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NEOPLASIA
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
Anna Dimberg, Fuad Bahram, Inger Karlberg, Lars-Gunnar Larsson, Kenneth Nilsson, and Fredrik Öberg
All-trans retinoic acid (ATRA) is a potential therapeutic agent for the treatment of
hematopoietic malignancies, because of
its function as an inducer of terminal differentiation of leukemic blasts. Although the
efficacy of ATRA as an anticancer drug
has been demonstrated by the successful
treatment of acute promyelocytic leukemia (APL), the molecular mechanisms of
ATRA-induced cell cycle arrest of myeloid
cells have not been fully investigated. In this
study, we show that the onset of ATRAinduced G0/G1 arrest of human monoblastic
U-937 cells is linked to a sharp down-
regulation of c-Myc and cyclin E levels and
an increase in p21WAF1/CIP1 expression. This
is followed by an increase in p27Kip1 protein
expression due to enhanced protein stability. The importance of an early decrease in Myc expression for these events
was demonstrated by the failure of a
U-937 subline with constitutive exogenous expression of v-Myc to cell cycle
arrest and regulate cyclin E and p27Kip1 in
response to ATRA. Preceding the initiation of G1 arrest, a transient rise in retinoblastoma protein (pRb), p107, and cyclin
A levels was detected. Later, a rapid fall in
the levels of cyclins A and B and a coordinate dephosphorylation of pRb at Ser780,
Ser795, and Ser807/811 coincided with
the accumulation of cells in G1. These
results thus identify a decrease in c-Myc
and cyclin E levels and a posttranscriptional up-regulation of p27Kip1 as important early changes, and position them in
the complex chain of events regulating
ATRA-induced cell cycle arrest of myeloid cells. (Blood. 2002;99:2199-2206)
© 2002 by The American Society of Hematology
Introduction
Hematopoietic tumors often arise as a consequence of uncontrolled
proliferation of immature blasts, failing to terminally differentiate
into mature blood cells.1 A hallmark of terminal differentiation is an
irreversible arrest in the G0/G1 phase of the cell cycle. This arrest
involves the coordinate regulation of signals that negatively control
the cell cycle machinery and inhibit the G1/S transition. At the heart
of the cell cycle lies the regulation of cyclin-dependent kinases
(CDKs), which control the phosphorylation of several substrates,
including retinoblastoma protein (pRb) family members, and thus
the transcriptional activation or repression of E2F target genes
important for cell cycle progression.2-4 The G1/S transition is
believed to be sequentially controlled by D-type cyclins, which
activate CDK4 and CDK6, and subsequently by cyclin E, which
associates with CDK2.5 CDK activity is dependent on association
with cyclins and subsequent phosphorylation-dephosphorylation
events, and may be inhibited by 2 different groups of CDK
inhibitors (CKIs), namely the Ink4 family and the Cip/Kip family.6
Thus, growth arrest associated with differentiation could be
achieved by several mechanisms including the down-regulation of
cyclins or up-regulation of CKIs or both. The critical events leading to a
cell cycle arrest are, however, likely to be specific for each mode of
induction of differentiation and may differ for each particular cell type.
Proliferation and differentiation of hematopoietic cells can be
regulated by a number of physiologic agents, including all-trans
retinoic acid (ATRA). ATRA treatment triggers terminal differentiation and growth arrest of several established human myeloid cell
lines in vitro and has also proven to be effective in the clinical
treatment of acute promyelocytic leukemia (APL) by inducing
differentiation and apoptosis of the immature blasts.7,8 Although
the biologic effects of ATRA are well characterized, the molecular
mechanisms regulating these processes are largely unknown. The
cell cycle arrest associated with ATRA-induced differentiation may
involve regulation of the expression of both cyclins and CKIs,
affect phosphorylation status of CDKs, and ultimately trigger
dephosphorylation of pRb pocket proteins. However, although
previous studies on ATRA-induced growth arrest of myeloid cells
have implicated individual proteins, for example, p21WAF1/CIP1,9-11 a
more complete investigation of the regulation of the cell cycle
machinery is required to understand the relative importance of
different events during the differentiation process.
To gain further insight into the ATRA-induced events leading to
G0/G1 arrest during terminal differentiation of human myeloid
cells, we have performed a comprehensive analysis of the regulation of CKIs, cyclins, CDKs, and pRb pocket proteins during
terminal differentiation. The well-characterized human U-937 cell
line12 was chosen as a model system. This established model of
monocytic differentiation can be induced to undergo terminal
differentiation by 12-O-tetradecanoyl phorbol-13-acetate (TPA),
From the Department of Genetics and Pathology, Rudbeck Laboratory,
Uppsala University, Uppsala, Sweden, and the Department of Plant Biology,
Uppsala Genetic Center, Swedish University of Agricultural Sciences, Uppsala,
Sweden.
Reprints: Fredrik Öberg, Department of Genetics and Pathology, Rudbeck
Laboratory, Uppsala University, S-751, 85 Uppsala, Sweden; e-mail:
[email protected].
Submitted June 14, 2001; accepted November 14, 2001.
Supported by the Swedish Cancer Society, the Magnus Bergwall Foundation,
the HKH Lovisa/Tielman Foundation, the Hans von Kantzow Foundation, the
King Gustaf V’s 80 Year Foundation, and the Åke Wiberg Foundation.
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
2199
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DIMBERG et al
vitamin D3, and ATRA, resulting in G0/G1-arrested cells with
distinct phenotypes.13-15 By kinetically correlating messenger RNA
(mRNA) and protein expression levels of important cell cycle
regulatory genes with the cell cycle distribution in U-937 cells after
ATRA induction, we determined the relative order of these events.
This enabled us to identify a temporal pattern of changes in gene
expression: (1) a rapid up-regulation of p21 and down-regulation of
c-Myc; (2) a down-regulation of cyclin E and up-regulation of p27,
at the onset of the cell cycle arrest; (3) an early, transient increase in
cyclins, pRb, and p107 expression; and finally (4) down-regulation
of cyclins A and B and dephosphorylation of pRb, coincidental to
the accumulation of cells in the G1 phase of the cell cycle. Taken
together our results demonstrate that a series of complex regulatory
events lead to an irreversible cell cycle arrest associated with
ATRA-induced myeloid differentiation, in which a rapid reduction
of c-Myc and cyclin E levels in conjunction with a posttranscriptional increase in p27 expression appears to be pivotal.
Materials and methods
Cell culture and ATRA induction
U-937-1,12,16 HL-60,17 NB-4,18 U-937-myc-2,19 containing the OK10
v-myc oncogene as part of the retroviral construct MMCV-neo, and the
parental control U-937-GTB cells19 were maintained in RPMI 1640
medium supplemented with 10% fetal bovine serum (Sigma, St Louis,
MO), glutamine, and antibiotics in a humidified 5% CO2 in air atmosphere
at 37°C. ATRA (Sigma) was dissolved in ethanol-dimethyl sulfoxide (1:5)
at 50 mM concentration and stored in aliquots at ⫺70°C. Inductions were
done on exponentially growing cells using 1 ␮M ATRA for the indicated
time periods.
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
plate probes hCC-2 (p130, pRb, p107, p53, p57, p27, p21, p19, p18, p16,
p14/15, L32, GAPDH) and hCYC-1 (cyclin A, cyclin B, cyclin C, cyclin
D1, cyclin D2, cyclin D3, cyclin A1, L32, GAPDH) were used to analyze
expression of CKI and cyclin mRNA.
Cell lysates and Western blot analysis
Cells were collected by centrifugation at 1500 rpm, washed once with PBS,
lysed in 1% NP-40, 0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, and 5 mM EDTA
with complete protease inhibitor (Boehringer Mannheim, Mannheim,
Germany), 1 mM Dithiotreitol (DTT), 1 mM phenylmethylsulfonyl fluoride
(PMSF), 0.1 mM NaVO3, 10 mM NaF, 50 ␮M NaMoO4, and 1 mM ZnCl2,
and incubated on ice for 15 minutes. Cell lysates were spun 15 minutes at
14 000 rpm at 4°C in a chilled microcentrifuge, supernatants were
collected, and the protein content was determined using the Bio-Rad protein
assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s recommendations. Equal amounts (10-15 ␮g) of extracts were fractionated on Novex NuPAGE (4%-12%, 10%) precast gels using the Novex
electrophoresis and blotting system (Novex, San Diego, CA). The membrane (Hybond-C extra, Amersham, Uppsala, Sweden) was blocked in 5%
dry milk in .1% Tween-Tris buffered Saline (TTBS) for 1 hour at room
temperature. Incubation with primary antibody was done at 4°C overnight,
and then the membranes were washed 5 times in TTBS, and incubated with
secondary horseradish peroxidase (HRP)–linked antibodies (Dako) for 1
hour at room temperature. After washing the membranes extensively in
TTBS, antibody binding was detected using enhanced chemoluminescence
plus (Amersham Pharmacia Biotech, Uppsala, Sweden). Primary antibodies
used were ␣-cyclin A (C-19), ␣-cyclin B (H-20), ␣-p27 (C-19), ␣-cyclin D2
(C-17), ␣-cyclin D3 (C-16), ␣-cyclin E (HE12), ␣-p107 (C-18), ␣-p130
(211.6), and ␣-actin (I-19) (Santa Cruz Biotechnology, Santa Cruz, CA) and
the Phosphoplus Rb (Ser780, Ser795, Ser807/811) antibody kit (Cell
Signaling Technology, Beverly, MA). Western blots were performed with
extracts from at least 3 independent ATRA-induced kinetic experiments and
routinely stained by Ponceau S to control for protein loading differences.
Cell cycle and immunofluorescence analysis
Analysis of cell cycle phase distribution and preparation of nuclei were
performed according to Vindelov et al.20 Briefly, cells were induced with 1
␮M ATRA for the indicated time, washed once in phosphate-buffered saline
(PBS), and treated with 0.03 mg/mL trypsin (Sigma) for 10 minutes at room
temperature. Then RNase A (Sigma, 0.08 mg/mL) and trypsin inhibitor
(Sigma, 0.5 mg/mL) were added, and cells were incubated for an additional
10 minutes at room temperature. Finally, prepared nuclei were stained with
propidium iodide (PI; Sigma, 0.2 mg/mL). Stained nuclei were analyzed
using a FACScan (Becton Dickinson, Mountain View, CA) and the
MacCycle software (Phoenix Flow Systems, San Diego, CA).
The surface antigen expression was analyzed by immunofluorescence
using flow cytometry (FACScan; Becton Dickinson). Briefly, the cells were
washed twice with PBS, incubated with phycoerythrin (PE)–conjugated
antibodies on ice for 30 minutes and finally washed twice with PBS plus
0.5% fetal calf serum (FCS). The PE-conjugated antibodies used were Leu
M5 (CD11c) (Becton Dickinson) and IgG2b (Dako, Glostrup, Denmark).
Statistical analysis
Statistical analysis was done by analysis of variance (ANOVA) followed by
multiple comparison by the Fisher method using the StatView computer
program. An asterisk denotes P ⬍ .05 (Figures 4 and 6).
RNA isolation and RNAse protection assay
Total RNA was isolated using the RNAgents Total RNA Isolation System
(Promega, Madison, WI) according to the manufacturer’s instructions.
RNA concentration was determined by measuring the absorbance at 260 nm
using a spectrophotometer.
The RNAse protection assay (RPA) was done with 7 ␮g of total RNA
using the RiboQuant Multi-Probe RNase Protection Assay system (Pharmingen, Becton Dickinson, Stockholm, Sweden) according to the manual and
32P-UTP (Sigma) labeled multitemplate probes (Pharmingen). Multitem-
Assay for p27Kip1 protein stability
Exponentially growing U-937 cells and cells induced to differentiate in
medium containing 1 ␮M ATRA for 72 hours were analyzed. To inhibit new
protein synthesis the cells were treated with 100 ␮g/mL cycloheximide
(Sigma) at time zero and p27 degradation followed for 8 hours. Whole cell
lysates were prepared at indicated times and subjected to Western blot
analysis as described above using primary antibodies directed against p27
or actin. The bands were quantified and the p27 protein half-life was
estimated from the slope of the curve.
Immunoprecipitation
Cell lysates were prepared as described above and equal amounts of protein
were incubated with rabbit pan-Myc antibodies IG921 and protein Aagarose beads at 4°C overnight. The immunocomplexes were washed 3
times in lysis buffer, and boiled at 95°C for 5 minutes prior to electrophoresis on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) gel. The fractionated protein was transferred from the gel to a
nitrocellulose membrane and subjected to immunoblotting as described,
using biotin-coupled C-33 monoclonal pan-Myc antibodies (Santa Cruz
Biotechnology) followed by streptavidin-HRP conjugates. The C-33 antibodies were conjugated with 10 ␮g biotin-X-NHS (Calbiochem-Novabiochem, La Jolla, CA) per milligram antibody in 0.1 M NaHCO3, pH 7.4, 0.1
M NaCl for 1 hour at room temperature followed by dialysis in PBS.
In vitro kinase assay
Whole cell lysates were prepared as described above, with the exception of
the use of 150 mM NaCl, 0.5% NP-40, and 50 mM Tris pH 6.8 lysis buffer.
Equal amounts of protein were subjected to immunoprecipitation with 2 ␮g
CDK2, CDK4, or CDK6 antibodies for 2 to 3 hours at 4°C, and then protein
G-Sepharose beads were added before a second incubation 1 to 2 hours at
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BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
REGULATION OF ATRA-INDUCED CELL CYCLE ARREST
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4°C. The immunoprecipitates were washed 3 times in lysis buffer and 3
times in kinase buffer (20 mM Tris pH 7.5, 4 mM MgCl2). The beads were
suspended in 10 ␮L reaction buffer containing 4 mM Tris, pH 7.5, 0.8 mM
MgCl2, 1 ␮g recombinant pRb (769, Santa Cruz) and 32P-␥-adenosine
triphosphate, and incubated for 30 minutes at 37°C. Prior to loading on 4%
to 12% NuPAGE gels (Novex), the samples were suspended in sample
buffer with reducing agent (Novex), boiled at 95°C for 5 minutes, and
briefly centrifuged. The amount of labeled pRb was quantified using a Fuiji
Bas 2000 Imaging System (Fuiji, Tokyo, Japan).
Results
ATRA-induced G0/G1 arrest in U-937 cells is associated with
reduced levels of cyclins A, B, D3, and E
The kinetics of ATRA-induced cell cycle arrest were determined by
flow cytometric analysis of PI-labeled nuclei. U-937 cells were
induced by ATRA and harvested at the indicated times; cell cycle
distribution was analyzed as described in “Materials and methods”
(Figure 1A). Cells started to accumulate in the G0/G1 phase of the
cell cycle between 24 and 48 hours of ATRA treatment, and the
percentage of cells in the G0/G1 phase continued to increase to over
90% after 96 hours of induction. To further analyze the kinetics of
ATRA-induced differentiation, the expression of the surface antigen p150.95/CD11c, indicative of monocyte/macrophage differentiation,22 was determined by FACS analysis after 24, 48, 72, and 96
hours of ATRA treatment (Figure 1B). The expression of CD11c
inversely correlated with the percentage of cycling cells (S⫹G2/M
phase cells), coupling differentiation to accumulation of cells in the
G0/G1 phase of the cell cycle.
Cell cycle progression is controlled by cyclins and CKIs, which
determine the activity of cyclin-dependent kinases. To determine
the expression of cyclins during U-937 G0/G1 arrest after ATRA
induction, RPA was performed using RNA from untreated or
treated cells and a multitemplate cyclin probe (h-CYC-1). A
marked reduction in cyclin A and cyclin B mRNA was observed
after 72 hours of ATRA treatment (Figure 2A). Both cyclin A and
cyclin B levels are associated with the G2 phase of the cell cycle
and are down-regulated in G1.2 The levels of cyclins C, A1, D1, and
D2 mRNA were not appreciably affected by ATRA treatment when
Figure 2. Cyclins A, B, D3, and E are down-regulated during ATRA-induced
differentiation of U-937 cells. (A) Cells were induced by ATRA and harvested at the
indicated times, and total mRNA was prepared. The regulation of cyclin mRNA during
ATRA treatment was analyzed by RPA using the hCYC-1 multiprobe template.
(B) The protein levels of cyclins A, B, D2, D3, and E during ATRA-induced
differentiation were determined by Western blot analysis using whole cell lysates.
Figure 1. U-937 cells arrest in the G0/G1 phase of the cell cycle and up-regulate
the differentiation marker CD11c in response to ATRA treatment. (A) The graph
indicates the percentage of cells in the S, G2, and M phases during ATRA
differentiation. U-937 cells were grown in the presence of ATRA and collected at the
indicated times, and the distribution of cells in different cell cycle phases was
determined by flow cytometry of PI-stained nuclei. Mean ⫾ SD (n ⫽ 4). (B) Induction
of CD11c expression during ATRA-induced differentiation. Cells were induced by
ATRA, collected at the indicated times, and analyzed by flow cytometry. Data are
presented as CD11c mean fluorescence intensity (MFI) corrected for background
binding by subtracting the MFI value for isotype-specific IgG2b control antibody.
Mean ⫾ SD (n ⫽ 3).
comparing several experiments, whereas cyclin D3 mRNA levels
were slightly down-regulated during differentiation.
Western blot analysis confirmed a sharp decrease in both
cyclin A and B protein levels occurring after 72 hours of ATRA
induction (Figure 2B). The levels of cyclins are controlled both
by transcriptional and posttranscriptional mechanisms; therefore, the protein levels of G0/G1 cyclins D1, D2, and D3 were
also analyzed. Whereas cyclin D1 was not detectable in these
cells, the level of cyclin D2 was unchanged, and the cyclin D3
protein level was decreased slightly during differentiation.
Cyclin E is known to play a role in the G0/G1 transition, but there
was no probe for this cyclin in the RPA template set used.
Instead, the cyclin E protein level was determined by Western
blot analysis. Interestingly, coinciding with the onset of cell
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DIMBERG et al
cycle arrest, cyclin E was found to be down-regulated after 24 to
48 hours of ATRA stimulation (Figure 2B).
CKIs p21WAF1/CIP1 and p27Kip1 are up-regulated by ATRA
treatment of U-937 cells, correlating to the onset of G0/G1 arrest
The levels of CKIs were monitored using a multitemplate CKI
probe (hCC-2; Figure 3A). An increase in p21 mRNA was detected
after 24 hours of ATRA induction, as previously reported by Liu et
al.9 In addition, we could detect a slight down-regulation of p16, a
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
CDK inhibitor associated with D-type cyclins. The levels of p130,
pRb, p53, p57, p27, p19, p18, and p14/15 mRNA remained unchanged.
The RPA analysis showed that p27 mRNA levels remained
unchanged after ATRA induction. However, p27 up-regulation can
occur by posttranscriptional mechanisms, and has previously been
reported to be induced by vitamin D3 treatment of U-937 cells.23
Therefore, the p27 protein level was monitored during ATRAinduced differentiation of U-937 cells. Western blot analysis
showed an increase in p27 protein starting after 48 hours of
induction (Figure 3B), coinciding with cell cycle arrest. To
determine if ATRA treatment affected p27 protein stability, the
half-life of p27 was analyzed at 72 hours of induction by following
protein levels after 1, 2, 4, 6, and 8 hours after blocking protein
synthesis by cycloheximide treatment. The estimated half-life of
p27 was approximately 3 to 4 hours in exponentially growing
U-937 cells, whereas there was a clear stabilization of p27 protein
in cells differentiated by ATRA for 72 hours, consistent with
reported observations in ATRA-differentiated neuroblastoma cells.24
Changes in the levels of cyclins and CKIs are reflected by
down-regulation of CDK 2–, 4–, and 6–associated
activity and dephosphorylation of pRb
Figure 3. ATRA-induced cell cycle arrest is associated with up-regulation of
CKIs p21 and p27. (A) RPA analysis of different CKIs during ATRA-induced
differentiation was done using the hCC-2 multitemplate probe and total mRNA from
ATRA-treated cells. (B) ATRA-induced regulation of p27 protein levels was determined by Western blot analysis using whole cell lysates of ATRA-treated cells
harvested at the indicated times. (C) To determine the turnover of p27 protein, cells
were grown with ATRA or left untreated, cycloheximide (CHX) was added after 72
hours of induction, and cells were harvested at 0, 1, 2, 4, 6, and 8 hours after CHX
treatment. Whole cell lysates were prepared and analyzed by Western blot using
antibodies directed against p27 or actin.
Cyclins and CKIs regulate the activity of CDKs, which in turn
regulate phosphorylation of several substrates, including pRb.
Phosphorylation of specific pRb serine or threonine residues has
been shown to regulate binding to different targets, including E2F
and the c-Abl tyrosine kinase.25,26 Because we observed downregulation of cyclins A, B, D3, and E and up-regulation of p21 and
p27, which may potentially alter the activities of CDKs 2, 4, and 6,
the activities of these CDKs were determined through an in vitro
kinase assay using pRb as a substrate. A significant (P ⬍ .05)
reduction in both CDK4, CDK6, and, most markedly, CDK2
activity was detected after 72 hours of ATRA stimulation (Figure
4A). Next, to directly determine the kinetics of changes in pRb
phosphorylation associated with ATRA-induced differentiation,
cells were induced by ATRA, whole cell lysates were prepared at
the indicated times, and Western blot analysis was performed using
antibodies directed against pRb phosphorylated on Ser780 or
Ser795, important for binding to E2F,25 or Ser-807/811, regulating
binding to c-Abl,26 or total levels of pRb (Figure 4B). As expected,
differentiation as the result of ATRA treatment was linked to the
disappearance of hyperphosphorylated pRb, specifically involving
dephosphorylation of Ser780, Ser795 and Ser807/811 with very
similar kinetics.
The other members of the retinoblastoma family of pocket
proteins, p107 and p130, have also been shown to be important in
cell cycle regulation and differentiation.27,28 Similar to the situation
for pRb, the function of p130 in cell cycle regulation is determined
by both the actual protein level and the phosphorylation status. To
determine if there was a change in the level of p130 or its
phosphorylation status during differentiation, whole cell lysates of
ATRA-induced cells were subjected to electrophoresis on a lowdensity gel followed by Western blot analysis using p130 antibodies (Figure 4C). There was a slight increase in p130 levels
transiently after 24 hours of ATRA treatment, and a drop in both
phosphorylated and total p130 after 120 hours of induction.
Interestingly, p107 protein levels were regulated in a similar
fashion, increasing transiently after 24 hours of ATRA induction, a
point before any apparent accumulation in the G1 phase of the cell
cycle can be observed, and the p107 expression thereafter continues to decline throughout the 96-hour period of ATRA induction
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protein was increased in both NB-4 and HL-60 cells, whereas
the cyclin E level was decreased (Figure 5).
Down-regulation of c-Myc precedes the G0/G1 arrest and is
necessary for regulation of cyclin E and p27Kip1
Previous reports have identified c-Myc as a regulator of cyclin
E-CDK2 activity through induction of cyclin E expression and
inhibition of the association of p27 with cyclin E-CDK2 complexes.33,34 Also, cyclin E-CDK2 complexes have been shown to
regulate p27 protein levels through phosphorylation on Thr187.35
This phosphorylation is a prerequisite for the proteosomedependent degradation of p27.36 Therefore, the observed ATRAinduced decrease in cyclin E levels and subsequent increase in p27
protein may be due to a down-regulation of c-Myc. To determine
the protein levels of c-Myc during differentiation, lysates from
ATRA-treated cells were immunoprecipitated using pan-Myc antibodies, and analyzed by Western blot. A decrease in the c-Myc
protein levels was initiated after 12 hours of ATRA treatment and
continued throughout differentiation (Figure 6A), preceding U-937
cell cycle arrest and changes in the levels of cyclin E and p27. This
raised the question of whether there is a causal relationship
between the reduction of Myc and the down-regulation of cyclin E,
and the following up-regulation of p27. To address this possibility
we used a U-937 subline constitutively expressing v-Myc, U-937myc-2.19,37 The U-937-myc-2 and the parental control cells,
U-937-GTB, were induced by ATRA and the cell cycle distribution
was analyzed during 96 hours. Only a small reduction in the
percentage of cycling cells was observed for the U-937-myc-2
cells, whereas U-937-GTB cells became G1 arrested (Figure 6B).
Figure 6C shows that U-937-myc-2 cells fail to down-regulate the
expression of cyclin E and up-regulate p27 at 72 hours after
induction, suggesting that the down-regulation of c-Myc is a
prerequisite for these changes.
Figure 4. ATRA treatment of U-937 cells results in decreased CDK activity
and a reduction in hyperphosphorylated pRb. (A) CDK2, 4, and 6 kinase
activity was measured in an in vitro kinase assay using pRb as a substrate. Cells
were grown in the presence or absence of ATRA for 72 hours, whole cell lysates
were prepared, and the activities of CDK2, CDK4, and CDK6 were determined
as described in “Materials and methods.” The graph indicates quantification of
the bands correlated to control. Mean ⫾ SD (n ⫽ 4). The asterisk indicates
significant differences from the corresponding controls (P ⬍ .05). (B-D) The levels
of total pRb or Ser780, Ser795, or Ser807/811 phosphorylated pRb (B), p130 (C),
or p107 (D) during ATRA-induced differentiation were determined by Western
blot of whole cell lysates prepared at the indicated times using specific
antibodies.
studied (Figure 4D). However, only p107 appeared to be regulated
on the mRNA level (Figure 2B).
ATRA induces down-regulation of cyclin E and up-regulation of
p27KIP1 in the ATRA-responsive human myeloid leukemic
cell lines HL-60 and NB-4
Next, we wanted to determine whether the regulation of p27 and
cyclin E, similar to the more well-established changes in
Myc29,30 and p21,9-11 is a common event linked to cell cycle
arrest during myeloid differentiation induced by ATRA. Therefore, we studied the regulation of these proteins in the ATRAsensitive human myeloid cell lines HL-60 and NB-4. HL-60
cells arrest in the G0/G1 phase of the cell cycle in response to
ATRA treatment, whereas NB-4 cells arrest in a non–G1restricted manner, at all points in the cell cycle31,32 (and data not
shown). After 96 hours of ATRA stimulation, the level of p27
Discussion
The successful treatment of APL patients with ATRA demonstrates
that external signals can overcome the block of differentiation in
myeloid leukemic cells in vivo.7 Although leukemias and lymphomas represent a diverse group of tumors that can arise as a
consequence of several different genetic changes, extensive work
done in established leukemic cell lines suggests that ATRA or
synthetic retinoid derivatives may be effective as therapeutic
agents in the treatment also of other types of myeloid malignancies
than APL. Identifying the critical subset of cell cycle control genes
involved in ATRA-induced growth arrest of myeloid cells is
important for the understanding of the molecular events underlying
the therapeutic potential of ATRA treatment.
Figure 5. HL-60 and NB-4 cells respond to ATRA treatment by down-regulation
of cyclin E and up-regulation of p27. Cells were induced by ATRA for 96 hours,
whole cell lysates were prepared, and the protein levels of cyclin E and p27 were
analyzed by Western blot.
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DIMBERG et al
Figure 6. Exogenous expression of Myc inhibits ATRA-induced cell cycle arrest
and the associated down-regulation of cyclin E and up-regulation of p27.
(A) The expression of c-Myc is down-regulated in response to ATRA treatment in
U-937 cells. Cells were treated with ATRA for 0, 12, 24, 48, and 72 hours as indicated;
whole cell lysates were prepared and subjected to immunoprecipitation using
pan-Myc antibodies. The immunocomplexes were collected, separated by SDSPAGE, and analyzed by Western blot using biotin-coupled pan-Myc antibodies and
streptavidin-HRP. (B) U-937-myc-2 cells and the parental subline U-937-GTB were
grown in the presence of ATRA, collected at the indicated times, and the distribution
of cells in different cell cycle phases was determined by flow cytometry of PI-stained
nuclei. Mean ⫾ SD (n ⫽ 3). The asterisk indicates significant differences from the
corresponding U-937-GTB controls. (C) The protein levels of cyclin E and p27 after
72 hours of ATRA treatment in U-937-GTB and U-937-myc-2 cells were analyzed by
Western blot.
Much research interest has been focused on the CKIs with
respect to the molecular events initiating a G1 cell cycle arrest.
During normal myeloid differentiation from CD34⫹ hematopoietic
progenitors a progressive increase in p21 mRNA and protein is
observed.38 Similarly, an early ATRA-induced up-regulation of p21
has been described in leukemic cell lines, for example, U-937,
HL-60, and NB-4 cells.9-11,39 The promoter of p21 has been shown
to contain a retinoic acid response element (RARE), directly
linking activation of the RAR/RXR nuclear receptor complex to
induction of p21 expression.9 However, the ATRA-induced level of
p21 protein in U-937 cells is relatively low as compared to
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
TPA-induced U-937 cells, and only detectable after immunoprecipitation in our system (data not shown). Therefore, we expected that
other CKIs would also be up-regulated by ATRA and that they
would contribute to the G0/G1 arrest. The level of p27 mRNA was
found to be constant during differentiation. However, the half-life
for the p27 protein increased in ATRA-differentiated U-937 cells.
This indicates that the mechanism for p27 up-regulation is, at least
partially, due to a stabilization of the p27 protein, consistent with
findings in ATRA-induced neuroblastoma.24 During hematopoiesis
a low expression of p27 protein is detected in early progenitors and
continues throughout myeloid differentiation with increased levels
or subcellular redistribution (or both) at later stages.40,41 Moderate
levels of p27 protein are found in undifferentiated U-937 cells, and
it is likely that the observed reduction in cyclin E levels contributes
to the following increase in p27 stability after ATRA treatment,
because cyclin E-CDK2 complexes can target p27 for degradation
through phosphorylation on Thr187.35,36 The general relevance of
cyclin E and p27 regulation for differentiation-associated growth
arrest of myeloid cells was confirmed in ATRA-induced NB-4 and
HL-60 cells.
Down-regulation of c-Myc is an important early event that has
been connected to terminal differentiation and growth arrest of
several cell types,29,30 including U-937.19,37 We observe a decrease
in c-Myc levels within 12 hours of ATRA stimulation and a
continued down-regulation throughout differentiation. Myc plays a
dual role in regulating the activity of cyclin E–CDK2 complexes,
primarily via the inhibition of p27, either by induction of cyclin D1
and cyclin D2 resulting in p27 sequestration33,34,42,43 or by direct
repression of the p27 promoter,44 but also through direct or indirect
transcriptional activation of the cyclin E gene.34 Consequently,
changes resulting from the early down-regulation of c-Myc may
directly affect the cell cycle machinery and be important for the
initiation of G0/G1 arrest. Two observations place c-Myc upstream
of the other changes in cell cycle control proteins. First, the kinetics
of c-Myc down-regulation is rapid and precedes subsequent
changes in cyclin E and p27. Second, we provide evidence that
constitutive expression of a v-myc gene prevents reduction of
cyclin E and increase in p27 and the consecutive G1 arrest.
An early reduction in CDK activity, initiating the G0/G1 arrest in
response to ATRA treatment of U-937 cells, appears to be largely
dependent on the marked down-regulation of cyclin E in combination with up-regulation of p21 and p27. This is later followed by a
fall in cyclin A and B expression, coincident with the accumulation
of cells in G1 (Figure 7A). The observed pattern of cyclin E, cyclin
A, and cyclin B expression in ATRA-induced U-937 cells is
reminiscent of that in cells undergoing myeloid differentiation from
normal hematopoietic progenitors, with a high expression during
the proliferative phase followed by down-regulation at the terminal
stage.45 In contrast, the sustained expression of cyclin D2 and the
marginal reduction of cyclin D3 levels during ATRA-induced cell
cycle arrest is clearly different as compared to normal myeloid
differentiation or, for example, to the G1 arrest that is induced in
response to the withdrawal of mitogenic signals (mainly affecting
D-type cyclins).5 This difference may either be the consequence of
the leukemic nature of the U-937 cells or a particular feature of
ATRA-induced differentiation.
Changes in the expression of cyclins and CKIs in response to
ATRA treatment were followed by down-regulation of CDK2,
CDK4, and CDK6 activity and dephosphorylation of pRb at
specific serine residues. The most apparent dephosphorylation of
pRb occurs after 72 hours of induction, although an elevation in
hypophosphorylated pRb is evident already after 24 hours of ATRA
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BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
REGULATION OF ATRA-INDUCED CELL CYCLE ARREST
Figure 7. Kinetics of the expression of cell cycle regulatory proteins and
dephosphorylation of pRB, associated with ATRA-induced differentiation.
(A) The temporal changes in protein expression of the indicated cell cycle regulators
based on the quantification of Western blot data from Figures 2B and 3B; cyclin A (Œ),
cyclin B (⽧), cyclin E (F), p27 (췦). (B) Kinetics of pRb dephosphorylation at specific
sites based on the quantification of Western blot data from Figure 4B; Ser780 (⽧),
Ser795 (Œ), Ser807/811 (F). Quantified data are displayed with the peak value for
each series arbitrarily set to 100. For cycling cells the actual percentage of cells in the
S⫹G2/M phases of the cell cycle is shown.
induction, when taking the increase in total pRb into account
(Figure 4 and Figure 7B). During the cell cycle progression, pRb is
sequentially phosphorylated by different cyclin-CDK complexes.46
Down-regulation of cyclin E precedes dephosphorylation of pRb at
Ser780, Ser795, and Ser807/811, and could therefore potentially
affect other phosphoacceptor sites. Phosphorylation of pRb by
cyclin E–CDK2 has been suggested to prevent E2F binding;
therefore, a reduction in cyclin E–CDK2 activity would be
predicted to inhibit E2F function and expression of S-phase
genes.3,47 Thus, the presence of hypophosphorylated pRb could
2205
potentially be important to preclude re-entry into S phase and
thereby establish an irreversible cell cycle exit in the terminally
differentiated cell.
The intimate link between terminal differentiation and cell
cycle arrest makes it difficult to assign a particular function of
an individual protein to either of the 2 processes. Also, several
of the cell cycle regulatory proteins may have dual functions.
For instance, the function of pRb family proteins has also been
linked to differentiation in various cell systems.27 Interestingly,
expression of an antisense pRb construct in U-937 cells, which
decreased pRb levels, resulted in defects in ATRA-induced
differentiation, but not cell cycle arrest.48 Up-regulation of p21
and p27 may also impinge on the differentiation process; for
example, up-regulation of p21 in APL cells has been reported to
be important for commitment to differentiation, but not required
for cell cycle arrest.39 Forced expression of p21 or p27 has been
suggested to induce markers of differentiation in U-937 cells,49
supporting this notion. Typically, it appears that during induced
differentiation myeloid cells traverse one or several cell cycles,
before the associated G1 arrest is established.39,49,50 Moreover,
the proliferative burst after vitamin D3 treatment of U-937 cells
is accompanied by a transient up-regulation of cyclins, p27, and
p21.49 In agreement with this observation, we observed a
transient up-regulation of cyclins A and D2, as well as pRb and
p107 within the first 24 hours of ATRA treatment. A transient
up-regulation of p107 has recently been suggested to be
important for adipocyte differentiation.51 It remains to be shown
if similar links between the transient up-regulation of cell cycle
control proteins and terminal differentiation exist in ATRAinduced U-937 cells.
The main new finding in this paper is that the initiation of
ATRA-induced cell cycle arrest in U-937 cells is preceded by
down-regulation of c-Myc, necessary for a coordinate fall in cyclin
E levels and up-regulation of p27. Although a schematic blueprint
of differentiation-associated regulation of cell cycle control proteins in myeloid cells has been drawn by this study, the molecular
mechanism of ATRA-induced regulation of cyclins, CDKs, and
CKIs warrants further investigation
Acknowledgment
We thank Lina Dimberg for critical reading of the manuscript.
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2002 99: 2199-2206
doi:10.1182/blood.V99.6.2199
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
Anna Dimberg, Fuad Bahram, Inger Karlberg, Lars-Gunnar Larsson, Kenneth Nilsson and Fredrik Öberg
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