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Cell Growth & Differentiation Vol. 10, 695-704, October 1999
© 1999 American Association for Cancer Research


Articles

Regulation of p27Kip1 Accumulation in Murine B-Lymphoma Cells: Role of c-Myc and Calcium1

Dubravka Donjerkovic, Liying Zhang and David W. Scott2

Department of Immunology, Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855 [D. D., L. Z., D. W. S.], and Molecular and Cellular Oncology Program, George Washington University, Washington, DC 20037 [D. D., D. W. S.]

Abstract

IgM cross-linking induces G1 arrest and apoptosis in murine B-lymphoma cells. It prevents pRb phosphorylation by decreasing cyclin-dependent kinase 2 activity via the up-regulation of cyclin kinase inhibitor p27Kip1. Anti-IgM also causes an increase in cytosolic free calcium and a loss of c-myc mRNA and protein. This down-regulation of c-Myc is prevented by CD40L, which rescues cells from anti-IgM-induced apoptosis. In this study, we addressed the mechanism(s) of anti-IgM-induced p27Kip1 accumulation. We examined effects of early events in B-cell receptor-mediated signaling, c-Myc down-regulation, and an increase in free calcium on p27Kip1. Down-regulation of c-myc alone had no effect on p27Kip1; neither did an increase in free calcium alone. Together, these two events led to p27Kip1 induction, growth arrest, and apoptosis. CD40L, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and cyclosporin A all prevented anti-IgM-induced p27Kip1 accumulation, suggesting that both the decrease in c-Myc expression and an increase in free calcium are necessary for p27Kip1 up-regulation.

Introduction

Murine B-lymphoma cells, the proliferation of which is inhibited by cross-linking mIgM,3 have been useful models for analyzing normal B-cell activation and tolerance, as well as for studying neoplastic proliferation. WEHI-231, ECH408, and CH31 cells are prototypical of such "immature" B-lymphoma cells in which membrane IgM cross-linking leads to G1 growth arrest (1) and subsequent apoptosis (2 , 3) .

Anti-IgM treatment of these cells leads to an accumulation of the hypophosphorylated, active, growth-suppressive form of the retinoblastoma gene product, pRb, in the late G1 phase of the cell cycle (4) . pRb is a nuclear phosphoprotein and is a potent cell cycle regulator (reviewed in Ref. 5 ). It suppresses cell growth by binding to a variety of cellular proteins such as the transcription factor complex, E2F. These interactions are regulated by cell cycle-dependent pRb phosphorylation (6, 7, 8) . Phosphorylation of pRb in middle to late G1 inactivates this protein, the E2F transcription factor is released from the pRb/E2F complex, and cells can progress into S phase.

pRb is phosphorylated in vitro on multiple serine and threonine residues by CDK/cyclin complexes (reviewed in Ref. 9 ). In early to mid-G1, Cdk4 and Cdk6 in complex with D-type cyclins phosphorylate pRb (reviewed in Ref. 10 ). As the cells progress toward S phase, the Cdk2/cyclin E and Cdk2/cyclin A complexes become active and are responsible for G1 to S transition (11, 12, 13) . Previously, we have shown that mIgM cross-linking prevents the formation of the active Cdk2/cyclin E and Cdk2/cyclin A kinase complexes in WEHI-231 lymphoma cells (14) . Therefore, anti-IgM blocks the G1 to S transition by modulating Cdk/cyclin complexes that phosphorylate pRb.

There are at least three mechanisms of CDK regulation (reviewed in Ref. 15 ). The active kinase complex consists of the catalytic subunit (CDK) and the regulatory subunit, cyclin (cyclin A or cyclin E in the case of Cdk2). To be active, the catalytic subunit needs to be phosphorylated on Thr-160, in the case of Cdk2 (16) . Finally, CDKs are regulated by CKIs [Kip/Cip family members in the case of Cdk2 (reviewed in Ref. 17 )]. p27Kip1, up-regulated upon mIgM cross-linking in WEHI-231 cells (14) , is a Mr 27,000 nuclear protein, structurally related to p21Cip1, another Kip/Cip family member (18 , 19) . Levels of p27Kip1 are increased in a variety of cells arrested in G1 by different stimuli, such as macrophages arrested by cAMP (20) , fibroblasts arrested by lovastatin (21) or by serum withdrawal (22) , and Mv1Lu mink epithelial cells arrested by transforming growth factor ß (23) .

p27Kip1 is mainly regulated on the protein level. In some systems, p27Kip1 is redistributed from Cdk4 complexes to Cdk2 complexes, resulting in late G1 arrest (21 , 24) . In some systems, such as in lovastatin-arrested HeLa cells and in density-arrested fibroblasts, an increased rate of translation and decreased rate of protein degradation is observed (25) . p27Kip1 degradation occurs via ubiquination (26) , because p27Kip1 is phosphorylated on Thr-187 by Cdk2/cyclin E (27, 28, 29, 30) , and this phosphorylated form is then targeted for ubiquination and degradation (31) . Furthermore, p27Kip1 ubiquination and degradation seems to be restricted to the cytoplasmic compartment because a novel protein Jab1 binds p27Kip1 in the nucleus and shuttles it to the cytoplasm, where p27Kip1 becomes ubiquitinated and degraded (32) .

There are several lines of evidence that suggest c-Myc as a negative regulator of p27Kip1. For example, induction of c-MycER fusion protein by 4-OH-tamoxifen in Rat1 fibroblasts leads to Cdk2/cyclin E activation. This is a result of: (a) inhibition of p27Kip1 binding to the Cdk2/cyclin E complexes (33) ; (b) p27Kip1 release from the Cdk2/cyclin E complexes (27) ; and (c) p27Kip1 degradation (34) . Furthermore, retroviral expression of p27Kip1 induces G1 arrest in parental Rat1 cells but not in Rat1 cells that ectopically express c-Myc (35) . Additionally, coexpression of Ras and c-Myc leads to cyclin E-associated kinase activity, S-phase induction and, mostly important, p27Kip1 loss (36) . In several experimental systems, there is an inverse correlation between c-Myc and p27Kip1 expression. For example, there is a correlation between c-Myc overexpression and p27Kip1 down-regulation in mammary epithelial cells (37) , whereas there is an increased p27Kip1 expression in Rat1 cells deficient in c-Myc (38) . There is also experimental evidence that c-Myc negatively regulates p27Kip1 in lymphocytes. IL-2 induces c-Myc in T cells (39) , and complete stimulation of T cells (T-cell receptor engagement and IL-2 receptor engagement) down-regulates p27Kip1 (40) . In B cells, mIgM cross-linking induces c-Myc (41) , and complete stimulation of B cells (BCR engagement and CD40 engagement) down-regulates p27Kip1 (42) .

In murine B-lymphoma cells, there is also an inverse correlation between c-Myc and p27Kip1 levels. Anti-IgM-induced growth arrest and apoptosis of WEHI-231 cells is regulated in part by c-Myc (43, 44, 45, 46) . Down-regulation of c-Myc is necessary for anti-IgM-induced growth arrest and apoptosis because overexpression of exogenous c-Myc renders WEHI-231 cells resistant to anti-IgM (46) . Furthermore, c-myc transcription in WEHI-231 lymphoma cells is regulated by NF{kappa}B (47) , and the inhibition of NF{kappa}B (leading to c-Myc down-regulation) induces apoptosis in WEHI-231 cells (48) . Anti-IgM treatment leads to an increase in c-myc mRNA within 1–2 h, a decrease to below the baseline level at 4–8 h (49) , and complete disappearance by 24 h in unsynchronized cells (43) . Anti-IgD, on the other hand, causes a stable increase in c-myc mRNA in IgD-expressing B-lymphoma cell lines (50) .4 Furthermore, only anti-IgM (but not anti-IgD), leads to p27Kip1 accumulation and to growth arrest and apoptosis (44 , 45 , 51) . Therefore, the decrease in c-Myc strongly correlates with anti-IgM-induced p27Kip1 accumulation, late G1 arrest, and apoptosis in anti-IgM-sensitive murine B-lymphoma cells.

A very early event in BCR signaling is an increase in cytosolic free calcium, which is a result of both influx of extracellular calcium and release of calcium from intracellular storage. In WEHI-231 cells, anti-IgM treatment results in an increase in free cytosolic calcium seconds to minutes after the BCR cross-linking (52 , 53) .

In this study, we examined the mechanism(s) of p27Kip1 accumulation in B-lymphoma cells arrested by mIgM cross-linking. We studied the effects of the early events in mIgM signaling, in particular, the role of calcium and of c-Myc, on p27Kip1. Our data indicate that neither the decrease in c-Myc nor the increase in cytoplasmic free calcium per se is sufficient for the significant up-regulation of p27Kip1. Rather, the loss of c-Myc, when accompanied by an increase in cytosolic free calcium, both of which are induced by mIgM cross-linking, was able to induce p27Kip1 accumulation, growth arrest, and apoptosis.

Results

CKI p27Kip1 Is Regulated on Both mRNA and Protein Levels in B-Lymphoma Cells.
All of the experiments presented in this study were done using WEHI-231, ECH408, and CH31 B-lymphoma cells in parallel. All three cell lines undergo G1 growth arrest and apoptosis and up-regulate p27Kip1 upon anti-IgM cross-linking, and results obtained with these cell lines were very similar.

The majority of experimental evidence suggests that p27Kip1 is regulated posttranscriptionally (mainly by degradation). However, Han et al. (54) reported an increase in p27Kip1 mRNA upon mIgM cross-linking in WEHI-231 cells. To determine whether p27Kip1 is regulated on the mRNA level, we isolated total RNA from the control WEHI-231, CH31, and ECH408 cells and from the cells treated with anti-IgM for 24 h (WEHI-231) or 20 h (ECH408 and CH31). As shown on Fig. 1ACitation , mRNA levels are slightly elevated (1.7-fold increase) upon anti-IgM treatment in WEHI-231 and in CH31 cells but not in ECH408 cells. It is possible, however, that p27Kip1 mRNA increases upon BCR cross-linking at earlier time points. Indeed, a time course experiment with WEHI-231 cells (Fig. 1B)Citation shows approximately a 3-fold increase in p27Kip1 mRNA levels 12 h after the treatment. This increase is transient, because p27Kip1 mRNA levels decline at 12–16 h after BCR cross-linking. Western blot analysis of the cells treated with anti-IgM revealed an increase in p27Kip1 protein (Fig. 1A)Citation in all three cell lines. This increase in p27Kip1 protein is much more dramatic than the increase in mRNA, suggesting that p27Kip1 is regulated not only at the mRNA but also on the protein level.



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Fig. 1. p27Kip1 is regulated on the protein and mRNA levels. A, exponentially growing cells were cultured for 24 h (WEHI-231) or for 20 h (ECH408 and CH31) with anti-IgM (1 µg/ml). Relative integrated density values were obtained by dividing the absolute values of anti-IgM-treated or untreated cells with the ones of the untreated cells. p27Kip1 mRNA levels were determined by Northern blot analysis, whereas p27Kip1 protein levels were determined by Western blot analysis of the total cell lysates. B, exponentially growing WEHI-231 cells were cultured with anti-IgM (1 µg/ml) for the indicated periods of time, and p27Kip1 mRNA levels were determined by Northern blot analysis. Relative integrated density values for p27Kip1 mRNA were obtained by dividing the absolute values of anti-IgM-treated or untreated cells with the ones of the untreated cells. Resulting values were then further divided by the relative integrated density values for ß-actin mRNA to correct for the unequal loading.

 
Caspase Inhibitor Z-VAD-FMK Rescues Cells from Anti-IgM-induced Apoptosis But Has No Effect on c-Myc, p27Kip1, or G1 Growth Arrest.
Caspases, a family of cystein proteases that cleave after aspartic acid residues, are effectors of apoptotic cell death (reviewed in Ref. 55 ) and have been implicated recently in regulation of Kip/Cip CKIs. Levkau et al. (56) reported that both p21Cip1 and p27Kip1 are cleaved at the COOH terminus by caspase 3 in human umbilical vein endothelial cells undergoing growth factor deprivation-induced apoptosis. Furthermore, caspase 3 is activated in CH31 (57) as well as in WEHI-231 cells,5 and it is possible that p27Kip1 is negatively regulated by caspase 3 in this model system. To examine the role of caspases in p27Kip1 regulation, we treated cells with anti-IgM in the presence or absence of a synthetic peptide inhibitor of caspases, Z-VAD-FMK. As shown in Fig. 2ACitation by the flow cytometric analysis of the cell cycle, at 24 h, Z-VAD-FMK prevents anti-IgM-induced apoptosis in WEHI-231 cells as well as in CH31 (57) but has no effect on G1 growth arrest, c-Myc, and p27Kip1 in WEHI-231 cells (Fig. 2B)Citation . These data suggest that caspase activation is on a separate pathway(s) from, or downstream of, c-Myc down-regulation, p27Kip1 accumulation, and G1 growth arrest in this experimental system.



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Fig. 2. Caspase inhibitor Z-VAD-FMK rescues cells from anti-IgM-induced apoptosis but has no effect on G1 growth arrest, c-Myc, and p27Kip1. A, WEHI-231 cells were cultured for 24 h with anti-IgM (1 µg/ml), either in the absence (upper row) or in the presence (lower row) of Z-VAD-FMK (50 µM). Cells were stained with PI and analyzed on a FACScalibur flow cytometer using Cell Quest software. Percentages of apoptotic and G1 cells are shown above each bar. B, cells were cultured as in 2A. Levels of c-Myc and p27Kip1 protein were determined by Western blot analysis.

 
c-Myc Down-Regulation Precedes p27Kip1 Accumulation.
Previous data from our laboratory (43, 44, 45) , as well as from Wu et al. (46) , suggested that anti-IgM-induced growth arrest and apoptosis of WEHI-231 cells is regulated by c-Myc. Furthermore, a decrease in c-Myc strongly correlates with anti-IgM-induced p27Kip1 accumulation, late G1 arrest, and apoptosis in anti-IgM-sensitive murine B-lymphoma cells. If indeed c-Myc is a negative regulator of p27Kip1 in this model system, anti-IgM-induced down-regulation of c-Myc should precede p27Kip1 accumulation mediated by BCR cross-linking. As shown by Western blot analysis in Fig. 3Citation , down-regulation of c-Myc below the baseline level (after a transient increase) occurs as early as 4 h after the treatment. On the other hand, p27Kip1 protein cannot be detected until 8 h after the treatment and is not dramatically up-regulated until 8–12 h after the treatment. Additionally, an increase in p27Kip1 mRNA is not observed until 6 h after BCR cross-linking (Fig. 1B)Citation . Therefore, anti-IgM-mediated c-Myc down-regulation occurs before p27Kip1 mRNA increase and protein accumulation.



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Fig. 3. c-Myc down-regulation precedes p27Kip1 accumulation. Exponentially growing WEHI-231 cells were cultured with anti-IgM (1 µg/ml) for the indicated periods of time. c-Myc and p27Kip1 protein levels were determined by Western blot analysis of the total cell lysates.

 
An Increase in Cytosolic Free Calcium, Together with the Decrease in c-Myc, Induces Apoptosis.
To test whether the loss of c-Myc is sufficient for p27Kip1 up-regulation, we took a pharmacological approach and down-regulated c-Myc by using the drug FR901228. FR901228 is a newly characterized fungal metabolite isolated from Chromobacterium violaceum (58) . It is used as an antitumor agent that inhibits c-myc expression in fibroblasts while having no effect on Ha-ras or ß-actin (59) . Recently, Wang et al. (60) showed that FR901228 inhibits c-myc expression in lymphoid cells but has no effect on cyclin B1, Cdc2, IL-2, Fas, and Grb-2. We cultured WEHI-231 and ECH408 cells for 24 and 20 h, respectively, in the presence of FR901228 and determined the effect of the drug on c-Myc and on the cell cycle-related proteins that play a role in the regulation of the G1 phase of the cell cycle. As expected, Western blot analysis showed that FR901228 down-regulated c-Myc in both cell lines (Fig. 4)Citation . It only slightly decreases E2F-1 in WEHI-231, but not in ECH408 cells, and has little or no effect on Cdk2, Cdk4, and cyclin D2 expression. Flow cytometric analysis of the cell cycle shows that FR901228 per se does not induce G1 growth arrest or apoptosis (Fig. 5A)Citation when used at nontoxic levels (<=0.3 ng/ml).



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Fig. 4. Fungal metabolite FR901228 downregulates c-Myc. Cells were cultured for 24 h (WEHI-231) or for 20 h (ECH408) with anti-IgM (1 µg/ml) or with FR901228 (0.3 ng/ml). The effects of FR901228 on c-Myc, as well as E2F-1, cyclin D2, Cdk2, and Cdk4 were determined by Western blot analysis using polyclonal or monoclonal antibodies. Relative integrated density values were obtained by dividing the absolute values of FR901228-treated cells with the ones of the untreated cells.

 


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Fig. 5. Cotreatment with FR901228 and ionomycin mimics anti-IgM signal for growth arrest and apoptosis and up-regulates p27Kip1. A, ECH408 cells were cultured for 20 h with anti-IgM (1 µg/ml), with FR901228 (0.3 ng/ml), with ionomycin (0.3 µM), or with FR901228 and ionomycin together. PI staining and cell cycle analysis followed. Percentages of apoptotic and G1 cells are shown. B, ECH408 cells were treated as in A and lysed in NP40 lysis buffer. Fifty µg of total protein per lane were loaded. Levels of p27Kip1 protein were determined by Western blot analysis.

 
To test whether the down-regulation of c-Myc is sufficient for the accumulation of the CDK inhibitor p27Kip1, we determined p27Kip1 protein levels in FR901228-treated cells by immunoblotting (Fig. 5B)Citation . Reproducibly, FR901228 alone does not up-regulate p27Kip1, indicating that some other event(s), in addition to c-Myc down-regulation, provided by mIgM-mediated signaling is responsible for p27Kip1 accumulation.

Because down-regulation of c-Myc by FR901228 had no effect on p27Kip1, we next examined what other signal(s) provided by anti-IgM are involved in p27Kip1 regulation. It has been well established that calcium ionophores (such as ionomycin) and protein kinase C activators (such as phorbol myristate acetate) can mimic anti-IgM signaling in B cells (61) . Therefore, cells were treated with FR901228 (to down-regulate c-Myc) and with either ionomycin (to mimic anti-IgM-induced increase in intracellular calcium) or with phorbol myristate acetate (to mimic anti-IgM-induced activation of protein kinase C). There is no additive effect between FR901228 and PMA in the induction of p27Kip1 expression, growth arrest, and/or apoptosis (data not shown). In contrast, the combination of FR901228 and ionomycin can mimic anti-IgM treatment and induce significant apoptosis (Fig. 5A)Citation . Similar to FR901228, ionomycin alone does not induced G1 growth arrest or apoptosis when used at nontoxic concentrations (<=300 nM).

A Decrease in c-Myc Protein Expression, Combined with the Increase in Cytosolic Free Calcium, Results in p27Kip1 Up-Regulation.
To determine whether down-regulation of c-Myc together with an increase in cytoplasmic free calcium can lead to p27Kip1 accumulation, we did Western blot analysis of p27Kip1 levels in cells treated with ionomycin alone, FR901228 alone, or with the two drugs in combination. The concentration of ionomycin used in this study (0.3 µM) resulted in an increase in cytosolic free calcium comparable with BCR-mediated free calcium increase.5 As shown in Fig. 5BCitation , ionomycin alone has no effect on p27Kip1, nor does it affect c-Myc (data not shown). Although neither FR901228 nor ionomycin alone induces p27Kip1 accumulation, the amount of p27Kip1 in cells treated with both drugs together is similar to levels observed in anti-IgM-treated cells (Fig. 6B)Citation . These results suggest that the decrease in c-Myc protein, when accompanied by an increase in cytosolic free calcium, can be sufficient for the accumulation of the CKI p27Kip1 in B-lymphoma cells.



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Fig. 6. CsA and BAPTA-AM prevent anti-IgM-induced p27Kip1 accumulation. WEHI-231 cells were cultured for 24 h with anti-IgM (1 µg/ml) in the presence or absence of either CsA (100 ng/ml) or BAPTA-AM (5 µM). p27Kip1 protein levels were determined by Western blot analysis.

 
Cyclosporin A and BAPTA-AM Prevent Anti-IgM-induced p27Kip1 Accumulation.
Cyclosporin A (CsA) is a well-known immunosuppressant that inhibits calcineurin (reviewed in Ref. 62 ), a calcium-dependent enzyme that dephosphorylates NFATc, thus enabling it to enter the nucleus and initiate transcription (reviewed in Ref. 63 ). BAPTA-AM is a commonly used calcium chelator. Both CsA and BAPTA-AM can, therefore, prevent processes that are downstream from, and are dependent on, an increase in cytoplasmic free calcium. If anti-IgM-induced p27Kip1 accumulation is one such process, it should be prevented by these drugs. To test this hypothesis, we cotreated cells with anti-IgM and CsA or with anti-IgM and BAPTA-AM. Indeed, as shown by Western blot analysis in Fig. 6Citation , both drugs prevented anti-IgM-induced p27Kip1 up-regulation, confirming that an increase in cytosolic free calcium is necessary for the p27Kip1 accumulation upon IgM cross-linking. Further studies need to be done to determine the possible role of NFAT in the regulation of p27Kip1 expression.

CD40L Rescues Cells Treated with FR901228 and Ionomycin from Apoptosis and Prevents p27Kip1 Up-Regulation.
Signaling through CD40 rescues WEHI-231 cells from anti-IgM-mediated apoptosis (64) . CD40L treatment induces Bcl-xL expression (65, 66, 67) and also prevents anti-IgM-induced down-regulation of NF{kappa}B/Rel and c-Myc (68 , 69) , as well as up-regulation of p27Kip1 (54) . To further confirm that down-regulation of c-Myc is necessary for p27Kip1 accumulation, we simultaneously treated cells with CD40L and anti-IgM, or with CD40L, FR901228, and ionomycin. As shown in Fig. 7ACitation , CD40L rescues ECH408 cells from anti-IgM-induced apoptosis, as well as from FR901228 and ionomycin-induced apoptosis. Moreover, CD40L prevents up-regulation of p27Kip1 induced by anti-IgM, as well as by FR901228 and ionomycin (Fig. 7B)Citation , confirming that down-regulation of c-Myc is necessary for p27Kip1 accumulation.



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Fig. 7. CD40L rescues cells from FR901228 and ionomycin-mediated apoptosis and prevents p27Kip1 up-regulation. A, ECH408 cells were cultured for 20 h with anti-IgM (1 µg/ml) or with FR901228 (0.3 ng/ml) and ionomycin (0.3 µM), either in the absence (upper row) or in the presence (lower row) of CD40L (6 µg/ml). Cells were stained with PI and analyzed on a FACScalibur flow cytometer. Percentages of apoptotic and G1 cells are shown. B, cells were cultured as in A, lysed in NP40 lysis buffer, and 50 µg of total protein were loaded in each lane. Levels of p27Kip1 protein were determined by Western blot analysis.

 
Discussion

Cross-linking of IgM on the surface of murine B-lymphoma cells induces an increase in cytosolic free calcium, down-regulation of c-Myc, and accumulation of p27Kip1. All of the above lead to a decrease in kinase activity of Cdk2/cyclin E and Cdk2/cyclin A complexes and consequently to an accumulation of the active, growth-suppressive, hypophosphorylated form of the retinoblastoma gene product, pRb. The ultimate result is late G1 growth arrest and apoptosis. In our hands, p27Kip1 is the only Kip/Cip family member whose expression increases upon anti-IgM treatment. Despite its importance in anti-IgM-mediated growth arrest and apoptosis, very little is known about the mechanisms of p27Kip1 accumulation after mIgM cross-linking. The objective of this study was to determine the mechanism(s) by which p27Kip1 is regulated, in murine B-lymphoma cells, after BCR cross-linking.

We have related events that are upstream of p27Kip1 in the mIgM-mediated process of growth arrest and apoptosis that might be responsible for p27Kip1 accumulation. Our studies of the kinetics of this process clearly show an accumulation of p27Kip1 protein as early as 8–12 h after anti-IgM treatment of unsynchronized cells (Fig. 3)Citation . An increase in cytosolic free calcium is observed within seconds to minutes after treatment with anti-IgM (52 , 53) , whereas a decrease in c-myc message and protein levels occurs 4 h after treatment (Fig. 3)Citation . Therefore, based on these kinetics, it is possible that the changes in c-Myc and in calcium play a role in p27Kip1 regulation upon mIgM cross-linking.

Extensive experimental data from our laboratory and Sonenshein’s group suggest that down-regulation of c-myc plays a crucial role in growth arrest and apoptosis in murine B-lymphoma cells (43, 44, 45, 46, 47, 48, 49) . Furthermore, evidence from other experimental systems, such as fibroblasts, as well as mature T and B cells, implicates c-Myc as a negative regulator of p27Kip1 (27 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) . To prove the necessity of c-Myc down-regulation in anti-IgM-induced p27Kip1 accumulation, we had to down-regulate c-Myc levels in the cells. Unfortunately, we could not use the antisense approach to down-regulate c-Myc, because c-myc antisense oligonucleotides actually stabilize c-myc message and protein levels in WEHI-231 cells (43) 6 and block growth arrest and apoptosis. This is, at least partially, due to the mitogenic effect of the unmethylated CpG motifs that are present in these oligodeoxynucleotides. It has been well established that unmethylated CpG dinucleotide, flanked by two 5' purines and two 3' pyrimidines, triggers B-cell activation (70) . Additionally, unmethylated CpG-containing oligonucleotides maintain high c-Myc levels and rescue WEHI-231 cells from anti-IgM-induced G1 arrest and apoptosis (71) . As an alternative pharmacological approach, we used the antitumor drug FR901228, which has been shown to inhibit c-Myc expression in murine fibroblasts (59) and in murine T-hybridoma cells (60) . Furthermore, it dramatically down-regulates c-Myc (Fig. 4)Citation in murine B-lymphoma cells while having little or no effects on other cell cycle-related proteins such as Cdk2, Cdk4, and cyclin D2, the most abundant D-type cyclin in these cells (Fig. 4)Citation . Importantly, FR901228 alone cannot induce p27Kip1 accumulation at the concentration that significantly down-regulate c-Myc (Figs. 4Citation and 5BCitation ), suggesting that, besides down-regulation of c-Myc, another signaling event provided by BCR cross-linking is also necessary for anti-IgM-induced accumulation of p27Kip1.

In addition to FR901228, we have used TPCK, a nonspecific protease inhibitor, to down-regulate c-Myc. Used extensively by Wu et al. (46 , 48) , TPCK is known to prevent the degradation of I{kappa}B, thus modulating NF{kappa}B and indirectly regulating c-myc transcription. We obtained virtually identical results with TPCK as we observed with FR901228 in terms of p27Kip1 up-regulation. In other words, TPCK alone is not sufficient to up-regulate p27Kip1 at the concentration that profoundly downregulates c-Myc (data not shown).

It is well established that CD40L can provide signals to prevent anti-IgM-driven growth arrest and apoptosis in B- lymphoma cells and that these signals maintain high c-Myc levels (64 , 68 , 69) . When we simultaneously treated cells with FR901228 and ionomycin in the presence of CD40L (to maintain c-Myc), cells were rescued from apoptosis (Fig. 7A)Citation . Under these conditions, p27Kip1 does not accumulate (Fig. 7B)Citation , a result confirming that the down-regulation of c-Myc is necessary for p27Kip1 accumulation. Finally, we are presently establishing WEHI-231 cells that overexpress exogenous c-myc under the control of an inducible promoter because stable transfectants are not viable.7 We predict that anti-IgM treatment will not induce p27Kip1 in these cells that are inducibly expressing exogenous c-Myc (or at least not as much as in parental WEHI-231 cells). However, it is not clear whether they will be resistant to apoptosis, based on recent report that induction of c-MycER enhances anti-IgM-induced apoptosis in WEHI-231 cells (72) .

In general, in primary cells (such as fibroblasts or even mature B cells) as well as in other cell lines (such as T-hybridoma cells) c-Myc overexpression leads to cell death (reviewed in Ref. 73 ). Although seemingly different, we propose that in mature B cells and immature B-lymphoma cells, the responses to c-Myc are essentially similar, the difference being quantitative. Resting, mature B cells have low basal levels of c-Myc that transiently increase upon BCR cross-linking (41) . However, unless T-cell help is provided (via CD40, for example), mature B cells will not proliferate. In WEHI-231 cells, BCR cross-linking also leads to transient increase in c-Myc, which is then followed by its profound down-regulation, growth arrest, and apoptosis (43 , 49 , 50) . As in mature B cells, WEHI-231 cells are rescued from growth arrest and apoptosis by CD40 engagement, which prevents c-Myc levels from falling (64 , 68) .

Furthermore, according to the dual signal model, c-Myc overexpression promotes pro-death signaling pathway(s). Unless a survival signal is applied (which can be substituted by Bcl-2 overexpression, for example), the affected cell dies (74 , 75) . In our hands, constitutive, stable, ectopic overexpression of c-Myc has been unsuccessful (all surviving clones express equal, albeit high, levels of c-Myc as parental cells). Wu et al. (46) reported an ectopic, constitutive c-Myc expression in WEHI-231 cells. However, basal expression of c-Myc in cells transfected with exogenous c-Myc is equal to basal c-Myc levels in parental cells. This suggests that there is a threshold amount of c-Myc that these cells can tolerate, and that transfectants expressing high levels of exogenous c-Myc are eliminated via apoptosis during the selection process. It also suggests that this threshold level of c-Myc is maintained in transfected cells, possibly by suppression of the endogenous c-Myc transcription by exogenous c-Myc. These transfectants also maintain c-Myc levels upon BCR cross-linking, because exogenous c-Myc expression is driven by retroviral promoter and is not regulated by BCR-mediated signaling. Recently, Hagiyama et al. (72) reported that overexpression of c-Myc (using the MycER inducible system) induces apoptosis in WEHI-231 cells, suggesting that, similar to primary cells and other cell lines, B-lymphoma cells are also sensitive to c-Myc overexpression.

In general, complete elimination of c-Myc is also lethal for primary cells as well as for cell lines, with the exception of the recently established c-Myc deficient, immortalized fibroblasts (38) . Therefore, either overexpression (relative to the basal levels) or complete elimination of c-Myc would result in apoptosis in both immature B-lymphoma and mature B cells. However, B-lymphoma cells express higher basal levels of c-Myc and can tolerate higher c-Myc levels, while being more sensitive to c-Myc down-regulation. Mature B cells, on the other hand, express low basal levels of c-Myc and are more sensitive to c-Myc overexpression while less sensitive to its down-regulation.

Why do B-lymphoma cells tolerate high levels of c-Myc? One possible explanation has been suggested by Wu et al. (76) . In WEHI-231 cells, positive transcriptional regulation by c-Myc via E box elements seems to be inferior to the c-Myc-mediated transcriptional repression via Inr elements, possibly because of the presence of dominant-negative Max protein, dMax (77) . If the main pathway of c-Myc regulation in WEHI-231 cells is transcriptional repression via Inr elements, down-regulation of c-Myc would lead to activation of Inr-regulated genes and to apoptosis (assuming that Inr-regulated genes encode for proapoptotic proteins). According to this model, in mature B cells, c-Myc-mediated transcriptional activation pathway would dominate; therefore, overexpression of c-Myc in normal B cells leads to apoptosis (via activation of E-box-regulated genes, the products of which are proapoptotic). If this is true, cells in which c-Myc-mediated transcriptional repression dominates over transcriptional activation (WEHI-231 cells, for example) can tolerate higher levels of c-Myc but are more sensitive to its down-regulation. Conversely, cells in which transcriptional activation dominates (most primary cells, including mature B cells) are more sensitive to c-Myc overexpression. Hence, we propose that either overexpression of c-Myc [relative to the basal levels (72) ] or down-regulation of c-Myc [relative to the basal levels (46) ] could result in apoptosis in both cell types.

Besides c-Myc down-regulation, another important, early event in anti-IgM signaling that might be involved in p27Kip1 regulation is an increase in cytosolic free calcium. To look at the effect of calcium increase on p27Kip1, we elevated cytoplasmic calcium levels using calcium ionophore ionomycin. In WEHI-231 cells, an ionomycin concentration of 0.3 µM results in an increase in cytosolic free calcium, which is comparable with the BCR-mediated increase.5 Ionophore-mediated G1 arrest has been reported in several different murine or human B-lymphoma cells (78 , 79) , and an ionophore-mediated increase in p27Kip1 expression has been reported in prostatic cancer cell line (80) . However, in our study, ionomycin alone does not induce G1 arrest, apoptosis, or p27Kip1 when used in nontoxic concentrations that give equal increase in cytosolic free calcium as BCR cross-linking (<=0.3 µM). As indicated, only when a Ca2+ increase is accompanied by down-regulation of c-Myc does p27Kip1 protein increase, similar to anti-IgM-treated cells. The intracellular calcium chelator BAPTA-AM, as well as CsA, prevents anti-IgM-induced p27Kip1 accumulation (Fig. 6)Citation , further supporting the role of calcium in p27Kip1 regulation in this system. However, under these conditions, we were unable to prevent apoptosis (data not shown). This suggests that another default pathway(s) that can lead to growth arrest and apoptosis (such as the induction of another CKI, for example) exists in these cells and is activated when the p27Kip1 pathway is disabled. In agreement with this, p27Kip1 deficient mice have normal numbers of B cells, and their B cells are fully functional (81 , 82) , suggesting that there is a p27Kip1-independent pathway by which these B cells undergo growth arrest.

Finally, in contrast to human umbilical vein endothelial cells undergoing growth factor deprivation-induced apoptosis (56) , caspases do not affect native, intact p27Kip1 protein levels in murine B-lymphoma cells after mIgM cross-linking (Fig. 2B)Citation . This suggests that p27Kip1 is either upstream of caspase activation or on a completely separate signaling pathway.

Fig. 8Citation represents our working model. Neither FR901228 nor ionomycin alone can induce accumulation of p27Kip1. However, the combination of c-Myc down-regulation (induced by FR901228) and the increase in cytosolic free calcium (induced by ionomycin), can induce growth arrest and apoptosis, and importantly, p27Kip1 accumulation to the levels induced by mIgM. Anti-IgM-induced accumulation of p27Kip1 can be prevented by the calcium chelator BAPTA-AM, as well as by the calcineurin inhibitor CsA, confirming that an increase in free cytosolic calcium and activation of calcium-dependent enzymes are necessary for anti-IgM-mediated p27Kip1 accumulation. Finally, CD40L protects cells from both anti-IgM- and FR901228 and ionomycin-induced apoptosis and prevents p27Kip1 accumulation, confirming that down-regulation of c-Myc is necessary for anti-IgM-induced p27Kip1 accumulation.



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Fig. 8. Proposed model for membrane IgM-mediated p27Kip1 accumulation. IgM cross-linking down-regulates c-Myc (which can be mimicked by FR901228) and increases intracellular free calcium (which can be mimicked by ionomycin). These two events together then lead to p27Kip1 up-regulation and ultimately to G1 growth arrest and apoptosis. c-Myc down-regulation can be prevented by the costimulatory signal via CD40/CD40L engagement. An increase in calcium can be prevented by the calcium chelator BAPTA-AM, whereas the activation of calcium-dependent enzyme calcineurin can be inhibited by CsA. Treatment with either CD40L, BAPTA-AM, or CsA prevents anti-IgM-induced p27Kip1 accumulation.

 
Our future goal is to define the next step(s) in the c-Myc-mediated branch and in the Ca2+-mediated branch of mIgM signaling (such as NFAT, for example) that lead to the accumulation of p27Kip1, growth arrest, and apoptosis in these cells. Our preliminary data5 suggest the involvement of PI3K as well as p70S6K in these processes.

In summary, in this study we examined mechanisms of p27Kip1 regulation in murine B-lymphoma cells. p27Kip1 is a haplo-insufficient tumor suppressor protein (83) , the expression of which is down-regulated in many human cancers (reviewed in Ref. 84 ). Its role in the regulation of the mammalian cell cycle has been well established, whereas recent experimental data suggest its role in apoptosis as well. Even though it has been known for several years that anti-IgM treatment leads to p27Kip1 up-regulation (which strongly correlates with Cdk2 inactivation, pRb underphosphorylation, G1 growth arrest, and apoptosis), little is known about p27Kip1 regulation in this model system. Our results demonstrate a relationship between Ca2+ mobilization and c-myc oncogene down-regulation (both of which are early events in anti-IgM signaling) and p27Kip1 expression. Further knowledge of the mechanisms by which p27Kip1 is regulated will help us to better understand the complex molecular events that lead to the loss of this important tumor suppressor, resulting in uncontrolled proliferation and the inability of cells to die.

Materials and Methods

Cell Culture.
WEHI-231, CH31, and ECH408 murine B-lymphoma cells are routinely maintained in our laboratory as tissue culture lines in RPMI 1640 (Bio-Whittaker, Walkersville, MD) supplemented with 5% fetal bovine serum, glutamine, and 2-mercaptoethanol. They are passaged every other day, regularly checked for Mycoplasma contamination, and new stocks are thawed regularly (every 30 passages) to avoid phenotypic drift. Cells were treated with indicated concentrations of polyclonal goat anti-mouse IgM (µ chain-specific; Southern Biotechnology Associates, Inc., Birmingham, AL), FR901228 (Ref. 58 ; Fujisawa Pharmaceutical Co., Osaka, Japan, a gift of Dr. Y. Shi, American Red Cross, Rockville, MD), ionomycin (Sigma Chemical Co., St. Louis, MO), CD40L (plasma membrane preparation from Sf21 cells expressing mouse CD40L, a gift from Dr. M. R. Kehry, Boehringer Ingelheim, Ridgefield, CT), cyclosporin A (Sandoz, Dorval, Quebec, Canada), BAPTA-AM (Molecular Probes, Eugene, OR), and Z-VAD-FMK (Enzyme Systems Products, Livermore, CA). Cells were incubated for 24 h (WEHI-231) or for 20 h (CH31 and ECH408) at a density of 2.5 x 105 cells/ml at 37°C with 7% CO2.

Cell Cycle Analysis.
One million cells were washed with cold PBS and fixed in 70% ice-cold ethanol for at least 1 h at 4°C, washed with cold PBS, resuspended in 0.5 ml of PBS containing 10 µg/ml RNase (Sigma), and incubated for 30 min at 37°C. PI (Sigma) was added at 100 µg/ml final concentration, and cells were analyzed on a FACScalibur flow cytometer (Becton-Dickinson). Data were analyzed by Cell Quest software.

Western Blot Analysis.
Five million cells were lysed in lysis buffer [50 mM Tris HCl (pH 7.4), 200 mM NaCl, 2 mM EDTA, 0.5% NP40, 50 mM NaF, 0.5 mM sodium orthovanadate, 20 mM sodium pyrophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride] for at least 10 min on ice. Lysates were clarified by centrifugation at 10,000 x g for 10 min, and protein concentration was determined by BCA protein assay kit (Pierce, Rockford, IL), according to the manufacturer’s instructions. Samples were boiled, and equal amounts of total protein (50 µg/lane) were then electrophoresed in reducing 12% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Bio-Rad Laboratories), which were blocked for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. Primary antibodies used in this study, their sources and concentrations, were as follows: polyclonal rabbit anti-mouse p27Kip1, C-19 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:200 dilution), monoclonal rat anti-mouse cyclin D2, 34B1-3 (Santa Cruz; 1:100 dilution), polyclonal rabbit anti-mouse E2F-1, C-20 (Santa Cruz; 1:1000 dilution), polyclonal rabbit anti-mouse Cdk2, M2 (Santa Cruz; 1:100 dilution), polyclonal rabbit anti-mouse Cdk4, C-22 (Santa Cruz; 1:100 dilution), and polyclonal rabbit anti-human c-Myc (Upstate Biotechnology, Lake Placid, NY; 1:500 dilution). Primary antibody probing was followed by a 1-h incubation at room temperature with horseradish peroxide-conjugated secondary antibody (polyclonal goat anti-rabbit IgG; Boehringer-Mannheim, Mannheim, Germany; 1:5000 dilution) and detection, using an enhanced chemiluminescence system (Boehringer-Mannheim) according to kit specifications. Polyclonal rabbit anti-rat IgG (a gift from Dr. Achsah Keegan, American Red Cross, Rockville, MD) was used at 1:4000 dilution for cyclin D2 Western blots before using horseradish peroxide-conjugated secondary antibody. NIH Image software was used for densitometric analysis.

Northern Blot Analysis.
Total cellular RNA was isolated from twenty million cells using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. RNA was quantitated spectrophotometrically, equivalent amounts (20 µg) were run on 1.2% formaldehyde/agarose gel with 1 µg/ml ethidium bromide, and transferred to the nylon membrane Nytran Plus (Schleicher & Schuell, Keene, NH). Murine p27Kip1 cDNA (kindly provided by Dr. Tony Hunter, Salk Institute, La Jolla, CA) was radioactively labeled using High Prime labeling kit (Boehringer Mannheim). The probe was purified using Nick Columns (Pharmacia Biotech). Membranes were stripped and reprobed with radioactively labeled murine ß-actin cDNA. Membranes were UV cross-linked, prehybridyzed for at least 2 h at 42°C, hybridyzed overnight at 42°C, and washed, and mRNA levels were detected by autoradiography.

Acknowledgments

We thank Drs. Carolyn Mueller, Achsah Keegan, and Wendy Davidson for critical reading of the manuscript and Therese Grdina and Bourke Maddox for technical assistance. We also thank Drs. Marylin Kehry, Yufang Shi, Tony Hunter, and Achsah Keegan for providing reagents.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by USPHS Grant CA55644. Back

2 To whom requests for reprints should be addressed at Immunology Department, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Phone: (301) 517-0335; Fax: (301) 517-0344; E-mail: scottd{at}usa.redcross.org Back

3 The abbreviations used are: mIgM, membrane IgM; mIgD, membrane IgD; CDK, cyclin-dependent kinase; CKI, cyclin dependent kinase inhibitor; RB, retinoblastoma; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; CsA, cyclosporin A; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; Z-VAD-FMK, carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone; IL, interleukin; BCR, B-cell receptor; NF{kappa}B, nuclear factor {kappa}B; PI3K, phosphatidylinositol 3-kinase; PI, propidium iodide; NFATc, nuclear factor of activated T cells (cytoplasmic). Back

4 S. Liu, G. Carey, W. Davidson, and D. Scott. Signaling via the BCR ITAM leads to downregulation of c-Myc, growth arrest and apoptosis, manuscript in preparation. Back

5 G. Carey and D. W. Scott, unpublished results. Back

6 G. Sonenshein, personal communication. Back

7 T. Grdina, E. Behre, and D. Scott, unpublished results. Back

Received for publication 4/19/99. Revision received 8/13/99. Accepted for publication 8/31/99.

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