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INTRODUCTION |
Classical NF-
B1 is a
heterodimer composed of two protein subunits, p50 and p65, that are
members of the Rel family of transcription factors (1). This family
consists of five known members, c-Rel, RelA (p65), RelB, p50, and p52
(2-6), and is identified by a characteristic N-terminal 300 amino acid
Rel homology domain. This region contains sequences for DNA binding,
dimerization, and nuclear localization. Association of Rel family
dimers with a second family of proteins, the I
Bs, is responsible for
the cytoplasmic sequestration of inactive NF-
B in unstimulated cells (7).
NF-
B was originally identified as a B cell-specific transcription
factor that bound to a decameric sequence within the intronic enhancer
of the immunoglobulin (Ig)
light chain gene (8). It had been shown
previously that the
B site within this enhancer is critical for
enhancer activity in reporter assays (9) and that NF-
B activation
controlled the ability of this enhancer to activate transcription
(10-12). During early B cell development, Ig
loci undergoing gene
rearrangement were also transcriptionally active (13-15), and this
activation correlated with the presence of active nuclear NF-
B (8,
10). These observations led to the hypothesis that NF-
B played a
critical role in the activation of Ig
locus transcription and
rearrangement during early B cell development (9-11, 14, 16). It was
later appreciated that NF-
B was in fact a ubiquitously expressed,
inducible factor (17), responsible for the activation of a diverse
array of genes (1), but its role in B cell development continues to be
of significant interest.
The role of NF-
B in the developmental regulation of gene expression
has been studied by generating mice deficient in individual Rel family
members (18-23). Whereas none of the single knock-out mice showed any
obvious defect in early B cell development, a recent report of mice
deficient in both p50 and p52 revealed an essential role for NF-
B in
the generation of mature B cells (24). Interestingly, mice deficient in
either p50 or c-Rel have normal numbers of mature B cells, but these
cells fail to activate appropriately in response to antigen receptor
stimulation leading to humoral immunodeficiency (18, 20). The role of
RelA in B cell development could not be assessed since deficiency in
this factor proved lethal by day 15 of embryogenesis, although adoptive
transfer experiments suggested that B cell expression of RelA was not
required for normal development (21, 25). Evaluation of RelA-deficient pre-morbid fetuses attributed death to massive liver cell apoptosis. Thus, whereas their precise role in early B cell development remains uncertain, genetic studies implicate Rel family members in the regulation of both cell activation and cell death.
A variety of other studies has demonstrated a role for NF-
B in both
protection from and induction of apoptosis (26-32). These reports
encompass a range of both cell types and apoptosis-inducing stimuli. Additional observations have suggested a role for NF-
B in
growth arrest and differentiation (24, 25, 33-37), and recently NF-
B transcription has been correlated with cell cycle progression (38). These observations lead to the conclusion that the cell type and
context of an NF-
B-inducing stimulus are critical determinants in
the outcome of a signal that can lead to proliferation,
differentiation, or death.
In an effort to clarify the effects of NF-
B on cell proliferation
and viability during B cell development, we utilized the tetracycline-regulated expression system (39, 40) to examine the
individual effects of either RelA or c-Rel overexpression in a pro-B
cell line. Whereas elevated levels of RelA resulted in a G1
cell cycle arrest followed by the induction of apoptosis, the
overexpression of c-Rel did not affect cell growth or viability. Both
the transactivating potential and the DNA binding specificity of RelA
were required for these effects. To investigate cell type specificity,
RelA was also overexpressed in immature and mature B lymphoma cell
lines. Interestingly, elevated levels of RelA in these lymphoma cell
lines did not result in apoptosis. From these observations, we conclude
that RelA expression can result in cell growth arrest, leading to the
induction of apoptosis, and that this apoptotic potential may be
developmentally stage-specific.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The pro-B cell line, 220-8, the immature B
cell lymphoma, WEHI 231, and the mature B cell lymphoma M12 were
cultured in RPMI 1640 with glutamine, supplemented with 10% fetal
bovine serum (Gemini Biological Products), 50 µg/ml
penicillin/streptomycin, and 10
4 M
-mercaptoethanol.
Cells were transfected by electroporation, and stable transfectants
were selected in either mycophenolic acid or G418 (Life Technologies,
Inc.). The transfectant pools were single cell-cloned by limiting
dilution into antibiotic-containing media to establish clonal
populations. All selections were performed and cultures maintained in
the presence of 1 µg/ml tetracycline (Sigma) to repress tTA
expression. Expression was induced by harvesting cells by
centrifugation and then replating them into media lacking tetracycline.
Expression Constructs--
The gpt expression
cassette from pSV2-gpt was cloned into the pTet-tTA plasmid
((41) a gift from Dr. David Schatz) (pTet-tTAgpt) to allow
establishment of stable transfectants. Correspondingly, the neomycin
drug resistance cassette from pGK-neo was cloned into the target
plasmid of the inducible system, pTet-splice (41), to create
pTetspliceneo (pTSN). Finally, a hemagglutinin epitope tag (HA-tag)
consisting of three tandem HA epitopes (a generous gift from Dr. Susan
Michaelis) was cloned into the EcoRV site of the pTSN
polylinker to generate pTSN.flu. When HindIII was used as a
cloning site, an in-frame C-terminal HA epitope tag was generated. A
custom-designed linker (Life Technologies, Inc.), containing a stop
codon, was added at the end of the HA epitope. All cDNA fragments
were then cloned into the HindIII site of pTSN.flu. In each
case, translational reading frame was confirmed by DNA sequencing.
The murine relA cDNA (a gift from Dr. Sankar Ghosh) was
digested with Bsu36I and blunted with mung bean nuclease
(Boehringer Mannheim), truncating the gene at the 3' end of the open
reading frame. Custom-made oligonucleotide linkers (Life Technologies, Inc.) were designed to maintain the reading frame and ligated to the 3'
end. A fragment containing the relA open reading frame was
then cloned into pTSN.flu. The RelA TADt construct, containing a
deletion of the C-terminal transactivation domains, was cloned using a
polymerase chain reaction strategy, resulting in a gene fragment
missing the 79 C-terminal amino acids. The c-rel cDNA (a
gift from Sankar Ghosh) was excised by DraI digestion
(eliminating the last two amino acids of the reported open reading
frame), and then cloned into pTSN.flu using oligonucleotide adapters. The mutated relA cDNA (RRPA) obtained from Dr. Sankar
Ghosh was similarly cloned into pTSN.flu. The
relA-c-rel chimeric genes were created using a
polymerase chain reaction protocol. The 79 C-terminal amino acids
(amino acid 471-549) of RelA were fused to the N terminus (433 amino
acids) of c-Rel to generate c-Rel.TAD, and the C-terminal 135 amino
acids of the c-Rel protein (amino acids 434-568) were fused to the N
terminus (470 amino acids) of the RelA cDNA. These gene fusions
were then cloned into pTSN.flu.
Electrophoretic Gel Mobility Shift Assays--
Nuclear extracts
were prepared using an Nonidet P-40 lysis method (42). Briefly, cells
were spun down and washed once in 1× PBS, resuspended in hypotonic
buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µl/ml aprotinin (Sigma)), and incubated for 15 min on ice. The cells
were then lysed in 0.5% Nonidet P-40 and the nuclei pelleted. The
nuclei were salt-extracted (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, and 1 µl/ml aprotinin) and incubated with vigorous shaking
for 15 min at 4 °C. Nuclear remnants were removed by centrifugation,
and the supernatant containing extracted protein was stored in aliquots
at
80 °C. Protein concentration was determined via a BCA
colorimetric assay (Pierce).
5-10 µg of nuclear extract was added to 2 µl of 10× binding
buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 50%
glycerol), 20 µg of bovine serum albumin, 6 µg of poly(dI-dC), 3 mM GTP, and 1% Nonidet P-40 (included only in NF-
B DNA
binding analysis), and 105 cpm polynucleotide kinase (New
England Biolabs) end-labeled oligonucleotide probe. A final salt
concentration of 10 mM was established by adding the
appropriate amount of 1 M NaCl to the volume of the protein
extract. The NF-
B probe used in these experiments was 5'
TAACAGAGGGGACBBBCCGAGAGCCA (B indicates BrdUrd nucleotides). The
quality of all extracts was monitored by gel mobility shift assay using
an octamer binding site oligonucleotide: 5'
GCCTCATTTGCATGGACTTAGCTTGTCCATGCAAATGAGG. The 20-µl binding reactions
were incubated 10 min at room temperature, loaded onto a 4%
polyacrylamide gel (that had been pre-run for 60 min at 150 V with
buffer recirculation) in 0.25× TBE, and electrophoresed at 150 V for
3-4 h. The gel was dried under vacuum and exposed to a PhosphorImager
screen (Molecular Dynamics) for 12-36 h.
Where indicated, supershift analysis was performed by incubating
anti-RelA antibody (Santa Cruz Biotechnology) or anti-c-Rel antibody
(Santa Cruz Biotechnology) with the protein extract for 60 min at
4 °C. The binding buffer was then added, and the reaction was
incubated for an additional 10 min at room temperature before gel loading.
Western Blotting--
Whole cell extracts were prepared by
harvesting 5 × 105-106 cells, washing
once in 1× PBS, and then lysing in sample buffer (10% glycerol, 3%
SDS, 62.5 mM Tris pH 6.8). The samples were then mixed with
an equal volume of bromphenol blue loading dye (containing 1 M
-mercaptoethanol), boiled, and then electrophoresed on
a 7.5% SDS-polyacrylamide gel. The gel was blotted onto a 0.45-µm nitrocellulose membrane (Protran, Schleicher & Schuell) by
electroelution. The blots were stained with Ponceau S to assess protein
transfer and then blocked 30 min to overnight in 5% powdered
milk/PBS-T (1× PBS, 0.1% Tween 20). Primary antibodies included
anti-c-Rel and anti-RelA (listed above), used at 1:1000 dilutions in
5% powdered milk/PBS-T and anti-HA-epitope (Boehringer Mannheim, clone
12CA5) used at a 1:400 dilution in 5% milk/PBS-T. Incubations with the primary antibodies were 1-3 h at room temperature, and subsequent washes were done in PBS-T. Secondary antibody incubations, either anti-mouse Ig or anti-rabbit Ig, were performed at 1:3000 dilution in
5% milk/PBS-T for 30-60 min. The blots were imaged using
chemiluminescence (ECL; Amersham Pharmacia Biotech).
Cell Viability Assay--
Cell viability was assessed via the
trypan blue exclusion properties of a cell culture. Cultures were
analyzed in duplicate as follows: 4 × 105 cells were
harvested by centrifugation (to remove tetracycline) and resuspended in
20 ml of complete media lacking tetracycline. Control cultures were
similarly prepared with the addition of tetracycline to a final
concentration of 1 µg/ml. 1.5-ml aliquots were removed from cultures
at the indicated time points. Each aliquot of cells was harvested by
centrifugation and resuspended in a small volume (varying with expected
cell numbers) of PBS, diluted 1:1 with a trypan blue solution (Life
Technologies, Inc.), and counted using a hemacytometer. The total
number of cells in 1.5 ml was calculated and recorded as the cell
number for the appropriate time point.
Annexin V Staining--
106 cells were pelleted,
washed in wash buffer (1× PBS, 3% fetal bovine serum, and 10 mM Hepes, pH 7.4), resuspended in 100 µl of wash buffer,
and split into duplicate tubes. Each sample was pelleted and
resuspended in wash buffer supplemented with either 2 mM
CaCl2 or 2 mM EDTA. The FITC-Annexin V reagent
(CLONTECH) was added at a 1:20 dilution, and
samples were incubated for 30 min on ice. The cells were then washed
once with 1.5 ml of wash buffer with or without CaCl2 and
resuspended in 500 µl of the appropriate wash buffer. 7-AAD was added
at a concentration of 1 µg/ml, and cells were incubated 10 min at
room temperature before analysis on a FACScan (Becton Dickinson). Data
were analyzed using CellQuest software (Becton Dickinson).
Cell Cycle Analysis--
Cell cycle analysis was performed as
reported previously (43). Cell cultures were pulsed with 30 µM bromodeoxyuridine (BrdUrd; Sigma B5002) for 60 min.
3 × 106 cells were pelleted and washed in PBS, 5 mM EDTA. The cells were fixed in methanol, pelleted,
resuspended in 1 ml of 2 N HCl, 0.2 mg/ml pepsin, and
incubated at room temperature for 30 min. 3 ml of 0.1 M
sodium tetraborate, pH 8.5 (Sigma), was added to neutralize the HCl,
the cells were pelleted, washed once in IFA (0.009 M Hepes/0.15 M NaCl, pH 7.7, 4% heat-inactivated newborn
calf serum (Life Technologies, Inc.), 0.1% sodium azide), washed once
in IFA + 0.5% Tween 20, resuspended in anti-BrdUrd antibody
(PharMingen Clone 3D4) diluted 1:1000 in IFA + 0.5% Tween 20, and
incubated for 30 min at room temperature. The cells were then washed in IFA, 0.5% Tween 20 and resuspended in 100 µl of
anti-mIgG1 (PharMingen) diluted 1:200 in IFA, 0.5% Tween
20 and incubated for 30 min at room temperature. A final wash was done
in IFA, 0.5% Tween 20, and the cells were resuspended in PBS, 20 µg/ml RNase and incubated for 15 min at room temperature. 7-AAD was
added at a final concentration of 1 µg/ml, and the samples were
analyzed using the FACScan and CellQuest software.
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RESULTS |
Tetracycline-regulated Expression of RelA and c-Rel--
An
inducible expression system was used to individually overexpress the
RelA and c-Rel proteins in the Abelson virus-transformed pro-B cell
line 220-8 (14). In this system, the DNA binding domain of the Tet
repressor protein (TetR), whose DNA binding is inhibited in the
presence of tetracycline, is fused to the transactivation domain of the
herpesvirus VP16 protein, resulting in a tetracycline-regulated
transactivator protein (tTA) (39). When Tet operator sequences are
positioned upstream of a minimal promoter, the binding of tTA results
in transcriptional activation of a downstream target gene. In the
presence of tetracycline, tTA protein cannot bind its operator
sequences, and the target gene is not transcribed. By placing the tTA
gene itself under control of the tTA-inducible promoter, an
autoregulatory loop is established resulting in tight control of target
gene expression (41).
A vector containing tTA expression and mycophenolic acid resistance
cassettes was used to generate stable 220-8 transfectants. Single cell
clones were screened for inducible expression of the tTA fusion protein
and a clone expressing high levels of tTA10 was identified for use in
subsequent experiments (data not shown). A second vector, in which
either the relA or c-rel cDNA (fused in-frame
with an HA epitope tag) was placed under the regulatory control of tet
operator sequences, was transfected into tTA10, and stable
transfectants were selected for G418 resistance. Single cell clones
were analyzed by Western blot for inducible expression of either RelA
or c-Rel protein. In the presence of tetracycline, no detectable
protein was made, but upon removal of tetracycline, protein expression
was seen as early as 5 h after antibiotic removal, and expression
was stable for up to 5 days (Fig. 1,
A and B). Levels of the induced RelA and c-Rel
proteins were similar to the levels of the corresponding endogenous
proteins (Fig. 1 and data not shown).

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Fig. 1.
Time course of induced Rel protein
expression. A, two RelA-transfected pro-B cell clones,
RelA.4 and RelA.18, were induced to express the RelA protein by removal
of tetracycline. At the indicated time points, 106 cells
were removed and whole cell lysates made. Half of each sample was
analyzed by Western blotting, using an anti-p65 antibody.
RelA-transfected clones were compared with both the original 220-8 cell
line and with the parental tTA-expressing cell line, tTA10. The
upper arrow indicates transfected RelA protein, and the
lower arrow indicates the endogenous RelA protein.
Transfected RelA is larger due to its C-terminal epitope tag.
B, c-Rel clone.19 was analyzed as representative of
c-Rel-overexpressing clones, in a protein expression time course,
similar to the protocol outlined above. The Western blot was developed
with an anti-HA antibody (Boehringer Mannheim), and the
arrow indicates the c-Rel-HA fusion protein. C,
antibody supershift analysis of induced NF- B site binding complexes.
Left panel, nuclear extracts were prepared from 220-8 cells
cultured in the absence (lane 1) or presence (lanes
2-4) of LPS. These extracts were analyzed by electrophoretic
mobility shift without (lanes 1 and 2) or with
(lanes 3 and 4) preincubation with anti-RelA or
anti-c-Rel antisera as indicated. Right panel, nuclear
extracts were prepared from a RelA-transfected clone cultured in the
presence (lane 5) or absence (lanes 6-8) of
tetracycline. These extracts were analyzed by electrophoretic mobility
shift without (lanes 5 and 6) or with
(lanes 7 and 8) preincubation with anti-RelA or
anti-c-Rel antisera as indicated. The specific NF- B DNA binding
complex is indicated by the arrow in each panel.
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The appearance of NF-
B binding activity in nuclear extracts
paralleled the accumulation of induced protein (Fig. 1C),
with maximal binding detected after 72 h of induction. Supershift
analysis of these complexes using specific antibodies identified the
presence of both RelA and c-Rel in these induced
B site-binding
complexes (Fig. 1C and data not shown). Thus, induction of
either RelA or c-Rel protein resulted in both activation and nuclear
translocation of NF-
B in the absence of an additional stimulus.
NF-
B can also be induced in untransfected cells by culturing them in
the presence of bacterial lipopolysaccharide (LPS). LPS induces nuclear
NF-
B complexes containing predominantly RelA (Fig. 1C)
and p50 (35, 36, 44).
Overexpression of RelA, but Not c-Rel, Leads to Cell
Death--
During our initial analysis of induced RelA or c-Rel
protein expression, we noted a significant amount of cell death in
pro-B cell clones expressing the RelA construct, but not in those
cultures expressing either the c-Rel construct or the parental tTA
construct itself. Untransfected LPS-treated 220-8 cultures failed to
show increased cell death, however (data not shown). A cell viability analysis, utilizing trypan blue exclusion, revealed a striking loss of
cells upon induction of the RelA protein (Fig.
2). In contrast, cells expressing either
the tTA regulatory protein or the c-Rel protein show only a modest
decrease in cell growth compared with control 220-8 clones.

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Fig. 2.
RelA induction is associated with cell
death. The viability of parental, RelA-expressing and
c-Rel-expressing clones was assessed by a cell counting analysis.
Duplicate cultures were seeded at a starting cell concentration of
1 × 104 cells/ml, either in presence or absence of
tetracycline. Aliquots were removed and counted every 12 h. The
total number of trypan blue-excluding cells within the 1.5-ml aliquot
was calculated. Duplicate culture calculations were averaged at each
time point and are represented on the graphs as single points. The
profile of cultures growing in the presence of tetracycline is
indicated by the filled squares, and the profile of cultures
growing under inducing conditions (the absence of tetracycline) is
indicated by the open circles. tTA10, the parental clone is
shown in the upper left hand panel. Three independent
RelA-overexpressing clones (RelA.10, RelA.21, and RelA.25) and two
independent c-Rel-overexpressing clones (c-Rel.8 and c-Rel.31) were
examined.
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A kinetic analysis of the effect of RelA expression on cell number
revealed a time lag between protein expression and cell death. Although
we could detect RelA as soon as 5 h after the removal of
tetracycline, with its expression peaking at 36 h (Fig. 1,
A and B), significant cell loss was not seen
until 72 h. Interestingly, this onset of cell loss coincided with
the maximal induction of nuclear
B site binding activity (Fig.
1C and data not shown). This implies that the observed cell
death might depend upon the nuclear translocation of RelA and
subsequent activation of cellular gene expression.
RelA-induced Cell Death Is Apoptotic--
To characterize cell
death in this system, we stained cells with FITC-conjugated recombinant
Annexin V (Fig. 3). Annexin V is a
protein that binds phosphatidylserine. During the early stages of
apoptosis, phosphatidylserine, which is primarily localized to the
inner leaflet of plasma membranes in healthy cells, is exposed on the
outer leaflet where it can be detected by Annexin V binding (45, 46).
Cells were also stained with 7-AAD, a DNA-binding dye that stains cells
that have lost their membrane integrity (a late event in both apoptotic
and non-apoptotic cell death). Induction of tTA expression in control
cultures resulted in a moderate increase in cell death. When cultures
expressing elevated levels of RelA were compared with parental
cultures, we found that clones overexpressing RelA had 2-3-fold more
Annexin V binding 7-AAD excluding cells, indicative of the induction of apoptosis (Fig. 3). We detected twice the number of dead cells in
the RelA-expressing clones as we did in control cultures. In addition,
we performed a gel electrophoretic analysis of DNA purified from these
cultures. RelA expression was associated with internucleosomal cleavage
of DNA characteristic of apoptosis (data not shown).

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Fig. 3.
Annexin V staining of Rel-A-overexpressing
cells. A, parental tTA10 and two RelA-transfected
clones were induced to express protein by removal of tetracycline.
After 4 days of growth under induction conditions, 5 × 106 cells were double-stained with Annexin V-FITC and 7-AAD
and analyzed by flow cytometry. Induced cultures were compared with the
corresponding cultures growing in the presence of tetracycline (only
tTA10 is shown; the other uninduced cultures were identical to tTA10).
Annexin V stains apoptotic cells, and 7-AAD stains dead cells
regardless of their mechanism of death. B, the table was
generated using CellQuest software to quantify the fraction of Annexin
V-FITC /7-AAD (live), Annexin
V-FITC+/7-AAD (apoptotic), and Annexin
V-FITC+/7-AAD+ (dead) cells from A,
above.
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A G1 Cell Cycle Arrest Precedes Induction of
Apoptosis--
The lag between induction of RelA expression and the
apparent onset of apoptosis led us to investigate whether cell cycle progression is affected by elevated levels of RelA protein. Parental (tTA10) and RelA-transfected clones were induced by removal of tetracycline. After various lengths of time, bromodeoxyuridine (BrdUrd)
was added to the media, and incubation was continued for another hour.
Cells continuing to cycle will incorporate BrdUrd into their DNA during
S phase. At each time point, cells were permeabilized, stained with
7-AAD and FITC-conjugated anti-BrdUrd antibody, and analyzed by flow
cytometry (Fig. 4A). Whereas
the parental and RelA overexpressing clones showed indistinguishable cell cycle distributions in the presence of tetracycline, the induction
of RelA overexpression led to a dramatic G1 cell cycle arrest (Fig. 4A). The fraction of RelA-expressing cells in
G1 increased from 45 to 75%, whereas the parental clone
remained essentially unchanged (Fig. 4B). Furthermore,
apoptotic cells, characterized by their sub-G1 DNA content,
accumulated in the RelA-expressing culture and not in the control
culture (apoptotic cells were not included in quantitative analyses of
cell cycle distribution). In RelA overexpressing cultures, as shown in
Fig. 4B, the fraction of cells in S phase decreased much
more rapidly than the fraction of apoptotic cells (as defined by
sub-G1 DNA content) increased, confirming the impression
that cell cycle arrest precedes apoptosis in this system.

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Fig. 4.
Cell cycle analysis of RelA-overexpressing
cells. A, a parental tTA10 culture and a representative
RelA-overexpressing culture (RelA.21) were induced by removal of
tetracycline and cultured for up to 5 days. Each day aliquots of cells
were pulsed with BrdUrd for 60 min, harvested, permeabilized, and
stained sequentially with a murine anti-BrdUrd antibody, an anti-mouse
IgG1 FITC-conjugated secondary reagent and 7-AAD. The staining profiles
of the uninduced and 5-day-induced cultures are shown. The boxed
regions indicate the various cell cycles stages (G1,
S, and G2/M) and cells with sub-G1 DNA content
(A). B, the subpopulations of cells in the tTA10
and RelA.21 cultures in each stage of the cell cycle before induction
and on each of 5 days after induction were quantified using CellQuest
software and the gates indicated in A, above. Cells with
sub-G1 DNA content are generally considered apoptotic. The
percentage of cells in S phase was calculated exclusive of the fraction
of dead cells (box A) in each culture.
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Induction of Apoptosis Requires the Transactivating Potential of
RelA--
Since Rel family members heterodimerize, it was possible
that RelA overexpression induced cell cycle arrest and apoptosis indirectly, by altering the distribution of various Rel family dimers.
Alternatively, RelA might provoke these phenomena by directly altering
target gene expression. To delineate the importance of transcriptional
transactivation by RelA for the induction of G1 cell cycle
arrest and apoptosis, we generated 220-8 clones inducibly expressing a
truncation mutant of RelA that lacks the previously characterized
C-terminal transactivation domain (amino acids 471-549) (47, 48). When
the growth of clones expressing this truncated protein was examined, no
obvious defects were noted beyond the mild growth retardation observed
with the parental tTA clone (Fig. 5).

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Fig. 5.
Mutant RelA proteins do not affect pro-B cell
growth. The cell viability assay was performed as in Fig. 2.
Growth in the presence of tetracycline is indicated by the filled
squares, and growth under inducing conditions is indicated by the
open circles. TADT 19 and TADT 31 are clones expressing the
transactivation domain truncation mutant RelA protein, and RRPA 6 and
RRPA 12 are clones expressing the protein kinase A site mutant RelA
protein.
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To corroborate this observation, we studied a previously characterized
RelA transactivation mutant, RRPA (49). This point mutation alters a
protein kinase A phosphorylation site that is essential for the
transactivation potential of RelA. We verified inducible expression of
the RRPA mutant RelA by Western blot and its ability to bind DNA by
mobility shift analysis (data not shown). In contrast to the phenotype
associated with overexpression of wild-type RelA, overexpression of the
RRPA mutant protein did not result in any proliferative defects (Fig.
5). Cell growth, in both the presence and absence of tetracycline, was
similar to the parental control. These results strongly suggest that
the transactivating potential of the RelA protein is necessary for the
apoptotic phenotype seen in clones overexpressing RelA.
Both the DNA Binding and Transactivation Domains of RelA Are
Specifically Required for Induction of Apoptosis--
Whereas the
preceding experiments showed that transactivation potential was
necessary for RelA to induce growth arrest and apoptosis, it
remained uncertain whether the requirement was specific for the RelA
transactivation domain. The amino acid sequences of the c-Rel and RelA
transactivation domains are quite distinct from one another, suggesting
that differences in transactivation may underlie the effects of
distinct Rel family dimers. To test this idea, we generated two
RelA/c-Rel chimeric transcription factors. In c-RelTAD, the
transactivation domain of RelA was replaced by the analogous domain of
c-Rel (the C-terminal 156 amino acids (50)), and in the chimera
RelATAD, the transactivation domain of c-Rel was replaced by the
analogous domain of RelA (the 79 C-terminal amino acids). We
transfected these chimeric proteins into tTA10 cells under tetracycline
regulation and analyzed cell viability following induction (Fig.
6). Control experiments confirmed that
both chimeric proteins were able to specifically bind DNA in
vitro and to transactivate an NF-
B-dependent
reporter construct in vivo (data not shown).

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Fig. 6.
RelA-c-Rel chimeric proteins do not affect
pro-B cell growth. This cell viability assay was performed as in
Fig. 2. Growth in the presence of tetracycline is indicated by the
filled squares, and growth under conditions of protein
induction is indicated by the open circles. The upper
panels represent the growth profiles of cells expressing a
chimeric protein composed of the RelA DNA binding domain and the c-Rel
transactivation domain (RelATAD.2 and RelATAD.5). The lower
panels represent the growth profile of cells expressing a chimeric
protein composed of the c-Rel DNA binding domain and the RelA
transactivation domain (c-RelTAD.5 and c-RelTAD.6).
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Surprisingly, neither chimeric protein reproduced the striking growth
arrest and apoptotic phenotype observed with wild-type RelA
overexpression. These results support the conclusion that both the RelA
transactivation domain and its DNA binding and dimerization domain are
critical for this phenotype.
Overexpression of RelA in B Cell Lymphoma Cell Lines Does Not Cause
Apoptosis--
Recent reports have suggested a role for NF-
B in the
protection of fibroblasts from TNF-
-induced apoptosis and
immature B cells from anti-IgM-induced apoptosis. To explore the
possibility that the effect of RelA on cell cycle progression and
apoptosis differed at different stages of development, we established
the tetracycline-regulated expression system in two B cell lymphoma lines, WEHI 231 and M12. Whereas the 220-8 cell line is representative of the pro-B cell stage of development, WEHI 231 has been used as a
model for the immature B cell stage and M12 for the mature B cell stage
of development. We found that overexpression of RelA in WEHI 231 or in
M12 does not alter cell growth (Fig. 7).
Expression levels and kinetics of RelA protein induction in the WEHI
and M12 cell clones were similar to those in the 220-8 transfectants (data not shown) and thus cannot account for the difference in phenotypes. Multiple attempts to generate additional distinct pro-B
cells lines expressing RelA were unsuccessful. We believe that this was
due to the extreme toxicity of even modest amounts of excess RelA to
these cells, despite tightly regulated expression. This discrepancy in
the effects of RelA overexpression in pro-B and immature B cells
suggests that there may be a developmental basis for the susceptibility
of pro-B cells to the induction of apoptosis by RelA.

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Fig. 7.
RelA overexpression does not affect the
growth of WEHI 231 B lymphoma cells. This cell viability assay was
performed as in Fig. 2. The tTA-regulated expression system was
established in the immature B cell lymphoma WEHI 231 and the mature B
cell lymphoma M12. Two RelA-expressing clones from each cell line were
analyzed. Growth in the presence of tetracycline is indicated by the
filled squares, and growth under conditions of protein
induction is indicated by the open circles.
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DISCUSSION |
NF-
B was initially discovered because of its ability to bind a
site in the Ig
locus intronic enhancer. Subsequently,
NF-
B-binding sites were found in transcriptional regulatory elements
associated with a broad array of genes involved in immune or
inflammatory responses (1). A number of recent experiments perturbing
the composition of NF-
B/Rel family dimers have revealed previously unsuspected roles for this family of transcription factors in regulating both cell division and apoptosis (21, 29, 32, 51).
The levels of the various Rel family members and their contributions to
NF-
B DNA binding activity are normally controlled by both
transcriptional and post-translational mechanisms. For example, NF-
B
activation can lead to increased synthesis of I
B
(but not
I
B
) (52-54), p50 (55), and c-Rel (56-58). This web of
interactions among the Rel family members and their inhibitors has
contributed to difficulty in identifying the unique functions of each
family member. We used an inducible gene expression system to disrupt
normal cellular regulation of the Rel family, leading to the
overexpression of either RelA or the c-Rel protein in a pro-B cell
line. The inappropriate regulation of these proteins resulted in
stimulation of nuclear NF-
B DNA binding activity. Whereas elevated
levels of RelA led to the induction of a G1 cell cycle
arrest followed by apoptosis, overexpression of c-Rel had little effect
on cellular proliferation or cell death.
Since RelA is known to interact with several I
B family members (7,
59-61), it is possible that its overexpression induced apoptosis
indirectly by altering the cytoplasmic localization of various NF-
B
complexes imposed by the I
B proteins. The recently cloned I
B
(60, 62, 63) has been reported to selectively interact with RelA
homodimers, and both I
B
and I
B
are known to interact with
RelA-containing heterodimers. The sequestration of various I
Bs
through interaction with inappropriately regulated RelA protein might
lead to nuclear translocation and target gene activation. The
overexpression of c-Rel, however, did not cause apoptosis although it
might be expected that a similar redistribution phenomenon would also
result from elevated levels of this protein subunit. The specificity of
the RelA-induced cell cycle arrest suggests that the initiation of this
signaling pathway is a direct result of RelA-specific target gene activation.
Our failure to observe cell cycle arrest or apoptosis in cells
expressing the RelA transactivation domain truncation mutant protein,
TADt, or the protein kinase A phosphorylation site point mutant
protein, RRPA, confirm the conclusion that the effects of RelA
overexpression were due to its transcriptional transactivation potential. It is important to realize that the kinetics of the cell
cycle arrest differ significantly from the induction of apoptosis. Whereas the cell cycle arrest was detectable after less than 24 h
of protein induction, apoptosis was not observed until after 72-96 h,
suggesting that the cells might be dying as a result of their inability
to continue normal cell cycle transit. This interpretation suggests a
role for RelA in the regulation of cell cycle proteins. The involvement
of c-Rel in the regulation of p53 and p21 transcript stability and E2-F
DNA binding activity has been reported (51). We examined each of these
cell cycle regulators in our system, as well as the transcriptional
regulation of the c-myc gene, whose role in cell division
and apoptosis has been extensively investigated (64-68), and we were
unable to identify any significant perturbations (data not shown).
Alternatively, it is possible that RelA contributes directly to the
transcriptional activation of proteins involved in the regulation of
apoptosis. A recent report showed that RelA was required for the
induction of FasL expression in a human T cell line, for example (69).
The time lag we observed in the onset of cell death after RelA
induction might reflect the time necessary to activate the expression
of FasL and for FasL to interact with Fas and activate the apoptosis
pathway. Identification of the downstream target gene(s) whose altered
expression results in the induction of this G1 cell cycle
arrest and apoptosis awaits further investigation.
Several lines of investigation into the relationship between NF-
B
and apoptosis have supported distinct functions in both the protection
from and the induction of programmed cell death. Deletion of the
relA gene in mice results in embryonic lethality. Massive
hepatocyte apoptosis was observed in pre-morbid fetuses, implicating
RelA in the prevention of cell death. Experiments with fibroblasts from
RelA knock-out mice showed that these cells were sensitized to
TNF-
-induced apoptosis (29). Although the viability of wild-type
fibroblasts was unaffected by TNF-
stimulation, up to 80% of
TNF-
-stimulated relA
/
fibroblasts were
killed. Apoptosis in this system could be inhibited by the expression
of exogenous RelA, suggesting that TNF-
stimulation of NF-
B
activity was involved in the protection of fibroblasts from apoptosis
during TNF-
signaling. Similar experiments examining this TNF-
signal in macrophage, T cell, and fibrosarcoma lines corroborated the
observations in fibroblasts (30, 32). Additional work done with the
immature B cell line, WEHI 231, which expresses surface immunoglobulin
(sIgM), also defined a role for NF-
B in the protection from
apoptosis induced by surface receptor cross-linking (31). The mechanism
of NF-
B protection from these apoptotic stimuli remains unclear.
A role for NF-
B in the induction of apoptosis has also been
observed. A report examining the expression of c-Rel in the developing avian embryo found that cells undergoing apoptosis expressed elevated levels of c-Rel protein. When c-Rel overexpression was induced by viral
transduction into primary avian hematopoietic cells, the cells
underwent an uncharacterized growth arrest that was followed by the
induction of programmed cell death (26). Transduction of
c-rel into the corresponding primary fibroblasts appeared to extend the lifespan of these cells, leading to the speculation that the
biological effects of c-Rel overexpression were cell type-specific.
NF-
B activation was also required in the induction of apoptosis
following the infection of AT-3 cells (a prostate carcinoma line) with
Sindbis virus. Blocking NF-
B DNA binding activity inhibited the
induction of apoptosis accompanying this viral infection (27).
Interestingly, this requirement for NF-
B was not seen in
Sindbis-virus induced apoptosis of N18 (neuroblastoma) cells, again
emphasizing the differential effects of NF-
B activation depending on
cell type. The activation of nuclear NF-
B DNA binding activity is
also observed in serum-starved 293 cells (a human embryonic kidney cell
line), concomitant with apoptosis of these cells (28). Transfection of
a dominant-negative mutant of the RelA subunit, truncated at its
C-terminal transactivation domains, attenuated the observed cell death,
suggesting the importance of transcriptional activation by NF-
B in
this model of apoptosis. These reports encompass a range of cell types
and apoptosis-inducing stimuli and suggest that both the cell type and
context of an NF-
B-activating signal are critical in determining
whether cells are protected from or induced to undergo apoptosis.
We addressed the paradoxical roles of NF-
B in apoptosis by examining
both the cell type specificity and the signaling context of its
pro-apoptotic activity in transfected B lineage cells. LPS stimulation
of transformed precursor B cells (including those used in our studies)
results in the nuclear translocalization of NF-
B, composed primarily
of the p50/RelA heterodimer, and does not result in any obvious
alteration in proliferation or induction of apoptosis (9, 17). When we
induced transfected pro-B cells to overexpress RelA, we also observed
the activation of nuclear NF-
B DNA binding complexes similar to
those seen with LPS stimulation. However, under these conditions, cells
experience growth arrest and subsequently undergo apoptosis. The
explanation for these disparate results may lie in compensatory
signaling pathways induced by LPS treatment that might counteract the
induction of growth arrest by nuclear NF-
B. Stimulating
RelA-overexpressing clones with LPS concomitant with RelA induction
does not rescue these cells, however (data not shown), suggesting that
the RelA-induced NF-
B signal is dominant in the induction of cell
death under these conditions.
We approached the cell type specificity issue by overexpressing RelA in
the immature B cell line WEHI 231 and the mature B cell line M12. The
overexpression of RelA in these cells did not result in either cell
cycle arrest or induction of apoptosis. In addition to representing
distinct stages of B cell development, 220-8, WEHI 231, and M12 cells
represent different states of cellular NF-
B. In most cells, NF-
B
is present in an inactive cytoplasmic form, but in several subsets of
cells, including mature B cells and macrophages (17, 33), nuclear
NF-
B DNA binding activity is constitutive. It is at the immature
stage of B cell development, represented by WEHI 231, that this
constitutive activity is acquired. The 220-8 line represents an earlier
stage in B cell development that precedes this constitutive activation.
During the analogous stage of development in the bone marrow, B cells
that do not successfully rearrange the Ig heavy chain locus and
synthesize heavy chain protein are eliminated via an apoptotic
mechanism (70). NF-
B may be the factor responsible for initiation of
this apoptotic pathway. Similarly, specific populations of developing B
cells within the avian bursa of Fabricius (71) are highly susceptible to the induction of cell death. 220-8 cells might represent a stage of
development that is the mammalian equivalent of avian B cell
populations that exploit NF-
B-induced apoptosis to perform cellular
selection. The WEHI 231 and M12 cell lines, having progressed beyond
this susceptible stage, would not be expected to be sensitive to the
apoptosis-inducing function of NF-
B.