|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2907-2917
Subcellular and Cell-Cycle Expression Profiles of CDK-Inhibitors in
Normal Differentiating Myeloid Cells
By
B. Yaroslavskiy,
S. Watkins,
A.D. Donnenberg,
T.J. Patton, and
R.A. Steinman
From the Departments of Medicine and Cell Biology, University of
Pittsburgh School of Medicine, Pittsburgh, PA.
 |
ABSTRACT |
A central question in hematopoiesis is how cell-cycling behavior
changes during the emergence of the differentiated state. To further
understand what genetic regulators might couple proliferation status to
differentiation, we studied the expression of the cell-cycle inhibitors
p21 and p27 during the in vitro differentiation of normal
CD34+ blast cells along the myeloid lineage. We find p27
but not p21 to be expressed in freshly harvested resting
CD34+ cells. Thereafter, p21 levels peak concurrent with
cellular proliferation and then decline in expression as cells undergo
terminal differentiation. In contrast, p27 levels are fairly constant
but the subcellular localization of p27 changes from nuclear expression
to predominantly cytoplasmic expression and finally to perinuclear
localization at progressive stages of differentiation. This report
discusses the implications of these findings.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
A CENTRAL FEATURE of differentiation is
the transition from quiescent progenitor cells to a pool of
proliferating precursor cells, giving rise to nonproliferating,
differentiated progeny. The emergence of terminally differentiated
cells in several lineages is coupled to a transition out of the mitotic
pool. Genetic regulators responsible for the coupling of proliferation
status to the emergence of the differentiated phenotype are under
investigation. Previous studies from our laboratory showed that the p21
(WAF1) cell-cycle inhibitor is upregulated during myeloid
differentiation in cell line models.1
p21 and other inhibitors of G1-phase cyclin-dependent kinases such as
p27 could conceivably contribute to downmodulation of the proliferating
potential of differentiating progeny. p21 and p27 share a similar
domain involved in cdk and cyclin binding, and both have been shown to
mediate growth arrest when overexpressed,2,3 contribute to
restriction point G1 arrest,4-6 and are upregulated in
myeloid differentiation models.1,7,8 However, the roles of
p21 and p27 during myelopoiesis may be distinct rather than overlapping. p21 has been shown either to promote or inhibit cell cycle
progression on a stoichiometric basis,9,10 operating as a
cycling-cdk assembly factor at low concentrations. p27 levels have been
shown to decrease and p21 levels to increase in lymphocytes stimulated
to exit from quiescence11 and on stimulation of factor depleted Mo7e cells with stem cell factor (SCF) and
granulocyte-macrophage colony-stimulating factor
(GM-CSF).12 Moreover, p21 knockout mice are defective in
steel-factor-driven myelopoiesis.12 Experiments in other
systems suggest that terminal differentiation might involve a
predominant role for p21; keratinocytes from p21-knockout
mice but not p27-knockout mice exhibit a significant
decrease in differentiation markers.13
As an initial step in establishing the role of these proteins during
differentiation of normal hematopoietic precursor cells, we have
analyzed the expression of these cdk inhibitors (cdki's) in normal
umbilical cord CD34+ cells stimulated to differentiate
along the myeloid lineage. Morphologic and cell cycle profiles of cells
expressing high levels of p21 and p27 were determined. Image analysis
of immunostained cytospins and fluorescence-activated cell sorter
(FACS) analysis showed that p21 expression is absent in freshly
harvested CD34+ cells and increases from 2 to 6 days after
exposure to SCF, granulocyte-colony-stimulating factor (G-CSF), and
interleukin-3 (IL-3) p21 levels peak before terminal differentiation
and fall within 11 days of exposure to cytokines. p27 is expressed both
in freshly harvested and in differentiated cells, but its subcellular
distribution is altered during differentiation; this suggests that p27
may serve different functions at different stages of myeloid
differentiation. Neither the expression of p21 nor p27 is restricted to
G0/G1 phase, as demonstrated by dual flow determination of cdki
expression and cell-cycle partitioning. These results suggest that p21
and p27 have distinct roles during the myeloid differentiation process.
The changing expression and subcellular patterns of these cdki's
during myelopoiesis indicate a complex function for p21 and p27
throughout differentiation, rather than a limited role mediating growth
arrest in the final stages of terminal differentiation.
| |
MATERIALS AND METHODS |
Cells.
CD34+ cells were prepared as previously
described.14 In brief, human umbilical cord blood (CB)
samples were obtained in accordance with institutional guidelines, and
low density mononuclear cells were isolated by Ficoll-Paque (1.077 g/mL) density gradient centrifugation. CB mononuclear cells were washed
twice in phosphate buffered saline (PBS) and resuspended in PBS + 0.6%
anticoagulant citrate dextrose (ACD) for magnetic labeling and
separation. Cells were incubated with blocking reagent (human IgG) and
QBEND/10 anti-CD34 antibody for 15 minutes at 4°C, then washed in
PBS/ACD-A followed by incubation with a secondary antibody-magnetic
microbead conjugate for an additional 15 minutes at 4°C. The
percentage of CD34+ cells generally exceeded 95% as
determined by flow cytometric analysis. The CD34+ cells
were cultured in serum-free StemPro media (Life Technologies, Gaithersburg, MD) and supplemented with 50 ng/mL kit ligand (R&D Systems, Minneapolis, MN), 100 ng/mL IL-3 (R & D Systems), and 30 ng/mL
G-CSF (Neupogen; Amgen, Inc, Thousand Oaks, CA). When indicated,
CD34+ cells were prepared by negative selection by using
Stem Sep columns (StemCell Technologies, Inc, Vancouver, Canada) and by
using a CD34+ enrichment cocktail per the manufacturer's
protocol. Use of negative selection to enrich CD34+ cells
yielded purity of roughly 70% CD34+ cells by flow
cytometric analysis. Neutrophils were harvested after spinning plasma,
removing residual erythrocytes with hypotonic washes, followed by
Ficoll-Paque (1.077 g/mL) density gradient centrifugation for 30 minutes at 400 g and collection of the pellet in PBS.
Lymphocytes were purified and washed from ficoll interface. Purity of
all cells (generally >95%) was verified by Wright-Giemsa staining.
Flow cytometry.
Cells harvested at various time points adjusted to 1 × 106 per mL were fixed with 1% paraformaldehyde and kept in
75% ethanol at  20°C. Before staining, cells were permeabilized
with 0.25% Triton-X 100 and 40 µg/mL digitonin for 5 minutes,
followed by washing. Cells were then incubated with monoclonal
antihuman p21 or antihuman p27 (Neomarkers, Freemont, CA) at a
concentration of 1:50 for 1 hour on ice, followed by incubation with
biotinylated horse antimouse antibody (Vector Laboratories, Burlingame,
CA) at 1:200 for 40 minutes on ice and extraavidin-fluorescein
isothiocyanate (FITC) conjugate at 1:100 (Sigma, St Louis, MO) for 30 minutes on ice. After antibody staining, the cells were prepared for
DNA staining by addition of RNAse and propidium iodide and incubated on
ice according to standard techniques. Additional antibodies used for
surface characterization of differentiation were CD71FITC (DAKO,
Carpinteria, CA), CD34PE (Becton Dickinson, San Jose, CA), CD11bPE
(DAKO), and CD15FITC (Becton Dickinson), which were all used according
to manufacturer's instructions.
Cells were also stained in suspension for confocal microscopy analysis.
Cells were fixed and permeabilized for flow cytometry. For two-color
staining, cells were incubated with p27 polyclonal antibody (Santa
Cruz, sc-528, directed at aa 191-198) at 1:50 dilution for 1 hour
followed by goat anti-rabbit Alexa-488 (Molecular Probes, Eugene, OR)
at 1:500 dilution for 1 hour. After staining for p27, the cells were
incubated with monoclonal anti-Ki67 (Oncogene Research Products,
Cambridge, MA) for 1 hour followed by goat anti-mouse Cy3-conjugated
secondary antibody (Jackson Immunoresearch Labs, Inc, West Grove, PA)
at a 1:3,000 dilution for 1 hour. Suspension cells were doubly stained
for p27 and CD34 or CD71 by incubating negatively selected cells with
phycoerythrin (PE) conjugated CD34 (anti-HPCA-2, Becton Dickinson) or
with CD71 FITC (Dako, Inc, Denmark) for 20 minutes followed by fixation
and permeabilization. The CD34+ stained cells were then
incubated with polyclonal rabbit anti-p27 (Santa Cruz) at 1:50 dilution
for 1 hour after goat anti-rabbit Alexa-488 (Molecular Probes) at 1:500
dilution. The CD71-stained cells were then incubated with monoclonal
anti-p27 (Neomarkers) at 1:50 dilution, followed by anti-mouse Cy3
(Jackson Immunoresearch Labs) at 1:3,000 dilution for 50 minutes.
Alternatively, as indicated, cells were stained with p27 goat
polyclonal (Santa Cruz, sc527, directed at N terminal aa 2-21) at 1:50
dilution, followed by donkey anti-goat Cy3 (Jackson Immunoresearch
Labs) at 1:500 dilution.
Immunocytochemistry.
Cytospins were stained by fixation in 2% paraformaldehyde and then
permeabilized in 0.1% Triton X-100 and 40 µg/mL digitonin, followed
by staining for 1 hour with monoclonal antibody (MoAb) to p21
(supernatant CP36, courtesy of Brian Dynlacht, at a ratio of 1:80) or
Neomarkers anti-p27 Ab-1 at a ratio of 1:50, followed by four PBS
washes, incubation for 30 minutes with biotinylated goat anti-mouse IgG
antibody (Vector Laboratories) again, followed by three washes.
Immunodetection was enabled by a final 30-minute incubation with
Cy3-conjugated-streptavidin at 1:12,000 (Jackson Immunochemicals, West
Grove, PA), followed by three PBS washes. Cells were counterstained
with Hoechst. In some experiments, the cells were incubated with BrDU
for 8 hours before harvest and the slides stained indirectly with
anti-BrDU antibody (Amersham, Arlington Heights, IL), biotinylated
horse antimouse antibody, and streptavidin AP-conjugate, developed with
AEC (Amersham). For BrDU staining and for p27 peroxidase staining,
slides were fixed in 50% ethanol for 30 minutes at 4°C, treated with
0.1 N HCl and 0.7% Triton, and heat denatured in H2O.
Confocal microscopy.
To maximize spatial resolution in all three axes, confocal microscopy
was used to examine the precise distribution of p27 in cells. After
immunohistochemical labeling, cell suspensions were placed onto charged
coverslips mounted within a Leica TCS-NT confocal
microscope (Leica, Deerfield, IL) and allowed to settle. Subsequently, the cells were scanned with a 100× plan apochromat objective at 1,024 × 1,024 pixel resolution, with a magnification factor of 2× at the scan head. To maximize Z-axis, resolution scans
were performed with a small pinhole such that the resolution from a
measured point spread function in the Z-axis was less than twice the
X-Y resolution (0.35 um). Each scan is the Kalman average of four
sequential scans through the middle plane of the cells.
Image analysis.
p21 staining structures in a field were rendered to a grey scale with
the Hoechst-stained nucleus as a mask to segregate cells. Profiles of
the p21 labeling are extracted and the intensity quantified by using
Optimas (Media Cybernetics, Silver Spring, MD).
| |
RESULTS |
To characterize p21 and p27 expression during myelopoiesis, we set up
an in vitro culture system of CD34+ cells simulating
myeloid differentiation. CD34+ cells were harvested by
immunoaffinity purification of mononuclear cells obtained from fresh
umbilical cord blood. The CD34+ cells were cultured in
StemPro (Life Technologies, Gaithersburg, MD) serum-free medium
supplemented with 100 ng/mL G-CSF, 50 ng/mL SCF, and 50 ng/mL IL-3. At
various time points after the initiation of culture, aliquots of cell
suspension were taken for flow cytometric analysis and for cytospins.
As shown in Fig 1A, these culture conditions restrict progeny to the myeloid lineage, with mostly myelocytes arising after 4 days in culture and bands and neutrophils predominating after 11 days. Morphological differentiation was accompanied by the expected development of cell-surface markers as
shown in Fig 1B. CD71, CD15, and CD11b were used to characterize the
emergence of differentiating cells. CD71, a marker of proliferating cells, appeared at 4 days in culture. Single positive CD15 cells were
followed by CD15+ CD11b+ and
CD15 CD11b+ cells emerging at day 7 as
indicated. This progression of markers is similar to previous
reports.15
View larger version (16K):
[in this window]
[in a new window]
| Fig 1.
(A) Differentiation of CD34+ cells in
StemPro culture medium. Morphology of cells stimulated to differentiate
into granulocytes by kit ligand, interleukin-3 (IL-3), and
granulocyte-colony-stimulating factor (G-CSF) in StemPro serum-free
medium. (B) Surface marker expression by CD34+ cells
differentiating under culture conditions. (C) Cell-cycle distribution
of differentiating CD34+ cells overtime determined by
propidium iodide staining.
|
|
Differentiation was accompanied by entry of cells into cycle, peaking
at 4 to 5 days, followed by a gradual decrease in the percentage of
cells in S-phase (Fig 1C). Staining after a 12-hour BrdU pulse
confirmed that the percentage of BrdU positive cells declined from 45%
at day 6 to 16% at day 10 (data not shown).
p21 and p27 during myelopoiesis.
To first characterize the expression of p21 and p27 in quiescent
CD34+ blasts and their differentiating progeny, we
immunostained cytospins of CD34+ blasts and of
CD34+ cells cultured in cytokines for 6 days. Freshly
harvested CD34+ were subjected to immunohistochemical
staining for p21 or p27 after overnight culture in low serum to
facilitate immunoaffinity bead detachment. Similar results were
obtained with immediate staining of cells after chymopapain treatment.
As Fig 2 shows, CD34+ blasts
were negative for p21 expression when using a sensitive detection
system consisting of a biotinylated secondary antibody and streptavidin
conjugated Cy3 tertiary staining. Staining with a monoclonal anti-p21
antibody CP36 (courtesy of Brian Dynlacht, MGH, Boston,
MA) is shown; similar results were obtained with several
commercial antibodies. In contrast to p21, p27 expression is readily
evident in nuclei of these cells and can be detected by using either a
fluorescent secondary antibody (shown here) or with peroxidase
enzymatic detection (see Fig 4). As noted above, the CD34+
cells are in G0/G1 phase. The absence of p21 and presence of p27 in the
nuclei of these cells suggests that p27, not p21 is contributing to
growth arrest of these cells, which is consistent with findings in
quiescent lymphocytes.16
View larger version (106K):
[in this window]
[in a new window]
| Fig 2.
Expression of p21 and p27 in CD34+ blasts
and in differentiating cells. (A-F) Cytospins of positively selected
CD34+ blasts maintained overnight in low serum after
harvest were stained for p21 or p27 as described (Neomarkers MoAbs).
Some beads used in the immunoisolation are apparent in the isotype
control. Nuclear staining of p27 is evident. Cytospins prepared after 6 days of culture in SCF, G-CSF, and IL-3 were simultaneously stained.
Nuclear staining of p21 and cytoplasmic staining of p27 is evident.
Cells are counterstained with Hoechst. (G-L) Staining of negatively
selected CD34+ cells freshly harvested (G) or after 4 days of culture (H-L). (G) Confocal micrograph of cells doubly stained
for CD34 (phycoerythrocin, red) and p27 (Alexa-488). A field containing
a CD34 cell is shown, enlarged for detail. (H)
Photograph of cells doubly stained for p27 (Cy3, red) and CD71
(fluorescein isothiocyanate [FITC]). After days of culture, 86% of
the cells were positive for CD71. (I) Confocal micrograph of cells
doubly stained for Ki-67 (Cy3) and p27 (Alexa-48). Nuclear Ki-67 and
cytoplasmic p27 is evident. Overlapping nuclear signals are yellow. (J)
Isotype control for (I). All isotype controls were similarly negative.
(K, L) Confocal microscopy of cells for p27, using C-terminal (G) and
N-terminal (L) (Santa Cruz) antibodies.
|
|
Several interesting findings emerge from staining of cells
differentiated for 6 days (predominantly myelocytes under our culture conditions). p21 protein is upregulated in the maturing cells, which is
consistent with our previous findings14 and is
predominantly localized to the nucleus. Surprisingly, the cells develop
strong p27 immunoreactivity in the cytoplasm as they differentiate. A reticular staining pattern is evident. The persistent expression of p27
contrasts with downmodulation of p27 protein levels in lymphocytes and
in fibroblasts stimulated to enter the cell cycle.4,16 The
cytoplasmic localization of p27 is distinct from the predominantly nuclear localization reported for p27 transfected into mink lung epithelial cells.17 In that study, forced cytoplasmic
expression of p27 eliminated cdk4 sequestration by cytoplasmic p15. The
persistence of p27 expression in proliferating and differentiating
myeloid cells was also confirmed by Western blotting and Northern
blotting (Fig 3). A single band
of the correct size is recognized in differenting cells by the
monoclonal used in immunostaining; the identity of an additional,
slowly migrating band most prominent in CD34+ blasts is
under investigation. This band is repeatable with other antibodies; we
note that ubiquitinated p27 gives a prominent band at this
position.18 p27 message is constant during
differentiation (Fig 3) in contrast to p21 message, which is
upregulated as we have previously reported.1,14
View larger version (73K):
[in this window]
[in a new window]
| Fig 3.
p27 message and protein levels during myelopoiesis. (A)
Persistence of p27 message in differentiating CD34+
cells. Aliquots of cells grown in the presence of SCF, G-CSF, and IL-3
for 7 or 10 days were harvested for RNA and a Northern blot probed for
p27 message as shown. (B) Freshly harvested UC CD34+
cells were cultured in the presence of cytokines for 12 days. At days
0, 6, 9, and 12 aliquots of 1 million cells were directly lysed in
laemmli buffer and fractionated on a 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gel for Western blot
analysis. Neutrophils (N) and lymphocytes (L) from the same cord sample
were analyzed. p27 immunoblot is shown; p21 levels were undetectable in
this assay. (C) p27 immunoblot of CD34+ blasts
fractionated for nuclear (N) and cytoplasmic expression of p27.
|
|
The relocalization of p27 in differentiating cells was confirmed by
using confocal microscopy as well as by using antibodies recognizing
distinct epitopes and by staining different fixation conditions (Figs 2
and 4). Confocal microscopy and Western
blotting (Fig 3C) was used to verify that cytospin staining showed p27 in the nuclei of CD34+ blasts rather than in overlying
cytoplasm. Figure 2G shows a representative confocal image of a
CD34+ and CD34 cell present in a population
of cells enriched for CD34+ by negative selection. Nuclear
p27 (green) is present in the CD34+ cell; nuclear and
cytoplasmic p27 is evident in the larger CD34 cell. p27
persistence and localization was also examined in cells stimulated to
proliferate by 4 days of culture in G-CSF, SCF, and IL-3 (Fig 2H-2L).
In Fig 2H, cells stained both for p27 and for the CD71 (transferrin
receptor), a marker of proliferation, are photographed. A smaller blast
and a larger cell positive both for CD71 (green) and p27 (red) are
shown. Figure 2I shows confocal microscopy of cells doubly stained for
the proliferation marker Ki-67 (red) and for p27 (green).
Isotype-control-stained cells are shown in Fig 2J. The presence of p27
in Ki-67+ cells is evident. Moreover, whereas Ki-67 only
stains the cell nucleus, p27 is also present in the cytoplasm;
cytoplasmic p27 appears to be associated with cellular proliferation.
Figures 2K and 2L are representative confocal micrographs of
differentiating cells after 4 days of culture stained with C-terminal
or N-terminal p27 antibodies, respectively. Both nuclear and
cytoplasmic staining is evident when using antibodies specific for
either epitope. The relocalization of p27 in differentiating cells was
confirmed by using peroxidase as an alternative staining and fixation
procedure. A third pattern of immunoreactivity was seen as cells moved
into the postmitotic compartment, with nuclear p27 staining and
perinuclear accentuation in metamyelocytes and bands (Fig 4).
View larger version (206K):
[in this window]
[in a new window]
| Fig 4.
Altered localization of p27 in differentiating myeloid
cells. CD34+ cells were differentiated for 0 (A, E), 6 (B), 9 (C, F), or 15 (D, G) days and aliquots taken at various
intervals for p27 immunostaining by using peroxidase detection.
Cytospins shown have been indirectly stained either with isotype
control (E-F) or anti-p27 antibody (Neomarkers Ab-1; A-D). Altered
patterns of p27 immunoreactivity are evident.
Fig. 5.
p21
expression in differentiating myeloid cells. Immunostained cytospins of
CD34+ cells cultured for 2 days (top), 6 days (middle),
or 11 days (bottom). Cells are stained for p21 expression (red, left)
by using indirect Cy-3 conjugated antibody staining. Cell nuclei are
counterstained with Hoechst (blue) shown as the same field on the
left.
|
|
In contrast to p27, p21 staining remained predominantly nuclear
throughout differentiation. Whereas almost all cells were p27+, varying only in staining pattern, the proportion of
cells exhibiting p21 positivity varied markedly during differentiation.
p21-expressing cells begin to arise 3 days after cytokine stimulation
of resting CD34+ cells; the p21 signal peaks at 6 days,
just before the emergence of terminally differentiated cells, and then
the proportion of cells expressing p21 begins to fall off (Fig
5). Image
analysis of the stained samples using quantitative laser confocal
microscopy gated to Hoechst-positive fields was used to quantitate
nuclear p21 (see Materials and Methods). The percentage of nuclei
positive for p21 was determined at 0, 3, 6, and 11 days after cytokine stimulation indicated a 31% decline in the percentage of
p21-expressing cells between culture days 6 and 11 (Fig
6A). The signal intensity of positive cells
was also quantitated by averaging the signal within the nucleus and
multiplying by the surface area encompassed by the signal (see
Materials and Methods). There is a slight decline also in the intensity
of the p21 signal in the positive cells (Fig 6B).
View larger version (12K):
[in this window]
[in a new window]
| Fig 6.
Analysis of p21 expression during differentiation. Image
analysis of cytospins shown in Fig 6 as described in the text. (A)
Proportion of cells positive for p21. Cy3 staining over Hoechst nuclei
corrected for background signal are quantified as a percentage of
nuclei present. (B) Intensity of p21 staining. p21 fluorescence
intensity at different days. Cy3 signal extracted and intensity
collected for the mask area for each cell as described in the text.
Intensities shown in the figure represent the mean sum of all cell
profiles generated.
|
|
An independent quantitation of changes in the expression of p21 and p27
was undertaken with flow cytometry. Cells were permeabilized and cdki
expression determined by indirect immunofluorescence as detailed in
Materials and Methods. The cell line K-562, which does not express p21,
was used as a negative control. Representative staining of p21 and p27
in CD34+ cells cultured for 5 days in the presence of SCF,
IL-3, and G-CSF is shown in Fig 7. Similar
results were seen for two different p21 moAbs (Neomarker p21 moAb-2 and
moAb-3) and two p27 antibodies (Neomarker moAb-1 and Transduction
Laboratories Ab-1 [Transduction Laboratories, Lexington, KY]; not
shown). Figure 8 shows the
kinetics of p21 and p27 in CD34+ cells over 19 days in
culture. The data indicate high levels in the percentage of cells
positive for p21 and p27 from days 5 to 11, during which proliferation
is occurring. Consistent with image analysis, p21 levels decline
sharply starting at day 9, concurrent with increasing number of cells
in the postmitotic compartment. p27 levels, in contrast, remain fairly
constant.
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Detection of p21 and p27 by flow cytometry. Cord
blood-derived CD34 cells differentiated for 5 days were permeabilized
for staining of nuclear cdki's p21 and p27. Cells were stained with
(Neomarkers) monoclonal p21 Ab-2 (left) or Ab-3 (center), or with p27
Ab-1 (Neomarkers) as indicated by blackened histogram; isotype control
is shown as open histogram. Similar staining of p21 K562
cells with anti-p21 antibodies showed no shift from isotype control
(not shown).
|
|
View larger version (13K):
[in this window]
[in a new window]
| Fig 8.
Kinetics of cdk-inhibitor expression during myelopoiesis.
Flow cytometric quantitation of expression of the p21 and p27 cdki's
in differentiating CD34+ cells analyzed overtime.
Representative results of two experiments. Samples shown are derived
from the same cord.
|
|
The fall off in p21 immunoreactive protein contrasts with Northern blot
results which indicate a significant upregulation of p21 message as
cells proceed through terminal differentiation.14 Conceivably, the native protein could be sequestered into
immunologically inaccessible complexes at late stages of
differentiation; alternatively, posttranscriptional regulation of p21
could alter its production or stability,19 as has been seen
in other systems. We have not been able to quantify p21 levels in these
cells with Western blotting because of limited sensitivity to the
levels in these cells.
The above analyses all indicated that the cdk's p21 and p27 are not
limited in their expression to growth-arrested myeloid precursor cells,
because p21 and p27 are expressed at high levels in populations of
cells that are growing rapidly. To determine whether p21 and p27
expression was primarily limited to cells in G0/G1 or in cells
synthesizing DNA, we doubly stained cells differentiated for various
lengths of times for p21 or p27 and for DNA content and subjected the
stained cells to FACS analysis. This enabled us to determine cell cycle
partitioning of cells that expressed one of these cdki with cells that
did not by gating separately on cdki expressing and nonexpressing cells
and helped determine DNA profiles of both subpopulations. Dual staining
procedures were first optimized by simultaneously staining log-phase
K562 cells for the expression of cyclin A and propidium iodide. The vast majority (94%) of cells were positive for cyclin A and were markedly enriched for S-phase cells (33% in G0/G1, 61% in S-phase) compared with cyclin A nonexpressors (70% in G0/G1, 17% in S-phase) (Fig 9).
View larger version (31K):
[in this window]
[in a new window]
| Fig 9.
Partitioning of cyclin A-expressing cells within the
cell cycle. K562 cells were double stained for cyclin A expression
(indirect FITC-staining) and for DNA content (propidium iodide [PI]).
This was a test of our ability to simultaneously determine the
expression of an intranuclear antigen by using indirect staining and
DNA content. (Top) Flow histogram, PI staining on y-axis;
FITC-staining on x-axis. Vertical line denotes 98%-negative isotype
control gate. Note most negative cells at left edge of histogram.
(Bottom) Cell-cycle distribution of cyclin A-negative cells (as
represented by arrow connecting to histogram gate). (Middle) Cell-cycle
distribution of cyclin A+ cells. G0/G1 and G2 DNA
contents are highlighted in black; S-phase is highlighted with hash
marks.
|
|
Similar analysis was done with CD34+ cells, which were
differentiated for 5 days and simultaneously stained for propidium
iodide and either p27 or p21. In agreement with cytospin staining
results, 94% of cells were positive for p27 (Fig
10). Interestingly, the cell-cycle
profile of the p27+ cells was nearly identical to that of
the 6% of cells that did not express p27 (Table
1). p21 was expressed by 61% of the cells; 39% were p21 negative (Fig 10). Again, there was little difference in
the cell-cycle expression of p21-expressors and nonexpressors. The intensity of the p21 and p27 signals in positive cells
was equally bright in all cell-cycle compartments.
View larger version (53K):
[in this window]
[in a new window]
| Fig 10.
Partitioning of p27-expressing and p21-expressing cells
within the cell cycle. Cord blood CD34+ cells
differentiated for 5 days were analyzed simultaneously for p27
expression and cell-cycle distribution (left) or for p21 expression and
cell-cycle distribution (right). Ninety-three percent of cells were
positive and 7% were negative for p27. Sixty-one percent of cells were
positive and 39% were negative for p21. Isotype gates and
representation of cellular populations are as in Fig 10. p27 (left) and
p21 (right)-negative cells at bottom; p27 (left) and p21
(right)-positive cells in middle. As is evident, the cell-cycle profile
of cdki-positive and -negative cells is similar.
|
|
| |
DISCUSSION |
This study was designed to investigate the expression patterns of the
cdki's p21 and p27 in normal differentiating myeloid cells by using an
in vitro culture system employing SCF, G-CSF, and IL-3. We have found
disparate patterns of expression of p21 and p27 both in terms of the
prevalence of expression in cells at different stages of
differentiation and in the localization of the cdki within the cell.
An advantage of the culture system used was that it recapitulated the
progressive appearance of differentiated progeny seen in normal
myelopoiesis. Because the cellular population was always somewhat
heterogeneous as it moved through stages of maturation, this enabled
the comparison at a single time point of cells expressing and those not
expressing a given cdki.
Freshly harvested CD34+ cells were in G0/G1 phase and
expressed p27 in their nucleus but not p21. This is similar to the cdki expression pattern of lymphocytes and corresponds to in vitro models in
which quiescent MO7e cells express little p21 in the absence of
cytokine activation.12 In these and other
systems,20 cell-cycle entry is associated with
downmodulation of p27 protein; it is interesting that this does not
appear to occur as the CD34+ cells give rise to precursor
cells in our study. A prominent p27 signal is present upon
immunoblotting of cellular extracts at different stages of
differentiation and is roughly equal to levels seen in neutrophils and
in lymphocytes. This suggests that the total level of p27 in cells
remains relatively constant during myelopoiesis.
Although p27 levels may not change much, a distinct role for p27 in
inhibiting the proliferation of myeloid precursors is suggested by the
leukocytosis manifested by p27 knockout mice.21 Whereas
these mice exhibit increased numbers of mature hematopoietic cells, the
proportions between lineage compartments is normal, suggesting that p27
action is focused on progenitors and early precursors. Because p27
appears not to be eliminated in proliferating myeloid cells, the major
determinant of p27 action may relate more to the binding complexes it
forms than to its precise level of expression. It has been recognized
that in replicating cells, p27 is sequestered into inactive complexes
with cyclin D-cdk422; overexpression of p15 results in
redistribution of p27 into inhibitory complexes with cdk2.
The finding that p27 changes its subcellular distribution pattern at
different stages of myelopoiesis is fascinating, as it suggests a clear
mechanism for p27 to switch binding partners and alter its function.
Sequestration of p27 out of the nucleus, for example, would be an
efficacious way of promoting cell-cycle entry. Conceivably,
redistribution of a significant proportion of p27 to the cytoplasm
could facilitate formation of cytoplasmic complexes with cdk4 and
enable myeloid proliferation mediated by active nuclear cyclinD-cdk2
complexes. In one study, forced expression of a p27 construct lacking a
nuclear localization signal resulted in cytoplasmic expression of p27
and led to cytoplasmic p27-cdk4 complex formation.17 Our
results raise the possibility that a shift from cdk2 to cdk4-p27
complexes could occur in a stage-specific manner through p27
redistribution. It has recently been reported that p27 is redistributed
into the cytoplasm in Barret's associated
adenocarcinoma,23 suggesting that aberrant appropriation of
such subcellular control mechanisms could occur in cancer.
Alternatively, novel binding partners might be accessible to
cytoplasmic p27, which would engage it in pathways distinct from cell-cycle control. This would also be consistent with the lack of
difference in cycling behavior of p27-expressing cells and p27
nonexpressing cells after 5 days in culture (Fig 10). It will be
interesting to examine co-localization of p27 binding partners concurrent with these shifts in p27 compartmentalization to test whether p27 acts to shuttle cell cycle or differentiation-regulatory proteins between compartments.
The results of our analysis of p21 expression are interesting in two
respects. We show in normal myeloid cells that p21 upregulation is
associated with proliferation during differentiation. This extends
findings reported in the MO7e in vitro model of myeloid differentiation.12 The expression of p21 is low in
growth-arrested CD34+ blast cells and begins to increase
around day 2, which is concurrent with cellular expression of CD71
(transferrin receptor), a marker of proliferation. Thereafter, the
pattern of p21 expression is inversely proportional to the percentage
of cells in G0/G1 (Fig 8), indicating a coupling of p21 to
proliferation of myeloid precursor cells. Several laboratories have
showed a stoichiometric activity of p21, in which it acts as a
cyclin-cdk-assembly factor and promotes proliferation at low
concentrations but is inhibitory at high concentrations.9,10 We note that in our system the
intensity of p21 signal within positive cells also peaked at day 6, suggesting that the proliferating precursor cells are not simply
expressing p21 at a subthreshold concentration for growth arrest (Fig
8). Moreover, fractionation of p21-expressing cells by DNA content did
not indicate any difference in the intensity of p21 expression between
S-phase and G0/G1-phase cells. It is conceivable that p21 function in
these cells is largely related to the differentiation process. That
would be consistent with the lack of difference in the cell-cycle
profiles of p21-expressing cells and nonexpressing cells. Conceivably,
a portion of the p21 expressed in these cells could be sequestered in
complexes with other binding partners such as casein kinase
II,24 SAPK,25 E2F,25 gadd45, or Myd 118,26 which may not as directly relate to cell-cycle
effects as to differentiation. Additional studies will be needed to
address this concept.
We also find discrepant expression of p21 message, which increases
throughout terminal differentiation14 and p21 protein, and
appears to decrease in the last stages of differentiation. Because we
are studying p21 in its native state, it is conceivable that
alterations in protein interactions render p21 inaccessible to our
antibodies at this late stage. Alternatively, ubiquitination or
translational inhibitory pathways could be activated at this stage. The
primary role for p21 could be in choreographing the transition into the
differentiated state rather than in maintaining it. Such findings have
been reported in muscle differentiation, in which p18 succeeds p21 in
playing a dominant role in sustaining growth arrest of terminally
differentiated cells.27 Intriguingly, Di Cunto et
al28 have recently reported a similar fall off in p21-protein expression during terminal differentiation of keratinocytes and have shown, moreover, that forced p21-protein expression at this
stage inhibits terminal differentiation markers. Whether similar
stage-specific restrictions on p21 expression occur in terminal
myelopoiesis will require further investigation.
| |
ACKNOWLEDGMENT |
We thank Brian Dynlacht for the gift of CP36 monoclonal anti-p21
antibody, Julie Goff for CD34+ cells, Eric Loeffert and
Donna Shields and for their technical assistance, and Vera Donnenberg
for helpful discussions.
| |
FOOTNOTES |
Submitted April 28, 1998; accepted December 28, 1998.
Supported in part by grants from Highmark Blue Cross/Blue Shield, the
American Cancer Society (JFRA-594), and the National Institute of
Health (HL54172-01) to R.A.S.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to R.A. Steinman, MD, PhD, E1052 BST,
University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213;
e-mail: Steinman+{at}pitt.edu.
| |
REFERENCES |
1.
Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, El-Houseini ME:
Induction of p21 (WAF1/CIP1) during differentiation.
Oncogene
9:3389, 1994[Medline]
[Order article via Infotrieve]
2.
El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B:
WAF1, a potential mediator of p53 tumor suppression.
Cell
75:817, 1993[Medline]
[Order article via Infotrieve]
3.
Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massague J:
Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.
Cell
78:59, 1994[Medline]
[Order article via Infotrieve]
4.
Coats S, Flanagan WM, Nourse J, Roberts JM:
Requirement of p27(kip1) for restriction point control of the fibroblast cell cycle.
Science
272:877, 1996[Abstract]
5.
Rivard N, Lallemain G, Bartek J, Pouyssegur J:
Abrogation of p27(kip1) by cdna antisense suppresses quiescence (g(0) state) in fibroblasts.
J Biol Chem
271:18337, 1996[Abstract/Free Full Text]
6.
Nakanishi M, Adami GR, Robetorye RS, Noda A, Venable SF, Dimitrov D, Pereira-Smith OM, Smith JR:
Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.
Proc Nat Acad Sci USA
92:4352, 1995[Abstract/Free Full Text]
7.
Wang QM, Jones JB, Studzinski GP:
Cyclin-dependent kinase inhibitor p27 as a mediator of the g(1)-s phase block induced by 1,25-dihydroxyvitamin d-3 in hl60 cells.
Cancer Res
56:264, 1996[Abstract/Free Full Text]
8.
Liu M, Lee MH, Cohen M, Bommakanti M, Freedman LP:
Transcriptional activation of the cdk inhibitor p21 by vitamin d-3 leads to the induced differentiation of the myelomonocytic cell line u937.
Genes Dev
10:142, 1996[Abstract/Free Full Text]
9.
Labaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E:
New functional activities for the p21 family of CDK inhibitors.
Genes Dev
11:847, 1997[Abstract/Free Full Text]
10.
Zhang H, Hannon GJ, Beach D:
p21-containing cyclin kinases exist in both active and inactive states.
Genes Dev
8:1750, 1994[Abstract/Free Full Text]
11.
Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee M-H, Massague J, Crabtree GR, Roberts JM:
Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin.
Nature
372:570, 1994[Medline]
[Order article via Infotrieve]
12.
Mantel C, Luo Z, Canfield J, Braun S, Deng C, Broxmeyer HE:
Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo.
Blood
88:3710, 1996[Abstract/Free Full Text]
13.
Missero C, Cunto FD, Kiyokawa H, Koff A, Dotto GP:
The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.
Genes Dev
10:3065, 1996[Abstract/Free Full Text]
14.
Steinman RA, Huang J-P, Yaroslavskiy B, Goff J, Ball ED, Nguyen A:
Regulation of p21(Waf1) during myelopoiesis.
Blood
91:4531, 1998[Abstract/Free Full Text]
15.
Terstappen LW, Safford M, Konemann S, Loken MR, Zurlutter K, Buchner T, Hiddemann W, Wormann B:
Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis.
Leukemia
5:757, 1991
16.
Solvason N, Wu WW, Kabra N, Wu XY, Lees E, Howard MC:
Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature b lymphocytes.
J Exp Med
184:407, 1996[Abstract/Free Full Text]
17.
Reynisdottir I, Massague J:
The subcellular locations of p15(Ink4b) and p27(Kip1) coordinate their inhibitory interactions with cdk4 and cdk2.
Genes Dev.
11:492, 1997[Abstract/Free Full Text]
18.
Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M:
Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27 [see comments].
Science
269:682, 1995[Abstract/Free Full Text]
19.
Maki CG, Howley PM:
Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation.
Mol Cell Biol
17:355, 1997[Abstract]
20.
Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE:
Cyclin E-CDK2 is a regulator of p27kip1.
Genes Dev
11:1464, 1997[Abstract/Free Full Text]
21.
Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM:
A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(kip1)-deficient mice.
Cell
85:733, 1996[Medline]
[Order article via Infotrieve]
22.
Reynisdottir I, Polyak K, Iavarone A, Massague J:
Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta.
Genes Dev
9:1831, 1995[Abstract/Free Full Text]
23.
Singh SP, Lipman J, Goldman H, Ellis FH, Aizenman L, Cangi MG, Signoretti S, Chiaur DS, Pagano M, Loda M:
Loss or altered subcellular localization of p27 in Barrett's associated adenocarcinoma.
Cancer Res
58:1730, 1998[Abstract/Free Full Text]
24.
Gotz C, Wagner P, Issinger OG, Montenarh M:
P21(waf1/cip1) interacts with protein kinase ck2.
Oncogene
13:391, 1996[Medline]
[Order article via Infotrieve]
25.
Shim J, Lee H, Park J, Kim H, Choi EJ:
A non-enzymatic p21 protein inhibitor of stress-activated protein kinases.
Nature
381:804, 1996[Medline]
[Order article via Infotrieve]
26.
Vairapandi M, Balliet AG, Fornace AJ, Hoffman B, Liebermann DA:
The differentiation primary response gene myd118, related to gadd45, encodes for a nuclear protein which interacts with pcna and p21(waf1/cip1).
Oncogene
12:2579, 1996[Medline]
[Order article via Infotrieve]
27.
Franklin DS, Xiong Y:
Induction of p18(INK4C) and its predominant association with CDK4 and CDK6 during myogenic differentiation.
Mol Biol Cell
7:1587, 1996[Abstract]
28.
Di Cunto F, Topley G, Calautti E, Hsiao J, Ong L, Seth PK, Dotto GP:
Inhibitory function of p21 (CIP1/WAF1) in differentiation of primary mouse keratinocytes independent of cell cycle control.
Science
280:1069, 1998[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
R. Bedi, J. Du, A. K. Sharma, I. Gomes, and S. J. Ackerman
Human C/EBP-{epsilon} activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation
Blood,
January 8, 2009;
113(2):
317 - 327.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Acosta, N. Ferrandiz, G. Bretones, V. Torrano, R. Blanco, C. Richard, B. O'Connell, J. Sedivy, M. D. Delgado, and J. Leon
Myc Inhibits p27-Induced Erythroid Differentiation of Leukemia Cells by Repressing Erythroid Master Genes without Reversing p27-Mediated Cell Cycle Arrest
Mol. Cell. Biol.,
December 15, 2008;
28(24):
7286 - 7295.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-A. Cao, A. J. Wagers, H. Karsunky, H. Zhao, R. Reeves, R. J. Wong, D. K. Stevenson, I. L. Weissman, and C. H. Contag
Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors
Blood,
December 1, 2008;
112(12):
4494 - 4502.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-H. Ling, T. Li, Z. Yuan, M. Haigentz Jr., T. K. Weber, and R. Perez-Soler
Erlotinib, an Effective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Induces p27KIP1 Up-Regulation and Nuclear Translocation in Association with Cell Growth Inhibition and G1/S Phase Arrest in Human Non-Small-Cell Lung Cancer Cell Lines
Mol. Pharmacol.,
August 1, 2007;
72(2):
248 - 258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Verhelle, L. G. Corral, K. Wong, J. H. Mueller, L. Moutouh-de Parseval, K. Jensen-Pergakes, P. H. Schafer, R. Chen, E. Glezer, G. D. Ferguson, et al.
Lenalidomide and CC-4047 Inhibit the Proliferation of Malignant B Cells while Expanding Normal CD34+ Progenitor Cells
Cancer Res.,
January 15, 2007;
67(2):
746 - 755.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Munoz-Alonso, J. C. Acosta, C. Richard, M. D. Delgado, J. Sedivy, and J. Leon
p21Cip1 and p27Kip1 Induce Distinct Cell Cycle Effects and Differentiation Programs in Myeloid Leukemia Cells
J. Biol. Chem.,
May 6, 2005;
280(18):
18120 - 18129.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. H. Min, J.-W. Cheong, J. Y. Kim, J. I. Eom, S. T. Lee, J. S. Hahn, Y. W. Ko, and M. H. Lee
Cytoplasmic Mislocalization of p27Kip1 Protein Is Associated with Constitutive Phosphorylation of Akt or Protein Kinase B and Poor Prognosis in Acute Myelogenous Leukemia
Cancer Res.,
August 1, 2004;
64(15):
5225 - 5231.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. H. Min, J.-W. Cheong, M. H. Lee, J. Y. Kim, S. T. Lee, J. S. Hahn, and Y. W. Ko
Elevated S-Phase Kinase-Associated Protein 2 Protein Expression in Acute Myelogenous Leukemia: Its Association with Constitutive Phosphorylation of Phosphatase and Tensin Homologue Protein and Poor Prognosis
Clin. Cancer Res.,
August 1, 2004;
10(15):
5123 - 5130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Denicourt and S. F. Dowdy
Cip/Kip proteins: more than just CDKs inhibitors
Genes & Dev.,
April 15, 2004;
18(8):
851 - 855.
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Gadhoum, M.-P. Leibovitch, J. Qi, D. Dumenil, L. Durand, S. Leibovitch, and F. Smadja-Joffe
CD44: a new means to inhibit acute myeloid leukemia cell proliferation via p27Kip1
Blood,
February 1, 2004;
103(3):
1059 - 1068.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Witko-Sarsat, S. Canteloup, S. Durant, C. Desdouets, R. Chabernaud, P. Lemarchand, and B. Descamps-Latscha
Cleavage of p21waf1 by Proteinase-3, a Myeloid-specific Serine Protease, Potentiates Cell Proliferation
J. Biol. Chem.,
November 27, 2002;
277(49):
47338 - 47347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Dimberg, F. Bahram, I. Karlberg, L.-G. Larsson, K. Nilsson, and F. Oberg
Retinoic acid-induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27Kip1
Blood,
March 15, 2002;
99(6):
2199 - 2206.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Cheng, H. Shen, N. Rodrigues, S. Stier, and D. T. Scadden
Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21Cip1/Waf1 or p27Kip1
Blood,
December 15, 2001;
98(13):
3643 - 3649.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Lin, S. Lim, M. A. Viani, M. Sapp, and M. S. Lim
Down-Regulation of Telomerase Activity in Malignant Lymphomas by Radiation and Chemotherapeutic Agents
Am. J. Pathol.,
August 1, 2001;
159(2):
711 - 719.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. Kastner, H. J. Lawrence, C. Waltzinger, N. B. Ghyselinck, P. Chambon, and S. Chan
Positive and negative regulation of granulopoiesis by endogenous RAR{alpha}
Blood,
March 1, 2001;
97(5):
1314 - 1320.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Iida, K. Matsumoto, H. Tomita, T. Nakajima, A. Akasawa, N. Y. Ohtani, N. L. Yoshida, K. Matsui, A. Nakada, Y. Sugita, et al.
Selective down-regulation of high-affinity IgE receptor (Fc{epsilon}RI) {alpha}-chain messenger RNA among transcriptome in cord blood-derived versus adult peripheral blood-derived cultured human mast cells
Blood,
February 15, 2001;
97(4):
1016 - 1022.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Pierelli, M. Marone, G. Bonanno, S. Mozzetti, S. Rutella, R. Morosetti, C. Rumi, S. Mancuso, G. Leone, and G. Scambia
Modulation of bcl-2 and p27 in human primitive proliferating hematopoietic progenitors by autocrine TGF-beta 1 is a cell cycle-independent effect and influences their hematopoietic potential
Blood,
May 15, 2000;
95(10):
3001 - 3009.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Cheng, N. Rodrigues, H. Shen, Y. Yang, D. Dombkowski, M. Sykes, and D. T. Scadden
Hematopoietic Stem Cell Quiescence Maintained by p21cip1/waf1
Science,
March 10, 2000;
287(5459):
1804 - 1808.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Walsh, M. Murphy, M. Silverman, R. Odze, D. Antonioli, H. Goldman, and M. Loda
p27 Expression in Inflammatory Bowel Disease-Associated Neoplasia : Further Evidence of a Unique Molecular Pathogenesis
Am. J. Pathol.,
November 1, 1999;
155(5):
1511 - 1518.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION