|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 244-251
Unbalanced Expression of Bcl-2 Family Proteins in
Follicular Lymphoma: Contribution of CD40 Signaling in Promoting
Survival
By
Paolo Ghia,
Vassiliki A. Boussiotis,
Joachim L. Schultze,
Angelo
A. Cardoso,
David M. Dorfman,
John G. Gribben,
Arnold S. Freedman, and
Lee M. Nadler
From the Department of Adult Oncology, Dana-Farber Cancer Institute;
the Departments of Medicine and Pathology, Brigham and Women's
Hospital; and Harvard Medical School, Boston, MA.
 |
ABSTRACT |
Although highly responsive, advanced stage follicular lymphoma (FL)
is not curable with conventional treatment. This relative resistance is
thought to be due to the t(14;18) that results in the constitutive
overexpression of the death-inhibiting protein bcl-2. However, the
observation that FL cells are sensitive to treatment in vivo and prone
to apoptosis on in vitro culture questions whether bcl-2 alone is
responsible for the pathogenesis and clinical behavior of this disease.
Therefore, multiple genes are likely to be involved in both the
lymphomagenesis and the clinical course of FL. We examined whether
expression of other bcl-2 family genes might also be operative. Here,
we show that FL cells display a different pattern of expression of
bcl-2 family proteins from normal germinal center (GC) B cells that are
thought to be their normal counterpart. FL cells express the
death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas
GC B cells express bcl-xL and mcl-1 but also the
proapoptotic proteins bax- and bad. Although maintaining
constitutive levels of bcl-2 and mcl-1, FL cells are not protected from
apoptosis when cultured in vitro. Their propensity to undergo apoptosis
is temporally associated with downregulation of bcl-xL.
More importantly, activation of FL cells via CD40 not only prevents
downregulation but increases the level of bcl-xL expression
and results in promotion of survival. These results support the
hypothesis that the overexpression of bcl-2 is not the only
antiapoptotic mechanism responsible for the pathogenesis of FL.
Survival of FL cells is determined by a number of death-inhibiting
proteins, among which bcl-xL appears to have the most
critical role. Moreover, these findings are consistent with the
hypothesis that, although FL cells are malignant, they respond to
microenvironmental signals such as CD40L that appear to contribute to
their survival through the upregulation of death-inhibiting proteins.
| |
INTRODUCTION |
THE CLINICAL ENTITY of follicular
lymphoma (FL) is an enigma for both the clinician and
biologist.1 Within the non-Hodgkin's lymphomas, this
malignancy is among the most sensitive to low-dose oral alkylating
agents, low-dose external beam radiation, and steroids. However,
although the overwhelming majority of patients with advanced stage FL
achieve a very good partial remission, many fewer achieve complete
remission and few, if any, are cured using conventional aggressive
combination chemotherapy.2,3 These clinical observations
support the hypothesis that significant biologic heterogeneity exists
within each patient's FL and that both chemosensitive and
chemoresistant populations might account for these findings.
This clinical enigma has been further complicated by the discovery that
the t(14;18), observed in most FLs,4 encodes for the
bcl-2 gene,5,6 a major suppressor of
apoptosis.7,8 Because B cells from bcl-2
transgenic mice do not die in vitro and result in a lymphoproliferative
state in vivo,9 it is contended that the constitutive
expression of bcl-2 by FL cells is likely to be responsible for both
the genesis of the malignancy as well as its relative chemoresistance.
However, the uniform expression of bcl-2 by all FL cells contrasts with
their exquisite sensitivity to treatment in vivo and their propensity
to undergo apoptosis spontaneously in vitro and provides support for
the notion that bcl-2 expression cannot by itself explain the
pathogenesis and/or the clinical behavior of this malignancy.
Since the cloning of bcl-2 from FL cells, many other genes
have been identified that positively and negatively contribute to cell
death and these molecules are referred to as the extended bcl-2 family
of cell death proteins.10 The best characterized of these
include the death suppressors bcl-xL11 and
mcl-1,12 the death regulator bad,13 and the
death effectors bax-,14
bcl-xS,11 and bak.15-17 Although
the molecular mechanism(s) by which these proteins prevent or induce
cell death has yet to be elucidated, it has been shown that the
survival of a cell is tightly regulated by their expression and
association.10 Death is promoted when bax- is in excess,
such that bax-bax homodimers are formed. Both bcl-2 and
bcl-xL prevent the accumulation of free excess bax- by
forming bcl-2-bax and bcl-xL-bax heterodimers. Similarly,
mcl-1 is thought to function like bcl-2 and
bcl-xL18,19 and appears to be relevant for the
survival of normal peripheral blood B cells.20 Death can
also be promoted by death regulators such as bad that bind to bcl-2 and
bcl-xL, thereby displacing bax- from its heterodimeric
form, allowing its homodimerization, and thereby inducing
apoptosis.13 The relevance of the above-noted pathways with
regard to chemosensitivity and/or resistance of malignant cells
is rapidly increasing. Supporting this notion is the observation that
bcl-xL expression dramatically reduces sensitivity of tumor
cells to chemotherapeutic agents, both in vitro and in
vivo.21,22 Similarly, reduced expression of bax- by
solid tumors appears to correlate with chemoresistance and translates
into shorter clinical survival.23-25
In the present report, we examined the expression of death suppressor,
regulator, and effector proteins in FL cells. Here, we show that FL
cells display a unique phenotype of predominantly antiapoptotic protein
expression compared with the balance of antiapoptotic and proapoptotic
proteins observed in normal germinal center (GC) B cells. Moreover, we
show that the apoptosis of FL cells occurs in vitro despite their high
constitutive bcl-2 levels and, in contrast, is associated with
decreasing expression of bcl-xL. Finally, we show that one
mechanism that can prevent the decrease in bcl-xL
expression is signaling via the CD40 molecule, suggesting that this
pathway might be relevant to the survival of these cells in vivo. Taken
together, these observations suggest that microenvironmental signals
can alter the expression of survival proteins, thereby contributing to
the clinical and biologic behavior of FL cells.
| |
MATERIALS AND METHODS |
Cells.
Lymph nodes were obtained from seven patients undergoing diagnostic
biopsies who were diagnosed with follicular, small cleaved cell type
lymphoma (working formulation). The malignant B cells were carrying the
t(14;18) translocation, as shown by polymerase chain reaction
(PCR).26,27 Tonsils were obtained, as discarded tissues,
from children undergoing tonsillectomy. All tissue samples from
patients were obtained following institutional guidelines at
Dana-Farber Cancer Institute (Boston, MA), Brigham and Women's
Hospital (Boston, MA), and Children's Hospital (Boston, MA).
Organs were cut with a scalpel blade and incubated two times with
Collagenase IV and DNAse I (Sigma, St Louis, MO) for two rounds of 15
minutes at 37°C, 5% CO2. They were then passed through a
fine wire mesh to prepare a single-cell suspension. Mononuclear cells
were isolated by Ficoll-hypaque gradient (density, 1.077 g/mL;
Pharmacia, Uppsala, Sweden). When not used directly for experiments at
the time of preparation, they were frozen in liquid nitrogen.
Purification of normal and neoplastic B cells.
Mononuclear cells were first submitted to E rosetting with sheep red
blood cells. Residual non-B cells were next removed from the
E population by incubation with a cocktail of anti-CD3,
-CD11b, -CD14, -CD56 monoclonal antibodies (MoAbs), as
described,26,27 followed by magnetic beads coated with goat
antimouse IgG and IgM antibodies (BioMags; Perspective Biosystems,
Framingham, MA) and application of a magnetic field. The purity of the
isolated B cells was always greater than 97%. Total tonsillar B cells
were further FACS-sorted into three different subsets, as described
below.
Antibodies.
Surface staining of normal and neoplastic tissue cells was performed
using the following antibodies: phycoerythrin (PE)-labeled anti-CD3 and
fluorescein isothiocyanate (FITC) anti-CD4; biotin (BIO)-labeled
anti-CD8; FITC-labeled anti-CD10 (CALLA); anti-CD14; FITC-, PE-, or
BIO-labeled anti-CD19; PE-labeled anti-CD20; and PE-labeled anti-CD56
(Coulter, Miami, FL); FITC-labeled anti-CD24 (Immunotech, Marseille,
France); and PE-labeled anti-CD38 (Becton Dickinson and Co, Mountain
View, CA). BIO-labeled anti-human IgD was purchased from Southern
Biotechnology (SBA, Birmingham, AL). Polyclonal
FITC-conjugated rabbit anti-human IgM, anti-IgG, anti-IgA and anti-IgD,
anti-human  light chain, and polyclonal PE-conjugated rabbit
anti- light chain were purchased from Dako (Carpenteria, CA).
Polyclonal BIO-conjugated goat anti-human IgD [F(ab)2]
and streptavidin-PE were obtained from SBA. Streptavidin-tricolor was
purchased from Caltag Laboratories (South San Francisco, CA).
Cytokines.
Recombinant human interleukin-1 (IL-1; 5 ng/mL; Endogen,
Cambridge, MA), IL-2 (50 U/mL; Genetics Institute, Cambridge, MA), IL-3
(5 ng/mL), IL-6 (10 ng/mL), IL-11 (10 ng/mL; Genetics Institute), IL-4
(2 ng/mL), IL-7 (10 ng/mL; Immunex, Seattle, WA), and IL-10 (10 ng/mL;
Genzyme, Cambridge, MA) were supplemented to the medium separately or
in combination (IL-2 + IL-10).
Cell surface staining and cell sorting.
Three-color immunofluorescence analysis was used for identification of
neoplastic populations in the pathological samples and
identification of different B-cell populations within the tonsillar
mononuclear cells. Tonsillar B cells were stained with BIO-labeled
anti-IgD (followed by streptavidin TRICOLOR), PE-labeled
anti-CD38, and FITC-labeled anti-CD19 antibodies. Naive B cells were
identified as being CD19+, IgD+,
CD38 cells; GC cells being CD19+,
IgD, CD38+; and memory B cells being
CD19+, IgD,
CD38.28 These three different subpopulations
were isolated by FACS-sorting. Cell surface immunofluorescence, flow
cytometric analysis, and cell sort were performed, as previously
described,29 on a Coulter Elite (Coulter Co) at 4°C. Only
cells exhibiting low forward angle and low right angle light scattering
properties (lymphoid gate) were analyzed and, when needed, sorted.
Immunohistochemistry.
Cryostat sections of lymph nodes involved by FL were fixed in acetone
for 10 minutes, washed with PBS, and incubated with anti-CD3, -CD20
(Coulter), and -CD40L (Pharmingen, San Diego, CA) MoAbs and the
follicular dendritic cells (FDC)-specific MoAb, DRC-1 (Dako) for 30
minutes. Slides were then washed with PBS, incubated with BIO horse
antimouse antibody (Vector Laboratories, Burlingame, CA), and then
incubated with avidin-biotinylated peroxidase complex (Vector
Laboratories) for 40 minutes, followed by reaction with
diaminobenzidine/hydrogen peroxide. Sections were subsequently stained
with 2% methyl green.
Cell culture.
Purified FL and GC cell populations were cultured in RPMI 1640
(Mediatech, Herndon, VA) supplemented with 10% fetal calf serum (FCS;
PAA Laboratories Inc, Newport Beach, CA), 2 mmol/L L-glutamine and 15
µg/mL gentamicin (GIBCO BRL, Gaithersburg, MD), in the presence or in
the absence of soluble CD40 ligand (CD40L)30 and cytokines.
Soluble CD40 ligand was a generous gift of Dr Peter Lane (Basel,
Switzerland).
Western blotting.
At the time of the initiation of culture and after 24 and 48 hours of
culture, B cells were harvested (in 2 cases, lymphoma cells were also
harvested after 3, 4, 5, and 7 days) and cell lysates were prepared
with lysis buffer containing 10 mmol/L Tris-HCl (pH 7.6), 5 mmol/L
EDTA, 50 mmol/L NaCl, aprotinin (5 µg/mL), pepstatin (1 µg/mL),
soybean trypsin inhibitor (2 µg/mL) 1 mmol/L phenylmethylsulfonyl
fluoride, and 1% NP-40 (Sigma). Whole cell lysates
(3 × 106 B-cell equivalents per lane or equivalent
amount of protein per lane) were analyzed on 12% gels in sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Immunoblotting was performed using MoAbs or polyclonal antisera against
bcl-2 (1:1,500, mouse monoclonal antibody; DAKO); bcl-x (1:1,000,
rabbit polyclonal antiserum; Transduction Laboratories);
bad (1:250, mouse MoAb; Transduction Laboratories, Lexington, KY); bax
and mcl-1 (1:500, rabbit polyclonal antiserum; Santa Cruz, Santa Cruz,
CA). As secondary reagent, horseradish peroxidase-antimouse IgG
F(ab )2 (1:3,000; Amersham, Arlington Heights, IL) or
horseradish peroxidase-antirabbit IgG (1:10,000; Biorad,
Hercules, CA) were used in the immunoblots.
Immunodetection was performed using the Renaissance (TM) enhanced
chemiluminescence system (NEN, Boston, MA).
Quantitative assessment of apoptosis.
Quantitative assessment of apoptosis on normal and neoplastic B cells
was determined by terminal deoxynucleotidil transferase (TdT)-mediated
dUTP-FITC nick end labeling (TUNEL)31 (Boehringer Mannheim
GmbH, Mannheim, Germany) (see Fig 3) and with an Annexin V-based
apoptosis detection kit (R&D Systems, Flanders, NJ).32
View larger version (18K):
[in this window]
[in a new window]
| Fig 3.
Quantitative assessment of apoptosis in FL cells cultured
in media alone. FL cells were cultured as described and the percentage
of apoptotic cells was assessed by TUNEL staining in flow cytometry.
The figure shows the percentage of cells (R1 = 23.6%) TUNEL
positive after 4 days of culture in one representative sample. Similar
results were obtained from all seven of the patients studied.
|
|
Cell cycle analysis.
Determination of the percentage of FL cells at each stage of the cell
cycle was performed by assessment of DNA content after staining with
ethidium bromide, according to an established protocol.33
| |
RESULTS |
Distinct patterns of Bcl-2 related proteins are expressed
in functionally distinct B-cell populations.
After an encounter with antigen, recirculating naive B cells migrate
into secondary lymphoid organs where they downregulate their Ig
receptors and differentiate into GC B cells.34 Within the
GC, antigen-specific B cells rapidly mutate their Ig variable region
genes. After positive selection by interaction with antigen presented
by follicular dendritic cells, antigen-specific B cells clonally expand
and then either differentiate into antibody-secreting plasma cells or
long-lived memory B cells.34 The above-noted
differentiative sequence results in functionally distinct populations
of B cells that display unique cell surface phenotypic
patterns,28 including (1) naive B,
sIgD+CD38; (2) GC B,
IgDCD38+; and (3) memory B,
sIgDCD38. Purified B cells from three
human tonsils were prepared and then analyzed by three-color FACS
analysis (CD19, sIgD, and CD38) for B-cell populations. Of the total
CD19+ cells, 45% to 55% displayed a naive, 30% to 45%
displayed a GC, and 10% to 15% displayed a memory phenotype. Each of
the above populations were sorted with a resulting purity of greater
than 97%. Cell lysates from each population were prepared and analyzed
by Western blot (3 × 106 cell equivalents or equivalent
amount of protein per lane) for the expression of death suppressor
(bcl-2, bcl-xL, and mcl-1), death regulator (bad), and
death inducer (bax-) proteins. Cell lysates
from Jurkat cells, grown in log phase, were
used as positive controls. As shown in Fig 1A, naive B cells expressed
levels of mcl-1 and bcl-2 comparable to that seen in Jurkat, but
bcl-xL was below the level of detection. Compared with
Jurkat, bax- was virtually undetectable and bad expression was
approximately 10-fold less. In contrast, GC B cells express high levels
of bcl-xL, mcl-1, and bax- and detectable levels of bad
but, as previously observed, do not have detectable bcl-2 expression.
Finally, memory B cells, like naive B cells, express both bcl-2 and
mcl-1 but, in addition, they also express bcl-xL, although
at lower levels than GC B cells. Bax- is undetectable and bad is
comparable to the low levels seen in naive B cells. These results show
that functionally distinct B-cell populations show discrete patterns of
bcl-2 family protein expression and are associated with the biologic
proclivity of these cells to survive and/or die in vivo.
View larger version (44K):
[in this window]
[in a new window]
| Fig 1.
Pattern of expression of bcl-2 family members in human
tonsillar B-cell populations (A) and FL cells (B). Samples from (A)
FACS-sorted CD38IgD+ naive B cells,
CD38+IgD GC B cells, and
CD38IgD memory B cells and (B) samples
from highly purified FL cells of five representative patients were
lysed and analyzed by SDS-PAGE and Western blotting using: anti-mcl-1
polyclonal antiserum (first panel); anti-bcl-x polyclonal antiserum
(second panel); anti-bcl-2 MoAb (third panel); anti-bax polyclonal
antiserum (fourth panel); or anti-bad MoAb (fifth panel). Expression of
the different proteins was quantitated in each lane using a Scanner
phosphoimager (Alpha Innotech Corp, San Leonardo, CA). Jurkat cell line
(last lane in A and B), grown in log phase, was used as control. A
total of 3 × 106 cell equivalents were used per test.
Similar results were obtained when experiments were performed on
tonsils or FL cells using an equivalent amount of protein per lane.
|
|
Unbalanced expression of bcl-2 family proteins in FL.
Because FL has been considered to be the neoplastic counterpart of GC B
cells,35 we examined the expression of the identical bcl-2
family proteins in seven patients with FL. For these experiments, lymph
nodes that were highly infiltrated by FL cells (>65%), as assessed
by immunohistochemistry, were selected and B cells were isolated (CD19
> 97%). Phenotypic analysis showed that B cells from each of these
nodes were homogeneously expressing CD19, CD10, and CD38 and expressing
monoclonal light chain. Moreover, all patients in this study expressed
the t(14;18), as shown by an amplifiable PCR product for the bcl-2/IgH
rearrangement. Using the identical approach used to analyze normal
B-cell populations, FL B cells were lysed and analyzed for bcl-2 family
protein expression by Western blotting (Fig 1B). As expected, all FLs
studied expressed homogeneous, high levels of bcl-2 protein. In
addition, both bcl-xL and mcl-1 were also expressed in FL
cells at levels comparable to those observed in normal GC B cells. In
contrast to GC B cells, both bax- and bad are either undetectable or
at very low levels.
In vitro apoptosis of FL cells is temporally associated with
downregulation of bcl-xL, which is prevented by CD40
activation.
Consistent with previous reports,36 GC B cells cultured in
media alone underwent apoptosis within 18
hours (>90% specific death; Fig 2A). In contrast, in vitro cultured
FL cells have been reported to survive for 24 to 48 hours and then die
by apoptosis over the next 5 to 7 days.37 We confirmed
these results in samples from all seven patients and a representative
cell survival curve is depicted in Fig 2B. Spontaneous FL cell death in
vitro was mediated by apoptosis, as determined by DNA nick end labeling
(TUNEL assay), to detect single-strand DNA fragmentation (Fig 3).
Apoptosis was also confirmed in all the samples with
the Annexin V method (data not shown). We
attempted to determine whether the inability of FL cells to survive in
vitro was due to the absence of a known cytokine; therefore, we
repeated these experiments in the presence of functional concentrations
of cytokines including IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10,
and IL-11 and the combination of IL-2 and IL-10 (known to deliver a
differentiative signal to GC B cells).38 As shown in Fig
2B, none of the conditions described above significantly altered the FL
cell survival curve.
View larger version (20K):
[in this window]
[in a new window]
| Fig 2.
Onset of apoptosis in GC (A) and FL cells (B) cultured in
media alone or in the presence of cytokines: prolongation of survival
in the presence of soluble CD40L. FACS-sorted GC B cells (A) or
purified FL cells of a representative patient (B) were cultured in
media alone or in the presence of several cytokines (see Materials and
Methods) or soluble CD40L for the indicated time and apoptosis was
assessed quantitatively by Trypan Blue exclusion at the indicated time
points. Similar results were obtained with GC cells isolated from two
other tonsils and with FL cells from six additional patients.
|
|
Because FL cells express high constitutive levels of death suppressors
bcl-2, bcl-xL, and mcl-1 but no death effectors bax- or
bad, it appears paradoxical that these cells undergo apoptosis in
vitro. Therefore, we postulated that proclivity to apoptosis might be
associated with a change in the expression of bcl-2 family proteins
during in vitro culture of FL cells. FL cells were cultured for 24 and
48 hours, and expression of the same bcl-2 family members was examined.
FL cells cultured in media showed stable high expression of bcl-2 over
the 48-hour culture period (Fig 4A).
Likewise, the mcl-1 protein was not altered over the same time
interval. In contrast, bcl-xL showed a rapid decline with
threefold decrease from basal level within 24 hours. Neither bax-
nor bad was induced over this time interval (data not shown). Identical
results were observed when, in three cases, the cells were also
analyzed after culture in the presence of the above-mentioned cytokines
(data not shown). These results suggest that the rapid decrease in
bcl-xL is associated with the in vitro death of FL cells.
Moreover, they suggest that the high constitutive expression of bcl-2
alone is insufficient to prevent apoptosis in vitro.
View larger version (31K):
[in this window]
[in a new window]
| Fig 4.
Changes in bcl-xL expression in FL cells (A)
and GC (B) upon culture in media alone or in the presence of sCD40L;
bcl-2 levels are not affected. Highly purified FL cells (A) and
FACS-sorted CD38+ GC B cells (B) were cultured in media
alone or in the presence of soluble CD40L. At the indicated time
intervals, cells were isolated, cell lysates were prepared, and 3 ×
106 cells were analyzed by SDS-PAGE and Western blotting
using anti-mcl-1 polyclonal antiserum (first panel), anti-bcl-x
polyclonal antiserum (second panel), and anti-bcl-2 MoAb (third
panel). Expression of the different proteins was quantitated in each
lane using a Scanner phosphoimager (Alpha Innotech Corp).
|
|
Previous studies have suggested that culture of both normal and
neoplastic B cells with CD40L improves survival in vitro. We therefore
sought to determine whether CD40 signaling would alter the expression
of bcl-2 family proteins in GC and FL cells and whether such an effect
would be temporally associated with protection from apoptosis. Purified
GC B and FL cells were isolated as described above and cultured in the
presence of media or soluble CD40L. As shown in Fig 2A and B, this
resulted in significantly improved survival. After 12 to 18 hours of
culture of GC cells in media alone, Western blot analysis showed a
dramatic downregulation of mcl-1 and bcl-xL protein
expression (Fig 4B). However, at this time point, virtually all cells
were dead (Fig 2A). In contrast, CD40 signaling dramatically suppressed
apoptosis and, at 18 hours, greater than 80% of the cells were alive
(Fig 2A). Western blot analysis of CD40-activated GC B cells showed no
change in the expression of bcl-2, which remained below the level of
detection (Fig 4B). Mcl-1 expression did not significantly change at
both 12 and 18 hours. In contrast, consistent with previous
reports,39 CD40-mediated activation maintained and
upregulated the expression of bcl-xL (Fig 4B).
We next examined whether CD40 signaling altered the expression of bcl-2
family proteins in FL cells as it did in GC cells. After CD40
activation, although survival was prolonged (Fig 2B), no increase in
cycling cells was observed and less than 2% of either freshly isolated
or CD40-activated FL cells were in S and G2/M stages of the cell cycle
(data not shown). Western blot analysis showed no significant change in
mcl-1 and bcl-2 at 24 and 48 hours of culture (Fig 4A). In contrast,
the baseline bcl-xL expression was significantly
upregulated at 24 and 48 hours (Fig 4A). However, in two cases, when
the analysis was extended up to 7 days, bcl-xL protein
began to decline after 4 days and restimulation at that time did not
prolong the survival of FL cells and it did not upregulate
bcl-xL protein expression (data not shown).
These results show that, although neoplastic, FL cells are still
capable of responding to a physiologic CD40-mediated signal. The above
results suggest that FL B cells might also receive such signals in vivo
resulting in upregulation of bcl-xL.
CD40 ligand-positive T cells infiltrate neoplastic follicles in
patients with FL.
CD40 ligand-positive T cells have been reported to be present in
significant numbers in the follicles of FL.40 We confirmed
the findings in lymph node sections stained with lineage-restricted
MoAbs and MoAbs to CD40L, from six of the seven FL patients studied.
Neoplastic follicles showed infiltrating T cells that were
immunoreactive for CD40L (data not shown).
| |
DISCUSSION |
In the present report, we show that highly enriched FL cells display a
unique pattern of expression of bcl-2 family proteins compared with the
major functional populations of human B cells. Unlike GC B cells, which
are thought to be the normal cellular counterparts of FL
cells,35 FL cells express only death-suppressor proteins
(bcl-2, bcl-xL, and mcl-1), whereas normal GC B cells also
express bax- and bad. Although all FL cells studied express high,
constitutive levels of bcl-2, this did not protect them from undergoing
apoptosis when cultured in vitro. In contrast, rapid downregulation of
bcl-xL was associated with the propensity of FL cells to
undergo apoptosis in vitro. Activation of FL cells via CD40, but not
via any cytokine receptor tested, resulted in both maintenance and
upregulation of bcl-xL and promotion of survival. These
findings are consistent with the hypothesis that, although FL cells are
malignant, they respond to microenvironmental signals that appear to
contribute to their in vivo survival through the selective induction of
death protective proteins, predominantly of bcl-xL.
Because FL and GC B cells share a common follicular organization
(including presence of FDCs) as well as a unique cell surface phenotype
characterized by the expression of CD19, CD20, CD10, and CD38, FL cells
have long been considered to be the neoplastic counterpart of GC B
cells.35 Similarly, both FL and GC B cells undergo somatic
hypermutation and clonal expansion.34,41 In contrast to
these similarities, FL and GC B cells behave differently in vivo and in
vitro. In vivo, FL cells are largely G0 B cells with
little, if any, propensity to die, whereas most GC B cells are actively
cycling and are prone to apoptotic death. In vitro, both populations
undergo apoptosis, with FL cells dying more slowly than GC B cells. Our
results provide insight into the molecular events responsible for these
differences. Although GC B cells do not express bcl-2, they do express
the death suppressor bcl-xL and the death inducers bax-
and bad. The balance of these death-regulatory proteins appear to be
responsible, at least in part, for the propensity of GC B cells to
either undergo apoptosis or alternatively to differentiate and survive
as memory B cells, depending on the signals they receive from their
microenvironment. In contrast, highly enriched FL cells express only
death-suppressor proteins (bcl-2, bcl-xL, and mcl-1) and no
significant levels of either bax- or bad. Therefore, FL cells appear
to have an even more efficient mechanism with which to counter
physiological death signal(s) because they express both potent death
suppressors without any death effectors. Consistent with this
hypothesis is the observation that, in bcl-x/bcl-2 transgenic
mice, mature B cells are more effectively protected from death signals
than they are in transgenic mice expressing either death suppressor
alone.42 The capacity of
bcl-2+bcl-xL+bax FL
cells to undergo apoptosis in vitro questions the current model in
which bax- is required to mediate death and bcl-2/bcl-xL
protect from apoptosis by heterodimerization with
bax-.14,43 In support of this observation are studies in
bax-deficient and bcl-2-deficient mice showing
that bcl-2 may not always act through interaction with bax-, because
the affected tissues of
baxbax mice are not
identical to the affected tissues of
bcl-2bcl-2.44
Moreover, heterodimerization with bax- is also not always required
for bcl-xL to exert its death-repressing
activity.45,46 Other death-promoting proteins that have
been recently cloned, such as bak15-17 and
bik,47,48 can interact with bcl-2 and bcl-xL
and therefore might be critical for the survival-promoting activity of
these proteins.
Without question, bcl-2 plays an important role in normal and
neoplastic B-lymphocyte survival in vivo. With increasing age,
bcl-2-deficient mice display profound B and T
lymphopenia.44 Conversely, increased B-cell survival in
vivo and in vitro is observed in bcl-2-Ig-transgenic
mice.9 In these mice, mature B cells accumulate with age,
which is thought to be due to expansion of the memory B-cell pool
rather than altered B-cell selection within the GC.49
Although these mice uniformly develop B-cell hyperplasia, none develops
low-grade FL and only some progress to high-grade lymphoma, after
acquisition of a c-myc rearrangement.50 These results are
consistent with the hypothesis that a bcl-2 translocation
alone does not appear to be sufficient to induce FL or any other
neoplasm. In agreement with this is the observation that clones
harboring a PCR-amplifiable bcl-2 translocation can be
detected in the secondary lymphoid organs51 and peripheral
blood of normal individuals.52 Interestingly, the frequency
of these translocations appears to increase significantly with
age.53
Increasing evidence supports the conclusion that bcl-2 is not
sufficient to regulate apoptosis in all cell types or in response to
all stimuli. Expression of bcl-2 does not protect the phenotypically
immature B-cell lymphoma WEHI-231 from anti-µ-induced cell
death.54 Similarly, anti-µ-induced apoptosis of a
Burkitt cell line is not accompanied by modulation of bcl-2
expression.55 Likewise, our results show that, despite
constitutive high levels of bcl-2, FL cells undergo apoptosis in vitro.
Moreover, no modifications in bcl-2 protein level can be detected
accompanying the onset of apoptosis, whereas a rapid downregulation of
bcl-xL is observed. This result parallels the previous
observations in T cells in which a dramatic decrease in
bcl-xL, but not bcl-2 protein levels, precedes apoptosis
induced by IL-2 withdrawal.46
CD40/CD40L interaction is thought to play a key role for proliferation,
isotype switching, memory formation, and rescue from apoptosis in both
normal and malignant B cells.36,55-58 In all of the studies
described above, a dissociation exists between the rescue from
apoptosis achieved by CD40 stimulation and induction of bcl-2. In fact,
bcl-2 is induced relatively late during CD40-mediated GC B cells
rescue.36 Similarly, bcl-2 is not induced in Burkitt's
lymphoma or WEHI 231 cell lines rescued by CD40 activation after
anti-µ-induced apoptosis.36,55 In contrast, it has been
recently suggested that upregulation of bcl-xL could be a
key event in CD40-mediated survival in both normal tonsillar B
cells39 and the immature B-cell lymphoma WEHI-231
cells.59
To date, the outcome of CD40 ligation of FL cells has been
controversial. Intermediate and high-grade lymphoma B-cell lines appear
to be inhibited in their growth by either anti-CD40 antibody or soluble
CD40 ligand,60 whereas FL cells appear to be stimulated to
grow.37 We show here that activation of FL cells by the
soluble form of CD40L, but not via any cytokine receptor thus far
tested, results in upregulation of bcl-xL and protection of
apoptosis in vitro. In this respect, FL B cells appear to behave as
normal GC B cells. Therefore, FL cells are capable of receiving
microenvironmental signals that might contribute to their survival. The
biologic relevance of these findings is supported by the observation
that CD40L-expressing CD3+/CD4+ T cells are
admixed with FL cells within the neoplastic follicles,40 as
we also confirmed in infiltrated lymph nodes from all patients tested.
We have also confirmed these findings by flow cytometric analysis, in
which approximately 70% to 80% of the T cells were CD4+
and 10% of these cells coexpress CD40L on their cell surface (data not
shown). Therefore, CD40L-positive T cells might provide signals via
CD40 recapitulating in vivo our in vitro findings. However, CD40L on T
cells may not provide the only viability signal to FL cells. The
physical interaction between FDC and GC B lymphocytes, as well as FDC
and FL cells, has been shown to rescue the latter from apoptotic cell
death.61,62 However, the pattern of CD40L expression in the
GCs is clearly distinct from that of FDC. Therefore, the molecular
interactions that regulate the antiapoptotic effect of FDCs are
unknown.
Our results suggest that microenvironmental signals are at least in
part responsible for the modulation of FL survival in vivo. Blockade of
such signals may facilitate the entry of FL cells into the death
pathway(s). Precise definition of such signals and pathways might
potentially provide novel approaches to alter the sensitivity of FL to
therapy as well as to develop novel treatment modalities for this
disease.
| |
FOOTNOTES |
Submitted May 20, 1997;
accepted September 3, 1997.
Supported by National Institutes of Health Grants No. P01-CA66996 and
CA55207 (A.S.F). P.G. is supported by a fellowship from the
American-Italian Cancer Foundation; by Toby S. Meyerson, Esq of Paul,
Weiss, Rifkind, Wharton & Garrison; and by Jerry I. Speyer, President,
Tishman Speyer Properties, Inc.
Address correspondence to Paolo Ghia, MD, PhD, Dana-Farber Cancer
Institute, D738, 44 Binney St, Boston, MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful for the expert technical assistance of J. Borus for the
PCRs. We also acknowledge the help of Michelle Yoon. We also
acknowledge Dr F. Caligaris-Cappio for critical reading of the
manuscript. We are thankful to Toby S. Meyerson, Esq of Paul, Weiss,
Rifkind, Wharton & Garrison, and Jerry I. Speyer,
President Tishman Speyer Properties Inc for their support to P.G.
| |
REFERENCES |
1.
Longo DL:
What's the deal with follicular lymphomas?
J Clin Oncol
11:202,
1993[Free Full Text]
2.
Dana BW,
Dahlberg S,
Nathwani BN,
Chase E,
Coltman C,
Miller TP,
Fisher RI:
Long-term follow-up of patients with low-grade malignant lymphomas treated with doxorubicin-based chemotherapy or chemoimmunotherapy.
J Clin Oncol
11:644,
1993[Abstract]
3.
Rohatiner A,
Lister TA:
Management of follicular lymphoma.
Curr Opin Oncol
6:473,
1994[Medline]
[Order article via Infotrieve]
4.
Tsujimoto Y,
Cossman J,
Jaffe E,
Croce CM:
Involvement of the bcl-2 gene in human follicular lymphoma.
Science
228:1440,
1985[Abstract/Free Full Text]
5.
Tsujimoto Y,
Finger LR,
Yunis J,
Nowell PC,
Croce CM,
Stein H,
Gerdes J,
Mason DY:
Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation.
Science
226:1097,
1984[Abstract/Free Full Text]
6.
Cleary ML,
Smith SD,
Sklar J:
Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation.
Cell
47:19,
1986[Medline]
[Order article via Infotrieve]
7.
Hockenbery D,
Nunez G,
Milliman C,
Schreiber RD,
Korsmeyer SJ:
Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.
Nature
348:334,
1990[Medline]
[Order article via Infotrieve]
8.
Korsmeyer SJ:
Bcl-2 initiates a new category of oncogenes: Regulators of cell death.
Blood
80:879,
1992[Free Full Text]
9.
McDonnell TJ,
Deane N,
Platt FM,
Nunez G,
Jaeger U,
McKearn JP,
Korsmeyer SJ:
bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation.
Cell
57:79,
1989[Medline]
[Order article via Infotrieve]
10.
Yang E,
Korsmeyer SJ:
Molecular thanatopsis: A discourse on the BCL2 family and cell death.
Blood
88:386,
1996[Free Full Text]
11.
Boise LH,
Gonzalez-Garcia M,
Postema CE,
Ding L,
Lindsten T,
Turka LA,
Mao X,
Nunez G,
Thompson CB:
bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.
Cell
74:597,
1993[Medline]
[Order article via Infotrieve]
12.
Kozopas KM,
Yang T,
Buchan HL,
Zhou P,
Craig RW:
MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2.
Proc Natl Acad Sci USA
90:3516,
1993[Abstract/Free Full Text]
13.
Yang E,
Zha J,
Jockel J,
Boise LH,
Thompson CB,
Korsmeyer SJ:
Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death.
Cell
80:285,
1995[Medline]
[Order article via Infotrieve]
14.
Oltvai ZN,
Milliman CL,
Korsmeyer SJ:
Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.
Cell
74:609,
1993[Medline]
[Order article via Infotrieve]
15.
Chittenden T,
Harrington EA,
O'Connor R,
Flemington C,
Lutz RJ,
Evan GI,
Guild BC:
Induction of apoptosis by the Bcl-2 homologue Bak.
Nature
374:733,
1995[Medline]
[Order article via Infotrieve]
16.
Farrow SN,
White JH,
Martinou I,
Raven T,
Pun KT,
Grinham CJ,
Martinou JC,
Brown R:
Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K.
Nature
374:731,
1995[Medline]
[Order article via Infotrieve]
17.
Kiefer MC,
Brauer MJ,
Powers VC,
Wu JJ,
Umansky SR,
Tomei LD,
Barr PJ:
Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak.
Nature
374:736,
1995[Medline]
[Order article via Infotrieve]
18.
Sato T,
Hanada M,
Bodrug S,
Irie S,
Iwama N,
Boise LH,
Thompson CB,
Golemis E,
Fong L,
Wang HG,
Reed JC:
Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system.
Proc Natl Acad Sci USA
91:9238,
1994[Abstract/Free Full Text]
19.
Sedlak TW,
Oltvai ZN,
Yang E,
Wang K,
Boise LH,
Thompson CB,
Korsmeyer SJ:
Multiple Bcl-2 family members demonstrate selective dimerizations with Bax.
Proc Natl Acad Sci USA
92:7834,
1995[Abstract/Free Full Text]
20.
Lomo J,
Smeland EB,
Krajewski S,
Reed JC,
Blomhoff HK:
Expression of the Bcl-2 homologue Mcl-1 correlates with survival of peripheral blood B lymphocytes.
Cancer Res
56:40,
1996[Abstract/Free Full Text]
21.
Minn AJ,
Rudin CM,
Boise LH,
Thompson CB:
Expression of bcl-xL can confer a multidrug resistance phenotype.
Blood
86:1903,
1995[Abstract/Free Full Text]
22.
Dole MG,
Jasty R,
Cooper MJ,
Thompson CB,
Nunez G,
Castle VP:
Bcl-xL is expressed in neuroblastoma cells and modulates chemotherapy-induced apoptosis.
Cancer Res
55:2576,
1995[Abstract/Free Full Text]
23.
Krajewski S,
Blomqvist C,
Franssila K,
Krajewska M,
Wasenius VM,
Niskanen E,
Nordling S,
Reed JC:
Reduced expression of proapoptotic gene BAX is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma.
Cancer Res
55:4471,
1995[Abstract/Free Full Text]
24.
McConkey DJ,
Chandra J,
Wright S,
Plunkett W,
McDonnell TJ,
Reed JC,
Keating M:
Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX.
J Immunol
156:2624,
1996[Abstract]
25.
Bargou RC,
Wagener C,
Bommert K,
Mapara MY,
Daniel PT,
Arnold W,
Dietel M,
Guski H,
Feller A,
Royer HD,
Dorken B:
Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.
J Clin Invest
97:2651,
1996[Medline]
[Order article via Infotrieve]
26.
Gribben JG,
Freedman AS,
Neuberg D,
Roy DC,
Blake KW,
Woo SD,
Grossbard ML,
Rabinowe SN,
Coral F,
Freeman GJ,
Ritz J,
Nadler LM:
Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma.
N Engl J Med
325:1525,
1991[Abstract]
27.
Schultze JL,
Cardoso AA,
Freeman GJ,
Seamon MJ,
Daley J,
Pinkus GS,
Gribben JG,
Nadler LM:
Follicular lymphomas can be induced to present alloantigen efficiently: A conceptual model to improve their tumor immunogenicity.
Proc Natl Acad Sci USA
92:8200,
1995[Abstract/Free Full Text]
28.
Liu YJ,
Barthelemy C,
de Bouteiller O,
Arpin C,
Durand I,
Banchereau J:
Memory B cells from human tonsils colonize mucosal epithelium and directly present antigen to T cells by rapid upregulation of B7-1 and B7-2.
Immunity
2:239,
1995[Medline]
[Order article via Infotrieve]
29.
Ghia P,
Gratwohl A,
Signer E,
Winkler TH,
Melchers F,
Rolink AG:
Immature B cells from human and mouse bone marrow can change their surface light chain expression.
Eur J Immunol
25:3108,
1995[Medline]
[Order article via Infotrieve]
30.
Lane P,
Brocker T,
Hubele S,
Padovan E,
Lanzavecchia A,
McConnell F:
Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation.
J Exp Med
177:1209,
1993[Abstract/Free Full Text]
31.
Gorczyca W,
Bigman K,
Mittelman A,
Ahmed T,
Gong J,
Melamed MR,
Darzynkiewicz Z:
Induction of DNA strand breaks associated with apoptosis during treatment of leukemias.
Leukemia
7:659,
1993[Medline]
[Order article via Infotrieve]
32.
Koopman G,
Reutelingsperger CP,
Kuijten GA,
Keehnen RM,
Pals ST,
van Oers MH:
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
Blood
84:1415,
1994[Abstract/Free Full Text]
33.
Ghia P,
ten Boekel E,
Sanz E,
de la Hera A,
Rolink A,
Melchers F:
Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci.
J Exp Med
184:2217,
1996[Abstract/Free Full Text]
34.
MacLennan IC:
Germinal centers.
Annu Rev Immunol
12:117,
1994[Medline]
[Order article via Infotrieve]
35.
Stein H,
Gerdes J,
Mason DY:
The normal and malignant germinal centre.
Ballieres Clin Haematol
11:531,
1982
36.
Holder MJ,
Wang H,
Milner AE,
Casamayor M,
Armitage R,
Spriggs MK,
Fanslow WC,
MacLennan IC,
Gregory CD,
Gordon J:
Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl-2 induction.
Eur J Immunol
23:2368,
1993[Medline]
[Order article via Infotrieve]
37.
Johnson PW,
Watt SM,
Betts DR,
Davies D,
Jordan S,
Norton AJ,
Lister TA:
Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40/stromal cell system.
Blood
82:1848,
1993[Abstract/Free Full Text]
38.
Arpin C,
Dechanet J,
Van Kooten C,
Merville P,
Grouard G,
Briere F,
Banchereau J,
Liu YJ:
Generation of memory B cells and plasma cells in vitro.
Science
268:720,
1995[Abstract/Free Full Text]
39.
Tuscano JM,
Druey KM,
Riva A,
Pena J,
Thompson CB,
Kehrl JH:
Bcl-x rather than Bcl-2 mediates CD40-dependent centrocyte survival in the germinal center.
Blood
88:1359,
1996[Abstract/Free Full Text]
40.
Carbone A,
Gloghini A,
Gruss HJ,
Pinto A:
CD40 ligand is constitutively expressed in a subset of T cell lymphomas and on the microenvironmental reactive T cells of follicular lymphomas and Hodgkin's disease.
Am J Pathol
147:912,
1995[Abstract]
41.
Zelenetz AD,
Chen TT,
Levy R:
Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection.
J Exp Med
176:1137,
1992[Abstract/Free Full Text]
42.
Grillot DA,
Merino R,
Pena JC,
Fanslow WC,
Finkelman FD,
Thompson CB,
Nunez G:
bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice.
J Exp Med
183:381,
1996[Abstract/Free Full Text]
43.
Yin XM,
Oltval ZN,
Korsmeyer SJ:
BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax.
Nature
369:321,
1994[Medline]
[Order article via Infotrieve]
44.
Veis DJ,
Sorenson CM,
Shutter JR,
Korsmeyer SJ:
Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.
Cell
75:229,
1993[Medline]
[Order article via Infotrieve]
45.
Cheng EH,
Levine B,
Boise LH,
Thompson CB,
Hardwick JM:
Bax-independent inhibition of apoptosis by Bcl-XL.
Nature
379:554,
1996[Medline]
[Order article via Infotrieve]
46.
Broome HE,
Dargan CM,
Krajewski S,
Reed JC:
Expression of Bcl-2, Bcl-x, and Bax after T cell activation and IL-2 withdrawal.
J Immunol
155:2311,
1995[Abstract]
47.
Boyd JM,
Gallo GJ,
Elangovan B,
Houghton AB,
Malstrom S,
Avery BJ,
Ebb RG,
Subramanian T,
Chittenden T,
Lutz RJ,
Chinnadurai G:
Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins.
Oncogene
11:1921,
1995[Medline]
[Order article via Infotrieve]
48.
Han J,
Sabbatini P,
White E:
Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.
Mol Cell Biol
16:5857,
1996[Abstract]
49.
Smith KG,
Weiss U,
Rajewsky K,
Nossal GJ,
Tarlinton DM:
Bcl-2 increases memory B cell recruitment but does not perturb selection in germinal centers.
Immunity
1:803,
1994[Medline]
[Order article via Infotrieve]
50.
McDonnell TJ,
Korsmeyer SJ:
Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14;18).
Nature
349:254,
1991[Medline]
[Order article via Infotrieve]
51.
Limpens J,
de Jong D,
van Krieken JH,
Price CG,
Young BD,
van Ommen GJ,
Kluin PM:
Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia.
Oncogene
6:2271,
1991[Medline]
[Order article via Infotrieve]
52.
Limpens J,
Stad R,
Vos C,
de Vlaam C,
de Jong D,
van Ommen GJ,
Schuuring E,
Kluin PM:
Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals.
Blood
85:2528,
1995[Abstract/Free Full Text]
53.
Liu Y,
Hernandez AM,
Shibata D,
Cortopassi GA:
BCL2 translocation frequency rises with age in humans.
Proc Natl Acad Sci USA
91:8910,
1994[Abstract/Free Full Text]
54.
Cuende E,
Ales-Martinez JE,
Ding L,
Gonzalez-Garcia M,
Martinez C,
Nunez G:
Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells.
EMBO J
12:1555,
1993[Medline]
[Order article via Infotrieve]
55.
Wang H,
Grand RJ,
Milner AE,
Armitage RJ,
Gordon J,
Gregory CD:
Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: Relationship to the cell-cycle and role of the retinoblastoma protein.
Oncogene
13:373,
1996[Medline]
[Order article via Infotrieve]
56.
Liu YJ,
Joshua DE,
Williams GT,
Smith CA,
Gordon J,
MacLennan IC:
Mechanism of antigen-driven selection in germinal centres.
Nature
342:929,
1989[Medline]
[Order article via Infotrieve]
57.
Nakayama K,
Nakayama K,
Dustin LB,
Loh DY:
T-B cell interaction inhibits spontaneous apoptosis of mature lymphocytes in Bcl-2-deficient mice.
J Exp Med
182:1101,
1995[Abstract/Free Full Text]
58.
Marshall-Clarke S,
Owen G,
Tasker L:
Ligation of CD40 with soluble CD40 ligand reverses anti-immunoglobulin-mediated negative signalling in murine B lymphoma cell lines but not in immature B cells from neonatal mice.
Immunology
87:624,
1996[Medline]
[Order article via Infotrieve]
59.
Choi MS,
Boise LH,
Gottschalk AR,
Quintans J,
Thompson CB,
Klaus GG:
The role of bcl-XL in CD40-mediated rescue from anti-mu-induced apoptosis in WEHI-231 B lymphoma cells.
Eur J Immunol
25:1352,
1995[Medline]
[Order article via Infotrieve]
60.
Funakoshi S,
Longo DL,
Beckwith M,
Conley DK,
Tsarfaty G,
Tsarfaty I,
Armitage RJ,
Fanslow WC,
Spriggs MK,
Murphy WJ:
Inhibition of human B-cell lymphoma growth by CD40 stimulation.
Blood
83:2787,
1994[Abstract/Free Full Text]
61.
Petrasch S,
Kosco M,
Perez-Alvarez C,
Schmitz J,
Brittinger G:
Proliferation of non-Hodgkin-lymphoma lymphocytes in vitro is dependent upon follicular dendritic cell interactions.
Br J Haematol
80:21,
1992[Medline]
[Order article via Infotrieve]
62.
Lindhout E,
Mevissen ML,
Kwekkeboom J,
Tager JM,
de Groot C:
Direct evidence that human follicular dendritic cells (FDC) rescue germinal centre B cells from death by apoptosis.
Clin Exp Immunol
91:330,
1993[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
J. A. Burger, P. Ghia, A. Rosenwald, and F. Caligaris-Cappio
The microenvironment in mature B-cell malignancies: a target for new treatment strategies
Blood,
October 15, 2009;
114(16):
3367 - 3375.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-J. Goval, C. Thielen, C. Bourguignon, R. Greimers, E. Dejardin, Y. S. Choi, J. Boniver, and L. de Leval
The prevention of spontaneous apoptosis of follicular lymphoma B cells by a follicular dendritic cell line: involvement of caspase-3, caspase-8 and c-FLIP
Haematologica,
August 1, 2008;
93(8):
1169 - 1177.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Luqman, S. Klabunde, K. Lin, G. V. Georgakis, A. Cherukuri, J. Holash, C. Goldbeck, X. Xu, E. E. Kadel III, S. H. Lee, et al.
The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells
Blood,
August 1, 2008;
112(3):
711 - 720.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Travert, P. Ame-Thomas, C. Pangault, A. Morizot, O. Micheau, G. Semana, T. Lamy, T. Fest, K. Tarte, and T. Guillaudeux
CD40 Ligand Protects from TRAIL-Induced Apoptosis in Follicular Lymphomas through NF-{kappa}B Activation and Up-Regulation of c-FLIP and Bcl-xL
J. Immunol.,
July 15, 2008;
181(2):
1001 - 1011.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Quijano, A. Lopez, A. Rasillo, S. Barrena, M. Luz Sanchez, J. Flores, C. Fernandez, J. M. Sayagues, C. S. Osuna, N. Fernandez, et al.
Association between the proliferative rate of neoplastic B cells, their maturation stage, and underlying cytogenetic abnormalities in B-cell chronic lymphoproliferative disorders: analysis of a series of 432 patients
Blood,
May 15, 2008;
111(10):
5130 - 5141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. M. Barbe-Tuana, D. Klein, H. Ichii, D. M. Berman, L. Coffey, N. S. Kenyon, C. Ricordi, and R. L. Pastori
CD40-CD40 Ligand Interaction Activates Proinflammatory Pathways in Pancreatic Islets
Diabetes,
September 1, 2006;
55(9):
2437 - 2445.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Peeva, J. Gonzalez, R. Hicks, and B. Diamond
Cutting Edge: Lupus Susceptibility Interval Sle3/5 Confers Responsiveness to Prolactin in C57BL/6 Mice
J. Immunol.,
August 1, 2006;
177(3):
1401 - 1405.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Gururajan, R. Chui, A. K. Karuppannan, J. Ke, C. D. Jennings, and S. Bondada
c-Jun N-terminal kinase (JNK) is required for survival and proliferation of B-lymphoma cells
Blood,
August 15, 2005;
106(4):
1382 - 1391.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. G. Gribben
Death of follicular lymphoma cells--the long and the short of it
Blood,
January 15, 2004;
103(2):
374 - 374.
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-L. Zhao, M. E. Daneshpouy, N. Mounier, J. Briere, C. Leboeuf, L.-F. Plassa, E. Turpin, J.-M. Cayuela, J.-C. Ameisen, C. Gisselbrecht, et al.
Prognostic significance of bcl-xL gene expression and apoptotic cell counts in follicular lymphoma
Blood,
January 15, 2004;
103(2):
695 - 697.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Katz, C. Lord, C. A. Ford, S. B. Gauld, N. A. Carter, and M. M. Harnett
Bcl-xL antagonism of BCR-coupled mitochondrial phospholipase A2 signaling correlates with protection from apoptosis in WEHI-231 B cells
Blood,
January 1, 2004;
103(1):
168 - 176.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. W. C. J. van de Donk, D. Schotte, M. M. J. Kamphuis, A. M. W. van Marion, B. van Kessel, A. C. Bloem, and H. M. Lokhorst
Protein Geranylgeranylation Is Critical for the Regulation of Survival and Proliferation of Lymphoma Tumor Cells
Clin. Cancer Res.,
November 15, 2003;
9(15):
5735 - 5748.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Younes and M. E. Kadin
Emerging Applications of the Tumor Necrosis Factor Family of Ligands and Receptors in Cancer Therapy
J. Clin. Oncol.,
September 15, 2003;
21(18):
3526 - 3534.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Jinquan, H. H. Jacobi, C. Jing, A. Millner, E. Sten, L. Hviid, L. Anting, L. P. Ryder, C. Glue, P. S. Skov, et al.
CCR3 Expression Induced by IL-2 and IL-4 Functioning as a Death Receptor for B Cells
J. Immunol.,
August 15, 2003;
171(4):
1722 - 1731.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sanchez-Beato, A. Sanchez-Aguilera, and M. A. Piris
Cell cycle deregulation in B-cell lymphomas
Blood,
February 15, 2003;
101(4):
1220 - 1235.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Linderoth, M. Jerkeman, E. Cavallin-Stahl, S. Kvaloy, and E. Torlakovic
Immunohistochemical Expression of CD23 and CD40 May Identify Prognostically Favorable Subgroups of Diffuse Large B-cell Lymphoma: A Nordic Lymphoma Group Study
Clin. Cancer Res.,
February 1, 2003;
9(2):
722 - 728.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hase, Y. Kanno, H. Kojima, C. Morimoto, K. Okumura, and T. Kobata
CD27 and CD40 Inhibit p53-independent Mitochondrial Pathways in Apoptosis of B Cells Induced by B Cell Receptor Ligation
J. Biol. Chem.,
November 27, 2002;
277(49):
46950 - 46958.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. T. de la Fuente, B. Casanova, J. V. Moyano, M. Garcia-Gila, L. Sanz, J. Garcia-Marco, A. Silva, and A. Garcia-Pardo
Engagement of {alpha}4{beta}1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine
J. Leukoc. Biol.,
March 1, 2002;
71(3):
495 - 502.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Husson, E. G. Carideo, D. Neuberg, J. Schultze, O. Munoz, P. W. Marks, J. W. Donovan, A. C. Chillemi, P. O'Connell, and A. S. Freedman
Gene expression profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays
Blood,
January 1, 2002;
99(1):
282 - 289.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Klein, Y. Tu, G. A. Stolovitzky, M. Mattioli, G. Cattoretti, H. Husson, A. Freedman, G. Inghirami, L. Cro, L. Baldini, et al.
Gene Expression Profiling of B Cell Chronic Lymphocytic Leukemia Reveals a Homogeneous Phenotype Related to Memory B Cells
J. Exp. Med.,
December 3, 2001;
194(11):
1625 - 1638.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Z. Rassidakis, A. H. Sarris, M. Herling, R. J. Ford, F. Cabanillas, T. J. McDonnell, and L. J. Medeiros
Differential Expression of BCL-2 Family Proteins in ALK-Positive and ALK-Negative Anaplastic Large Cell Lymphoma of T/Null-Cell Lineage
Am. J. Pathol.,
August 1, 2001;
159(2):
527 - 535.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. Zhou, N. B. Levy, H. Xie, L. Qian, C.-Y. G. Lee, R. D. Gascoyne, and R. W. Craig
MCL1 transgenic mice exhibit a high incidence of B-cell lymphoma manifested as a spectrum of histologic subtypes
Blood,
June 15, 2001;
97(12):
3902 - 3909.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Hyjek, A. Chadburn, Y. F. Liu, E. Cesarman, and D. M. Knowles
BCL-6 protein is expressed in precursor T-cell lymphoblastic lymphoma and in prenatal and postnatal thymus
Blood,
January 1, 2001;
97(1):
270 - 276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Corcione, L. Ottonello, G. Tortolina, P. Facchetti, I. Airoldi, R. Guglielmino, P. Dadati, M. Truini, S. Sozzani, F. Dallegri, et al.
Stromal Cell-Derived Factor-1 as a Chemoattractant for Follicular Center Lymphoma B Cells
J Natl Cancer Inst,
April 19, 2000;
92(8):
628 - 635.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. C.T. Aguiar, Y. Yakushijin, S. Kharbanda, S. Tiwari, G. J. Freeman, and M. A. Shipp
PTPROt: An Alternatively Spliced and Developmentally Regulated B-Lymphoid Phosphatase That Promotes G0/G1 Arrest
Blood,
October 1, 1999;
94(7):
2403 - 2413.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. G. Wickremasinghe and A. V. Hoffbrand
Biochemical and Genetic Control of Apoptosis: Relevance to Normal Hematopoiesis and Hematological Malignancies
Blood,
June 1, 1999;
93(11):
3587 - 3600.
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Voorzanger-Rousselot, M.-C. Favrot, and J.-Y. Blay
Resistance to Cytotoxic Chemotherapy Induced by CD40 Ligand in Lymphoma Cells
Blood,
November 1, 1998;
92(9):
3381 - 3387.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract