During the endomitotic cell cycle of
megakaryocytic cell lines, the levels of cyclin B1 and the activity of
cyclin B1-dependent Cdc2 kinase, although detectable, are
reduced as compared with megakaryocytes undergoing a mitotic cell
cycle. The levels of cyclin A, however, are comparable during both cell
cycles. The expression of cyclin B1 mRNA is also equivalent in
proliferating and polyploidizing cells. In the current study, we found
that the rate of cyclin B1 protein degradation is enhanced in
polyploidizing megakaryocytes. This finding has led us to further
investigate whether the ubiquitin-proteosome pathway responsible for
cyclin B degradation is accelerated in these cells. Our data indicate that polyploidizing megakaryocytic cell lines and primary bone marrow
cells treated with the megakaryocyte proliferation- and ploidy-promoting factor, the c-Mpl ligand, display increased activities of the ubiquitin-proteosome pathway, which degrades cyclin B, as
compared with proliferating megakaryocytic cell lines or diploid bone
marrow cells, respectively. This degradation has all the hallmarks of a
ubiquitin pathway, including the dependence on ATP, the appearance of
high molecular weight conjugated forms of cyclin B, and inhibition of
the proteolytic process by a mutated form of the ubiquitin-conjugating
enzyme Ubc4. Our studies also indicate that the ability to degrade
cyclin A is equivalent in both the mitotic and endomitotic cell cycles.
The increased potential of polyploid megakaryocytes to degrade cyclin B
may be part of the cellular programming that leads to aborted
mitosis.
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INTRODUCTION |
During the mitotic cell cycle, cyclin B steadily accumulates at
interphase and is rapidly degraded before the cells exit mitosis (reviewed in Ref. 1). Cyclin B degradation is essential for progression
through to the next cell cycle. The specificity of cyclin B degradation
is determined by the 9-amino acid motif conserved among the N termini
of B-type cyclins (cyclin Box) (reviewed in Refs. 1 and 2). Mutation of
the conserved arginine in the cyclin box inhibits ubiquitination of
cyclins in Xenopus extracts, indicating that the cyclin box
serves as a signal for ubiquitination (3, 4). Cyclin A and B2, but not
B1, require binding to p34Cdc2 for their proteolysis (5, 6). While some
components involved in cyclin B ubiquitination and degradation have
been identified, such as the ubiquitin-conjugating enzymes Ubc4 (4) and
Ubc10 (7) and enzymes Cdc16p, Cdc26p, Cdc27p, and Cdc23p in yeast
(reviewed in Ref. 8), much has yet to be explored.
Megakaryocytes undergo a unique cell cycle resulting in DNA replication
in the absence of complete mitosis and cytokinesis. We and others found
that during this cell cycle, DNA synthesis is not continuous, but
rather consists of an S phase (of approximately 9 h) and a gap (of
approximately 1-2 h) (9-11). Studies aiming to investigate the nature
of the gap phase during endomitosis led to the conclusion that mitosis
is initiated but aborted at entry to anaphase (13). It was found that
polyploid megakaryocytes derived from bone marrow cells treated with
the ploidy-promoting factor, the c-Mpl ligand (reviewed in Ref. 12),
can condense their chromosomes when treated with nocodazole (13). In
this study, polyploid megakaryocytes were also found to display early features of prometaphase, such as spindle formation but not any characteristics of anaphase. Because of the rarity of megakaryocytes in
the bone marrow and the inability to obtain a pure population of
diploid megakaryocytes to be used for synchronization and for following
biochemical parameters during the development of high ploidy cells, we
and others have resorted to megakaryocytic cell lines. It was found
that during the endomitotic cell cycle in MegT and HEL cells the
activity of the mitotic kinase, cyclin B1-dependent Cdc2
kinase, is reduced as compared with the activity during the mitotic
cell cycle in these cells (10, 14). Under these conditions, however,
the level of cyclin B1 protein is reduced, while the level of cyclin B1
mRNA is comparable in proliferating and polyploidizing
megakaryocytes (10). Carrow et al. (15) detected cyclin B1
and a cyclin B1- and Cdc2-dependent kinase activities in
nonsynchronized, high ploidy bone marrow megakaryocytes treated with
the c-Mpl ligand, although it has not yet been investigated how the
level of activity of these kinases compares with that in proliferating
diploid megakaryocytes or other bone marrow cells. In this context, it
should be mentioned that while cyclin B levels peak prior to the early
stages of mitosis, some level of cyclin B and cyclin
B-dependent Cdc2 kinase are also detected during S phase
and are essential for progression through this phase of the yeast cell
cycle (16). Early stages of mitosis, such as chromosome condensation,
could be initiated by the
never-in-mitosis (NIMA)-type kinases
in the absence of cyclin B-dependent Cdc2 kinase activity
(17, 18). Abrogation of mitosis during polyploidization could result
from a variety of reasons, including a premature degradation of cyclin
B. This latter possibility could lead to the detection of lower levels
of this cyclin during endomitosis. In the current study, we sought to
investigate whether the activity of the ubiquitin-proteosome pathway
responsible for cyclin B degradation is altered in polyploidizing
megakaryocytes. Our data indicate that polyploidization is associated
with increased activity of the ubiquitin pathway responsible for the
recognition and proteolysis of cyclin B. This feature may be a part of
cellular programming that leads megakaryocytes to abort mitosis during
polyploidization.
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MATERIALS AND METHODS |
Culture Conditions--
MegT cells (clone 37C1) were grown in a
liquid culture, as described before (19). To induce ploidy, 1 × 106 cells were seeded into a 75-cm2 culture
flask and incubated in 5% CO2 at 39.5 °C for 4 or 5 days (10, 19). Cells were counted by hemocytometer, and cell viability was followed by staining with trypan blue. The attached cells as well
as the detached cells were separately collected and subjected to
various analyses. A clone (Y10/L8057) of the mouse megakaryocytic cell
line L8057 (20), selected by limited dilution based on its ability to
express acetylcholinesterase (a unique marker for rodent
megakaryocytes) (21) and to respond to 25 ng/ml
PEG-rHuMGDF1 (generous gift
of Amgen, Inc. Thousands Oaks, CA) by polyploidizing. Acetylcholinesterase activity was assayed as described before (9).
Culture conditions for this cell line were as described elsewhere (20).
Rat bone marrow cells were isolated from the femurs also as described
before (22). Cells were cultured in 5% CO2 at 37 °C in
a liquid culture under conditions that were previously shown to support
maturation and ploidy of primary megakaryocytes (22, 23). The cells
were cultured in the presence of Iscove's modification of Dulbecco's
medium (IMDM) and 20% horse serum supplemented with penicillin (2000 units/ml), streptomycin (200 µg/ml), L-glutamine (0.592 mg/ml), and when indicated with 50 ng/ml PEG-rHuMGDF for 3 days prior
to collection of large mature megakaryocytes.
Purification of Megakaryocytes--
Polyploidizing
megakaryocytes of varying sizes (20-µm diameter and higher) were
isolated from bone marrow cells derived from rat femurs (22) and
cultured for 3 days in the presence of 50 ng/ml of PEG-rHuMGDF, to
promote megakaryopoiesis, and in the presence of a mixture of growth
factors including 1 unit/ml erythropoietin (Amgen), 10 units/ml of
granulocyte-macrophage colony-stimulating factor, and 5 units/ml of
stem cell factor (Genzyme Corp., Boston, MA) to promote proliferation
and survival of different bone marrow lineages, as we described before
(22). The isolation procedure involved mesh filtration (nylon mesh
screens from Spectramesh, Spectrum Medical, Inc., Los Angeles, CA). To
this end, clumps were initially removed by filtration through a
200-µm filter followed by filtration with a 17-µm filter, which
allowed diploid cells to pass through the mesh. Cells retained on the
filter were retrieved by washing the inverted filter with
phosphate-buffered saline (136 mM NaCl, 8 mM
Na2HPO4, 2.6 mM KCl, 1.4 mM KH2PO4, pH 7.4). This procedure
was repeated twice. We confirmed by counting cells under the light
microscope and by staining for the unique marker for megakaryocytes,
acetylcholinesterase (21), that megakaryocytes were enriched to account
for 60-70% of bone marrow cells (see Fig. 8A), a level
comparable with the purification method involving elutriation (24).
Because of the size and content of megakaryocytes, in comparison with
the small diameter bone marrow cells, the enrichment of megakaryocytic
material (e.g. protein) would be substantially higher.
Western Blotting--
Y10/L8057 cells grown in suspension were
collected by centrifugation (380 × g, 5 min) in growth
medium, and MegT cells adhering to the culture dish were stripped from
the plate with a cell scraper, while nonadhering cells were collected
separately by centrifugation. Cells were washed twice with cold
phosphate-buffered saline and then lysed in lysis buffer (0.5% Nonidet
P-40, 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml n-tosyl-L-phenylalanine chloromethyl
ketone, 10 µg/ml soybean trypsin inhibitor) followed by
centrifugation at 15,000 × g for 5 min. For Western
blotting, 10 µg of lysed proteins were separated by 7.5 or 12%
SDS-polyacrylamide gel electrophoresis (25) and electrophoretically
transferred from the gel onto a nitrocellulose membrane (Schleicher & Schull) in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. The membrane was stained
with 0.1% Ponceau S (Sigma) in 5% acetic acid to visualize protein
bands. The membrane was destained with water, washed in TBS (10 mM Tris (pH 8.0), 150 mM NaCl) and blocked for
1 h with TBST (Tris-buffered saline with 0.1% Tween 20)
containing 5% dry milk. The blot was incubated for 1 h at room
temperature in the presence of 10 ml of TBST supplemented with a
monoclonal antibody to cyclin B1 (GNS-1 diluted to 0.2 µg/ml),
generated against the N-terminal domain of cyclin B between amino acids
1 and 21 (Pharmingen, San Diego, CA). This antibody cross-reacts with
the corresponding murine protein, as tested by the manufacturers and by
ourselves (10). When indicated, a polyclonal antibody to cyclin B1,
which does not block exclusively the N-terminal region of cyclin B, was
used (26). A limited amount of this antibody was available to us, and
thus most of the studies were performed with the commercial monoclonal
antibody to cyclin B1. The blot was washed four times, each time for 10 min, and incubated for 1 h with appropriate horseradish peroxidase-labeled secondary antibody (Amersham Corp.) at a 1:1500 to
1:3000 dilution in TBST. The blot was washed four times (each for 10 min) with TBST, and the Enhanced Chemiluminescence system (Amersham)
was used for detection of proteins, as instructed by the
manufacturer.
Preparation of Cytosolic Extract and Degradation
Assay--
Cells were washed twice with phosphate-buffered saline by
centrifugation (380 × g, 5 min). The volume of the
pelleted cells was estimated, and the cells were washed again by
centrifugation in the presence of 5 volumes of hypotonic buffer (10 mM Tris (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethanesulfolyl fluoride). Cells were resuspended
in 2 volumes of hypotonic buffer and left on ice for 10 min prior to
breaking by strokes in a glass homogenizer. Cells were spun down for 10 min in a microcentrifuge. Protein assay was performed on the
supernatant as described in Ref. 10. Since adequate degradation was
obtained with this preparation (27), we did not need to further clean
the cytoplasmic extract by high speed centrifugation (100,000 × g). Degradation of recombinant cyclin B was monitored as
follows. Cytosolic extract (25 µg of protein) was added to a reaction
mixture containing the following components in a final volume of 25 µl: 12 mM Tris (pH 7.5), 60 mM KCl, 3.5 mM MgCl2, 1 mM ATP (or ATP
S), 20 mM creatine phosphate, 50-200 ng of recombinant cyclin B1
or cyclin A (Pharmingen), purified from salt by buffer exchange with an
Amicon 30-kDa Filtration Unit (Amicon Beverly, MA). The reaction was
carried out at 30 °C for the indicated time. When indicated, the
cellular extract was pretreated with 0.15 units of apyrase (Sigma) for
20 min at 30 °C, after which the reaction was processed in the
absence of ATP or creatine phosphate. Ubc4 and Ubc2 were obtained as
described elsewhere (28). Point mutations in Ubc4 and Ubc2 were
generated by oligonucleotide-directed mutagenesis as described in Ref.
28.
Ploidy Analysis--
Bone marrow was collected from femurs of
rats as described before (22, 23). Marrows were suspended in 0.5 ml of
CATCH buffer and incubated for 60 min at 4 °C with 4A5 monoclonal
antibody ascites (29) (generous gift of Sam Burstein, University of
Oklahoma Health Sciences Center). The cells were then incubated with
fluorescein-conjugated goat anti-rat IgG(Fab
)2 (Biosource
International, Camarillo, CA) for 30 min at 4 °C. Staining with
propidium iodide was followed by determination of DNA content of 4A5
positive cells using a statistical package on a FACScan flow cytometer
(Becton-Dickinson) as we described previously (10). Y10/L8057 cells
were stained with propidium iodide and subjected to ploidy analysis
also as described before (10).
 |
RESULTS |
Levels of Cyclin B1 and Cdc2 and the Half-life of Cyclin B1 in the
Proliferating and Polyploidizing Megakaryocytic Cells
Y10/L8057--
In the current study, we assayed for cyclin B1 and Cdc2
levels during polyploidization of a megakaryocytic cell line,
Y10/L8057, which we subcloned from L8057 cells (20) (see "Materials
and Methods"), based on its ability to polyploidize in response to the megakaryocyte ploidy-promoting factor PEG-rHuMGDF (Fig.
1). In the presence of PEG-rHuMGDF or of
phorbol 12-myristate 13-acetate (PMA), a moderate or large fraction,
respectively, of the cells underwent polyploidization, while the rest
remained in the 2N proliferative mode (Fig. 1). Western blot analyses
indicated that during polyploidization induced by these agents, the
levels of cyclin B1, but not of Cdc2, were reduced (Fig.
2). We next explored the possibility that
reduced levels of cyclin B reflected an accelerated degradation of this
protein in polyploidizing cells. As shown in Fig.
3, the half-life of cyclin B1 in
polyploid Y10/L8057/L8057 cells was substantially shorter than in
proliferating cells. The enhanced degradation and reduced levels of
cyclin B1 in polyploidizing megakaryocytes were not a result of cell
cycle arrest, since cells induced to polyploidize displayed high rates
of DNA synthesis (Table I).

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Fig. 1.
The ploidy status of cultured Y10/L8057
cells. Y10/L8057 cells (1.5 × 105 cells/ml) were
cultured in IMDM containing 10% FCS alone or supplemented with 50 ng/ml PEG-rHuMGDF or 50 nM PMA for 1, 2, and 3 days. Cells were collected, stained with propidium iodide, and analyzed by flow
cytometry, as detailed under "Materials and Methods." No significant change in ploidy was observed in cells cultured with PEG-rHuMGDF until 3 days of culturing with the stimuli (for which data
are presented). Titration experiments indicated that PEG-rHuMGDF was
saturating at a concentration of 50 ng/ml (not shown).
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Fig. 2.
The levels of cyclin B and Cdc2 in Y10/L8057
cells undergoing a mitotic or an endomitotic cell cycle. Y10/L8057
cells were cultured in IMDM medium containing 10% FCS alone
(lane 1), supplemented with 50 ng/ml PEG-rHuMGDF (lane
2), or supplemented with 50 nM PMA (lane 3)
for the time indicated. Cells were collected and subjected to Western
blotting with a monoclonal antibody to cyclin B1 or Cdc2, as indicated
under "Materials and Methods." Equal loading of protein was
confirmed by staining the nitrocellulose membrane with Ponceau S, as
described before (10) (not shown). The data presented are
representative of three experiments.
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Fig. 3.
Half-life of cyclin B in proliferating or
polyploidizing Y10/L8057 cells. A, Y10/L8057 cells were
cultured for 3 days in IMDM medium containing 10% FCS alone, during
which they undergo a mitotic cell cycle (Proliferating
Cells), or supplemented with 50 nM PMA
(Polyploid Cells). Cyclohexamide (500 µg/ml) was added to
the culture, and cells were collected at the indicated times and
subjected to Western blot analysis with a monoclonal antibody to cyclin
B1, as described under "Materials and Methods." B,
the nitrocellulose membrane from panel A was stained with
Ponceau S to confirm equal loading of protein per lane. The data shown are representative of three experiments.
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Table I
Ploidy analysis of megakaryocytes derived from liquid cultures of rat
bone marrow cells treated with PEG-rHuMGDF
Liquid cultures of rat bone marrow cells were incubated in the absence
or presence of 50 ng/ml PEG-rHuMGDF, as indicated, for 3 days.
Megakaryocyte number was determined by counting acetylcholine esterase-positive cells, and ploidy analysis was performed as detailed
under "Materials and Methods." The data are representative of two
experiments.
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Accelerated Degradation of Recombinant Cyclin B by a
Ubiquitin-Proteosome Extract Derived from Polyploidizing Y10/L8057
Cells--
A shortened half-life of cyclin B could result from an
enhanced activity of the ubiquitin-proteosome pathway in polyploidizing cells. To test this possibility, cytosolic extracts were prepared from
proliferating megakaryocytes or cells undergoing endomitosis. These
extracts were incubated with recombinant cyclin B1, and cyclin B1
degradation was followed. As shown in Fig.
4, a significant degradation of
recombinant cyclin B was displayed by extracts from highly
polyploidizing Y10/L8057 cells (treated with PMA), less from cells
displaying moderate polyploidization (treated with PEG-rHuMGDF), but
not in proliferating cells. A high level of cyclin B1 degradation (in
PMA-treated cells) was dependent on the addition of ATP to the
reaction, as typical of a ubiquitin-dependent degradation
(30) (Fig. 4). Cellular ATP was sufficient, however, to promote a low
level of cyclin B degradation in the PEG-rHuMGDF-treated cells.

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Fig. 4.
Degradation of recombinant cyclin B1 by
extracts derived from polyploid and diploid Y10/L8057 cells.
Recombinant cyclin B1 was incubated for 30 min at 30 °C with cell
extracts derived from Y10/L8057 cells cultured under conditions
described in Fig. 1 in the presence of IMDM medium containing 10% FCS
alone (lane 1), supplemented with 50 ng/ml PEG-rHuMGDF
(lane 2) or with 50 nM PMA (lane 3),
in the presence or absence of 1 mM ATP, as indicated. The
reaction was subjected to Western blotting with a monoclonal antibody
to cyclin B1, all as detailed under "Materials and Methods." The
blot in panel A was briefly stained with Ponceau S to
confirm equal loading of protein in each lane (not shown). The data are representative of three experiments.
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Degradation of Cyclin A Is Not Accelerated in Polyploidizing
Y10/L8057 Cells--
Cyclin A protein is highly expressed in the
megakaryocytic cell line, MegT, in HEL cells (10, 31) and in Y10/L8057
cells undergoing polyploidization (Fig.
5A). Since cyclin A is also subjected to degradation via the ubiquitin pathway (reviewed in Ref.
32), we examined whether the ability of polyploid megakaryocytes to
degrade this cyclin is accelerated as was the case for cyclin B1.
Interestingly, and in accordance with the detection of high levels of
cyclin A in polyploid megakaryocytes, the ability of this latter cell
type to degrade recombinant cyclin A was comparable with the rate
displayed by proliferating cells (Fig. 5B).

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Fig. 5.
Degradation of Cyclin A in cell extracts
derived from Y10/L8057 cells. A, Western blots of cell
extracts derived from Y10/L8057 cells cultured for different days in
IMDM medium containing 10% FCS alone or supplemented with 50 nM PMA, as indicated, using anti cyclin A. B,
cell extracts, derived from Y10/L8057 cells cultured for 3 days without
(lane 1) or with 50 nM PMA (lane 2) or with 50 ng/ml PEG-rHuMGDF (lane 3), were incubated at
30 °C for 30 min in the presence of 1 mM ATP and in the
absence or presence of recombinant cyclin A (rec-cyclin A)
as indicated. The reactions were then subjected to Western blot
analysis using anti-cyclin A antibody. The data are representative of
two experiments.
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Accelerated Degradation of Recombinant Cyclin B by a
Ubiquitin-Proteosome Extract Derived from Polyploidizing MegT
Cells--
MegT cells are megakaryocytes that have been immortalized
with the temperature-sensitive large T antigen (19). At the permissive temperature (34 °C), the cells proliferate. At the nonpermissive temperature (39.5 °C), a fraction of the cells remain adhering to
the dish and undergo a mitotic cell cycle, while the other fraction (in
which T antigen is destroyed) detaches from the plate and undergoes an
endomitotic cell cycle (10). During this endomitotic process, the level
of cyclin B protein, but not of cyclin A protein nor of cyclin B
mRNA, is reduced, as compared with proliferating cells (10).
Cytosolic extracts were prepared from these proliferating megakaryocytes or from cells undergoing endomitosis. These extracts were incubated with recombinant cyclin B1, and cyclin B1 degradation was followed. As shown in Fig.
6A, a significant degradation
of recombinant cyclin B was displayed by extracts prepared from
polyploidizing cells. Cyclin B degradation was prevented in the
presence of apyrase, which degrades ATP, or in the presence of the
nonhydrolyzable (by the proteosomes) ATP analog, ATP
S. An additional
hallmark of a ubiquitin-dependent degradation is the
appearance of high molecular weight conjugates of cyclin B and
ubiquitin. To detect these high molecular weight complexes, we also
used a polyclonal antibody to cyclin B protein. This antibody, in
contrast to the monoclonal antibody used in Fig. 6A, is not
directed against the N-terminal domain of cyclin B and thus should
recognize ubiquitin-bound cyclin B. Indeed, as shown in Fig.
6B, high molecular weight conjugates of recombinant cyclin B
appeared only in polyploid cells, depending on the availability of
ATP.

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Fig. 6.
Degradation of recombinant cyclin B by
extracts derived from polyploid and diploid MegT cells. A,
recombinant cyclin B1 was incubated for 30 min at 30 °C in the
presence or absence of ATP, as indicated, with cell extracts derived
from MegT cells cultured at 34 °C (proliferating cells) (lane
1), from adhering MegT cells cultured at 39.5 °C (proliferating
cells) (lane 2), or from MegT cells in suspension cultured
at 39.5 °C and in which T antigen is inactivated (polyploidizing
cells) (lane 3). The reactions were subjected to Western
blotting using a monoclonal antibody to cyclin B1. Incubation of
recombinant cyclin B1 alone, in the absence of cell extract, is shown
in lane C. 5-10-s exposure of the blot was sufficient for
detecting the bands by the ECL system, while prolonged exposure did not
yield additional bands. The data are representative of four
experiments. B, the blot in panel A was briefly
stained with Ponceau S to confirm equal loading of protein in each
lane. C, Western blot analysis of a filter loaded with
samples as in panel A, using a polyclonal antibody to cyclin
B1. To detect bands by the ECL method, the exposure of x-ray film to
the membrane was carried out for 20 min.
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PEG-rHuMGDF-treated Primary Bone Marrow Cells Display an Increased
Ability to Degrade Recombinant Cyclin B--
To test that the
phenotype observed in Y10/L8057 and MegT cells is not exclusive to
these cell lines, we prepared extracts from primary rat bone marrow
cells cultured for 3 days in the absence or presence of PEG-rHuMGDF
(reviewed in Ref. 12). Under our liquid culture conditions, PEG-rHuMGDF
did indeed increase the frequency and ploidy of megakaryocytes (Table
II). A cellular extract prepared from
PEG-rHuMGDF-treated bone marrow cells degraded recombinant cyclin B
more efficiently than extract from diploid bone marrow cells (Fig.
7). This was accompanied by a large shift of cyclin B to high molecular weight forms. It is technically impossible at this stage to purify significant quantities of
proliferating, small megakaryocytes from primary bone marrow, thus
preventing us from comparing the extent of cyclin B degradation in
diploid versus polyploid megakaryocytes. We were, however,
able to obtain a preparation of highly enriched polyploid
megakaryocytes from bone marrow cells cultured with PEG-rHuMGDF (Fig.
8A). As shown in Fig.
8B, the rate of degradation of recombinant cyclin B1 by an
extract derived from large megakaryocytes is significantly higher than
the rate of degradation by diploid bone marrow cells. We realize that
these results, although indicative, do not demonstrate conclusively
that polyploidizing primary megakaryocytes display enhanced activity of
the ubiquitin-proteosome pathway as compared with diploid
megakaryocytes.
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Table II
DNA synthesis in proliferating and polyploidizing Y10/L8058 cells
Y10/L8057 cells were cultured in the absence or presence of 50 nM PMA, as indicated, and incorporation of
[3H]thymidine to DNA was determined as detailed under
"Materials and Methods." The data are representative of two
experiments performed in duplicate.
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Fig. 7.
Degradation of recombinant cyclin B1 by bone
marrow cells treated with PEG-rHuMGDF. A, recombinant cyclin
B1 was incubated for 30 min at 30 °C in the presence of ATP alone
(lane C) or with cell extracts derived from bone marrow
cells cultured for 3 days in the absence (BM), or presence
of 50 ng/ml PEG-rHuMGDF (BM + MGDF). The reactions were then
subjected to Western blot analysis using a polyclonal antibody to
cyclin B. B, the blot in panel A was stained with
Ponceau S to confirm equal loading of protein to each lane. The data
are representative of two experiments
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Fig. 8.
Enrichment of megakaryocytic cells from bone
marrow cultures treated with PEG-rHuMGDF. A, phase contrast
microscopy of partially purified large megakaryocytes (average size of
30-50 µm in diameter) derived from PEG-rHuMGDF-treated bone marrow
cells, recognized morphologically and confirmed by staining for
acetylcholin esterase, as we described before (9) (not shown) (original magnification × 200). B, recombinant cyclin B1 was
incubated for the indicated time at 30 °C in the presence or absence
of ATP with cell extracts derived from rat bone marrow cells depleted of megakaryocytes or cell extracts derived from a preparation of
enriched megakaryocytes. The reactions were subjected to Western blotting using a monoclonal antibody to cyclin B1. C, the
blot in panel B was stained with Ponceau S to confirm equal
loading of protein to each lane. The data are representative of two
experiments.
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Megakaryocyte-induced Degradation of Cyclin B Depends on the
Ubiquitin-conjugating Enzyme Ubc4--
It has been reported that
cyclin B degradation in other systems is mediated via the
ubiquitin-conjugating enzyme Ubc4 (4). We thus used a mutated form of
Ubc4 in which cysteine 85 was replaced by serine. Such a protein was
shown to act in vitro as a dominant negative mutant (28).
Our data indicated that mutated Ubc4, but not mutated Ubc2 protein,
slowed the rate of degradation of recombinant cyclin B by purified
megakaryocytes (Fig. 9), indicating that
Ubc4 mediates cyclin B degradation in this cell type.

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Fig. 9.
Inhibition of megakaryocyte-mediated
degradation of cyclin B1 by the mutated form of Ubc4. Recombinant
cyclin B1 was incubated at 30 °C in the presence or absence of ATP
for the indicated time with cell extracts derived from megakaryocytes
enriched from bone marrow cultures treated with 50 ng/ml PEG-rHuMGDF.
Incubations were carried out in the absence (C) or presence
of 10 µM mutated Ubc4 or Ubc2, as indicated, and then
subjected to Western blot analysis using a monoclonal antibody to
cyclin B1.
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DISCUSSION |
In some systems investigated, the lack of cyclin B1 alone is
sufficient to drive endoreduplication. Endoreduplication in some Drosophila cell types is indeed associated with the lack of
cyclin B1 (33). Also, the metaphase II arrest in mouse oocytes is
controlled through destruction of cyclin B1 (34). Certain treatments,
such as with inhibitors of protein kinases in mammalian cells or high levels of the protein encoded by rum1, which inhibits the
mitotic kinase in fission yeast, block M phase and induce repeats of S phase (35-37). We found previously that polyploidization in
megakaryocytic cell lines is associated with reduced levels of cyclin
B1 protein, but not of cyclin A or of cyclin B1 mRNA, as compared
with the levels in diploid cells (9, 10). In this context, it is
important to note that although cyclin B is important for the
G2/M transition, low levels of cyclin B may also be needed
for the S phase of yeast cell cycles (16, 38). In the current study, we
found that cyclin B1 half-life in polyploidizing cells is decreased, as
compared with that in proliferating cells. We thus explored the
possibility that a reduced level of cyclin B1 during endomitosis
reflects an enhanced ability of these cells to degrade this cyclin,
since this may result in abrogated mitosis (13). We found that
polyploidization in the megakaryocytic cell lines MegT and Y10/L8057 or
in primary megakaryocytes was associated with an enhanced ability of
the cells to degrade recombinant cyclin B1 protein, as compared with the diploid proliferating megakaryocytic cell lines or
nonmegakaryocytic bone marrow cells, respectively. Also, an extract of
total bone marrow cells treated with the ploidy-promoting factor, the
c-Mpl ligand, exhibited an augmented ability to degrade cyclin B, as compared with nontreated bone marrow cells. This proteolytic process displayed all of the hallmarks of a ubiquitin-dependent
degradation (39), such as formation of high molecular weight complexes
of cyclin, dependence on ATP, and competition by the mutated form of
the ubiquitin-conjugating enzyme Ubc4.
Many components are involved in ubiquitination and degradation of
cyclin B. Cyclin B ubiquitination is regulated via the
ubiquitin-conjugating enzymes Ubc4 (4) and UbcX/Ubc10 (7, 40), and on
ubiquitin ligase (E3) activity, the latter associated with a complex of proteins termed the anaphase-promoting complex (APC) or cyclosome (2,
41, 42). Together with the ubiquitin-activating enzyme, these
components promote polyubiquitination of cyclin B, which targets the
protein to proteolysis by the 26 S proteosome. Besides Cdc16 and Cdc27,
the identities of the components of APC in mammalian cells remain to be
explored. The APC determines the specificity of degradation of
different cyclins. King and colleagues (4) concluded that different
cyclins are recognized by different components of the ubiquitination
machinery. It was found that the cyclin A destruction box cannot
functionally substitute for its B-type counterpart, indicating the
existence of different components of the APC. Since cyclin A levels are
elevated during the G1/S phase of the mitotic or
endomitotic cell cycles in megakaryocytes (10) and since our current
study demonstrates that polyploidizing megakaryocytes do not display an
enhanced ability to degrade recombinant cyclin A, it is reasonable to
assume that a cyclin B1-specific component of the APC may be
up-regulated during polyploidization. Future identification of the APC
components will allow a further examination of this process, and one
may speculate that ploidy-promoting factors may affect the expression
of such an APC-specific component.
We thank Carl Jackson, Richard Ashmaun, and
Sam Lucas for skillful ploidy analysis of rat bone marrow cells.