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(Received for publication, June 13, 1995; and in revised form, November
29, 1995) From the
The platelet precursor, the megakaryocyte, matures to a
polyploid cell as a result of DNA replication in the absence of mitosis
(endomitosis). The factors controlling endomitosis are accessible to
analysis in our megakaryocytic cell line, MegT, generated by targeted
expression of temperature-sensitive simian virus 40 large T antigen to
megakaryocytes of transgenic mice. We aimed to define whether
endomitosis consists of a continuous phase of DNA synthesis (S) or of S
phases interrupted by gaps. Analysis of the cell cycle in MegT cells
revealed that, upon inactivation of large T antigen, the cells shifted
from a mitotic cell cycle to an endomitotic cell cycle consisting of
S/Gap phases. The level of the G
The development of hematopoietic cells consists of few stages:
the commitment of pluripotent stem cells to differentiate rather than
to remain in the resting G The major events common to all eukaryotic cell cycles
are the replication of chromosomes during S phase and their segregation
during mitosis. The dependences of S phase and mitosis on each other
ensure orderly progression through the cell cycle (Hartwell and
Weinert, 1989). However, in some developmental situations, chromosome
replication and segregation can be uncoupled. For example, a
G Past studies of megakaryocyte development, endomitosis, and
maturation have been hampered because of the rarity of megakaryocytes
in bone marrow and because of the lack of a pure megakaryocytic cell
line that can enter and complete a normal maturation cycle. Different
leukemia cell lines derived from hematopoietic progenitors have been
used in studying the biochemistry as well as gene regulation of the
megakaryocytic lineage. Some human erythroleukemic cell lines exhibit
myeloid, erythroid, as well as megakaryocytic markers (Martin and
Papayannopoulou, 1982; Tabilio et al., 1983; Ravid et
al., 1993a), while other cell lines (Greenberg et al.,
1988; Sledge et al., 1986; Adachi et al., 1991) are
enriched with megakaryocytic markers, but require exposure to a
substance such as phorbol 12-myristate 13-acetate in order to ploidize.
This later agent, being pluripotent, may induce changes in cyclin
expression unrelated to megakaryocyte ploidy, as demonstrated on a
nonmegakaryocytic cell line HL60 (Akiyma et al., 1993). We
have recently generated a megakaryocytic cell line, MegT, by targeted
expression of the temperature-sensitive form of large T antigen in
megakaryocytes of transgenic mice, via the platelet factor four
tissue-specific promoter (Ravid et al., 1993b). MegT cells
which become polyploid upon inactivation of the oncogene were used to
determine the role of different cyclins in promoting endomitosis. Our
data suggest that once large T antigen is degraded, the cells undergo
endomitosis while containing low levels of cyclin B1 and low activity
of the mitotic kinase.
For Western
blotting, 10 µg of lysed proteins were separated on 7.5% or 12%
SDS-PAGE (Laemmli and Favre, 1973) and electrophoretically transferred
from the gel onto an Immobilon-P membrane (Millipore) in a buffer
containing 25 mM Tris, 192 mM glycine, and 20%
methanol. The membrane was washed in TBS (10 mM Tris, pH 8.0,
150 mM NaCl) and blocked for 1 h with TBST (TBS with 0.05%
Tween 20) containing 5% dry milk. The blot was washed four times for 5
min with TBST and incubated for 1 h at 4 °C in the presence of 10
ml of TBST supplemented with one of the following primary antibodies
raised against human proteins: rabbit polyclonal antibodies to cyclin A
(diluted 1:2000) (a gift of Dr. Tony Hunter at Salk Institute),
anti-cyclin B1 monoclonal antibody (GNS-1 diluted to 0.2 µg/ml),
anti-Cdc2 monoclonal antibody (A17 diluted to 0.5 µg/ml), anti-SV40
large T antigen monoclonal antibody (PAb 101 diluted to 1 µg/ml)
(all from Pharmingen, San Diego, CA), rat antibody to human cyclin D3
(diluted to 0.2 µg/ml) (Oncoge Science, Uniondale, NY). All these
antibodies cross-react with the corresponding mouse proteins, as tested
by the manufacturers. The blot was washed four times, each time for 10
min, and incubated for 1 h with horseradish peroxidase-labeled
appropriate secondary antibody (1:1500 to 1:3000 dilution in TBST
(Amersham). 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.
Figure 1:
Determination of
mRNA for different cyclins in MegT cells. MegT cells were synchronized
by serum starvation at 34 °C. At zero time and at successive time
points after serum addition, a portion of the cells was subjected to
nuclei staining and flow cytometry analysis (Ravid et al.,
1993b) to determine the cell cycle point using the CellFit program, as
described under ``Materials and Methods.'' A full cell cycle
was completed within 24 h. RNA prepared from synchronized MegT cells at
different time points (hours) was subjected to Northern blot analysis.
Total RNA (15 µg/lane) was electrophoresed on 1% agarose gel which
was then blotted onto nitrocellulose. The blot was probed with
different cDNAs encoding different cyclins as well as with
Figure 2:
Phase-contrast photomicrographs of MegT
cells. MegT cells were cultured at 34 °C (A) or at 39.6
°C (B and C) for 4 days. The cells adhering to
the dish (B) and the detached cells (C) in suspension
were collected separately and viewed by phase-contrast microscope at a
magnification of
Figure 3:
Ploidy analysis of MegT cells. MegT cells
were cultured at 34 °C or at 39.5 °C for 4 days. The cells
adhering to the dish and the detached cells in suspension were
collected separately and subjected to nuclei staining and flow
cytometry analysis as described under ``Materials and
Methods.'' The abscissa shows the DNA content on a
logarithmic scale, determined based on fluorescence due to propidium
iodide staining, and the ordinate reflects the number of cells
at each DNA value (linear).
Of most
interest was the observation that the level of large T antigen, as
revealed by Western blot analysis, was notably reduced in the cells in
suspension (polyploid cells) but not in the adhering cells (mostly
diploid cells) cultured at the same nonpermissive temperature (Fig. 4A). We then determined the level of components
of the mitosis promoting factor (MPF), being cyclin B and Cdc2, in both
fractions of cells. As shown in Fig. 4A, the level of
cyclin B1 protein, but not of Cdc2, was significantly reduced in the
polyploid cells. Northern blot analysis indicated, however, that the
level of cyclin B1 mRNA was not changed significantly in polyploid
cells (Fig. 4B). In order to establish whether a
reduced level of cyclin B is a property of a T-antigen mutant cell line
or rather a property of polyploid megakaryocytes, we determined the
level of T antigen and of cyclin B in immortal fibroblasts (AR5)
transformed by origin-defective SV40 encoding a heat-labile T antigen
(Hubbard-Smith et al., 1992). At high temperature, these cells
display a reduced growth rate, but no hyperploidy (Resnick-Silverman et al., 1991). Western blot analyses (Fig. 5) indicated
that, although T antigen was degraded in AR5 cells incubated at high
temperature, the level of cyclin B was not altered.
Figure 4:
The levels of cyclin B1 and Cdc2 in MegT
cells. A, Western blot analysis of MegT cells cultured at 39.6
°C (lanes 1 and 2) or at 34 °C (lanes
3). Blots, loaded in each lane with 10 µg of protein prepared
from adhering cells (lanes 2 and 3) or detached cells (lane 1), were reacted with antibodies to large T antigen or
to Cdc2 or to cyclin B1 proteins. Equal loading of protein in each lane
was confirmed by brief staining of the blot with 0.1% Ponceau S (not
shown) followed by destaining prior to reacting with the indicated
antibody. Large T antigen, cyclin B1, and Cdc2 appeared with the
molecular masses of 82, 55, and 34 kDa, respectively, on the blot.
These results are representative of four experiments performed. B, Northern blot analysis of MegT cells cultured at 34 °C
or at 39.5 °C and collected as described in A. RNA
concentration was determined by absorbance at 260 nm before loading on
the agarose gel. Equal loading was confirmed by ethidium bromide
staining of the ribosomal bands 28 S (5 kb) and 18 S (2.5kb) shown in
the figure. C, the blot was probed with cDNA encoding mouse
cyclin B1. Two splicing forms of cyclin B1 were detected (1.6 kb and
2.5 kb), as also described before (Chapman and Wolgemuth,
1992).
Figure 5:
The
levels of cyclin B1, Cdc2, and T antigen in immortal fibroblasts. A, Western blot analysis of AR5 cells (fibroblasts transformed
by heat-labile tsA58 T antigen) cultured at 34 °C (lane 1)
or at 39.5 °C (lane 2). Blots, loaded in each lane with 10
µg of protein, were reacted with antibodies to large T antigen or
to Cdc2 or to cyclin B1 proteins. Equal loading of protein in each lane
was confirmed by brief staining of the blot with 0.1% Ponceau S (not
shown) followed by destaining prior to reacting with the indicated
antibody. These results are representative of two experiments
performed.
Figure 6:
DNA synthesis and mitotic kinase activity
in synchronized MegT cells. Synchronized MegT cells were subjected at
different time points to pulse labeling for 1 h with radiolabeled
thymidine and were then collected for determination of incorporation of
thymidine into DNA in the adhering (A) mitotic, and detached (B) endomitotic cells cultured at 39.5 °C. The inset shows histone phosphorylation by the mitotic kinase isolated at
the indicated points of the cell cycle, all as described under
``Materials and Methods.'' Results are from a representative
experiment, out of three performed.
Figure 7:
The levels of different cyclins in
synchronized MegT cells. Cell lysates were prepared separately from
adhering (A) mitotic and detached (B) endomitotic
MegT cells, cultured at 39.5 °C, at different hours postrelease
from synchronization. The cell lysates were subjected to Western blot
analysis, using 12% acrylamide gel. Equal loading of protein prepared
from endomitotic cells in suspension (C) and adhering mitotic (D) cells (10 µg of protein/lane) was confirmed by brief
staining (1-2 min) of the blots with 0.1% Ponceau S in 5% acetic
acid followed by destaining in water for 2 min and photography,
followed by a 10-min rinse in water prior to reaction with the
indicated antibodies. Lane M contains a ladder of molecular
mass markers. Large T antigen, cyclin A, cyclin B1, and Cdc2 appeared
with the molecular masses of 82, 60, 55, and 34 kDa, respectively, on
the blot.
Figure 8:
Cyclin D3 levels in MegT cells. Cell
lysates were prepared separately from adhering (A) mitotic,
and detached (B) endomitotic MegT cells, cultured at 39.5
°C, at different hours postrelease from synchronization. The cell
lysates were subjected to Western blot analysis, using 10% acrylamide
gel. Equal loading of protein prepared from endomitotic cells in
suspension (C) and adhering mitotic (D) cells (10
µg of protein/lane) was confirmed by brief staining (1-2 min)
of the blots with 0.1% Ponceau S in 5% acetic acid followed by
destaining in water for 2 min and photography, followed by a 10-min
rinse in water prior to reaction with cyclin D3 antibody. Cyclin D3
appeared with a molecular mass of 33 kDa on the
blot.
Endomitosis, involving DNA replication in the absence of
mitosis, can occur in three types of cells: those in which the
endoreplicated chromosomes are not synapsed or visible, those in which
cyclic condensation of the chromosomes is observed, and, in some cases,
those in which multinucleate cells have been referred to as polyploid
also. In the case of the megakaryocytic lineage, the endoreduplicated
DNA is all concentrated in one nucleus (Metcalf, 1989). However, it is
not certain yet if polyploid megakaryocytes enter only prophase
involving chromosome condensation or prophase as well as metaphase,
involving chromosome condensation and spindle formation but skip
anaphase, or whether the cells skip all stages in the G In a previous study, we
generated several clones of megakaryocytic cell lines by targeted
expression of the temperature-sensitive form of large T antigen in
transgenic mice, via the tissue-specific platelet factor four (PF4)
promoter (Ravid et al., 1993b). These cell lines express
several megakaryocytic markers, such as the glycoprotein GPIIb, and
acetylcholine esterase, and at the permissive temperature adhere to the
dish and contain high levels of large T antigen (Ravid et al.,
1993b). In the current study, we have chosen to investigate a clone
which allowed us to perform analyses of the mitotic and endomitotic
cell cycle at the same nonpermissive temperature. When MegT cells
(clone 37C1) were cultured at the nonpermissive temperature, only a
fraction of the cells (about 30%) had undetectable levels of large T
antigen and solely those cells appeared as round cells in suspension
with high ploidy nuclei. Because of the leaky nature of this
conditional oncogene, large T antigen may not have been completely
destroyed in the rest of the cells which remained adhering to the dish.
Nevertheless, this feature was taken as an experimental advantage,
since it allowed us to compare levels of different cyclins in diploid
and polyploid megakaryocytes cultured at the same elevated temperature.
Our results indicated that cells expressing high levels of large T
antigen were unable to initiate endoreduplication. In a recently
published study, additional transgenic mice carrying the
temperature-sensitive large T antigen under the control of the PF4
promoter have been generated (Robinson et al., 1994). Upon
aging, some of these mice developed megakaryocytic leukemias displaying
aberrations in megakaryocyte morphology and low platelet counts.
Transgenic mice in which megakaryocytes reached high ploidy level
seemed to express minute amounts of the oncogene (Robinson et
al., 1994). We aimed to define whether the megakaryocytic cell
cycle during endomitosis consists of a continuous S phase or of Gap/S
phases and to determine which cyclins are involved in this process.
Pulse-labeling of MegT cells with [ Although two types of cyclin B were
described in mammalian cells, B1 and B2 (Chapman and Wolgemuth, 1993),
it is not clear yet which of these cyclins plays a role in different
stages of mitosis in eukaryotic cells and if they are able to
substitute for each other. While the level of cyclin B1 protein was
reduced during endomitosis in MegT cells, we were unable to determine
the level of cyclin B2 protein because of the lack of an antibody that
recognizes mouse cyclin B2. It should be pointed out, however, that
cyclin B1 mRNA is the predominant one in MegT cells (Fig. 1).
Nevertheless, we do not exclude the possibility that small amounts of
B2 mRNA may lead to a significant amount of B2 protein in MegT cells.
In many systems tested, the lack of cyclin B1 alone is sufficient to
drive endoreduplication. Endoreduplication in some Drosophila cell types is indeed associated with a lack of cyclin B1 (Lehner
and O'Farrell, 1990). Also, the metaphase II arrest in mouse
oocytes is controlled through destruction of cyclin B1 (Kubiak et
al., 1993). Initiation of endoreduplication in different systems
depends on the availability of Cdc2 kinase and cyclin B, both composing
an active M phase promoting factor (MPF). Thus, certain treatments,
such as 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
(Usui et al., 1991; Moreno and Nurse, 1994). In the
filamentous fungus Aspergillus nidulans, the NIMA protein
kinase is required in addition to MPF for the M phase (O'Connell et al., 1994). Overexpression of this kinase in different
systems, including human cells, resulted in chromatin condensation
without other aspects of mitosis (O'Connell et al.,
1994). Although functional homologues of NIMA in mammalian cells have
not been described yet, some kinases have been reported to have
homology to the catalytic domain of NIMA (Schultz and Nigg, 1993).
These studies suggest that not all steps of mitosis are regulated by
the MPF kinase. The fission yeast Schizosaccharomyces cerevisiae temperature sensitivity cut8-563 mutation causes
chromosome overcondensation and short spindle formation in the absence
of cytokinesis, thus leading to the identification of a gene (cek1
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4266-4272
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/S cyclin, cyclin A, as
well as of the G phase cyclin, cyclin D3, were elevated at
the onset of DNA synthesis, either in MegT cells undergoing a mitotic
cell cycle or during endomitosis. In contrast, the level of the mitotic
cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle
while not detectable during endomitosis. Comparable levels of the
mitotic kinase protein, Cdc2, were detected during the mitotic cell
cycle or during endomitosis; however, cyclin B1-dependent Cdc2 kinase
activity was largely abolished in the polyploid cells. Fibroblasts
immortalized with the same heat-labile oncogene do not display reduced
levels of cyclin B1 upon shifting to high temperature nor do they
become polyploid, indicating that reduced levels of cyclin B1 is a
property of megakaryocytes and not of the T-antigen mutant. We conclude
that cellular programming during endoreduplication in megakaryocytes is
associated with reduced levels of cyclin B1.
phase or to proliferate, lineage
restriction and maturation of these committed cells, and synthesis of
cell-specific gene products. Although the mechanisms by which cells
withdraw from the stem cell pool are presently unknown, it was
suggested that the initial steps may be stochastic (Suda et
al., 1983). Thereafter, growth factors support progenitor cells to
develop along particular differentiation pathways (Metcalf, 1989). In
the megakaryocytic lineage, endomitosis involving DNA synthesis in the
absence of mitosis, as well as platelet production, are stimulated by
thrombopoietin, recently isolated (Kaushansky et al., 1994;
Wendling et al., 1994; Kuter et al., 1994; de Sauvage et al., 1994; Chang et al., 1995). The regulation of
the cell cycle and of endomitosis in this cell type has not been
explored yet./S cycle takes place without any intervening mitosis in
cells during the early development of the Drosophila embryo
(Smith and Orr-Weaver, 1991), as may also be the case in ploidizing
megakaryocytes. However, in most cells, the dependence of S phase upon
mitosis (M) and of M on S phase are strictly observed. The regulation
points of the cell cycle are the G/S and G
/M
transitions, for which the kinase activity of Cdk2 or Cdc2,
respectively, is crucial. These kinases are controlled through
association with regulatory subunits known as cyclins (Nasmyth, 1990;
Evans et al., 1983; Westendorf et al., 1990; Pines
and Hunter, 1990). The G/M transition is dependent on the
activity of the Cdc2 kinase, activation of which requires association
of Cdc2 with B-type cyclins and dephosphorylation on tyrosine (Draetta et al., 1989; King et al., 1994). Regulators of the
G/S transition include the cyclin B1-dependent Cdc2 kinase
(or its homologue Cdc28 in Saccharomyces cerevisiae)
associated to cyclin A (Pines and Hunter, 1990; Bartlett and Nurse,
1990). In clams, these two different cyclins, A and B, show a similar
periodicity in their synthesis and degradation, but in mammalian cells
the levels of cyclin A rise near the beginning of S phase and the
levels of cyclin B peak at the entry to mitosis (Evans et al.,
1983; Westendorf et al., 1990; Pines and Hunter, 1990). The
regulation of the G phase was studied first in S.
cerevisiae, where it was found that cyclins, cln1, cln2, and cln3 (CLN genes), control
progression through G, by modulating the activity of Cdc28
kinase (Hadwiger et al., 1989; Wittenberg et al.,
1990). Subsequently, equivalents to these G cyclins have
been identified, cyclins D1, D2, and D3 (reviewed by Reed, 1991).
Culture Conditions
MegT cells (clone 37C1) were
grown in a liquid culture, all as described before (Ravid et
al., 1993b). To induce ploidy, 1 
10
cells were
seeded into a 75-cm culture flask and incubated in 5%
CO at 39.5 °C for 4-5 days (Ravid et
al., 1993b). Cells were counted by hemocytometer, and cell
viability was followed by staining with Trypan Blue. For
synchronization experiments, cells were cultured for 24 h at the
indicated temperature after which the cells were shifted to a medium
containing 0.1% horse serum. Forty eight hours later, fresh medium
containing 20% horse serum was added to the cells. The attached cells
as well as the detached cells were collected separately at different
time points and subjected to various analyses. Immortal human
fibroblasts transformed by heat-labile (tsA58) T antigen (generous gift
of Dr. Harvey Ozer) were cultured as described elsewhere (Hubbard-Smith et al., 1992).Immunoprecipitation and Western
Blotting
Immunoprecipitation and Western blot analyses were
performed essentially as described before (Xiong et al.,
1992). To this end, MegT cells adhering to the culture dish and
nonadhering cells were collected separately by trypsinization or by
spinning down cells (380 g, 5 min) in the medium,
respectively. Cells were washed twice with cold phosphate-buffered
saline (PBS) ()(136 mM NaCl, 8 mM NaHPO
, 2.6 mM KCl, 1.4 mM KHPO, pH 7.4) by centrifugation and 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. Lysates, each containing 100
µg of protein in 400 µl of lysis buffer, were precleared by
incubation with normal mouse IgG and 40 µl of Zysorbin (fixed and
killed Staphylococcus aureus protein A, Zymed Laboratories,
Inc., San Francisco, CA) for 1 h at 4 °C followed by centrifugation
at 15,000 g for 5 min. Antibody was added to the
clarified lysate and incubated for 1 h at 4 °C. Protein A-Sepharose
CL-4B (Pharmacia Biotech Inc.) was added at a volume of 40 µl and
incubated at 4 °C for an additional hour. Immunoprecipitates were
washed three times with lysis buffer, resuspended in sodium dodecyl
sulfate (SDS) sample buffer, and separated on 12% SDS-polyacrylamide
gel (SDS-PAGE) (Laemmli and Favre, 1973). Protein assays were done as
recommended by the manufacturer (Bio-Rad Laboratories).Kinase Assays
Kinase assays were done essentially
as described before (Koff et al., 1991). Immunoprecipitates
containing the GNS-1 antibody (see above), which do not block the
binding of Cdc2 to cyclin B, and protein A-Sepharose CL-4B were washed
in kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl, 1 mM dithiothreitol). The beads were
resuspended in 50 µl of kinase buffer containing 2.5 µg of
histone H1 (Boehringer Mannheim, Germany), 5 µCi of
[-
P]ATP (3000 Ci/mmol, DuPont NEN), 30
µM ATP (Sigma). After a 20-min incubation at 25 °C, an
equal volume of 2
SDS sample buffer was added. The sample was
boiled for 5 min and centrifuged at 15,000 g for 5
min. 20 µl of the sample was loaded on 12% SDS-PAGE (Laemmli and
Favre, 1973). The gel was dried and subjected to autoradiography.DNA Synthesis
MegT cells were seeded in a 25-cm
flask at a concentration of 0.5 10 cells/5 ml of
medium (Ravid et al., 1993b). The cells were either cultured
at 34 °C or at 39.5 °C, as indicated. Cells were efficiently
synchronized by serum starvation, as described above, and, at different
time points after addition of serum, they were subjected to pulse
labeling with [
H]thymidine (15 µCi/5 ml)
(DuPont NEN) for 1 h. At the end of the pulse labeling, the medium was
collected, and the cells in suspension were spun down (380 g, 5 min) and washed twice with PBS. The adhering cells also
were washed with PBS. The adhering cells were extracted on the dish,
and the nonadhering cells were extracted in a tube, both with 1.5 ml of
5% trichloroacetic acid for 30 min at room temperature. Trichloroacetic
acid was discarded, and the cells were washed with an additional 2 ml
of trichloroacetic acid. Cells were dissolved in 0.5 ml of 1 N NaOH which was then collected to a scintillation vial containing 5
ml of scintillation mixture (Fisher Scientific) and counted in a
scintillation counter.Flowcytometer Analysis
Cells were scraped off the
plate with PBS or collected in medium, when in suspension, and pelleted
for 5 min at 380 g. Cells washed with PBS were
resuspended in propidium iodide (0.05 µg/ml in 0.1% sodium citrate,
pH 7.4) essentially as described before (Ravid et al., 1993b).
Uniform nuclear staining was achieved over 16 h at 4 °C. Just
before flow cytometry analysis, the cells were treated with
ribonuclease at a final concentration of 0.05 mg/ml, for 30 min at room
temperature. Cells were filtered through a 100-µm mesh, diluted
with propidium iodide solution to a concentration of 10 cells/ml, and subjected to flow cytometry analysis on a FACScan
system (Becton Dickinson). Data were collected and analyzed by CellFit
program (Becton Dickinson, San Jose, CA).Northern Blot Analysis
RNA isolation and Northern
blot analyses were performed as described before (Ravid et
al., 1993b). The blots were probed with different human cDNAs
encoding different cyclins, each recognizing the corresponding mouse
cDNA (a gift of Dr. Emma Lees, Massachusetts General Hospital Cancer
Center), or with mouse cDNA generated by the polymerase chain reaction
using DNA primers based on published sequences (Chapman and Wolgemuth,
1992). The blots were washed under low stringency for 60 min at 59
°C with 2 SSC (Ravid et al., 1993b). Blots
subjected to repeated hybridizations were first stripped by immersing
the blot for 10 min in a preboiled solution of 0.1% SDS (w/v in
HO). Probing with actin cDNA was performed last to confirm
equal loading of RNA in each lane.
Expression of Messages for Different Cyclins in MegT
Cells
Proliferating MegT cells were synchronized by serum
deprivation, and RNA was prepared from cells harvested at different
time points of the cell cycle. The RNA was subjected to Northern blot
analysis using different cyclin cDNAs as probes. As shown in Fig. 1, MegT cells expressed cyclins A, B1, D1, and D3 mRNAs,
but to a lesser extent B2 mRNA, and not D2 mRNA. It should be pointed
out that the B2 and D2 cDNAs used as probes detected the corresponding
mRNA from mouse total bone marrow, confirming that the probes used were
capable of recognizing the murine messages (not shown). Indeed, as
described for other systems, B-type and D-type cyclins are
differentially expressed in different tissues (Chapman and Wolgemuth,
1992, 1993; Matsushime et al., 1991).
-actin
cDNA to confirm equal loading of RNA. X-ray films exposed to the blots
were developed after 48-h exposure, except for the ones probed with
actin and large T antigen cDNAs which were exposed for 5 h. Two
splicing forms of cyclin B1 (1.6 kb and 2.5 kb) and two of cyclin A
(about 2.2 kb and 3.6 kb) were detected, as also described before
(Chapman and Wolgemuth, 1992; Samejima and Yanagida,
1994).
Correlation between the Level of Large T Antigen and of
the Mitotic Cyclin in Diploid and Polyploid MegT Cells
We noted
that once MegT cells were shifted to the temperature which is not
permissive for stability of large T antigen, a fraction of the cells
remained adhering to the dish while the other fraction detached from
the dish. The nonadhering cells, representing 34 + 9% (n = 6) of the cells at 4 days postculturing, appeared as
round and larger cells. At the permissive temperature (34 °C), all
cells remained adhering to the culture dish (Fig. 2). In the
current study, we performed separate ploidy analyses on the detached
and adhering cells cultured at the same nonpermissive temperature as
compared to MegT cells cultured at 34 °C. Under the later
conditions, all cells were adhering to the dish and consisted of 2N
(diploid) and 4N cells (Fig. 3). At the nonpermissive
temperature, the fraction of cells attached to the plate also consisted
of 2N and 4N cells (Fig. 3B). In contrast, the majority
of the detached cells were 4N and 8N cells (Fig. 3C).
In a previous study, we found that the megakaryocyte-promoting factor,
thrombopoietin (Kuter et al., 1994), did not have a
significant effect on the ploidy state of MegT cells. ()Recently, these cells were also analyzed for their ability
to express c-mpl, the receptor for thrombopoietin (Wendling et al., 1994). We found that the level of expression of
c-mpl in MegT cells was low, revealed only by the polymerase
chain reaction, but not by Northern blot analysis (not shown). This
indicated that signaling pathways critical for reaching a ploidy state
higher than 8-16N are not active in these cells.
100.
The Activity of the Mitotic Kinase during the Cell Cycle
in Synchronized MegT Cells
Pulse labeling with radiolabeled
thymidine was used to determine the profile of DNA synthesis in MegT
cells, synchronized by serum deprivation and cultured at the
nonpermissive temperature. Upon release from synchronization, at
successive time points, the adhering and nonadhering cells were labeled
for 1 h with [H]thymidine and collected
separately. During 40 h in culture, two cycles of DNA synthesis were
observed in MegT cells adhering to the dish, each spanning about 18 h.
The value of [H]thymidine incorporation at the
first peak of DNA synthesis was lower than the peak value during the
second cycle, and the cell number doubled at the end of the second
cycle, all as expected during a mitotic cell cycle (Fig. 6A). In contrast, each cycle of DNA synthesis in
the detached cells was completed within 10 h with a short gap between
the S phases (Fig. 6B). The cell number remained
constant at the end of both cycles, as expected during
endoreduplication. The second peak of DNA synthesis reached a value for
[H]thymidine incorporation lower than the one
expected in case all cells would have undergone endoreduplication. It
should be pointed out, however, that also in the case of primary bone
marrow megakaryocytes only a fraction of the cells continues
endomitosis for several cycles to reach a high ploidy state (Rovolic,
1974). Immunoprecipitation of equal amounts of proteins prepared from
the mitotic and endoreduplicating MegT cells revealed that the activity
of the mitosis promoting factor (MPF), consisting of cyclin B-dependent
Cdc2 kinase, was high during mitosis in the replicating MegT cells
while hardly detectable in the polyploid cells (inset in Fig. 6).
The Level of Different Cyclins in Synchronized MegT
Cells
We also sought to determine the level of G phase, G/S, and M phase cyclins at the onset and
offset of DNA synthesis in synchronized MegT cells cultured at the
nonpermissive temperature. As shown in Fig. 7A, Western
blot analyses revealed that during the mitotic cell cycle the level of
the G/S cyclin, cyclin A, was slightly higher at the onset
of DNA synthesis while elevation of cyclin B1 level started at S phase,
as expected before entry to mitosis. During the endomitotic cell cycle (Fig. 7B), the levels of cyclin A and of the protein
Cdc2 were significantly high, but cyclin B1 was barely detectable. As
to the large T antigen, it was readily detectable during the mitotic
cell cycle, but, undetectable in the polyploid cells. These results
further suggested that turning off the oncogene was a prerequisite for
shifting from a mitotic cell cycle to endomitosis. It should be also
pointed out that, during the mitotic cell cycle, the level of cyclin A
cycled moderately, while it did not seem to cycle during the
endomitotic cell cycle. It is possible that we were unable to detect a
rapid transient decrease in cyclin A during the short G phase of the
endomitotic cell cycle. The level of the G phase protein,
cyclin D3, for which a high level of mRNA was detected (Fig. 1)
was also determined. Cyclin D3 level was high at time points
corresponding to the G phase (Fig. 6) during the
mitotic cell cycle while moderate changes were observed in the level of
this cyclin during endomitosis (Fig. 8).
and
M phases to directly enter a G phase. While electron
microscopic analyses of different stages of mitosis in megakaryocytes
are underway, we sought to investigate the cyclin composition of
megakaryocytes undergoing endomitosis.H]thymidine
revealed that during endomitosis DNA synthesis was not continuous, but,
rather, interrupted by a short gap. The level of the G phase cyclin, cyclin D3, cycled during the mitotic cell cycle in
MegT cells and rose at the gap phase during endomitosis. This latter
result further suggested the existence of a G phase during
endomitosis in megakaryocytes. During this process of
endoreduplication, the whole cell cycle was quite short, spanning about
10 h, in correlation with a previous study in rat primary bone marrow
megakaryocytes (Odell et al., 1968). Further cyclin analyses
in MegT cells revealed no significant differences in the level of
cyclin A and in the level of Cdc2 protein during a mitotic or
endomitotic cell cycle. However, while the cyclin B1 level rose at the
onset of mitosis in continuously doubling MegT cells, it was hardly
detectable in the polyploid cells. In accordance, the activity of
cyclin B1-dependent kinase was low. Interestingly, the reduced level of
cyclin B1 could not be attributed to low level transcription, as the
level of cyclin B1 mRNA was not reduced in polyploid cells. The
mechanism of cyclin degradation, a highly selective process, is not
well understood. The amino-terminal sequences have been shown to play a
critical role in targeting cyclins to the ubiquitin degradation pathway
(Hershko et al., 1991), the activation of which occurs at the
onset of anaphase. It is plausible then that cyclin B1 is either not
stable or not translated in polyploid megakaryocytes. We confirmed that
a reduced level of cyclin B1 is a property of polyploid megakaryocytes,
rather than a property of our particular T-antigen cell line, by
analyzing immortal fibroblasts transformed with the
temperature-sensitive T antigen. At the nonpermissive temperature,
these latter cells are not polyploid (Resnick-Silverman et
al., 1991) and display a reduced level of T antigen, but not of
cyclin B1. In accordance with these results is our observation that
primary megakaryocytes derived from mouse bone marrow lack cyclin B1,
as revealed by lack of staining (via immunohistochemistry) with cyclin
B1 antibody (not shown).) encoding a novel protein kinase which
complements the mutation that blocks anaphase (Samejima and Yanagida,
1994). The model proposed by O'Connell et al.(1994)
involves condensation of chromatin by a NIMA homologue while other
mitotic events down to anaphase are regulated by MPF. Anaphase is
regulated by inactivation of MPF and may be regulated also by other
kinases such as cek1
analogues. If so, our
model of polyploid MegT cells with significantly reduced levels of
cyclin B1 may be used for studying the roles of different
mitosis-related kinases in endomitosis.
))
We thank Tony Hunter and Emma Lees for valuable
insight, Dimitry Kamen for helpful technical assistance, and Harvey
Ozer for the generous gift of immortal SVtsA/HF-A cells.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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