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Tumor Biology |
University Medical Center Charité, Humboldt University, Robert-Rössle-Klinik, Department of Hematology, Oncology, and Tumor Immunology [S. M., B. D., M. Y. M.], and Max-Delbrück-Center for Molecular Medicine [A. R., K. B., B. D.], D-13125 Berlin, Germany
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ABSTRACT |
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INTRODUCTION |
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1 and PLC-2 have been described previously
(7)
. Although these events after CD20 ligation are well
characterized, further downstream events in the CD20 signaling cascade
are not known. Due to the broad expression pattern of the CD20 antigen throughout B-cell differentiation, expression on more than 90% of B-cell non-Hodgkin-lymphomas (8) , and lack of antigen shedding (9) , CD20 appeared to be an ideal target for immunotherapeutic approaches in the treatment of B-cell malignancies. Thus, a variety of antibodies have been used in clinical trials as naive antibodies (9) or radioimmunoconjugates (10) . The mouse-human chimeric antibody IDEC-C2B8 has recently been approved for clinical application (11 , 12) . This antibody recognizes the B1 epitope of the CD20 molecule. Thus far, two major effector mechanisms are hypothesized, CDC and ADCC (12) ; however, the role of CDC and ADCC for in vivo tumor regression has not been clearly shown. In patients treated with an i.v. infusion of IDEC-C2B8, a dose-dependent, rapid depletion of B cells could be observed (11) . Although early B-cell depletion is observed in nearly all patients, only 40–60% show a clinical response of longer duration (13) . In addition, a significant number of patients will have progressive disease during antibody therapy (14) . This observation raised questions concerning the molecular and cellular mechanisms underlying tumor regression and the development of resistance to anti-CD20 treatment, underscoring the necessity to understand signaling events occurring after CD20 ligation as well as the function of the molecule. Because it had been reported that murine anti-CD20 antibodies can induce apoptosis after extensive cross-linking (15) , we were interested in investigating the capacity of IDEC-C2B8 to induce apoptosis and the molecular mechanisms underlying this process. For this purpose, we took advantage of B-cell lymphoma models reflecting different stages of lymphomagenesis: induction of apoptosis was studied in human Burkitt’s lymphoma cell lines, which are sIgM positive and reflect a more immature stage of differentiation. In addition, we analyzed more mature B-cell lymphoma cell lines carrying the t(14;18) translocation (16 , 17) , which is typical for follicular lymphoma, one of the main clinical targets of anti-CD20 treatment.
Apoptosis is a tightly regulated process of cell death, which is
controlled by the interplay of a number of positive and negative
regulatory factors including members of the Bcl-2 gene family
(18)
. The execution of the death pathway is mediated by
activated caspases (cysteinyl aspartate-specific proteinases; Ref.
19
). Previous findings concerning B-cell apoptosis result
from studies investigating the BCR-mediated cell death of B-cell lines.
We and others have shown the involvement of Bcl-2 family members
Bax-
and Bcl-xL and effector caspase-3 after
BCR-mediated cell death (20, 21, 22)
. The cleavage of specific
caspase target proteins like D4-GDI and transcription factor SP-1 was
identified as a result of caspase-3-like activity (22)
.
Another group of enzymes involved in mediating mitogenic or
apoptotic signals is the MAPKs (for a recent summary, see Ref.
23
). All three types of MAPKs, ERKs [p44 (ERK1) and p42
(ERK2)], c-Jun NH2-terminal kinases
(stress-activated PKs), and p38, modulate a distinct set of
transcription factors and have been proposed to modulate BCR signaling.
MAPK activity in B lymphocytes is shown for activation of different
receptors as CD19, CD22, or CD40 (24, 25, 26)
. However, the
role of MAPKs in the signaling processes of B cells is not yet clear,
but activation of ERKs might be required for induction of BCR-mediated
apoptosis, whereas stress-activated PK activity might mediate mitogenic
signals (27)
.
We report that extensively cross-linked anti-CD20 antibody IDEC-C2B8 is
a potent inducer of apoptosis in both of the lymphoma cell line models
and that, among sIgM-positive Burkitt’s lymphoma cell lines, only cell
lines susceptible to BCR-mediated apoptosis were sensitive to
anti-CD20-mediated apoptosis. Investigation of signaling events after
antigen cross-linking further sustained these observations, revealing
that both anti-CD20- and BCR-mediated apoptosis are dependent on
caspase-3 and involve similar responses of the ERGs Berg36 and c-myc
and up-regulation of bax- as well as MAPK and AP-1 activation.
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MATERIALS AND METHODS |
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-thioglycerol, 100 units/ml
penicillin, and 100 µg/ml streptomycin. All supplements were
purchased from Life Technologies, Inc. (Karlsruhe, Germany).
Antibodies.
Anti-CD20-mediated apoptosis was induced using the following
antibodies: (a) anti-CD20 mAb IDEC-C2B8 (generously provided
by Dr. U. Riedel; Hoffmann-LaRoche, Freiburg, Germany); and
(b) anti-CD20 antibody B9E9 (Coulter Immunotech, Hamburg,
Germany). Cross-linking of soluble anti-CD20 antibody B9E9 was achieved
using goat antimouse IgG F(ab')2 antibody
fragments (GAM; Dianova, Hamburg, Germany). BCR-mediated apoptosis was
induced as described previously using goat antihuman IgM
F(ab')2 antibody fragments (Dianova; Ref.
28
). The murine IgG1 antibody HD20 (kindly provided by
Gerhard Moldenhauer; Deutsches Krebsforschungszentrum, Heidelberg,
Germany) as well as a human anti-EpCAM IgG1 antibody (kindly
provided by Torsten Dreier, Micromet GmbH, Martinsried, Germany) served
as a control. All antibodies used in cell culture experiments were
devoid of NaN3.
[3
H]Thymidine Incorporation.
Proliferation of cells in the absence or presence of immobilized or
soluble anti-CD20 and control antibodies was determined by the use of
[3
H]thymidine incorporation. Ten thousand cells
were incubated in 200 µl of culture medium in 96-well flat-bottomed
microtiter plates (Falcon; Becton Dickinson, Heidelberg, Germany). One
µCi of [3
H]thymidine (Amersham, Braunschweig,
Germany) per well was added, and cell cultures were continued for an
additional 20 h. The cells were harvested onto glass filters
(UniFilterTM-96, GF/C; Packard, Dreieich, Germany), and the
incorporated radioactivity was measured using TopCount Counter
(Canberra Packard TFM, Dreieich, Germany). All assays were performed in
triplicate. Statistical analysis was performed using Student’s
t test.
Immunofluorescence and Flow Cytometry.
Surface antigen expression was analyzed by flow cytometry using
standard protocols. Cells were preincubated with 1% BSA (fraction V;
Roth, Karlsruhe, Germany) and 1% human IgG (Venimmun; Cention,
Marburg, Germany) in PBS for 20 min. Direct immunofluorescence staining
was performed using FITC-conjugated mAbs against CD20 (Becton
Dickinson). sIgM expression was detected indirectly by an anti-IgM
antibody (Becton Dickinson) and a FITC-conjugated goat antimouse IgG
antibody (Jackson ImmunoResearch; Dianova). Cells were washed twice and
resuspended in 1x PBS containing 1% BSA and 2 µg/ml propidium
iodide (Sigma, Deisenhofen, Germany) for gating on viable cells.
Immunofluorescence was determined using a FACScan flow cytometer and
CELLQuest software (Becton Dickinson). Quantification of surface
antigen expression was performed by determining the MFI.
Induction and Analysis of Apoptosis.
Anti-CD20-mediated apoptosis was induced by two different methods.
First, mAb IDEC-C2B8 was immobilized by coating the antibody (20
µg/ml PBS) on non-tissue culture-treated 6-well plates
(Falcon; Becton Dickinson) overnight at 4°C. For induction of
apoptosis, the cell lines were incubated in these pretreated plates.
Second, cells were incubated with 10 µg/ml anti-CD20 antibody B9E9
for 30 min; subsequently, anti-CD20 antibody was cross-linked by 20
µg/ml goat antimouse (GAM) antibody F(ab')2
fragments. Control reactions were performed using the murine IgG1
antibody HD20 and a human anti-EpCAM IgG1 antibody. BCR-mediated
apoptosis was induced with 1.3 µg/ml goat antihuman IgM
F(ab')2 fragments. After induction of apoptosis,
cells were stained with acridine orange (5 µg/ml; Sigma) and observed
by fluorescence microscopy. The number of cells revealing
characteristic features of apoptosis (fragmented or condensed nuclei)
were enumerated, and the percentage was determined per total of 100
cells. All assays were performed in triplicate. Statistical analysis
was performed using Student’s t test.
Measurement and Inhibition of Caspase Activity.
Caspase-3 activation was detected by Western blot analysis (see below).
Caspase-3 family activity was measured using the ApoAlert CPP32 Assay
Kit (Clontech, Heidelberg, Germany). After the CD20 antigen was
triggered, 1 x 106 cells were
washed once in 1x PBS and resuspended in 50 µl of chilled cell lysis
buffer. Thereafter, cells were incubated for 10 min on ice, followed by
the addition of 50 µl of 2x reaction buffer containing the caspase
substrate DEVD-p-NA to a final concentration of 50
µM. On proteolytic cleavage of DEVD-p-NA by caspase
activity, free p-NA was detected in a spectrophotometer at 400 nm.
Caspase activity was blocked using the cell-permeable irreversible caspase-3 family inhibitor z-DEVD-fmk (Calbiochem, Bad Soden, Germany) or the caspase-1 inhibitor ac-YVAD-cmk (Calbiochem). Samples were preincubated for 30 min using different concentrations of the caspase inhibitor. After this, apoptosis was induced, and cells were analyzed at 12 and 20 h as described above. Assays were performed in triplicate. Statistical analysis was performed using Student’s t test.
Protein Preparation and Immunoblotting.
Cells from different time points after the induction of apoptosis were
washed once in 1x PBS. Adherent cells were lysed directly on the cell
culture plate. Cells were lysed for 30 min at 4°C [using lysis
buffer containing 20 mM Tris-acetate (pH 7.4), 10
mM sodium glycerophosphate, 50 mM sodium
fluoride, 5 mM Na PPi, 1% Triton
X-100, 0.1 mM EDTA, 1 mM EGTA, and 0.2
mM PMSF] or for 5 min at 80°C [using lysis buffer
containing 1% SDS, 10 mM Tris-HCL (pH 7.5), and 2
mM EDTA (pH 8.0)]. Samples were then centrifuged (14,000
rpm, 3 min). The amount of protein was measured by quantitative protein
assay (Pierce, Rockford, IL). Protein samples (35 µg/lane) were
subjected to 10% or 12% SDS-PAGE and transferred onto nitrocellulose
filters (Schleicher and Schuell, Dassel, Germany). After Ponceau S
staining, the filters were blocked [1% nonfat dry milk, 0.1% Triton
X-100, 150 mM NaCl, 50 mM Tris (pH 7.5)] and
incubated with 1:1000 diluted primary antibodies [polyclonal anti-PARP
(N-20), polyclonal anti-Ly-GDI (C-20), polyclonal anti-SP1 (PEP 2),
polyclonal anti-Bcl-2 (
C-21), polyclonal
anti-Bcl-xL/S (S-18), polyclonal anti-Bad (N-20),
and polyclonal anti-Bax (P19) (all from Santa Cruz, Heidelberg,
Germany); polyclonal anti-caspase-3 (PharMingen, Hamburg, Germany);
polyclonal anti-phospho-c-Jun (Ser63), polyclonal
anti-phospho-p38 MAPK
(Thr180/Tyr182), and
monoclonal anti-phospho-p44/p42 MAPK
(Thr202/Tyr204; all from
New England Biolabs, Beverly, MA)]. Thereafter, filters were incubated
with horseradish peroxidase-conjugated secondary antibodies (dilution,
1:15000; Santa Cruz). Bands were visualized using the enhanced
chemiluminescence system (Amersham).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay.
Nuclear extracts were prepared as described previously
(29)
. Briefly, 1 x 106 cells were collected, washed with 10 ml of
PBS, and pelleted by centrifugation. PBS was removed, and the cell
pellet was resuspended in 400 µl of cold buffer A [10 mM
HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1
mM EGTA, 1 mM DTT, and 0.5 mM
PMSF]. The cells were allowed to swell on ice for 15 min, after which
5 µl of a 10% solution of NP40 (Boehringer, Mannheim,
Germany) were added, and the tube was vortexed for 10 s.
The homogenate was centrifuged for 30 s in a microfuge. The
nuclear pellet was resuspended in 50 µl of ice-cold buffer C [20
mM HEPES (pH 7.9), 0.4 M NaCl, 25% glycerol, 1
mM EDTA, 1 mM EGTA, 1 mM DTT, and 1
mM PMSF], and the tube was rocked at 4°C for 15 min. The
nuclear extract was centrifuged for 5 min in a microfuge at 4°C, and
the supernatant was frozen at -70°C. For electrophoretic mobility
shift analysis, 5 µg of nuclear protein were incubated in 10
mM Tris (pH 7.5), 50 mM NaCl, 5 mM
MgCl2, 10 mM DTT, 1 mM
EDTA, 5% glycerol, 1 µg of BSA, and 1 µg of poly(dI-dC)
with 20,000 cpm of radioactive 32P-labeled probe
for 15 min at room temperature, followed by separation of the
DNA-protein complexes on 4% acrylamide gels. For supershift analysis,
1 µg of antibody [c-Fos, c-Jun, and (c-Jun, JunB, and JunD)-specific
antibody; all from Santa Cruz] was added to the nuclear extract and
incubated for 15 min at 4°C before the addition of reaction buffer
and labeled probe. The following double-stranded oligonucleotide was
used as a probe: 5'-AGCTAGCATGAGTCAGACAC-3' and
5'-AGCTGTGTCTGACTCATGCT-3' (human Col-AP-1; Ref. 30
). A
double-stranded oligonucleotide with a mutated AP-1 consensus binding
site served as a control.
Northern Blot Analysis.
Total RNA was prepared using guanidinium isothiocyanate lysis
and CsCl gradient centrifugation. Cells were lysed in a 4 M
guanidinium solution [4 M guanidinium isothiocyanate, 20
mM sodium acetate (pH 5.2), 0.1 mM DTT, and
0.5% N-lauryl-sarcosine]. The viscosity of the solution
was reduced by drawing the lysate 10 times through a 20-gauge needle.
The cell lysate was layered on top of a 5.7
M CsCl cushion [5.7 M CsCl
and 100 mM EDTA (pH 8.0)]. Thereafter, the tube
was centrifuged for 18–22 h in an ultracentrifuge (Beckmann L-60;
Beckmann, München, Germany) at 31,000 rpm (150,000 x g). After centrifugation, the supernatant was
removed carefully, and the RNA pellet was dissolved, ethanol
precipitated, and quantified spectrophotometrically. For Northern
analysis, 10 µg of total RNA were subjected to gel electrophoresis on
a 1.1% formaldehyde-1.2% agarose gel and transferred to a nylon
membrane (Appligene, Heidelberg, Germany). After UV cross-linking, the
membrane was prehybridized (ExpressHyb solution; Clontech) at 68°C
for 30 min. The blots were hybridized with
[-32P]dCTP-labeled random prime-labeled DNA
probes (c-myc, Berg36, and glyceraldehyde-3-phosphate dehydrogenase)
overnight at 68°C. Membranes were washed for 40 min at room
temperature in 2x SSC and 0.05% SDS and then washed for 40 min at
50°C in 0.5% SSC and 0.1% SDS.
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RESULTS |
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shows the time course of
the antiproliferative effect of IDEC-C2B8. In comparison with the
medium control and control antibodies, CD20-dependent inhibition of
proliferation could be detected as soon as 2 h after
incubation. Maximal inhibition (80% relative to the medium
control; P < 0.00008) was reached after
10–12 h of incubation. This contrasts with the soluble antibody (5–25
µg/ml), which showed only a slight decrease of proliferation (data
not shown).
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. After 24 h, more
than 70% of the cells demonstrated fragmented nuclei (IDEC-C2B8
versus HD20, P < 0.00002).
Similar results were generated by cross-linking of the mouse anti-CD20
antibody B9E9 on BL41-s cells using goat antimouse
F(ab')2 fragments and using immobilized mouse
monoclonal anti-CD20 antibody B9E9 (data not shown). Again, only a weak
increase with 15–20% apoptotic cells cultured in the presence of
soluble anti-CD20 mAb (10 µg/ml) was detectable; the control
antibodies HD20 and human anti-EpCAM IgG1 had no effect (data not
shown). Furthermore, as a model for follicular lymphoma, the two
t(14;18)-positive lymphoma cell lines, SU-DHL-4 and K422, were
investigated. Both cell lines were sensitive to soluble and extensively
cross-linked anti-CD20 antibody, with 20–35% apoptotic cells after
incubation on immobilized IDEC-C2B8 for 20 h (Fig. 1B
;
IDEC-C2B8 versus HD20, P < 0.0003; data not shown).
Increase of the Bcl-2 Gene Family Member Bax during
Anti-CD20-mediated Apoptosis.
Recently, it was reported that extensively cross-linked murine
anti-CD20 mAbs are able to induce B-cell apoptosis (15)
.
However, the mechanisms leading to apoptosis by these antibodies are
not known. Because members of the Bcl-2 family have been shown to be
involved in different processes of B-cell apoptosis, we studied the
protein expression of the proapoptotic members Bax and Bad and the
antiapoptotic members Bcl-2 and Bcl-xL after
induction of anti-CD20-mediated apoptosis. The investigated BL60-2
cells demonstrated significant Bax up-regulation after 2 h of
stimulation, followed by a decrease in protein expression level after
6–8 h (Fig. 2A)
. No
significant changes of the other Bcl-2 family members investigated were
observed.
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. The
detection of the larger SP1 cleavage fragment of
Mr 68,000 was possible as early as
4–6 h after induction of apoptosis. Six h after CD20 triggering by
immobilized IDEC-C2B8, PARP degradation was detectable. Fig. 2B
shows the cleavage of PARP using an antibody recognizing
the Mr 24,000 cleavage product. No
cleavage products were detectable after incubation of the cells with
the control antibodies.
CD20-induced Apoptosis Activates Caspase-3.
The results mentioned above indicated the involvement of caspase-3
family in anti-CD20-induced apoptosis. Indeed, activation of caspase-3
was detectable in the analyzed cell lines BL60-2 and SU-DHL-4 by
Western blot analysis after extensive cross-linking of IDEC-C2B8.
Cleavage of the Mr 32,000 procaspase-3
generated the p24 intermediate autocatalytically cleaved to active
caspase-3 (Fig. 2B)
. Measurement of caspase-3 family
activity by analysis of cleavage of the caspase-substrate DEVD-p-NA
underlined the observed time course of activation (data not shown).
Furthermore, inhibition of caspase-3 by the cell-permeable irreversible
caspase-3 family inhibitor z-DEVD-fmk inhibited apoptosis in a
dose-dependent manner, with almost complete inhibition at 200
µM (Fig. 2C
; P < 0.0001). In contrast to these results, the interleukin
1ß-converting enzyme inhibitor ac-YVAD-fmk did not block apoptosis
(data not shown), suggesting that caspase-1 is not involved in
anti-CD20-mediated apoptosis.
Correlation between Anti-CD20- and BCR-mediated Apoptosis.
In all experiments studying CD20-dependent cell death in Burkitt’s
lymphoma cell lines, we used BCR-mediated apoptosis as a positive
control for apoptosis sensitivity of the investigated cell lines.
Surprisingly, we observed a strict correlation between anti-CD20- and
BCR-mediated apoptosis in the investigated Burkitt’s lymphoma
cell lines. This led us to perform experiments comparing BCR- and
CD20-mediated induction of apoptosis. CD20-dependent apoptosis occurred
in the BCR-sensitive Burkitt’s lymphoma cell lines BL60-2 and BL41-s,
but not in the resistant variant BL41-r. Cells resistant to
BCR-mediated apoptosis were clearly resistant to CD20-mediated
apoptosis. There was no correlation between antigen expression level
and sensitivity or resistance to the apoptotic stimuli. The results are
summarized in Table 1
; the number of
apoptotic cells after anti-CD20- and anti-BCR-mediated apoptosis and
the MFI of CD20 and IgM expression are indicated.
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. In contrast to these changes observed
after extensive cross-linking of CD20 antigen, the c-myc expression
level remained nearly unaltered, and Berg36 mRNA showed only a weak
increase in the presence of soluble IDEC-C2B8 (10 µg/ml; Fig. 3C
). These data support our observation of a correlation
between BCR- and anti-CD20-mediated apoptosis in sIgM-positive cell
lines.
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are not known. In contrast, BCR activation is known to
induce functional relevant PLC--dependent p44/p42 MAPK-activation
(25)
and increased AP-1 activity (33)
.
Therefore, we focused on comparison of MAPK and AP-1 activation after
CD20 and BCR triggering. After culturing BL60-2 cells on immobilized
IDEC-C2B8, a strong induction of p44/p42 phosphorylation, which
correlates with induction of activity, was detectable using an
anti-phospho-p44/p42 MAPK mAb. Comparable with p44/p42 MAPK induction
after BCR ligation, the phosphorylation of p42 (ERK2) was more
pronounced, as described previously for BCR-mediated apoptosis
(Fig. 4A
; Ref.
25
). Furthermore, soluble anti-CD20 antibody also showed a
weak induction of p44/p42 phosphorylation. Modulation of p44/p42
activity might therefore not only be induced by extensive CD20
cross-linking. The cell lines BJAB and Daudi, which are both resistant
to anti-CD20- and BCR-mediated apoptosis, showed different levels of
basal phosphorylated p44/p42 MAPK that remained unaltered on extensive
CD20 or BCR cross-linking (Fig. 4A)
. Because no changes in
c-jun or p38 MAPK phosphorylation were detected by use of
phospho-specific antibodies (data not shown), these MAPKs might not be
involved in the signaling events leading to the induction of apoptosis
after CD20 or sIgM triggering. In addition, the sIgM-negative
t(14;18)-positive cell line SU-DHL-4 also revealed p44/42 activation
following CD20 triggering, suggesting that p44/42 activity can be
regulated by CD20 in the absence of the sIgM (Fig. 4A)
.
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. This increase was more pronounced after
BCR triggering. A supershift analysis revealed predominantly fos and,
to a weaker extent, junB and junD but almost no c-jun as components of
the AP-1 complex (Fig. 4C)
. Again, soluble anti-CD20
antibody was also able to induce AP-1 DNA binding activity, although to
a much lesser degree in comparison with the extensively cross-linked
antibody (Fig. 4B)
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DISCUSSION |
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It was reported that mAbs directed against B-cell surface receptors
with strong signaling activity such as CD19 were the most effective
antibodies in an in vivo B-cell lymphoma therapy model
(34)
. These results suggested that the signaling activity
of the antigen might be responsible for lymphoma inhibition rather than
effector cell recruitment. Indeed, antiproliferative effects were also
reported for IDEC-C2B8 (35)
. In contrast, recently
published data show the importance of FcR-positive cells for
antibody-mediated antitumor activity (36)
. The mechanisms
of antitumor activity of IDEC-C2B8 are not clear; therefore, it was our
aim to study effects of anti-CD20 mAb IDEC-C2B8 on human lymphoma cell
lines.
Immobilized or extensively cross-linked anti-CD20 antibody IDEC-C2B8
resulted in potent induction of apoptosis in both of the B-cell
lymphoma cell line models investigated. Thus induction of apoptosis was
observed in the t(14;18)-positive lymphoma cell lines SU-DHL-4 and K422
as well as in the Burkitt’s lymphoma cell lines BL60-2 and BL41 (Fig. 1B)
. In contrast, soluble antibody led only to a weak
induction of apoptosis. In vivo cross-linking of anti-CD20
mAb might be achieved through FcR-positive cells (15)
.
It has been demonstrated recently that BALB/c nude mice bearing a
xenotransplanted human Burkitt’s lymphoma could be cured by IDEC-C2B8
application, whereas nearly no tumor regression could be observed in
FcR-/- nu/nu mice (36)
. Our findings might
reflect this observation. In our experimental system, extensive
cross-linking of the antibody is leading to strong induction of
apoptosis in the absence of effector cells. Because extensive antibody
cross-linking is shown to enforce the signaling activity of the
recognized antigen (see above, compare soluble and extensively
cross-linked antibody; Ref. 37
), this mechanism might
occur in the presence of FcR-positive cells through Fc-FcR
interaction in addition to effector cell-related mechanisms.
Postulating that induction of apoptosis contributes to the prolonged
remission of lymphomas after treatment with IDEC-C2B8, we investigated
key regulators of programmed cell death. Rapid induction of apoptosis
could be observed after extensive CD20 cross-linking with a significant
increase in Bax protein expression (Fig. 2A)
. Furthermore,
CD20-dependent apoptosis involved activation of caspase-3 activity,
resulting in cleavage of the caspase-3 family target proteins PARP and
SP1 (22
, 38)
, and could be completely abrogated by
inhibitors of caspase 3 (Fig. 2, B and C)
.
During BCR-triggered apoptosis, constitutively expressed Bcl-2 and
Bcl-xL protein levels remained unaltered, whereas
a strong induction of Bax- was detectable in the human Burkitt’s
lymphoma cell line BL41 (39)
. Furthermore, overexpression
of Bax- sensitized BL41 cells to BCR-mediated apoptosis
(21)
. A similar pattern of Bcl-2 family gene expression is
described in this work for anti-CD20-mediated apoptosis. This suggests
that, as in BCR-mediated apoptosis, up-regulation of Bax might be an
early key event in CD20-mediated apoptosis.
Dysregulated expression of Bcl-2 gene family members has been reported in different malignancies and is believed to contribute to the pathogenesis of these tumors and to influence the response to treatment (40, 41, 42) . It is therefore conceivable that dysregulated Bcl-2 family protein expression in lymphomas (43) , resulting in the inability to undergo apoptosis on certain stimuli, might contribute to resistance to IDEC-C2B8. The slower onset of apoptosis induction in the t(14;18)-positive cell lines compared with the Burkitt’s lymphoma cell lines could be explained by the high level of bcl-2 expression detectable in t(14;18)-positive cell lines.
One of the most interesting findings in our study is the observation
that there is a tight correlation between anti-CD20- and BCR-mediated
apoptosis (Table 1)
with regard to the sensitivity of the Burkitt’s
lymphoma cell lines studied. Both t(14;18)-positive lymphoma cell
lines, which showed no sIgM expression, were sensitive to
anti-CD20-mediated apoptosis. Among the Burkitt’s lymphoma cell lines,
all of which showed sIgM expression, only cell lines sensitive to
BCR-mediated apoptosis were sensitive to anti-CD20-mediated apoptosis.
Recently, it has been suggested that PTKs are involved in
anti-CD20-mediated apoptosis (15)
. CD20 is associated with
the Src family PTKs Lyn, Fyn, and Lck (6
, 44)
, which are
also activated after BCR activation (45)
. Furthermore,
CD20 signaling appeared to involve, in part, phosphorylation substrates
similar to those observed after sIgM stimulation (7)
.
These data and the observation of induction of anti-CD20-mediated
apoptosis in Burkitt’s lymphoma cells susceptible to sIgM-mediated
apoptosis could be interpreted as the result of similar signaling
events after both stimuli. To support this hypothesis, we compared the
changes of two ERGs, c-myc and Berg36, in Northern blot analysis (Fig. 3)
. Down-regulation of c-myc has been shown to be critically involved
in the process of BCR-mediated apoptosis (31)
, and an
increase of Berg36 RNA has been shown to occur after calcium
ionophore-induced apoptosis in Burkitt’s lymphoma cell lines
(32)
. Similar kinetics were seen after sIgM induction and
extensive CD20 cross-linking with a strong induction of Berg36 and a
decrease of c-myc. The down-regulation of c-myc RNA does not differ
from previously published results showing c-myc mRNA induction after
anti-CD20 stimulation (46)
because these data were
generated using antibody 1F5, which is, in contrast to all known
anti-B1 anti-CD20 antibodies (including IDEC-C2B8), able to activate B
cells (3
, 4)
.
Furthermore, BCR- and CD20-mediated signaling was able to induce
activation of the MAPKs p44/p42 (Fig. 4)
. MAPK p44/p42 activity was
shown to mediate BCR-induced apoptosis in WEHI 231 B cells
(25)
. Activation of the p44/p42 MAPKs by the BCR is
dependent on activation of PLC-2 (47)
. CD20- and
BCR-initiated signaling activate PLC-1 and PLC-2 (7
, 48)
, which might explain the similar pattern of MAPK activation.
Because activation of PLC-2 after BCR signaling requires Syk
activity (49)
, and this PTK is not directly associated
with the CD20 molecule (6)
, anti-CD20-mediated PLC-2
activation might differ from that observed after BCR signaling.
Furthermore, it was shown that BCR-mediated ERK activation involves the
RAS/Raf-1/MAPK/ERK kinase signaling pathway (50
, 51)
. It
will be important to determine the function of this signaling pathway
after CD20 ligation. The similar composition of the induced AP-1
complex after CD20 and BCR ligation (Fig. 4, B and C)
underlines the similarity of the signaling pathways after
extensive CD20 and BCR ligation. As described previously for
BCR-mediated apoptosis, this complex is predominantly composed of c-fos
(33)
. Nevertheless, in the more mature t(14;18)-positive
B-cell lymphoma cell lines, anti-CD20-mediated apoptosis occurs
independent of the presence of sIgM-associated signaling events.
Possibly, only a subgroup of the more immature lymphoma cells is
sensitive to anti-CD20-mediated apoptosis, whereas sensitivity in
the more mature t(14;18)-positive B-cell lymphoma cells is a
more general phenomenon.
We propose that IDEC-C2B8 treatment failure might thus be explained not only by lack or modulation of CD20 antigen expression but also by dysregulated signaling and execution pathways of apoptosis. The involvement of p44/p42 MAPK and AP-1 in CD20 signaling may contribute to new insights in the signaling cascade after CD20 ligation, and it will be interesting to further investigate a functional relationship between CD20- and BCR-mediated signaling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Deutsche Forschungsgemeinschaft
Grant Ma-1664/1-3 and Verbund Klinisch-Biomedizinische Forschung Grant
KBF 01 GB 9609/4. 
2 To whom requests for reprints should be
addressed, at Max-Delbrück-Center for Molecular Medicine, FG
Dörken, D-13125 Berlin, Germany. Phone: 4930-94062720; Fax:
4930-94063124.
3 The abbreviations used are: BCR, B-cell
receptor; mAb, monoclonal antibody; sIgM, surface IgM; MAPK,
mitogen-activated protein kinase; AP-1, activator protein 1; ERK,
extracellular signal-regulated kinase; PLC, phospholipase C; CDC,
complement-dependent cytotoxicity; ADCC, antibody-dependent cellular
cytotoxicity; MFI, mean fluorescence intensity; p-NA,
p-nitroanilide; PMSF, phenylmethylsulfonyl fluoride;
PARP, poly(ADP-ribose) polymerase; ERG, early response gene; PK,
protein kinase; EpCAM, epithelial cell adhesion molecule; z-DEVD-fmk,
z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH2 F; FcR, Fc
receptor; ss, supershift; PTK, protein tyrosine kinase.
Received 12/29/99. Accepted 10/17/00.
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). As a
control, BL60-2 cells were incubated on uncoated plates (
), and the
plates were coated with the murine IgG1 antibody HD20 (
) and a human
anti-EpCAM IgG1 antibody (
; hum. IgG1;
all 20 µg/ml). After 2, 10, and 20 h, 1 µCi of
[3H]thymidine was added per well, and the culture was
continued for an additional 20 h. Results are the mean of
triplicate measurements; error bars denote SDs.
B, left panel, BL60-2 cells were
incubated for 2 (
), 8 (
), and 20 (
