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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3711-3723
Endomitosis of Human Megakaryocytes Are Due to Abortive Mitosis
By
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.
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ABSTRACT |
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.
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INTRODUCTION |
PLATELETS 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 phases (G1+G2) 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 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
CD34+ 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.
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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  and  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 CD34+ cells.
CD34+ 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.
CD34+ 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
CD34+ 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
CD34+ cells.
CD34+ 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 -Minimum Eagle Medium
(-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 granulocyte-macrophage
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 GM-CSF 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  20°C, and
rehydrated in phosphate-buffered 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 5× 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 (1× PBS without Ca2+ or
Mg2+, 0.1% Triton X-100). The antibodies used included
mouse antibodies (a mixture of 2 MoAbs directed towards and 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
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 20°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 MK-specific 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 CD41+ 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. CD41+ 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 × 105 to 5 × 106 cells lysed in Laemmli buffer (0.125 mmol/L
Tris, pH 6.8, 4% sodium dodecyl sulfate [SDS], 20% glycerol, 10%
mercaptoethanol, 1 µg/mL aprotinin, and 0.02% bromophenol blue)
were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE;
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, 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/( -32P)ATP [10 µCi (-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.
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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 CD34+ 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 three-color 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.
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| Fig 1.
Polyploid MKs have several centrioles.
CD34+ 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 × 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 × 2,000.
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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 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.
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| Fig 2.
Polyploid MKs in prometaphase. Cultures were performed as
described in Fig 1, except that nocodazole (1 µg/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 × 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 × 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 × 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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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 anti-vWF (TRITC) antibodies and the Hoechst
dye correspond to an anaphase with chromosomes having migrated in
proximity of the asters.
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| 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 × 1,000 for [a] through [j] and × 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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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.
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).
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| 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.
|
|
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.
CD41+ 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, 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.
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| 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 anti-cyclin 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 × 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.
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| 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
>4N MKs. The quality of the sorted cells was checked by examination with a fluorescence microscope (original magnification × 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 CD41+ cells before
sorting is shown with 14% >4N 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 >4N 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.
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| 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, >4N MKs. (b) Lane 1, 2N-4N MKs; lane 2, >4N 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, >4N MKs. (d) Lane 1, 2N-4N MKs;
lane 2, >4N MKs.
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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.
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| 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 >4N)
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 ( -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 [(-32P)ATP, histone H1
with control IgG1 immunoprecipitate]. Lane 1, (-32P)ATP
histone H1 with control IgG1 immunoprecipitate as a negative control;
lane 2, 2N-4N MKs; lane 3, >4N MKs; lane 4, unfractionated MKs (5 × 106 cells); lane 5, unfractionated MKs (1 × 106 cells); lane 6, unfractionated MKs (5 × 105 cells).
|
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| |
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 CD34+ 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 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 pseudo-metaphase. 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 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 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 investigators 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 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.
| |
FOOTNOTES |
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.
| |
ACKNOWLEDGMENT |
The authors thank J.L. Nichol (Amgen, Thousand Oaks, CA) for the gift
of PEG-rhuMGDF and stem cell factor; D. Cossman (Immunex, 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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Abstract
Introduction
Abstract