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Cyclin D3 Is Essential for Megakaryocytopoiesis
By Zhengyu Wang, Ying Zhang, Dimitry Kamen, Emma Lees, and Katya Ravid
mitosis, includingS phases interrupted by short gaps. Using
A normal cell cycle in most eukaryotic cells consists of a
immunohistochemistry, we showed that mature megatightly regulated sequence of phases includingDNA synthekaryocytes express the GI phase cyclin and cyclin D3, but
and a gap(GI). In
sis (S) followed by a gap(G2). mitosis (M),
not the mitotic cyclin, cyclin BI. Under culture conditions
the megakaryocytic lineage,the cells undergo endomitosis,
that selectively promote megakaryocytopoiesis. antisense
which involves DNA synthesis in the absence of mitosis,
oligonucleotides designed
to suppress cyclinD3 expression,
thus giving rise to polyploidcells. We aimed at defining
but not sense oligonucleotidesor antisense oligonucleotides
of a continuwhether the megakaryocytic cell cycle consists
to cyclin BI, dramatically suppress endomitosis and abroous S phase or of G l / S phases and at determining which
gate megakaryocyte development. Our results indicate
that
cyclins are involved in this process. Studies were performed
endoreduplicationin megakaryocytes is associated
with IOW
in primary cultures of mouse bone marrow cells. DNA synlevels of or the absence of cyclin BI, whereas progression
thesis in megakaryocytes was followed by determining incorporation of a DNA precursor, bromodeoxyuridine (BrdU), through this process depends on the GI phase for which
into the cells by in situ staining for BrdU. These experimentscyclin D3 is crucial.
showedthat no more than 15% of the recognizable megakar- 0 1995 by The American Societyof Hematology.
yocytes in normal bone marrow are in the process of endo-
T
HE MEGAKARYOCYTE is unique among bone marrow (BM) cells in possessing a polyploid nucleus. This
cell is characterized by its large size, is rare in the BM (about
0.1% of total BM cells), and has a nucleus that contains up
to 32 times the diploid amount of DNA. At some stage after
commitment to the megakaryocytic lineage, megakaryocyte
precursors stop cell division but continue to replicate their
DNA, a process called endomitosis, followed by cytoplasmic
maturation and platelet fragmentation.’ Little is known about
the nature of the cell cycle in this unique cell type and what
regulates it.
In all eukaryotic cells, progression through the cell cycle
is regulated by cyclin-dependent protein kinases’ (CDKs),
which are essential for the start of the S phase: and Cdc2,
which is essential for the start of m i t ~ s i s B-type
.~
cyclins
are synthesized during the S and G2 phases.’ The binding
of cyclin B to Cdc2 induces phosphorylation and activation
of the complex that is essential for the G2 to M transition.6
Activation of Cdc2 at the end of G2 is mediated by the
phosphatase C d ~ 2 5This
. ~ process is followed by activation
of the cyclin destruction system that results in the degradation of cyclin B at the end of mitosis, the release of inactive
Cdc2, and entry into interpha~e.~.’
Cdk2, like Cdc2, is regulated by its association with cyclins. Cdk2 associates with
cyclin E in the middle of G1: followed by formation of a
complex with cyclin A at the start of S phase.” Other studies
also show that cyclin D, which is differentially expressed in
different cell types, can regulate G1 progression via interaction with Cdk2, Cdk4, or
In the present study, we have used immunohistochemistry
to follow DNA synthesis in primary BM megakaryocytes.
Our results indicated that only a small portion of BM megakaryocytes were actively involved in endomitosis, including
a short G1 phase followed by an S phase. We have determined the presence of different cyclins in cells undergoing
endomitosis and used antisense oligonucleotides to show
their role in promoting this unique cell cycle.
MATERIALS AND METHODS
Reagents and animals. FVB mice were purchased from Charles
River Breeding Laboratories (Wilmington, MA). Acetylthiocholine
iodide, bromcdeoxiuridine, fluorodeoxyuridine, adenosine, sodium
citrate, theophylline, and antirat IgG tetramethylrhodamine isothiocyanate (TRITS) conjugate were from Sigma (St Louis, MO). COUBlood, Vol 86, No 10 (November 15). 1995: pp 3783-3788
marinphenylmaleimide was from Molecular Probes Inc (Junction
City, OR). Nylon mesh screens were from Spectramesh, Spectrum
Medical, Inc (Los Angeles, CA). Goat antimouse IgG conjugated
to fluorescein isothiocyanate (FITC), culture media, and sera were
purchased from GIBCO Laboratories (Grand Island, NY). Oligonucleotides and S-oligonucleotides were from Midland Laboratories
(Midland, TX). Mouse antibody to bromodeoxyuridine (BrdU) and
BrdU staining kit were from Zymed Laboratories (South San Francisco, CA). Taq polymerase and polymerase chain reaction (PCR)
reagents were obtained from Perkin Elmer Labs (Foster City, CA).
Crystal Mount was from Fisher Scientific (Pillsburgh, PA). Mouse
monoclonal antibody (MoAb) to cyclin B1 was from Pharmingen
(San Diego, CA). Rat MoAbs to cyclin D3 or cyclin D1 (both
recognizing mouse proteins) were from Oncogene Science (Uniondale, NY). Hematopoietic growth factors were from R&D Systems
(Minneapolis, MN).
Cultures of BM cells. Mouse BM cells were isolated from the
femurs as described before,l4.l5except that deoxyribonuclease was
omitted from the preparation buffer. Cells were cultured in 5% CO,
at 37°C in a liquid culture under conditions that were shown before
to support maturation and ploidy of primary megakaryocyte^.'^^'^
The cells were cultured in the presence of Iscove’s modification of
Dulbecco’s medium (IMDM) and 20% horse serum, unless otherwise indicated, supplemented with penicillin (2,000 U/mL), streptomycin (200 p&mL), and L-glutamine (0.592 mg/mL).
DNA synthesis. Mouse BM cells were cultured for overnight,
as described above. Subsequently, 10 pmoVL BrdU and 1 pmoVL
fluorodeoxyuridine were added to the medium. At different time
points, cells were collected and spun down with a cytospin on a
polylysine-coated slide. Exposure of cells to BrdU at this concentration for 24 hours is not toxic to cells.’6The cells were fixed in 75%
From the Department of Biochemistry, Cancer Research Center,
Boston University School of Medicine, Boston, MA: and the Massachusetts General Hospital Cancer Center, Charlstown, MA.
Submitted February I, 1995; accepted July 18, 1995.
Supported by NIHLBI Grant No. HL53080-02 to K.R. K.R. is an
Established Investigator of the American Heart Association.
Address reprint requests to Katya Ravid, DSc, PhD, Biochemistry
K225, Boston University School of Medicine, 80 East Concord St,
Boston, MA 02118.
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.
0 1995 by The American Society of Hematology.
0006-4971/95/8610-0015$3.00/0
3783
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3784
WANG ET AL
ethanol for 15 minutes at 4°C and then washed twice with phosphatebuffered saline (PBS; 136mmoVLNaC1/8mmoVL
NazHPOd2.6
mmoI/L KC1/1 .4 m m o m KH2PO4,pH 7.4). Incorporation of BrdU
to the cells was determined by using the Zymed BrdU staining kit
(Zymed Laboratories). Fixed cells were stained with an antibody to
BrdU followed by staining with a strepavidin-biotin system, all as
described by the manufacturer. This method was shown before to
be sensitive, detecting DNA synthesis in normal or malignant cells
in S phase." We used as positive controls intestine in S phase
mounted on a slide, which was provided by the manufacturer in the
BrdU staining kit.
Immunohistochemistry. Cytospun BM cells were fixed and
treated, as described by the Zymed BrdU staining kit, with 5% goat
serum for 15 minutes at room temperature to block nonspecific
binding. The serum was drained (not washed) and the cells were
incubated with 200 p L of PBS containing one of the following first
antibodies, as indicated: 1 p L of mouse MoAb to human cyclin B 1 ;
2 pL of rabbit polyclonal antibody to human cyclin B (gift of Dr
Tony Hunter); 1 pL of mouse MoAb to BrdU; 1 pg of rat MoAb
to mouse cyclin D3 or D l . All of these antibodies cross-react with
the corresponding proteins from mouse (Tony Hunter [The Salk
Institute, San Diego, CA], personal communication [November
19941 and as tested by the manufacturers). Cross-reactivity of human
cyclin B1 antibody with mouse cyclin B was also tested by us on
a mouse cell line (data not shown). The cells were incubated for 1
hour at room temperature with the first antibody, washed three times
with PBS, and incubated for 1 hour at room temperature with a
secondary antibody, either antirat, antirabbit, or antimouse IgG conjugated to a fluorochrome. The slides were washed three times with
PBS, mounted with Crystal Mount, and examined with phase and
fluorescent microscopes.
AntisenseoligonucleotidesinculturesofBMcells. Phosphothioester oligonucleotides (S-oligos) were ethanol-precipitated, washed
twice with 70% ethanol, and dissolved in sterile PBS at a concentration of about 10 mmol/L. BM cells were depleted of megakaryocytes
by filtration through a 250-pm mesh filter followed by passing the
eluent through a 17-pmmesh filter. This procedure resulted in depletion of more than 90% of the megakaryocytes in BM, as indicated
by staining with the megakaryocyte-specific marker, acetylcholinesterase.'* The BM cells were cultured for 16 hours at a concentration
of 3 X IO3 cells/mL medium in l-cm diameter plates in the presence
of IMDM and dialyzed and heat-inactivated (30 minutes at 56°C)
20% fetal bovine serum supplemented with penicillin (2,000 U/mL),
streptomycin (200 pg/mL). and L-glutamine (0.592 mg/mL). Under
these culture conditions, high ploidy megakaryocytes are easily iden'~
were added to the culture at
tified 3 days after c ~ l t u r i n g .S-oligos
a concentration of 15 pmol/L; 24 hours after the first addition, additional oligomers were added at a concentration of 10 ymol/L. Mouse
BM cells cultured for 3 days in the presence or absence of the
indicated oligomers were collected and stained for viability with
trypan blue, counted with a hemocytometer, and subjected to acethylcholinesterase assay or to immunohistochemistry. Protein was determined as described hefore."
Acethylcholinesteruse uctivity. Acethylcholinesterase activity
was determined fluorometrically in cell lysate using acetylthiocholine iodide as a substrate in conjugation with coumarinphenylmaleimide, as described by Ishibashi et al,?" or by in-situ staining of cells
spun down on slides using acetylthiocholine iodide as a substrate."
RESULTS
DNA synthesis in BM megakaryocytes. DNA synthesis
in BM megakaryocytes was followed by determining incorporation of a DNA precursor, BrdU, into the cells. To this
end, mouse BM cells werecultured in the presence of BrdU
for up to 24 hours in a liquid culture system that promotes
megakary~cytopoiesis.'~."
The BM cellswere stained at different time points in situ with an antibody to BrdU and the
percentage of megakaryocytes activelysynthesizing DNA
was determined. Megakaryocytes were easily identified on
the basis of size and morphology. These experiments were
performedonnonsynchronized
cells becauseany attempt
to synchronize primary BM cells, eg, serum
starvation or
lovastatin,2' resulted
in
major
cell
death. To ascertain
whether DNA synthesis in megakaryocytes is continuous or
interrupted by a gap, we determined the percentage of cells
that were BrdU-positive as a function of time of exposure
to BrdU. After 30 and 60 minutes of incubation, 7% and
IO%, respectively, of the megakaryocytes were BrdU-positive. After 90 minutes of incubation and at following successivetime points (3, 6, 16, or 24 hours of incubation), a
maximum of 15% of the recognizable megakaryocytes were
BrdU-positive, whereas most of the large mature cells were
BrdU-negative (Fig 1). The time taken to observe a maximal
percentage of cells that are BrdU-positive is approximately
the length of the gap phase preceding the S phase. Cells in
~~~
Fig 1. DNA synthesis in BM megakaryocytes. Mouse BM cells werecultured in a liquid culture for 24 hours in the presence of BrdU, all as
described in the Materialsand Methods. Cells were spun
down on a slide and stained with antibody to BdrU followed by a peroxidase staining.
containing about
Results arefrom one representative experiment of
three performed (A). In every experiment,two slides were analyzed, each
1.5 x I O 5 cells. Arrows point to some megakaryocytes of different sizes. Megakaryocytes were identified on the basis of morphology with a
phase-contrast microscope underhigh magnification (B). Bar: 100 p m (A), 20 p m (B).
Fig 2. Expression of cyclin proteins. Cytospun BM cells (A) were subjected to double immunofluorescence staining with a mixture of rat
antibody to cyclin D3 and mousa antibody to cyclin B, followed by reaction with a mixture of antirat Ig (IgG) conjugatedto TRITS (B; cyclin
D3) and antimouse IgG conjugated to FITC(C; cyclin B). In a separate experiment, a reaction with rabbit polyclonal antibody to cyclin B1
followed by staining with antirabbit IgG yielded a
result identical to the one prerrentedin (C) (datanot shown). As a control, cells were stained
only with antirat IgG conjugatedto TRITS (D). Cytorpun BM cells (E) were also subjectedto immunofluorescence staining with a rat antibody
to TRiTS (F;cyclin Dl). Cells were analyzed
with a phase-contrast microscope
to cyclin D1 followed by areactionwith antirat Ig (IgG) conjugated
(A and E)and a fluorescent microscope
(B, C, D, and F). The results are representatives of examinationof two experiments, with each having
0.8 to 2.0 x I O 5 cells analyzed per slidein triplicate. The arrows point to a megakaryocyteidentified on the basis of morphology.Bar, 40 pm.
Fig 3. Effect of cyclin D3 antisense and sense oligomers on megakaryocytes.A comparison analysisof cDNA sequences of murine cyclins
Dl, D2, and D3 on the GenBank database'2M," showed few regions with low homology. Sequencesin a low-homology region (AI are presented
5' to 3', with the number onthe left indicating the position in relation to the translation start. Mouse BM cells werecultured in the absence
(B1 or presence (C) of cyclin D3 antisense oligomer (5'-CAGGCGGAGTAAACTCT-3'),which was designedto hybridize to mRNA in the region
cytospunand stainedfor acetylcholinesterase
shown in (Al. or in the presence ofthe corresponding cyclin D3 sense oligomer (D). The cells were
activity (giving rise to a brown product), followed by staining with hematoxylin. Culture conditions and other details are described in the
Materials and Methods. Theresults are from one representative experiment offive performed. Bar, 100 pm.
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E
D
3
A
Cyclin D l
21 1,
GAGCCATGCTTAAGACT
Cyclin D2
81,
AGAACCTGTTGACCATC
Cyclin D3
169,
AGAGITTACTCCGCCTG
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3786
the early stage of the gap phase will join the BrdU-positive
population at the end of G1 phase, upon which a maximal
number of cells will become BrdU-positive. Based on this
rationale, we conclude that the G1 phase is less than 90
minutes in length. We also conclude that the majority of the
mature megakaryocytes in normal BM are beyond the process of DNA synthesis and ploidization. These results represent averages derived from six preparations, each of about
150,000 cells, from three different experiments, with a standard deviation of up to 2%. A total of approximately 900
megakaryocytes were examined. In other experiments, in
which the mature megakaryocytes were filtered out of the
BM (see Materials and Methods) before culturing in the
presence of BrdU, BrdU was incorporated into the majority
of the newly developing megakaryocytes (data not shown).
Determination of expression of different cyclins in megakaryocytes. D-type cyclins regulate G1 progression,whereas
cyclin B is essential for mitosis in different eukaryotic
Because of the rarity of megakaryocytes in BM,
we used immunohistochemistry to detect the presence of
these cyclins in individual cells. As shown in Fig 2, megakaryocytes stained strongly with an antibody to cyclin D3
but not with cyclin B antibody. About 10% of the megakaryocytes displayed staining with anticyclin D3, a value corresponding to the percentage of cells undergoing endomitosis
(Fig 1). Positive staining with either cyclin B or cyclin D3
antibodies was occasionally observed in nonmegakaryocytes
(Fig 2B and C). An antibody to cyclin Dl weakly stained
about 10% of the megakaryocytes (Fig 2F). The intensity
of staining observed in different preparations reacted with
anticyclin D1 varied, but was consistently weaker than the
staining displayed with anticyclin D3. These results represent the examination of several fields, each containing 0.8
to 2 X IO5 cells, as detailed in Fig 2. The unavailability of
an antibody for cyclin D2 that exhibits staining in immunohistochemistry studies prevented us form exploring the presence of this cyclin in megakaryocytes.
The use of antisense oligonucleotides to determine the role
of cyclin D3 in endomitosis. Because cyclin D3 appeared to
be highly expressed in megakaryocytes, antisense oligomers
were used to determine its role in promoting megakaryocytopoiesis. A synthetic 17-mer oligonucleotide designed to hybridize with unique sequence elements in cyclin D3 mRNA
(Fig 3A) was added to a culture of primary BM cells depleted
of megakaryocytes. Mature megakaryocytes were removed
from the culture before the addition of the antisense oligomers to follow-up potential effects on early and later stages
of endomitosis. Under these culture conditions, the megakaryocytes develop from precursors and undergo endomitosis
to form high ploidy cells that survive in the culture for more
than 5 days.I4*lS
Immunohistochemistry of BM cells was used
to establish that cyclin D3 protein was inhibited by treatment
with the antisense oligomer. Quantitative analyses by Westem or Northern blots were not practical to perform, because
megakaryocytes represent less than 1% of total BM cells.
Megakaryocytes were identified by morphology and by
in-situ staining for acetylcholinesterase, which was uniquely
expressed in murine megakaryocyte^.'^ Antisense to cyclin
D3 (ASD3) caused a 75% decrease in the total number of
megakaryocytes and resulted in the emergence
of small mega-
WANG ET AL
karyocytes, as compared with nontreated cells or cells cultured with sense oligonucleotides (SD3) (Fig 3B, C, and D).
Also, pulse-labeling experiments with BrdU (for 2 hours) in
BM cells cultured for 48 hours in the presence or absence
of ASD3 showedthat 2% and 12%, respectively, of the
recognizable megakaryocytes synthesized DNA. These results indicated that cyclin D3 is essential for thenormal
progression through the cell cycle in megakaryocytes. The
level of acetylcholinesterase activity in cell lysates prepared
from BM cells correlated well with the in-situ staining for
this enzyme, ie, acetylcholinesterase activity was significantly reduced in cells treated with ASD3 but not in cells
exposed to an antisense oligonucleotide designed to suppress
expression of cyclin B (Table 1). Results similar to those
described above were obtained with a different cyclin D3
antisense oligonucleotide designed to hybridize with mouse
cyclin D3 mRNA along 17 bp immediately downstream to
the translation start (results not shown). The antisense oligonucleotides did not significantly affect the total number of
BM cells in culture. Because our culture conditions were
originally selected to selectively promote megakaryocytopoiesis and less the proliferation of other BM cells," these
studies described above did not exclude the possibility that
ASD3 or ASB had the potential to abrogate the cell cycle
of other subtypes ofBM cells. Indeed, in the presence of
hemopoietic growth factors (interleukin-3 [IL-31, [L-6,
erythropoietin, and granulocyte-macrophage colony-stimulating factor) that support growth of myeloid and erythroid
cells in c ~ l t u r e , ' ~the
. ' ~total number of BM cells decreased
when exposed to ASD3 orASB (Table l), indicating that
the mitotic cell cycle in some other lineages depends on
cyclins D3 or B. As also noticed in Table 1, in the presence
of hemopoietic growth factors, ASB abrogated megakaryocytopoiesis to some extent. This is likely to be caused by an
inhibitory effect of ASB on growth factors-induced conversion of precommitted cells (undergoing a cyclin B-dependent
mitotic cell cycle) to megakaryocytes.
DISCUSSION
The megakaryocyte is an unusual BM cell characterized
by its ability to synthesize DNA in the absence of mitosis,
thus forming a polyploid nucleus. Ploidy analyses of normal
murine BM cells show a reproducible pattern of ploidy with
a fixed ratio between megakaryocytes of different ploidy
classes, including less than 5% 2N (diploid), 17% 8N, 61%
16N, 19% 32N, and 0.7% 64N ceIls.'4.22It is not certain yet
whether polyploid megakaryocytes enter prophase as well
as metaphase, which involves chromosome condensation and
spindle formation in the absence of anaphase, or rather skip
most of the stages in M phase to directly enter a GI phase.
Early ultrastructural studies suggested that megakaryocytes
undergo metaphase during endomitosis; however, there was
no clear reference to nuclear breakdown." Thus, although
further electron microscopic analyses of different stages of
mitosis in megakaryocytes are underway, we aimedat examining the role of different cyclins in promoting endomitosis.
Basic questions regarding cell cycle regulationin the megakaryocytic lineage have been difficult to
address because of
thelackof a pure megakaryocytic cell line that mimics the
full developmental processof the megakaryocytic lineage. The
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3181
CYCLIN D3AND MEGAKARYOCYTOPOIESIS
currentlyavailablecelllinesexhibitmultilineagepmperties
andor lowploidy states orrequireaddition ofmultipotent
agents suchas phorbol esters to induce ploidy.w27
In the current
study,weusedprimarycultures
of mouseBMcellsunder
conditions found before"^'^ to promote megakaryocyte ploidy
to study some aspects of the megakaryocytx cell cycle. Because megakaryocytes are rare in the
BM, comprising only
0.1% of total BM cells, we used in-situ staining to observe
DNA synthesis. Experiments involving the usage of the DNA
precursor BrdU showed that only a maximum of 15% of all
mouse BM megakaryocytes were actively synthesizing DNA.
The rest of the megakaryocytes, which were mostly large
mature cells, were in a permanent resting phase. Itnotisclear yet
which gene product is involved in turning off DNA synthesis
inthesematuremegakaryocytes
in normalBM.Cellcycle
inhibitors in other systems, suchas the p27 protein that mediates signals of transforming growth factor p and inhibits entry
into S phase or the p21 protein,28r" mayplay aroleinthe
megakaryocytic cell cycle. We are currently pursuing a lineof
investigation to explore the potential role ofsuch cell cycle
inhibitors in megakaryocytes.
Ode11 et a130determined the labeling index of megakaryocyte endomitotic figures after a single injection of radiolabeled thymidine to rats. These investigators concluded that
the length of the S phase in megakaryocytes is about 7 to 8
hours and that the generation cycle time, which was defined
as the time required for one complete round of DNA synthesis and endomitosis, was 9.3 hours. These findings are consistent with our estimation of the length of the gap phase
preceding DNA synthesis in mouse BM megakaryocytes as
being less than 90 minutes in length. It is of interest to note
that a shortened cell cycle consisting of GUS phases was
also observed in some cells of Drosophila embryo^.^' In the
current study, we aimed at exploring the role of different
cyclins in promoting the endomitotic cell cycle in megakaryocytes. Immunohistochemistry was used to show that the G1
phase cyclin, cyclin D3, was highly expressed in megakaryocytes. Experiments involving the use of antisense oligonucleotides were performed to determine the dependancy of the
megakaryocytic cell cycle on cyclin D3 and, hence, on the
G1 phase. Antisense oligonucleotides to different cyclins
were successfully used to suppress cyclins in other systems.32
Our data indicated that antisense oligomers to cyclin D3
abrogated megakaryocyte development. These results further
pointed to the importance of the G1 phase for normal progression through the endomitotic cell cycle in megakaryocytes. According to a preliminary report, cyclin D3 was
highly expressed also in a human erythroleukemia cell line
(HEL) induced to ploidize by phorbol esters.33D-type cyclins
are differentially expressed in various cell lineages ina
highly growth-factor-dependent manner." Thus, it would
be interesting to explore the inducibility of cyclin D3 in
megakaryocytes by the potent megakaryocytic factor, thrombopoietin, which was recently purified and characterized?""
During a normal cell cycle, S phase progression depends
on completion of the M phase.37However, certain treatments,
such as inhibitors of protein kinases in mammalian cells or
high level of the protein encoded by ruml, which inhibits
the mitotic kinase in fission yeast, block M phase and induce
repeats of S p h a ~ e . ~In
' . ~certain
~
Drosophila cells, endore-
Table 1. Effect of Cyclin D3 Antisense Oligonucleotidm
on Megakaryocytopoiesir
Additions
Experiment 1
None
ASD3
ASB
Experiment 2
None
None
ASD3
ASB
Total BM Cells 1%)
Acetylcholinesterase
Activity
100
100
81
100
26
93
100
175
105
96
100
130
46
98
1%)
BM cells were cultured in the presence of antisense oligonucleotides to cyclin D (ASD3; described in the legend to Fig 4) or in the
presence of antisense oligonucleotides to cyclin B (ASE) (5"TCCTAGTGACCCTGAGCG-3'). designed to hybridize to cyclin B mRNA immediately downstream to the translation start." Acetylcholinesterase
activity, a unique marker for megakaryocytes, was determined as fluorescence emission per microgram of protein. The results in experiment 1 are of a representative experiment shown in Fig 4. In the five
experiments performed, the highest percentage of activity observed
with ASD3 was 35%. The results in Experiment 2, except those for
None, represent data obtained in the presence of a cocktail of growth
factors that support proliferation of myeloid and erythroid cells in
c u l t ~ r e , including
'~
erythropoietin (1 U/mL), interleukin-3 (IL-3; 5 ng/
ml), IL-6 (5ng/mL), and granulocyte-macrophage colony-stimulating
factor (5ng/mL). These data are representative of two experiments.
Other details are described in the Materials and Methods.
duplication appears to be associated with the lack of cyclin
B protein.@It has been suggested that the absence of cyclin
B in these Drosophila cells prevents M phase and, as a
consequence, redirects the cell back to a repeated round of
S phase. As also recently reported, the metaphase I1 arrest
in mouse oocytes is controlled by destruction of cyclin B.4'
In accordance with these reports is our observation that
mouse megakaryocytes undergoing endomitosis either lack
or contain a low level of cyclin B1. It is yet to be explored
whether low-level transcriptiodtranslation or rather rapid
degradation regulates the level of cyclin B in polyploid
megakaryocytes. Although we were unable to directly determine Cdc2 kinase activity in megakaryocytes, in all eukaryotic cells tested so far the activation of this mitotic kinase
depends on availability of high levels of cyclin B.6 O'Conne11 et a14' suggested that different stages of mitosis may be
regulated by different kinases, ie, condensation of chromatin
is regulated by a NIMA kinase homologue whereas other
mitotic events are regulated by Cdc2 kinase. It is plausible
then that early stages of mitosis occur in megakaryocytes
undergoing endomitosis. Because resting cells of all lineages
lack cyclin B and yet the cells do not ploidize, we suggest
that reduced levels of cyclin B 1 alone may not be sufficient
to drive endomitosis, but, in conjugation with an increase in
the G1 phase-promoting cyclin, cyclin D3, endomitosis in
megakaryocytes progresses normally.
ACKNOWLEDGMENT
We thank Tony Hunter for a valuablediscussion, Paul Toselli for
assistance with the fluorescent micrographs, and Denise M. Revah
for typing the manuscript.
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3788
WANG ET AL
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1995 86: 3783-3788
Cyclin D3 is essential for megakaryocytopoiesis
Z Wang, Y Zhang, D Kamen, E Lees and K Ravid
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