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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1971-1978
Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic
Lineage Commitment of Human Bone Marrow Progenitor Cells
By
Gösta Bergh,
Mats Ehinger,
Inge Olsson,
Sten Eirik W. Jacobsen, and
Urban Gullberg
From the Department of Hematology, and the Stem Cell Laboratory,
Department of Internal Medicine, University of Lund, Lund, Sweden.
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ABSTRACT |
The retinoblastoma gene product (pRb) is involved in both cell cycle
regulation and cell differentiation. pRb may have dual functions during
cell differentiation: partly by promoting a cell cycle brake at
G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates
differentiation of leukemic cell lines involving mechanisms other than
the induction of G1 arrest. In the present study, we
investigated the role of pRb in differentiation of human bone marrow
progenitor cells. Human bone marrow cells were cultured in a
colony-forming unit-granulocyte-macrophage (CFU-GM)
assay. The addition of antisense RB oligonucleotides ( -RB), but not
the addition of sense orientated oligonucleotides (SO) or scrambled
oligonucleotides (SCR), reduced the number of colonies staining for
nonspecific esterase without affecting the clonogenic growth. Monocytic
differentiation of CD34+ cells supported by FLT3-ligand
and interleukin-3 (IL-3) was correlated to high levels of
hypophosphorylated pRb, whereas neutrophilic differentiation, supported
by granulocyte colony-stimulating factor (G-CSF) and stem cell factor
(SCF), was correlated to low levels. The addition of -RB to liquid
cultures of CD34+ cells, supported with FLT3-ligand and
IL-3, inhibited monocytic differentiation. This was judged by
morphology, the expression of CD14, and staining for esterase.
Moreover, the inhibition of monocytic differentiation of
CD34+ cells mediated by -RB, which is capable of
reducing pRb expression, was counterbalanced by an enhanced
neutrophilic differentiation response, as judged by morphology and the
expression of lactoferrin. CD34+ cells incubated with
oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken
together, these data suggest that pRb plays a critical role in the
monocytic and neutrophilic lineage commitment of human bone marrow
progenitors, probably by mechanisms that are not strictly related to
control of cell cycle progression.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE RETINOBLASTOMA protein (pRb) is
believed to play a key role in the regulation of the cell cycle. The
activity of pRb depends on the degree of phosphorylation, which is cell
cycle phase dependent. When hypophosphorylated, pRb mediates a brake at
the G1-phase of the cell cycle by inhibiting the E2F family of transcription factors, which activate genes for progression from
G1- to S-phase (reviewed in Weinberg1).
Recently, mechanisms by which hypophosphorylated pRb silences the
transcription of mitotic genes were described. pRb recruits a histone
deacetylase to E2F, thus creating a complex that probably by modulating
local chromatin structure prevents expression from E2F-regulated
genes.2,3 In addition to inhibiting cell proliferation, pRb
also promotes specific differentiation programs.4-6 Mice
with targeted retinoblastoma gene (RB) function show defective
differentiation in tissues such as blood and brain. The hematopoietic
abnormalities described in the Rb  / mice include reduced
formation of hepatic blood islands, coupled with an increased
proportion of immature nucleated erythroid cells.7-9 A role
for pRb in human hematopoiesis has been implicated; high levels of pRb
are induced and sustained during erythroid differentiation, whereas pRb
is downregulated during granulocytic maturation.10 The
mechanisms governing hematopoietic lineage commitment and
differentiation are not fully understood. However, increasing evidence
indicates a role for transcription factors in orchestrating these
processes. The functions of hematopoietic transcription factors seem to
be modulated in a complex manner by a variety of signals and
interactions (reviewed in Shivdasani and Orkin11 and Tenen
et al12). In particular, pRb has been shown to interact
with hematopoietic transcription factors, including NF-IL-6, PU1, and
Elf1.13-15 Recently, we demonstrated that pRb is involved
in differentiation responses in leukemic cell lines involving
mechanisms distinct from those specifically linked to the control of
cell cycle progression.16 Data indicating that pRb plays a
critical role in the monocytic and neutrophilic lineage commitment of
normal human bone marrow progenitors are presented.
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MATERIALS AND METHODS |
Oligonucleotides.
Two phosphorothioate-modified oligodeoxynucleotides were designed to
target RB mRNA and designated antisense RB oligonucleotide 1 (AS1) and
antisense RB oligonucleotide 2 (AS2), respectively. Three
phosphorothioate-modified oligodeoxynucleotides were used as controls
for unspecific or toxic effects of the antisense oligos: 2 sense-orientated oligonucleotides (SO1 and SO2) and a scrambled oligonucleotide (SCR). The sequence of AS1 read 5' GGG GGT TTT GGG CGG CAT GAC 3', complementary to 21 nucleotides starting from position 3 to +18 bp in the RB mRNA coding
sequence.16 AS2 read 5' GTG AAC GAC ATC TCA TCT AGG
3', complementary to 21 nucleotides starting from position 465 to
485 bp in the RB mRNA coding sequence.10,17 The sequence of SO1 read 5' GTC ATG CCG CCC AAA ACC
CCC 3', corresponding to position 3 to +18 bp in
the RB cDNA coding sequence. The sequence of SO2 read 5' GGA TCT
ACT CTA CAG CAA GTG 3', corresponding to the reversed orientation
of AS2. The SCR sequence read 5' TAC TGG CTA AGC CTA GCA TGA
3' and corresponded to a randomly scrambled AS2.10
AS1, AS2, and SO1 were initially synthesized by Bio Molecular Resource
Facility, Lund University (Lund, Sweden). AS2, SO2, and SCR were later
synthesized by Cyber Gene AB (NOVUM, Huddinge, Sweden).
Hematopoietic growth factors.
The growth of unfractionated mononuclear bone marrow cells in
clonogenic assay (colony-forming unit-granulocyte-macrophage [CFU-GM]) was supported by 10% conditioned medium from
the bladder carcinoma cell line 5637. The liquid culture of
CD34+ bone marrow cells was supported by purified
recombinant human growth factors at predetermined optimal
concentrations. Interleukin-3 (IL-3) and FLT3-ligand (FL; both
available due to the generosity of Immunex, Seattle, WA) were used at
25 ng/mL and 50 ng/mL, respectively. Stem cell factor (SCF) and
granuloctye colony-stimulating factor (G-CSF; Amgen Inc, Thousand Oaks,
CA) were both used at 50 ng/mL.
Assay for granulocyte-macrophage progenitors.
Human bone marrow cells were obtained by gradient centrifugation and
cultured in agar as described.18 Colonies (>40 cells) were scored after 14 days of incubation and subsequently stained for
unspecific esterase, a marker of monocytic differentiation, as
described elsewhere.19
Liquid culture of human CD34+ bone marrow cells.
CD34+ bone marrow cells were initially isolated as
described elsewhere.20 Later, mononuclear bone marrow cells
obtained by gradient centrifugation were positively selected for CD34
expression by MidiMACS (Miltenyi Biotech, Auburn, CA), followed by
staining with antihuman CD34 fluorescein isothiocyanate (FITC; Becton
Dickinson, San Jose, CA) and antihuman CD38 phycoerythrin (PE) or
isotype control antibodies. CD34+CD38+ cells
were then sorted on FACSVanatage (Becton Dickinson).
The purity of CD34+ cells was greater than 90%, as
determined by flow cytometry. Isolated CD34+ cells were
grown in a defined complete Iscove's modified Dulbecco's medium
(IMDM) medium as previously described.20 Cells
incubated with FL plus IL-3, promoting predominantly monocytic
differentiation,20 were seeded at 5,000 cells/mL. Cells
stimulated with G-CSF plus SCF, to promote mainly granulocytic
differentiation,21 were seeded at 2,000 cells/mL. Cells
were expanded in a volume of 1 mL for 14 days at 37°C, 5%
CO2 in fully humidified air. Cells destined for
determination of pRb levels were cultured in a volume of 20 to 100 mL.
Flow cytometric evaluation of cell surface phenotype.
Cultured cells were analyzed for cell surface antigen expression as
described.20 Expression of CD14 antigen, a monocyte-related surface antigen,22 was analyzed using a mouse antihuman
CD14 PE monoclonal antibody (MoAb; Becton Dickinson).
Morphological evaluation of hematopoietic differentiation.
Cytospin preparations of cultured cells were stained with
May-Grünwald-Giemsa and examined with light microscopy.
Immunocytochemistry.
Lactoferrin was detected as described,23 with the following
modifications. Cytospin preparations of cultured cells were fixed in
4% formaldehyde in 0.1 mol/L phosphate buffer (pH 7.0) for 20 minutes.
Cells were then permeabilized by incubation for 30 minutes in
Tris-buffered saline (TBS; 50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl)
containing 1% Triton X-100. Unspecific binding was blocked by
incubating cells with TBS containing 1% bovine serum albumin (BSA;
Sigma, St Louis, MO). Polyclonal rabbit antibodies to
human lactoferrin (A106; DAKO D 306, Glostrup, Denmark) were diluted to
0.86 µg/mL in TBS containing 0.25% BSA and allowed to bind during 1 hour of incubation. The slides were then washed 3 times in TBS,
followed by incubation for 1 hour with alkaline phosphatase-conjugated
swine antirabbit Igs (D306; DAKO D 306) diluted 60-fold in TBS
containing 0.25% BSA. After 3 washes in TBS, alkaline phosphatase
activity was detected by Fast Red/Naphtol AS-MS (Sigma Fast; Sigma) as
a substrate. After washing in running tap water, slides were
counterstained in Mayers hematoxylin. All steps were performed at room temperature.
Western blot analysis.
Cells were lysed at 4°C in a buffer consisting of 50 mmol/L Tris
HCl (pH 8.0), 0.15 mol/L NaCl, 5 mmol/L EDTA (pH 8.0), and 0.5%
Nonidet P40 (KEBO, Stockholm, Sweden), including 1 tablet of a protease
inhibitor cocktail (Complete; Boehringer Mannheim, Mannheim, Germany)
per 50 mL of lysis buffer. Incubation on ice for 1 hour followed,
before approximately 10 seconds of vigorous vortexing. After lysis, the
DNA was removed by centrifugation at 37,500g for 1 hour at
4°C. The lysates were then subjected to specific
immunoprecipitation by the addition of 1 µg of mouse sc 102 anti-Rb
MoAb (Santa Cruz Laboratories, Santa Cruz, CA). Immunocomplexes were allowed to form and adsorbed to a mixture of
protein A- (Pharmacia, Uppsala, Sweden) and protein G-sepharose (Sigma)
at 4°C overnight. After centrifugation, the precipitate was washed
3 times with lysis buffer. The immunoprecipitated proteins were
separated on a 6% precast Tris-Glycine gel electrophoresis (Novex, San
Diego, CA). Proteins were electroforetically transferred to Immobilon-P
membranes (Millipore, Bedford, MA) in blotting buffert (39 mmol/L
glycin [pH 9.2], 48 mmol/L Tris, 1.3 mmol/L sodium dodecyl sulfate
[SDS], 20% methanol) at 20 V for 1 hour. After incubation in
blocking buffert (5% dry milk powder in PBS) for 0.5 hour, the
membrane was incubated for 2 hours with mouse sc 102 anti-Rb MoAb at
0.5 µg/mL in the following PBS buffer (pH 7.3 to 7.4): 0.137 mol/L
NaCl, 8 mmol/L Na2HPO4 × 2 H2O, 2.7 mmol/L KCl, 1.5 mmol/L
KH2PO4, and 0.05% Tween 20. The membrane was
then probed with alkaline phosphatase-conjugated rabbit antimouse IgG (DAKO A/S, Copenhagen, Denmark) diluted 1:500 in PBS buffer for 1 hour.
The specific proteins were then visualized with chromogenic substrates
(5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt [ICN] at 0.05 mg/mL and nitro blue tetrazolium [Sigma] at 0.1 mg/mL) in a blocking
buffert without BSA (10.6 mmol/L Na2CO3, 39.3 mmol/L NaHCO3) that contained 4 mmol/L MgCl2.
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RESULTS |
Effects of  -RB on growth and differentiation of unfractionated
mononuclear human bone marrow cells.
The inhibitory effects of AS1 on myelomonocytic differentiation of
U-937 cells16 led us to investigate the effects of
antisense RB oligonucleotides on the growth of normal human bone marrow progenitor cells. The total number of CFU-GM colonies formed was not
affected by the addition of AS1 at 2.5 to 10.0 µmol/L, compared with
the addition of oligo buffer only, except by a slight reduction at 5.0 µmol/L, indicating that AS1 did not exert toxic effects (Fig 1). However, reduced staining for
unspecific esterase, a marker of monocytic
differentiation,19 was observed after incubation with AS1,
compared with the addition of oligo buffer. However, the addition of
SO1 did not reduce staining for unspecific esterase compared with the
addition of oligo buffer (Table 1). Similar data were obtained using another set of oligonucleotides; the addition
of AS2 reduced staining for unspecific esterase compared with the
addition of buffer, whereas, after the addition of SO2 or SCR, positive
staining for unspecific estherase was comparable to addition of buffer
alone (Table 1). These data suggested that suppression of pRb inhibits
differentiation of myeloid progenitors along the monocytic lineage,
without affecting their clonogenic growth.
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| Fig 1.
Effects of oligonucleotides on the number of CFU-GM
colonies. Bone marrow cells were obtained by gradient centrifugation
and cultured in agar. Oligo buffer only ( ), antisense RB
oligonucleotides (AS1;  ), or sense orientated oligonucleotides (SO1;
 ) were added at indicated concentrations on days 0 and 7. Colonies
(>40 cells) were scored on day 14. Values shown are the percentages
of the number of colonies formed when oligo buffer only was added.
Results shown represent the mean values from 3 separate experiments
(bars ± SEM). The total number of colonies formed per dish with oligo
buffer added was 63 ± 7 (SEM).
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pRb levels during monocytic or neutrophilic differentiation of
CD34+ progenitor cells, respectively.
To further investigate the role of pRb in monocytic and neutrophilic
differentiation, purified CD34+ bone marrow cells were
cultured in the presence of FL plus IL-3, promoting predominantly
monocytic differentiation20
(Table 2) or, in the presence of G-CSF plus
SCF, favoring mainly neutrophilic differentiation21 (Table
2). On day 0, during uninduced conditions, the purified
CD34+ bone marrow cells expressed very low levels of pRb,
which is in line with previous findings10
(Fig 2A). In cells stimulated towards
neutrophilic differentiation with G-CSF plus SCF, a slight increase in
the level of pRb was detected on days 3 and 5. In contrast, in
CD34+ cells stimulated with FL plus IL-3, promoting
monocytic differentiation, a considerable increase in pRb levels was
detected at days 3 and 5 as compared with day 0 (Fig 2A and B).
Moreover, at day 3, the ratio between hyperphosphorylated pRb and
hypophosphorylated pRb seemed higher in cells stimulated with G-CSF
plus SCF than in cells supported with FL plus IL-3 (Fig 2A and B).
These data suggest that monocytic commitment, in contrast to
neutrophilic commitment, is correlated to a considerable upregulation
of levels of hypophosphorylated pRb. A similar, but less pronounced,
difference in pRb level between cells incubated with G-CSF plus SCF and
cells stimulated with FL plus IL-3 was observed also on days 7 and 14 (data not shown).
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| Fig 2.
(A) Levels of pRb during neutrophilic and monocytic
differentiation on days 0 and 3. Cells selected for CD34 expression
were incubated on day 0 with cytokines favoring neutrophilic (G-CSF and
SCF, both at 50 ng/mL) or monocytic (FL at 50 ng/mL and IL-3 at 25 ng/mL) differentiation, respectively. On day 0, 0.55 × 106 cells not stimulated with any cytokines were subjected
to protein extraction, followed by specific immunoprecipitation with an
anti-pRb antibody (sc-102; Santa Cruz), 6% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), and Western blot analysis. On day 3, 0.55 × 106 cells from cells incubated with G-CSF/SCF or
FL/IL-3, respectively, were subjected to protein extraction, followed
by specific immunoprecipitation with an anti-pRb antibody (sc-102;
Santa Cruz), 6% SDS-PAGE, and Western blot analysis. pRb of different
molecular weights are indicated with arrows (ppRb, hyperphosphorylated;
pRb, hypophosphorylated). (B) Levels of pRb during neutrophilic and
monocytic differentiation on days 3 and 5. Cells selected for CD34
expression were incubated on day 0 with cytokines, favoring
neutrophilic (G-CSF and SCF, both at 50 ng/mL) or monocytic (FL at 50 ng/mL and IL-3 at 25 ng/mL) differentiation, respectively. After 3 and
5 days of expansion, respectively, 1.5 × 106 cells were
subjected to protein extraction, followed by specific
immunoprecipitation with an anti-pRb antibody (sc-102; Santa Cruz), 6%
SDS-PAGE, and Western blot analysis. pRb of different molecular weights
are indicated with arrows (ppRb, hyperphosphorylated; pRb,
hypophosphorylated).
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-RB inhibit monocytic differentiation and enhance neutrophilic
differentiation of human CD34+ bone marrow progenitor
cells.
To analyze the indicated differences in pRb expression during monocytic
and neutrophilic differentiation (Fig 2A and B) in a functional way,
the effects of -RB on the growth and differentiation of
CD34+ cells were investigated. Constitutive suppression of
pRb expression by transfection with an antisense-RB construct inhibits
myelomonocytic differentiation of leukemic U-937 cells,16
and AS2 has been shown to downregulate RB mRNA levels in liquid culture
of human hematopoietic progenitors.10 Purified
CD34+ bone marrow cells were cultured in the presence of FL
plus IL-3 to promote monocytic differentiation. The addition of AS2,
but not of SO1, reduced the expression of the monocyte-related surface antigen CD1422 compared with incubation with FL/IL-3 alone
(Fig 3 and Table 2). The addition
of AS2, SO2, or SCR (Fig 4) to
CD34+ progenitors stimulated with FL plus IL-3 allowed a
similar growth rate, as did the addition of oligo buffer (Table 2).
Morphologically, FL/IL-3 stimulated predominantly the formation of
large cells with vacuolated cytoplasm and with nonlobulized nuclei
(hereafter referred to as monocytes; Fig 5A
and Table 2), confirming previous findings.20 The addition
of AS2, but not of SO2, SCR, or oligo buffer, to cells incubated with
FL/IL-3 resulted not only in reduced formation of monocytes, but also
in an increased fraction of cells with granulocytic morphology (Fig 5
and Table 2). The addition of AS2 or SO1 to CD34+ cells
supported by G-CSF/SCF resulted in cells with granulocytic morphology,
which is not different from cells incubated with G-CSF/SCF alone (Table
2). To further characterize the differentiation response mediated by
AS2, but not by SO2 or SCR, as judged by morphology (Fig 5), cells were
subjected to immunocytochemistry. The addition of AS2, but not of SO2
or SCR, to cells incubated with FL/IL-3 resulted in cells with positive
staining for lactoferrin, which is a granulocyte-specific protein with
iron-binding capacity stored in secondary granulae (reviewed in Levay
and Viljoen24; Fig 6). The
staining of normal neutrophils, used as positive control, was of
comparable intensity (Fig 6). The addition of AS2, but not of SO2 or
SCR, to CD34+ cells incubated with FL/IL-3 reduced positive
staining for unspecific esterase of 200 counted cells from 64% of
cells incubated with SO2 and 62% of cells incubated with SCR to 20%
of cells incubated with AS2.
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| Fig 3.
Effects of oligonucleotides on differentiation of
CD34+ cells induced with FL plus IL-3 assayed by
expression of CD14. CD34+ cells were seeded at a
concentration of 5,000 cells/mL and incubated in medium supplemented
with FL at 50 ng/mL plus IL-3 at 25 ng/mL to promote monocytic
differentiation. On days 0, 5, and 10, antisense RB oligonucleotides
(AS2) or sense-orientated oligonucleotides (SO1) were added, each time
at a concentration of 5.0 µmol/L. After 14 days, cells were assayed
for expression of CD14, a marker of monocytic differentiation. Values
shown are the percentages of cells expressing CD14. Results shown
represent the mean values from 3 separate experiments (bars ± SEM).
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| Fig 4.
Effects of oligonucleotides on the proliferation of bone
marrow cells selected for CD34 expression and incubated with FL plus
IL-3. CD34+ cells were seeded at a concentration of 5,000 cells/mL and incubated in medium supplemented with FL at 50 ng/mL and
IL-3 at 25 ng/mL to promote monocytic growth. On days 0, 5, and 10, antisense RB oligonucleotides (AS2), sense-orientated oligonucleotides
(SO2), scrambled oligonucleotides (SCR), or oligo buffer, respectively,
were added at a concentration of 5 µmol/L. On days 7 and 14, cells
were counted. Results shown represent the mean values from 3 separate
experiments (bars ± SEM). Results from SCR are from 2 independent
experiments. Viability was always greater than 90% (data not shown).
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| Fig 5.
Effects of oligonucleotides on differentiation
of CD34+ cells induced with FL plus IL-3 assayed by
morphology. Cells at an initial concentration of 5,000/mL were
incubated in medium supplemented with FL at 50 ng/mL and IL-3 at 25 ng/mL to promote monocytic differentiation. On days 0, 5, and 10, antisense RB oligonucleotides (AS2), sense-orientated oligonucleotides
(SO2), or scrambled oligonucleotides (SCR) were added, each time at a
concentration of 5.0 µmol/L. After 14 days, cytospin slides were
prepared and stained with May-Grünwald-Giemsa. Cells incubated
with FL, IL-3, and oligo buffer (A); with FL, IL-3, and SO2 (B); with
FL, IL-3, and SCR (C); or with FL, IL-3, and AS2 (D).
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| Fig 6.
Effects of oligonucleotides on differentiation of
CD34+ cells induced with FL plus IL-3 assayed by
immunocytochemistry. Cells at an initial concentration of 5,000/mL were
incubated in medium supplemented with FL at 50 ng/mL and IL-3 at 25 ng/mL to promote monocytic differentiation. On days 0, 5, and 10, antisense RB oligonucleotides (AS2), sense-orientated oligonucleotides
(SO2), scrambled oligonucleotides (SCR), or oligo buffer were added,
each time at a concentration of 5.0 µmol/L. After 14 days, cytospin
slides were subjected to immunochemistry using antibodies to human
lactoferrin, as described in Materials and Methods. Red-colored
cytoplasm indicates the presence of lactoferrin, a granulocyte-specific
protein. Cells incubated with FL, IL-3, and buffer (A); with FL, IL-3,
and SO2 (B); with FL, IL-3, and SCR (C); with FL, IL-3, and AS2 (D);
and human neutrophils (E).
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The analysis of pRb expression during granulocytic and monocytic
differentiation (Fig 2A and B); the effects of AS2 or SO1 on the
expression of CD14 (Fig 3); the effects of AS2, SO2, or SCR on
morphology (Fig 5) and on staining for lactoferrin (Fig 6) and
unspecific esterase; and also the results from the CFU-GM experiments
supported the conclusion that pRb is involved in human myeloid
differentiation. Taken together with the data on proliferation from
this part of the study (Fig 4), a role for pRb in monocytic and
neutrophilic lineage commitment of human bone marrow progenitor cells
is indicated, probably by mechanisms that are at least partly distinct from those involved in cell cycle control.
Effects of AS2 on the production of pRb.
To determine whether the effects of AS2 on differentiation of
CD34+ cells supported by FL/IL-3 were correlated to an
inhibition of the upregulation of pRb levels, as demonstrated in Fig
2A, pRb levels in these cells were assayed. The addition of AS2 was
correlated to a lower level of pRb compared with addition of oligo
buffer, SO2, or SCR, as judged by Western blot on days 3 and 5 (Fig 7). These results indicate that the
effects of AS2 on the differentiation of CD34+ bone marrow
progenitors were pRb specific. Effects of AS2 on pRb level were also
determined on days 7 and 14. A similar, but generally less pronounced,
difference in pRb level on days 7 and 14 between cells incubated with
AS2 and cells incubated with SO1 was repeatedly documented (data not
shown).
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| Fig 7.
Effects of AS2 on the production of pRb. Cells at an
initial concentration of 5,000/mL were incubated in medium supplemented
with FL at 50 ng/mL and IL-3 at 25 ng/mL to promote monocytic
differentiation. Oligo buffer, sense-orientated oligonucleotides (SO2),
scrambled oligonucleotides (SCR), or antisense RB oligonucleotides
(AS2) were added on days 0, 5, and 10, each time at a concentration of
5.0 µmol/L. On days 3 and 5, 1.5 × 106 cells from each
incubation were subjected to protein extraction, followed by specific
immunoprecipitation with an anti-pRb antibody (sc-102; Santa Cruz), 6%
SDS-PAGE, and Western blot analysis. pRb of different molecular weights
are indicated with arrows (ppRb, hyperphosphorylated; pRb,
hypophosphorylated).
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DISCUSSION |
The aim of the present study was to investigate the role of pRb in
monocytic differentiation of human bone marrow cells. The data
presented show that monocytic differentiation is correlated to high
levels of hypophosphorylated pRb, whereas during neutrophilic differentiation, pRb levels are low. The low levels of pRb during neutrophilic differentiation are in line with previous
findings.10 Antisense RB oligonucleotides
(-RB) inhibited the formation of monocytic colonies in a CFU-GM
assay, with little effect on clonogenic growth, as well as inhibited
monocytic differentiation of CD34+ bone marrow progenitors
in liquid culture, again with no effect on cell growth. Furthermore,
the -RB-mediated inhibition of monocytic differentiation of
CD34+ cells was counterbalanced by a neutrophilic
differentiation response.
The exact mechanisms governing hematopoietic differentiation and
lineage commitment are not clear. External signals, such as cytokines,
are known to evoke responses affecting key cellular events such as
proliferation, differentiation, and survival. These signals are
mediated through complex regulation of specific genes, eg, genes
controlling cell cycle progression and cell-type-specific genes. The
molecular basis for regulation of hematopoiesis seems to depend on the
activities of different hematopoietic transcription factors (reviewed
in Shivdasani and Orkin11 and Tenen et al12). In this context, it is interesting to note that hematopoietic transcription factors, such as NF-IL6 (C/EBP ), PU1, and Elf1, have
been shown to interact with pRb.13-15
It has previously been demonstrated by both ourselves and other
researchers that pRb is involved in monocytic differentiation of
leukemic U937 cells.13,16 Our present results show that monocytic differentiation of normal hematopoietic progenitor cells is
dependent on pRb. This notion is based on the findings that monocytic
differentiation is associated with a strong upregulation of levels of
hypophosphorylated pRb (Fig 2A) and that, when bone marrow cells were
incubated with -RB, the appearance of monocytic characteristics,
such as morphology, the expression of CD14, and esterase positivity,
was inhibited. Moreover, a role for pRb in the choice between
neutrophilic and monocytic commitment was indicated. CD34+
cells incubated with cytokines that otherwise promote predominantly monocytic differentiation showed neutrophilic maturation, such as
neutrophilic morphology, and the appearance of lactoferrin as a
response to the addition of -RB.
How is it possible to understand that progenitors incubated with
cytokines (FL plus IL-3) normally promoting predominantly monocytic
differentiation not only respond with reduced monocytic differentiation, but also show a corresponding increase in neutrophilic differentiation when -RB are added (Figs 3, 5, and 6 and Table 2)?
Hypothetically, putative monocyte-specific pRb-dependent transcription
factors are not able to activate their target genes optimally when
upregulation of pRb is suppressed in the presence of -RB. Given the
importance of transcription factors of the C/EBP family in neutrophil
differentiation,25-28 it is particularly interesting that
pRb can interact with several members of this family.6,13
For example it has been shown that pRb can interact physically with
C/EBPs to increase binding to DNA and transactivation of target genes
during terminal adipocyte differentiation.6 Recently, it
was reported that the transcription factor C/EBP is upregulated
during granulocytic differentiation and rapidly downregulated during
the alternative monocytic pathway.29 This finding indicates
that C/EBP serves as a myeloid differentiation switch, acting on
bipotential precursors and directing them to mature granulocytes. In
the present study, the levels of pRb during granulocytic commitment and
differentiation were low (Fig 2A and B), which has also been reported
by others.10 This may contradict an activating role for pRb
in C/EBP-mediated transcription during granulocytic differentiation.
Data presented in this study rather suggest that pRb suppresses
activation of neutrophilic transcription factors, in analogy to the
ability of pRb to suppress activity of other transcription
factors.1-3
We did not find any effect on proliferation of progenitor cells
mediated by the addition of antisense Rb oligonucleotides (Fig
4). In line with this, it was recently reported that the main
regulation of the E2F family of transcription factors in hematopoietic
cells may be mediated by the pRb-related pocket protein
p130.30 This report lends support to our finding that pRb
may be primarily involved in other processes than cell cycle control in
hematopoietic cells, eg, lineage commitment and cell differentiation.
Antisense techniques have been successfully used for functional
analysis of various genes, including RB.17 However,
antisense strategies suffer from an inborn vagueness. Any observed
biological effect correlated to the use of antisense oligonucleotides
might be nonspecific, ie, not due to sequence-specific targeting of the
mRNA in question.31 However, in the present study, AS2
seemed to act in a sequence specific way, insofar as it was able to
reduce pRb levels in the cells investigated (Fig 7). However, it cannot be formally excluded that AS2 mediates neutrophilic commitment through
pRb-independent mechanisms and that the observed reduction of pRb in
cells incubated with -RB compared with cells incubated with oligo
buffer, SO2, or SCR (Fig 7) merely reflects pRb levels when neutrophils
are formed (Fig 2A and B and Table 2). Nonantisense mechanisms,
including cytotoxic breakdown effects of antisense oligonucleotides,
have recently been demonstrated to be the cause of the
antiproliferative effects of antisense oligonucleotides, which has been
shown in a number of studies.32 However, the absence of
antiproliferative effects or toxic effects of -RB on progenitor
cells in this study argue in favor of a sequence-specific action of the
-RB. In this work, -RB affected the differentiation, but not the
proliferation, of CD34+ cells supported by FL/IL-3 (Figs 3,
4, 5, and 6 and Table 2). This suggests that the proliferation and the
differentiation induced by FL/IL-3 on hematopoietic cells may be
mediated through separate signaling pathways. AS2 was not able to
inhibit the minor monocytic growth of CD34+ cells supported
by G-CSF/SCF (Table 1). This may reflect that the minor population of
monocytic progenitors recruited by G-CSF/SCF is not dependent on pRb.
In partial contrast to our results, growth-stimulatory effects of
-RB on early, but not on late, hematopoietic progenitor cells have
been reported.17 The reason for these differences in
effects of -RB on growth is unclear, but could be that the growth
stimulation used in the present work is not favoring very early progenitors.
Williams et al33 have reported extensive contribution of
pRb-deficient cells to the tissues of adult RB / RB +/+
chimeric mice, including mature erythrocytes and neurons. This
observation questions an intrinsic cell autonomous requirement for pRb
in the differentiation of the tissues targeted in the RB
/ mice, eg, erythrocytes and the central nervous system.
Instead, it suggests that the phenotype of the RB / mice
is due to extrinsic effects of pRb-deficient cells other than
precursors of erythroid or neuronal cells, eg, surrounding stromal
cells. However, the results in the present study argue for cell
autonomous intrinsic effects of pRb in myeloid differentiation, because
nonmyeloid cells, eg, stromal cells, are probably absent in our
experimental settings.
The present study suggests that pRb is crucial for monocytic commitment
and differentiation in humans. The RB / mice show hematopoietic defects: a high proportion of nucleated immature erythroid cells compared with the wild-type mouse. However, no specific
defect in monocytic maturation in RB / mice has
been reported. Lee et al7 report that myeloid cells are
present in mutant embryos. Monocytes constitute a minor proportion of the total blood cell population in the wild-type animal and the nucleated blood cell population of the mutated animal is totally dominated by immature erythrocytes.7 These circumstances
may bring about difficulties in demonstrating a putative specific defect in monocytic differentiation in the pRb-deficient mice. Also,
pRb deficiency targets different cells in different species; humans are
highly susceptible to development of retinoblastoma, whereas mice
develop pituitary tumors.9
To conclude, our data suggest that pRb is involved in the monocytic and
neutrophilic lineage commitment of human bone marrow progenitor cells,
probably by mechanisms not directly linked to pRb-mediated cell cycle regulation.
| |
ACKNOWLEDGMENT |
The authors thank Ellinor Johnsson for skillfully and enthusiastically
making the most beautiful immunocytochemistry stainings and Gunilla
Naumann for help and expert guidance with esterase stainings.
| |
FOOTNOTES |
Submitted March 19, 1998; accepted May 18, 1999.
Supported by grants from the Swedish Cancer Society, the Swedish
Society of Paediatric Cancer, The Swedish Medical Research Council
(Project No. 11546), the Georg Danielsson Foundation, the Gunnar, Arvid
and Elisabeth Nilsson Foundation, the John Persson Foundation, the John
and Augusta Persson Foundation, Funds of Lunds Sjukvårdsdistrikt, the
Swedish Society for Medical Research, the Tobias Foundation, the Thelma
Zoegas Foundation, the Crafoord Foundation, the Foundation of Greta and
Johan Kock, the Alfred Österlund Foundation, and the Hipple
Cancer Research Center (Dayton, OH).
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 Gösta Bergh, MD, Research Dept. 2., E-block, University Hospital, S-221 85 Lund, Sweden.
| |
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