Ig to block nonspecific binding.
Subsequently, an antibody raised against mouse IgG conjugated to
magnetic beads was added for 15 minutes at 4°C. The CD341 cells were
retained on the column and were eluted by pressure using the plunger
supplied with the column. The purity was estimated by labeling with an
R-PE-labeled MoAb directed toward CD34 (HPCA-2, clone 8G2;
Becton Dickinson) and was about 90% after two passages through the
column.
In vitro liquid cultures of MKs from CD341 cells. CD341 cells were
grown for 7 to 10 days in Iscove’s modified Dulbecco’s medium
(GIBCO) containing penicillin/streptomycine/glutamine (250 U/mL,
250 µg/mL, 2 mmol/L; Sigma Chemical Co, St Louis, MO), 1.5%
deionized bovine serum albumin (BSA; Cohn fraction V; Sigma),
iron-saturated human transferrin (300 µg/mL; Sigma), and a mixture of
sonicated lipids (20 µg/mL) prepared as previously reported.4 The
medium was supplemented with PEG-rhuMGDF (10 ng/mL; a generous gift from J.L. Nichol, Amgen, Thousand Oaks, CA) either alone or
in combination with 50 ng/mL recombinant human stem cell factor
(SCF; a generous gift from Amgen). In some experiments, 1 µg/mL
nocodazole (Sigma) was added for 5 hours to the culture media to
synchronize the cells.
Cell lines. The UT-7 cell line32 was grown in a-Minimum Eagle
Medium (a-MEM; GIBCO) supplemented with 10% fetal calf serum
(GIBCO), penicillin/streptomycine/glutamine (250 U/mL, 250 U/mL, 2
mmol/L; Sigma), and 2 ng/mL human recombinant granulocytemacrophage colony-stimulating factor (GM-CSF; a generous gift from
Immunex, Seattle, WA). An Mpl-L–responsive UT-7 clone was also
used. This cell line was a gift from D. Duménil and F. Goncalvés
(INSERM U362, Institut Gustave Roussy, Villejuif, France) and was
obtained by the transfer of the human c-mpl coding sequence with a
retroviral vector (UT-7/c-mpl). By changing the cytokine from GMCSF to PEG-rhuMGDF, an increase in the MK phenotype could be
obtained resulting in the presence of some polyploid cells (5% of the
cells .4N). These cell lines were used as positive controls.
Immunolabeling for fluorescence microscopy. Cells were cytocentrifuged at 500 rpm for 4 minutes, fixed in methanol (Carlo Erba;
Rodano, Italy) for 5 minutes at 220°C, and rehydrated in phosphatebuffered saline (PBS). The cells were permeabilized for 5 minutes with
0.1% Tween 20 (Sigma) before incubation for 1 hour at room
temperature with the appropriate antibodies. The antibodies used
included hybridoma supernatant fluid directed against centrosomes or
centrioles diluted at 1:2, human antibody directed toward lamin B
(diluted at 1:500), and a rabbit antibody directed against vWF (54
ng/mL). After three washes with PBS, cells were incubated for 1 hour at
room temperature with the appropriate secondary antibodies (FITC
sheep antimouse IgG, TRITC goat antihuman or antimouse IgG, and
TRITC or FITC goat against rabbit IgG). DNA was labeled by Hoechst
33258 at 7.5 ng/mL (Hoechst 33258; Sigma) for 15 minutes in the dark.
The cell preparations were analyzed with a fluorescence microscope
equipped with the appropriate filter combinations (Zeiss, Oberkochen,
Germany) or with a paraconfocal microscope (CellScan; Bionis Atlantic, Clamart, France).33
Immunolabeling for confocal microcopy. Cells were centrifuged at
300g for 10 minutes at 37°C onto circular slides covered with
polylysine (1 mg/mL; Sigma). The cells were fixed for 5 minutes with a
microtubule stabilizing buffer MTSB (80 mmol/L K-PIPES, pH 6.5, 5
mmol/L EGTA, and 2 mmol/L MgCl2 for a 53 solution) containing 3%
paraformaldehyde and then permeabilized for 15 minutes with the same
buffer containing 0.2% saponin at room temperature. The immunostaining technique was the same as described above, except that dilution of
the antibodies and washes were performed in PBS-GT (13 PBS without
Ca21 or Mg21, 0.1% Triton X-100). The antibodies used included
mouse antibodies (a mixture of 2 MoAbs directed towards a and b
tubulins diluted at 60 µg/mL, anti-3F3 diluted at 1:1,000), rabbit
polyclonal antibodies (anti-vWF, anti-cyclin B1 diluted at 1:200), and
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ENDOMITOSIS OF HUMAN MEGAKARYOCYTES
human antibody (anti-kinetochores diluted at 1:500). DNA was labeled
with propidium iodide (10 µg/mL) for 30 minutes at room temperature
in the dark after 30 minutes of incubation with RNAse A (1 mg/mL,
preincubated for 15 minutes at 95°C to inactivate contaminating
DNAse; Boehringer Mannheim, Meylan, France).
Immunolabeling for flow cytometric analysis. Cultured cells were
washed in PBS before fixation in 80% ethanol. Cells were maintained
for at least 24 hours at 220°C, washed in PBS containing 1% BSA and
permeabilized with 0.25% Tween 20 (Sigma) at 4°C for 15 minutes.
After two centrifugations at 200g, cells were incubated at 4°C for 30
minutes with either the anti-cyclin B1 MoAb (2.5 µg/mL) or with
control IgG1 antibodies. The anti-cyclin B1 MoAb used in these
experiments (Pharmingen) has been widely used to detect cyclin B1 in
normal and leukemic cells by flow cytometry.34-36 The cells were then
washed twice and incubated with FITC-labeled sheep antimouse Ig for
30 minutes at 4°C. Finally, PBS containing 50 µg/mL propidium iodide
(Sigma) and 100 µg/mL RNase A was added to the cell pellet for
approximately 2 hours. Cell samples were analyzed on a FACSort
(Becton Dickinson) equipped with an argon laser (15 mW, 480 nm
excitation). FITC and propidium iodide were assigned to the FL1 and
FL3 channels, respectively, whereas the FL3A and FL3W channels
were used to exclude cell aggregates. For each sample, 10,000 cells
were acquired in the list mode and analyzed with the Cellquest software
package (Becton Dickinson).
Cell sorting of MKs. Cells were recovered after 8 days of culture
and incubated with the FITC-Tab MoAb directed against the MKspecific cell surface receptor CD41b (GpIIb) for 30 minutes at 4°C in
their culture medium. Cells were washed in PBS-EDTA and incubated
for an additional 2 hours with 0.01 mmol/L Hoechst 33342 (Sigma) at
37°C. MKs were sorted according to their DNA content using a
FACSVantage cytometer (Becton Dickinson) equipped with two argon
lasers (tuned to 488 and 360 nm, respectively, and operating at 500 mW;
Coherent Radiation, Palo Alto, CA) and a 200- or 300-µm nozzle. A
morphologic gate, including all the CD411 cells, was determined on
two-parameter histograms (side scatter [SSC] v forward scatter [FSC]).
Pulse processing using FSC width and UV emission was used to
exclude cell aggregates. The sorting gate was constructed using the
intersection of these gates. CD411 cells were sorted into a 2N/4N and a
.4N cell fraction at 500 cells/s at 4°C. The quality of the sorted cells
was confirmed by microcospic examination and flow cytometric
reanalysis. Less than 5% single cells in aggregates was observed in the
.4N cell fraction as determined by fluorescence microscopy.
Western blot analysis. Soluble proteins obtained from approximately 5 3 105 to 5 3 106 cells lysed in Laemmli buffer (0.125 mmol/L
Tris, pH 6.8, 4% sodium dodecyl sulfate [SDS], 20% glycerol, 10% b
mercaptoethanol, 1 µg/mL aprotinin, and 0.02% bromophenol blue)
were separated by SDS-polyacrylamide gel electrophoresis (SDSPAGE; 12%) and then transferred electrophoretically (130 mA, 90
minutes) onto a nitrocellulose membrane (Biorad, Hercules, CA) in a
buffer containing 25 mmol/L Tris, 192 mmol/L glycine, 0.01% SDS,
and 20% methanol. Nonspecific binding was inhibited by a preincubation with dried milk overnight at 4°C. The membranes were incubated
for 90 minutes at room temperature with the different primary antibodies, including a rabbit polyclonal antibody raised against human cyclin
B1 (0.1 µg/mL; Santa Cruz) and a mouse or a polyclonal antibody
directed against human CDK1 (1 µg/mL and 2 µg/mL, respectively).
After two washes with TBS-Tween (10 mmol/L Tris, pH 7.5, 150
mmol/L NaCl, 0.1% Tween, 0.02% NaN3), the membranes were
incubated for 30 minutes at room temperature with either goat antirabbit
or antimouse antibodies conjugated to horseradish peroxidase diluted at
1:5,000 and 1:10,000, respectively (Amersham), or with I125 protein A
(Amersham). The bands were developed with an enhanced chemiluminescence system (ECL kit; Amersham) or by autoradiography.
Immunoprecipitation and kinase assays. The cells were washed
once with PBS and incubated in lysis buffer (50 mmol/L Tris, pH 7.4,
3713
250 mmol/L NaCl, 5 mmol/L EDTA, 0.5% Nonidet P-40, and a mixture
of protease inhibitors purchased from Boehringer Mannheim) for 10
minutes on ice. After centrifugation at 15,000g for 5 minutes, the
supernatant was incubated with a mouse MoAb raised against human
cyclin B1 (GNS-1; 1 µg per 106 cells; Pharmingen) for 6 hours at 4°C.
Protein A-sepharose CL-4B (Pharmacia) was added (50 µL) and
incubated overnight at 4°C. The beads were washed three times with the
lysis buffer and twice with kinase buffer (50 mmol/L Tris-HCl, pH 7.4,
10 mmol/L MgCl2, 0.1 mg/mL BSA) by centrifugation at 2,500g for 5
minutes. The pellet was incubated in 50 µL kinase buffer with Histone
H1 (20 µg), a magnesium ATP/(g-32P)ATP [10 µCi (g-32P)ATP, 500 µmol/L
ATP], and protease inhibitor mixtures for 30 minutes at 25°C. The
reaction products were resuspended in SDS-PAGE buffer and loaded on
a 12% polyacrylamide gel followed by autoradiography.
RESULTS
In vitro MK culture. In the bone marrow, MKs represent
approximately 0.1% of the cell population, and only about 1%
of the MKs is undergoing endomitosis at a given time. For this
reason, human MKs were obtained using a two-step purification
of CD341 blood and bone marrow precursors, followed by in
vitro culture in the presence of PEG-rhuMGDF. MKs were fully
mature by day 12, although their average ploidy was much
lower than what has been described for MKs in the bone
marrow.4 MKs began to polyploidize after about 6 days of
culture, and most of the studies described below were performed between days 6 and 10 with culture samples containing
50% to 80% MKs (80% with PEG-rhuMGDF alone and 50%
with SCF plus PEG-rhuMGDF). By day 8, about 20% of the
MKs are proliferating, as demonstrated by propidium iodide
staining and flow cytometric analysis.
Presence of several centrosomes and centrioles in polyploid
MK. During the cell cycle, centrosomes and centrioles are
separated at the end of the G2 phase or in prophase.37 In five
experiments, cultures were studied at day 8 and the cells were
examined by microscopy after immunostaining using a threecolor method. MKs were identified by their reactivity with a
rabbit polyclonal anti-vWF antibody (FITC), centrosomes and
centrioles by murine MoAbs (TRITC), and DNA by the
Hoechst dye. As shown in Fig 1a and b, a large MK with a
polylobulated nucleus exhibited several centrioles that occasionally were clustered in the cytoplasm. Their precise number was
difficult to determine, because each spot was at the threshold of
detection. Therefore, para-confocal microscopy was used to
obtain better resolution. As shown in Fig 1c, several centrioles
(.16) were observed throughout the cytoplasm. The same
results were observed using an anti-centrosome MoAb, and up
to 32 centrosomes per MK could be identified. These findings
confirmed previous ultrastructural studies of MKs from the
bone marrow,38,39 indicating that, during polyploidization, MKs
go through several G2/early prophases.
MKs enter into mitosis. Cultures were treated with 1 µg/mL
nocodazole for 5 hours, which resulted in an accumulation of
about 20% of the MKs in prometaphase. Higher concentrations
of nocodazole or longer exposure time did not modify these
results. After staining with May-Grünwald Giemsa, polyploid
metaphases were observed in large MKs. As shown in Fig 2a,
more than 200 well-individualized chromosomes could be
identified in a single MK. The number of arms per chromosome
was normal. Similar findings were observed with fluorescent
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Fig 1. Polyploid MKs have several centrioles. CD341 cells purified from blood cytapheresis or bone marrow were grown in the presence of
PEG-rhuMGDF and SCF or PEG-rhuMGDF alone. At day 8, cells were recovered, cytocentrifuged, fixed, and permeabilized. Cells were
double-labeled by indirect immunofluorescence and the DNA was counterstained by the Hoechst dye. Cells were examined with a conventional
fluorescence microscope (a and b) and by confocal microscopy (c). (a) Observation with a combination of filters permitting simultaneous
visualization of FITC and Hoechst staining. Labeling with an MK-specific antibody directed toward vWF (FITC) shows a granular pattern. Original
magnification 3 1,000. (b) Centrioles appear as red spots (TRITC) in the cytoplasm. These spots (,16) appear to be clustered in the Golgi area of
this MK. (c) Centrioles (red) are better observed by a confocal microscopy and can be seen to be scattered in the cytoplasm of the MK. Original
magnification 3 2,000.
Fig 3. Presence of a spherical mitotic spindle in endomitotic MKs. Cultures were performed as described in Fig 1 in the absence of nocodazole.
Cells were centrifuged on a slide, fixed with a microtubule stabilizing buffer containing 3% formaldehyde, and then permeabilized with 0.2%
saponin. Cells were labeled with an anti-tubulin antibody (a, b, c, d, e, f, g, h, and k), an anti-vWF antibody (a), propidium iodide (c), an
anti-kinetochore antibody (i and k), and the 3F3 MoAb (j). Cells were then examined by confocal microscopy (original magnification 3 1,000 for [a]
through [j] and 3 1,500 for [k]). (a) Superimposition of a double staining with the anti-tubulin MoAb (FITC) and the anti-vWF (TRITC). (b)
Anti-tubulin staining shown alone. Note that the complex mitotic spindle has a spherical conformation. (C) Superimposition of a double staining
with the anti-tubulin MoAb (FITC) and propidium iodide. (d) Anti-tubulin staining shown alone. (e, f, g, and h) A mitotic spindle shown under
different angles. Spindle microtubules radiate from each aster to form a spherical conformation. (i and j) Double staining with the
anti-kinetochore antibody (TRITC) and 3F3 MoAb (FITC). Kinetochores are distibuted all around and the 3F3 labeling only colocalized with the
asters, as previously demonstrated during anaphase of a mitotic cycle.40 (k) Double staining with the anti-kinetochore antibody (TRITC) and the
anti-tubulin MoAb (FITC). The kinetochores are clustered around each aster, suggesting that chromosome segregation has occured.
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ENDOMITOSIS OF HUMAN MEGAKARYOCYTES
3715
Fig 2. Polyploid MKs in prometaphase. Cultures were performed as described in Fig 1, except that nocodazole (1 mg/mL) was added in some
experiments to 8-day culture for 5 hours. Cells were stained either by May-Grünwald Giemsa or labeled by antibodies (FITC or TRITC) and
counterstained by the Hoechst dye (blue) after cytocentrifugation and fixation. (a) This MK is blocked in pseudometaphase by nocodazole.
Well-condensed, individualized chromosomes are present. The cytoplasm already has a granular pattern with purple staining localized to one
pole of the cell that may correspond to granules (arrow). The nuclear envelope is not apparent. Original magnification 3 2,000. (b) Two large MKs
(appointed by arrow) are labeled by an anti-vWF antibody (FITC) and conterstained by Hoechst. These endomitotic MKs with condensed
chromosomes are blocked in prometaphase. Original magnification 3 1,000. (c) An MK in interphase is labeled by the human anti-lamin B
antibody (TRITC). The polylobulated nucleus is surrounded by a distinct linear red labeling. Original magnification 3 1,000. (d, e, and f) A
polyploid MK in spontaneous metaphase is shown. Expression of vWF (FITC; d), Hoechst (e), and lamin B (TRITC; f). Only a faint and dispersed
labeling is observed in TRITC, demonstrating that the nuclear envelope has disappeared.
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3716
probes using an anti-vWF antibody to identify MKs and
Hoechst staining to visualize condensed chromosomes (Fig 2b).
To determine if the nuclear envelope breaks down, a human
antibody directed against lamin B was used. This antibody
labeled the nuclear envelope in interphase MK by an intense
linear staining surrounding the nucleus (Fig 2c). In contrast,
there was either no labeling or only a faint dispersed labeling
corresponding to residual nuclear vesicles in endomitotic MK
(Fig 2d, e, and f) as well as in MKs blocked in prometaphase. In
comparison, a human antibody directed against the kinetochores showed a different pattern of labeling with numerous
spots in the nucleus.
Polyploid MKs have a complex spherical mitotic spindle.
These untreated cells were also studied to examine the mitotic
spindle using immunofluorescence labeling with a combination
of anti-tubulin murine MoAbs (FITC), rabbit anti-vWF antibody (TRITC), and the Hoechst dye or with anti-tubulin murine
MoAbs (FITC) and propidium iodide. More than 300 endomitotic MKs were observed in total. As shown in Fig 3, the mitotic
spindle was complex and contained several poles (Fig 3a
through d) whose number depended on the MK ploidy. As many
as 32 asters could be observed in these cells. As shown by
confocal microscopy in Fig 3b, e, f, g, and h, the spindle was
spherical. The spindle poles were localized around the sphere
and polar microtubules extended from one pole toward all the
others creating this spherical conformation. Some endomitosis
studied with the combination of anti-tubulin (FITC) and antivWF (TRITC) antibodies and the Hoechst dye correspond to an
anaphase with chromosomes having migrated in proximity of
the asters.
Chromosome separation occurs normally in endomitotic
MKs. To better characterize late stages of mitosis, cells were
labeled by MoAb directed toward the 3F3/2 phosphoepitope
and with an anti-kinetochore antibody. As previously shown,40
mitotic poles but not kinetochores expressed this phosphoepitope in metaphase or anaphase (data not shown). However, a
labeling was observed on larger spots that correspond to the
aster. This was also the case for endomitotic MKs (Fig 3i and j).
In these experiments, MKs were identified on their polyploidy
as cells expressing more than 100 kinetochores. This finding
implies that chromosomes from endomitotic MKs are correctly
aligned on the mitotic spindle and are able to segregate. To
determine if chromosome separation can occur, endomitotic
MKs were stained with anti-tubulin and anti-kinetochore antibodies. In normal anaphase figures, chromosomes were labeled
by the anti-kinetochore antibody close to the two spindle poles.
Similarly, kinetochores were clustered around each spindle pole
in endomitotic MKs (Fig 3k). This observation shows that
endomitosis in MKs is not due to a block in the metaphase/
anaphase transition and that chromosome segregation occurs in
anaphase. However, this chromatid segregation was limited due
to the complex spindle.
In rare MKs, the end of mitosis could be observed with
decondensation of the chromosomes and reformation of the
nuclear envelope, which suggests that only cytokinesis is lacking in
endomitotic MKs. Attempts to examine a larger number of MKs at
this stage of mitosis failed, because nocodazole treatment seems to
be irreversible in this cellular system.
VITRAT ET AL
MKs express cyclin B1 and CDK1. The studies noted above
showed that, during polyploidization, MKs enter into mitosis
that is associated with a complex spindle. We therefore examined the expression of cyclin B1. Its expression was investigated with an MoAb while DNA was stained by propidium
iodide. In the first set of experiments, a flow cytometry
technique was set up to exclude cell aggregates. Propidium
iodide staining was analyzed on the FL3H, FL3A, and FL3W
channels of the flow cytometer. The FL3H parameter was
determined with a logarithmic amplifier. The 2N peak was set in
channel 120 of FL3A to detect MKs with a ploidy ranging from
2N to 32N. Using a dot plot analysis with the FL3A and FL3W
parameters, a gate that excluded most cell aggregates (including
the 6N peak) was constructed (Fig 4A and B). Using this
approach, it was possible to simultaneously obtain a linear
(FL3A) and a logarithmic (FL3H) measurement of the ploidy
(Fig 4C through F).
This technique was used to determine the expression of
cyclin B1 as a function of ploidy. The anti-cyclin B1 MoAb
used in these experiments has been previously used to detect
cyclin B1 through the cell cycle in normal lymphocytes,
leukemic cells, solid tumor cells, and polyploid cells.34-36 A
specific labeling (Fig 4) was observed almost exclusively at the
level of the 8N, 16N, and 32N peaks, which may correspond to
either the G1 or G2/M phase of the cell cycle (using flow
cytometry, we cannot distinguish between the G2/M phase of
the lower ploidy classes and the G1 phase of the upper ploidy
classes). Interestingly, very few S phase cells were labeled with
this MoAb. The intensity of staining increased 1.7-fold between
the 8N and 16N cells. Therefore, there is an increase in cyclin
B1 level with ploidy up to 16N, but slightly less important than
the ploidy enhancement. In contrast, the level of cyclin B1
seems similar in 16N and 32N MKs.
To further examine the localization of cyclin B1 during
endomitosis, standard immunofluorescence analysis with the
anti-cyclin B1 MoAb and Hoechst dye was performed. In
endomitotic MKs, faint cyclin B1 staining delineated the
mitotic spindle (Fig 5a and b). In some MKs at late stages of the
endomitosis (anaphase), no cyclin B1 was detected, suggesting
a degradation of the protein by the proteasome (Fig 5c and d).
To clearly demonstrate that these polyploid cells correspond to
true MKs, a triple staining was performed (anti-cyclin B1,
Hoechst, and vWF) that confirms the results given above (Fig
5e, f, and g). To further characterize the cyclin B1 and CDK1 in
MKs, polyploid MKs were purified. Cultured MKs (day 9) were
labeled with an anti-CD41b MoAb (anti-GpIIb) and the 33342
Hoechst dye. CD411 MKs were sorted using a 200- or 300-µm
nozzle according to their ploidy into two cell fractions (2N/4N
and .4N cells). Cell aggregates were excluded using the pulse
processors of the cytometer. This approach allowed us to obtain
a relatively pure population of MKs (.95%) with a purity of
greater than 90% of the different ploidy classes. However, the
more rare 8N MKs were contaminated by MK aggregates,
which represented on average 5% of the cells as determined by
microscopic examination. Such a cell sorting is shown in Fig 6a
and b. Reanalysis of the sorted fractions confirmed these
microscopic examinations (Fig 6c, d, and e). The presence of
cyclin B1 and CDK1 proteins was determined by Western blot
analysis on cell lysates from purified MKs. As shown in Fig 7a,
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ENDOMITOSIS OF HUMAN MEGAKARYOCYTES
3717
Fig 4. Expression of cyclin B1 in polyploid MKs.
Cultures were performed as described in Fig 1. At
day 8, cells were labeled by an anti-cyclin B1 MoAb
(D and F) or an isotype control (FITC, FL1; C and E)
and propidium iodide (FL3). Cells (10,000 8N cells)
were acquired in a gate R1 (FL3A v FL3W; B) intersected with a gate R2 (FSC v SSC; A) to eliminate cell
aggregates and cell debris. Ploidy was studied using
simultaneously linear (FL3A; C and D) or logarithmic
(FL3H; E and F) amplifiers. A specific labeling with
the anti-cyclin B1 Moab is shown on (D) and (F) at
the level of the 8N, 16N, and 32N peaks. No labeling
is present in the S phases from 8N to 16N or from
16N to 32N.
the 62-kD cyclin B1 was detected in both MK fractions. The
analysis was performed on the same number of cells for both
MK fractions; the higher expression of cyclin B1 in the .4N
MKs than in the 2N/4N cells could be the reflect of higher
amounts of protein per polyploid MK. When the lanes were
loaded with an equal quantity of proteins from 2N/4N and
polyploid MKs, the same amounts of cyclin B1 were present in
both MK fractions (Fig 7b). Cyclin B1 was detected with the
same apparent molecular weight as in the controls (UT-7 c-mpl
cell line). In addition, a faint band with a lower molecular
weight (57 kD) was sometimes observed in MK samples, with
marked differences in intensity among experiments. This
band may correspond to the heavy chain of the Ig that was used
for MK purification. CDK1 was also detected on immunoblots
in both cell fractions and was expressed at a high level in
2N/4N MKs and polyploid MKs (Fig 7c and d). Two molecular
forms could be visualized as in control cell lines (UT7-c-mpl,
U937, and HL60). The lower molecular form corresponding to
dephosphorylated CDK1 was predominent in both MK fractions.
An H1 histone kinase activity is present in endomitotic MKs.
The presence of CDK1 and cyclin B1 in MKs does not
necessary mean that the two proteins associate in a functional
complex. Therefore, we examined the kinase activity of cyclin
B1 immunoprecipitates. For these experiments, cultures were
treated with 1 µg/mL nocodazole for 5 hours and subsequently
sorted as described into two cell fractions. Immunoprecipitation
was performed with the anti-cyclin B1 antibody and the H1
histone kinase activity of the immunoprecipitates studied. A
high level of H1 histone kinase activity was found in unsorted
cells, as well as in the 2N/4N and the polyploid (.4N) cell
fractions (Fig 8). The activity was in the same range in the
different cell fractions. These data show that cyclin B1 is
functional in endomitotic MKs.
DISCUSSION
The purpose of this study was to determine the cellular
mechanism of endomitosis in human MKs. Based on previously
described cell cycle abnormalities, three main hypothesis have
been proposed: (1) a continuous S phase that does not arrest
until the 2xN ploidy; (2) a succession of S and G1-G2 phases
without entry into mitosis; and (3) an abortive mitosis without
caryokinesis and cytokinesis. A morphologic approach was
performed to distinguish between these different possibilities.
The main difficulties of this type of approach are due to the
scarceness of endomitotic MKs. MKs were cultured from
human CD341 cells in the presence of PEG-rhuMGDF or the
combination of SCF plus PEG-rhuMGDF. This allows us to
obtain a large proportion of MKs (up to 80%), of which
approximately 20% are proliferating by day 8, as demonstrated
by propidium iodide staining and flow cytometric analysis.
However, on slides, less than 1% of these MKs are in M phase,
which explains why no complete observation of the endomitotic
process has yet been performed. In the rat and mouse, it has
been shown that the complete length of DNA synthesis is about
9.3 hours.2,19 It can be calculated from the present study that the
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3718
VITRAT ET AL
Fig 5. Localization of cyclin
B1 in endomitotic MKs. Cultures
were performed as described in
Fig 1 and cells were stained as
described in Fig 3 by an anticyclin B1 polyclonal antibody (b
and d; TRITC), an anti-vWF MoAb
(e; FITC), and the Hoechst dye (a
and c). They were examined by
conventional fluorescent microscopy (original magnification 3
500). In this polyploid MK in endomitosis (a), cyclin B1 staining
draws the mitotic spindle with
its multiple asters. In two MKs in
metaphase, cyclin B1 is detectable (arrows on the left; c and d).
In contrast, in a polyploid MK
with an anaphase figure (arrow
on the right; c and d) as shown
by segregation of the chromatids, cyclin B1 is undetectable. In
this endomitotic MK (f) expressing the vWF (e), cyclin B1 colocalized with the asters, as shown
by the arrows.
M phase of MK takes less than 30 minutes, if the doubling time
is similar in humans and mice. Thus, nocodazole was used to
observe a large number of MK in endomitosis. This agent has
permitted us to obtain an average of 20% of MKs in pseudometaphase. However, its blocking effect appeared to be irreversible, and we were therefore not able to observe a large fraction
of synchronized MKs in late phases of mitosis.
However, despite this limitation, our results clearly show that
endomitosis is an abortive mitosis for the three following
reasons. (1) The presence of several centrosomes or several
pairs of centrioles in polyploid MK demonstrates that MKs
during polyploidization undergo several G2/prophases, eliminating the possibility that polyploidization could be due to a
continuous S phase. (2) After nocodazole treatment, a significant fraction of MKs was in pseudo-metaphase, with a total
breakdown of the nuclear envelope and the presence of
condensed chromosomes with a normal number of arms per
chromosome. (3) Endomitosis was also observed in the absence
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ENDOMITOSIS OF HUMAN MEGAKARYOCYTES
3719
Fig 6. Examination of sorted MKs. After 8 days of culture, the cells were sorted on their CD41 expression and on their ploidy by Hoechst
staining into two cell fractions corresponding to 2N-4N and G4N MKs. The quality of the sorted cells was checked by examination with a
fluorescence microscope (original magnification 3 100) under UV light (a and b). The polyploid fraction with multilobulated nuclei (b) was
slightly contamined by some cell agregates. In the 2N-4N fraction (a), the cells were smaller. Cells were also reanalyzed by flow cytometry. A
typical experiment is shown in (c), (d), and (e). In (c), the ploidy histogram of the CD411 cells before sorting is shown with 14% G4N MKs. The
ploidy histogram of the 2N-4N cell fraction is shown in (d). The purity of this fraction is greater than 99.9%. In (e), the ploidy histogram of G4N is
shown with a purity of greater than 93%. It is noteworthy that contaminant cells (5%) are only 2N cells. The width of the peak is slightly larger
than in the unfractionated cells due to a slight efflux of the Hoechst during cell sorting.
of nocodazole and was characterized by a complex multipolar
mitotic spindle and a breakdown of the nuclear envelope. The
number of poles seemed to correlate with the MK ploidy.
Interestingly, the assembly of this spindle was spherical. Each
pole was localized at the periphery, with microtubules extending from one pole to another, creating this spherical conformation, but with apparently an absence of microtubules in the
center of this sphere. Each pole may be involved in several
Fig 7. Expression of cyclin B1 and CDK1 proteins in polyploid MKs. Experiments were performed as described in Fig 6. Western blot analysis
was performed with a polyclonal antibody directed against cyclin B1 and an MoAb against CDK1. The experiments were performed with the
proteins derived from the same number of cells (a and c) or with the same amounts of proteins (b and d). Expression of cyclin B1 in MKs (a and b).
(a) Lane 1, UT-7 cell line transfected with c-mpl (UT-7-c-mpl) and grown with PEG-rhuMGDF; lane 2, UT-7-mpl grown with PEG-rhuMGDF and
synchronized with nocodazole; lane 3, 2N-4N MKs; lane 4, G4N MKs. (b) Lane 1, 2N-4N MKs; lane 2, G4N MKs. Expression of CDK1 in MKs (c and
d). (c) Lane 1, UT-7-c-mpl cell line as positive control; lane 2, 2N-4N MKs; lane 3, G4N MKs. (d) Lane 1, 2N-4N MKs; lane 2, G4N MKs.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3720
VITRAT ET AL
Fig 8. Histone H1 kinase activity in polyploid MKs. Immunoprecipitation of cyclin B1 in the two fractions of MKs synchronized by nocodazole
and sorted on their ploidy (2N-4N and G4N) or in asynchronized MKs was performed with an MoAb raised against cyclin B1 (GNS-1;
Pharmingen). The histone H1 was phosphorylated by the immunoprecipitate in the presence of (g-32P)ATP and the phosphorylation was
determined by electrophoresis on a 12% polyacrylamide gel followed by autoradiography. The results were compared with a negative control
[(g-32P)ATP, histone H1 with control IgG1 immunoprecipitate]. Lane 1, (g-32P)ATP histone H1 with control IgG1 immunoprecipitate as a negative
control; lane 2, 2N-4N MKs; lane 3, G4N MKs; lane 4, unfractionated MKs (5 3 106 cells); lane 5, unfractionated MKs (1 3 106 cells); lane 6,
unfractionated MKs (5 3 105 cells).
chromosome movements along this sphere. It was difficult to
identify the different stages of mitosis in a precise manner.
However, it was clear from the nocodazole experiments that the
progression of the mitotic cycle up to metaphase is normal in
endomitotic MKs. It has been suggested previously that the
endomitosis could correspond to a lack in metaphase/anaphase
transition.28,29 To determine if anaphase occurs, we first determined whether chromosomes were able to align normally on
this complex spindle. For this purpose, the 3F3/2 antibody41 that
identifies a kinetochore phosphoepitope that signals chromosome alignment was used.40,42 Disappearance of this phosphoepitope on kinetochores of metaphase chromosome implies that
the cells are triggered to enter anaphase. Our results show that
the 3F3/2 phosphoepitope disappears from the kinetochores of
polyploid MK in metaphase, strongly suggesting that endomitosis is not a block in the metaphase/anaphase transition. Furthermore, labeling of kinetochores and tubulin clearly showed that
the chromosomes move toward each spindle pole and thus that
anaphase occurs normally in endomitotic MKs with a segregation of the chromosomes. However, chromatids remained close
to each other. This was essentially due to both the short length
and the complexity of the spindle. Chromosomes subsequently
decondensed, the spindle disassembled, and the nuclear envelope reformed all around the chromosomes, confining all
chromatids into a single nucleus. To make a detailed study of
the late stages of the endomitotic process (reformation of the
nuclear envelope), it will be necessary to synchronize the MK
cell cycle. Nevertheless, the results clearly show a major defect
in cytokinesis, although this does not seem to be the primary
defect explaining the endomitotic process of MK. Several genes
involved in cytokinesis have been isolated in the Drosophila.43-45 Defects of these genes led to the formation of
polyploid cells that contain multiple nuclei (2 to 6). Our results
indicate that the endomitotic process may be the consequence of
both a multipolar spherical spindle that limits chromatid
segregation and the absence of cytokinesis.
If endomitosis is really an abortive mitosis, each endomitotic
round should require the presence of a mitotic cyclin and its
associated CDK1 kinase activity (M phase promoting factor),
and we would expect that the cells were unable to enter mitosis
in the absence of CDK1 activity.24,46 Recently, several investiga-
tors have suggested that MK polyploidization is due to major
abnormalities in the CDK1 activity.19-23 A similar phenomenon
has been described in different models of endoreduplication,
which, in contrast to MK, leads to a 2xN ploidy in the absence of
mitosis.47 In favor of this hypothesis, two reports describe the
absence or very low level of cyclin B1 in MK.19,22 Our results
showing that endomitosis is an incomplete mitosis are in sharp
contrast to these findings, because the presence of a B-type
cyclin is absolutely required for cells to enter into mitosis. This
cyclin could be either cyclin B1 or another B-type cyclin. We
were able to detect cyclin B1 in MK using two techniques. First,
the protein could be detected by flow cytometry using an MoAb
that has been previously used to detect cyclin B1 in mitotic
cells. Cyclin B1 was essentially found at the 8N, 16N, and 32N
peaks of ploidy, and only in a minority of S phase endomitotic
MKs as observed for normal mitosis. Second, cyclin B1 protein
expression could be demonstrated by Western blot analysis in
polyploid MKs. Taken together, these results clearly show that
cyclin B1 is present in endomitotic MKs. Differences with the
previous studies might be explained by the fact that cyclin B1 is
only transiently expressed during the MK cell cycle that
requires the examination of a large number of MK and that these
previous studies were not performed with cultured MKs stimulated by Mpl-L. Cyclin B1 was concentrated in the spindle of
endomitotic MK, as previously described for mitosis.48 The
level of cyclin B1 increased a little less than the content in DNA
up to 16N, but seems constant between 16N and 32N. This may
explain that the modal ploidy level is 16N in MKs.
The presence of cyclin B1 by itself is not sufficient to enter
into mitosis, because the cyclin must be associated with CDK1.
It has been shown that two cell lines with an MK phenotype
(HEL and MEG-01), induced to polyploidization by phorbol
ester, lack a CDK1 kinase activity, despite the presence of both
CDK1 and cyclin B1.20,21 In one of these studies, it was
suggested that this was due to a downregulation of CDC25C, a
phosphatase necessary to activate CDK1,21 whereas the other
study suggests that the absence of kinase activity was due to the
inability of cyclin B1 to complex with CDK1.20 In another
model using a megakaryocytic cell line generated by targeted
expression of temperature-sensitive simian virus 40, the level of
cyclin B1-dependent CDK1 activity was greatly reduced in
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ENDOMITOSIS OF HUMAN MEGAKARYOCYTES
polyploid cells due to low cyclin B1 level. In human polyploid
MKs, we could detect a high CDK1 level. In addition,
endomitotic polyploid MKs possess a histone H1 kinase activity
associated to cyclin B1 that is not markedly different from
2N-4N MKs. In favor of our results, it has very recently been
shown that polyploid murine MK also express cyclin B1 and
that a histone H1 activity could be detected after suc1 purification.49 During mitosis, the CDK1/cyclin B complex associates
with the microtubules in the mitotic spindle and regulates
mitotic spindle formation.50 Because MKs present an abnormal
mitotic spindle, it is possible that the M-phase promoting factor
in MK cells could be partly different from other cells. It cannot
be excluded that cyclin B1 may associate with another CDK
than CDK1 or that other B-type cyclins with a different
expression during the cell cycle are also present in MKs.
Further studies will be required to precisely identify the
components of the H1 histone kinase activity in endomitotic
MKs.
The metaphase/anaphase checkpoint is regulated by a proteolytic system called cyclosome or anaphase-promoting complex51-55 that degrades several proteins such as topoisomerase II
or cyclin B1. Proteolysis of cyclin B leads to the inactivation of
CDK1 kinase activity at the end of metaphase. However,
chromosome segregation does not require the degradation of
cyclin B1.56 In contrast, preventing the inactivation of cyclin
B/CDK1 complexes blocked chromosome decondensation and
inhibited telophase chromosome movement.57 Recent experiments expressing a nondestrucible form of cyclin B into
prometaphase normal rat kidney cells have shown that the
primary effect of CDK1 inactivation is on the spindle dynamics
that regulate chromosome movement and cytokinesis.58 Therefore, an abnormality in the degradation of cyclin B could partly
explain the endomitotic process (multipolar spindle, limited
chromosome segregation, and absence of cytokinesis). Further
experiments on the M phase promoting factor of MK may allow
a better understanding of the endomitosis regulation. It has been
shown very recently in a human megakaryoblastic cell line that
ectopic expression of p21WAF1 induced megakaryocytic differentiation with an increase in ploidy, suggesting that CDK inhibitors may play an important role in the induction of the
endomitotic process.59
In conclusion, we have shown that endomitosis is an abortive
mitosis with an unusual multipolar spherical spindle and that a
functional histone H1 kinase activity associated with cyclin B1
is present in endomitotic MKs. These results have important
implications for the identification of the molecular mechanisms
that regulates the endomitotic process. Characterization of this
process may lead to new strategies to modify endomitosis and to
better control cell proliferation and platelet production.
NOTE ADDED IN PROOF
During revision, a similar observation to that presently
reported has been published using murine MKs stimulated by
Mpl-L60 and results were interpretated as a lack of anaphase B
in endomitotic MKs.
ACKNOWLEDGMENT
The authors thank J.L. Nichol (Amgen, Thousand Oaks, CA) for the
gift of PEG-rhuMGDF and stem cell factor; D. Cossman (Immunex,
3721
Seattle, WA) for GM-CSF; R. McEver (Oklahoma City, OK) for
providing Tab MoAb; J. Pines (Wellcome/CRC Institute, Cambridge,
UK) for the anti-cyclin B1 polyclonal antibody; M. Bornens (Institut
Curie, Paris, France) for the MoAbs against centrosomes and centrioles;
J.C. Brouet (Hôpital St Louis, Paris, France) for the human antibodies
against lamin B and kinetochores; and G.J. Gorbsky (Charlottesville,
VA) for the 3F3/2 MoAb. We are grateful to F. Beaujean (Hôpital Henri
Mondor, Créteil, France) for providing the cytapheresis samples and to
J.C. Châtain (Bionis, Clamart, France) and R. Hellio (Institut Pasteur,
Paris, France) for confocal microscopy assistance. We are indebted to S.
Burstein for improving the English manuscript.
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1998 91: 3711-3723
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
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