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Molecular and Cellular Biology, December 2000, p. 8684-8695, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Repression of c-myc Is Necessary but Not
Sufficient for Terminal Differentiation of B Lymphocytes In
Vitro
Kuo-I
Lin,1
Yi
Lin,1 and
Kathryn
Calame1,2,*
Department of
Microbiology1 and Department of
Biochemistry & Molecular Biophysics,2 College of
Physicians and Surgeons, Columbia University, New York, New York 10032
Received 25 May 2000/Returned for modification 30 June
2000/Accepted 4 September 2000
 |
ABSTRACT |
The importance of c-myc as a target of the Blimp-1
repressor has been studied in BCL-1 cells, in which Blimp-1 is
sufficient to trigger terminal B-cell differentiation. Our data show
that Blimp-1-dependent repression of c-myc is required for
BCL-1 differentiation, since constitutive expression of c-Myc blocked
differentiation. Furthermore, ectopic expression of cyclin E mimicked
the effects of c-Myc on both proliferation and differentiation,
indicating that the ability of c-Myc to drive proliferation is
responsible for blocking BCL-1 differentiation. However, inhibition of
c-Myc by a dominant negative form was not sufficient to drive BCL-1 differentiation. Thus, during Blimp-1-dependent plasma cell
differentiation, repression of c-myc is necessary but not
sufficient, demonstrating the existence of additional Blimp-1 target genes.
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INTRODUCTION |
In many cell lineages, the c-Myc
oncoprotein is critical for regulating growth control, apoptosis,
and/or differentiation, and its dysregulation also plays a causal role
in a wide variety of neoplasias (12, 17, 52). c-Myc is
induced by mitogens expressed during G1 to S progression
and required for efficient cell cycle progression (reviewed in
references 12 and 17). Ectopic
expression of c-Myc promotes reentry of fibroblasts into the cell cycle
in the absence of mitogenic stimulation (18). c-Myc
antisense oligonucleotides inhibit entry into S phase (24), and antagonism of c-Myc by Mad overexpression inhibits proliferation (54). Recent studies suggest that c-Myc's proliferative
effects may be due primarily to activation of genes required for cell growth rather than genes that regulate cell cycle progression (28,
29), although both kinds of targets probably play a role in
c-Myc-dependent proliferation. c-Myc expression also induces apoptosis
when mitogen is withdrawn from fibroblasts and in other cell contexts
(20, 49). Downregulation of c-Myc accompanies differentiation (19, 34), and ectopic expression of c-Myc blocks terminal differentiation in mouse erythroleukaemia cells, human
monoblastic cells (U-937), myeloid leukemic M1 cells, and postmitotic
murine keratinocytes (13, 25, 26, 35, 50). Additionally,
inhibition of c-Myc results in induction of differentiation in a human
promyelocytic leukemia cell line (HL-60) and in murine F9
teratocarcinoma cells (23, 25, 73) or reverse tumorigenesis in hematopoietic lineages of mice (21).
c-Myc belongs to the basic helix-loop-helix/leucine zipper family of
transcription factors (7). Heterodimerization between c-Myc
and its partner, Max, is obligatory for binding site-dependent activation of target genes (6, 7, 53). Genes containing Myc-Max binding sites have been identified and shown to be regulated by
Myc-Max (14). In addition, c-Myc regulates transcription of
other genes that lack Myc-Max binding sites, by binding at initiator
sequences (38) or by associating with other transcription proteins, including TFII-I (60), YY-1 (64-66),
and Miz-1 (62).
Consistent with its role in other cell lineages, c-Myc has been shown
to be important in normal B-cell lymphopoiesis and to be dysregulated
in many B-cell malignancies. During B-cell development, c-Myc levels
change in precise ways, suggesting that c-Myc is critical for the
highly regulated periods of cell proliferation that occur as B cells
mature (45). c-Myc is present at high levels in pro-B,
pre-BI, and pre-BII cells and falls when B cells become small, resting,
and surface immunoglobulin M (IgM)-positive immature B cells. c-Myc
levels rise again during antigen-induced proliferation of mature B
cells (45). Dysregulated expression of c-Myc in B cells is
often tumorigenic. For example, chromosomal translocations of the
c-myc gene to Ig gene loci are present in most human
Burkitt's lymphomas and murine plasmacytomas (37), and
transgenic mice expressing c-myc under the control of the Eµ heavy-chain intronic enhancer develop progressive,
stage-nonrestricted B-lymphoid neoplasias (1) preceded by
increased cell size of pretransformed B cells (28). Finally,
c-Myc expression declines as B cells differentiate into memory cells
(42) or Ig-secreting plasma cells (36). Although
the importance of c-Myc for growth control during normal B
lymphopoiesis and in B-cell tumors is clear, the significance, if any,
of c-myc repression during terminal B-cell differentiation
has not been carefully investigated.
The zinc finger protein Blimp-1 (B lymphocyte-induced maturation
protein-1) represses c-myc in B cells. Blimp-1 was first identified as a plasmacytoma-specific repressor factor that bound to a
negative element in the murine c-myc promoter (30,
39). Plasmacytoma-specific repressor factor was subsequently
identified as Blimp-1 (39). Blimp-1 was cloned by
subtractive hybridization of mRNAs induced when a mature B-cell
lymphoma line, BCL-1, differentiated into a plasma cell phenotype in
response to treatment with interleukin-2 (IL-2) and IL-5
(71). Blimp-1 is also induced in other in vitro models of
terminal B-cell differentiation and is expressed in murine plasmacytoma
lines, human myeloma lines, and in vivo human and murine plasma cells
(3a, 39, 55, 69; J. F. Piskurich et al.,
submitted for publication).
Significantly, ectopic expression of Blimp-1 is sufficient to drive
differentiation of BCL-1 cells to a plasma cell phenotype, evidenced by
induction of Syndecan-1 on the cell surface, J-chain expression, IgM
secretion, and increased cell size and granularity (39, 71).
Ectopic expression of Blimp-1 also downregulates endogenous c-Myc,
correlating well with the cessation of proliferation that occurs during
B-cell terminal differentiation (39). Inhibition of B-cell
differentiation by CD40 engagement is associated with downregulation of
Blimp-1 induced by cytokines in CH12 cells (55), further
supporting the role of Blimp-1 in terminal differentiation of B cells.
Thus, Blimp-1 appears to be a master regulator of terminal B-cell differentiation.
Given the established importance of c-Myc in growth regulation and
differentiation in other cell lineages, it seemed likely that the
previously observed Blimp-1-dependent repression of c-myc transcription was critical for terminal B-cell differentiation. We
wished to determine if c-myc repression was necessary and/or sufficient for plasma cell development, and if either were true, we
wished to determine which aspect of c-Myc function was important in
controlling plasma cell differentiation.
To address these questions experimentally, we have taken advantage of
the BCL-1 culture model for plasma cell differentiation. The ability of
BCL-1 lymphoma cells to differentiate to a plasma cell phenotype was
used early on to study expression of secreted versus membrane forms of
mu mRNA in response to lipopolysaccharide (LPS)
(75) and to identify and characterize factors for inducing plasma cell differentiation (8). Subsequently, BCL-1 cells have been exploited to study other aspects of plasma cell development, including induction of J chain transcription (44)
and the identification of BSAP as an IL-2-inducible repressor of
J chain transcription (59). Blimp-1 was
originally cloned and characterized based on its ability to trigger
differentiation of BCL-1 cells (46, 71). Therefore, BCL-1
cells provide a well-characterized model system for further analysis of
the role of Blimp-1 in plasma cell differentiation.
We report here that ectopic expression of c-Myc blocks terminal
differentiation of BCL-1 cells, showing that repression of c-myc by Blimp-1 is necessary for differentiation of mature
B cells into plasma cells. Furthermore, the importance of repressing c-Myc is related to the ability of c-Myc to promote proliferation, since ectopic expression of cyclin E, which mimics the proliferative effect of c-Myc but not other effects, also inhibits terminal differentiation of BCL-1 cells. However, inhibition of c-Myc by induction of a dominant negative form of c-Myc was not sufficient to
cause differentiation of BCL-1 cells, although it was sufficient to
inhibit proliferation. These data suggest that repression of c-myc transcription and cell cycle arrest are not sufficient
to cause BCL-1 differentiation and indicate that Blimp-1-dependent regulation of additional target genes is required for full plasma cell differentiation.
| |
MATERIALS AND METHODS |
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
-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 -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
[
-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.
| |
RESULTS |
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.
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FIG. 1.
Ectopic expression of Blimp-1 decreases endogenous
c-myc transcription. BCL-1 cells (2 × 105)
were infected with pGC-YFP or pGC-Blimp-YFP. Three days postinfection,
cells were sorted, and RNA was prepared from YFP-positive cells and
used for semiquantitative RT-PCR analysis. cDNA dilutions, as
indicated, were analyzed for c-myc and GAPDH
mRNAs.
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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).
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FIG. 2.
Effects of ectopic expression of c-Myc on BCL-1 terminal
differentiation. (A) Total c-Myc levels did not decline in Myc-BCL-1
transfectants in response to cytokine treatment. Two Myc-BCL-1 lines
(Myc#2 and Myc#5) and a control transfectant (#2) growing in log phase
were either treated with recombinant IL-2 and IL-5 for 3 days or left
untreated. Immunoblots to detect c-Myc and -actin in
cytokine-treated (+) and nontreated ( ) cells are shown. (B)
Percentage of cells in S phase analyzed by FACS analysis. Cells (two
control transfectants, #1 and #2, and two Myc-BCL-1 lines, #2 and #5)
with or without IL-2 and IL-5 treatment for 3 days were subjected to
BrdU and propidium iodide staining. One experiment, representative of
three, is shown. (C) Secreted IgM analyzed by ELISA from culture
supernatants after 3 days of cytokine treatment (solid bar) or
untreated (open bar). Experiments were performed at least three times,
and standard deviations are shown. (D) FACS profiles of surface
Syndecan-1 staining of control transfectant (#2) and Myc-BCL-1 lines
(#2 and #5) treated with IL-2 and IL-5 for 3 days (dark line) or left
untreated (fine line). Experiments were done at least four times, and
one set of representative profiles is shown.
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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 G
1 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.
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FIG. 3.
Ectopic expression of cyclin E promotes proliferation
and blocks BCL-1 differentiation after cytokine treatment. (A)
Immunoblots for cyclin E and -actin from the cell lysates of cyclin
E-BCL-1 (#4, #7, and #9) and a control transfectant. (B) Percentage of
cells in S phase as analyzed by BrdU and propidium iodide staining from
2 × 105 log-phase cells (control transfectant,
Myc-BCL-1, and cyclin E-BCL-1 cells) treated with cytokine for 3 days
(solid bar) or left untreated (open bar). Experiments were done at
least twice, and one representative result is shown. (C) Secreted IgM
measured by ELISA for control and cyclin E-BCL-1 (#4, #7, and #9) cells
treated with IL-2 and IL-5 for 3 days (solid bar) or left untreated
(open bar). Standard deviations were derived from three independent
experiments. (D) FACS profiles of Syndecan-1 staining for control
transfectant and cyclin E-BCL-1 (#4, #7, and #9) with (dark line) or
without (fine line) cytokine treatment for 3 days. Experiments were
done at least twice.
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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.
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FIG. 4.
Blimp-1 mRNA is induced in Myc-BCL-1 and cyclin E-BCL-1
cells after cytokine treatment. Total RNA (10 µg) from cells
(parental BCL-1 cells, Myc-BCL-1 #5, and cyclin E-BCL-1 #7) treated
with IL-2 and IL-5 for 0, 2, 10, 24, and 48 h was subjected to
RNase protection analysis using [-32P]UTP-labeled
riboprobes specific for mouse Blimp-1 and GAPDH. Numbers represent the
fold induction of Blimp-1 mRNA after normalization to the
level of GAPDH mRNA.
|
|
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.
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|
FIG. 5.
c-Myc is downregulated in cyclin E-BCL-1 cells after
IL-2 and IL-5 treatment. Log-phase control and cyclin E-BCL-1 #4, #7,
and #9 cell lines (2 × 105) were either left
untreated or treated for differentiation for 3 days, and cell lysates
were harvested for Western blotting analysis as described in Materials
and Methods. Protein (10 µg) was separated by SDS-8% PAGE, and
c-Myc and -actin levels were revealed by probing with specific
antibodies. Numbers represent the fold decrease in c-Myc after
normalization to -actin.
|
|
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).
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|
FIG. 6.
Induction of dominant negative c-Myc activity by 4-OHT
in MycDN-ER-BCL-1 cells. (A) Immunoblots for the detection of dominant
negative Myc-ER fusion protein. The position of the 83-kDa size marker
is shown on the left. N.S., nonspecific band. (B) Cell cycle status of
2 × 105 cells (control and MycDN-ER #4 and #6) left
untreated (open bar) or treated with cytokine (solid bar) or 500 nM
4-OHT (dotted bar) for 3 days. Results show the percentage of cells in
S phase determined by FACS after BrdU and propidium iodide staining.
Experiments were done twice, and one set of results is shown.
|
|
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.
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|
FIG. 7.
Cell cycle arrest by dominant negative c-Myc does not
cause differentiation of BCL-1 cells. (A) IgM secretion in control and
MycDN-ER-BCL-1 #4 and #6 cells after 3 days of cytokine or 4-OHT
treatment. The 2 × 105 cells were left untreated or
treated with recombinant IL-2 and IL-5 or 500 nM 4-OHT for 3 days, and
supernatants were collected and assayed by ELISA for IgM levels.
Results show the averages from three independent determinations.
Standard deviations are also shown. (B) Cell surface Syndecan-1
staining profiles analyzed by FACS of control (left panel),
MycDN-ER-BCL-1 #4 (middle panel), and MycDN-ER-BCL-1 #6 (right panel)
cells following cytokine or 4-OHT treatment. The cells were left
untreated (fine line) or treated with recombinant IL-2 and IL-5 (dashed
line) or 500 nM 4-OHT (dark line) for 3 days, and surface marker
staining using anti-Syndecan-1 antibody was performed. Experiments were
done three times, giving similar results.
|
|
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 G
1 (
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.
| |
DISCUSSION |
The data presented here reveal several important aspects of
terminal B-cell development in the context of the BCL-1 cell model. First, reduction of c-Myc is necessary for plasma cell differentiation, since constitutive expression of c-Myc blocks differentiation. This
establishes the functional importance of c-myc as a target of Blimp-1-dependent repression. Second, continued proliferation is
sufficient to block plasma cell differentiation, suggesting that cell
proliferation is the aspect of c-Myc function that must be repressed to
allow differentiation. Finally, neither blocking of c-Myc activity nor
cessation of proliferation is sufficient to trigger terminal
differentiation. This leads to the important conclusion that, in
addition to c-myc, Blimp-1 must regulate other target genes
in order to trigger plasma cell differentiation.
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-
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.
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|
FIG. 8.
Model for the roles of Blimp-1 and c-Myc in B-cell
terminal differentiation. II°, secondary. ODC, ornithine
decarboxylase. MHCII, major histocompatibility complex II.
|
|
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.
| |
ACKNOWLEDGMENTS |
We are grateful to R. Dalla-Favera, I. Greenwald, C. Angelin-Duclos, and D. H. Chang for critically reading the
manuscript and to the members of the Calame laboratory for helpful
discussions. We especially thank D. H. Chang for allowing use of
the pGC-Blimp-YFP construct and C. Tunyaplin for technical help on the
RNase protection assay. We thank D. Cobrinik for providing the
pSKmcyclinE construct, G. Littlewood for providing the pBabeMycNDERpuro
construct, G. Fathman for the pGC-IRES-YFP plasmid, and S. Goff for
cyclin E antibody. We thank J. Liao for excellent technical assistance.
This work was supported by grants GM29361 and AI 43576 to K. Calame.
Kuo-I Lin is a fellow of the Leukemia and Lymphoma Society (5332-00).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, HHSC 1202, Columbia University College of Physicians and Surgeons, 701 W. 168th St., New York, NY 10032. Phone: (212) 305-3504. Fax: (212) 305-1468. E-mail: KLC1{at}columbia.edu.
| |
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Abstract
Introduction
