Repression of c-myc Is Necessary but Not Sufficient for Terminal Differentiation of B Lymphocytes In Vitro

Cell culture and BCL-1 cell differentiation.

BCL-1 cells (CW13.20-3B3, ATCC CRL 1669) were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-Product, Inc.), gentamicin (20 μg/ml) (Gemini), and 50 μM β-mercaptoethanol. To induce differentiation, 2 ml of cells (10⁵ cells/ml) was placed in six-well plates and either left untreated or treated with recombinant mouse IL-2 and IL-5 (20 ng/ml) (R&D systems, Inc.) for 3 days. For induction of dominant negative c-Myc, 4-hydroxytamoxifen (4-OHT) (Sigma) dissolved in ethanol was added directly to culture medium to a final concentration of 500 nM. Lovastatin (LOV) (Calbiochem), for inhibiting the cell cycle, was dissolved in H₂O.

Plasmids.

The retroviral vector pGC-Blimp-YFP was cloned by blunt-ended ligation of a cDNA fragment of hemagglutinin (HA)-tagged Blimp-1 into the ClaI site of pGC-IRES-YFP (a kind gift from G. Fathman, Stanford). pSV2Myc and its control vector pSV2neo were described previously (39). For constructing the cyclin E expression vector, mouse cyclin E cDNA was cut with EcoRI from pSKmcyclinE and then ligated into the EcoRI site of pcDNA3.1Zeo (Invitrogen). For constructing the inducible dominant negative c-Myc expression vector, the cDNA of dominant negative human c-Myc fused to the mouse estrogen receptor (MycDN-ER) was cut with EcoRI from pBabeMycDNERpuro (a gift from G. Littlewood [40]) and ligated into the EcoRI site of pcDNA3.1Zeo.

Transfection and transduction.

Various retroviral vectors (10 μg) were transfected into Phoenix cells (a kind gift from G. Nolan, Stanford) using the calcium phosphate method. Virus supernatant was collected 48 h posttransfection and prepared essentially as previously described (10). Virus-infected cells were sorted by fluorescence-activated cell sorting (FACS) for yellow fluorescent protein (YFP) expression.

RT-PCR.

Reverse transcription (RT)-PCR on virus-transduced cells was performed essentially as previously described (4). Briefly, 250 ng of total RNA, isolated by the Trizol method (Gibco-BRL) from YFP⁺ cells, was digested with 10 U of DNase (Promega) for 1 h and subjected to cDNA synthesis. Mouse c-Myc was amplified using primers 5′-GGGCCAGCCCTGAGCCCCTAGTGC-3′ and 5′-ATGGAGATGAGCCCGACTCCGACC-3′. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using primers 5′-TTAGCACCCCTGGCCAAGG-3′ and 5′-CTTACTCCTTGGAGGCCATG-3′. Cycling conditions were 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C for 30 cycles. PCR products were analyzed on a 1.5% agarose gel.

BCL-1 transfectants stable transfectants.

A total of 10⁷ BCL-1 cells in log phase were transfected with 10 μg of experimental expression vectors or control vector by electroporation as previously described (39). Forty-eight hours after transfection, cells were placed in 96-well plates at limiting dilution and treated with geneticin (800 μg/ml) (Gibco-BRL) for selection of c-Myc and control transfectants or zeocin (400 μg/ml) (Invitrogen) for selection of cyclin E stable transfectants, inducible dominant negative c-Myc stable transfectants, and control transfectants. Cells were fed weekly with fresh medium containing antibiotics, and resistant clones were expanded for analysis.

Western blot analysis.

Whole-cell lysates were prepared in 20 mM Tris-HCl (pH 7.5)–10% glycerol–150 mM NaCl–1% NP-40–0.1% sodium dodecyl sulfate (SDS)–0.5% deoxycholate–2 mM dithiothreitol (DTT)–1 mM phenylmethylsulfonyl fluoride (PMSF)–proteinase inhibitor cocktail (Sigma) and centrifuged in a cold room at 13,000 rpm for 5 min. Supernatants were aliquoted and frozen for protein quantification and subsequent SDS-polyacrylamide gel electrophoresis (PAGE), and 20 μg of protein, quantified by the Bradford assay (Bio-Rad), was separated on an SDS–8% PAGE gel (for monitoring the expression of c-Myc and cyclin E) or SDS–6% PAGE gel (for monitoring the expression of MycDNER) and proteins were then electrotransferred onto a nitrocellulose membrane. Membranes were blocked with 5% dry milk in phosphate-buffered saline (PBS) plus 0.2% Tween 20 (PBS-T). Subsequently, membranes were blotted with a polyclonal anti-mouse c-Myc (39) diluted at the ratio of 1:3,000, polyclonal anti-cyclin E (Santa Cruz Biotechnology; M-20) diluted 1:100, monoclonal anti-human c-Myc (Santa Cruz Biotechnology; 9E10) diluted 1:100, or monoclonal anti-mouse β-actin (Sigma) diluted 1:3,000 in 2% dry milk–PBS-T. A peroxidase-conjugated goat anti-rabbit IgG (Boehringer Mannheim) or goat anti-mouse IgG (Boehringer Mannheim) was later used as the secondary antibody at a dilution of 1:10,000 in 2% dry milk–PBS-T. Subsequent enhanced chemiluminescence (NEN Life Science Product) was performed according to the manufacturer's suggestions.

RNase protection assay.

Total cellular RNA was isolated by the guanidinium thiocyanate procedure (11). RNase protection assays were performed as described (41). Briefly, antisense cRNA probes were generated using T3 or T7 RNA polymerase (Promega) with [α-³²P]UTP from cDNA templates, and 10 μg of total RNA was then hybridized with a 360-bp probe specific for the zinc finger 1 and 2 regions of mouse Blimp-1 and a 180-bp mouse GAPDH probe in 80% formamide–40 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] buffer (pH 6.4)–400 mM NaCl–1 mM EDTA overnight at 45°C. The reaction was then treated with RNase cocktail (Amicon) at 30°C for 30 min followed by treatment with 0.125 mg of proteinase K and 0.5% SDS at 37°C for 15 min. The RNA was analyzed on a denaturing 6% polyacrylamide–urea gel. Gel imaging was done by PhosphorImager, and band intensity was analyzed by ImageQuant software (Molecular Dynamics).

Flow cytometry.

For surface Syndecan-1 analysis, cells were harvested, washed once with PBS, and suspended in PBS containing 2% fetal bovine serum at 10⁶ cells/ml. Cells were then incubated with phycoerythrin-conjugated anti-mouse Syndecan-1 (Pharmingen) at 4°C for 30 min (1 μl of antibody per 10⁵ cells), washed two times with PBS, and then analyzed by FACScan (Becton Dickinson) and CellQuest software.

Cell cycle analysis.

5-Bromo-2′-deoxyuridine (BrdU) (Boehringer Mannheim) was added to cells at a final concentration of 10 μM for 2 h, and cells were harvested, washed once with PBS, and fixed in 70% ethanol for at least 12 h. Staining was performed according to the manufacturer's instructions (Pharmingen). In brief, 10⁶ cells/ml were denatured in 2 N HCl, neutralized by 0.1 M sodium borate (pH 8.5), and then stained with fluorescein isothiocyanate-conjugated mouse anti-human BrdU antibody (Pharmingen) at room temperature for 30 min. After staining with propidium iodide (10 μg/ml) (Boehringer Mannheim) in PBS for 20 min at room temperature, cells were analyzed using FACscan.

ELISA.

For the enzyme-linked immunosorbent assay (ELISA), cell supernatants were harvested by centrifugation of the culture medium from treated or untreated BCL-1 cells twice at 2,000 rpm. Supernatants were then serially diluted in duplicate in PBS containing 1% bovine serum albumin into 96-well plates coated with anti-mouse IgM (Southern Biotechnology Associates) and incubated for 1.5 h at room temperature. Captured IgM was further incubated with alkaline phosphatase-conjugated goat anti-mouse IgM (Southern Biotechnology Associates) diluted to 1:1,000 in PBS containing 1% bovine serum albumin for 1 h at room temperature. The alkaline phosphatase substrate p-nitrophenyl phosphate (Southern Biotechnology Associates) was dissolved according to the manufacturer's instructions at the concentration of 1 mg/ml. The plates were subsequently read on a plate reader (Molecular Devices). Mouse IgM (Southern Biotechnology Associates) was used for generating the standard curve.

Ectopic expression of c-Myc blocks terminal differentiation of BCL-1 cells in response to cytokine.

Ectopic expression of Blimp-1 is sufficient to cause terminal differentiation to a plasma cell phenotype in the BCL-1 model system (39, 71). We have previously shown that one target of Blimp-1-dependent transcriptional repression is the c-myc gene (39). c-Myc levels fall in BCL-1 cells upon induction of Blimp-1 (39). In splenocytes treated with lipopolysaccharide, Blimp-1 mRNA is induced and c-Myc levels decrease (3). To extend these findings, we monitored endogenous c-myc mRNA levels in BCL-1 cells expressing ectopic Blimp-1 using a retrovirus expressing a bicistronic mRNA for Blimp-1 and YFP. Three days after virus infection, about 50% of the cells were infected, as determined by FACS for YFP expression. c-myc mRNA was determined by semiquantitative RT-PCR in YFP⁺ BCL-1 cells infected with either Blimp-1-YFP virus or YFP-only control virus. GAPDH levels were used as an internal control for cDNA template. The data show that ectopic expression of Blimp-1 was sufficient to cause a decrease in endogenous c-myc mRNA of approximately fourfold (Fig. 1), formally demonstrating that Blimp-1 represses endogenous c-myc transcription in BCL-1 cells.

Since Blimp-1 represses c-myc and induces plasma cell differentiation in BCL-1 cells, we took advantage of the BCL-1 system to assess the functional importance of repressing c-myc transcription in the context of plasma cell differentiation. Stable transfectants of BCL-1 cells expressing c-Myc driven by a constitutive simian virus 40 promoter (Myc-BCL-1) were generated for this purpose. Stable transfectants were then treated with IL-2 and IL-5 to induce differentiation. As shown in Fig. 2A, after 3 days of IL-2 and IL-5 treatment, c-Myc declined to 60%. In two c-Myc-expressing cell lines, little or no decrease was observed following cytokine treatment: c-Myc declined to 84% in Myc#2 and remained at 100% in Myc#5. Similar results were found by analyzing c-myc mRNA levels in control and c-Myc transfectants (not shown).

Following treatment with IL-2 and IL-5, BCL-1 cells undergo terminal differentiation into plasma cells, characterized by cessation of cell proliferation, induction of surface marker Syndecan-1, and IgM secretion (39, 46, 71). We examined the effect of ectopic c-Myc expression on these phenotypic hallmarks of terminal differentiation. To analyze the cell cycle status of c-Myc and control transfectants, cells were treated with IL-2 and IL-5 for 3 days, pulsed with BrdU to monitor DNA replication, and stained with propidium iodide to monitor DNA content prior to analysis by FACS. In two control transfectants, after 3 days of IL-2 and IL-5 treatment, cells became quiescent (percentage of cells in S phase dropped from 25 to 5% in control #1 and from 44 to 16% in control #2), consistent with other studies showing that B cells undergo growth arrest during terminal differentiation (22) (Fig. 2B). In contrast, Myc-BCL-1 cells continued proliferating after 3 days of cytokine treatment; the percentage of cells in S phase was essentially the same as that of untreated cells (28% versus 25% in Myc#2, and 39% versus 43% in Myc#5) (Fig. 2B).

We next assayed the ability of these cells to differentiate following cytokine treatment by measuring the levels of secreted IgM and the plasma cell surface maker Syndecan-1. We found that ectopic expression of c-Myc blocks IgM secretion in response to IL-2 and IL-5 treatment. As shown in Fig. 2C, a control transfectant (#2) showed significant IgM production, but Myc-BCL-1 cells (#1 to #5) failed to secrete IgM. Syndecan-1 on the cell surface was measured by flow cytometry; its level increased about threefold in a control transfectant after 3 days of cytokine treatment (Fig. 2D, top panel, compare fine line to dark line). In contrast, Syndecan-1 remained unchanged in Myc-BCL-1 lines (#2 and #5 are shown) (Fig. 2D, middle and bottom panels). These data show that ectopic expression of c-Myc blocks terminal differentiation of BCL-1 cells, indicating that repression of c-myc by Blimp-1 is a necessary event for plasma cell differentiation in this culture model.

Ectopic expression of cyclin E blocks terminal differentiation of BCL-1 cells in response to cytokine.

c-Myc downregulation, accompanied by growth arrest, has been shown to be associated with terminal differentiation in many cell lineages in addition to B cells (16, 25, 34, 36, 57). However, the precise mechanism(s) by which c-Myc functions in terminal differentiation is not well understood. The BCL-1 system provides a way to explore this question because we can test which aspect of c-Myc function is responsible for the inhibition of BCL-1 terminal differentiation. One important facet of c-Myc function is to promote proliferation, either by regulating genes encoding cell cycle regulators or by regulating genes involved in growth and metabolism (14). However, c-Myc also regulates genes such as telomerase reverse transcriptase, albumin, Ig lambda, and terminal deoxynucleotidyl transferase that have no obvious role in proliferation (14). We reasoned that if the proliferation aspect of c-Myc's activity was critical for blocking BCL-1 differentiation, it should be possible to achieve the same effect by activating proliferation in a different way. Alternatively, if regulation of other c-Myc-dependent target genes was required, enforced proliferation would not be sufficient to inhibit BCL-1 terminal differentiation.

Cyclin E has been shown to promote the G₁ phase of the cell cycle (33) and to abrogate cell cycle arrest induced by overexpression of p16^INK4a, a cdk4 inhibitor (2). Thus, we generated stable BCL-1 cell lines constitutively expressing mouse cyclin E (cyclin E-BCL-1) driven by a cytomegalovirus promoter in order to keep BCL-1 cells in a proliferative state after IL-2 and IL-5 treatment. Immunoblots showed that while endogenous cyclin E was undetectable in control cells, cyclin E was clearly detectable in three cyclin E-BCL-1 lines (#4, #7, and #9) chosen for study (Fig. 3A). Cell cycle analysis, carried out as described in the legend to Fig. 3B, showed that a control transfectant showed a significant reduction in the percentage of cells in S phase following cytokine treatment (14 versus 41% in S phase) (Fig. 3B). However, the cytokine-treated cyclin E-BCL-1 lines behaved like the cytokine-treated Myc-BCL-1 lines and continued to cycle (Fig. 3B). Similar results were obtained in two additional independent experiments. These data show that ectopic expression of cyclin E mimics ectopic expression of c-Myc in BCL-1 cells with respect to keeping the cells in cycle following cytokine treatment.

Having established that both the Myc- and cyclin E-BCL-1 lines continued to proliferate following cytokine treatment, we asked whether cyclin E expression mimicked c-Myc with respect to inhibition of terminal differentiation. Interestingly, after 3 days of IL-2 and IL-5 treatment, we observed significant IgM secretion (increased up to about 10-fold) in a control transfectant. However, IgM secretion was dramatically impaired in all cyclin E-BCL-1 lines (#4, #7, and #9) (Fig. 3C). When the plasma cell surface marker Syndecan-1 was monitored by FACS (Fig. 3D), we observed only a slight increase in the cyclin E-BCL-1 lines compared to a control transfectant, in which surface Syndecan-1 increased about threefold.

In Table 1, the phenotypic characteristics of Myc-BCL-1 and cyclin E-BCL-1 lines after 3 days of IL-2 and IL-5 treatment are summarized and compared. We conclude that ectopic expression of cyclin E mimics ectopic expression of c-Myc and inhibits BCL-1 terminal differentiation. These data strongly support the view that it is the ability of c-Myc to drive proliferation that inhibits BCL-1 differentiation, and this aspect of c-Myc function must be repressed during B-cell differentiation.

Blimp-1 mRNA is induced by cytokine treatment of Myc- and cyclin E-BCL-1 cells.

We and others have shown that IL-2 plus IL-5 induces Blimp-1 mRNA in BCL-1 cells (39, 71) and that Blimp-1 is sufficient to drive their terminal differentiation. One potential explanation for the failure of Myc- and cyclin E-BCL-1 cells to differentiate is that Blimp-1 mRNA induction might be blocked. Therefore we examined the induction of Blimp-1 mRNA in these cells and in parental BCL-1 cells using an RNase protection assay (Fig. 4). In parental BCL-1 cells, the induction of Blimp-1 mRNA was detected after 2 h of IL-2 plus IL-5 treatment, and after 48 h, Blimp-1 mRNA was induced 2.7-fold. Blimp-1 mRNA increased similarly following cytokine treatment of the transfectants. Induction was observed after 2 h of cytokine treatment, and after 48 h Myc-BCL-1 line (#5) showed 5.3-fold induction and cyclin E-BCL-1 line (#7) 3.5-fold induction. Thus, induction of Blimp-1 mRNA in response to cytokine treatment occurs normally in lines ectopically expressing c-Myc or cyclin E. These data show that failure to induce Blimp-1 mRNA cannot explain the failure of the c-Myc- and cyclin E-expressing lines to differentiate. They also show that Blimp-1 mRNA levels are not subject to negative feedback by c-Myc or by proliferation.

We also examined c-Myc in the cyclin E-BCL-1 lines using immunoblotting. As shown in Fig. 5, in a control transfectant treated with IL-2 and IL-5 for 3 days, c-Myc protein levels decreased to 46% of those in untreated cells. c-Myc levels in all of the cyclin E BCL-1 lines (#4, #7, and #9) decreased similarly after treatment with cytokine (47, 50, and 39%, respectively). This shows that ectopic expression of cyclin E does not affect the normal decrease in c-Myc which occurs in response to cytokine-dependent induction of the Blimp-1 transcriptional repressor. It provides further evidence that normal regulation of c-Myc targets not related to cell cycle progression is insufficient to inhibit BCL-1 differentiation in conditions of enforced proliferation.

Expression of dominant negative c-Myc is not sufficient to drive terminal differentiation of BCL-1 cells.

Our data show that repression of c-myc by Blimp-1 is necessary for terminal differentiation of BCL-1 cells. We also wished to determine if repression of c-myc is sufficient to drive differentiation in this system. Our strategy was to express an inducible dominant negative form of c-Myc. The dominant negative c-Myc (MycDN) contained a deletion in the transactivation domain (amino acids 103 to 143) but retained the basic helix-loop-helix/zipper domains so that it could still associate with Max and bind DNA. Its dominant negative activity has been established in several previous studies, in which it was used to interfere with the function of endogenous c-Myc (9, 20, 40, 63). We used an inducible system in which dominant negative c-Myc was fused to a mutated estrogen receptor (MycDN-ER) and transcribed from the cytomegalovirus promoter. Thus, expression was constitutive and activity could be induced by treatment with the synthetic ligand 4-OHT (15, 40). Immunoblotting revealed that the MycDN-ER fusion protein was present in two lines chosen for study (MycDN-ER-BCL-1 #4 and #6) (Fig. 6A).

In other systems, the induction of MycDN-ER by 4-OHT has been verified by demonstrating a decreased percentage of cells in S phase (40). Therefore, we determined the cell cycle distribution of MycDN-ER-BCL-1 lines before and after induction of MycDN-ER with 500 nM 4-OHT (Fig. 6B). There was only a slight change in the percentage of cells in S phase (45% versus 53%) in a control transfectant after 4-OHT treatment (compare dotted bar to open bar). In contrast, the percentage of cells in S phase in MycDN-ER-BCL-1 transfectants after 4-OHT treatment declined consistently (from 46% to 22% in MycDN-ER #4 and from 52% to 39% in MycDN-ER #6). This shows that MycDN activity is inducible by 4-OHT in these lines and also shows that endogenous c-Myc appears to be more fully inhibited in line #4 than in line #6. We also determined the cell cycle status of these lines following cytokine treatment. Both of the MycDN-ER-BCL-1 lines and the control transfectant were growth arrested after IL-2 and IL-5 treatment (compare solid bar to open bar), indicating that the transfectants retained the ability to respond normally to cytokine treatment.

These transfectants were then analyzed to determine if inhibition of c-Myc was sufficient to drive differentiation to a plasma cell phenotype. MycDN-ER-BCL-1 cells were treated with 4-OHT or cytokine for 3 days and then analyzed for markers of the plasma cell phenotype. IgM secretion was analyzed by ELISA, and Fig. 7A shows that in both MycDN-ER-BCL-1 cells (#4 and #6), activation of MycDN-ER by 4-OHT did not induce IgM secretion, although they responded normally to cytokine treatment. Surface Syndecan-1 levels were also measured: in both MycDN-ER-BCL-1 cells and a control transfectant, Syndecan-1 was induced after IL-2 and IL-5 treatment (Fig. 7B, dashed line). However, 4-OHT alone treatment did not induce Syndecan-1 expression in either MycDN-ER #4 or #6 (compare dark line to dashed line). These results suggest that inhibition of c-Myc and the cessation of cell cycle progression that accompanies it are not sufficient to induce BCL-1 cell differentiation.

It remained a possibility that induction of MycDN did not inhibit cell cycle progression sufficiently to trigger terminal differentiation. Therefore, we sought to arrest BCL-1 cell cycle progression using LOV, which causes growth arrest at G₁ (56) and is sufficient to induce differentiation of human monocytic Mono Mac 6 cells (72). We treated BCL-1 cells with different concentrations of LOV (within a dose range that did not cause cell death) and analyzed the effect of LOV on cell cycle progression as well as on BCL-1 cell differentiation. The percentage of cells in S phase decreased from 36% to 12% after 3 days of 3 μM LOV treatment, compared to 17% in S phase following cytokine treatment. However, inhibition of cell cycle progression by LOV was not sufficient to cause BCL-1 cells to secrete IgM or to express Syndecan-1 on their surface (not shown), further indicating that inhibition of the cell cycle is not sufficient to drive terminal differentiation of BCL-1 cells.

Dissecting the mechanism(s) by which Blimp-1 acts as a master regulator of plasma cell differentiation.

Expression of Blimp-1 is sufficient to drive terminal differentiation of BCL-1 cells into plasma-like cells, providing an important experimental system for elucidating the changes in gene expression required for this event (39, 71). The c-myc gene was previously identified as a Blimp-1 target gene, and c-myc repression in BCL-1 correlates with cell cycle arrest (39) (Fig. 2A and B). The data presented here establish the functional relevance of c-myc repression, since enforced expression of c-Myc blocks the ability of these cells to differentiate (Fig. 2). It is interesting to note in this context that Blimp-1 mRNA was induced normally by cytokine treatment of BCL-1 clones expressing constitutive c-Myc (Fig. 4). Therefore, Blimp-1-dependent regulation of other possible target genes is not sufficient to overcome the inhibition by c-Myc.

The data in Fig. 7 show, however, that dominant negative c-Myc was not sufficient to trigger differentiation of BCL-1 cells. In contrast, inhibition of c-Myc activity can induce differentiation of some nonlymphoid cell lines (23, 25, 73). Also, overexpression of the cyclin-dependent kinase inhibitor p18^INK4c in human lymphoblastoid SKW cells caused differentiation (47). It is likely that differences in the developmental stage may account for the difference in the response of SKW and BCL-1 cells to cessation of the cell cycle.

It is formally possible that c-Myc activity was not sufficiently inhibited in any of our MycDN-ER-BCL-1 lines to induce differentiation. However, we also blocked BCL-1 cell proliferation with LOV, since our data showed that c-Myc's proliferative activity is critical for plasma cell differentiation (Fig. 3). Even when more complete cessation of cycle was achieved with LOV, this was not sufficient to induce BCL-1 differentiation. Thus, neither expression of MycDN-ER nor cessation of proliferation is sufficient to trigger terminal differentiation. These data, in conjunction with the observation that enforced proliferation blocks terminal differentiation, support the conclusion that repression of c-myc transcription is necessary but not sufficient to drive BCL-1 differentiation. We suggest that regulation of other target genes in addition to c-myc is required during the Blimp-1 program of terminal differentiation.

If other Blimp-1 target genes are important during BCL-1 differentiation, what might those target genes be and how might Blimp-1 affect their transcription? It seems most likely that Blimp-1 target genes will be repressed, since there is ample evidence that Blimp-1 is a transcriptional repressor. Its human homologue, PRDI-BF1, also functions as a transcriptional repressor of the interferon-β gene (27, 31). Blimp-1 represses transcription of a heterologous promoter in a GAL4 fusion assay (74). Blimp-1/PRDI-BF1 mediates transcriptional repression via the association with both hGroucho (58) and histone deacetylase (74) proteins. However, these data do not rule out the possibility that Blimp-1 might also activate transcription in the context of a currently unidentified target gene.

Several genes, including BCL-6 (48), CIITA (68), BSAP (5), A1 (32), and CD23 (61), are known to be transcriptionally repressed upon plasma cell differentiation and are good candidates to be direct targets of Blimp-1. Indeed, we have recently shown that the P3 promoter of the CIITA gene, encoding a coactivator required for class II MHC, Ii, and DM gene transcription, is directly repressed by Blimp-1 (Piskurich et al., submitted). In addition, there are likely to be Blimp-1 target genes as yet unidentified. Our current model for the roles of c-myc and other genes in Blimp-1-dependent plasma cell differentiation is summarized in Fig. 8.

Role of c-Myc in terminal B-cell differentiation.

Inhibition of plasma cell differentiation by constitutive expression of c-Myc (Fig. 2) is consistent with similar observations in other cell lineages in which ectopic c-Myc blocks differentiation (13, 25, 26, 35, 50). Furthermore, mice harboring an Eμ-c-myc transgene develop B-cell lymphomas (1), display abnormal apoptosis (51), and have little evidence of terminal differentiation (67), consistent with the requirement for c-myc repression during plasma cell development.

The mechanism(s) by which c-Myc blocks differentiation is not well understood. Our data show that proliferation is sufficient to inhibit BCL-1 differentiation, since the effect of constitutive c-Myc can be mimicked by constitutive cyclin E (Fig. 3). In the cyclin E-BCL-1 lines, Blimp-1 mRNA and c-Myc are normally induced or repressed, respectively, in response to cytokine treatment, so that other Blimp-1- or c-Myc-dependent target genes are presumably regulated normally. However, this is not sufficient to overcome the inhibition of differentiation caused by continued proliferation. Thus, c-Myc target genes that drive proliferation are the ones that must be correctly regulated to allow terminal B-cell differentiation. These target genes may regulate cell cycling directly or may regulate cell growth and metabolism or both (reviewed in reference 14). Cell cycle regulators Gadd45, Cdc25A, and cyclins A and E are induced by c-Myc, and c-Myc also regulates the activity of cyclin E-Cdk2 (43, 70). In addition, c-Myc target genes involved in metabolism and growth control, such as the genes for carbamoyl-phosphate synthase–aspartate carbamoyl transferase–dihydroorotase, ornithine decarboxylase, dihydrofolate reductase, thymidine kinase, lactate dehydrogenase A, H-ferritin, iron regulatory protein 2, eIF4E, and eIF2a, may be critical for cell proliferation (14, 28, 29). It will be interesting to establish stable BCL-1 lines expressing individual c-Myc target genes to examine their effect on differentiation. Similar studies in M1 myeloid leukemia cells indicated that ectopic expression of ornithine decarboxylase could not mimic c-Myc's effect on differentiation (25).

Results obtained with any cell line need to be interpreted cautiously with respect to extrapolation to mechanisms in normal cells. However, BCL-1 cells have provided an accurate model for studying many aspects of B-cell biology in the past. In addition, we have shown that induction of Blimp-1 mRNA, repression of c-myc mRNA, and cessation of cell cycling also occur upon lipopolysaccharide treatment of primary splenic B cells (3). We have also found that Blimp-1 is expressed in vivo in murine and human plasma cells (3a). Thus, the observations made in the BCL-1 cell model are likely to have important implications for terminal differentiation of B cells in vivo.