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HEMATOPOIESIS
From INSERM U 362, Institut Gustave Roussy, Villejuif,
France.
The cyclin-dependent kinase inhibitor p21Waf-1/Cip-1 is
expressed at high level during megakaryocyte differentiation, but its
precise function remains unknown. In this study, it is
confirmed that p21 was expressed at a high level in hypoploid
(2N and 4N) and polyploid (at least 8N) human megakaryocytes
derived from CD34+ cells. A high expression of
p27Kip1, p16, cyclin E, and cyclin D3 was also found in
both populations associated with a hypophosphorylated form of
retinoblastoma protein, suggesting that the majority of
hypoploid and polyploid megakaryocytes are
G1-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to
the murine model. The modal ploidy of megakaryocytes derived from
lineage-negative cells was 32N, and an elevated expression of p21
was found in high-ploidy megakaryocytes. In addition, p21 and p27 were
coexpressed in the majority of mature polyploid megakaryocytes. The
p21 was detected by immunofluorescence in megakaryocytes
derived from p53 Terminal megakaryocyte differentiation has many
original aspects.1,2 When the promegakaryoblast begins to
synthesize specific platelet proteins, megakaryocytic cells switch from
cell division (mitosis) to polyploidization (endomitosis). At the end of polyploidization, the megakaryocyte increases synthesis of specific
platelet proteins and the size of its cytoplasm, leading to enhanced
production of platelets.3 Finally, megakaryocytes form
proplatelets by extending long-branched pseudopods that will give rise
to platelets by fragmentation.4,5
Megakaryocyte polyploidization occurs by an original process called
endomitosis, which corresponds to an abortive mitosis in which
megakaryocytes skip late mitotic phases, anaphase B, telophase, and
cytokinesis.6,7 The endomitotic cell cycle is quite
similar to a normal cell cycle but with x successive G1/S/G2/M phases, leading to a
2xN ploidy.6,8 At present, the
molecular mechanisms regulating the switch from mitosis to endomitosis
are unknown. However, as in all the other cell lineages, cell-cycle
exit is associated with terminal differentiation.
The Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors,
which includes p21Cip1/Waf1, p27Kip1, and
p57Kip2, plays a crucial role in coupling cell-cycle arrest
with differentiation.9-14 Members of the Cip/Kip family
interact with both cyclins and cyclin-dependent kinases and inhibit all
CDK activities. However, their major effects are observed on the cyclin
E-CDK2 complex, leading to a block in the G1 phase.
Members of this family also have other effects on the cell biology. The
p21Cip1/Waf1 molecule, which has been first identified as a
p53 target,15,16 has many functions on cell cycle,
apoptosis, senescence, and differentiation. The p21 has opposing
effects on the cell cycle, being capable of blocking the cell cycle in
G1 and also in G2/M.17 More
surprisingly, p21 at low concentrations enhances the interaction
between cyclin D3 and CDK4 and facilitates G1
progression.18,19 The p21 has been also implicated in the
polyploidization of different cell models. The p21 induces hepatocyte
polyploidization mainly by inhibiting cytokinesis.20 In
other cell types, p21 overexpression leads to polyploidization by
favoring endoreplication21; however, p21 deficiency may
also lead to polyploidy.22
Recently, it has been shown that megakaryocytes express high levels of
p21 and p27 during differentiation.23,24 It has been
suggested that p21 is implicated in megakaryocyte polyploidization because overexpression of p21 in 2 cell lines with a megakaryocyte phenotype leads to nucleus polylobulation.25,26 In
addition, thrombopoietin, the humoral factor that regulates
ploidization and platelet production, increases p21 transcription and
expression through signal transducer and activator of transcription 5 (Stat5) activation.25
In this study, we have investigated the role of p21 on megakaryocyte
polyploidization by studying megakaryocytopoiesis of p21 nullizygote
mice (p21 Mice
Cell lines
Antibodies Phycoerythrin (PE)-murine antihuman CD41, PE-rat antimurine CD62P, anti-CD16/CD32 Fc(III/II), and fluorescein isothiocyanate (FITC)-rat antimurine CD41, all obtained from Pharmingen (San Diego, CA), were used for flow cytometric analysis. Monoclonal antibodies against ,  , and  tubulins were purchased
from Sigma (St Louis, MO). Isotype controls were obtained from
Becton Dickinson (Mountain View, CA) and Pharmingen.
The following rat monoclonal antibodies purchased from Pharmingen were used for depletion of lineage-positive cells: TER119, Gr-1, anti-CD11b (or Mac-1), B220, and anti-CD3. The following antibodies were used for Western blot and immunofluorescence: antihemagglutinin (HA) monoclonal antibody (mAb) (Boehringer Mannheim, Mannheim, Germany); anti-cyclin D3 mAb (Pharmingen); anti-cyclin E mAb (Pharmingen); anti-cyclin E polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-cyclin A mAb (Pharmingen); anti-cyclin B1 mAb (Pharmingen); anti-p21 mAb (Pharmingen); anti-p21 polyclonal antibody (Calbiochem, Meudon, France); anti-p27 polyclonal antibody (Pharmingen); anti-p16INKb polyclonal antibody (Pharmingen); anti-p57Kip2 polyclonal antibody (Santa Cruz Biotechnology); antiretinoblastoma protein (anti-Rb) mAb (Pharmingen); FITC antihuman Ki-67 mAb (Pharmingen); and an anti-von Willebrand factor (vWF) polyclonal antibody (Dako, Glostrup, Denmark). For indirect immunofluorescence, donkey tetrahodamine isothiocyanate (TRITC)-labeled antimouse, TRITC-labeled antigoat, or FITC-labeled antirabbit F(ab')2 fragments (Jackson Immunoresearch, West Grove, PA) were used. Progenitor cells and platelet isolation CD34+ cells were obtained either from the bone marrow of healthy patients undergoing hip surgery, with their informed consent, or from aliquots of cytapheresis from the peripheral blood of patients after mobilization. Cells were separated over a Ficoll-metrizoate gradient (Biochrom, Berlin, Germany), and CD34+ cells were purified by immunomagnetic selection (Miltenyi Biotec, Bergisch Gladbach, Germany).In mice, lineage-negative (lin Murine platelets were isolated from blood drawn by cardiac puncture and collected into an equal volume of acid dextrose citrate 1/10 diluted in buffered saline-glucose-citrate (BSGC). Plasma rich in platelets was prepared and laid over a 10-mL sepharose 4B column (Pharmacia Biotech, Buckinghamshire, United Kingdom) equilibrated with BSGC, pH 7.3. In vitro liquid cultures of megakaryocytes CD34+ cells were grown for 7 to 12 days in Iscoves modified Dulbecco medium (Gibco BRL) containing 1.5% deionized bovine serum albumin (Cohn fraction V; Sigma), iron-saturated human transferrin supplemented with selenium and insulin (Gibco BRL), and a mixture of sonicated lipids (20 µg/mL).28 The medium was supplemented with polyethylene glycol-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) (10 ng/mL) (Kirin Brewery, Tokyo, Japan), either alone or in combination with 50 ng/mL human stem cell factor (SCF) (Amgen, Thousand Oaks, CA).Murine lin Immunolabeling for fluorescence microscopy Cells were cytocentrifuged onto slides at 550 rpm for 4 minutes. Cells were fixed in 0.2% paraformaldehyde (Serva, Heidelberg, Germany) for 5 minutes at room temperature. They were permeabilized with 0.1% Triton X-100 (Sigma) prior to incubation with the antitubulin or the anti-p21, anti-p27, or anti-p57 antibodies. After 3 washes, cells were incubated with the appropriate secondary antibodies. DNA was finally labeled with 4'-6-diamidino-2-phenylindole-2HCl (DAPI) in Vectashield (Vector, Burlingame, CA), and slides were mounted.Flow cytometric analysis DNA analysis. To measure the ploidy of murine marrow megakaryocytes, cells were labeled with FITC-anti-CD41 mAb after preincubation with an anti-CD16/CD32 Fc(III/II), washed, and incubated in a hypotonic citrate solution containing 50 µg/mL propidium iodide (PI) (Sigma) for at least 2 hours (usually overnight) at 4°C. To measure the ploidy of cultured murine megakaryocytes and sorted human cultured CD41+ cells, cells were incubated in medium containing NP 40 (1/1000) and 50 µg/mL PI for at least 2 hours.DNA and nuclear protein analysis.
Cultured cells were washed in 1 × phosphate-buffered saline (PBS)
prior to fixation in 80% ethanol. Cells were maintained for at least
24 hours at Activation of platelets by thrombin Platelet activation by thrombin was studied as previously described.29 Platelets filtrated on a sepharose 4B column were adjusted at a concentration of 1 × 107/mL in BSGC (pH 7.3). Thrombin (Diagnostica Stago, Asnières, France) was serially diluted in BSGC and distributed into round tubes containing the PE-anti-CD62P mAb and FITC anti-CD41 mAbs. Tubes were incubated for 10 minutes at 37°C. The reaction was stopped by the addition of freshly prepared 0.6% paraformaldehyde. After 20 minutes of incubation at room temperature, pellets were suspended in 1 mL PBS and analyzed by flow cytometry.Cell sorting of megakaryocytes Cells recovered after 8 to 9 days for human cultures or after 2 days for murine cultures were incubated with the PE-anti-CD41 mAb for human cells or FITC-anti-CD41 mAb for mouse cells for 30 minutes at 4°C and for an additional 2 hours with 0.01 M Hoechst 33342 (Sigma) at 37°C. Megakaryocytes were sorted according to their DNA content by means of a FacsVantage cytometer (Becton Dickinson) equipped with 2 argon lasers (Coherent Radiation, Palo Alto, CA) tuned to 488 and 360 nm, respectively and a 200-µm nozzle. CD41+ cells were sorted into a 2N/4N and an at least 8N cell fraction at 500 cells per second at 4°C for human cells and into an at least 8N cell fraction for mouse cells.Retroviral vectors, virus production, and cell infection The following 2 constructs were used: Migr (long terminal repeat-internal ribosome entry site-enhanced green fluorescent protein [LTR-IRES-EGFP]) (a generous gift from J. Miller, Philadelphia, PA) and Migr-p21 (LTR-triple HA-p21-IRES-EGFP). The human p21 complementary DNA (cDNA) was obtained from C. Cayrol (Toulouse, France). A triple HA was inserted at the 5' end of the p21 cDNA by reverse transcriptase-polymerase chain reaction. The tagged p21 cDNA was sequenced and then inserted into the Migr retroviral vector.The retrovirus-producing cell line 293 EBNA (Invitrogen, Carlsbad, CA) was maintained in Dulbecco medium containing 10% fetal calf serum (Gibco BRL). Vesicular stomatite virus-G (VSV-G) pseudotyped retroviruses were produced by transient transfection of 293 EBNA cells by means of the Exgen reagent (Euromedex, Mundolsheim, France) and 3 plasmids: pCMV-G (VSV-G coding sequence); pCMV-gag-pol (both kindly supplied by Dr J. Morgenstern, Millenium, Boston, MA); and the Migr vector. Supernatant containing infectious retroviral particles was recovered and concentrated 60-fold by means of an Amicon (Millipore, Bedford, MA). Viral titers were determined by limiting dilution assay on NIH 3T3 cells according to the EGFP fluorescence, and ranged from 1 × 107 to 5 × 108 viral particles per milliliter. To infect murine cells, an ecotropic retrovirus was prepared. The Phoenix ecotropic packaging cell line obtained from the ATCC (Stanford, CA) was infected with the VSV-G Migr vector, and a stable polyclonal cell line was obtained. Human cells derived from CD34+ cells were infected at days
5 and 6 of culture by adding 10% viral supernatant.
Lin Human megakaryocytes were sorted on a FacsVantage after labeling with the PE anti-CD41 mAb. Cells expressing high levels of EGFP and CD41 were sorted. Their ploidy was subsequently measured after staining with PI. Infected UT-7 cells were sorted according to the intensity of EGFP fluorescence into 4 fractions: negative, low/medium, high, and very bright. In the mouse cells, infection was controlled under a fluorescent inverted microscope. Ploidy in the cells of at least 8N was directly measured after PI labeling. Western blot analysis Soluble proteins obtained from approximately 500 000 to 5 × 106 cells lysed in Laemmli buffer were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) and then transferred electrophoretically onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were incubated for 90 minutes at room temperature with the different primary antibodies. After 2 washes, membranes were incubated with either goat antirabbit or antimouse antibodies conjugated to horseradish peroxidase diluted at 1:5000 and 1:10 000, respectively (Amersham). The bands were developed with an enhanced chemiluminescence system (ECL kit; Amersham).
Expression of cycle-associated proteins in human megakaryocyte differentiation Bone marrow CD34+ cells were cultured in the presence of SCF and PEG-rHuMGDF, and at 8 to 9 days of culture, megakaryocytes were studied. As previously demonstrated,7,30 in most experiments megakaryocyte ploidy was low, with the majority of megakaryocytes with a 2N and 4N content (about 70%). Polyploid megakaryocytes were essentially 8N, and fewer than 10% of the megakaryocytes had a ploidy higher than 8N. In some experiments, ploidy was slightly higher, with up to 15% being 16N cells. Ploidy did not increase with longer times in culture, and a large majority of megakaryocytes were labeled by an anti-vWF antibody. Megakaryocytes were purified by cell sorting, dependent on their ploidy, into 2 cell fractions (2N/4N cells and cells of at least 8N) by double-staining with a FITC anti-CD41 mAb and Hoechst 33342 (Figure 1A). Purity of both fractions was greater than 90% after reanalysis as previously reported.7
Cells sorted at days 8 to 9 from 8 independent experiments were pooled in order to obtain a sufficient number of cells and to diminish the variability among samples. Expression of cell-cycle proteins isolated from both megakaryocyte fractions was studied by Western blots (Figure 1B). As previously reported, p21, p27, p16, cyclin E, and cyclin D3 were expressed at high levels in both fractions; in addition, p53 was expressed at high levels in both fractions (data not shown) while cyclin A was also detected but at a lower level in both fractions (Figure 1B). In addition, as we have previously reported,7 cyclin B1 was also present but at very low levels in comparison with cyclin D3 (Figure 1C, data obtained from another pool of sorted cells obtained with the same purity). These data show that many cell-cycle proteins associated with the G1 phase were present in the 2 asynchronous megakaryocyte cell populations, suggesting that the majority of cells were in the G1/S phase of the cell cycle. Detection of cyclin B1 in both fractions correlates with the presence of fewer than 1% of cells in mitosis or endomitosis when cells were observed on a slide with a DAPI staining. To confirm this hypothesis, we studied the expression and the
phosphorylation of Rb (Figure 2). Flow
cytometric analysis revealed that Rb was present in megakaryocytes of
all ploidy classes, demonstrating that this gene is normally expressed
during polyploidization (Figure 2A). In situ immunofluorescence
labeling demonstrated a patchy staining of Rb, which was localized to
the nucleus (Figure 2B). The phosphorylation status of the Rb protein
was studied by the migration pattern in 2N/4N and at least 8N
megakaryocytes (Figure 3A), by which we
observed a hypophosphorylated form of Rb, which was largely predominant
and accumulated in both cell fractions. This result clearly confirms
that most hypoploid and polyploid megakaryocytes were blocked at the
G1/S transition and have arrested their cell cycle. Then,
we studied expression of p21 according to the date of culture and,
thus, to megakaryocyte differentiation. At day 7 of culture, a minority
of megakaryocytes (17%) expressed p21 with a nuclear localization. At
days 9, 10, and 12 of culture, expression of the Ki-67 antigen and p21
protein was studied by double labeling on slides in 2 independent
experiments. On average, at day 9, 45% of megakaryocytes expressed
p21, and 18.5% the Ki-67 antigen. The percentage of megakaryocytes
coexpressing p21 and the Ki-67 antigen was approximately 15%. However,
in megakaryocytes expressing a very high level of p21, the Ki-67
antigen was nearly undetectable, confirming the reciprocal expression
between p21 and Ki-67.23 At day 10, 65% of megakaryocytes
expressed p21 at intermediate or high level with a nuclear
localization; 17.5% expressed Ki-67 antigen; and 2.7% coxpressed the
2 markers. We detected p21 both in the nucleus of small megakaryocytes
with a round or indented nucleus and in large megakaryocytes. At day 12, Ki-67 antigen was detected in fewer than 5% of the cells. Double
labeling was performed between p21 and cyclin D3, and nearly all
megakaryocytes (more than 90%) expressed both proteins with a nuclear
localization, further suggesting that mature hypoploid and polyploid
megakaryocytes are blocked in the G1 phase of the cell
cycle. Later in culture, p21 was expressed in a lower number of
megakaryocytes because p21 was no longer detected in the nucleus of
apoptotic megakaryocytes.
Therefore, one limitation of the in vitro human model of megakaryocyte differentiation is related to the fact that there is no correlation between the level of ploidy and the precise stage of maturation. Indeed, terminal differentiation leading to proplatelet formation can occur in low-ploidy megakaryocytes. Thus, to further elucidate the role of p21 during megakaryocyte differentiation, we switched to the murine model of differentiation in which it is known that the majority of megakaryocytes can reach a higher ploidy.31 Expression of Rb and p21 in murine megakaryocytes Murine marrow lin cells were purified and cultured
in the presence of SCF and PEG-rHuMGDF. At day 4 of culture, in
contrast to human cultures, the modal ploidy of murine megakaryocytes
was 32N (see below). Ploidy increased very quickly between day 2 and day 4, suggesting that megakaryocytes were partially synchronous in
their cell cycle. This was confirmed by studying expression of the
Ki-67 antigen because the large majority of megakaryocytes (94%)
express this antigen at day 2 of culture. Thus, megakaryocytes were
sorted at this date. However, in 2-day cultures, the number of
megakaryocytes was low, and only the cells of at least 8N could be
sorted in sufficient number to study the Rb phosphorylation (Figure
3B). In contrast to human megakaryocytes, Rb migrated as 2 molecular
species corresponding to a hyperphosphorylated and a hypophosphorylated
form. The 2 forms were expressed at an equivalent level, which is
consistent with the presence of a normal Rb regulation during an
endomitotic cell cycle. Expression of p21 was studied by flow cytometry
at day 4 of culture (Figure 4A), 2 days
later in culture than Rb phosphorylation studies; at this date of
culture, megakaryocytes were mature and had stopped their endomitotic
process. Expression of p21 was extremely elevated in megakaryocytes
with high ploidy. Indeed, by in situ immunofluorescence, we observed
that the great majority (greater than 90%) of megakaryocytes with a
polylobulated nucleus expressed p21. Labeling was localized in the
nucleus with a diffuse staining (Figure 4B). Most megakaryocytes (about
70%) expressed high levels of p21 as illustrated in Figure 4B, and a
minor fraction (about 20%) expressed p21 at a lower level. As in
humans, a reciprocal expression between p21 and Ki-67 antigen was
found. At day 2 of culture, Ki-67 antigen and p21 were detected in 94%
and 10% of polyploid murine megakaryocytes, respectively, and 5%
coexpressed both markers. The percentage of megakaryocytes positive for
Ki-67 antigen decreased the following days (72% and 20% being
positive at days 3 and 4, respectively).
Expression of p21 in megakaryocytes is not p53 dependent In human megakaryocytes of 2N/4N and at least 8N, p53 was expressed at high levels. Thus, we next studied whether p21 expression was regulated by p53 during megakaryocyte differentiation. Lin marrow cells from p53/ mice were
cultured in the presence of SCF and PEG-rHuMGDF for 4 days, and
expression of p21 was studied by immunofluorescence labeling on slides.
We detected p21 in the nucleus of almost all p53/
megakaryocytes with a fluorescence intensity quite similar to that
observed in wild-type megakaryocytes (Figure
5). This result demonstrates that p21
expression is not regulated by p53 during megakaryocyte
differentiation.
To more precisely investigate the role of p21 in megakaryocyte
differentiation, we studied the megakaryocytopoiesis of
p21 p21 / and wild-type CF1 mice (average,
1 270 000 ± 250 000/µL in wild-type and
1 460 000 ± 530 000/µL in p21/ mice), and no
difference was found in the platelet sensitivity to thrombin activation
in 4 repeated experiments (data not shown), suggesting that
p21/ platelets have a normal function.
Because it has been reported that p21 plays an important role in the
centrosome separation and mitotic spindle organization in several cell
types,22 we next studied whether p21 We next analyzed the ploidy of p21
Together, these results demonstrated that p21 Effects on the overexpression of p21 on the ploidy of murine and human megakaryocytes To further define the role of p21, overexpression in cell lines was performed. A bicistronic retrovirus (Migr) containing HA-tagged p21 cDNA under the control of murine stem cell virus (MSCV) LTR and EGFP was constructed to overexpress p21 in murine and human megakaryocytes. Ecotropic and VSV-G-pseudotyped retrovirus were used for infection of murine and human cells, respectively. Similar retroviruses containing a vector coding for EGFP alone were used as controls.The UT-7 cell line was first tested to confirm the functionality of the
transduced p21. Cells were sorted on the level of EGFP expression into
4 populations (negative, low/intermediate, high, and bright fluorescent
intensity). An increase in cells in G1 phase that
correlated with the intensity of the EGFP was observed. More than 85%
of the UT-7 cells expressing a very high level of EGFP were blocked in
G1, impeding cell growth. The level of p21 was increased at
least 15-fold in UT-7 cells after retroviral transduction (Figure
7B). This result indicates that p21
expression and function correlated with the expression of EGFP. Then,
lin
Here, we sought to determine the precise role of p21 during
megakaryocyte differentiation. In previous studies on p21 expression during megakaryocytic differentiation, both p21 messenger RNA and
protein were detected at a high level in megakaryocytes. Unlike p27,
which was expressed only in Ki67 In contrast, in the mouse system, megakaryocytes can reach a high ploidy level, up to 256N after 3 to 4 days of culture, with a coupling between terminal differentiation and ploidization. In this model, p21 was expressed at a very high level only in polyploid megakaryocytes. A good correlation was observed between p21 and p27 expression, with the majority of megakaryocytes expressing these 2 CDK inhibitors. Therefore, p21 and p27 expression is high only in mature megakaryocytes that have terminated their ploidization. This high expression of the p21 protein in murine megakaryocytes obtained in culture contrasts with previous in vivo studies in which only a minority (fewer than 15%) of megakaryocytes in the spleen synthesized p21 as measured by in situ hybridization.24 Possibly, this can be explained by the differences in the intensity of the cytokine stimulation and by the fact that cultures are nearly synchronized in early days. The p21 is regulated through p53-dependent and p53-independent
mechanisms.9,38 A large part of the p53 effect on cell cycle is mediated by p21, and thus p21 is responsible for the cell-cycle arrest induced by DNA damage.15,16 In contrast, regulation of p21 during differentiation has been shown to be p53
independent in many cellular models39 although it has been recently reported that p21 expression during erythroid differentiation was p53 dependent.40 We observed that p21 was expressed at
a similarly high level in wild-type and p53 The p21 and p27 can induce polyploidy in several cellular models by
inducing endoreduplication. The cell-cycle effect of p21 may be related
to the down-regulation of proteins involved in cytokinesis and mitosis
entry, including CDK1 and cyclin B1.42,43 It has been
suggested that overexpression of p21 may transiently block cells at the
G2/M transition17,21 and that cells then skip
mitosis and endoreplicate DNA. Rb plays a crucial role in this
regulation by preventing endoreplication, in which p21 overexpression induces polyploidy only in Rb To more precisely assess the role of p21 on megakaryocyte
polyploidization, we studied the effects of its inactivation or overexpression. The p21-deficient mice have a decreased number of
hematopoietic progenitors with a defect in their cytokine
responsveness.48,49 More recently, it has been shown that
p21 To better understand the role of p21, we subsequently switched to an
overexpression strategy. The p21 was transduced into a cell line with a
megakaryocyte phenotype (UT-7) by means of a retroviral vector, and as
expected, an arrest in G1 was correlated with the level of
p21 protein expression. These data are in contrast with results
previously obtained studying the transient expression of p21 in UT-7,
where p21 induced nuclear polylobulation26; however,
ploidy was not measured in these experiments. Re-expression of p21 in
p21 It is noteworthy that megakaryocyte and myogenic differentiation have several similarities, including polyploidization (by totally different mechanisms) and the same high expression of cyclin D3 and p21.51 In muscle differentiation, cyclin D3 is involved in G1 growth arrest and differentiation by sequestering CDKs and CDK inhibitors in a high molecular complex.51 Transgenic mice overexpressing cyclin D3 have megakaryocytes of higher ploidy than wild-type animals, demonstrating that cyclin D3 is involved in the endomitotic process.24 However, treatment of megakaryocyte cultures with a cyclin D3 antisense oligonucleotide inhibited maturation, suggesting that cyclin D3 is also involved in megakaryocyte differentiation.8,47 Similarly, p21 plays an important role in skeletal muscle differentiation, and the molecular mechanisms involved in thi |
Top
Abstract
Introduction
20°C, washed in PBS containing 1% bovine serum
albumin, and permeabilized with 0.25% Tween 20 (Sigma). Cells were
subsequently incubated with the anti-Rb mAb or anti-p21 polyclonal
antibody or with control antibodies and then with FITC-sheep antimouse
or rabbit immunoglobulin G. Finally, PBS containing 50 µg/mL PI
(Sigma) and 100 µg/mL RNase A was added to the cell pellet for
approximately 2 hours. Cell samples were analyzed on a FACSort
(Becton Dickinson).
