- Email: [email protected]
Involvement of Polyamines in B Cell Receptor–Mediated Apoptosis: Spermine Functions as a Negative Modulator
Experimental Cell Research 265, 174 –183 (2001) doi:10.1006/excr.2001.5177, available online at http://www.idealibrary.com on
Involvement of Polyamines in B Cell Receptor–Mediated Apoptosis: Spermine Functions as a Negative Modulator Takeshi Nitta,* Kazuei Igarashi,† Atsuya Yamashita,* Mikio Yamamoto,‡ and Naoki Yamamoto* ,1 *Department of Microbiology and Molecular Virology, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; †Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan; and ‡Department of Biochemistry II, National Defense Medical College, Saitama, Japan
INTRODUCTION The B cell lymphoma WEHI231 has been used as a model for studying clonal deletion of B cells on the basis of its ability to undergo growth arrest and apoptosis by B cell antigen receptor (BCR) cross-linking. To comprehensively analyze the genes involved in BCR-mediated apoptosis, we applied the technique of serial analysis of gene expression (SAGE) to WEHI231. Comparison of expression patterns revealed that BCR cross-linking caused coordinate changes in the expression of genes involved in polyamine metabolism. Polyamines are ubiquitous compounds required for cell proliferation and homeostasis. The coordinate expression of the polyamine-related genes was confirmed by semiquantitative reverse transcriptase– polymerase chain reaction analysis. During apoptosis, the genes involved in polyamine biosynthesis were downregulated, whereas those involved in polyamine catabolism were upregulated, suggesting that intracellular polyamines play a role in BCR-mediated apoptosis. Levels of intracellular putrescine, spermidine, and spermine were reduced after BCR crosslinking. These effects were prevented by concurrent CD40 stimulation, which blocked BCR-mediated apoptosis. Furthermore, addition of spermine could repress the BCR-mediated apoptosis by attenuating the mitochondrial membrane potential (⌬m) loss and activation of caspase-7 induced by BCR signaling. These findings strongly suggest that polyamine regulation is involved in apoptosis during B cell clonal deletion. © 2001 Academic Press
Key Words: apoptosis; B cell; polyamine; SAGE; spermine; WEHI231.
The murine immature B lymphoma cell line WEHI231 has been widely used as an in vitro model to study the regulation of B cell activation and apoptosis [1]. WEHI231 cells express surface immunoglobulin M (sIgM) 2 [1, 2] and undergo apoptosis after cross-linking of sIgM with anti-IgM antibody, which functions as a surrogate antigen [3, 4]. These effects can be blocked by concurrent stimulation of the CD40 receptor [5]. Apoptosis induced by signaling via antigen receptors could account for the mechanism by which self-reactive B cells are deleted. The observation that apoptosis is prevented by CD40 suggests that non–self-reactive B cells selectively proliferate by contact with activated T cells that express the CD40 ligand. Therefore, studies of B cell receptor (BCR)-mediated apoptosis of WEHI231 may provide information about the molecular mechanisms underlying clonal deletion of B cells. In the present study, we used serial analysis of gene expression (SAGE) [6] to comprehensively analyze changes in gene expression during BCR-mediated apoptosis in WEHI231 cells. SAGE allows qualitative and quantitative analysis of a large number of transcripts in any given cell or tissue without previous knowledge of the genes. Profiling of the gene expression pattern revealed that BCR cross-linking caused coordinate changes of the expression of a group of genes involved in metabolism of polyamines. Polyamines, namely putrescine, spermidine, and spermine, are ubiquitous and essential compounds in both eukaryotic and prokaryotic cells and are needed for cell growth and proliferation [7, 8]. The intracellular concentrations of polyamines are tightly regulated 2
1 To whom reprint requests should be addressed at Department of Microbiology and Molecular Virology, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Fax: 81-3-5803-0124. E-mail: yamamoto.mmb@ med.tmd.ac.jp.
0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: APCHA, N-(3-aminopropyl) cyclohexylamine; BCR, B cell receptor; OAZ, ornithine decarboxylase antizyme; ODC, ornithine decarboxylase; RT–PCR, reverse transcriptase–polymerase chain reaction; SAGE, serial analysis of gene expression; SAMDC, S-adenosylmethionine decarboxylase; sIgM, surface immunoglobulin M; SSAT, spermidine/spermine N 1 -acetyltransferase; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling.
174
POLYAMINE REGULATION IN B CELL APOPTOSIS
by biosynthesis, degradation, and transport [7, 8]. In the polyamine biosynthetic pathway, ornithine decarboxylase (ODC), the first and rate-limiting enzyme, catalyzes the decarboxylation of L-ornithine, leading to formation of putrescine. Spermidine synthase catalyzes the production of spermidine from putrescine, and subsequently spermine synthase catalyzes the production of spermine from spermidine, both by adding an aminopropyl group to the precursor. The donor of the aminopropyl moiety is decarboxylated adenosylmethionine, which is produced by S-adenosylmethionine decarboxylase (SAMDC). Catabolism of polyamines is essentially a reverse of their biosynthesis. Spermidine/ spermine N 1-acetyltransferase (SSAT) catalyzes the formation of acetylspermine and acetylspermidine, which are oxidized to spermidine and putrescine, respectively, by a constitutive enzyme polyamine oxidase. SSAT is induced when intracellular polyamines overaccumulate [9, 10]. Overaccumulation of cellular polyamines also induces a unique regulatory protein, ornithine decarboxylase antizyme (OAZ) [11, 12]. OAZ is a nonenzymatic protein that binds to ODC, inactivates it, and accelerates its degradation [11, 12]. OAZ also blocks cellular polyamine uptake and stimulates its excretion [13–15]. The highly regulated pathways of polyamine metabolism highlight the importance of these compounds in cell physiology. Polyamines are required for DNA replication, cell proliferation, cell homeostasis, and tumorigenesis. Indeed, the polyamine biosynthetic pathway is a target of some anticancer drugs [7, 8, 16]. Depletion of polyamines is often lethal, resulting in cell cycle arrest or apoptosis [17–19]. It has been reported that several forms of apoptosis, induced by cellular signaling, are closely related to polyamine regulation [19 – 21]. Our investigation revealed coordinate changes in the expression of genes involved in polyamine metabolism, suggesting an implication of polyamines in BCRmediated apoptosis. Our results suggest that modulation of intracellular polyamines plays a role in apoptosis during B cell clonal deletion. MATERIALS AND METHODS Cell culture. The WEHI231 (kindly provided by T. Tsubata) cell line was maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 M 2-mercaptoethanol, 10% (v/v) fetal bovine serum, 100 U/ml penicillin G, and 100 g/ml streptomycin. In experiments involving the addition of polyamines in cell culture, 1 mM aminoguanidine was added as an inhibitor of amine oxidase derived from fetal bovine serum. Aminoguanidine had no effect on the various parameters of the cell measured in this study. Reagents. Goat F(ab⬘)2 anti-mouse IgM (Southern Biotechnology Associates) and hamster anti-mouse CD40 monoclonal antibodies (Pharmingen) were used in this study. Rat anti-mouse HS1 monoclonal antibody was kindly provided by T. Watanabe [22, 23]. Putrescine 䡠 2HCl, spermidine 䡠 3HCl, and spermine 䡠 4HCl were pur-
175
chased from Sigma. N-(3-aminopropyl) cyclohexylamine (APCHA) was described previously [18]. Putrescine was used at 4 mM concentration, spermidine at 2 mM, spermine at 0.5 mM, and APCHA at 10 M, except in a dose–response examination. Cell cycle analysis. For cell cycle analysis, cells were fixed with 70% ethanol and treated with 0.5 mg/ml RNase A at 37°C for 20 min. Then cells were stained with 30 g/ml propidium iodide (Sigma) and analyzed by FACSCalibur (Becton Dickinson). Cell cycle profile of the living cell population was determined by means of ModFit LT (Becton Dickinson). Proportion of subdiploid cells in the total population was analyzed by Cell Quest (Becton Dickinson). Determination of samples for SAGE analysis. To determine the point when the G1 arrest and apoptosis occurred and to obtain the correct information of gene expression associated with cell cycle and apoptosis, we used a cell cycle synchronization system [24]. By treatment with thymidine and nocodazole, cells were synchronized at M phase. After restart of growth, control WEHI231 cells synchronously progressed cell cycle throughout growth. Cells treated with anti-IgM at 0 h, in contrast, were arrested at G1 phase 16 h after growth restart and could not enter into S phase, and subsequently apoptotic cells increased. Thus, G1-synchronized control cells at 12 h, and cells treated with anti-IgM for 16 h that were arrested at G1 phase and undergoing apoptosis, were determined to be analyzed by SAGE. SAGE analysis. Total RNAs were isolated with the RNeasy Mini kit (Qiagen) and cleaned by the treatment with DNase I (GenHunter). SAGE libraries were constructed from 50 g total RNA from control and anti-IgM–treated cells, as described previously [25, 26]. Fourteen– base pair sequence tags from each cDNA were concatemerized, cloned, and sequenced. PROGENEX software (Fujiyakuhin) was applied to extract SAGE tags and generate gene expression profiles [25, 26]. Transcripts corresponding to the obtained tags were identified by homology searching against database by the BLAST program. Reverse transcriptase–polymerase chain reaction (RT–PCR). For expression analysis of many genes on a limited amount of RNA, a convenient real-time RT–PCR was performed by SYBR Green PCR Core Reagents and ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). First-strand cDNAs were synthesized from DNase I–treated total RNA by MultiScribe Reverse Transcriptase (PE Applied Biosystems) using random hexamer as primers. cDNA from cells at 0 h was gradually diluted and used as a standard, and the amount of mRNA was relatively quantified by comparing threshold cycle of each sample with those of the standard. The threshold cycle was determined by detecting the increase in signal associated with an exponential growth of PCR product. Accuracy of the amplification of each gene and the absence of nonspecific amplification were confirmed by analyzing PCR products by polyacrylamide gel electrophoresis. All primers used for PCR were designed by PrimerExpress software (PE Applied Biosystems). A list of oligonucleotide primers is available from the authors upon request. Measurement of polyamine contents. Polyamine content was measured essentially according to the method described previously [27]. Cells were treated with 5% trichloroacetic acid, and the polyamines in the supernatant were separated on a high-performance liquid chromatography system. The precipitate was stored for measurement of protein contents. Western blot analysis. Tyrosine phosphorylation of HS1 after sIgM cross-linking in WEHI231 cells was analyzed according to previous report [22]. Cells were preincubated with or without putrescine, spermidine, or spermine at 37°C for 1 h and treated with anti-IgM at 37°C for 1 min. The cells were lysed and subjected to Western blot analysis with an anti-phosphotyrosine monoclonal antibody, PY20, labeled with horseradish peroxidase (TaKaRa), or with a rat monoclonal antibody to mouse HS1 followed by peroxidase-
176
NITTA ET AL.
labeled anti-rat IgG (TAGO Immunologicals). The blots were developed by the ECL system (NEN). DNA fragmentation assay. For the DNA laddering assay, cellular DNAs were prepared as described previously [28]. The samples were subjected to 2% agarose gel electrophoresis, followed by staining with ethidium bromide. In Situ Cell Death Detection kit, Fluorescein (Roche Diagnostics), based on terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method, was used for quantitative analysis of DNA fragmentation. Cells were fixed with 2% paraformaldehyde, permeabilized by 0.1% Triton X-100, and incubated with TUNEL reaction mixture at 37°C for 60 min in the dark. The cells were then analyzed by FACSCalibur. Enzyme assay for caspase activity. Caspase activation in WEHI231 cells was assessed by the CPP32/Caspase-3 Fluorometric Protease Assay Kit (MBL). Cells were lysed with cell lysis buffer, and the lysate was incubated with 50 M of the fluorescent substrate Asp–Glu–Val–Asp–7-amino– 4-trifluoromethyl coumarin at 37°C for 60 min. Cleaved substrate fluorescence was determined by luminescence spectrometry (LS50B; PE Applied Biosystems) with a 400-nm excitation filter and 505-nm emission filter. Caspase activity was determined as the fluorescence intensity normalized by protein amount. Mitochondrial membrane potential. After cell culture, cells were incubated with 40 nM of 3,3⬘-dihexyloxacarbocynine iodide [DiOC6(3); Sigma] for 15 min at 37°C, and then analyzed by FACSCalibur. Proportion of cells with depolarized mitochondria was analyzed by Cell Quest.
RESULTS
SAGE Analysis Revealed Coordinate Expression of Polyamine-Related Genes Gene expression was studied by SAGE analysis in control WEHI231 cells and cells stimulated with antiIgM antibody. We obtained 10,236 and 10,351 tags representing 4578 and 4720 unique transcripts for control and anti-IgM–treated cells, respectively. Profiles of relative gene expression patterns were generated by comparing the occurrence of each tag between two samples. In the gene expression profiles, one of the most differentially expressed tags corresponded to the gene that encodes spermidine synthase, an enzyme involved in polyamine biosynthesis (eight tags in control vs. one tag in anti-IgM; Table 1). In light of this, we looked at the profiles for tags corresponding to other genes whose products regulate polyamine metabolism. Expression of six genes was found to be altered (Table 1). Interestingly, four genes downregulated by antiIgM encoded enzymes involved in polyamine biosynthesis, whereas two genes upregulated by anti-IgM encoded an enzyme in the catabolic route (SSAT) or a regulatory protein inhibiting the polyamine biosynthesis (OAZ). These results suggest that expression of genes that are involved in polyamine metabolism is altered in response to BCR stimulation in WEHI231 B cells.
TABLE 1 SAGE Profile of Genes Involved in Polyamine Metabolism Frequency Gene description
Accession no. a L19311
CACAATGTAAGCTT
Spermidine synthase ODC Spermine synthase SAMDC
TCCGACGAGCGGCT TTAGAGTTTCTGTT
OAZ SSAT
Tag sequence AGCACCGCCTATGC ATTGGCAGAATGGG GCCAAAGAAGGGAG
a b c
Control b
IgM c
8
1
M10624 AF031486
12 3
5 2
Z14986 S50054 U52822 L10244
1
0
5 0
12 1
Matched GenBank accession number. The number of each tag in 10,236 transcript is indicated. The number of each tag in 10,351 transcript is indicated.
Coordinate Changes in the Expression of PolyamineRelated Genes by BCR or CD40 Signals Treatment of WEHI231 with anti-IgM arrested the cell cycle at G1 by 24 h, and the cells subsequently underwent apoptosis. The G1 arrest and apoptosis were prevented by concurrent treatment with antiCD40 antibody, which, by itself, had no effect (Figs. 1A, 1B). To confirm the results from the SAGE analysis, we performed semiquantitative RT–PCR analysis on the genes involved in polyamine metabolism. The relative changes in mRNA levels of six genes, including spermidine synthase, spermine synthase, SAMDC, ODC, SSAT, and OAZ, were determined after treatment with anti-IgM, anti-CD40, or anti-IgM plus anti-CD40 (Fig. 1C). Mouse 18S ribosomal RNA was used as an indicator of total RNA amount (data not shown). Anti-IgM caused significant reduction of the mRNA levels of spermidine synthase, spermine synthase, and SAMDC. Downregulation of these genes was prevented by treatment with anti-CD40. In the case of spermidine synthase, the CD40 by itself had an effect on mRNA levels, increasing the level of mRNA by about twofold (Fig. 1C). The expression of ODC, the rate-limiting enzyme in the polyamine biosynthesis, was slightly induced by anti-IgM at 2 h, and after that, it was not significantly affected. In cells treated with anti-CD40 or anti-IgM plus anti-CD40, induction of the ODC expression was observed at 12 h. A different profile was seen with SSAT and OAZ. These mRNAs were immediately upregulated by antiIgM treatment, and their expression remained elevated for 24 h. These effects could be prevented by anti-CD40 treatment. Collectively, in cells treated with anti-IgM, the genes involved in polyamine synthesis were repressed, and the genes involved in the catabolic
POLYAMINE REGULATION IN B CELL APOPTOSIS
177
FIG. 1. Coordinate changes of expression of polyamine metabolism regulatory genes during BCR-mediated apoptosis in WEHI231 cells. Cells were cultured in the absence (closed squares) or presence of 5 g/ml anti-IgM (open squares), 1 g/ml anti-CD40 (open triangles), or 5 g/ml anti-IgM plus 1 g/ml anti-CD40 (open circles). At the time indicated, cells were harvested and the cell cycle profile was determined by flow cytometry analysis. Percentages of cells in G1 phase (A) and subdiploid cells (B) were determined as described under Materials and Methods. Total RNA was isolated from cells at the time indicated, and 1 g of RNA was subjected to semiquantitative RT–PCR (C). The amount of mRNA of each gene was expressed relative to the amount at 0 h. Values represent the means ⫾ SD of three individual experiments. OAZ, ornithine decarboxylase antizyme; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SPM, spermine; SSAT, spermidine/spermine N 1 -acetyltransferase.
pathway were upregulated. With the exception of ODC, the results obtained from RT–PCR are consistent with those from the SAGE profile. The extent of changes of genes did not always agree between two methods, most probably because the number of tags in our SAGE analysis was not enough (approximately 10,000 tags for each sample) to correctly quantify the expression
levels of genes. Concurrent stimulation of CD40 prevented changes in the expression of these genes. Changes in Intracellular Polyamine Levels during Apoptosis in WEHI231 Cells We measured the intracellular concentrations of three polyamines—putrescine, spermidine, and sperm-
FIG. 2. Changes of polyamine contents during the cell cycle arrest and apoptosis by anti-IgM and their restoration by anti-CD40. Culture conditions and symbols represented were same as in Fig. 1. Cells were harvested and intracellular concentration of putrescine (PUT), spermidine (SPD), and spermine (SPM) were determined by high-performance liquid chromatography analysis. Values represent the means ⫾ SD of three individual experiments.
178
NITTA ET AL.
ine—which are the final cellular products of the genes described above (Fig. 2). The level of putrescine decreased rapidly after anti-IgM treatment and was undetectable by 24 h. The level of spermidine was also reduced by anti-IgM treatment, but the rate of decline was slower than that of putrescine. The depletion of putrescine and spermidine was partially prevented by concurrent stimulation of CD40. The level of spermine was slightly decreased up to 24 h after anti-IgM treatment, but subsequently decreased to about half of control by 36 h, when cells were completely arrested at G1 and apoptosis progressed. The reduction of spermine level was completely prevented by treatment with antiCD40. Thus, all three polyamines were significantly decreased during G1 arrest and apoptosis caused by BCR cross-linking, although the time course and magnitude was different for each polyamine. The depletion of polyamines could be prevented by concurrent signaling via CD40. These results suggest that polyamines, especially spermine, play some role in controlling apoptosis of WEHI231 cells. Inhibitory Effect of Spermine on BCR-Mediated Apoptosis Several types of apoptosis or growth arrest have been reported to be attenuated by the addition of exogenous polyamines [19 –21]. We tested whether exogenous polyamines could prevent the apoptosis induced by anti-IgM treatment in WEHI231 cells. DNA ladder formation induced by anti-IgM was significantly reduced by exogenously added spermine, but not putrescine or spermidine (Fig. 3A). The inhibitory effect of spermine on anti-IgM–induced apoptosis and its dose-dependent response were confirmed by measuring the extent of DNA fragmentation by in situ TUNEL assay (Figs. 3B, 4A). The increase of the frequency of subdiploid cells after anti-IgM treatment was also reduced in the presence of exogenous spermine, although the G1 arrest was not affected (Fig. 4B). Uptake of exogenous polyamines was confirmed by measuring intracellular polyamine levels (Table 2). Extracellular addition of each polyamine increased its own intracellular level but decreased levels of the other polyamines, probably due to feedback regulation of the biosynthetic enzymes. To determine whether the exogenous spermine interferes with the cross-linking of sIgM or not, we analyzed the tyrosine phosphorylation of HS1. The HS1 protein, a substrate of nonreceptor type protein–tyrosine kinase, is phosphorylated immediately after cross-linking of sIgM, and its phosphorylation is a key step in BCR-mediated apoptosis in WEHI231 cells [22, 23]. Anti-IgM induced tyrosine phosphorylation of HS1 protein, and the presence of exogenous polyamines did
FIG. 3. Effects of exogenous polyamines on BCR-mediated apoptosis in WEHI231 cells. WEHI231 cells pretreated with putrescine (PUT), spermidine (SPD), or spermine (SPM) for 1 h were cultured with anti-IgM (5 g/ml) for 36 h. Total cellular DNAs from 2 ⫻ 10 5 cell equivalents were subjected to DNA laddering assay (A). Percentage of cells with fragmented DNA was determined by flow cytometry analysis with in situ TUNEL reaction (B). Each column represents the mean ⫾ SD of at least three individual experiments. Statistical differences were assessed by Student’s t test. *P ⬍ 0.01 compared with anti-IgM–treated cells. Effect of exogenous polyamines on tyrosine phosphorylation of HS1 protein was analyzed by Western blotting (C) with anti-phosphotyrosine (upper panel) or anti-mouse HS1 (lower panel).
not alter the phosphorylation of HS1 (Fig. 3C). These results indicate that spermin protected apoptosis by reasons other than interfering with sIgM cross-linking and initial step of BCR signaling pathway. To further confirm the inhibitory effect of spermine on apoptosis, APCHA, an inhibitor of spermine synthase [18], was added to cells treated with anti-IgM. Enforced depletion of intracellular spermine by APCHA increased the frequency of subdiploid cells (Table 2; Fig. 4B) and DNA fragmentation (Fig. 4C) induced by anti-IgM. The effect of APCHA was reversed by exogenously added spermine. APCHA alone did not
POLYAMINE REGULATION IN B CELL APOPTOSIS
179
FIG. 4. Effects of exogenous spermine or APCHA on BCR-mediated apoptosis in WEHI231. Cells pretreated or untreated with spermine (SPM) or N-(3-aminopropyl) cyclohexylamine (APCHA) for 1 h were cultured with or without anti-IgM (5 g/ml) and/or anti-CD40 (1 g/ml) for 36 h. In panel A, SPM was used at the concentration indicated. DNA fragmentation was quantified by flow cytometry analysis with in situ TUNEL reaction (A and C). Each column represents the mean ⫾ SD of at least three individual experiments. **P ⬍ 0.05 and *P ⬍ 0.01 compared with anti-IgM–treated cells. Panel B shows cell cycle profile analyzed by flow cytometry. Values above horizontal bars represent the percentages of subdiploid cells in the total cell population. Percentages of cells at G1 phase in the living cell population are denoted on the right side of the G1 peak.
induce apoptosis, probably because of a compensatory effect by an increase in spermidine level. The effects of spermine on apoptosis were correlated with caspase activation and mitochondrial alteration. Caspases, a family of cysteine proteases, act as effectors of apoptosis (reviewed in Thornberry and Lazebnik [29]). BCR-mediated apoptosis of WEHI231 is dependent on the activation of caspase-7/Mch3, a member of the CPP32/caspase-3 family [30, 31]. The activation of CPP32/caspase-3 in BCR-mediated apoptosis is closely associated with mitochondrial dysfunction, including loss of the mitochondrial membrane potential (⌬m) [30, 32, 33]. An increase in the level of cellular spermine reduced the activity of caspase-7/Mch3 induced by anti-IgM, whereas depletion of spermine by APCHA enhanced activation of
caspase-7/Mch3 (Table 3; Fig. 5A). The percentage of cells with depolarized mitochondria was also significantly reduced by spermine and increased by APCHA (Fig. 5B). Anti-CD40 also prevented the apoptotic changes including DNA fragmentation, caspase-7/ Mch3 activation, and ⌬m loss induced by anti-IgM (Figs. 4C, 5). DISCUSSION
WEHI231 cells have been extensively used as an in vitro model to understand the mechanisms controlling cell death and survival in clonal deletion during B cell development. However, the cellular mechanisms involved in BCR signaling, in which many factors are orchestrated to execute the death program, are not
180
NITTA ET AL.
TABLE 2 Effects of Exogenous Polyamines or APCHA on Intracellular Polyamine Contents of WEHI231 Cells a Polyamine concentration (pmol/g protein) Variable
PUT
SPD
SPM
Control IgM IgM ⫹ PUT (4 mM) IgM ⫹ SPD (2 mM) IgM ⫹ SPM (0.1 mM) IgM ⫹ SPM (0.2 mM) IgM ⫹ SPM (0.5 mM) IgM ⫹ APCHA IgM ⫹ APCHA ⫹ SPM (0.5 mM) SPM (0.5 mM) APCHA APCHA ⫹ SPM (0.5 mM)
2.1 ⫾ 0.2 ⬍0.1 72.1 ⫾ 19.0 4.0 ⫾ 1.1 ⬍0.1 ⬍0.1 0.3 ⫾ 0.1 0.1 ⫾ 0.04
14.5 ⫾ 0.9 1.3 ⫾ 0.4 6.2 ⫾ 1.5 39.3 ⫾ 9.1 0.4 ⫾ 0.1 0.8 ⫾ 0.1 1.4 ⫾ 0.4 7.6 ⫾ 1.2
20.4 ⫾ 1.5 10.2 ⫾ 3.4 11.2 ⫾ 3.4 6.2 ⫾ 0.2 15.6 ⫾ 0.8 20.3 ⫾ 1.5 26.3 ⫾ 3.1 5.4 ⫾ 0.2
0.7 ⫾ 0.02 0.2 ⫾ 0.1 0.3 ⫾ 0.1
1.4 ⫾ 0.1 2.3 ⫾ 1.4 31.1 ⫾ 3.6
18.9 ⫾ 0.6 32.9 ⫾ 4.1 5.0 ⫾ 0.1
0.4 ⫾ 0.1
1.7 ⫾ 0.2
22.9 ⫾ 0.6
a Culture conditions were same as in Figs. 3 and 4. Cells were harvested at 36 h, and intracellular concentrations of putrescine (PUT), spermidine (SPD), and spermine (SPM) were determined by high-performance liquid chromatography analysis. Values represent the means ⫾ SD.
fully understood. In this study, we demonstrate that BCR signaling leads to a decrease in intracellular polyamines by coordinate changes in the expression of genes involved in polyamine metabolism. It is well established that ODC is a key enzyme in polyamine regulation. In WEHI231 cells, however, expression of ODC mRNA is less affected by anti-IgM or anti-CD40 treatment than is the expression of the other polyamine regulatory genes. Coordinate changes TABLE 3 Effects of Exogenous Spermine or APCHA on Intracellular Polyamine Contents of WEHI231 Cells a Polyamine concentration (pmol/g protein) Variable
PUT
SPD
SPM
Control IgM IgM ⫹ SPM IgM ⫹ APCHA IgM ⫹ APCHA ⫹ SPM IgM ⫹ CD40
2.7 ⫾ 0.4 ⬍0.1 0.2 ⫾ 0.1 ⬍0.1 0.2 ⫾ 0.1 2.1 ⫾ 1.3
16.8 ⫾ 1.2 3.8 ⫾ 0.2 1.2 ⫾ 0.2 12.8 ⫾ 0.9 1.8 ⫾ 0.2 13.4 ⫾ 0.9
19.3 ⫾ 1.1 15.3 ⫾ 1.6 24.6 ⫾ 1.9 7.2 ⫾ 0.1 23.2 ⫾ 0.5 20.1 ⫾ 2.1
a Culture conditions were same as in Fig. 5. Cells were harvested at 30 h, and intracellular concentrations of putrescine (PUT), spermidine (SPD), and spermine (SPM) were determined by high-performance liquid chromatography analysis. Values represent the means ⫾ SD.
FIG. 5. Effect of exogenous spermine or N-(3-aminopropyl) cyclohexylamine (APCHA) on caspase-7/Mch3 activity and ⌬m collapse. Cells pretreated or untreated with spermine (SPM) or APCHA for 1 h were cultured with or without anti-IgM (5 g/ml) and/or anti-CD40 (1 g/ml). Cells were harvested at 30 h and were analyzed as described under Materials and Methods. Each column represents the mean ⫾ SD of three individual experiments. **P ⬍ 0.05 and *P ⬍ 0.01 compared with anti–IgM-treated cells.
in the expression of spermidine synthase, spermine synthase, SAMDC, and SSAT are probably responsible for the changes in intracellular spermidine and spermine levels after anti-IgM treatment. The decrease in intracellular putrescine after anti-IgM treatment in WEHI231 may be due to a posttranscriptional downregulation of ODC by OAZ, which is rapidly induced by anti-IgM. Although the expression of OAZ is known to be regulated by a unique posttranscriptional machinery [11], enhanced transcription also has a significant effect on polyamine content and cell growth [34, 35]. In addition, it is likely that the regulatory gene or genes that encode cell-surface polyamine transporters are also involved in the control of intracellular polyamine levels during BCR-mediated apoptosis [36]. However, the genes encoding mammalian cell polyamine transporters have not yet been identified. OAZ also negatively regulates the intracellular polyamine levels by inhibiting uptake of polyamines and by promoting their excretion [13–15]. Induction of OAZ
POLYAMINE REGULATION IN B CELL APOPTOSIS
expression by BCR cross-linking and its repression by CD40 signaling might play important roles in regulating the polyamine levels in WEHI231 cells, although further analysis will be required to demonstrate the role of OAZ in BCR-mediated apoptosis. In any event, coordinate expression of the metabolic regulatory genes after BCR cross-linking suggests a commitment of the cell to actively decrease intracellular polyamines. CD40 signaling prevents the effects of antiIgM on the gene expression and polyamine levels. In particular, the intracellular spermine level was completely maintained by CD40 signaling, suggesting the particular importance of this polyamine in the reduction of the BCR-mediated apoptosis. Like the WEHI231 cell line, primary splenic B cells decreased the intracellular levels of polyamines after apoptosis induction by anti-IgM, but concurrent antiCD40 treatment recovered them (data not shown). These alterations of intracellular polyamines were also preceded by coordinate expression of the metabolic regulatory genes in splenic B cells. These results suggest that the regulation of cellular polyamines was closely associated with apoptosis and survival of B cells in clonal deletion in vivo. Exogenous spermine attenuated but did not completely prevent BCR-mediated apoptotic changes, including DNA fragmentation and hypodiploidy. Thus, spermine might be a mediator of the execution step but not a dominant regulator of apoptosis. It has been reported that exogenous spermine prevents DNA fragmentation during apoptosis in rat thymocytes [37] and in the mouse T cell line CTLL-2 [20]. It was suggested that the protective effect of spermine on DNA fragmentation is due to direct binding of spermine to DNA and modification of chromatin structure [38]. Spermine has also been shown to prevent apoptosis of cultured cerebellar neurons, and its inhibitory effect appears to be mediated at a site upstream of caspase activation [39]. Indeed, our results agree with the latter model. The level of intracellular spermine correlated with the extent of caspase-7/Mch3 activity induced by BCR signaling, suggesting that spermine plays a role in the cellular cascade involved in caspase activation. In BCRmediated apoptosis, activation of CPP32/caspase-3 family caspase or caspases, which function as effector caspases, is preceded by mitochondrial dysfunction [30, 32]. It has been proposed that the alteration in mitochondrial function plays a critical role in activation of the caspase cascade, possibly due to a release of cytochrome c and/or other caspase activators from mitochondria into the cytosol [29, 33]. Our results that the ⌬m loss was prevented by spermine and enhanced by its depletion indicate that spermine has a protective effect on mitochondrial alteration during apoptosis.
181
Many other biochemical studies have demonstrated that spermine inhibits the ⌬m collapse in vitro [40, 41], suggesting that spermine directly affects mitochondria. Thus, it is suggested that depletion of intracellular spermine by BCR signaling plays a role in the reduction of the mitochondrial function to accelerate caspase activation and apoptosis. BCR-mediated apoptosis is defective in NZB and NZB/W mice that are prone to systemic autoimmune diseases, and it is therefore thought to be one of the key pathogenic events of autoimmune diseases [42]. It has been suggested that polyamines also play an important role in autoimmune diseases. Serum polyamine levels, cellular polyamine levels, or both are elevated in patients with rheumatoid arthritis [43] or systemic lupus erythematosus [44] and in NZB/W mice [45]. Moreover, inhibition of polyamine biosynthesis was reported to produce a remarkable improvement in several clinical symptoms of NZB/W mice [45]. In this study, we demonstrate that polyamine regulation is implicated in the control of BCRmediated apoptosis of B cells. Therefore, WEHI231 cells may be a useful tool to study the role and function of polyamine not only in cellular apoptotic cascade but also in the pathogenesis and chemotherapy of autoimmune diseases. We thank Dr. Keith Williams for kind suggestions and help in preparing this manuscript. We also thank Dr. Takeshi Tsubata for providing the WEHI231 cell line, Dr. Takeshi Watanabe for providing anti-mouse HS1 antibody, and Dr. Kazuhiro Nishimura for exceptional assistance with high-performance liquid chromatography analysis. We are also grateful to Drs. Shoji Yamaoka and Norio Yamamoto for helpful discussions. This work was supported by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Drug ADR Relief, R&D Promotion, and Products Review of Japan, grants from the Japan Health Science Foundation, and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation.
REFERENCES 1.
2. 3.
4.
5.
6.
Boyd, A. W., and Schrader, J. W. (1981). The regulation of growth and differentiation of a murine B cell lymphoma. J. Immunol. 126, 2466 –2469. Ralph, P. (1979). Functional subsets of murine and human B lymphocyte cell lines. Immunol. Rev. 48, 107–121. Benhamou, L. E., Cazenave, P. A., and Sarthou, P. (1990). Anti-immunoglobulins induce death by apoptosis in WEHI-231 B lymphoma cells. Eur. J. Immunol. 20, 1405–1407. Hasbold, J., and Klaus, G. G. (1990). Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas. Eur. J. Immunol. 20, 1685–1690. Tsubata, T., Wu, J., and Honjo, T. (1993). B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature 364, 645– 648. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270, 484 – 487.
182
NITTA ET AL. 22.
Fukuda, T., Kitamura, D., Taniuchi, I., Maekawa, Y., Benhamou, L. E., Sarthou, P., and Watanabe, T. (1995). Restoration of surface IgM-mediated apoptosis in an anti-IgM–resistant variant of WEHI-231 lymphoma cells by HS1, a protein–tyrosine kinase substrate. Proc. Natl. Acad. Sci. USA 92, 7302– 7306.
Fogel-Petrovic, M., Kramer, D. L., Ganis, B., Casero, R. A. Jr., and Porter, C. W. (1993). Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N 1acetyltransferase—A gene uniquely regulated by polyamines and their analogs. Biochim. Biophys. Acta 1216, 255–264.
23.
Yamanashi, Y., Fukuda, T., Nishizumi, H., Inazu, T., Higashi, K., Kitamura, D., Ishida, T., Yamamura, H., Watanabe, T., and Yamamoto, T. (1997). Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis. J. Exp. Med. 185, 1387–1392.
Suppola, S., Pietila¨, M., Parkkinen, J. J., Korhonen, V.-P., Alhonen, L., Halmekyto¨, M., Porter, C. W., and Ja¨nne J. (1999). Overexpression of spermidine/spermine N 1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: Evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue. Biochem. J. 338, 311–316.
24.
Ishida, T., Kobayashi, N., Tojo, T., Ishida, S., Yamamoto, T., and Inoue, J. (1995). CD40 signaling-mediated induction of Bcl-XL, Cdk4, and Cdk6. Implication of their cooperation in selective B cell growth. J. Immunol. 155, 5527–5535.
25.
Ryo, A., Suzuki, Y., Ichiyama, K., Wakatsuki, T., Kondoh, N., Hada, A., Yamamoto, M., and Yamamoto, N. (1999). Serial analysis of gene expression in HIV-1–infected T cell lines. FEBS Lett. 462, 182–186.
26.
Ryo, A., Kondoh, N., Wakatsuki, T., Hada, A., Yamamoto, N., and Yamamoto, M. (2000). A modified serial analysis of gene expression that generates longer sequence tags by nonpalindromic cohesive linker ligation. Anal. Biochem. 277, 160 –162.
27.
Igarashi, K., Kashiwagi, K., Hamasaki, H., Miura, A, Kakegawa, T., Hirose, S., and Matsuzaki, S. (1986). Formation of a compensatory polyamine by Escherichia coli polyamine-requiring mutants during growth in the absence of polyamines. J. Bacteriol. 166, 128⫺134.
28.
Ishizawa, M., Kobayashi, Y., Miyamura, T., and Matsuura, S. (1991). Simple procedure of DNA isolation from human serum. Nucleic Acids Res. 19, 5792.
29.
Thornberry, N. A., and Lazebnik, Y. (1998). Caspases: Enemies within. Science 281, 1312–1316.
30.
Doi, T., Motoyama, N., Tokunaga, A., and Watanabe, T. (1999). Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231. Int. Immunol. 11, 933–941.
31.
Bra´s, A., Ruiz-Vela, A., Buitrago, G. G. D., and Martı´nez, A. C. (1999). Caspase activation by BCR cross-linking in immature B cells: Differential effects on growth arrest and apoptosis. FASEB J. 13, 931–944.
32.
Bouchon, A., Krammer, P. H., and Walczak, H. (2000). Critical role for mitochondria in B cell receptor–mediated apoptosis. Eur. J. Immunol. 30, 69 –77.
33.
Green, D. R., and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281, 1309 –1312.
34.
Murakami, Y., Matsufuji, S., Miyazaki, Y., and Hayashi, S. (1994). Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth. Biochem. J. 384, 183–187.
35.
Iwata, S., Sato, Y., Asada, M., Takagi, M., Tsujimoto, A., Inaba, T., Yamada, T., Sakamoto, S., Yata, J.-I., Shimogori, T., Igarashi, K., and Mizutani, S. (1999). Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation. Oncogene 18, 165– 172.
36.
DeBenedette, M., Olson, J. W., and Snow, E. C. (1993). Expression of polyamine transporter activity during B lymphocyte cell cycle progression. J. Immunol. 150, 4218 – 4224.
37.
Bru¨ne, B., Hartzell, P., Nicotera, P., and Orrenius, S. (1991). Spermine prevents endonuclease activation and apoptosis in thymocytes. Exp. Cell Res. 195, 323–329.
7.
Pegg, A. E. (1988). Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48, 759 –774.
8.
Marton, L. J., and Pegg, A. E. (1995). Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 35, 55–91.
9.
10.
11.
12.
13.
14.
Matsufuji, S., Matsufuji, T., Miyazaki, Y., Murakami, Y., Atkins, J. F., Gesteland, R. F., and Hayashi, S. (1995). Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80, 51– 60. Ivanov, I. P., Gesteland, R. F., and Atkins, J. F. (1998). A second mammalian antizyme: Conservation of programmed ribosomal framshifting. Genomics 52, 119 –129. Mitchell, J. L. A., Judd, G. G., Bareyal-Leyser, A., and Ling, S. Y. (1994). Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem. J. 299, 19 –22. Suzuki, T., He, Y., Kashiwagi, K., Murakami, Y., Hayashi, S., and Igarashi, K. (1994). Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells. Proc. Natl. Acad. Sci. USA 91, 8930 – 8934.
15.
Sakata, K., Kashiwagi, K., and Igarashi, K. (2000). Properties of a polyamine transporter regulated by antizyme. Biochem. J. 347, 297–303.
16.
Meyskens, F. L. Jr., and Gerner, E. W. (1999). Development of difluoromethylornithine (DFMO) as a chemoprevention agent. Clin. Cancer Res. 5, 945–951.
17.
Nakaike, S., Kashiwagi, K., Terao, K., Iio, K., and Igarashi, K. (1988). Combined use of alpha-difluoromethylornithine and an inhibitor of S-adenosylmethionine decarboxylase in mice bearing P388 leukemia or Lewis lung carcinoma. Jpn. J. Cancer Res. 79, 501–508.
18.
He, Y., Shimogori, T., Kashiwagi, K., Shirahata, A., and Igarashi, K. (1995). Inhibition of cell growth by combination of ␣-difluoromethylornithine and an inhibitor of spermine synthase. J. Biochem. 117, 824 – 829.
19.
Schipper, R. G., Pening, L. C., and Verhofstad, A. A. J. (2000). Involvement of polyamines in apoptosis. Facts and controversies: Effectors or protectors? Semin. Cancer Biol. 0, 55– 68, doi:10.1006/scbi.2000.0308.
20.
Min, A., Hasuma, T., Yano, Y., Matsui-Yuasa, I., and Otani, S. (1995). Regulation of apoptosis of interleukin 2– dependent mouse T-cell line by protein tyrosine phosphorylation and polyamines. J. Cell. Physiol. 165, 615– 623.
21.
Thomas, T., Balabhadrapathruni, S., Gardner, C. R., Hong, J., Faaland, C. A., and Thomas T. J. (1999). Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: Alterations in polyamine biosynthesis and the expression of p21/CIP1/ WAF1. J. Cell. Physiol. 179, 257–266.
POLYAMINE REGULATION IN B CELL APOPTOSIS 38.
Snyder, R. D. (1989). Polyamine depletion is associated with altered chromatin structure in HeLa cells. Biochem. J. 260, 697–704.
39.
Harada, J., and Sugimoto, M. (1997). Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons. Brain Res. 753, 251–259.
40.
Tassani, V., Biban, C., Toninello, A., and Siliprandi, D. (1995). Inhibition of mitochondrial permeability transition by polyamines and magnesium. Biochem. Biophys. Res. Commun. 207, 661– 667.
41.
Tassani, V., Campagnolo, M., Toninello, A., and Siliprandi, D. (1996). The contribution of endogenous polyamines to the permeability transition of rat liver mitochondria. Biochem. Biophys. Res. Commun. 226, 850 – 854.
Received October 12, 2000 Revised version received January 23, 2001 Published online March 16, 2001
42.
183
Kozono, Y., Kotzin, B. L., and Holers, G. M. (1996). Resting B cells from New Zealand Black mice demonstrate a defect in apoptosis induction following surface IgM ligation. J. Immunol. 156, 4498 – 4503. 43. Flescher, E., Bowlin, T. L., Ballester, A., Houk, R., and Talal, N. (1989). Increased polyamines may downregulate interleukin 2 production in rheumatoid arthritis. J. Clin. Invest. 83, 1356 – 1362. 44. Puri, H., Campbell, R. A., Puri, V., Harner, M. H., Talwalker, Y. B., Musgrave, J. E., Bartos, F., Bartos, D., and Loggan, B. (1978). Serum-free polyamines in children with systemic lupus erythematosus. Adv. Polyamine Res. 2, 359 –367. 45. Thomas, T. J., and Messner, R. P. (1991). Difluoromethylornithine therapy of female NZB/W mice. J. Rheumatol. 18, 215– 222.
Involvement of Polyamines in B Cell Receptor–Mediated Apoptosis: Spermine Functions as a Negative Modulator Takeshi Nitta,* Kazuei Igarashi,† Atsuya Yamashita,* Mikio Yamamoto,‡ and Naoki Yamamoto* ,1 *Department of Microbiology and Molecular Virology, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; †Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan; and ‡Department of Biochemistry II, National Defense Medical College, Saitama, Japan
INTRODUCTION The B cell lymphoma WEHI231 has been used as a model for studying clonal deletion of B cells on the basis of its ability to undergo growth arrest and apoptosis by B cell antigen receptor (BCR) cross-linking. To comprehensively analyze the genes involved in BCR-mediated apoptosis, we applied the technique of serial analysis of gene expression (SAGE) to WEHI231. Comparison of expression patterns revealed that BCR cross-linking caused coordinate changes in the expression of genes involved in polyamine metabolism. Polyamines are ubiquitous compounds required for cell proliferation and homeostasis. The coordinate expression of the polyamine-related genes was confirmed by semiquantitative reverse transcriptase– polymerase chain reaction analysis. During apoptosis, the genes involved in polyamine biosynthesis were downregulated, whereas those involved in polyamine catabolism were upregulated, suggesting that intracellular polyamines play a role in BCR-mediated apoptosis. Levels of intracellular putrescine, spermidine, and spermine were reduced after BCR crosslinking. These effects were prevented by concurrent CD40 stimulation, which blocked BCR-mediated apoptosis. Furthermore, addition of spermine could repress the BCR-mediated apoptosis by attenuating the mitochondrial membrane potential (⌬m) loss and activation of caspase-7 induced by BCR signaling. These findings strongly suggest that polyamine regulation is involved in apoptosis during B cell clonal deletion. © 2001 Academic Press
Key Words: apoptosis; B cell; polyamine; SAGE; spermine; WEHI231.
The murine immature B lymphoma cell line WEHI231 has been widely used as an in vitro model to study the regulation of B cell activation and apoptosis [1]. WEHI231 cells express surface immunoglobulin M (sIgM) 2 [1, 2] and undergo apoptosis after cross-linking of sIgM with anti-IgM antibody, which functions as a surrogate antigen [3, 4]. These effects can be blocked by concurrent stimulation of the CD40 receptor [5]. Apoptosis induced by signaling via antigen receptors could account for the mechanism by which self-reactive B cells are deleted. The observation that apoptosis is prevented by CD40 suggests that non–self-reactive B cells selectively proliferate by contact with activated T cells that express the CD40 ligand. Therefore, studies of B cell receptor (BCR)-mediated apoptosis of WEHI231 may provide information about the molecular mechanisms underlying clonal deletion of B cells. In the present study, we used serial analysis of gene expression (SAGE) [6] to comprehensively analyze changes in gene expression during BCR-mediated apoptosis in WEHI231 cells. SAGE allows qualitative and quantitative analysis of a large number of transcripts in any given cell or tissue without previous knowledge of the genes. Profiling of the gene expression pattern revealed that BCR cross-linking caused coordinate changes of the expression of a group of genes involved in metabolism of polyamines. Polyamines, namely putrescine, spermidine, and spermine, are ubiquitous and essential compounds in both eukaryotic and prokaryotic cells and are needed for cell growth and proliferation [7, 8]. The intracellular concentrations of polyamines are tightly regulated 2
1 To whom reprint requests should be addressed at Department of Microbiology and Molecular Virology, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Fax: 81-3-5803-0124. E-mail: yamamoto.mmb@ med.tmd.ac.jp.
0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: APCHA, N-(3-aminopropyl) cyclohexylamine; BCR, B cell receptor; OAZ, ornithine decarboxylase antizyme; ODC, ornithine decarboxylase; RT–PCR, reverse transcriptase–polymerase chain reaction; SAGE, serial analysis of gene expression; SAMDC, S-adenosylmethionine decarboxylase; sIgM, surface immunoglobulin M; SSAT, spermidine/spermine N 1 -acetyltransferase; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling.
174
POLYAMINE REGULATION IN B CELL APOPTOSIS
by biosynthesis, degradation, and transport [7, 8]. In the polyamine biosynthetic pathway, ornithine decarboxylase (ODC), the first and rate-limiting enzyme, catalyzes the decarboxylation of L-ornithine, leading to formation of putrescine. Spermidine synthase catalyzes the production of spermidine from putrescine, and subsequently spermine synthase catalyzes the production of spermine from spermidine, both by adding an aminopropyl group to the precursor. The donor of the aminopropyl moiety is decarboxylated adenosylmethionine, which is produced by S-adenosylmethionine decarboxylase (SAMDC). Catabolism of polyamines is essentially a reverse of their biosynthesis. Spermidine/ spermine N 1-acetyltransferase (SSAT) catalyzes the formation of acetylspermine and acetylspermidine, which are oxidized to spermidine and putrescine, respectively, by a constitutive enzyme polyamine oxidase. SSAT is induced when intracellular polyamines overaccumulate [9, 10]. Overaccumulation of cellular polyamines also induces a unique regulatory protein, ornithine decarboxylase antizyme (OAZ) [11, 12]. OAZ is a nonenzymatic protein that binds to ODC, inactivates it, and accelerates its degradation [11, 12]. OAZ also blocks cellular polyamine uptake and stimulates its excretion [13–15]. The highly regulated pathways of polyamine metabolism highlight the importance of these compounds in cell physiology. Polyamines are required for DNA replication, cell proliferation, cell homeostasis, and tumorigenesis. Indeed, the polyamine biosynthetic pathway is a target of some anticancer drugs [7, 8, 16]. Depletion of polyamines is often lethal, resulting in cell cycle arrest or apoptosis [17–19]. It has been reported that several forms of apoptosis, induced by cellular signaling, are closely related to polyamine regulation [19 – 21]. Our investigation revealed coordinate changes in the expression of genes involved in polyamine metabolism, suggesting an implication of polyamines in BCRmediated apoptosis. Our results suggest that modulation of intracellular polyamines plays a role in apoptosis during B cell clonal deletion. MATERIALS AND METHODS Cell culture. The WEHI231 (kindly provided by T. Tsubata) cell line was maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 M 2-mercaptoethanol, 10% (v/v) fetal bovine serum, 100 U/ml penicillin G, and 100 g/ml streptomycin. In experiments involving the addition of polyamines in cell culture, 1 mM aminoguanidine was added as an inhibitor of amine oxidase derived from fetal bovine serum. Aminoguanidine had no effect on the various parameters of the cell measured in this study. Reagents. Goat F(ab⬘)2 anti-mouse IgM (Southern Biotechnology Associates) and hamster anti-mouse CD40 monoclonal antibodies (Pharmingen) were used in this study. Rat anti-mouse HS1 monoclonal antibody was kindly provided by T. Watanabe [22, 23]. Putrescine 䡠 2HCl, spermidine 䡠 3HCl, and spermine 䡠 4HCl were pur-
175
chased from Sigma. N-(3-aminopropyl) cyclohexylamine (APCHA) was described previously [18]. Putrescine was used at 4 mM concentration, spermidine at 2 mM, spermine at 0.5 mM, and APCHA at 10 M, except in a dose–response examination. Cell cycle analysis. For cell cycle analysis, cells were fixed with 70% ethanol and treated with 0.5 mg/ml RNase A at 37°C for 20 min. Then cells were stained with 30 g/ml propidium iodide (Sigma) and analyzed by FACSCalibur (Becton Dickinson). Cell cycle profile of the living cell population was determined by means of ModFit LT (Becton Dickinson). Proportion of subdiploid cells in the total population was analyzed by Cell Quest (Becton Dickinson). Determination of samples for SAGE analysis. To determine the point when the G1 arrest and apoptosis occurred and to obtain the correct information of gene expression associated with cell cycle and apoptosis, we used a cell cycle synchronization system [24]. By treatment with thymidine and nocodazole, cells were synchronized at M phase. After restart of growth, control WEHI231 cells synchronously progressed cell cycle throughout growth. Cells treated with anti-IgM at 0 h, in contrast, were arrested at G1 phase 16 h after growth restart and could not enter into S phase, and subsequently apoptotic cells increased. Thus, G1-synchronized control cells at 12 h, and cells treated with anti-IgM for 16 h that were arrested at G1 phase and undergoing apoptosis, were determined to be analyzed by SAGE. SAGE analysis. Total RNAs were isolated with the RNeasy Mini kit (Qiagen) and cleaned by the treatment with DNase I (GenHunter). SAGE libraries were constructed from 50 g total RNA from control and anti-IgM–treated cells, as described previously [25, 26]. Fourteen– base pair sequence tags from each cDNA were concatemerized, cloned, and sequenced. PROGENEX software (Fujiyakuhin) was applied to extract SAGE tags and generate gene expression profiles [25, 26]. Transcripts corresponding to the obtained tags were identified by homology searching against database by the BLAST program. Reverse transcriptase–polymerase chain reaction (RT–PCR). For expression analysis of many genes on a limited amount of RNA, a convenient real-time RT–PCR was performed by SYBR Green PCR Core Reagents and ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). First-strand cDNAs were synthesized from DNase I–treated total RNA by MultiScribe Reverse Transcriptase (PE Applied Biosystems) using random hexamer as primers. cDNA from cells at 0 h was gradually diluted and used as a standard, and the amount of mRNA was relatively quantified by comparing threshold cycle of each sample with those of the standard. The threshold cycle was determined by detecting the increase in signal associated with an exponential growth of PCR product. Accuracy of the amplification of each gene and the absence of nonspecific amplification were confirmed by analyzing PCR products by polyacrylamide gel electrophoresis. All primers used for PCR were designed by PrimerExpress software (PE Applied Biosystems). A list of oligonucleotide primers is available from the authors upon request. Measurement of polyamine contents. Polyamine content was measured essentially according to the method described previously [27]. Cells were treated with 5% trichloroacetic acid, and the polyamines in the supernatant were separated on a high-performance liquid chromatography system. The precipitate was stored for measurement of protein contents. Western blot analysis. Tyrosine phosphorylation of HS1 after sIgM cross-linking in WEHI231 cells was analyzed according to previous report [22]. Cells were preincubated with or without putrescine, spermidine, or spermine at 37°C for 1 h and treated with anti-IgM at 37°C for 1 min. The cells were lysed and subjected to Western blot analysis with an anti-phosphotyrosine monoclonal antibody, PY20, labeled with horseradish peroxidase (TaKaRa), or with a rat monoclonal antibody to mouse HS1 followed by peroxidase-
176
NITTA ET AL.
labeled anti-rat IgG (TAGO Immunologicals). The blots were developed by the ECL system (NEN). DNA fragmentation assay. For the DNA laddering assay, cellular DNAs were prepared as described previously [28]. The samples were subjected to 2% agarose gel electrophoresis, followed by staining with ethidium bromide. In Situ Cell Death Detection kit, Fluorescein (Roche Diagnostics), based on terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method, was used for quantitative analysis of DNA fragmentation. Cells were fixed with 2% paraformaldehyde, permeabilized by 0.1% Triton X-100, and incubated with TUNEL reaction mixture at 37°C for 60 min in the dark. The cells were then analyzed by FACSCalibur. Enzyme assay for caspase activity. Caspase activation in WEHI231 cells was assessed by the CPP32/Caspase-3 Fluorometric Protease Assay Kit (MBL). Cells were lysed with cell lysis buffer, and the lysate was incubated with 50 M of the fluorescent substrate Asp–Glu–Val–Asp–7-amino– 4-trifluoromethyl coumarin at 37°C for 60 min. Cleaved substrate fluorescence was determined by luminescence spectrometry (LS50B; PE Applied Biosystems) with a 400-nm excitation filter and 505-nm emission filter. Caspase activity was determined as the fluorescence intensity normalized by protein amount. Mitochondrial membrane potential. After cell culture, cells were incubated with 40 nM of 3,3⬘-dihexyloxacarbocynine iodide [DiOC6(3); Sigma] for 15 min at 37°C, and then analyzed by FACSCalibur. Proportion of cells with depolarized mitochondria was analyzed by Cell Quest.
RESULTS
SAGE Analysis Revealed Coordinate Expression of Polyamine-Related Genes Gene expression was studied by SAGE analysis in control WEHI231 cells and cells stimulated with antiIgM antibody. We obtained 10,236 and 10,351 tags representing 4578 and 4720 unique transcripts for control and anti-IgM–treated cells, respectively. Profiles of relative gene expression patterns were generated by comparing the occurrence of each tag between two samples. In the gene expression profiles, one of the most differentially expressed tags corresponded to the gene that encodes spermidine synthase, an enzyme involved in polyamine biosynthesis (eight tags in control vs. one tag in anti-IgM; Table 1). In light of this, we looked at the profiles for tags corresponding to other genes whose products regulate polyamine metabolism. Expression of six genes was found to be altered (Table 1). Interestingly, four genes downregulated by antiIgM encoded enzymes involved in polyamine biosynthesis, whereas two genes upregulated by anti-IgM encoded an enzyme in the catabolic route (SSAT) or a regulatory protein inhibiting the polyamine biosynthesis (OAZ). These results suggest that expression of genes that are involved in polyamine metabolism is altered in response to BCR stimulation in WEHI231 B cells.
TABLE 1 SAGE Profile of Genes Involved in Polyamine Metabolism Frequency Gene description
Accession no. a L19311
CACAATGTAAGCTT
Spermidine synthase ODC Spermine synthase SAMDC
TCCGACGAGCGGCT TTAGAGTTTCTGTT
OAZ SSAT
Tag sequence AGCACCGCCTATGC ATTGGCAGAATGGG GCCAAAGAAGGGAG
a b c
Control b
IgM c
8
1
M10624 AF031486
12 3
5 2
Z14986 S50054 U52822 L10244
1
0
5 0
12 1
Matched GenBank accession number. The number of each tag in 10,236 transcript is indicated. The number of each tag in 10,351 transcript is indicated.
Coordinate Changes in the Expression of PolyamineRelated Genes by BCR or CD40 Signals Treatment of WEHI231 with anti-IgM arrested the cell cycle at G1 by 24 h, and the cells subsequently underwent apoptosis. The G1 arrest and apoptosis were prevented by concurrent treatment with antiCD40 antibody, which, by itself, had no effect (Figs. 1A, 1B). To confirm the results from the SAGE analysis, we performed semiquantitative RT–PCR analysis on the genes involved in polyamine metabolism. The relative changes in mRNA levels of six genes, including spermidine synthase, spermine synthase, SAMDC, ODC, SSAT, and OAZ, were determined after treatment with anti-IgM, anti-CD40, or anti-IgM plus anti-CD40 (Fig. 1C). Mouse 18S ribosomal RNA was used as an indicator of total RNA amount (data not shown). Anti-IgM caused significant reduction of the mRNA levels of spermidine synthase, spermine synthase, and SAMDC. Downregulation of these genes was prevented by treatment with anti-CD40. In the case of spermidine synthase, the CD40 by itself had an effect on mRNA levels, increasing the level of mRNA by about twofold (Fig. 1C). The expression of ODC, the rate-limiting enzyme in the polyamine biosynthesis, was slightly induced by anti-IgM at 2 h, and after that, it was not significantly affected. In cells treated with anti-CD40 or anti-IgM plus anti-CD40, induction of the ODC expression was observed at 12 h. A different profile was seen with SSAT and OAZ. These mRNAs were immediately upregulated by antiIgM treatment, and their expression remained elevated for 24 h. These effects could be prevented by anti-CD40 treatment. Collectively, in cells treated with anti-IgM, the genes involved in polyamine synthesis were repressed, and the genes involved in the catabolic
POLYAMINE REGULATION IN B CELL APOPTOSIS
177
FIG. 1. Coordinate changes of expression of polyamine metabolism regulatory genes during BCR-mediated apoptosis in WEHI231 cells. Cells were cultured in the absence (closed squares) or presence of 5 g/ml anti-IgM (open squares), 1 g/ml anti-CD40 (open triangles), or 5 g/ml anti-IgM plus 1 g/ml anti-CD40 (open circles). At the time indicated, cells were harvested and the cell cycle profile was determined by flow cytometry analysis. Percentages of cells in G1 phase (A) and subdiploid cells (B) were determined as described under Materials and Methods. Total RNA was isolated from cells at the time indicated, and 1 g of RNA was subjected to semiquantitative RT–PCR (C). The amount of mRNA of each gene was expressed relative to the amount at 0 h. Values represent the means ⫾ SD of three individual experiments. OAZ, ornithine decarboxylase antizyme; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SPM, spermine; SSAT, spermidine/spermine N 1 -acetyltransferase.
pathway were upregulated. With the exception of ODC, the results obtained from RT–PCR are consistent with those from the SAGE profile. The extent of changes of genes did not always agree between two methods, most probably because the number of tags in our SAGE analysis was not enough (approximately 10,000 tags for each sample) to correctly quantify the expression
levels of genes. Concurrent stimulation of CD40 prevented changes in the expression of these genes. Changes in Intracellular Polyamine Levels during Apoptosis in WEHI231 Cells We measured the intracellular concentrations of three polyamines—putrescine, spermidine, and sperm-
FIG. 2. Changes of polyamine contents during the cell cycle arrest and apoptosis by anti-IgM and their restoration by anti-CD40. Culture conditions and symbols represented were same as in Fig. 1. Cells were harvested and intracellular concentration of putrescine (PUT), spermidine (SPD), and spermine (SPM) were determined by high-performance liquid chromatography analysis. Values represent the means ⫾ SD of three individual experiments.
178
NITTA ET AL.
ine—which are the final cellular products of the genes described above (Fig. 2). The level of putrescine decreased rapidly after anti-IgM treatment and was undetectable by 24 h. The level of spermidine was also reduced by anti-IgM treatment, but the rate of decline was slower than that of putrescine. The depletion of putrescine and spermidine was partially prevented by concurrent stimulation of CD40. The level of spermine was slightly decreased up to 24 h after anti-IgM treatment, but subsequently decreased to about half of control by 36 h, when cells were completely arrested at G1 and apoptosis progressed. The reduction of spermine level was completely prevented by treatment with antiCD40. Thus, all three polyamines were significantly decreased during G1 arrest and apoptosis caused by BCR cross-linking, although the time course and magnitude was different for each polyamine. The depletion of polyamines could be prevented by concurrent signaling via CD40. These results suggest that polyamines, especially spermine, play some role in controlling apoptosis of WEHI231 cells. Inhibitory Effect of Spermine on BCR-Mediated Apoptosis Several types of apoptosis or growth arrest have been reported to be attenuated by the addition of exogenous polyamines [19 –21]. We tested whether exogenous polyamines could prevent the apoptosis induced by anti-IgM treatment in WEHI231 cells. DNA ladder formation induced by anti-IgM was significantly reduced by exogenously added spermine, but not putrescine or spermidine (Fig. 3A). The inhibitory effect of spermine on anti-IgM–induced apoptosis and its dose-dependent response were confirmed by measuring the extent of DNA fragmentation by in situ TUNEL assay (Figs. 3B, 4A). The increase of the frequency of subdiploid cells after anti-IgM treatment was also reduced in the presence of exogenous spermine, although the G1 arrest was not affected (Fig. 4B). Uptake of exogenous polyamines was confirmed by measuring intracellular polyamine levels (Table 2). Extracellular addition of each polyamine increased its own intracellular level but decreased levels of the other polyamines, probably due to feedback regulation of the biosynthetic enzymes. To determine whether the exogenous spermine interferes with the cross-linking of sIgM or not, we analyzed the tyrosine phosphorylation of HS1. The HS1 protein, a substrate of nonreceptor type protein–tyrosine kinase, is phosphorylated immediately after cross-linking of sIgM, and its phosphorylation is a key step in BCR-mediated apoptosis in WEHI231 cells [22, 23]. Anti-IgM induced tyrosine phosphorylation of HS1 protein, and the presence of exogenous polyamines did
FIG. 3. Effects of exogenous polyamines on BCR-mediated apoptosis in WEHI231 cells. WEHI231 cells pretreated with putrescine (PUT), spermidine (SPD), or spermine (SPM) for 1 h were cultured with anti-IgM (5 g/ml) for 36 h. Total cellular DNAs from 2 ⫻ 10 5 cell equivalents were subjected to DNA laddering assay (A). Percentage of cells with fragmented DNA was determined by flow cytometry analysis with in situ TUNEL reaction (B). Each column represents the mean ⫾ SD of at least three individual experiments. Statistical differences were assessed by Student’s t test. *P ⬍ 0.01 compared with anti-IgM–treated cells. Effect of exogenous polyamines on tyrosine phosphorylation of HS1 protein was analyzed by Western blotting (C) with anti-phosphotyrosine (upper panel) or anti-mouse HS1 (lower panel).
not alter the phosphorylation of HS1 (Fig. 3C). These results indicate that spermin protected apoptosis by reasons other than interfering with sIgM cross-linking and initial step of BCR signaling pathway. To further confirm the inhibitory effect of spermine on apoptosis, APCHA, an inhibitor of spermine synthase [18], was added to cells treated with anti-IgM. Enforced depletion of intracellular spermine by APCHA increased the frequency of subdiploid cells (Table 2; Fig. 4B) and DNA fragmentation (Fig. 4C) induced by anti-IgM. The effect of APCHA was reversed by exogenously added spermine. APCHA alone did not
POLYAMINE REGULATION IN B CELL APOPTOSIS
179
FIG. 4. Effects of exogenous spermine or APCHA on BCR-mediated apoptosis in WEHI231. Cells pretreated or untreated with spermine (SPM) or N-(3-aminopropyl) cyclohexylamine (APCHA) for 1 h were cultured with or without anti-IgM (5 g/ml) and/or anti-CD40 (1 g/ml) for 36 h. In panel A, SPM was used at the concentration indicated. DNA fragmentation was quantified by flow cytometry analysis with in situ TUNEL reaction (A and C). Each column represents the mean ⫾ SD of at least three individual experiments. **P ⬍ 0.05 and *P ⬍ 0.01 compared with anti-IgM–treated cells. Panel B shows cell cycle profile analyzed by flow cytometry. Values above horizontal bars represent the percentages of subdiploid cells in the total cell population. Percentages of cells at G1 phase in the living cell population are denoted on the right side of the G1 peak.
induce apoptosis, probably because of a compensatory effect by an increase in spermidine level. The effects of spermine on apoptosis were correlated with caspase activation and mitochondrial alteration. Caspases, a family of cysteine proteases, act as effectors of apoptosis (reviewed in Thornberry and Lazebnik [29]). BCR-mediated apoptosis of WEHI231 is dependent on the activation of caspase-7/Mch3, a member of the CPP32/caspase-3 family [30, 31]. The activation of CPP32/caspase-3 in BCR-mediated apoptosis is closely associated with mitochondrial dysfunction, including loss of the mitochondrial membrane potential (⌬m) [30, 32, 33]. An increase in the level of cellular spermine reduced the activity of caspase-7/Mch3 induced by anti-IgM, whereas depletion of spermine by APCHA enhanced activation of
caspase-7/Mch3 (Table 3; Fig. 5A). The percentage of cells with depolarized mitochondria was also significantly reduced by spermine and increased by APCHA (Fig. 5B). Anti-CD40 also prevented the apoptotic changes including DNA fragmentation, caspase-7/ Mch3 activation, and ⌬m loss induced by anti-IgM (Figs. 4C, 5). DISCUSSION
WEHI231 cells have been extensively used as an in vitro model to understand the mechanisms controlling cell death and survival in clonal deletion during B cell development. However, the cellular mechanisms involved in BCR signaling, in which many factors are orchestrated to execute the death program, are not
180
NITTA ET AL.
TABLE 2 Effects of Exogenous Polyamines or APCHA on Intracellular Polyamine Contents of WEHI231 Cells a Polyamine concentration (pmol/g protein) Variable
PUT
SPD
SPM
Control IgM IgM ⫹ PUT (4 mM) IgM ⫹ SPD (2 mM) IgM ⫹ SPM (0.1 mM) IgM ⫹ SPM (0.2 mM) IgM ⫹ SPM (0.5 mM) IgM ⫹ APCHA IgM ⫹ APCHA ⫹ SPM (0.5 mM) SPM (0.5 mM) APCHA APCHA ⫹ SPM (0.5 mM)
2.1 ⫾ 0.2 ⬍0.1 72.1 ⫾ 19.0 4.0 ⫾ 1.1 ⬍0.1 ⬍0.1 0.3 ⫾ 0.1 0.1 ⫾ 0.04
14.5 ⫾ 0.9 1.3 ⫾ 0.4 6.2 ⫾ 1.5 39.3 ⫾ 9.1 0.4 ⫾ 0.1 0.8 ⫾ 0.1 1.4 ⫾ 0.4 7.6 ⫾ 1.2
20.4 ⫾ 1.5 10.2 ⫾ 3.4 11.2 ⫾ 3.4 6.2 ⫾ 0.2 15.6 ⫾ 0.8 20.3 ⫾ 1.5 26.3 ⫾ 3.1 5.4 ⫾ 0.2
0.7 ⫾ 0.02 0.2 ⫾ 0.1 0.3 ⫾ 0.1
1.4 ⫾ 0.1 2.3 ⫾ 1.4 31.1 ⫾ 3.6
18.9 ⫾ 0.6 32.9 ⫾ 4.1 5.0 ⫾ 0.1
0.4 ⫾ 0.1
1.7 ⫾ 0.2
22.9 ⫾ 0.6
a Culture conditions were same as in Figs. 3 and 4. Cells were harvested at 36 h, and intracellular concentrations of putrescine (PUT), spermidine (SPD), and spermine (SPM) were determined by high-performance liquid chromatography analysis. Values represent the means ⫾ SD.
fully understood. In this study, we demonstrate that BCR signaling leads to a decrease in intracellular polyamines by coordinate changes in the expression of genes involved in polyamine metabolism. It is well established that ODC is a key enzyme in polyamine regulation. In WEHI231 cells, however, expression of ODC mRNA is less affected by anti-IgM or anti-CD40 treatment than is the expression of the other polyamine regulatory genes. Coordinate changes TABLE 3 Effects of Exogenous Spermine or APCHA on Intracellular Polyamine Contents of WEHI231 Cells a Polyamine concentration (pmol/g protein) Variable
PUT
SPD
SPM
Control IgM IgM ⫹ SPM IgM ⫹ APCHA IgM ⫹ APCHA ⫹ SPM IgM ⫹ CD40
2.7 ⫾ 0.4 ⬍0.1 0.2 ⫾ 0.1 ⬍0.1 0.2 ⫾ 0.1 2.1 ⫾ 1.3
16.8 ⫾ 1.2 3.8 ⫾ 0.2 1.2 ⫾ 0.2 12.8 ⫾ 0.9 1.8 ⫾ 0.2 13.4 ⫾ 0.9
19.3 ⫾ 1.1 15.3 ⫾ 1.6 24.6 ⫾ 1.9 7.2 ⫾ 0.1 23.2 ⫾ 0.5 20.1 ⫾ 2.1
a Culture conditions were same as in Fig. 5. Cells were harvested at 30 h, and intracellular concentrations of putrescine (PUT), spermidine (SPD), and spermine (SPM) were determined by high-performance liquid chromatography analysis. Values represent the means ⫾ SD.
FIG. 5. Effect of exogenous spermine or N-(3-aminopropyl) cyclohexylamine (APCHA) on caspase-7/Mch3 activity and ⌬m collapse. Cells pretreated or untreated with spermine (SPM) or APCHA for 1 h were cultured with or without anti-IgM (5 g/ml) and/or anti-CD40 (1 g/ml). Cells were harvested at 30 h and were analyzed as described under Materials and Methods. Each column represents the mean ⫾ SD of three individual experiments. **P ⬍ 0.05 and *P ⬍ 0.01 compared with anti–IgM-treated cells.
in the expression of spermidine synthase, spermine synthase, SAMDC, and SSAT are probably responsible for the changes in intracellular spermidine and spermine levels after anti-IgM treatment. The decrease in intracellular putrescine after anti-IgM treatment in WEHI231 may be due to a posttranscriptional downregulation of ODC by OAZ, which is rapidly induced by anti-IgM. Although the expression of OAZ is known to be regulated by a unique posttranscriptional machinery [11], enhanced transcription also has a significant effect on polyamine content and cell growth [34, 35]. In addition, it is likely that the regulatory gene or genes that encode cell-surface polyamine transporters are also involved in the control of intracellular polyamine levels during BCR-mediated apoptosis [36]. However, the genes encoding mammalian cell polyamine transporters have not yet been identified. OAZ also negatively regulates the intracellular polyamine levels by inhibiting uptake of polyamines and by promoting their excretion [13–15]. Induction of OAZ
POLYAMINE REGULATION IN B CELL APOPTOSIS
expression by BCR cross-linking and its repression by CD40 signaling might play important roles in regulating the polyamine levels in WEHI231 cells, although further analysis will be required to demonstrate the role of OAZ in BCR-mediated apoptosis. In any event, coordinate expression of the metabolic regulatory genes after BCR cross-linking suggests a commitment of the cell to actively decrease intracellular polyamines. CD40 signaling prevents the effects of antiIgM on the gene expression and polyamine levels. In particular, the intracellular spermine level was completely maintained by CD40 signaling, suggesting the particular importance of this polyamine in the reduction of the BCR-mediated apoptosis. Like the WEHI231 cell line, primary splenic B cells decreased the intracellular levels of polyamines after apoptosis induction by anti-IgM, but concurrent antiCD40 treatment recovered them (data not shown). These alterations of intracellular polyamines were also preceded by coordinate expression of the metabolic regulatory genes in splenic B cells. These results suggest that the regulation of cellular polyamines was closely associated with apoptosis and survival of B cells in clonal deletion in vivo. Exogenous spermine attenuated but did not completely prevent BCR-mediated apoptotic changes, including DNA fragmentation and hypodiploidy. Thus, spermine might be a mediator of the execution step but not a dominant regulator of apoptosis. It has been reported that exogenous spermine prevents DNA fragmentation during apoptosis in rat thymocytes [37] and in the mouse T cell line CTLL-2 [20]. It was suggested that the protective effect of spermine on DNA fragmentation is due to direct binding of spermine to DNA and modification of chromatin structure [38]. Spermine has also been shown to prevent apoptosis of cultured cerebellar neurons, and its inhibitory effect appears to be mediated at a site upstream of caspase activation [39]. Indeed, our results agree with the latter model. The level of intracellular spermine correlated with the extent of caspase-7/Mch3 activity induced by BCR signaling, suggesting that spermine plays a role in the cellular cascade involved in caspase activation. In BCRmediated apoptosis, activation of CPP32/caspase-3 family caspase or caspases, which function as effector caspases, is preceded by mitochondrial dysfunction [30, 32]. It has been proposed that the alteration in mitochondrial function plays a critical role in activation of the caspase cascade, possibly due to a release of cytochrome c and/or other caspase activators from mitochondria into the cytosol [29, 33]. Our results that the ⌬m loss was prevented by spermine and enhanced by its depletion indicate that spermine has a protective effect on mitochondrial alteration during apoptosis.
181
Many other biochemical studies have demonstrated that spermine inhibits the ⌬m collapse in vitro [40, 41], suggesting that spermine directly affects mitochondria. Thus, it is suggested that depletion of intracellular spermine by BCR signaling plays a role in the reduction of the mitochondrial function to accelerate caspase activation and apoptosis. BCR-mediated apoptosis is defective in NZB and NZB/W mice that are prone to systemic autoimmune diseases, and it is therefore thought to be one of the key pathogenic events of autoimmune diseases [42]. It has been suggested that polyamines also play an important role in autoimmune diseases. Serum polyamine levels, cellular polyamine levels, or both are elevated in patients with rheumatoid arthritis [43] or systemic lupus erythematosus [44] and in NZB/W mice [45]. Moreover, inhibition of polyamine biosynthesis was reported to produce a remarkable improvement in several clinical symptoms of NZB/W mice [45]. In this study, we demonstrate that polyamine regulation is implicated in the control of BCRmediated apoptosis of B cells. Therefore, WEHI231 cells may be a useful tool to study the role and function of polyamine not only in cellular apoptotic cascade but also in the pathogenesis and chemotherapy of autoimmune diseases. We thank Dr. Keith Williams for kind suggestions and help in preparing this manuscript. We also thank Dr. Takeshi Tsubata for providing the WEHI231 cell line, Dr. Takeshi Watanabe for providing anti-mouse HS1 antibody, and Dr. Kazuhiro Nishimura for exceptional assistance with high-performance liquid chromatography analysis. We are also grateful to Drs. Shoji Yamaoka and Norio Yamamoto for helpful discussions. This work was supported by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Drug ADR Relief, R&D Promotion, and Products Review of Japan, grants from the Japan Health Science Foundation, and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation.
REFERENCES 1.
2. 3.
4.
5.
6.
Boyd, A. W., and Schrader, J. W. (1981). The regulation of growth and differentiation of a murine B cell lymphoma. J. Immunol. 126, 2466 –2469. Ralph, P. (1979). Functional subsets of murine and human B lymphocyte cell lines. Immunol. Rev. 48, 107–121. Benhamou, L. E., Cazenave, P. A., and Sarthou, P. (1990). Anti-immunoglobulins induce death by apoptosis in WEHI-231 B lymphoma cells. Eur. J. Immunol. 20, 1405–1407. Hasbold, J., and Klaus, G. G. (1990). Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas. Eur. J. Immunol. 20, 1685–1690. Tsubata, T., Wu, J., and Honjo, T. (1993). B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature 364, 645– 648. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270, 484 – 487.
182
NITTA ET AL. 22.
Fukuda, T., Kitamura, D., Taniuchi, I., Maekawa, Y., Benhamou, L. E., Sarthou, P., and Watanabe, T. (1995). Restoration of surface IgM-mediated apoptosis in an anti-IgM–resistant variant of WEHI-231 lymphoma cells by HS1, a protein–tyrosine kinase substrate. Proc. Natl. Acad. Sci. USA 92, 7302– 7306.
Fogel-Petrovic, M., Kramer, D. L., Ganis, B., Casero, R. A. Jr., and Porter, C. W. (1993). Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N 1acetyltransferase—A gene uniquely regulated by polyamines and their analogs. Biochim. Biophys. Acta 1216, 255–264.
23.
Yamanashi, Y., Fukuda, T., Nishizumi, H., Inazu, T., Higashi, K., Kitamura, D., Ishida, T., Yamamura, H., Watanabe, T., and Yamamoto, T. (1997). Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis. J. Exp. Med. 185, 1387–1392.
Suppola, S., Pietila¨, M., Parkkinen, J. J., Korhonen, V.-P., Alhonen, L., Halmekyto¨, M., Porter, C. W., and Ja¨nne J. (1999). Overexpression of spermidine/spermine N 1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: Evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue. Biochem. J. 338, 311–316.
24.
Ishida, T., Kobayashi, N., Tojo, T., Ishida, S., Yamamoto, T., and Inoue, J. (1995). CD40 signaling-mediated induction of Bcl-XL, Cdk4, and Cdk6. Implication of their cooperation in selective B cell growth. J. Immunol. 155, 5527–5535.
25.
Ryo, A., Suzuki, Y., Ichiyama, K., Wakatsuki, T., Kondoh, N., Hada, A., Yamamoto, M., and Yamamoto, N. (1999). Serial analysis of gene expression in HIV-1–infected T cell lines. FEBS Lett. 462, 182–186.
26.
Ryo, A., Kondoh, N., Wakatsuki, T., Hada, A., Yamamoto, N., and Yamamoto, M. (2000). A modified serial analysis of gene expression that generates longer sequence tags by nonpalindromic cohesive linker ligation. Anal. Biochem. 277, 160 –162.
27.
Igarashi, K., Kashiwagi, K., Hamasaki, H., Miura, A, Kakegawa, T., Hirose, S., and Matsuzaki, S. (1986). Formation of a compensatory polyamine by Escherichia coli polyamine-requiring mutants during growth in the absence of polyamines. J. Bacteriol. 166, 128⫺134.
28.
Ishizawa, M., Kobayashi, Y., Miyamura, T., and Matsuura, S. (1991). Simple procedure of DNA isolation from human serum. Nucleic Acids Res. 19, 5792.
29.
Thornberry, N. A., and Lazebnik, Y. (1998). Caspases: Enemies within. Science 281, 1312–1316.
30.
Doi, T., Motoyama, N., Tokunaga, A., and Watanabe, T. (1999). Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231. Int. Immunol. 11, 933–941.
31.
Bra´s, A., Ruiz-Vela, A., Buitrago, G. G. D., and Martı´nez, A. C. (1999). Caspase activation by BCR cross-linking in immature B cells: Differential effects on growth arrest and apoptosis. FASEB J. 13, 931–944.
32.
Bouchon, A., Krammer, P. H., and Walczak, H. (2000). Critical role for mitochondria in B cell receptor–mediated apoptosis. Eur. J. Immunol. 30, 69 –77.
33.
Green, D. R., and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281, 1309 –1312.
34.
Murakami, Y., Matsufuji, S., Miyazaki, Y., and Hayashi, S. (1994). Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth. Biochem. J. 384, 183–187.
35.
Iwata, S., Sato, Y., Asada, M., Takagi, M., Tsujimoto, A., Inaba, T., Yamada, T., Sakamoto, S., Yata, J.-I., Shimogori, T., Igarashi, K., and Mizutani, S. (1999). Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation. Oncogene 18, 165– 172.
36.
DeBenedette, M., Olson, J. W., and Snow, E. C. (1993). Expression of polyamine transporter activity during B lymphocyte cell cycle progression. J. Immunol. 150, 4218 – 4224.
37.
Bru¨ne, B., Hartzell, P., Nicotera, P., and Orrenius, S. (1991). Spermine prevents endonuclease activation and apoptosis in thymocytes. Exp. Cell Res. 195, 323–329.
7.
Pegg, A. E. (1988). Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48, 759 –774.
8.
Marton, L. J., and Pegg, A. E. (1995). Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 35, 55–91.
9.
10.
11.
12.
13.
14.
Matsufuji, S., Matsufuji, T., Miyazaki, Y., Murakami, Y., Atkins, J. F., Gesteland, R. F., and Hayashi, S. (1995). Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80, 51– 60. Ivanov, I. P., Gesteland, R. F., and Atkins, J. F. (1998). A second mammalian antizyme: Conservation of programmed ribosomal framshifting. Genomics 52, 119 –129. Mitchell, J. L. A., Judd, G. G., Bareyal-Leyser, A., and Ling, S. Y. (1994). Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem. J. 299, 19 –22. Suzuki, T., He, Y., Kashiwagi, K., Murakami, Y., Hayashi, S., and Igarashi, K. (1994). Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells. Proc. Natl. Acad. Sci. USA 91, 8930 – 8934.
15.
Sakata, K., Kashiwagi, K., and Igarashi, K. (2000). Properties of a polyamine transporter regulated by antizyme. Biochem. J. 347, 297–303.
16.
Meyskens, F. L. Jr., and Gerner, E. W. (1999). Development of difluoromethylornithine (DFMO) as a chemoprevention agent. Clin. Cancer Res. 5, 945–951.
17.
Nakaike, S., Kashiwagi, K., Terao, K., Iio, K., and Igarashi, K. (1988). Combined use of alpha-difluoromethylornithine and an inhibitor of S-adenosylmethionine decarboxylase in mice bearing P388 leukemia or Lewis lung carcinoma. Jpn. J. Cancer Res. 79, 501–508.
18.
He, Y., Shimogori, T., Kashiwagi, K., Shirahata, A., and Igarashi, K. (1995). Inhibition of cell growth by combination of ␣-difluoromethylornithine and an inhibitor of spermine synthase. J. Biochem. 117, 824 – 829.
19.
Schipper, R. G., Pening, L. C., and Verhofstad, A. A. J. (2000). Involvement of polyamines in apoptosis. Facts and controversies: Effectors or protectors? Semin. Cancer Biol. 0, 55– 68, doi:10.1006/scbi.2000.0308.
20.
Min, A., Hasuma, T., Yano, Y., Matsui-Yuasa, I., and Otani, S. (1995). Regulation of apoptosis of interleukin 2– dependent mouse T-cell line by protein tyrosine phosphorylation and polyamines. J. Cell. Physiol. 165, 615– 623.
21.
Thomas, T., Balabhadrapathruni, S., Gardner, C. R., Hong, J., Faaland, C. A., and Thomas T. J. (1999). Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: Alterations in polyamine biosynthesis and the expression of p21/CIP1/ WAF1. J. Cell. Physiol. 179, 257–266.
POLYAMINE REGULATION IN B CELL APOPTOSIS 38.
Snyder, R. D. (1989). Polyamine depletion is associated with altered chromatin structure in HeLa cells. Biochem. J. 260, 697–704.
39.
Harada, J., and Sugimoto, M. (1997). Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons. Brain Res. 753, 251–259.
40.
Tassani, V., Biban, C., Toninello, A., and Siliprandi, D. (1995). Inhibition of mitochondrial permeability transition by polyamines and magnesium. Biochem. Biophys. Res. Commun. 207, 661– 667.
41.
Tassani, V., Campagnolo, M., Toninello, A., and Siliprandi, D. (1996). The contribution of endogenous polyamines to the permeability transition of rat liver mitochondria. Biochem. Biophys. Res. Commun. 226, 850 – 854.
Received October 12, 2000 Revised version received January 23, 2001 Published online March 16, 2001
42.
183
Kozono, Y., Kotzin, B. L., and Holers, G. M. (1996). Resting B cells from New Zealand Black mice demonstrate a defect in apoptosis induction following surface IgM ligation. J. Immunol. 156, 4498 – 4503. 43. Flescher, E., Bowlin, T. L., Ballester, A., Houk, R., and Talal, N. (1989). Increased polyamines may downregulate interleukin 2 production in rheumatoid arthritis. J. Clin. Invest. 83, 1356 – 1362. 44. Puri, H., Campbell, R. A., Puri, V., Harner, M. H., Talwalker, Y. B., Musgrave, J. E., Bartos, F., Bartos, D., and Loggan, B. (1978). Serum-free polyamines in children with systemic lupus erythematosus. Adv. Polyamine Res. 2, 359 –367. 45. Thomas, T. J., and Messner, R. P. (1991). Difluoromethylornithine therapy of female NZB/W mice. J. Rheumatol. 18, 215– 222.