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
Activins, members of the transforming growth factor-b 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 cellcycle 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. (Molecular Endocrinology 11: 1044–
1052, 1997)
INTRODUCTION
Activins were originally isolated as factors from ovarian fluid that stimulate the secretion of FSH from pi0888-8809/97/$3.00/0
Molecular Endocrinology
Copyright © 1997 by The Endocrine Society
tuitary cells (1, 2). These factors are members of the
transforming growth factor-b (TGFb) family and are
encoded by two closely related genes, activin-bA and
activin-bB. They exist as homodimers (bAbA, bBbB) or
a heterodimer (bAbB) of the gene products and have
been designated as activin A, activin B, and activin AB,
respectively (3). Recently, two new activin b-chains
(activin-bC, activin-bD) 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 (7–10), and negative cell growth of various cell
types including gonadal cells and adrenal cells (11–
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 reg1044
Mechanism of G1 Arrest by Activin A
ulatory 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 D1related 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 (25–29) and p27KIP1
(30–32) contain highly homologous regions in the Nterminal portion responsible for CDK-inhibitory activity
(30, 33–35). 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 TGFb (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 TGFb-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 growthinhibitory 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.
RESULTS
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
1045
Fig. 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.
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.
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).
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
MOL ENDO · 1997
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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).
Activin A Induces Expression of p21CIP1/WAF1
Fig. 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.
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-29deoxy-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.
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).
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.
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.
Mechanism of G1 Arrest by Activin A
1047
Table 1. Activin A Increases the Population of G1 Phase in BCL-2-Expressing HS-72 Cells
BCL-2 Clones
HS-72B-5
%G1
%S
%G2/M
%Apo
HS-72B-6
HS-72B-12
0
12
24
36
0
12
24
36
0
12
24
36
45
43
13
6
78
12
5
4
69
17
4
10
71
17
4
9
43
47
6
4
84
8
4
4
80
11
4
5
76
10
3
11
45
44
5
6
81
9
4
6
80
9
5
6
78
8
4
10
BCL-2-expressing HS-72 cells (HS-72B-5, HS-72B-6, HS-72B-12) were exposed to activin A (50 ng/ml) for indicated times and
analyzed for the cell-cycle distribution. Numbers below clones indicate hours of incubation.
Fig. 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.
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.
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
Fig. 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).
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/WAF1bound CDK4 and CDK2 by immunoprecipitation. AntiCDK4 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
MOL ENDO · 1997
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Fig. 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.
untreated cells contained none of these, showing that
activin A increased levels of CDK4 and CDK2 complexed
with p21CIP1/WAF1.
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 CDK2associated Rb kinase activity in HS-72 cells (data not
shown).
Effect of Cyclin D2 Overexpression on Activin AInduced 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 SRa promoter (pMKcyl-2). HS-72 cells transfected
with the plasmid without cyclin D2 cDNA (HMK45)
were used as control cells. Cyclin D2-transfected cells
Vol 11 No. 8
Fig. 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(2)] was used as negative control.
(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) (57–63%) than that in
HMK45 cells (40–48%), 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.
Mechanism of G1 Arrest by Activin A
Fig. 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 antip21CIP1/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.
Fig. 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.
DISCUSSION
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
1049
Fig. 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 6 SD from triplicate experiments. Closed bar, Percent
cells in the S phase; open bar, the G2/M phase; hatched bar, the
G1 phase.
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
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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.
TGFb, a prototype of the TGFb family, causes G1
arrest by inhibiting Rb phosphorylation (42). Rb appears to be a common target of TGFb and activin A for
the growth inhibition. TGFb 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 3–6 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.
MATERIALS AND METHODS
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 Iscove’s modified Dulbecco’s medium (IMDM,
GIBCO-BRL, Gaithersburg, MD) supplemented with 10%
FBS, 100 mg/ml of streptomycin, and 100 U/ml of penicillin.
Human BCL-2-expressing HS-72 cells (HS-72B-5, HS72B-6, HS-72B-12) (18) were cultured in IMDM containing
10% FBS and 450 mg/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 3
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-29deoxy-uridine labeling and detec-
Vol 11 No. 8
tion kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s 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
SRa 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 mfarads. Forty-eight
hours after electroporation, the cells were suspended in complete IMDM medium containing 450 mg/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 mg/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 3 g. The
protein concentration of the supernatants was determined,
and 20 mg 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 manufacturer’s 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 mg of either anti-CDK4
(C-22) or anti-p21CIP1/WAF1 sera (C-19) at 4 C for 1 h and then
incubated with 20 ml 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 (G3–245) was
purchased from PharMingen (San Diego, CA). Anti-CDK2 (M20), anti-CDK4 (C-22), anti-CDK6 (C-21), anti-p21CIP1/WAF1 (C19), 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 (72–13G), anti-cyclin D2 (34B1–3), and anticyclin D3 (18B6–10) monoclonal antibodies were purchased
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antip21CIP1/WAF1 monoclonal antibody (Ab-4) was obtained from
Calbiochem-Novabiochem Corp. (Cambridge, MA).
Mechanism of G1 Arrest by Activin A
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 3 106) 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 b-glycerophosphate, 1 mM NaF, and 0.1 mM sodium
orthovanadate, incubated on ice for 10 min, and centrifuged at
12,000 3 g for 10 min. Protein extracts were immunoprecipitated for 2 h with 3 mg of either anti-CDK4 (C-22) or anti-CDK2
rabbit sera (M-20) and then for 2 h with 30 ml 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 ml of kinase buffer containing 2 mg of glutathione
S-transferase-Rb fusion protein (GST-Rb) (Santa Cruz Biotechnology Inc.), 100 mM cold ATP, 2.5 mM EGTA, 10 mM b-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 manufacturer’s instruction. Five micrograms of total RNA were electrophoresed in a formaldehyde-agarose gel and blotted on a
nylon membrane (Hybond-N1, 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 [a-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 mg 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, 59-GCTGCAGACAGACTGGCCAG-39;
p16INK4a reverse primer, 59-AGGCATCGCGCACATCCAGC-39;
p15INK4b forward primer, 59-CCTGGAAGCCGGCGCAGATC-39;
p15INK4b reverse primer, 59-GCGTGTCCAGGAAGCCTTCC-39.
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.
1051
Received October 3, 1996. Re-revised April 2, 1997.
Accepted April 7, 1997.
Address requests for reprints to: Kenji Yamato, Department of Oral Science, The National Institute of Infectious
Diseases, 1–23-1 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.
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