Activin A Induction of Cell-Cycle Arrest Involves Modulation of Cyclin D2 and p21CIP1/WAF1 in Plasmacytic Cells
Kenji Yamato,
Takeyoshi Koseki,
Masahiro Ohguchi,
Masahiro Kizaki,
Yasuo Ikeda and
Tatsuji Nishihara
Department of Oral Science (K.Y., T.K., M.O., T.N.) The
National Institute of Infectious Diseases Tokyo 162, Japan,
Department of Molecular Cellular Oncology/Microbiology (K.Y.)
Faculty of Dentistry Tokyo Medical and Dental University Tokyo
113, Japan
Division of Hematology (M.K., Y.I.) Keio
University Medical School Tokyo 160, Japan
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ABSTRACT
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Activins, members of the transforming growth
factor-ß family, have been implicated in the regulation of growth and
differentiation of various types of cells. We have recently found that
activin A induces apoptotic cell death of plasmacytic cells including B
cell hybridoma cells and myeloma cells. In the present study, we
demonstrated that activin A caused cell-cycle arrest in the G1 phase
before appearance of apoptotic cells in mouse B cell hybridoma cells.
Phosphorylation of retinoblastoma protein (Rb) and in vitro
Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in
activin A-treated cells. Analysis of expression of genes regulating Rb
phosphorylation revealed that activin A suppressed cyclin
D2, the sole D-type cyclin gene expressed in the hybridoma cells,
and activated p21CIP1/WAF1 but had no
effect on expression of cyclin-dependent kinases (CDK2,
CDK4, CDK6) and other CDK inhibitors
(p27KIP1,
p16INK4a,
p15INK4b). Modulation of cyclin D2 and
p21CIP1/WAF1 expression resulted in a
decrease in level of cyclin D2-CDK4 complex and an increase in level of
CDK4 complexed with p21CIP1/WAF1. Moreover,
overexpression of cyclin D2 partially abrogated inhibition of Rb
phosphorylation and G1 arrest in the hybridoma cells.
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INTRODUCTION
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Activins were originally isolated as factors from ovarian fluid
that stimulate the secretion of FSH from pituitary cells (1, 2). These
factors are members of the transforming growth factor-ß (TGFß)
family and are encoded by two closely related genes,
activin-ßA and activin-ßB. They exist as
homodimers (ßAßA, ßBßB) or a heterodimer (ßAßB) of the gene
products and have been designated as activin A, activin B, and activin
AB, respectively (3). Recently, two new activin ß-chains
(activin-ßC, activin-ßD) have been reported, and the biological
activities of these remain to be determined (4, 5). In addition to
regulation of reproductive endocrine system, activins are implicated in
regulation of erythroid differentiation (6), mesoderm induction of
embryo (7, 8, 9, 10), and negative cell growth of various cell types
including gonadal cells and adrenal cells (11, 12, 13, 14). Recently, activin A
has been independently isolated from cultured media of activated mouse
macrophages (15, 16) and mouse bone marrow stromal cells (17) as a
factor inhibiting the growth of plasmacytic cells including mouse B
cell hybridoma cells and mouse and human myeloma cells. Although the
plasmacytic cell growth-inhibitory activity of activin A has been shown
to be mediated, in part, by inducing apoptotic cell death (15, 16, 18),
the precise mechanism by which activin A exerts its negative growth
effect has not been elucidated.
Retinoblastoma protein (Rb) controls the cell-cycle progression at the
G1 to S transition in response to extracellular signals for growth
inhibition (review in Ref. 19). Hypophosphorylated Rb (pRb) binds to
the E2F transcription factor and cancels its ability to activate genes
required for entry into S phase. Phosphorylation of Rb abrogates its
binding to E2F, allowing E2F to activate the genes (20). Rb is
phosphorylated by catalytic subunits of cyclin-dependent kinase
(CDK)4/CDK6 and CDK2 complexed with specific regulatory subunits of
cyclins D and E, respectively. Mouse cyclin D1 has been cloned as a
delayed early response gene induced by growth stimulation, and cyclins
D2 and D3 have been isolated as cyclin D1-related genes (21). The three
D-type cyclins are differentially expressed in various cell types (21, 22). In response to mitogenic stimuli, cyclin D accumulates and
complexes with CDK4 and CDK6 to form catalytically active kinases in
the mid-G1 phase (21, 23) and phosphorylate Rb, the sole characterized
physiological substrate (22). With cell-cycle progression toward the G1
to S transition, cyclin E-CDK2 kinase activity increases and also
participates in the regulation of Rb phosphorylation.
Recently inhibitors of CDKs have been identified (review in Ref. 24).
p21CIP1/WAF1 (25, 26, 27, 28, 29) and p27KIP1 (30, 31, 32)
contain highly homologous regions in the N-terminal portion responsible
for CDK-inhibitory activity (30, 33, 34, 35). p21CIP1/WAF1 and
p27KIP1 cancel the kinase activities of cyclins D-CDK4 and
that of cyclin E-CDK2 by direct binding to these catalytically active
kinase complexes. Expression of p21CIP1/WAF1 is
induced by physiological agents including TGFß (36, 37).
p16INK4a (38) and p15INK4b (39) contain 4-fold
repeated ankyrin motifs and bind to CDK4 and CDK6 but not to cyclins or
other CDKs. These complexes possess no kinase activity.
p15INK4b has been cloned as a
TGFß-responsive gene, and accumulation of the gene product
causes direct inactivation of CDK4/CDK6 and then redistributes
p27KIP1 from CDK4/CDK6 to CDK2, leading to the inhibition
of CDK2 kinase (37, 40).
The present study was undertaken to determine the precise
mechanism by which activin A exerts its growth-inhibitory activity in
plasmacytic cells. Our data showed that activin A inhibits the growth
of plasmacytic cells by causing G1 arrest, which is followed by
apoptotic cell death. We also presented evidence that the G1 arrest is
induced by suppression of CDK4-mediated Rb phosphorylation through
combined modulation of cyclin D2 and
p21CIP1/WAF1 in hybridoma cells.
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RESULTS
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Activin A-Induced Growth Inhibition Is Caused by G1 Arrest and
Subsequent Apoptosis
The effect of activin A on the growth of plasmacytic cells was
studied using HS-72 mouse B cell hybridoma cells highly sensitive to
activin A-induced apoptosis (16, 18). The cells were cultured with
various concentrations of activin A, and cell growth was monitored by
MTT (3-[4,5-dimethylthiazol 2-yl]-2,5-diphenyltetrazolium bromide)
assay (Fig. 1
). In the absence of activin A, HS-72 cells
showed continuous growth with a doubling time of 16 h. Cultivation
with activin A suppressed the cell growth in a dose-dependent manner.
The growth inhibition was observed at as low as 6.3 ng/ml, and the
maximal effect was seen at 50 ng/ml of activin A. When the cells were
exposed to more than 12.5 ng activin A per ml, gradual viability loss
was observed after 20 h.

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Figure 1. Activin A Suppresses the Growth of HS-72 Hybridoma
Cells
The cells were cultured with various concentrations of activin A, and
cell viability was monitored by MTT assay at the times indicated.
Closed square, 0 ng/ml of activin A; closed
circle, 6.3 ng/ml; closed triangle, 12.5 ng/ml;
open square, 25 ng/ml; open circle, 50
ng/ml; open triangle, 100 ng/ml.
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The effect of activin A on the cell-cycle progression was investigated
in HS-72 cells. The cells were incubated with activin A (50 ng/ml) for
12, 26, and 36 h and analyzed for the cell-cycle distribution by
flow cytometry (Fig. 2
). Cultivation with activin A for
12 h increased the population of the cells in the G1 phase from
43% to 79% with reduction of those in the S phase from 46% to 6%.
At 26 h after treatment, the cell-cycle distribution of HS-72
cells was essentially the same as that seen at 12 h after
treatment, except that the proportion of the cells with hypodiploid DNA
representing apoptotic cells increased from 11% to 37%. At 36 h,
the proportion of apoptotic cells reached 75%. This was consistent
with the observation that loss of cell viability occurred after
treatment with activin A for 20 h (Fig. 1
).

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Figure 2. Cell Cycle Analysis of HS-72 Cells Cultured with
Activin A
HS-72 cells were cultured in the absence or presence of 50 ng/ml of
activin A for 12 h, 26 h, and 36 h and then stained with
propidium iodide. DNA content was analyzed by flow cytometry.
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The increased population of the G1 phase in activin A-treated cells
might represent G1-arrested cells or those that were not G1-arrested
but committed to apoptosis and subsequently detected as hypodiploid
cells. To explore these possibilities, we examined the effect of
activin A on cell cycle of BCL-2-expressing HS-72 cells resistant to
activin A-induced apoptosis (HS-72B-5, HS-72B-6, HS-72B-12) (18). As
shown in Table 1
, activin A caused accumulation of the
G1 cells with only a slight increase in the apoptotic population in all
three BCL-2-expressing clones. Furthermore, 5-bromo-2'deoxy-uridine
(BrdU) incorporation assay revealed that exposure to 50 ng/ml of
activin A for 12 h decreased the rate of DNA synthesis in HS-72
cells by 5-fold. These results demonstrated that the accumulation of G1
cells in activin A-treated HS-72 cells was caused by G1 arrest but not
by apoptosis of cells that had already committed to cell division.
Induction of G1 arrest was also observed in other B cell hybridoma
(HS-2, HS-4, HS-5, HS-6, HS-9) and SP2/0 myeloma cells sensitive to
activin A-induced apoptosis (data not shown), showing that activin A
exerted plasmacytic cell growth inhibition by inducing both G1 arrest
and apoptosis.
Activin A Induces Rb Hypophosphorylation and Suppression of
Cyclin D2
To investigate the mechanism by which activin A caused G1 arrest
in hybridoma cells, the phosphorylation state of Rb was studied by
immunoblot analysis. Exponentially growing HS-72 cells contained both
hyperphosphorylated (ppRb) and hypophosphorylated forms of Rb (pRb)
(Fig. 3
). Activin A (50 ng/ml) decreased levels of ppRb
and increased those of pRb in the cells at 6 h after treatment.
The cells cultured with activin A for 12 h and 24 h contained
mostly pRb. Thus, activin A caused hypophosphorylation of Rb in HS-72
cells. To determine the basis for Rb hypophosphorylation, we examined
the effect of activin A on the expression of D-type cyclins and CDKs in
HS-72 cells. As shown in Fig. 3
, levels of CDK4 and CDK6 were not
altered by activin A (50 ng/ml). Subtypes of D-type cyclins expressed
in HS-72 cells were determined by immunoblot analysis using monoclonal
antibodies specific for cyclin D1, cyclin D2, and cyclin D3. HS-72
cells did not express either cyclin D1 or cyclin D3 (data not shown)
but contained cyclin D2. Activin A decreased the levels of cyclin D2 by
3-fold and 25-fold at 6 h and 12 h after exposure,
respectively. Expression of cyclin D2 was barely detectable in the
cells cultured with activin A for 24 h (Fig. 3
).

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Figure 3. Expression of Rb, Cyclin D2, CDK4, and CDK6 in
HS-72 Cells after Treatment with Activin A
HS-72 cells were cultured with activin A (50 ng/ml) for the times
indicated and analyzed for Rb, cyclin D2, CDK4, and CDK6 by
immunoblotting. pRb, Hypophosphorylated Rb; ppRb, hyperphosphorylated
Rb.
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Activin A Induces Expression of
p21CIP1/WAF1
Immunoblot analysis showed that untreated HS-72 cells contained
undetectable levels of p21CIP1/WAF1 (Fig. 4
). Upon exposure to activin A (50 ng/ml), expression of
p21CIP1/WAF1 was detected as early as 6 h, and its
level increased with time. HS-72 cells constitutively expressed
p27KIP1, and its level was not altered by activin A for
24 h (Fig. 4
). Immunoblot analysis of untreated cells with
anti-CDK2 antibody detected doublet bands with fast and slow
mobilities, representing active and inactive forms of CDK2,
respectively (41) (Fig. 4
). Exposure to activin A had no effect on
expression of CDK2, but increased levels of cyclin E, a regulatory
subunit of CDK2 (Fig. 4
).

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Figure 4. The Effect of Activin A on Expression of
p21CIP1/WAF1, p27KIP1, CDK2, and Cyclin E
HS-72 cells were cultured with activin A (50 ng/ml) for the indicated
times and examined for levels of p27KIP1 and
p21CIP1/WAF1, CDK2, and cyclin E by immunoblotting.
p21CIP1/WAF1 was detected by anti-mouse
p21CIP1/WAF1 monoclonal antibody (Ab-4).
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Effect of Activin A on Expression of Cyclin D2 mRNA and
p21CIP1/WAF1 mRNA
We found that activin A modulated expression of cyclin D2 and
p21CIP1/WAF1 in HS-72 cells. To determine whether
alteration of their expression occurred at the level of mRNA, Northern
blot analysis was performed (Fig. 5
). Proliferating
HS-72 cells expressed high levels of cyclin D2 mRNA. Activin
A decreased its levels as early as 6 h after treatment. The
minimal level (24%) of the transcript was detected at 12 h after
treatment. Untreated HS-72 cells expressed faint levels of
p21CIP1/WAF1 mRNA (Fig. 5
). Temporal
accumulation of p21CIP1/WAF1 mRNA was seen at
3 h after treatment. After treatment for 6 h, its levels
gradually decreased with time and returned to baseline at 12
h.

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Figure 5. Activin A Modulates Levels of Cyclin
D2 mRNA and p21CIP1/WAF1 mRNA
HS-72 cells exposed to activin A (50 ng/ml) for various times were
analyzed for levels of cyclin D2 mRNA and
p21CIP1/WAF1 mRNA by Northern blotting. The
blot was serially probed with
p21CIP1/WAF1 cyclin D2 and
GAPDH cDNA fragments.
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Activin A Does Not Induce Either
p16INK4a or
p15INK4b Expression
The effect of activin A on levels of p15INK4b and
p16INK4a was investigated in HS-72 cells by immunoblot
analysis. HS-72 cells expressed P16INK4a, and activin A did
not increase its level (Fig. 6A
). Expression of
p15INK4b was not detected in HS-72 cells cultured with or
without activin A (data not shown). To confirm the lack of
p15INK4b induction by activin A, we undertook
semiquantitative RT-PCR analysis. As shown in Fig. 6B
, RT-PCR
amplification of RNA samples from the untreated cells gave rise to
bands of p16INK4a and
p15INK4b cDNA fragments with expected sizes
whereas no band was detected in negative controls. Specificity of the
PCR was confirmed by the sequencing of PCR products. Serial dilution of
cDNA samples of the untreated cells caused a proportional decrease in
intensities of the bands, showing that the PCR condition employed in
the experiment was adequate for semiquantification of
p16INK4a and p15INK4b
mRNA. Levels of p16INK4a and
p15INK4b fragments were similar between
untreated and activin A-treated cells when compared at the same-fold
dilution, showing that activin A did not enhance the expression of
either p16INK4a mRNA or
p15INK4b mRNA in HS-72 cells.

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Figure 6. Activin A Does Not Alter Expression of
p16INK4a and p15INK4b
A, Effect of activin A on p16INK4a expression. HS-72 cells
were exposed to activin A (50 ng/ml) for indicated times and examined
for expression of p16INK4a by immunoblotting. B,
Semiquantitative RT-PCR analysis of p15INK4b
mRNA and p16INK4a mRNA levels in HS-72 cells
cultured with activin A. cDNA samples were obtained by RT reaction of
total RNA from untreated cells (0 h) and the cells treated with activin
A (50 ng/ml) for either 4 h or 8 h. Undiluted (1:1) and
diluted cDNA samples (1:10, 1:100) were subjected to PCR amplification
using specific primers for p16INK4a and
p15INK4b. Total RNA without RT reaction
[RT(-)] was used as negative control.
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Effect of Activin A on Levels of Cyclin D2-CDK4 Complex and Those
of p21CIP1/WAF1-Bound CDK4 and CDK2
We showed that activin A decreased levels of cyclin D2 and
increased those of p21CIP1/WAF1. Because cyclin D2
positively regulates CDK4 kinase activity by forming a binary complex
with CDK4 and p21CIP1/WAF1 negatively regulates kinase
activities of both CDK4 and CDK2 by binding with cyclin D-CDK4 and
cyclin E-CDK2, respectively, we examined the effect of activin A on the
levels of cyclin D2-CDK4 complex and those of
p21CIP1/WAF1-bound CDK4 and CDK2 by immunoprecipitation.
Anti-CDK4 immunoprecipitates from untreated HS-72 cells contained
cyclin D2, the levels of which decreased to 50% and 8% at 6 h
and 12 h, respectively, after the treatment (Fig. 7A
). The level of p21CIP1/WAF1 was faint in
anti-p21CIP1/WAF1 immunoprecipitate from the cells cultured
without activin A and increased by exposure to activin A for 6 h
and 12 h (Fig. 7B
). Anti-p21CIP1/WAF1
immunoprecipitates from activin A-treated cells contained CDK4, cyclin
D2, and CDK2 whereas those from untreated cells contained none of
these, showing that activin A increased levels of CDK4 and CDK2
complexed with p21CIP1/WAF1.

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Figure 7. Activin A Decreases Level of Cyclin D2-CDK4 Complex
and Increases Levels of p21CIP1/WAF1-Bound CDK4 and CDK2
Anti-CDK4 (A) and anti-p21CIP1/WAF1 immunoprecipitates (B)
from untreated HS-72 cells (0 h) and cells cultured with activin A (50
ng/ml) for 6 h and 12 h were analyzed by immunoblotting using
anti-CDK4, anti-CDK2 and anti-p21CIP1/WAF1 rabbit IgG, and
anti-cyclin D2 monoclonal antibody. IgH indicates bands of Ig heavy
chains. Exposed films were scanned by a densitometer, and intensity of
each band was normalized to that of IgH.
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To determine whether these effects of activin A contributed to
inhibition of Rb phosphorylation by CDK4 and CDK2, we assayed in
vitro Rb kinase activities of anti-CDK4 and anti-CDK2
immunoprecipitates from activin A-treated and untreated HS-72 cells. As
demonstrated in Fig. 8
, anti-CDK4 immunoprecipitate from
proliferating HS-72 cells caused phosphorylation of Rb at Ser780. The
CDK4-associated Rb kinase activity significantly decreased at 6 h
after treatment and was undetectable at 12 h, showing that activin
A inhibited Rb kinase activity of CDK4. Rb kinase assay of CDK2 using
an antibody reactive to Rb phosphorylated by CDK2 revealed that activin
A had no effect on CDK2-associated Rb kinase activity in HS-72 cells
(data not shown).

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Figure 8. Activin A Inhibits Rb Kinase Activity of CDK4 in
HS-72 Cells
GST-Rb was incubated with anti-CDK4 immunoprecipitates from HS-72 cells
exposed to activin A for 0 h, 6 h, and 12 h in the presence of cold ATP
and immunoblotted. Control reaction contained immunoprecipitate with
normal rabbit IgG instead of anti-CDK4 immunoprecipitate.
Phosphorylated Rb was detected by anti-phospho-Rb (ser780) antibody.
The blot was reprobed with anti-GST antibody.
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Effect of Cyclin D2 Overexpression on Activin A-Induced G1 Arrest
in Hybridoma Cells
To determine whether down-modulation of cyclin D2 was involved in
activin A-induced G1 arrest, HS-72 cells expressing high levels of
exogenous mouse cyclin D2 (HD37.1, HD37.5, HD37.8) were
established by introducing cyclin D2-expression plasmid
driven by the SR
promoter (pMKcyl-2). HS-72 cells
transfected with the plasmid without cyclin D2 cDNA (HMK45)
were used as control cells. Cyclin D2-transfected cells (HD37.8,
HD37.5) and HMK45 control cells were treated with either 5 ng/ml or 50
ng/ml of activin A for 12 h and then analyzed for Rb
phosphorylation. As shown in Fig. 9A
, HD37.8 and HD37.5
cells expressing high levels of cyclin D2 were less sensitive to
activin A-mediated cyclin D2 suppression as compared with control HMK45
cells. Activin A-induced hypophosphorylation of Rb was partially
blocked in HD37.8 cells and HD37.5 cells. Similar results were also
obtained in HD37.1 cells (data not shown). The proportion of cells in
the S1 phase was higher in cyclin D2-transfected cells (HD37.8, HD37.1,
HD37.5) (5763%) than that in HMK45 cells (4048%), showing that
overexpression of cyclin D2 increased the population of the S phase
(Fig. 9
, B and C). Accumulation of G1 phase cells and reduction of S
phase cells occurred in cyclin D2-transfected cells, but to a lesser
extent than in HMK45 cells, suggesting that expression of exogenous
cyclin D2 partially blocked activin A-induced G1 arrest of
HS-72 cells.

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Figure 9. Overexpression of Cyclin D2 Partially Blocks
Activin A-Induced Rb Hypophosphorylation and G1 Arrest
A, Rb phosphorylation in cyclin D2-transfected HS-72 cells.
cyclin D2-transfected cells (HD37.8, HD37.5) and control HMK45 cells
were treated with activin A (5 ng/ml, 50 ng/ml) for 12 h and
examined for cyclin D2 and Rb by immunoblotting. Effect of activin A on
cell cycle of cyclin D2-transfected HS-72 cells in Exp 1 (B) and Exp 2
(C). Cyclin D2-transfected cells [HD37.8 (B), HD37.1, and HD37.5 (C)]
and HMK45 control cells were cultured with activin A (5 ng/ml, 50
ng/ml) for 12 h and analyzed for cell-cycle distribution. The
results (B) are given as the mean ± SD from
triplicate experiments. Closed bar, Percent cells in the
S phase; open bar, the G2/M phase; hatched
bar, the G1 phase.
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DISCUSSION
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In the present study, we demonstrated that activin A can inhibit
the plasmacytic cell growth by arresting the cell cycle in the G1 phase
and subsequently inducing apoptosis. Because activin A inhibited Rb
phosphorylation and Rb kinase activity of CDK4 in hybridoma cells, it
is most likely that activin A causes G1 arrest by blocking
CDK4-mediated Rb phosphorylation, which is indispensable for the
cell-cycle progression at the G1 to S transition (19). The Rb kinase
activity of CDK4 is positively regulated by D-type cyclins and
negatively regulated by CDK inhibitors including p16INK4a,
p15INK4b, p21CIP1/WAF1, and p27KIP1
(19, 24). We showed that HS-72 cells expressed cyclin D2 as the sole
regulatory subunit of CDK4 and that activin A suppressed cyclin
D2 in the hybridoma cells. In addition, activin A activated
p21CIP1/WAF1 but had no effect on expression of
CDKs (CDK4, CDK6, CDK2) and other CDK inhibitors in HS-72 cells.
Modulation of expression of these genes resulted in decreased levels of
cyclin D2-CDK4 complex and increased levels of
p21CIP1/WAF1-bound CDK4, suggesting that activin A blocks
Rb kinase activity of CDK4 by altering expression of cyclin
D2 and p21CIP1/WAF1. Restoration of
decreased cyclin D2 level by expression of exogenous cyclin
D2 partially blocked activin A-induced Rb hypophosphorylation and
G1 arrest in the hybridoma cells. Thus, these results suggest that
cyclin D2 suppression alone is not sufficient but requires
concomitant activation of p21CIP1/WAF1 for
activin A-induced G1 arrest. We found that activin A also increased
levels of p21CIP1/WAF1-bound CDK2. However, in
vitro Rb kinase activity of CDK2 was not inhibited in the cells
cultured with activin A (data not shown). Activin A enhanced
expression of cyclin E, which might counteract the inhibitory
effect of accumulated p21CIP1/WAF1 on CDK2 kinase activity.
Since G1 arrest preceded apoptosis in activin A-treated hybridoma
cells, alteration of cyclin D2 and
p21CIP1/WAF1 expression might be involved in
activin A-induced apoptosis.
TGFß, a prototype of the TGFß family, causes G1 arrest by
inhibiting Rb phosphorylation (42). Rb appears to be a common target of
TGFß and activin A for the growth inhibition. TGFß exerts this
effect by activating p15INK4b (37, 39) and, in
some cell types, p21CIP1/WAF1 in addition to
p15INK4b (36). We found that activin A activated
p21CIP1/WAF1, but failed to induce the
expression of p15INK4b in the hybridoma cells.
Whether the lack of p15INK4b induction is caused
by the difference of receptor structures or cell types remains to be
clarified. Activin A caused the transient accumulation of
p21CIP1/WAF1 mRNA between 36 h after treatment
whereas a gradual and persistent increase in level of
p21CIP1/WAF1 protein was seen after 6 h. These
observations suggest that activin A-induced
p21CIP1/WAF1 expression may involve both
transcriptional activation of the gene and stabilization of the gene
product. Activin A is produced by activated monocyte/macrophage-like
cells (6, 15) and bone marrow stromal cells (17) and inhibits the
growth of various plasmacytic cell lines at physiological
concentrations. This raises the possibility that activin A is involved
in the regulation of both endocrine and immune systems.
In summary, we demonstrated that activin A exerted the plasmacytic cell
growth-inhibitory activity by inducing G1 arrest and subsequent
apoptosis. Molecular analysis of cell cycle-regulating genes showed
that modulation of cyclin D2 and
p21CIP1/WAF1 might be responsible for activin
A-induced G1 arrest in B cell hybridoma cells.
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MATERIALS AND METHODS
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Cell Culture
Mouse B cell hybridoma cells (HS-2, HS-4, HS-5, HS-6, HS-72,
HS-9) (16, 18) and SP2/0 mouse myeloma cells were cultured in Iscoves
modified Dulbeccos medium (IMDM, GIBCO-BRL, Gaithersburg, MD)
supplemented with 10% FBS, 100 µg/ml of streptomycin, and 100 U/ml
of penicillin. Human BCL-2-expressing HS-72 cells (HS-72B-5, HS-72B-6,
HS-72B-12) (18) were cultured in IMDM containing 10% FBS and 450
µg/ml of G418.
MTT Assay and BrdU Incorporation Assay
Growth property of HS-72 cells was examined by MTT assay as
described previously (43). Briefly, cells were suspended in IMDM
containing 5% FBS and antibiotics at a density of 2 x
105 cells/ml and then dispensed in 96-well plates with or
without activin A. After various incubation times at 37 C, cell
viability was determined by colorimetric assay with MTT (Sigma
Chemical Co., St. Louis, MO). BrdU incorporation was measured by a
5-bromo-2'deoxy-uridine labeling and detection kit (Boehringer
Mannheim, Mannheim, Germany) according to the manufacturers
instruction. Recombinant human activin A (44) was a kind gift from Y.
Eto (Ajinomoto Co., Kawasaki, Japan).
Plasmid and Transfection
pcN9-cyl2 containing the full-length coding region of the mouse
cyclin D2 cDNA (21) was kindly provided by H. Matsushime
(Nippon Roche Research Center). The mouse cyclin D2 cDNA was
excised from pcN9-cyl2 by EcoRI digestion and subcloned into
the EcoRI site of pMKITneo carrying the SR
promoter and the neo gene (a generous gift from K. Maruyama,
Tokyo Medical and Dental University). The resultant mouse cyclin
D2 expression plasmid was designated pMKcyl2. Cells were
transfected with either pMKcyl2 or pMKITneo by electroporation using an
Electroporator II (Invitrogen, San Diego, CA) at 500 V/cm, 1000
µfarads. Forty-eight hours after electroporation, the cells were
suspended in complete IMDM medium containing 450 µg/ml of G418 and
dispensed into 96-well plates at 50 cells per well. Stable
transfectants were screened for cyclin D2 expression by immunoblotting,
and single cell clones expressing high levels of cyclin D2 were
obtained by limiting dilution.
Cell-Cycle Analysis
Cells were suspended in hypotonic solution [0.1% Triton X-100,
1 mM Tris-HCl (pH 8.0), 3.4 mM sodium citrate,
0.1 mM EDTA] and stained with 5 µg/ml of propidium
iodide. DNA content was analyzed by a FACScan (Becton Dickinson, San
Jose, CA). Population of cells in each cell cycle phase was determined
by a CellFIT software (Becton Dickinson).
Antibodies, Immunoblot, and Immunoprecipitation Analyses
Cells were dissolved in 50 mM Tris-HCl (pH 6.8), 2%
SDS, boiled for 5 min, and then centrifuged at 12,000 x
g. The protein concentration of the supernatants was
determined, and 20 µg of extracted proteins were separated in 12.5%
polyacrylamide gels containing 0.1% SDS, and then electroblotted on
polyvinyldine fluoride membranes. For analysis of Rb, 7.5%
polyacrylamide gels were used. Immunodetection was performed using a
ECL Western blotting detection system (Amersham International, Little
Chalfont, UK) according to the manufacturers instruction. Expression
of individual proteins were detected by different filters. Relative
levels of bands were determined by densitometric analysis. Blots were
stained with Coomassie brilliant blue and confirmed to contain a
similar amount of protein extract on each lane.
For immunoprecipitation, cells were lysed in the lysis buffer
[1% NP-40, 50 mM Tris-HCl (pH 8.0), 150 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride]. Four hundred
micrograms of protein extracts were reacted with 1 µg of either
anti-CDK4 (C-22) or anti-p21CIP1/WAF1 sera (C-19) at 4 C
for 1 h and then incubated with 20 µl of protein G-sepharose
beads (Pharmacia LKB Biotechnology Inc., Piscataway, NJ).
Immunoprecipitates were washed four times with the lysis buffer and
analyzed by immunoblotting. Anti-Rb monoclonal antibody (G3245) was
purchased from PharMingen (San Diego, CA). Anti-CDK2 (M-20), anti-CDK4
(C-22), anti-CDK6 (C-21), anti-p21CIP1/WAF1 (C-19),
anti-p27KIP1 (N-21), and anti-cyclin E (M-20) rabbit IgG
and anti-p16INK4a (M-156-G) and anti-p15INK4b
goat IgG (M20-G) and anti-cyclin D1 (7213G), anti-cyclin D2
(34B13), and anti-cyclin D3 (18B610) monoclonal antibodies were
purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Anti-p21CIP1/WAF1 monoclonal antibody (Ab-4) was obtained
from Calbiochem-Novabiochem Corp. (Cambridge, MA).
In Vitro Rb Kinase Assay
In vitro Rb kinase assay was performed
according to DeGregori et al with a minor modification (45).
Briefly, cells (2 x 106) were suspended in IP buffer
[50 mM HEPES (pH 7.5), 150 mM NaCl, 1
mM EDTA, 2.5 mM EGTA, 1 mM
dithiothreitol, 0.1% Tween 20] containing 10% glycerol, 0.1
mM phenylmethylsulfonylfluoride, 10 mM
ß-glycerophosphate, 1 mM NaF, and 0.1 mM
sodium orthovanadate, incubated on ice for 10 min, and centrifuged at
12,000 x g for 10 min. Protein extracts were
immunoprecipitated for 2 h with 3 µg of either anti-CDK4 (C-22)
or anti-CDK2 rabbit sera (M-20) and then for 2 h with 30 µl of
50% protein G-Sepharose beads. The immunoprecipitates were washed four
times with IP buffer and twice with kinase buffer [50 mM
HEPES (pH 7.5), 10 mM MgCl2, 1 mM
dithiothreitol], resuspended in 30 µl of kinase buffer containing 2
µg of glutathione S-transferase-Rb fusion protein (GST-Rb)
(Santa Cruz Biotechnology Inc.), 100 µM cold ATP, 2.5
mM EGTA, 10 mM ß-glyserophosphate, 0.1
mM sodium orthovanadate, and 1 mM NaF and
incubated at 30 C for 30 min with occasional mixing. The reactions were
added by SDS-PAGE sample buffer, boiled, and immunoblotted.
Phosphorylated Rb was detected by anti-phospho-Rb rabbit sera (Medical
and Biological Laboratories Co., Nagoya, Japan) (46).
Northern Blot
Total RNA was extracted from cells using an Isogen RNA
extraction kit (Nippon Gene, Tokyo, Japan) according to the
manufacturers instruction. Five micrograms of total RNA were
electrophoresed in a formaldehyde-agarose gel and blotted on a nylon
membrane (Hybond-N+, Amersham). Mouse cyclin D2, mouse
p21CIP1/WAF1, and human GAPDH cDNA
fragments were isolated from pcN9-cyl2, pCMW35T3, and pKS321 (47),
respectively, and labeled with [
-32P]dCTP using a
Multiprime DNA labeling system (Amersham). Hybridization and washing
were performed as described previously (48), and x-ray films were
exposed to the blot. Relative levels of bands were determined by
densitometry and normalization to those of GAPDH.
Semiquantitative RT-PCR
Complementary DNA was synthesized from 1 µg of total RNA.
Undiluted and diluted cDNA samples (1:10, 1:100) were subjected to PCR
amplification with forward and reverse primers specific for mouse
p16INK4a or p15INK4b cDNA
(49) using a RNA PCR core kit (Perkin-Elmer, Norwork, CT). PCR was
performed by incubating at 94 C for 4 min, and then 35 cycles of
denaturing at 94 C for 1 min, annealing at 60 C for 45 sec, and
extension at 72 C for 2 min, followed by heating at 72 C for 10 min.
PCR products were electrophoresed in 2% agarose gels and visualized by
ethidium bromide staining. PCR primers used in the experiment were as
follows:
p16INK4a forward primer,
5'-GCTGCAGACAGACTGGCCAG-3';
p16INK4a reverse primer,
5'-AGGCATCGCGCACATCCAGC-3';
p15INK4b forward primer,
5'-CCTGGAAGCCGGCGCAGATC-3';
p15INK4b reverse primer,
5'-GCGTGTCCAGGAAGCCTTCC-3'.
 |
ACKNOWLEDGMENTS
|
---|
We thank Y. Eto for activin A, H. Matsushime for mouse
cyclin D2 cDNA, K. Maruyama for pMKITneo, T. Tokino for
mouse p21CIP1/WAF1 cDNA, and H. Sugino,
Tokushima University, for helpful discussions. Anti-phospho-Rb
antibodies were generous gifts from T. Moritsu, Medical and Biological
Laboratories Co.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Kenji Yamato, Department of Oral Science, The National Institute of Infectious Diseases, 123-1 Toyama, Shinjuku-ku, Tokyo 162, Japan.
This work was supported in part by grants-in-aid from the Ministry of
Education, Science, and Culture of Japan and from the Ministry of
Health and Welfare of Japan and Keio University Special Grant-in-aid
for Innovative Collaborative Research Projects.
Received for publication October 3, 1996.
Accepted for publication April 7, 1997.
 |
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