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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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.

 
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. 2Go). 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. 1Go).



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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.

 
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 1Go, 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.


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Table 1. Activin A Increases the Population of G1 Phase in BCL-2-Expressing HS-72 Cells

 
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. 3Go). 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. 3Go, 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. 3Go).



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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.

 
Activin A Induces Expression of p21CIP1/WAF1
Immunoblot analysis showed that untreated HS-72 cells contained undetectable levels of p21CIP1/WAF1 (Fig. 4Go). 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. 4Go). 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. 4Go). Exposure to activin A had no effect on expression of CDK2, but increased levels of cyclin E, a regulatory subunit of CDK2 (Fig. 4Go).



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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).

 
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. 5Go). 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. 5Go). 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.

 
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. 6AGo). 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. 6BGo, 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.

 
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. 7AGo). 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. 7BGo). 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.

 
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. 8Go, 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.

 
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{alpha} 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. 9AGo, 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. 9Go, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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 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 SR{alpha} 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 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 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 (G3–245) 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 (72–13G), anti-cyclin D2 (34B1–3), and anti-cyclin D3 (18B6–10) 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 manufacturer’s 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 [{alpha}-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, 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.

Received for publication October 3, 1996. Accepted for publication April 7, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Vale W, Rivier J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from ovarian follicular fluid. Nature 321:732–734
  2. Ling N, Ying SY, Ueno N, Shimasami S, Esch F, Hota M, Guillemin R 1986 Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin. Nature 321:779–782[Medline]
  3. Massagué J 1987 The TGF-ß family of growth and differentiation factors. Cell 49:437–438[Medline]
  4. Høtten G, Neidhardt H, Schneider C, Pohl J 1995 Cloning of a new member of the TGF-beta family: a putative new activin beta C chain. Biochem Biophys Res Commun 206:608–613[CrossRef][Medline]
  5. Oda S, Nishimura S, Murakami K, Ueno N 1995 Molecular cloning and functional analysis of a new activin beta subunit: dorsal mesoderm-inducing activity in Xenopus. Biochem Biophys Res Commun 206:581–588[CrossRef]
  6. Eto Y, Tsuji T, Takezawa M, Takano Y, Yokogawa Y, Shibai H 1987 Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095–1103[Medline]
  7. Green JB, Smith JC 1990 Graded changes in dose of a Xenopus activin A-homologue elicit stepwise transitions in embryonic cell fate. Nature 347:391–394[CrossRef][Medline]
  8. Smith JC, Price BMJ, Van Nimmen K, Huylebroeck D 1990 Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin-A. Nature 345:729–731[CrossRef][Medline]
  9. Sokol S, Melton DA 1991 Pre-existent pattern in Xenopus pole cells revealed by induction with activin. Nature 351:409–411[CrossRef][Medline]
  10. Smith JC 1993 Mesoderm-inducing factors in early vertebrate development. EMBO J 12:4463–4470[Medline]
  11. Gonzalez-Manchon C, Vale W 1989 Activin A, inhibin and TGF-ß modulate growth of two gonadal cell lines. Endocrinology 125:1666–1672[Abstract]
  12. Kojima I, Ogata E 1989 Dual effect of activin on cell growth in Balb/c 3T3 cells. Biochem Biophys Res Commun 159:1107–1113[Medline]
  13. Spencer SJ, Rabinovici J, Jaffe RB 1990 Human recombinant activin-A inhibits proliferation of human fetal adrenal cells in vitro. J Clin Endocrinol Metab 71:1678–1680[Abstract]
  14. McCarthy S, Bicknell R 1993 Inhibition of vascular endothelial cell growth. J Biol Chem 268:23066–23071[Abstract/Free Full Text]
  15. Nishihara T, Okahashi N, Ueda N 1993 Activin A induces apoptotic cell death. Biochem Biophys Res Commun 197:985–991[CrossRef][Medline]
  16. Nishihara T, Ohsaki Y, Ueda N, Koseki T, Eto Y 1995 Induction of apoptosis in B lineage cells by activin A derived from macrophages. J Interferon Cytokine Res 15:509–516[Medline]
  17. Brosh N, Sternberg D, Honigwachs-Sha’anani J, Lee B-C, Shav-Tal Y, Tzehoval E, Shulman LM, Tolendo J, Hacham Y, Carmi P, Jiang W, Horn F, Burstein Y, Zipori D 1995 The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. J Biol Chem 270:29594–29600[Abstract/Free Full Text]
  18. Koseki T, Yamato K, Krajewski S, Reed JC, Tsujimoto Y, Nishihara T 1995 Activin A-induced apoptosis is suppressed by overexpression of Bcl-2. FEBS Lett 376:247–250[CrossRef][Medline]
  19. Weinberg RA 1995 The retinoblastoma protein and cell cycle control. Cell 81:323–330[Medline]
  20. Nevins JR 1992 E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science 258:424–429[Medline]
  21. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ 1991 Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 65:701–713[Medline]
  22. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato J, Livingston DM 1993 Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell 73:487–497[Medline]
  23. Matsushime H, Ewen ME, Strom DK, Kato J, Hanks SK, Roussel MF, Sherr CJ 1992 Identification and properties of an atypical catalytic subunit (p34PSK-3/cdk4) for mammalian D type G1 cyclins. Cell 71:323–334[Medline]
  24. Sherr CJ, Roberts JM 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149–1163[CrossRef][Medline]
  25. El-Deiry WS, Tokino T, Velculescu VE, Levy R, Parsons R, Trent JM, Lin D, Mercer E, Kinzler KW, Vogelstein B 1993 WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825[Medline]
  26. Gu Y, Turek CW, Morgan DO 1993 Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366:707–710[CrossRef][Medline]
  27. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ 1993 The p21 cdk-interacting protein Cip 1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816[Medline]
  28. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D 1993 p21 is a universal inhibitor of cyclin kinases. Nature 366:701–704[CrossRef][Medline]
  29. Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR 1994 Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 211:90–98[CrossRef][Medline]
  30. Polyak K, Kato J, Solomon MJ, Sherr CJ, Massagué J, Roberts JM, Koff A 1994 p27kip1, a cyclin-cdk inhibitor, links transforming growth factor-ß and contact inhibition to cell cycle arrest. Genes Dev 8:9–22[Abstract]
  31. Polyak K, Lee M, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massagué J 1994 Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular mitogenic signals. Cell 78:59–66[Medline]
  32. Toyoshima H, Hunter T 1994 p27, a novel inhibitor of G1 cyclin/cdk protein kinase activity, is related to p21. Cell 78:67–74[Medline]
  33. Chen J, Jackson PK, Kirschner MW, Dutta A 1995 Separate domains of p21 involved in the cdk kinase and PCNA. Nature 374:386–388[CrossRef][Medline]
  34. Luo Y, Hurwiz J, Massagué J 1995 Cell-cycle inhibition by independent CDK and PCNA binding domains in p21CIP1. Nature 375:159–161[CrossRef][Medline]
  35. Nakanishi M, Robetorge RS, Adami GR, Pereira-Smith DM, Smith JR 1995 Identification of the active region of the DNA synthesis inhibitory gene p21sdi1/CIP1/WAF1. EMBO J 14:555–563[Abstract]
  36. Elendary A, Berchuck A, Davis P, Havrilesky L, Bast JRC, Iglehart JD, Marks JR 1994 Transforming growth factor ß1 can induce CIP1/WAF1 expression independent of the p53 pathway in ovarian cancer cells. Cell Growth Differ 5:1301–1307[Abstract]
  37. Reynisdóttir I, Polyak K, Massagué J 1995 Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-ß. Genes Dev 9:1831–1845[Abstract]
  38. Serrano M, Hannon GJ, Beach D 1993 A new regulatory motif in cell cycle control causing specific inhibition of cyclin D/cdk4. Nature 366:704–707[CrossRef][Medline]
  39. Hannon GJ, Beach D 1994 p15INK4b is a potential effector of TGF-ß-induced cell cycle arrest. Nature 371:257–261[CrossRef][Medline]
  40. Peters G 1994 Stifled by inhibitions. Nature 371:204–205[CrossRef][Medline]
  41. Gu Y, Rosenblatt J, Morgan DO 1992 Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J 11:3995–4005[Abstract]
  42. Laiho M, CeCaprio JA, Ludlow JW, Livingston DM, Massagué J 1990 Growth inhibition by TGFß linked to suppression of retinoblastoma protein phosphorylation. Cell 62:175–185[Medline]
  43. Mosmann T 1983 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63[CrossRef][Medline]
  44. Murata M, Eto Y, Shibai H, Sakai M, Muramatsu M 1988 Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin ßA chain. Proc Natl Acad Sci USA 85:2434–2338[Abstract]
  45. DeGregori J, Leone G, Ohtani K, Miron A, Nevins JR 1995 E2F-1 accumulation bypasses a G1 arrest resulting from the inhibition of G1 cyclin-dependent kinase activity. Genes Dev 9:2873–2887[Abstract]
  46. Kitagawa M, Higashi H, Jung H-K, Suzuki-Takahashi I, Ikeda M, Tamai K, Kato J, Segawa K, Yoshida E, Nishimura S, Taya Y 1996 The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. EMBO J 15:7060–7069[Abstract]
  47. Tokunaga K, Nakamura Y, Sakata K, Fujimori K, Ohkubo M, Sawada K, Sakiyama S 1987 Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers. Cancer Res 47:5616–5619[Abstract]
  48. Sugito S, Yamato K, Sameshima Y, Yokota J, Yano S, Miyoshi I 1991 Adult T-cell leukemia: structures and expression of the p53 gene. Int J Cancer 49:880–885[Medline]
  49. Quelle DE, Ashmun RA, Hannon G, Rehberger PA, Trono D, Richter KH, Walker C, Beach D, Sherr CJ, Serrano M 1995 Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene 11:635–645[Medline]