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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1348-1358
Retinoic Acid Prevents Phosphorylation of pRB in Normal Human B
Lymphocytes: Regulation of Cyclin E, Cyclin A, and p21Cip1
By
Soheil Naderi and
Heidi Kiil Blomhoff
From the Department of Medical Biochemistry, Institute group of Basic
Medical Sciences, Faculty of Medicine, University of Oslo, Oslo,
Norway.
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ABSTRACT |
The mechanisms underlying the growth-inhibitory effect of retinoids
on normal human B lymphocytes are not well understood. We addressed
this issue by examining the effect of retinoic acid on the cell cycle
machinery involved in G1/S transition. When retinoic acid was
administered to B cells stimulated into mid to late G1 by anti-IgM
antibodies (anti-µ) and Staphylococcus aureus crude cell
suspension (SAC), the phosphorylation of pRB required for S-phase entry
was prevented in a time- and dose-dependent manner. Thus, 2-hour
treatment with retinoic acid at the optimal concentration of 1 µmol/L
prevented phosphorylation of pRB, and effects were noted at
concentrations as low as 10 nmol/L. Based on our results, we suggest
that the rapid effect of retinoic acid on pRB phosphorylation is due
primarily to the reduced expression of cyclin E and cyclin A in late
G1. This could lead to the diminished cyclin E- and cyclin
A-associated kinase activities noted as early as 2 hours after
addition of retinoic acid. Furthermore, our results imply that the
transient induction of p21Cip1 could also be involved.
Thus, retinoic acid induced a rapid, but transient increased binding of
p21Cip1 to CDK2. The retinoic acid receptor (RAR) agonist
TTNPB mimicked the key events affected by retinoic acid, such as pRB
phosphorylation, cyclin E expression, and expression of
p21Cip1, whereas the RAR-selective antagonist Ro 41-5253 counteracted the effects of retinoic acid. This implies that retinoic
acid mediates its growth-inhibitory effect on B lymphocytes via the nuclear receptors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
AN IMPORTANT ASPECT of the in
vivo function of vitamin A is its protection against
infections.1,2 It is believed that this in part is due to
the requirement of retinoids in maintaining normal differentiation of
the epithelium along the body linings.3 The role of
retinoids on the immune system per se, however, is unclear. Although
most in vivo studies conclude that retinoids are required for a
functional immune system,1,4 high intake of vitamin A has
been reported to impair normal immune responses.5 In vitro
studies on cultured lymphocytes have shown both
stimulatory6,7 and inhibitory8,9 effects of
retinoids. We have previously documented that physiological levels of
retinoids inhibit the proliferation of human peripheral blood B
lymphocytes8 as well as of human B-cell precursors, murine
splenic lymphocytes, and murine B-cell precursors.10 Still,
however, it appears that the inhibitory effect of retinoids on normal
lymphocytes is a controversial issue.
To further strengthen our data on the inhibitory role of physiological
levels of retinoids on B-cell growth, we wished to examine the effect
of retinoids on the cell cycle machinery. Unraveling the components of
the cell cycle machinery has during recent years lead to a profound new
understanding of the mechanisms involved in cell cycle
progression.11,12 Furthermore, the identification of
cyclin-dependent kinases (CDKs) as the key driving force in cell cycle
progression has helped to identify important substrates that are
phosphorylated by CDKs. Among these key substrates, the protein product
of the retinoblastoma gene (pRB) appears as particularly important.12 pRB is a tumor suppressor gene that is
frequently inactivated in human tumors.13 The physiological
role of pRB in normal cells is, however, to prevent the cells from
entering into S phase from G1.12 This feature of pRB is
accomplished by its role as a pocket protein, binding transcription
factors such as E2F and DP that are known to regulate
S-phase-promoting genes.14 The phosphoprotein pRB is
active as a sequestrator of transcription factors only in its
hypophosphorylated state. Upon sequential phosphorylation in G1 by
CDK4/CDK6 and CDK2, pRB is inactivated, leading to release of E2F,
transcription of S-phase genes, and subsequent entry into S
phase.12 It is believed that pRB phosphorylation identifies
the restriction point in G1 of the cell cycle, as a point of no return
in stimulation of cells to proliferate.12 Thus, the
regulation of CDKs that are responsible for pRB phosphorylation appears
as a key event in the processes that determine the proliferative state
of a cell. The main regulation of CDK activity is at the level of
interaction with specific cyclins, CDK inhibitors (CKIs), and by
specific phosphorylation and dephosphorylation of critical amino acids
on the CDK catalytic subunit.11
In the present study, we have examined the effect of physiological
doses of all-trans-retinoic acid on regulation of the cell cycle machinery in human peripheral blood B lymphocytes, with specific
emphasis on the events leading to pRB dephosphorylation in the G1 phase
of the cell cycle. It has been an issue of debate whether or not
nuclear receptors mediate the effects of retinoids on growth,
differentiation, and apoptosis in lymphocytes,15 as well as
in other cell types.16 In this study we therefore included
a synthetic retinoid known to act via nuclear retinoic acid receptors
(RAR), which we recently showed could mimic the effects of
all-trans-retinoic acid on regulation of both growth and
apoptosis in human B-lymphocytes.17 Furthermore, we
included an RAR-selective antagonist, previously shown to counteract
the effects of retinoic acid in several different cell
systems.18
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MATERIALS AND METHODS |
Reagents and antibodies.
Retinoids are defined as retinol and derivatives of retinol, including
those without biological activity. Vitamin A is commonly used to define
naturally occurring retinoids with biological activity similar to that
of retinol, including retinol itself. Retinoic acid is the oxidized
form of retinol, and the isomer all-trans-retinoic acid was
purchased from Sigma Chemical Co (St Louis, MO). The RAR agonist
Ro13-7410 (TTNPB) and the RAR-selective antagonist Ro 41-5253 were
kindly provided by Dr M. Klaus (Hoffman-La Roche, Basel, Switzerland).
Anti-cyclin E (HE12; for Western blot analysis, HE111; for
immunoprecipitation), anti-cyclin A (C-19), anti-CDK2
[(M2)-G], anti-cyclin D2 (34B1-3), anti-cyclin D3 (D-7),
anti-CDK4 (C-2), anti-CDK6 (C-21),
anti-p21Cip1 (C-19), and anti-p27Kip1 (C-19)
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pRB
(G3-254) was obtained from Pharmingen (San Diego, CA).
Cell purification, cell culture, and proliferation assay.
Resting human B lymphocytes were isolated from platelet-depleted buffy
coats obtained from the Blood Bank (Ullevål Hospital, Oslo, Norway),
as described by Funderud et al.19 Each buffy coat (50 mL)
was diluted in 25 mL RPMI 1640 medium (GIBCO, Grand Island,
NY) containing 0.01 mol/L EDTA and mixed with 2 × 108 Dynabeads coated with anti-CD19 antibody (Product No.
111.04; Dynal, Oslo, Norway). After incubation of the mixture on a
rotating wheel for 1 hour at 4°C, the rosetted cells were attracted
to a samarium cobalt magnet and the nonrosetting cells were removed. Cell rosettes were subsequently washed five times with 10 mL RPMI 1640 medium containing 1% heat-inactivated fetal bovine serum (FBS; GIBCO)
to remove the residual nonrosetting cells. To detach the beads from the
cells, cell rosettes were cultured in RPMI 1640 medium containing 1%
FBS overnight at 37°C in a CO2 incubator, and then the
beads were attracted to a samarium cobalt magnet and the B cells were
recovered from the supernatant. Under these conditions, less than 1% T
cells, natural killer (NK) cells, and monocytes were detected in the
cell population.
B cells were cultured in RPMI 1640 medium supplemented with 1% FBS, 2 mmol/L glutamine, 125 U/mL penicillin, 125 µg/mL streptomycin at a
density of 1.5 × 106 cells/mL as previously
described.19 Cells were stimulated to enter the cell cycle
by the addition of the combination of anti-µ [37.5 µg/mL of
F(ab')2 fragments of rabbit polyclonal antibodies to
human IgM heavy chain; Dako, Copenhagen, Denmark] and 0.005% of
formalin-fixed SAC (Sigma). To examine the effect of retinoic acid or
the RAR-selective agonist TTNBP on B lymphocytes, different concentrations of retinoic acid or TTNBP were added to cell cultures for the indicated number of hours before harvesting the cells 32 hours
poststimulation. For measurement of DNA synthesis, cells were cultured
in microtiter plates at an initial density of 7.5 × 104 cells/0.2 mL. Cells were pulsed with 0.2 mCi of
[3H]thymidine (Amersham, Buckinghamshire,
UK) for the last 20 hours of a 68-hour incubation. The
cells were then harvested on a cell harvester and counted on a liquid
scintillation counter (Topcount; Packard, Meriden, CT).
Immunoblot analysis and immunoprecipitations.
Total cell lysates were prepared by lysis with Laemmli sample
buffer.20 After boiling the samples for 5 minutes, cellular debris was removed by centrifugation at 4°C and the protein content of the supernatant was determined by Bradford analysis (Bio-Rad, Hercules, CA). Equal amounts of proteins (30 to 100 µg) were resolved on a 7.5% (for pRB) or 10% to 12% (for other proteins)
polyacrylamide gel under reducing conditions, and transferred onto
nitrocellulose membrane (Amersham) using a semidry transfer cell
(Bio-Rad). After blocking in TBS-T (0.1% Tween 20) containing 5%
nonfat dry milk for 1 hour at room temperature, the membrane was
incubated with 1 µg/mL of the indicated antibody in TBS-T for 2 hours
at room temperature. The membrane was then washed four times with
TBS-T, incubated with a 1:6000 dilution of horseradish
peroxidase-linked secondary antibody (Bio-Rad), and the immunoreactive
proteins were visualized with the enhanced chemiluminescence detection system (Amersham).
For immunoprecipitations followed by immunoblotting, whole cell
extracts were prepared essentially as described by Gorospe et
al.21 In brief, the cells were lysed for 20 minutes on ice in Triton X-100 lysis buffer (50 mmol/L Tris [pH 7.5], 250 mmol/L NaCl, 0.1% Tween X-100, 10 µg of leupeptin per mL, 9.5 µg of
aprotinin per mL, 35 µg of phenylmethylsulfonyl floride per mL, 5 mmol/L NaF, 0.1 mmol/L ortho-vanadate, 10 mmol/L  -glycerophosphate), and the lysate was cleared by centifugation at 4°C. Cell lysates corresponding to equal amounts of protein (500 µg) were
immunoprecipitated for 2 hours at 4°C with the desired antibody.
The immunocomplexes were absorbed to 30 µL of a 1:1 slurry of protein
G sepharose beads (Pharmacia, Sweden) for 1 hour at 4°C, collected
by centrifugation at 2000g for 5 minutes, and washed twice with
the lysis buffer. The beads were then resuspended in Laemmli sample
buffer, boiled, and subjected to Western analysis.
Kinase assays.
For histone H1 kinase assays, whole cell extracts were prepared with
Triton X-100 lysis buffer as described.21 After preclearing with 20 µL of a 1:1 slurry of protein G-sepharose for 30 minutes at
4°C, 200 µg of each lysate was immunoprecipitated with 2 µg of
the indicated antibody as described above. After immunoprecipitation, the beads were washed twice with lysis buffer, and once with kinase buffer (50 mmol/L Tris [pH 7.5], 10 mmol/L MgCl2, 1 mmol/L dithiothreitol [DTT], 2 mmol/L EGTA, 1 mmol/L NaF, 0.1 mmol/L
ortho-vandate, 10 mmol/L -glycerophosphate). The beads were
resuspended in 15 µL of kinase buffer containing 30 µmol/L
adenosine triphosphate (ATP), 5 µg histone H1 (Upstate Biotechnology,
NY), 10 µCi of [ -32P]ATP per reaction mixture, and
incubated for 30 minutes at 30°C. Reactions were stopped with
addition of 7.5 µL 3× Laemmli sample buffer. The samples were
boiled for 5 minutes and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After
electrophoresis, gels were stained with Coomassie blue, dried, and
subjected to autoradiography.
pRB kinase assays were performed essentially as described previously by
Matsushime et al.22 In brief, whole cell extracts were
prepared by resuspending the cell pellets on ice in Tween 20 lysis
buffer (50 mmol/L HEPES [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, 2.5 mmol/L EGTA, 0.1 % Tween 20, 10% glycerol, 1 mmol/L DTT, 10 µg of
leupeptin per mL, 9.5 µg of aprotinin per mL, 35 µg of
phenylmethylsulfonyl fluoride per mL, 5 mmol/L NaF, 0.1 mmol/L
ortho-vandate, 10 mmol/L -glycerophosphate), and vortexed at
5-minute intervals for 20 minutes. After removing the insoluble material by centrifugation, the lysates were precleared by incubation with 25 µL of a 1:1 slurry protein G-sepharose for 30 minutes at
4°C and analyzed for protein content by the Bradford method (Bio-Rad). CDK6-associated complexes were recovered by
immunoprecipitation of 600 µg of cell lysates with
anti-CDK6 polyclonal antibodies. After washing the beads
twice with Tween 20 lysis buffer and three times with pRB kinase buffer
(50 mmol/L HEPES [pH 7.5], 10 mmol/L MgCl2, 1 mmol/L DTT,
2 mmol/L EGTA, 1 mmol/L NaF, 0.1 mmol/L ortho-vandate, 10 mmol/L -glycerophosphate), the reactions were initiated by addition
of 50 µL pRB kinase buffer containing 20 µmol/L ATP, 1 µg
glutathione S-transferase (GST)-tagged RB(GST-RB) (Santa Cruz
Biotechnology), and 10 µCi of [-32P]ATP per reaction
mixture and incubated for 30 minutes at 30°C. After addition of the
3× Laemmli sample buffer, samples were boiled and subjected to
SDS-PAGE analysis and autoradiography.
Northern blot analysis.
Poly (A)+ mRNA was purified from 25 × 106
cells for each treatment using Dynabeads oligo (dT)25
magnetic beads (Dynal, Oslo, Norway) following manufacturer's
instructions, fractionated on a formaldehyde/agarose gel, transferred
onto Hybond-N filter (Amersham, UK) in 20× SSC and
ultraviolet crosslinked.23 The filter was then hybridized
with the indicated cDNA probes at 42°C in 50% formamide, washed to
final stringency of 0.1× SSC, 0.1% SDS at 50°C, and
autoradiographed. The probes (p21Cip1, NotI fragment of
PC-WAF1-S 24; cyclin E, HindIII fragment of
Rc-cycE25; cyclin A, HindIII-XbaI fragment of
Rc-cycA25) were labeled with [ -32P]dCTP
(Amersham Megaprime Labeling System, Amersham) according to the
Amersham Megaprime protocol. The p21Cip1 probe was a kind
gift from B. Vogelstein (Baltimore, MD). The cyclin E and cyclin A
probes were kind gifts from R.A. Weinberg (Cambridge, MA).
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RESULTS |
Retinoic acid inhibits DNA synthesis also when added near the
restriction point in normal B-lymphocytes.
The purpose of the present study was to examine early changes in cell
cycle parameters upon treatment of normal human B lymphocytes with
retinoic acid. We have previously shown that the restriction point in
human B lymphocytes is located approximately 30 to 35 hours into G1,
ie, approximately 10 hours before S-phase entry.26 We
therefore measured the effect of retinoic acid on DNA synthesis when
added at the beginning of the cell culture, or when added after 32 hours of stimulation with anti-µ and SAC. As shown in Fig 1, retinoic acid inhibited the DNA
synthesis of B lymphocytes at both the physiological level (10 nmol/L)
and the pharmacological level (1 µmol/L), in agreement with our
previous report.8 Thus at the optimal dose of 1 µmol/L
retinoic acid, DNA synthesis was reduced by 61%. Interestingly,
retinoic acid added as late as 32 hours after stimulation of the cells
still inhibited the DNA synthesis, although the effect was somewhat
reduced.
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| Fig 1.
Effect of retinoic acid on DNA synthesis. Human B cells
were isolated and stimulated with  µ (37.5 µg/mL) and SAC
(0.005%) as described in Material and Methods. Cells (0.75 × 106 cells/mL) were cultured in 96-well culture plates in
triplicates. Retinoic acid (10 or 1000 nmol/L) was added at the
indicated times (T) after stimulation. [3H]Thymidine
incorporation (mean ± SD) was measured as described in Material and
Methods. One representative experiment of three is shown. µ,
F(ab')2 fragment of rabbit antihuman IgM; SAC,
Staphylococcus aureus crude cell suspension; RA, retinoic
acid.
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Retinoic acid prevents phosphorylation of pRB in a dose- and
time-dependent manner in late G1.
The phosphorylation of pRB by CDK2 in complex with cyclin E is believed
to be a key event in the regulation of S-phase entry, and appears to
define the restriction point in late G1.12 It was therefore
of particular interest to examine whether retinoic acid would prevent
phosphorylation of pRB as part of its growth-inhibitory effect on human
B lymphocytes. The Western blot in Fig 2
shows the kinetics of pRB phosphorylation during stimulation of
B-lymphocytes with anti-µ and SAC. The results showed that the
maximum level of pRB phosphorylation was obtained after 42 hours of
stimulation (ie, approximately at the time of entrance into S
phase26). When the cells entered the G1 phase of the
following cell cycle after stimulation for 70 hours, pRB again appeared
in its hypophosphorylated state.
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| Fig 2.
Expression and phosphorylation of pRB in stimulated human
B cells. Freshly isolated human B cells (1.5 × 106
cells/mL) were stimulated with a combination of µ (37 µg/mL) and
SAC (0.005%) for the indicated times. Total cell lysates were prepared
and 30 µg per lane was subjected to Western blot analysis as
described in Material and Methods. Hypophosphorylated pRB and
hyperphosphorylated pRB (ppRB) were detected with the RB-PMG3-245
monoclonal antibody. The lane indicated as 0 hrs refers to a sample
harvested before mitogenic stimulation of the cells. µ,
F(ab')2 fragment of rabbit antihuman IgM; SAC,
Staphylococcus aureus crude cell suspension.
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We examined the effect of retinoic acid on pRB phosphorylation by
adding different concentrations of retinoic acid to the cell cultures
together with the stimulatory agent at time 0. The cells were harvested
after 32 hours, and the level of pRB phosphorylation was assessed by
Western blot analysis. As shown in Fig 3A,
phosphorylation of pRB was prevented by retinoic acid at low
concentrations. In fact, concentrations as low as 1 to 10 nmol/L, ie,
physiological concentrations,27 showed considerable
inhibition of pRB phosphorylation. However, an even stronger effect on
pRB phosphorylation was noted at 1 µmol/L of retinoic acid, and this
concentration was therefore used in the following experiments.
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| Fig 3.
Retinoic acid inhibits pRB phosphorylation. (A) Human B
cells (1.5 × 106 cells/mL) were stimulated with µ
(37 µg/mL) and SAC (0.005%) for 32 hours in the absence (control) or
presence of retinoic acid at the indicated concentrations. Total cell
lysates were prepared and 30 µg of total protein was analyzed for pRB
protein by Western blotting. The lane indicated as 0 hrs refers to a
sample harvested before mitogenic stimulation of the cells. (B) Left
panel: Cells (1.5 × 106 /mL) were stimulated with µ
(37 µg/mL) and SAC (0.005%) and harvested at the indicated times
after stimulation. Right panel: Cells were stimulated with µ (37 µg/mL) and SAC (0.005%). Retinoic acid (1000 nmol/L) was then added
to the cell cultures at each indicated time before harvesting the cells
at 32 hours. Total cell lysates were prepared and subjected to Western
blot analysis for pRB. µ, F(ab')2 fragment of
rabbit antihuman IgM; SAC, Staphylococcus aureus crude cell
suspension; RA, retinoic acid.
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Because addition of retinoic acid at time 0 appeared to partially but
not totally prevent pRB phosphorylation, we assessed the effect of
retinoic acid when administered to the cells later in G1. To do so,
retinoic acid was added to the cultures at different times during G1,
but before the restriction point. All the cells were harvested at 32 hours, and the phosphorylation state of pRB was analyzed by Western
blotting. In the right panel of Fig 3B the effect of retinoic acid on
pRB phosphorylation is shown. Note that the phosphorylation patterns of
pRB shown in the right panel should be compared with that of the
32-hour control in the left panel, because all the cell cultures were
harvested at 32 hours. Any reduced level of pRB phosphorylation in
retinoic acid-treated cells as compared with that of the 32-hour
control would indicate that retinoic acid has prevented pRB
phosphorylation. The other controls in the left panel of Fig 3B show
the phosphorylation state of pRB at the various times in G1 when
retinoic acid was added.
The results in Fig 3B indicated that pRB phosphorylation was prevented
by retinoic acid in a time-dependent manner. When retinoic acid was
added to the cultures between 8 and 2 hours before harvesting the cells
at 32 hours into G1, retinoic acid appeared to totally prevent further
pRB phosphorylation. When added earlier in G1, ie, between time 0 and
16 hours into G1, the phosphorylation of pRB was only partially
prevented. These results are in agreement with our previous findings
that retinoic acid blocks B lymphocytes in mid to late G1, ie, after
approximately 24 hours of stimulation into G1.8
Retinoic acid inhibits the kinase activity of CDK2.
The phosphorylation of pRB in G1 is mediated by CDK4 and CDK6 in
complex with cyclin D, and later in G1 by CDK2 in complex with cyclin E
and cyclin A.12 The prevention of pRB phosphorylation in
the presence of retinoic acid could therefore be due to inhibition of
one or more of these complexes. We therefore examined the effect of
retinoic acid on the activities of CDK4, CDK6, and CDK2. As shown in
Fig 4A, the activation-induced increase in
CDK2 activity was reduced by retinoic acid in a time-dependent manner.
Again an effect was noted even when retinoic acid was added as late as
2 hours before harvesting the cells. No effect of retinoic acid was
noted on the kinase activity of CDK6 (Fig 4B), whereas CDK4 kinase
activity in our hands was below detectable levels in the isolated
B-lymphocytes using GST-RB as substrate (data not shown).
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| Fig 4.
Effect of retinoic acid on G1 CDKs. Mitogenic stimulation
and retinoic acid treatment of the cells were performed as described in
the legend to Fig 3B. Whole cell extracts were prepared as described in
Material and Methods. (A) Equal amounts of whole cell extracts (200 µg) were immunoprecipitated with antibody to CDK2 followed by kinase
assay with histone H1 as substrate as described in Material and
Methods. (B) Cells were stimulated with µ (37 µg/mL) and SAC
(0.005%) and harvested at 32 hours. For retinoic acid treatment of the
cells, retinoic acid (1000 nmol/L) was added to the cell cultures at
each indicated time before harvesting the cells at 32 hours. CDK6 was
immunprecipitated from 600 µg of whole cell extracts and the
immunoprecipitates were then assayed for kinase activity with GST-RB as
substrate as described in Materials and Methods. + peptide,
background activity was determined by blocking the CDK6 antibody with
the specific antigenic peptide. µ, F(ab')2
fragment of rabbit antihuman IgM; SAC, Staphylococcus aureus
crude cell suspension; RA, retinoic acid.
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Retinoic acid changes the protein expression of cyclin E, cyclin A,
and p21Cip1.
The inhibitory effect of retinoic acid on the CDK2 activity could be
due to either reduced levels of cyclins or changes in the level of
CKIs. By Western blot analysis we measured the kinetics of retinoic
acid-mediated changes in protein levels of the various G1 cyclins,
CDKs, and CKIs. As presented in the left panel of Fig 5A, we first analyzed the levels of G1
cyclins during progression through G1, showing that cyclin D2 and D3
were expressed before cyclin E, which was expressed before cyclin A. The expression of cyclin A was in particular distinct, appearing at 24 hours and exhibiting a substantial increase at 30 to 32 hours after stimulation.
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| Fig 5.
Effect of retinoic acid on the expression of various cell
cycle regulatory proteins in human B cells. (A) Mitogenic stimulation
and retinoic acid treatment of the cells were performed as described in
the legend to Fig 3B. Total cell lysates were prepared and equal
amounts of total protein (60 µg) were subjected to Western blot
analysis with antibodies against the proteins indicated on the right.
(B) Cells (1.5 × 106 cells/mL) were stimulated for 16 or
24 hours before addition of retinoic acid (1000 nmol/L). The cells were
then treated with retinoic acid for the indicated times and analyzed
for the expression of p21Cip1 by Western blot analysis.
µ, F(ab')2 fragment of rabbit antihuman IgM;
SAC, Staphylococcus aureus crude cell suspension; RA, retinoic
acid.
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The effect of retinoic acid on the level of the various cell cycle
proteins are shown in the right panel of Fig 5A. Retinoic acid
prevented the activation-induced increase in cyclin E and cyclin A
levels in a time-dependent manner that could explain the reduced kinase
activity of CDK2. The levels of cyclin D2, cyclin D3, CDK2, CDK4, and
CDK6 were not affected by retinoic acid. We did not detect cyclin D1 in
the B cells (data not shown).
We did not detect protein expression of the CKIs
p15/p16Ink4 in the isolated human B lymphocytes (data not
shown). The level of CKI p27Kip1 was high in unstimulated
cells, in accordance with previous reports on other cell
types.28,29 Upon stimulation with anti-µ and SAC, the
expression of p27Kip1 was gradually reduced. Treatment of
the cells with retinoic acid did not prevent the activation-induced
decline in the levels of p27Kip1.
More surprising were the changes noted in the protein levels of
p21Cip1. As reported in other cell types,28,30
the level of p21Cip1 was low in unstimulated cells.
Stimulation of the cells was accompanied by induction of
p21Cip1, and it was further enhanced by retinoic acid when
added 2 hours before harvesting the cells. However, the effect of
retinoic acid on the expression of p21Cip1 was only
transient. Retinoic acid present for more than 4 to 8 hours reduced the
level of p21Cip1 to that observed in control cells (Fig
5A).
The transient expression of p21Cip1 induced by retinoic
acid occurs in all parts of G1.
The transient increased expression of p21Cip1 that was
observed when retinoic acid was added to the stimulated cells 2 to 4 hours before the restriction point in G1 could represent a window in G1
in which retinoic acid could induce the expression of
p21Cip1. Alternatively, the noted effect could
represent a general ability of retinoic acid to transiently increase
the level of p21Cip1 independent of the position in the
cell cycle in which retinoic acid was added to the cultures. To
distinguish between these two possibilities, we added retinoic acid to
the cell cultures at various times in G1, and followed the expression
of p21Cip1 for the next 8 to 16 hours. Interestingly, we
showed that retinoic acid was able to transiently increase the level of
p21Cip1 independent of the position in G1 (Fig 5B). The
immunoblot presented in Fig 5B was also reprobed with cyclin E and
cyclin A, and reduced levels of both cyclins were noted when retinoic
acid was added as early as 16 hours into G1 (data not shown).
Retinoic acid reduces the kinase activity associated with cyclin E
and cyclin A.
Near the restriction point in G1, pRB is phosphorylated by CDK2 in
complex with cyclin E and later in complex with cyclin A.12
The inhibition of CDK2 kinase activity noted in the presence of
retinoic acid could therefore be due to reduced expression of cyclin E
and/or cyclin A. Therefore, we measured the kinase activity associated
with cyclin E and cyclin A. As shown in the right panel of
Fig 6, retinoic acid prevented activation
of the kinase coimmunoprecipitated with either cyclin E or cyclin A, as
compared with that of control cells stimulated for 32 hours in the
absence of retinoic acid.
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| Fig 6.
Retinoic acid decreases the kinase activity associated
with cyclin E and cyclin A. Mitogenic stimulation and retinoic acid
treatment of the B cells were performed as described in the legend to
Fig 3B. Whole cell extracts were prepared, immunoprecipitated with
antibodies against cyclin E (top) or cyclin A (bottom), and then the
associated kinase activity of the immunoprecipitates was determined
with histone H1 as substrate. IP, immunoprecipitating antibody; µ,
F(ab')2 fragment of rabbit antihuman IgM; SAC,
Staphylococcus aureus crude cell suspension; RA, retinoic
acid.
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The effect of retinoic acid on the binding of p21Cip1 to
CDK2.
To examine whether the transient increased expression of
p21Cip1 resulted in a transient increase in its binding to
CDK2, we performed coimmunoprecipitation studies. CDK2 was
immunoprecipitated from B cells stimulated in the presence or absence
of retinoic acid. As shown in Fig 7, the
expression of CDK2 was unchanged by retinoic acid in agreement with the
Western blot analysis in Fig 5A. The level of p21Cip1
coimmunoprecipitated with CDK2 did not, however, follow the expression of CDK2, but rather paralleled the expression of p21Cip1
itself. The CDK2 present in unstimulated cells contained small amounts
of p21Cip1. Entry into G1 was accompanied by a moderate
increase in the amount of p21Cip1 associated with CDK2, and
it remained constant throughout G1. Analysis of CDK2 immunoprecipitates
from retinoic acid-treated cells showed an increase in the amount of
p21Cip1 associated with the CDK2 complexes. At 2 hours
after retinoic acid treatment of the cells, the amount of
p21Cip1 that associated with CDK2 was at its peak level,
and it then declined gradually upon longer exposure of the cells to
retinoic acid. Thus, at 8 hours the level of p21Cip1
associated with CDK2 had decreased to control levels. The converse experiment was also performed by immunoprecipitation with
anti-p21Cip1 antibodies and then immunoblotting for CDK2.
Whereas p21Cip1 immunoprecipitated from stimulated cells
contained a constant amount of CDK2, there was a substantial increase
in the amount of CDK2 associated with p21Cip1 at 2 hours
after retinoic acid treatment (data not shown). Retinoic acid had no
effect on the level of p27Kip1 associated with CDK2 (data
not shown).
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| Fig 7.
Retinoic acid induces a transient increase in the
association of p21Cip1 with CDK2 complexes. Mitogenic
stimulation and retinoic acid treatment of the B cells were performed
as described in the legend to Fig 3B. Whole cell extracts were
immunoprecipitated with antibody against CDK2. The immunoprecipitates
were then examined for CDK2 (top) and p21Cip1 (bottom)
levels by Western blot analysis. IP, immunoprecipitating antibody; W,
Western blot antibody; µ, F(ab')2 fragment of
rabbit antihuman IgM; SAC, Staphylococcus aureus crude cell
suspension; RA, retinoic acid.
|
|
Effects of retinoic acid on mRNA levels of cyclin E, cyclin A, and
p21Cip1.
The changes noted in expression of the various cell cycle components
were also measured at the mRNA level. By Northern blot analysis, we
analyzed the steady state levels of cyclin E, cyclin A, and
p21Cip1 mRNAs isolated from cells treated with or without
retinoic acid. As shown in Fig 8, the mRNA
levels of cyclin E and A were low in unstimulated cells, and both
increased upon stimulation of the cells. Retinoic acid prevented the
increase in mRNA expression of both cyclin E and A, and the kinetics
was comparable to that of the protein levels.
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| Fig 8.
Effect of retinoic acid on cyclin E, cyclin A, and
p21Cip1 mRNA expression. Mitogenic stimulation and retinoic
acid treatment of the B cells were performed as described in the legend
to Fig 3B. mRNA from 25 × 106 cells was extracted and
analyzed by Northern blotting using 32P-labeled cyclin E,
cyclin A, and p21Cip1 probes as described in Materials and
Methods. As a loading control we have shown the residual rRNA levels
present in the different lanes. µ, F(ab')2
fragment of rabbit antihuman IgM; SAC, Staphylococcus aureus
crude cell suspension; RA, retinoic acid.
|
|
The changes in the expression of p21Cip1 mRNA did also
follow that of the protein expression. Thus, the level of
p21Cip1 mRNA was low in unstimulated cells, and its level
increased upon stimulation of the cells into G1. Furthermore, retinoic
acid induced a rapid (within 2 hours) increase in the level of
p21Cip1 mRNA, which declined to control levels within 8 hours of treatment.
The RAR agonist TTNPB mimics and the RAR-selective antagonist Ro
41-5253 counteracts the effects of retinoic acid on the cell cycle
machinery.
To exclude the possibility that the effects of retinoic acid on the
cell cycle machinery were independent of the nuclear retinoic acid
binding receptors, we analyzed the effect of the RAR-selective agonist
TTNPB on some of the key events affected by retinoic acid. We have
previously shown that an RAR agonist could inhibit proliferation of B
lymphocytes,17 and in the present study we show that TTNPB
grossly mimicked the effect of retinoic acid on phosphorylation of pRB,
expression of cyclin E, and p21Cip1
(Fig 9). Thus, TTNPB (100 nmol/L) inhibited
both the pRB phosphorylation and cyclin E expression, whereas it
induced a transient increase in the level of p21Cip1.
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| Fig 9.
The RAR-specific agonist TTNPB mimics the effects of
retinoic acid. Left panel: B cells (1.5 × 106/mL) were
stimulated with µ (37 µg/mL) and SAC (0.005%) and harvested at
the indicated times after stimulation. Right panel: Cells were
stimulated with µ (37 µg/mL) and SAC (0.005%). TTNPB (100 nmol/L) was then added to the cell cultures at each indicated time
before harvesting the cells at 32 hours. Total cell lysates were
prepared and subjected to Western blot analysis for pRB (top), cyclin E
(middle), and p21Cip1 (bottom). µ,
F(ab')2 fragment of rabbit antihuman IgM; SAC,
Staphylococcus aureus crude cell suspension.
|
|
To further confirm the involvement of nuclear receptors in the actions
of retinoic acid on the cell cycle machinery, we used the RAR
antagonist Ro 41-5253. As shown in Fig
10A, Ro 41-5253 (1 µmol/L) counteracted the inhibitory effect of
retinoic acid on DNA synthesis. Furthermore, Ro 41-5253 prevented both
the retinoic acid-mediated inhibition of pRB phosphorylation (Fig 10B)
and the transient induction of p21Cip1 (Fig 10C). Note that
we in the experiments presented in Fig 10 used retinoic acid at the
concentration of 10 nmol/L, because the concentration of dimethyl
sulfoxide (DMSO) in the solution of 1 µmol/L Ro 41-5253 was slightly
toxic to the cells in the presence of higher concentrations of retinoic
acid.
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| Fig 10.
The RAR-selective antagonist Ro 41-5253 blocks the
effects of retinoic acid. (A) The effect of Ro 41-5253 on DNA synthesis
was measured as described in Materials and Methods, using Ro 41-5253 at
a concentration of 1 µmol/L, and retinoic acid at the concentration
of 10 nmol/L. (B) B cells (1.5 × 106/mL) were stimulated
with µ (37 µg/mL) and SAC (0.005%) for 32 hours in the absence
or presence of retinoic acid (10 nmol/L). For Ro 41-5253 treatment of
the cells, Ro 41-5253 (0.5 or 1 µmol/L) was added to the cell
cultures alone or together with retinoic acid and the cells were
harvested at 32 hours. Total cell lysates were prepared and subjected
to Western blot analysis for pRB as described in Material and Methods.
(C) Ro 41-5253 (1 µmol/L) was added to the cell cultures alone or
together with retinoic acid (10 nmol/L) at the indicated times before
harvesting the cells at 32 hours. Total cell lysates were prepared and
subjected to Western blot analysis for p21Cip1. µ,
F(ab')2 fragment of rabbit antihuman IgM; SAC,
Staphylococcus aureus crude cell suspension.
|
|
| |
DISCUSSION |
The present study is to our knowledge the first attempt to elucidate
the effect of retinoic acid on the cell cycle machinery in normal
cells, and in lymphocytes particularly. Our previous published data on
the growth-inhibiting effect of retinoids on human
B-lymphocytes8,10 are supported by several reports showing the inhibitory effect of retinoids on lymphoid
growth.5,31,32 However, most reports regard retinoids
as important activators of lymphoid proliferation.1-3
Therefore, we found it important to examine the mechanisms whereby
retinoic acid exerts its growth-inhibiting effects on normal B cells.
In our previous report, we showed that retinoids, including retinoic
acid, blocked the cell cycle progression of human B lymphocytes in mid
to late G1.8 We defined the point of inhibition to be somewhere between 24 hours into G1 and the restriction point, the
restriction point being located 30 to 35 hours into G1 of human B
lymphocytes.26 In the present study, we wished to define the part of the cell cycle machinery that was affected by retinoic acid. Therefore, we added retinoic acid to the B-lymphocyte cultures at
different times during G1, and all the cells were harvested at 32 hours. Because phosphorylation of pRB is one of the critical events in
the regulation of G1/S transition,12 we first studied the
effect of retinoic acid on this component of the cell cycle machinery.
Indeed we observed that concentrations of retinoic acid as low as 1 to
10 nmol/L, ie, physiological levels, were able to prevent pRB
phosphorylation. Effects were noted even when retinoic acid was added
as late as 2 hours before the restriction point in G1.
The key regulators of pRB phosphorylation in early to mid G1 are the
cyclin-dependent kinases CDK4 and CDK6, whereas CDK2 has a role in late
G1 and at the G1/S transition.12 These kinases are
activated through their binding to specific cyclins and are inhibited
by specific CKIs.11 We examined the effects of retinoic acid on the various G1 kinases, and we found that the activity of CDK2
was reduced. We did not detect CDK4 activity in the isolated B cells,
and CDK6 activity was not affected by retinoic acid. The inhibition of
CDK2 activity could be due to reduced levels of cyclin E or cyclin A,
or it could be due to the increase in the level of one of the CKIs. We
observed that retinoic acid prevented expression of both cyclin E and
cyclin A, and we also noted a transient increase in the expression of
the CKI p21Cip1 2 to 4 hours after addition of retinoic
acid. The increased expression of p21Cip1 was associated
with increased binding to CDK2. No effects were observed on the levels
of cyclin D2 and cyclin D3, or on the level of p27Kip1.
Importantly, both the transient induction of p21Cip1 and
the reduced expression of cyclin E and A could also be detected when
retinoic acid was added to the cells early in G1. Thus it appears that
these events could be the cause rather than the result of the cell
cycle arrest mediated by retinoic acid in mid to late G1.
Effects on the cell cycle machinery have also been noted in other cell
systems that are growth-inhibited by retinoids. Zhou et
al33 have reported an inhibitory effect of retinoids on the expression of D-type cyclins and CDKs, whereas, in agreement with our
present results on B lymphocytes, retinoic acid has been shown to
reduce the level of cyclin E in HL-60 cells34 and in
bronchial epithelial cell lines.35 Effects of retinoic acid
on CKIs have also been reported. Increased levels of
p27Kip1 upon treatment with retinoic acid have been shown
in astrocytoma cells36 as well as in neuroblastoma
cells.37 Increased levels of p27Kip1 are,
however, commonly observed in growth-inhibited cells.28,38 This is usually a late event in growth inhibition that is observed several hours after the cells have received the growth- inhibitory signal. It is therefore possible that the increase in the levels of
p27Kip1 is the result rather than the cause of growth
inhibition. We did also note a small increase in p27kip1
levels upon treatment of B cells with retinoic acid for 32 hours, but
we regard this to be a secondary effect.
Retinoic acid has, in agreement with our present results, been reported
to increase the level of p21Cip1. For instance, Liu et al
39 showed that differentiation of U937 cells with retinoic
acid was associated with induced expresssion of p21Cip1.
However, in MCF-7 cells treated with retinoic acid the level of
p21Cip1 was reduced.40 Thus, it appears that
the effect of retinoic acid on p21Cip1 depends on the cell
system. It is, however, puzzling that the increase in the expression of
p21Cip1 and in its binding to CDK2 that we observed was
only transient, whereas the activity of CDK2 was permanently reduced. A
similar transient induction of p21Cip1 was observed at the
mRNA level in HL60R cells that were growth inhibited by a novel
synthetic retinoid, CD437,16 and by TGF- in epithelial
cells.41 It is, however, important to note that the
relation between the level of p21Cip1 and its role as a CKI
is not a simple one. In fact, it has recently been reported that
p21Cip1 at low concentrations can function as an assembly
factor between cyclins and CDKs, and thereby is required for activation
of CDKs.42,43,44 This could explain the moderate increase
in the expression of p21Cip1 that was noted upon
stimulation of the B cells into G1. At higher concentrations, however,
p21Cip1 acts as a CKI.11 Therefore, it appears
that the ability of p21Cip1 to act as an inhibitor or
activator of CDKs depends on the relative levels of cyclins, CDKs, and
of p21Cip1 itself. It is reasonable to assume that the
early retinoic acid-mediated increase in the expression of
p21Cip1 could lead to the rapid inhibition of CDK2 activity
that we observed. Upon longer (more than 4 hours) exposure to retinoic
acid, the reduction in CDK2 activity could be due to the substantial
decrease in cyclin E and cyclin A levels.
It has been an issue of debate whether or not nuclear receptors mediate
all the effects of retinoids in vivo. In target cells, vitamin A is
metabolized into retinoic acid, and retinoic acid mediates its effect
via nuclear RARs.45,46 However, vitamin A can also be
metabolized into retro-retinoids such as anhydroretinol (AR) and
14-hydroxy-4, 14 retro-retinol (14-HRR), which do not function as
regulators of transcription.15 AR has been shown to act as
a growth inhibitor for lymphocytes,47 whereas 14-HRR appears to be required for normal lymphocyte
proliferation.15 Although retinoic acid does not appear to
be metabolized into retro-retinoids in vivo, it was still important to
examine whether or not nuclear retinoid-binding receptors mediate the
effects of retinoic acid on the cell cycle machinery. We have
previously shown that normal B cells express the nuclear receptors
RAR, RAR, and retinoid X receptor (RXR), and that the
RAR agonist TTNBP could mimic the effect of retinoic acid on growth and
apoptosis in normal human B lymphocytes.17 In the present
study, TTNBP essentially functioned as retinoic acid in regulating the
components of the cell cycle machinery. It prevented pRB
phosphorylation and cyclin E expression, and transiently induced
p21Cip1 expression.
To do the opposite experiments, ie, to block the effects of retinoic
acid, we used the RAR-selective antagonist Ro 41-5253. Ro 41-5253 prevented the inhibition of both DNA synthesis and of pRB
phosphorylation, and it prevented the transient increase in the
expression of p21Cip1 induced by retinoic acid. It has been
shown that the gene coding for p21Cip1 possesses a
RAR/RXR-responsive element in its promoter,39 and in the
present study we showed that retinoic acid altered the expressions of
cyclin E, cyclin A, and p21Cip1 at the mRNA levels. Thus,
the present findings support the notion that retinoic acid mediates its
effects on the cell cycle machinery at the transcriptional level via
nuclear RARs.
Taken together, the present study firmly establishes the inhibitory
role of retinoic acid on the cell cycle progression of normal human B
lymphocytes. The key event of pRB phosphorylation near the restriction
point in G1 is prevented by retinoic acid, and it appears that this
effect is primarily due to downregulation of cyclin E, cyclin A, and
induced expression of p21Cip1. We are currently in the
process of analyzing the effects of the various retro-retinoids on the
cell cycle machinery in normal B lymphocytes, to further unravel the
role of retinoids in lymphoid proliferation.
| |
ACKNOWLEDGMENT |
The authors thank Hilde R. Haug for excellent technical assistance.
| |
FOOTNOTES |
Submitted October 8, 1998; accepted April 13, 1999.
Supported by The Norwegian Cancer Society, The Norwegian Research
Council, Freia Research Foundation, and Jahre Research Foundation.
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 correspondence to Heidi Kiil Blomhoff, PhD, Department of
Medical Biochemistry, Institute Group of Basic Medical Sciences,
Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern,
N-0317, Oslo, Norway; email: h.k.blomhoff{at}basalmed.uio.no.
| |
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Development,
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[Abstract]
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S. Vuocolo, E. Purev, D. Zhang, J. Bartek, K. Hansen, D. R. Soprano, and K. J. Soprano
Protein Phosphatase 2A Associates with Rb2/p130 and Mediates Retinoic Acid-induced Growth Suppression of Ovarian Carcinoma Cells
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[Abstract]
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A. Ertesvag, N. Engedal, S. Naderi, and H. K. Blomhoff
Retinoic Acid Stimulates the Cell Cycle Machinery in Normal T Cells: Involvement of Retinoic Acid Receptor-Mediated IL-2 Secretion
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[Abstract]
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J. M. Miano and B. C. Berk
Retinoids: New Insight Into Smooth Muscle Cell Growth Inhibition
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TOP
ABSTRACT
INTRODUCTION