* Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan; and Department of Dermatology, Nagoya University School of Medicine Showa-ku, Nagoya 466, Japan
Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.
MEGAKARYOCYTES are unique among mammalian
marrow cells in that they leave the diploid (2N)
state to differentiate, synthesizing 4-64 times
the normal DNA content (Odell et al., 1970 The process of polyploidization has been thought to be
mechanistically divided into three steps: endomitosis, endoreduplication, and nuclear restitution (Therman et al.,
1983 Past studies on megakaryocyte differentiation and platelet production have been hampered because of the rarity
of megakaryocytes in bone marrow, the lack of megakaryocyte-specific differentiation factor, TPO, and the lack of
a useful polyploidization-inducible megakaryocytic cell
line. Some erythroleukemic and/or megakaryocytic cell
lines exhibit megakaryocytic markers (Tabilio et al., 1983 We therefore attempted to clarify the molecular and cellular mechanism of TPO-induced polyploidization of megakaryocytes in vitro by immunofluorescent microscopic studies. We found that during polyploidization, multiple spindle
poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during
prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. The two spindle poles in
anaphase were located in close proximity during anaphase,
so that the reassembling nuclear envelope may enclose all
the sister chromatids in a single nucleus at anaphase, followed by the skipping of telophase and cytokinesis. We
thus found that polyploidization is not simply caused by
skipping mitosis, and here we discuss possible molecular
mechanisms of TPO-induced polyploidization in bone marrow megakaryocytes.
Antibodies
A monoclonal antibody specific to Preparation of Megakaryocytes
Bone marrow cells were freshly prepared from BDF1 mice (6- to 8-wk-old
females) by flushing marrow cavities with Iscove's modified Dulbecco's
medium (IMDM) through 26-gauge needles. Cells (1 × 106 cells/ml) were
washed and cultured with bone marrow stromal cells for 2 wk in IMDM
containing 10% FCS and the recombinant mouse TPO (50 U/ml) as described previously (Nagahisa et al., 1996 Indirect Immunofluorescence Microscopy
Smear samples of cultured megakaryocytes were fixed with 100% methanol for 2 min at room temperature and then washed with PBS for 10 min
at room temperature. Cells on coverslips were stained with primary antibodies for 2 h at 37°C. After rinsing with PBS five times, secondary antibodies were applied for 1 h at 37°C. The coverslips were washed with PBS
five times and mounted in 90% glycerol/PBS containing 0.1% p-phenylenediamine and 0.2 µg/ml 4 Multiple Mitotic Spindle Poles Were Formed during
Polyploidization of Bone Marrow Megakaryocytes
To study the TPO-induced polyploidization process of primary megakaryocytes, we immunostained a number of
bone marrow megakaryocytes, which were cultured in the
presence of TPO for 2 wk, with antibodies against various
kinds of intracellular proteins regulating mitosis. A normal
cell division requires a functional bipolar spindle, but how
mitotic spindle poles are organized during polyploidization of megakaryocytes has not been characterized. Therefore, first of all, we stained the megakaryocytes with anti-
We next stained the centrosomes with antibody against
In large, matured megakaryocytes that have multilobed
nuclei and are just ready to form proplatelets, however, no
mitotic spindle pole was recognized by anti- Nuclear Membrane Is Broken Down
during Polyploidization
As described above, megakaryocytes were found to enter
mitosis. In normal mammalian cells, concomitant with this
entry into mitosis, the nuclear envelope breaks down and
disappears, and then upon exit from mitosis, the nuclear
envelope reassembles to form the nucleus. It has been postulated, however, that polyploidization was caused by the
skipping of mitosis after each round of DNA replication
(Long, M.W. 1993. 8th Symposium of Molecular Biology of Hematopoiesis. 196; Datta et al., 1996
Characterization of Mitosis during
Megakaryocyte Polyploidization
We next studied how mitosis progresses during polyploidization of megakaryocytes. Mitosis is classically described
as consisting of five major phases: prophase, prometaphase,
metaphase, anaphase, and telophase.
The first sign that a cell is about to enter mitosis is a period called prophase. Fig. 3 A, a, shows that the chromatin,
which was diffuse in interphase, slowly condenses into
well-defined chromosomes in a polyploidizing megakaryocyte. The cytoplasmic microtubules that were part of the
interphase cytoskeleton disassemble, and the main component of the mitotic apparatus, the mitotic spindle, begins
to form (Fig. 3 A, b-d). Anticentriole antibody (Fig. 3 A,
c) and anti-
Prometaphase starts abruptly with disruption of the nuclear envelope. Stainings of a megakaryocyte in prometaphase
with DAPI showed the condensation of the chromosomes
(Fig. 3 B, a). Stainings with anti- In metaphase, the chromosomes move to points equidistant from the poles. To learn how the chromosomes align
in polyploid megakaryocytes in mitosis, we stained the
cells with DAPI (Fig. 3 C and D, a) and anti- Anaphase begins abruptly as the paired kinetochores on
each chromosome separate, allowing each chromatid to be
pulled toward the spindle pole it faces. Two categories of
movement can generally be distinguished. In anaphase A,
the sister chromatids separate from each other and move
toward the poles. In anaphase B, the polar microtubules
elongate and the two poles of the spindle move farther
apart. To see the megakaryocytes in anaphase, we stained the separating sister chromatids with anticentromere antibody (Fig. 3 E, c), which recognizes a specific centromere
DNA sequence. Anticentromere antibody staining clearly
showed that many bundles of the sister chromatids (c)
were moving toward multiple centrosomes (b-d), indicating that anaphase A is entirely visible in polyploidizing megakaryocytes. It was observed, however, that the pair of
spindle poles in anaphase were located in closer compared
with the other cell types and that they remained stationary
and did not move farther apart as the other cell types normally behaved during anaphase. In addition, we never observed the megakaryocytes in telophase or cytokinesis.
To see the chromosome movement in more detail, we
looked further at the polyploidizing megakaryocytes stained
with anticentromere antibody, anti-
These observations indicate that the polyploidizing
megakaryocytes actually progress through normal prophase,
prometaphase, metaphase, and up to anaphase A, but not
to anaphase B, telophase, or cytokinesis, and that the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus because of the lack of outward
movement of the spindle poles during anaphase. It is now
obvious that polyploidization of megakaryocytes is not simply due to the skipping of mitosis, abnormal chromosome arrangement, or abnormal number or location of
centrosomes.
The hypothesis that G1-S-G2-G1 phases continue without
entry into mitosis during polyploidization of megakaryocytes has been propounded on the basis of results obtained
by chemical-induced polyploidization of megakaryocytic
cell lines (Long, M.W. 1993. 8th Symposium of Molecular
Biology of Hematopoiesis. 196; Datta et al., 1996 Our findings suggest that polyploidizing megakaryocytes have a unique regulatory mechanism in anaphase.
One possibility is that factors regulating anaphase B, such
as microtubule motor proteins, might be involved in this
polyploidization process since we observed that the pair of
spindle poles in anaphase were located in close proximity
to each other, probably because of the lack of outward
movement of spindle poles during anaphase. In normal cells, the polar microtubules elongate much more, and a
nuclear reformation occurs by the fusion of vesicles bound
to daughter chromosomes and separately encloses each set
of daughter chromosomes at telophase. In megakaryocytes, however, sets of daughter chromosomes are not separated far enough to enclose an individual set. Consequently, the reassembling nuclear envelope may enclose
all the sister chromatids into a single nucleus at anaphase,
thereafter skipping telophase and cytokinesis. Recent
work shows that a number of cellular functions are carried
out by various types of kinesin and kinesin-related motor
proteins, and members of four of the eight kinesin subfamilies play crucial roles in cell division (Moore and Endow,
1996 Taken together, a model of the polyploidization process
of megakaryocytes is shown in Fig. 5. Here, we describe a
megakaryocyte polyploidizing from 4N to 8N as an example (lower panel). Two centrosomes duplicate to form four
centrosomes in a cell, and the cell normally enters the first
step of mitosis, prophase. The chromatin condenses into
chromosomes, and the mitotic spindles are formed. The
nuclear envelope is normally disrupted, and mitotic spindles enter the nucleus at prometaphase. At metaphase,
two planes of the aligned chromosomes cross at right angles, and four pairs of spindle poles are beautifully aligned
between the crossed chromosomes. In anaphase, the sets
of sister chromatids separate each other and move toward
each pole. However, the pair of spindle poles in this stage
is located at a closer distance than normal cells (Fig. 5, upper panel), and the pair of spindle poles stay fixed and do
not move farther apart as normal cells do. The reassembling nuclear envelope encloses all the sister chromatids
into one nucleus because of the lack of outward movement
of the spindle poles during anaphase. Without telophase
or cytokinesis, the cell with ploidy 8N goes into another
round of cell cycle.
Replication in eukaryotic cells is precisely regulated so
that all the DNA is replicated once during a single S phase
(Laskey et al., 1989 It has been postulated that polyploidization of megakaryocytes is caused by skipping mitosis either as a result
of reduction of cyclin B and/or Cdc2 or by diminished kinase activity of the complex (Gu et al., 1993 Endomitosis is generally defined as reproduction of nuclear elements not followed by chromosome movement or
cytoplasmic division and is morphologically characterized
as occurring within an intact nuclear envelope, while there
is some degree of mitotic structural change. This definition
has also been used for a mechanism of polyploidization of
megakaryocytes (Ebbe, 1976 The regulation of polyploidization of megakaryocytes is
totally enigmatic, as is its biological significance. It is reasonable to assume that some evolutionary advantage derives from the ability to make platelet-producing cells in
this manner, but what this advantage could be is a still
mystery. One might presume that higher-ploidy cells could
produce more platelets than lower-ploidy cells, or that actual production and release is more efficient from a single
large cell than from several smaller ones, but none of these
suppositions has been proven. It is known that megakaryocyte DNA content is related to megakaryocyte cell size
and thus to the eventual number of platelets produced. It
is also clear that megakaryocytes synthesize increased
amounts of DNA before increases in cytoplasmic volume
and cytoplasmic maturation. Here, we described the cellular aspects of TPO-induced megakaryocyte polyploidization, and the mechanism of polyploidization can now be
investigated at molecular levels. The results shown here
will be helpful in understanding the unresolved puzzles regarding polyploidization.
) in a single cell. Although this process is initiated after the proliferative phases of development, it precedes development of
the earliest morphologically recognizable cell, the megakaryoblast (Long et al., 1982a
,b; Williams and Jackson,
1982
). While DNA replication is abnormal in the sense of
not having a typical 2N-4N cycle, the process of polyploidization in these cells is tightly regulated. With each replicative event, the entire DNA content is duplicated such
that megakaryocytes have multiples of a normal diploid
DNA content (i.e., 4N, 8N, 16N, 32N, etc., where 2N is the
normal nuclear DNA content of a cell in G0/G1 phase of
the cell cycle). In these cells, therefore, the regulation of
DNA synthesis is released from its normal cell cycle control, but global control is retained over the total amount of
DNA replicated. The nature of polyploidy is the key to
understanding the biology of megakaryocytes themselves
and their role in platelet production. However, the regulation of this process remains totally unknown.
). Endomitosis is generally defined as reproduction of
nuclear elements not followed by chromosome movement
or cytoplasmic division and is morphologically characterized as occurring in the presence of an intact nuclear membrane. This definition implies that while there is some degree of mitotic structural change, it occurs within an intact
nuclear envelope. The definition has also been cited as the
mechanism of polyploidization of megakaryocytes (Ebbe,
1976
), although it has not been known whether endomitotic megakaryocytes retain an intact nuclear membrane.
Therefore, it has simply been assumed that polyploidization of megakaryocytes is due to the skipping of mitosis after each round of DNA replication. It is well known that
the cell division of eukaryotes is regulated by a complex with a maturation promoting factor (MPF or Cdc2-cyclin
B complex). The level of Cdc2 is constant throughout the
cell cycle, but cyclin B accumulates during the G2 phase
and is destroyed by proteasome after anaphase (Draetta et
al., 1989
; Solomon et al., 1990
; Hunt et al., 1992
). Therefore, polyploidization of megakaryocytes has been postulated to be caused by either reduction of cyclin B and/or Cdc2 or diminished kinase activity of the complex. It was
indeed reported that primary megakaryocytes and the
phorbol ester-induced megakaryocytic cell line MegT lack
cyclin B (Gu et al., 1993
; Wang et al., 1995
; Zhang et al.,
1996
). Inactivation of the Cdc2-cyclin B complex due to
the marked reduction of Cdc2 was also described in phorbol ester-induced HEL cells (Datta et al., 1996
). It has not
been proved, however, that this hypothesis is applicable
for thrombopoietin (TPO)1-induced polyploidization of
megakaryocytes.
; Ogura et al., 1985
; Sledge et al., 1986
; Greenberg et al.,
1988
; Adachi et al., 1991
; Ravid et al., 1993
; Datta et al.,
1996
; Hudson et al., 1996
; Takada et al., 1996
; Kikuchi et
al., 1997
) but cannot induce polyploidization or require exposure to substances such as phorbol esters to polyploidize. Polyploidization induced by chemical agents is a
widely observed phenomenon, but its mechanism varies
depending on the drugs and cell lines. TPO is a recently identified cytokine that specifically regulates proliferation and maturation of megakaryocytes (Bartley et al., 1994
; de
Sauvage et al., 1994
; Kaushansky et al., 1994
; Kuter et al.,
1994
; Wendling et al., 1994
) and actually stimulates polyploidization of primary immature megakaryocytes in vitro
(Broudy et al., 1995
; Debili et al., 1995
; Angchaisuksiri et
al., 1996
). The availability of TPO and its capacity to induce the proliferation and differentiation of megakaryocyte progenitor cells has allowed megakaryocyte numbers to be expanded in vitro. This culture system has allowed us
to investigate the cellular and molecular aspects of the
polyploidization of these cells.
Materials and Methods
-tubulin was purchased from Sigma
Chemical Co. (St. Louis, MO). A rabbit polyclonal antibody specific to
COOH-terminal peptides of Xenopus
-tubulin, which recognizes mouse
-tubulin as well, was provided by Dr. H. Masuda at RIKEN. Autoantibodies against centromere and centriole were identified with indirect immunofluorescence studies with commercial prefixed HEP-2 cell slides
(Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) as described (Muro et al., 1990
). Anti-RanBP2 antiserum 551 (Yokoyama et
al., 1995
) was provided by Dr. T. Nishimoto at Kyushu University, Fukuoka, Japan, and a rabbit anti-MCM3 antiserum was provided (Kubota et al.,
1994
) by Dr. H. Takisawa at Osaka University (Osaka, Japan). The FITC-
labeled F(ab
)2 fragment was purchased from Zymed Laboratories (San
Francisco, CA) and Cy3-conjugated F(ab
)2 fragment was obtained from
Jackson ImmunoResearch Laboratories (West Grove, PA).
). Recombinant mouse TPO was
prepared from the supernatants of COS-7 cells transfected with mouse
TPO cDNA in expression vector pME18 (Nagata et al., 1995
). In 14 d with
TPO, various stages of megakaryocytes, which had ploidy between 2N and
128N, were produced in the liquid culture, although few were generated
without TPO. Most of the large suspension cells were confirmed to be
megakaryocytes by immunostaining with megakaryocyte/platelet-specific
antibody Pm-1 (Nagata et al., 1995
) and CD61 (PharMingen, San Diego, CA).
,6-diamidino-2-phenylindole dihydrochloride (DAPI). As a negative control, application of the primary antibodies
was omitted from the procedure mentioned above. The cells were observed under a fluorescence microscope (model BX60-34-FLBD1; Olympus Corp., Lake Success, NY) at a final magnification of 1,500, and photographs were taken using Fuji film (ASA 400; Tokyo, Japan).
Results
-tubulin antibody to learn the organization of mitotic
spindles. Surprisingly, we found that multiple mitotic spindle poles were formed in all megakaryocytes in mitosis.
The photograph in Fig. 1 A shows a megakaryocyte forming 32 spindle poles. The number of spindle poles in a
megakaryocyte varies from 4 to 64, or even much more,
but we were unable to count the exact number formed
when it exceeded 32 because of the abundance.
Fig. 1.
Multiple mitotic
spindle poles formation during
TPO-induced polyploidization
of primary megakaryocytes.
Mitotic spindle poles were
detected by immunofluorescent light microscopy in TPO-induced primary mouse megakaryocytes. Megakaryocytes
cultured with TPO were fixed
in methanol for probing with
anti--tubulin antibody (A),
anti-
-tubulin antibody (B),
and anticentriole antibody
(C), followed by incubation
with an FITC-labeled F(ab
)2
fragment (A) or a Cy3-conjugated F(ab
)2 fragment (B
and C).
[View Larger Version of this Image (37K GIF file)]
-tubulin, a well-characterized component of the centrosomes (Zheng et al., 1991
; Joshi, 1994
). Anti-
-tubulin
staining of the megakaryocytes clearly confirmed that
multiple centrosomes were formed in megakaryocytes in
mitosis (Fig. 1 B). In animal cells, the core of the centrosome has a pair of centrioles. Therefore, we also
stained the centrioles with anticentriole antibody (Fig. 1
C) and confirmed that multiple centrosomes were produced in mitotic megakaryocytes. These photographs also
show megakaryocytes bearing 32 centrosomes.
-tubulin antibody staining (data not shown). The immunostaining of the
matured megakaryocytes with antibodies against
-tubulin
and centrioles also confirmed that there were no visible
centrosomes (data not shown), indicating that multiple
centrosomes disappear as megakaryocytes finally mature
for some unknown reason and by an unknown mechanism.
), and thus polyploidization has been thought to occur within an intact nuclear envelope. Whether or not the nuclear membrane
breaks down as megakaryocytes polyploidize has never
been examined. We therefore stained the nuclear membrane in megakaryocytes with anti-RanBP2 antibody
(anti-551). RanBP2 is a nuclear pore complex protein, and
thus anti-551 antibody that specifically recognizes RanBP2
can clearly stain the nuclear envelopes as described
(Yokoyama et al., 1995
). As shown in Fig. 2 A, c, anti-551
antibody staining clearly showed a lobulated nuclear surface of a megakaryocyte in interphase. The whole cell including cytoplasm, however, was stained with anti-551 antibody in a mitotic megakaryocyte forming eight mitotic
spindle poles (Fig. 2 B, c), indicating that the nuclear
membrane was broken down as the megakaryocyte entered
mitosis and was reassembled in interphase. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to the skipping of mitosis and that it
does not occur within an intact nuclear envelope.
Fig. 2.
Nuclear membrane is broken down during polyploidization of megakaryocytes. Megakaryocytes in interphase (A) or in mitosis (B) were stained with DAPI (a), or probed with anti--tubulin antibody (b) or anti-RanBP2 antibody (anti-551) (c), followed by incubation with an FITC-labeled F(ab
)2 fragment (b) or a Cy3-conjugated F(ab
)2 fragment (c). d shows triple stainings of the same cells.
[View Larger Versions of these Images (61 + 62K GIF file)]
-tubulin antibody (Fig. 3 A, b) stainings confirmed that multiple mitotic spindle poles were formed
and assembled outside the nucleus in a megakaryocyte in
prophase, as described above.
Fig. 3.
Polyploidizing megakaryocytes in various stages of mitosis. A number of primary megakaryocytes treated with TPO were
stained with DAPI, anti--tubulin antibody, anti-
-tubulin antibody, anticentriole antibody, or anticentromere antibody. (A) Megakaryocyte in prophase was stained with DAPI (a), anti-
-tubulin antibody (b), anticentriole antibody (c), or all three (d). (B) Megakaryocyte in prometaphase was probed with DAPI (a), anti-
-tubulin antibody (b), anti-
-tubulin antibody (c), or all three (d). (C and
D) Megakaryocytes in metaphase, with centrosomes numbering four (C) and eight (D), were stained with DAPI (a), anti-
-tubulin antibody (b), anticentriole antibody (c), or all three (d). (E) Megakaryocyte in anaphase A was stained with DAPI (a), anti-
-tubulin antibody (b), anticentromere antibody (c), or all three (d).
[View Larger Versions of these Images (31 + 50 + 50 + 36 + 55K GIF file)]
-tubulin (Fig. 3 B, b) and
anti-
-tubulin (Fig. 3 B, c) antibodies demonstrated that
mitotic spindles entered the nucleus and that the microtubules radiated out from the multiple centrosomes, indicating that the nuclear membrane was broken down as described above.
-tubulin antibody (Fig. 3 C and D, b). Here, we show two megakaryocytes forming four (Fig. 3 C) or eight (Fig. 3 D) mitotic
spindle poles. As shown in the figures (a-d), we clearly observed that two or four planes of the aligned chromosomes
crossed at right angles. Staining with anti-
-tubulin (b)
and anticentriole (c) antibodies demonstrated that four or
eight pairs of spindle poles were symmetrically located on
either side of each face of the plate between the crossed
chromosomes (a-d).
-tubulin antibody,
and DAPI at all stages of mitosis (Fig. 4). In interphase,
the centromeres were dispersed in the entire area of the
nucleus (Fig. 4 A). In prophase and prometaphase, the
chromatin condensed into well-defined chromosomes, and
staining of the centromeres (Fig. 4 B) showed that the
chromosomes began to align for metaphase. The multiple
mitotic spindle poles were formed at this stage. Fig. 4, C-E,
shows a megakaryocyte polyploidizing from ploidy 4N to
8N at the stage just before metaphase, metaphase, and
anaphase A, respectively. At the stage just before metaphase, two planes of the aligned chromosomes crossed at right angles, and two pairs of spindle poles were symmetrically located in close proximity to this crossing on either side of
each face of the plate (Fig. 4 C). The centromeres were located around the crossing and moving to points equidistant from the poles. In metaphase, two pairs of spindle
poles moved outward and stretched the sister chromatids
tightly toward the poles; thus, the centromeres were located just in the middle of the crossed chromosome planes
between the two poles (Fig. 4 D). In anaphase A, the sister
chromatids were pulled toward the spindle poles so that
four sets of centromeres were moving toward each pole
(Fig. 4 E). No set, however, could be separated far enough
to be enclosed by individual nuclear envelopes. Fig. 4 F
shows a megakaryocyte polyploidizing from ploidy 8N to 16N in anaphase A. The sets of centromeres were located
close to each centrosome, and none of the sets of chromosomes was separated completed. We found no megakaryocytes in telophase or cytokinesis.
Fig. 4.
Centromere movement during polyploidizing megakaryocytes. TPO-treated primary megakaryocytes were stained with anticentromere antibody (red, first column), anti--tubulin antibody (green, second column), DAPI (blue, third column), and triple staining (fourth column) during mitosis. (A) Megakaryocyte in interphase. (B) Megakaryocytes in prometaphase. (C) Megakaryocyte with
ploidy 8N at the stage just before metaphase. (D) Megakaryocyte with ploidy 8N in metaphase. (E) Megakaryocyte with ploidy 8N in
anaphase A. (F) Megakaryocyte with ploidy 16N in anaphase A.
[View Larger Version of this Image (70K GIF file)]
Discussion
). In this
study, we examined whether or not this hypothesis is applicable to the TPO-induced polyploidization of primary
megakaryocytes. Staining of a number of primary megakaryocytes with various antibodies against intracellular
components regulating cell division clearly demonstrated
that the formation of multiple mitotic spindle poles (centrosomes) and the rupture of the nuclear envelope are required for the polyploidization process of megakaryocytes. We further showed that megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase,
metaphase, and up to anaphase A, but not to anaphase B,
telophase, or cytokinesis. These observations indicate that
polyploidization is not simply caused by skipping mitosis,
and thus the hypothesis described above was proved to be
totally incorrect in naturally occurring polyploidization of
megakaryocytes, although it may be true in chemically induced polyploidization of some cell lines. It is also now
clear that polyploidization is not caused by either abnormal chromosome arrangement nor abnormal number or
location of centrosomes.
; Walczak and Mitchison, 1996
). One of these subfamilies, mitotic kinesin-like protein 1 (MKLP1), causes plus
end-directed sliding of microtubules over one another and
may mediate anaphase B spindle elongation. MKLP1 is a
plus end-directed human kinesein-related motor protein that bundles antiparallel microtubules and slides them
past each other at 4 µm/min, a velocity consistent with
anaphase B spindle elongation in vivo (Nislow et al.,
1992
). The association of MKLP1 with the midbody supports its proposed role in separating poles at anaphase B
by sliding the antiparallel interdigitating nonkinetochore microtubules past each other (Nislow et al., 1992
). Therefore, lack of outward movement of spindle poles during
anaphase B in polyploidizing megakaryocytes might be
due to the disregulation of MKLP1 in mitotic megakaryocytes. The abnormal regulation of polyploidizing megakaryocytes in anaphase B, especially regulation of MKLP1
activity in anaphase B, remains to be explained.
Fig. 5.
Schematic drawing of hypothetical mechanism of TPO-induced polyploidization of megakaryocytes. Upper panel shows
a normal mitosis, and lower panel shows the mitosis of megakaryocytes during polyploidization.
[View Larger Version of this Image (32K GIF file)]
). A licensing factor minichromosome
maintenance 3 (MCM3) has been suggested to regulate
once-per-cell-cycle DNA replication (Hennessy et al.,
1990
; Yan et al., 1993
; Kimura et al., 1994
). MCM3 is localized in the nucleus throughout the whole interphase and is
redistributed in the extrachromosomal region during mitosis. It is also known that MCM3 cannot be detected in the
cells that do not divide. We therefore stained polyploid megakaryocytes with anti-MCM3 antibody and found that
MCM3 was localized in the nucleus in interphase (data not
shown), indicating that these polyploid megakaryocytes
are ready to undergo another round of DNA replication.
It was found to be localized outside the chromatids at early
prophase and dispersed into the cytoplasm during prometaphase and anaphase A (data not shown). These observations suggest that DNA is replicated only once per
cell cycle in polyploidizing megakaryocytes and that the
megakaryocytes do enter mitosis, and the reassembling
nuclear envelope encloses all the sister chromatids in a single nucleus once per cell cycle. We were not able, however, to detect MCM3 either in cytoplasm or in nucleus of
the fully matured megakaryocytes (data not shown), indicating that these completely matured megakaryocytes
would no longer undergo another round of DNA synthesis.
; Wang et al., 1995
;
Datta et al., 1996
; Zhang et al., 1996
). In the TPO-induced
polyploidization process, however, megakaryocytes do enter mitosis, and thus the Cdc2-cyclin B complex must be
fully active during early mitosis. To confirm this, we examined whether the levels of Cdc2 and cyclin B are normally
regulated during polyploidization. Staining with anti-Cdc2 antibody showed that Cdc2 was constantly expressed during all stages of mitosis (data not shown). Staining with cyclin B antibody showed that cyclin B was clearly detected
at early mitosis in megakaryocytes, including those with a
ploidy >8N, and that the expression of cyclin B decreased
in anaphase A (data not shown). While these observations
are inconsistent with those described previously, we concluded that polyploidization is apparently not caused by
the lack of cyclin B since megakaryocytes driven by active
Cdc2-cyclin B complex actually do enter mitosis. On the other hand, however, we could not detect cyclin B in fully
matured megakaryocytes that no longer undergo polyploidization and are ready for proplatelet formation (data
not shown). These matured megakaryocytes had no centrosomes, nor could we detect any MCM3 anywhere in
these megakaryocytes. The phenotypes of the fully matured
megakaryocytes thus seem to be completely different from those of actively dividing normal cells and of polyploidizing megakaryocytes. We speculate that the previous controversial reports may describe only fully matured primary
megakaryocytes or chemically induced cell lines but not
actually polyploidizing primary megakaryocytes. It is still
possible that chemically induced polyploidization is caused
by the lack of Cdc2-cyclin B complex activity.
), although it has not been
known whether endomitotic megakaryocytes retain an intact nuclear membrane. Now we have clearly shown that
the nuclear membrane does break down as megakaryocytes enter mitosis to polyploidize, and that polyploidizing
megakaryocytes progress through mitosis up to anaphase
A. Thus, the term endomitosis was found to be no longer
applicable to the mechanism of polyploidization of megakaryocytes. Other terminology should be used to define
the megakaryocyte polypoidization process.
Received for publication 5 June 1997 and in revised form 7 July 1997.
Address all correspondence to Kazuo Todokoro, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1, Koyadai, Tsukuba, Ibaraki 305, Japan. Tel.: 81 298 36 9075. Fax: 81 298 36 9090. e-mail: todokoro{at}rtc.riken.go.jpWe thank Dr. T. Nishimoto for anti-RanBP2 antibody, Dr. H. Takisawa
for anti-MCM3 antibody, Dr. H. Masuda for anti--tubulin antibody, and
Drs. H. Takisawa, S. Hisanaga, M. Inagaki, K. Matsuzawa, S. Kotani, and
T. Nagasawa for valuable discussions.
This work was supported in part by a grant from the Mitsubishi Foundation, a Special Grant for Promotion of Research from RIKEN, and grants from the Ministry of Education, Science, and Culture of Japan.
DAPI, 66-diamidino-2-phenylindole;
MKLP1, mitotic kinesin-like protein 1;
TPO, thrombopoietin.
1. | Adachi, M., R. Ryo, T. Sato, and N. Yamaguchi. 1991. Platelet factor 4 gene expression in a human megakaryocytic leukemia cell line (CMK) and its differentiated subclone (CMK11-5). Exp. Hematol. 19: 923-927 |
2. | Angchaisuksiri, P., P.L. Carlson, and E.N. Dessypris. 1996. Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system. Br. J. Haematol. 93: 13-17 |
3. | Bartley, T.D., J. Bogenberger, P. Hunt, Y.S. Li, H.S. Lu, F. Martin, M.S. Chang, B. Samal, J.L. Nichol, S. Swift, et al . 1994. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell. 77: 1117-1124 |
4. |
Broudy, V.C.,
N.L. Lin, and
K. Kaushansky.
1995.
Thrombopoietin (c-mpl
ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases
megakaryocyte ploidy in vitro.
Blood.
85:
1719-1726
|
5. | Datta, N.S., J.L. Williams, J. Caldwell, A.M. Curry, E.K. Ashcraft, and M.W. Long. 1996. Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content. Mol. Biol. Cell. 7: 209-223 [Abstract]. |
6. |
Debili, N.,
F. Wendling,
A. Katz,
J. Guichard,
J. Breton-Gorius,
P. Hunt, and
W. Vainchenker.
1995.
The Mpl-ligand or thrombopoietin or megakaryocyte
growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood.
86:
2516-2525
|
7. | de Sauvage, F.J., P.E. Hass, S.D. Spencer, B.E. Malloy, A.L. Gurney, S.A. Spencer, W.C. Darbonne, W.J. Henzel, S.C. Wong, W.J. Kuang, et al . 1994. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature (Lond.). 369: 533-538 |
8. | Draetta, G., F. Luca, J. Westendorf, L. Brizuela, J. Ruderman, and D. Beach. 1989. Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF. Cell. 56: 829-838 |
9. | Ebbe, S.. 1976. Biology of megakaryocytes. Prog. Hemostasis Thromb. 3: 211-229 |
10. | Greenberg, S.M., D.S. Rosenthal, T.A. Greeley, R. Tantravahi, and R.I. Handin. 1988. Characterization of a new megakaryocytic cell line: the Dami cell. Blood. 72: 1968-1977 [Abstract]. |
11. | Gu, X.F., A. Allain, L. Li, E.M. Cramer, D. Tenza, J.P. Caen, and Z.C. Han. 1993. Expression of cyclin B in megakaryocytes and cells of other hematopoietic lineages. C. R. Acad. Sci. III. 316: 1438-1445 |
12. | Hennessy, K.M., C.D. Clark, and D. Botstein. 1990. Subcellular localization of yeast CDC46 varies with the cell cycle. Genes Dev. 4: 2252-2263 [Abstract]. |
13. | Hennessy, K.M., A. Lee, E. Chen, and D. Botstein. 1991. A group of interacting yeast DNA replication genes. Genes Dev. 5: 958-969 [Abstract]. |
14. |
Hudson, K.M.,
N.C. Denko,
E. Schwab,
E. Oswald,
A. Weiss, and
M.A. Lieberman.
1996.
Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins.
Blood.
88:
3465-3473
|
15. | Hunt, T., F.C. Luca, and J.V. Ruderman. 1992. The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo. J. Cell Biol. 116: 707-724 [Abstract]. |
16. |
Joshi, H.C..
1994.
Microtubule organizing centers and ![]() |
17. | Kaushansky, K., S. Lok, R.D. Holly, V.C. Broudy, N. Lin, M.C. Baily, J.W. Forstorm, M.M. Buddle, P.J. Oort, F.S. Hagen, et al . 1994. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature (Lond.). 369: 568-571 |
18. |
Kikuchi, J.,
Y. Furukawa,
S. Iwase,
Y. Terui,
M. Nakamura,
S. Kitagawa,
M. Kitagawa,
N. Komatsu, and
Y. Miura.
1997.
Polyploidization and functional
maturation are two distinct processes during megakaryocytic differentiation:
involvement of cyclin-dependent kinase inhibitor p21 in polyploidization.
Blood.
89:
3980-3987
|
19. |
Kimura, H.,
N. Nozaki, and
K. Sugimoto.
1994.
DNA polymerase ![]() |
20. | Kubota, Y, S. Mimura, S. Nishimoto, H. Takisawa, and H. Nojima. 1994. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 81: 601-609 . |
21. |
Kuter, D.J.,
D.L. Beeler, and
R.D. Rosenberg.
1994.
The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production.
Proc. Natl. Acad. Sci. USA.
91:
11104-11108
|
22. | Laskey, R.A., M.P. Fairman, and J.J. Blow. 1989. S phase of the cell cycle. Science (Wash. DC). 246: 609-614 |
23. | Long, M.W., N. Williams, and S. Ebbe. 1982a. Immature megakaryocytes in the mouse: physical characteristics, cell cycle status, and in vitro responsiveness to thrombopoietic stimulatory factor. Blood. 59: 569-575 [Abstract]. |
24. | Long, M.W., N. Williams, and T.P. McDonald. 1982b. Immature megakaryocytes in the mouse: in vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes. J. Cell Physiol. 112: 339-344 |
25. | Moore, J.D., and S.A. Endow. 1996. Kinesin proteins: a phylum of motors for microtubule-based motility. BioEssays. 18: 207-218 |
26. | Muro, Y., K. Sugimoto, T. Okazaki, and M. Ohashi. 1990. The heterogeneity of anticentromere antibodies in immunoblotting analysis. J. Rheumatol. 17: 1042-1047 |
27. |
Nagahisa, H.,
Y. Nagata,
T. Ohnuki,
M. Osada,
T. Nagasawa,
T. Abe, and
K. Todokoro.
1996.
Bone marrow stromal cells produce thrombopoietin and
stimulate megakaryocyte growth and maturation but suppress proplatelet
formation.
Blood.
87:
1309-1316
|
28. |
Nagata, Y.,
H. Nagahisa,
Y. Aida,
K. Okutomi,
T. Nagasawa, and
K. Todokoro.
1995.
Thrombopoietin induces megakaryocyte differentiation in hematopoietic progenitor FDC-P2 cells.
J. Biol. Chem.
270:
19673-19675
|
29. | Nislow, C., V.A. Lombillo, R. Kuriyama, and J.R. McIntosh. 1992. A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature (Lond.). 359: 543-547 |
30. | Odell, T.T., C.W. Jackson, and T.J. Friday. 1970. Megakaryocytopoiesis in rats with special reference to polyploidy. Blood. 35: 775-782 |
31. | Ogura, M., Y. Morishima, R. Ohno, Y. Kato, N. Hirabayashi, H. Nagura, and H. Saito. 1985. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood. 66: 1384-1392 [Abstract]. |
32. | Ravid, K., D.J. Kuter, D.L. Beeler, T. Doi, and R.D. Rosenberg. 1993. Selection of an HEL-derived cell line expressing high levels of platelet factor 4. Blood. 81: 2885-2890 [Abstract]. |
33. | Sledge, G.W., M. Glant, J. Jansen, N.A. Heerema, B.J. Roth, M. Goheen, and R. Hoffman. 1986. Establishment in long term culture of megakaryocytic leukemia cells (EST-IU) from the marrow of a patient with leukemia and a mediastinal germ cell neoplasm. Cancer Res. 46: 2155-2159 [Abstract]. |
34. | Solomon, M.J., M. Glotzer, T.H. Lee, M. Philippe, and M.W. Kirschner. 1990. Cyclin activation of p34cdc2. Cell. 63: 1013-1024 |
35. | Tabilio, A., P.G. Pelicci, G. Vinci, P. Mannoni, C.I. Civin, W. Vainchenker, U. Testa, M. Lipinski, H. Rochart, and J. Breton-Gorius. 1983. Myeloid and megakaryocytic properties of K-562 cell lines. Cancer Res. 43: 4569-4573 [Abstract]. |
36. | Takada, M., N. Morii, S. Kumagai, and R. Ryo. 1996. The involvement of the rho gen product, a small molecular weight GTP-binding protein, in polyploidization of a human megakaryocytic cell line, CMK. Exp. Hematol. 24: 524-530 |
37. | Therman, E., G.E. Sarto, and P.A. Stubblefield. 1983. Endomitosis: a reappraisal. Hum. Genet. 63: 13-18 |
38. | Walczak, C.E., and T.J. Mitchison. 1996. Kinesin-related proteins at mitotic spindle poles: function and regulation. Cell. 85: 943-946 |
39. |
Wang, Z.,
Y. Zhang,
D. Kamen,
E. Lee, and
K. Ravid.
1995.
Cyclin D3 is essential for megakaryocytopoiesis.
Blood.
86:
3783-3788
|
40. | Wendling, F., E. Maraskovsky, N. Debili, F. Christina, M. Teepe, M. Titeux, N. Methia, J. Breton-Gorius, D. Cosman, and W. Vainchenker. 1994. cMpl ligand is a humoral regulator of megakaryocytopoiesis. Nature (Lond.). 369: 571-574 |
41. | Williams, N., and H. Jackson. 1982. Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells. Cell Tiss. Kinet. 15: 483-494 |
42. | Yan, H., A.M. Merchant, and B.K. Tye. 1993. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes Dev. 7: 2149-2160 [Abstract]. |
43. | Yokoyama, N., N. Hayashi, T. Seki, N. Pante, T. Ohba, K. Nishii, K. Kuma, T. Hayashida, T. Miyata, U. Aebi, et al . 1995. A giant nucleopore protein that binds Ran/TC4. Nature (Lond.). 376: 184-188 |
44. |
Zhang, Y.,
Z. Wang, and
K. Ravid.
1996.
The cell cycle in polyploid megakaryocytes is associated with reduced activity of cyclin B1-dependent Cdc2 kinase.
J. Biol. Chem.
271:
4266-4272
|
45. |
Zheng, Y.,
M.K. Jung, and
B.R. Oakley.
1991.
![]() |