(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/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.
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 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.
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/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).
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 -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).
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 100.
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 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.
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 [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).
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) 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.