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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2068-2075
NEOPLASIA
Inducible loss of NF- B activity is associated with apoptosis
and Bcl-2 down-regulation in Epstein-Barr virus-transformed B
lymphocytes
Jean Feuillard,
Marino Schuhmacher,
Sylvie Kohanna,
Marianne Asso-Bonnet,
Frédérique Ledeur,
Raymonde Joubert-Caron,
Philippe Bissières,
Axel Polack,
Georg W. Bornkamm, and
Martine Raphaël
From Biochimie Cellulaire des Hémopathies Lymphoïdes,
Université Paris, Bobigny, France, and Institut für
Klinische Molekularebiologie and Tumorgenetik, München, Germany.
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Abstract |
The Epstein-Barr virus (EBV)-encoded latent membrane protein-1
induces NF- B activity by targeting IB . To
understand the role of NF-B activation in EBV-related oncogenesis,
we have subcloned mutated IB32/36A cDNA into a
pHEBo vector containing doxycycline regulatory sequences and stably
transfected this construct into a lymphoblastoid cell line. Two tightly
regulated clones were obtained in which IB32/36A
was inducible in a doxycycline dose-dependent manner. Levels of
inducible IB32/36A peaked at day 2. Inhibition of
NF-B activity was closely correlated with levels of inducible IB32/36A. Levels of 3 well-known
NF-B-dependent genes, CD54, p105, and endogenous IB, were
decreased when IB32/36A was induced, and
the growth of IB32/36A-induced EBV-infected cells was slightly reduced. Loss of NF-B activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was
strongly increased in IB32/36A-overexpressing
cells. Together these results show that it is possible to control
IB32/36A levels, ie, NF-B activity, in
EBV-infected B-lymphocytes using a doxycycline-inducible vector.
Moreover, our results indicate that NF-B can protect EBV-infected
cells from apoptosis by Bcl-2. Finally, our results suggest that a
cellular model with doxycycline-inducible IB32/36A may be useful in the identification of genuine NF-B target genes in EBV-infected B cells.
(Blood. 2000;95:2068-2075)
© 2000 by The American Society of Hematology.
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Introduction |
Epstein-Barr virus (EBV)-related B-cell
transformation involves cell reprogramming through the modulation of
critical cellular transcription factors. Such dysregulation may be the
result of direct interactions between viral proteins and cellular
transcription factors and from a rerouting of defined cellular signal
transduction pathways. For example, EBNA2
protein interacts directly with RBP-J, an inhibitory transcription
factor, converting it to a positive transcription regulator. Another
well-documented example is the activation of both the AP-1 and the
NF-B transcription factors by LMP-1, which mimics a constitutively
active receptor of the tumor necrosis factor (TNF) receptor family (see
Mosialos1 and Manet et al2 for review).
In mammalian cells, nuclear NF-B activity corresponds to
heterodimers and homodimers between the 5 Rel/NF-B proteins,
including p50/p105 (NF-B1), p65 (RelA), p52/p100 (NF-B2),
c-Rel, and RelB. These proteins share a common Rel homology domain,
involved in both dimerization and DNA binding to the consensus B
site. NF-B activation has been implicated in the positive
regulation of a variety of genes of the immune response, such as i-NOS,
IL-1, IL-2, IL-6, IL-8, tumor necrosis factor- (TNF-), major
histocompatibility complex (MHC) class 1 and 2, CD25, CD54, and CD80,
A20 protein, the proto-oncogene c-myc, and the Rel/NF-B
proteins itself, including NF-B1, NF-B2, c-Rel, and the
inhibitor IB.3 Moreover, even though NF-B has
been associated with the induction of apoptosis in some cellular
systems, there is increasing evidence for a role of NF-B
activation in the protection of cells against apoptosis.3,4
Rel/NF-B complexes containing RelA or c-Rel are usually trapped
within the cytoplasm of most cell types by interaction with inhibitory
proteins called IB, IB , and IB . Activation of NF-B depends on the phosphorylation and degradation of IBs, leading to the accessibility of nuclear transport signals and nuclear
translocation of the RelA- and c-Rel-containing dimers. Various cell
activation pathways such as the TNF-, IL-1, PKC, and
lipopolysaccharide signal transduction pathways converge at the IB
phosphorylation step through the activation of a specific, multisubunit
complex called the IB kinase (IKK) that contains at least 3 polypeptides directly involved in IB phosphorylation: IKK,
IKK,5-8 and NEMO.9 IKK and IKK are
able to phosphorylate IB and IB specifically on serine
residues 32/36 and 19/23 respectively, whereas the NEMO polypeptide
is thought to be involved in signal integration from a variety of
signaling pathways that converge at IB phosphorylation and
NF-B activation.
That constitutive NF-B activation can contribute directly to cell
transformation has been recently highlighted in the Tax model. Indeed,
transfection of NEMO into mutant cells unable to activate NF-B is
associated with both the restoration of NF-B activation and the
transformation capacity of Tax.9,10 Various studies suggest
a role of Rel/NF-B in oncogenesis. For example, v-Rel, a virally
truncated and mutated version of c-Rel, is responsible for fatal
hematologic diseases in chicken and turkey, and transgenic mice
expressing v-Rel have rapid T-cell lymphoblastic
disorders.11-13 In humans, the chromosomal translocation
t(10;14)(q24;q32) affects p52 and represents a recurrent chromosomal
rearrangement found in certain lymphoid malignancies14-16
of B-cell origin. Amplification of the REL locus has been reported to
be a feature of primary extranodal lymphoma.17
LMP-1, one of the major transforming proteins of EBV, contributes to
enhanced NF-B activation in EBV-infected cells.18 It
is a transmembrane protein consisting of a short amino terminal cytoplasmic region, 6 transmembrane domains, and 200 cytoplasmic carboxyterminal amino acids. Deletion experiments revealed that the
carboxyterminal cytoplasmic tail carries two domains, CTAR1 and CTAR2,
collectively responsible for LMP-1 tumorigenesis and the activation of
both NF-B19,20 and JNK kinase.21 CTAR1 is
responsible for approximately 30% and CTAR2 for approximately 70% of
LMP1-associated NF-B activation. The 6 transmembrane domains are
required for LMP-1 aggregation and the consequent signal transduction that, through the aggregation of TRADD and TRAF
molecules,22,23 allows LMP-1 to mimic a constitutively
active receptor of the TNF receptor family. The CTAR1 and CTAR2 domains
of LMP-1 contain binding sites for TRAF1 and TRADD molecules,
respectively.24,25 Both adaptor proteins can themselves
bind to TRAF2, which leads to NF-B activation through the
recruitment of the NF-B-inducing kinase (NIK) and to subsequent
IKK activation, which leads in turn to the phosphorylation and
degradation of IBs.26
The aim of this work was to design a cellular model that would allow us
functionally to analyze the role of NF-B and to identify putative
NF-B target genes in EBV-infected lymphocytes. For this purpose,
we chose to inhibit Rel/NF-B complexes specifically by stable
transfection of the cDNA encoding for IB mutated on serine 32 and 36 (IB32/36A).27 Furthermore, to
avoid selection bias for those clones able to escape the potentially
deleterious effects of IB overexpression, dominant-negative
IB32/36A was expressed in a conditional fashion
from a doxycycline-inducible promoter.28 To prevent vector
integration into the genomic DNA, which is difficult to obtain in
lymphoblastoid cells and may give rise to position effects, we used the
pHEBo backbone containing the EBV OriP sequences. The pHEBO construct
allows the vector to replicate episomally in the presence of
EBV-encoded nuclear protein-1 (EBNA1).29,30 Our results
showed that it was possible to obtain cells in which
IB32/36A is inducible after doxycycline treatment
in a dose-responsive manner. Levels of IB32/36A
induction were tightly correlated with those of NF-B inhibition.
In addition, we demonstrated that the inhibition of NF-B was
correlated with a decrease in CD54, p105/NF-B1, and endogenous
IB expression, implicating these genes as potentially
NF-B-regulated in EBV-infected cells. Finally, we showed that the
loss of NF-B activity in EBV-infected lymphocytes was associated
with a significant decrease of Bcl-2 protein expression and with a
dramatic increase in their sensitivity to apoptosis.
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Materials and methods |
Plasmid constructs
MS4A was derived by cloning the reverse tetracyclin
repressor-transactivator protein (rtTA28) and a
tetracycline regulatable promoter31 onto the pHEBo
vector.29 To achieve tight regulation, the cytomegalovirus
(CMV) promoter was replaced by the EBV-LMP2A promoter in which the
endogenous EBNA2 response element was
substituted by a heptamerized tet operator sequence. The
IB32/36A cDNA was excised from
pCMV-IB32/36A27 by HindIII and XbaI
digestion and subcloned into the MS4A vector at the Sfi1 sites
(MS4A-IB32/36A; see Figure
1). The 0.4SK-luc plasmid containing the
IB promoter with 3 B sites upstream of the luciferase gene
was a kind gift of A. Israël (Institut Pasteur, Paris,
France).32
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| Fig 1.
Schematic diagram of the
MS4A-IB32/36A vector.
The different cassettes are indicated in grey boxes: tetO7,
tetracycline-responsive elements; IB32/36A, c-DNA
encoding for IB32/36A super-repressor; rtTA,
cassette encoding for the doxycycline-responsive factor; EBV-OriP,
EBV-OriP sequences; Hygro, cassette encoding for hygromycin
resistance.
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Protein extracts and electromobility shift assay
Nuclear and cytoplasmic extracts were performed in parallel as
described.33 Gel shift experiments were performed with 20 µg nuclear proteins as described34 with the
PRE oligonucleotide containing the 2-B sites of the
human immunodeficiency virus enhancer.35
Western blot
Our standard gel conditions consisted of 10% SDS-PAGE without
stacking gel as described.36 Where indicated, a stacking
gel (4% polyacrylamide, 125 mmol/L glycine, pH 6.8) was
used according to standard procedures.37 Thirty micrograms
protein was fractionated and electrotransferred onto a polyvinylidene
difluoride membrane (Millipore, Bedford, MA) 0.8 mA/cm2
with the Hoefer semidry apparatus (Pharmacia Biotech, Orsay, France).
Antibodies used for Western blot analysis were MAD 10B for
IB38 (generous gift of R. Hay, University of St
Andrew, St Andrew, UK) diluted at 1/500, monoclonal antibody 124 for
Bcl-2 (Dako, Trappes, France) diluted at 1/100, rabbit immune serum no.
1140 diluted at 1/1500 for p105 (generous gift of N. Rice, NCI,
Frederick). Detection of the antigen-antibody complexes
was performed using a horseradish peroxidase-coupled antimouse or antirabbit antibody (Biorad, Ivry/Seine, France) diluted at 1/10 000,
followed by chemiluminescent visualization (Biolabs, Hitchin, UK).
Cell culture and cell transfection
One of our lymphoblastoid cell lines, named PRI, was cultured
continuously in RPMI 1640 with standard concentrations of
L-glutamine and antibiotics and 10% fetal calf serum.
Transfected cells were cultured in the presence of hygromycin
(Calbiochem, Meudon, France) at 300 µg/mL and 20% fetal calf serum.
Twenty million cells were electroporated with 20 µg
MS4A-IB32/36A at 250 V and 960 µF. After 4 days,
hygromycin was added at 50 µg/mL. Hygromycin concentration was
progressively increased up to 300 µg/mL during the first 2 weeks of
selection. After 4 weeks of culture in the presence of hygromycin,
transfected cells were plated at a limiting dilution on 96-well plates.
Screening of clones was based on the inducibility of IB
expression after doxycycline treatment. To induce IB overexpression with doxycycline, cells were washed once in RPMI and
resuspended in standard medium without hygromycin. Doxycycline (purchased from AP-HP, Paris, France and stored at  20°C at 1 mg/mL
in water) was then added at concentrations ranging from 0.01 µg/mL to
8 µg/mL (usually 2 µg/mL). Western blot analysis of IB
expression was performed 48 hours after doxycycline treatment. Transient transfections were conducted in triplicate with 20 µg 0.4SK-luc plasmid for 10 million cells. Cells were washed once in RPMI,
resuspended in standard medium, and electroporated at 250 V and 960 µF. Then the cells were resuspended in standard medium at
106 cells/mL, left 1 hour at 37°C, washed once, and
resuspended in standard medium without hygromycin and with or without
doxycycline at 2 µg/mL. Luciferase assays were performed after 2 days
(Promega, Madison, WI), according the manufacturer's instructions.
Immunolabelings and flow cytometry analysis
Antibodies tested are listed in Table 1.
The expression of cell-surface molecules was assessed by direct and
indirect immunofluorescence staining according standard protocols,
using a Coulter XL flow cytometer (Coulter, Margency, France).
Cell-cycle analysis and apoptosis detection
Cell-cycle analysis and detection of apoptosis were performed by
flow cytometry as described39 on the basis of BrdU
incorporation into DNA and propidium iodide staining. Fluorescence
analysis of chromatin condensation and fragmentation was conducted
after cytocentrifugation of 50 000 cells and DNA staining with the
Hoechst 33258 diluted at 20 µg/mL in phosphate-buffered
saline. Staining of cells with fluorescein isothiocyanate-conjugated
Annexin V (Pharmingen, San Diego, CA) was performed according to the
manufacturer's protocol.
| |
Results |
Characteristics of IB32/36A induction by
doxycycline in lymphoblastoid cells transfected with the
MSA4-IB32/36A vector
The MS4A-IB32/36A vector was generated by
subcloning the IB cDNA mutated at positions encoding for the
two serine 32 and 36 residues
(IB32/36A)27 into the MS4A vector,
whose transcriptional activity is positively regulated by the addition
of doxycycline. The MS4A construct consists of a pHEBo
backbone29 (with its EBV-OriP sequences) into which have
been subcloned both a promoter carrying the tetO7 tetracycline
responsive element and the cDNA encoding for rtTA protein28
(Figure 1).
After transfection of cells with the MS4A-IB32/36A
vector, cells were grown in the presence of hygromycin and cloned by limiting dilution. Twenty-four clones were screened by Western blotting
for IB32/36A induction after 48 hours of
doxycycline treatment at 2 µg/mL. Two clones were found to be
IB32/36A inducible. Then the NF-B activity of
IB32/36A-inducible clones was assessed by both
EMSA and luciferase assay after the transfection of
0.4SK-luc plasmid, which harbors the promoter of IB with its 3 B sites.32 A typical result is illustrated in Figure
2, which shows that a significant induction of
IB32/36A was correlated with a dramatic decrease of
NF-B activity in DNA binding and transcriptional activity. We next
set up a dose-response experiment (Figure 3). Results demonstrated
that IB32/36A was inducible by doxycycline in a
dose-dependent manner and that the decrease of NF-B binding activity was tightly correlated with the levels of
IB32/36A induction. In the following experiment,
induction of IB32/36A was performed at 2 µg/mL
doxycycline because doxycycline concentrations greater than 5 µg/mL
had been reported to be toxic. SDS-PAGE without a stacking
gel36 did not allow the separation of endogenous IB
from IB32/36A (Figures 2,
3). Endogenous
IB could, however, be distinguished from the transfected gene
product on the basis of its higher electrophoretic mobility if a
stacking gel was used.27 A comparison of both gel
conditions is depicted in Figure 4. Our results showed that there was
no detectable leakage of the tet07 promoter (Figure
4). Furthermore, levels of endogenous
IB decreased when IB32/36A
expression was induced by doxycycline (Figure 4), suggesting that
endogenous IB is a genuine target gene of NF-B in
EBV-infected cells.
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| Fig 2.
Characterization of the IB32/36A
inducibility after doxycycline treatment.
(A) Western blot detection of IB protein in
MS4A-IB32/36A-transfected cells with (+) or without
() doxycycline at 2 µg/mL. (B) NF-B binding activity assessed
by EMSA in MS4A-IB32/36A-transfected cells with (+)
or without () doxycycline at 2 µg/mL. (C) Schematic representation
of the transcriptional regulatory element of the IB promoter
with its 3 B sites in front of the luciferase gene (construct
0.4SK-luc). (D) Relative NF-B transcriptional activity in
MS4A-IB32/36A-transfected cells with (+) or without
() doxycycline at 2 µg/mL. Presented results correspond to the
mean of 3 transfection experiments with the 0.4SK-luc construct. Each
transfection experiment was conducted in triplicate.
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| Fig 3.
Dose-responsive inducibility of
IB32/36A by doxycycline.
MS4AIB32/36A-transfected cells were treated for
48 hours with various concentrations of doxycycline as indicated. Upper
panel: Western blot detection of IB. Lower panel:
NF-B-binding activity assessed by EMSA.
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| Fig 4.
Gel electrophoresis with or without stacking gel followed
by Western blot analysis of IB32/36A.
MS4A-IB32/36A-transfected cells were (lanes 1 and
3) or were not (lanes 2 and 4) treated with doxycycline for 48 hours.
SDS-PAGE and Western blot detection of IB was performed
without (lanes 1 and 2) or with (lanes 3 and 4) stacking gel.
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Finally, we established the kinetics of IB32/36A
induction. As shown in Figure 5, IB32/36A induction
was highest after 48 hours of doxycycline treatment at 2 µg/mL.
Levels of IB32/36A slowly decreased at day 4 and
day 7. Kinetics of the inhibition of NF-B binding activity
strictly correlated with the kinetics of IB32/36A
induction. Therefore, phenotypic analysis of doxycycline-treated cells
was systematically performed at day 2.
CD54, p105, and IB are putative target genes of
NF-B in EBV infected B cells
Loss of NF-B activity in the presence of
IB32/36A was associated with down-regulation of the
transcriptional activity of the IB promoter (Figure 2D) and
resulted in a significant decrease of endogenous IB levels
(Figures 4, 5). This strongly implicates IB to be a genuine target gene of NF-B in EBV-infected
cells. To further evaluate the function of the induced
IB32/36A protein, we analyzed the expression of
some genes that may be positively regulated by NF-B. Expression of
CD54 and p105 was decreased in these cells after 48 hours of
doxycycline treatment at 2 µg/mL (Figure
6). These results suggested that the CD54
and p105 genes may also be genuine target genes of NF-B in
EBV-immortalized lymphoblastoid cells.
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| Fig 5.
Kinetics of IB32/36A inducibility
after doxycycline treatment.
Cells were (+) or were not () treated with doxycycline at 2 µg/mL.
Times of protein extraction are indicated at the top of the figure (D0:
protein extraction was performed immediately after doxycycline
treatment; D1, D2, D4, and D7: protein extraction was performed
immediately after 1, 2, 4, and 7 days of doxycycline treatment,
respectively). Upper panel: Western blot detection of IB after
SDS-PAGE with stacking gel; mutated and endogenous IB are
indicated by the arrow. Lower panel: NF-B-binding activity
assessed by EMSA.
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| Fig 6.
Down-regulation of CD54 and p105 expression after
induction of IB32/36A.
Levels of CD54 membrane expression were assessed by flow cytometry on
cells treated (+ DoxyC) or not ( DoxyC) with doxycycline at 2 µg/mL for 48 hours (A). Levels of p105 expression were assessed by
Western blot on cells treated (+) or not () with doxycycline at 2 µg/mL for 48 hours (B).
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Functional consequences of the IB32/36A
induction in lymphoblastoid cells
Because NF-B has been reported to protect cells from
apoptosis,40-42 and because LMP-1 transfection has been
reported to enhance Bcl-2 expression in B cells,43,44 we
examined the relationship between the inducible inhibition of NF-B
and any changes in cell proliferation sensitivity to apoptosis.
Proliferation of doxycycline-treated cells was decreased slightly in
terms of the growth kinetics of cells in culture and the fraction of
cells in S-phase, as estimated by flow cytometry after BrdU
incorporation (not shown).
Spontaneous apoptosis did not increase significantly in
doxycycline-treated cells. However, treatment of cells with
daunorubicine, a DNA-intercalating chemotoxic drug that induces
apoptosis, clearly showed that IB32/36A
overexpression (ie, the loss of NF-B activity) can sensitize
EBV-infected B cells to apoptosis, as revealed by chromatin
condensation and DNA fragmentation (Figures 7A,
7B), sub-G1 peak induction (Figure 7C), and
annexin-V cell-surface fixation (Figure 7D). Finally, we found that the
loss of NF-B binding activity was correlated with a dramatic
decrease of Bcl-2 expression within 48 hours in EBV-infected B cells
(Figure 7E).
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| Fig 7.
Induction of apoptosis and expression of Bcl-2 in
MS4A-IB32/36A-transfected cells.
(A) Analysis of nuclear condensation after Hoechst 33258 staining.
Cells were (+ DoxyC) or were not ( DoxyC) pretreated with
doxycycline at 2 µg/mL for 24 hours. Daunorubicine (Dauno) was added
for 18 hours at the concentrations indicated. (B) Dose response to
apoptosis induction by daunorubicine in cells pretreated (+ DoxyC) or
not ( DoxyC) with doxycycline at 2 µg/mL for 24 hours.
Percentages of apoptotic cells were assessed by counting cells with
apoptotic nuclei under fluorescence microscope after Hoechst 33258 staining. (C) Flow cytometry analysis of DNA content after propidium
iodide staining of the DNA of cells pretreated (+ DoxyC) or not
( DoxyC) with doxycycline at 2 µg/mL for 24 hours and then
treated with daunorubicine at 0.2 µmol/L for 18 hours. (D) Annexin V
staining of cells pretreated (+ DoxyC) or not ( DoxyC) with
doxycycline at 2 µg/mL for 24 hours and then treated with
daunorubicine at 0.2 µmol/L for 18 hours. (E) Bcl-2 expression of
cells treated (+) or not () with doxycycline at 2 µg/mL for 48 hours was assessed by Western blot.
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Discussion |
The relationship between LMP-1 and NF-B activation has been
extensively studied, and every step between the autoaggregation of
LMP-1 and the activation of the IKK has been documented. LMP-1, acting
through TRAF molecules, activates several signal transduction pathways
including the NF-B,45 JNK,21 and
ras-MAPK46 activation cascades. Therefore, to
address the question of the specific role of NF-B in EBV-infected
B lymphocytes, our strategy was to specifically inhibit NF-B in
EBV-infected B cells.
Using the doxycycline-inducible system, we
obtained EBV-immortalized B lymphocytes in which it was
possible to inhibit NF-B. To our knowledge, this is the first
model that allows one to study the role of NF-B in
EBV-infected B lymphocytes. Establishment of stably transfected cells
is often a critical step in the analysis of the function of a
particular protein. In current stable transfection protocols,
a major bias is introduced by the fact that selection takes
place while the transfected gene of interest is expressed. If that
particular gene is toxic or induces cell-cycle arrest, establishment of
transfected clones is either impossible or will lead to the selection
of clones which, through mutation, have escaped the effect of the
protein to be studied. To circumvent this problem, various groups have
worked on and developed inducible vectors. Fusion of the protein of
interest to the hormone binding domain of the estrogen or
glucocorticoid receptor has provided a powerful tool with which to
study the induction of proliferation, differentiation, and
apoptosis.47-49 However, in this system,
interference of estrogen with NF-B cannot be excluded. Indeed, a
physical association between the activated glucocorticoid receptors and p65 has been reported,50 and estrogen molecules
may interact directly with glucocorticoid receptors.51
However, the tetracycline/doxycycline inducible system originally
designed by Bujard and collaborators31,52 has proven
itself useful for the analysis of the function of proteins whose
continuous expression may be deleterious for the cell or may
prevent selection in vitro.53 The system described by
Gossen and Bujard31 and Gossen et al52 consists
of two plasmids: one encodes the tetracycline repressor-VP16 or the
reversed tetracycline repressor-VP16 transactivator fusion protein; the
other plasmid encodes the gene of interest under the control of a
tetracycline-regulatable promoter. The major difficulties of this
system are that cells must be stably transfected twice and that the
level of expression is highly variable, depending on the site of vector
integration into the genomic DNA a phenomenon known as position effect.
To circumvent these problems, we cloned both the doxycycline-dependent
transcription factor rtTA and the tetracycline-regulatable sequences
onto the same derived episomal vector (Schuhmacher, manuscript in
preparation). pHEBo vectors can be easily transfected into EBV-infected
B lymphocytes and maintained episomally. They do not exhibit position
effects and, therefore, show little variation in the expression of the
transfected gene among different, stably transfected
cells.54,55 We have cloned the mutated
IB32/36A cDNA into this vector and have established
stably transfected EBV-immortalized cells that indeed express the
mutated IB32/36A cDNA in a doxycycline
dosage-dependent, inducible fashion. Furthermore IB32/36A was functional in terms of the inhibition
of NF-B binding, NF-B transcriptional activity, and
NF-B-dependent gene expression such as that of endogenous
IB, p105, and CD54. We obtained a tight correlation between
doxycycline concentrations in the medium, levels of
IB32/36A protein induction, and levels of NF-B
binding inhibition. These results indicated that
IB32/36A protein levels directly correlated with
the level of active rtTA molecules.
Conditional gene expression systems have also proven useful in the
analysis of the role of LMP-1 in EBV-driven proliferation. A
conditional LMP-1 gene has been introduced into a mini-EBV
background56 haboring a tetR-KRAB chimeric repressor
cassette and used to immortalize primary B cells.57
Inducible loss of LMP-1 expression was associated with the
proliferative arrest of these cells. Reinduction of LMP-1 expression
was associated with the reentry of the cells into the cell cycle and
the reactivation of the JNK1 and NF-B signal transduction pathways. These results do not clarify to which extent either JNK1 or
NF-B activation contributes to cellular proliferation. In our
cellular model, the inducible loss of NF-B was associated with
only minor effects on the cell cycle in terms of DNA synthesis and cell
growth in culture (not shown). These data could appear to suggest that
continuous LMP-1-induced NF-B activation was not required for
proliferation and that proliferation would be caused by the activation
of a different LMP-1-mediated signal transduction pathway. However,
this interpretation is not conclusive because analysis of the kinetics
of NF-B inhibition has revealed a gradual decrease in the level of
mutated IB32/36A protein over time. This decrease
in protein level was associated with a concomitant, gradual increase of
NF-B DNA binding activity to considerable, albeit lower, levels
than in the absence of doxycycline. An autoregulatory loop between
NF-B and endogenous IB is well established32
but was not expected for the mutated IB32/36A in
EBV-infected cells. Several possibilities may account for this phenomenon. For example, levels of rtTA-VP16 fusion protein may be
down-regulated after IB32/36A
overexpression. In fact, a search of cryptic B sites in the
MS4A-IB32/36A vector revealed the presence of 3 B consensus sequences (GGGRNNYYCC) within the CMV promoter of the
rtTA gene. On the other hand, the accumulation of
IB32/36A-mutated protein may allow its
phosphorylation on tyrosine 4258 or on its caboxyterminal
end,59 promoting its proteolytic degradation. This suggests
that over a longer period of time, a new equilibrium is reached in the
transfected cells between the inhibition of NF-B by the mutated
IB32/36A and the activation of NF-B by LMP-1,
resulting in significant and stable levels of NF-B activation,
though at a lower level than in absence of doxycycline. A relationship
between NF-B activation and cyclin D1 expression was reported
recently.60 Therefore, our results do not answer whether
EBV-immortalized B cells can in fact proliferate in the absence of
NF-B over a longer period of time. Furthermore, our results do not
exclude a role for NF-B in the initial steps of EBV
immortalization of primary B cells. Indeed, it is noteworthy that
acetylsalicylic acid, which inhibits NF-B, can block EBV-induced
B-cell proliferation in vitro.61
A series of recent publications points to a relationship between
NF-B activity and the protection of cells from
apoptosis.40-42,62,63 These results have been obtained in
various cell types, including EBV-negative B-cells and EBV-infected
lymphoblastoid cells.63-65 Here we show that the induction
of IB32/36A protein is associated with the
sensitization of cells to apoptosis in EBV-infected B-cells. Furthermore, we show that the loss of NF-B is correlated with a
significant decrease of the anti-apoptotic protein Bcl-2. Induction of
Bcl-2 by EBV in B cells has already been demonstrated.43,44 Transfection of these cells by LMP-1 is associated with an increase in
NF-B activity and the subsequent overexpression of
Bcl-2.44 A correlation between LMP-1 and Bcl-2 expression
has been noted in AIDS-related primary brain lymphomas.66
Antisense oligonucleotides against LMP-1 suppress Bcl-2 expression in
EBV-infected B cells.67 Bcl-2 induction by LMP-1 may be a
feature exclusive to B cells. Rowe et al44 have shown that
the transfection of LMP-1 into non-B-cell lines is associated with
NF-B activation and CD54 up-regulation without any effect on
Bcl-2. Furthermore, even in B cells, Bcl-2 up-regulation by LMP-1 does
not seem to be mediated directly by NF-B because after the
transfection of LMP-1, NF-B activation and CD54 occurred
simultaneously whereas Bcl-2 induction was delayed.44
Therefore, to date, a direct causal relationship between NF-B
activation and Bcl-2 up-regulation by LMP-1 has not been determined.
Our results clearly show that the loss of NF-B is associated with
a down-regulation of Bcl-2. This suggests that Rel/NF-B complexes
participate in the positive control of Bcl-2 gene expression in
EBV-infected B cells, most likely through intermediate target gene expression.
It is noteworthy that protection against apoptosis through NF-B
activation does not seem to be restricted to a particular cell type. A
recent report62 has shown that NF-B's anti-apoptotic effect is mediated by the up-regulation of TRAF1, TRAF2,
c-IAP1, and c-IAP2 molecules in the HT1080 fibrosarcoma cell line,
resulting in the inhibition of caspase-8 activation. No up-regulation
of Bcl-2 by NF-B activation was noted in this non-B-cell line.
Caspase-8 activation and Bcl-2 down-regulation are initial steps in the cascade of events leading to programmed cell death. The caspase-8 cascade is activated after the aggregation of death-domain proteins at
the membrane in response to external stimuli (see Vaux and Korsmeyer68 for review). It would be interesting to verify
whether EBV-mediated NF-B activation leads to the control of TRAF
and c-IAP gene expression in our cellular model. Bcl-2 down-regulation seems to be provoked by endogenous cell damage, such as DNA strand breaks after x-ray irradiation. Apoptosis induced by x-ray irradiation is mediated by p53 (see Levine69 for review). P53 exerts
both positive transcriptional control on the Bax gene70 and
negative control of Bcl-2 gene transcription.71 LMP-1 is
responsible for the up-regulation of the A20 protein, a 790-amino acid
protein with a unique zinc finger motif.18 A20 gene
expression is up-regulated by NF-B in response to TNF- and
protects cells against the cytotoxic effect of TNF-.72
LMP-1-mediated protection of cells from apoptosis occurs through A20
overexpression, which in turn inhibits p53.73 Therefore, it
can be hypothesized that the loss of NF-B in EBV-infected cells
could be responsible for the down-regulation of A20 gene transcription,
thus allowing p53 to inhibit Bcl-2 gene expression and to activate the
transcription of Bax. This in turn results in the down-regulation of
Bcl-2 gene expression and a sensitization of cells to apoptosis.
One of the next tasks to further our understanding of the role of
NF-B in EBV-related lymphomagenesis will be the identification of
NF-B target genes. MS4A-IB32/36A-transfected
cells may allow the identification of such genes in EBV-infected B
cells. Using bidimensional gel electrophoresis, we are performing a
comparative analysis of MS4A-IB32/36A-transfected
lymphoblastoid cells either overexpressing or not expressing
IB32/36A. Initial results show that at least 15 cytosolic polypeptides are down-regulated when NF-B is inhibited.
Thus, MS4A-IB32/36A-transfected B-lymphoblastoid cells may be a useful and valuable cellular model to help us in our
search for genuine NF-B target genes in the context of
EBV-transformed B lymphocytes.
| |
Acknowledgments |
We thank Hermann Bujard and Manfred Gossen for the gift of plasmids and
for sharing their results before publication. We also thank R. Hay
(University of St. Andrews, UK) and N. Rice (NCI, Frederick) for the kind gift of monoclonal antibodies and
immune sera. We thank Alain Israël (Institut Pasteur, Paris,
France) for the kind gift of the 0.4SK-luc plasmid and Charles Hall for critically reading the manuscript.
| |
Footnotes |
Submitted June 30, 1999; accepted October 24, 1999.
Supported by a EU grant (Pathogenesis of EBV-related
lymphoproliferative disorders in immunocompromised patients contract CHRX-CT94-0651) to M.R. and G.W.B. Partially supported by ANRS (contrat
98017), Association pour la Recherche sur le Cancer, Villejuif, France;
contrat 9348, Comité Départemental de la Ligue contre le
Cancer; BMBF (01 GE 9609/3); Die Deutsche Forschungsgemeinschaft; and
Fonds der Chemischen Industrie. J.F. was supported by a grant from the
Ligue Départementale Contre le Cancer (Seine Saint Denis).
Reprints: Jean Feuillard, Service d'Hématologie
Biologique, CHU Avicenne, 125 route de Stalingrad, 93009 Bobigny Cedex, France; e-mail: jean.feuillard{at}avc.ap-hop-paris.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction