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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3381-3387
Resistance to Cytotoxic Chemotherapy Induced by CD40 Ligand in
Lymphoma Cells
By
Nathalie Voorzanger-Rousselot,
M.-C. Favrot, and
Jean-Yves Blay
From Unité Cytokine et Cancer, Unité INSERM U453, Centre
Léon Bérard, Lyon, France.
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ABSTRACT |
The modulation of the cytotoxic effects of an anthracyclin by CD40L
was investigated in five non-Hodgkin's lymphoma (NHL) cell lines
(Daudi, Raji, BJAB, BL36, BL70). Incubation with doxorubicin (DOX)
increased in a dose-dependent manner the percentage of apoptosis in NHL
cells. Coculture with irradiated L cells expressing CD40L (CD40L L
cells), but not CDw32 (CDw32 L cells), significantly reduced (33% to
89%) the percentage of apoptosis in all five cell lines treated with
0.1 to 0.5 µg/mL of DOX, but in only three cell lines at 1 µg/mL.
Interleukin-10 (IL-10), IL-6, IL-2, or tumor necrosis factor (TNF)
induced no additive protective effects with CD40L L cells. In all five
cell lines, DOX induced a concentration-dependent increase of the
activity of the cysteine-protease caspase 3. Coculture with CD40L L
cells, but not with CDw32 L cells, inhibited (38% to 100%) the
activation of caspase 3 induced by 0.1 to 0.5 µg/mL of DOX in all
five NHL cell lines, but in only two cell lines at 1 µg/mL. Finally,
the antiproliferative effect of 0.1 to 0.5 µg/mL concentrations of
DOX was also partially abrogated on coculture with CD40L L cells in all
five cell lines, but in only two cell lines at 1 µg/mL. Cytokines,
either alone or in combination with CD40L L cells, did not affect
DOX-induced inhibition of proliferation. These results indicate that
CD40L inhibits the apoptosis and antiproliferative effect induced by
DOX and interferes with caspase 3 activation in B NHL cell lines.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ALTHOUGH NON-HODGKIN'S lymphoma (NHL)
cells are sensitive to cytotoxic chemotherapy, primary or secondary
chemoresistance frequently occurs and is the major cause of death of
these patients.1,2 The biological mechanisms by which
lymphoma cells acquire resistance to cytotoxic drugs are not fully
understood.3 Although markers associated with the
multi-drug resistance (MDR) phenotype, eg, P-glycoprotein
(P-gp), are likely to play a role in this phenomenon, in vivo,
chemoresistance of NHL is not consistently associated with P-gp
expression indicating the existence of additional
mechanisms.3
Apoptosis, or programmed cell death, is an active phenomenon dependent
on RNA and protein synthesis, which plays an essential role in many
normal processes, such as embryological development and immune cell
selection.4-8 Apoptosis can be induced by ionizing radiation in cancer treatment, engagement of cell surface molecules such as Fas and tumor necrosis factor (TNF)-R, or deprivement in growth
factors.7,9-12 Several cytotoxic agents act by inducing the
apoptosis of tumoral cells.13-17 It has recently been shown that apoptosis caused by doxorubicin (DOX) in a human T-leukemia cell
line is mediated via CD95L induction and subsequent activation of the
CD95 pathway.17
CD40, a member of the TNF receptor superfamily, is expressed on B
lymphocytes and interacts with a ligand (CD40L) expressed on activated
T cells.18,19 CD40L exerts a complex modulation of B-cell
apoptosis: it promotes the survival of germinal center B cell, but also
induces Fas expression thereby rendering the cells sensitive to FasL or
agonists.19-22 Several cytokines, in particular
interleukin-10 (IL-10), IL-6, and IL-2, have also been reported to
inhibit the apoptosis of normal or neoplastic B cells, in particular
induced by cytotoxic agents, or FasL,23-27 a phenomenon associated with a modulation of the expression of bcl-2, bcl-xL, and/or BAG.28-30 CD40 is expressed at the surface
of fresh B NHL cells and is able to trigger a proliferative signal in
the presence of IL-6 and IL-10.19,31 The biological effect
of a combination of cytokines and CD40L on the modulation of apoptosis
of neoplastic B-cell lines induced by cytotoxic agents is not known.
In the present report, the capacity of CD40L to modulate the apoptosis
of lymphoma cell lines induced by a cytotoxic agent was investigated.
CD40L-expressing L cells were found capable of partially inhibiting the
apoptosis and antiproliferative effect of DOX on lymphoma cells, as
well as the activation of the cysteine protease caspase
3.32,33 These results uncover a new mechanism of resistance
to cytotoxic agents conferred by adjacent nontumoral cells expressing
CD40L.
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MATERIALS AND METHODS |
Cell lines and culture conditions.
Lymphoma cell lines (Daudi, Raji, BJAB, BL36, BL70) were grown at
106 cells/mL in RPMI 1640 (GIBCO-BRL, Gaithersburg,
MD) containing 10% fetal calf serum (FCS), 100 U/mL
penicillin, 100 mg/mL streptomycin (GIBCO) and 2 mmol/L L-glutamine.
CDw32/Fc RII and CD40 ligand (CD40 L) transfected Ltk (-) cell line
(CDw32 L cells and CD40L L cells) were kindly provided by Dr F. Rousset
and Dr J. Banchereau (Schering-Plough, Dardilly, France). The CD40L L
cells were previously published and are capable of inducing normal
B-cell growth and Ig secretion.22 The expression of CD40L
on the cell line used in the present report was evaluated before the
initiation of these experiments using anti-CD40L labeling experiments.
CD40L was detectable at the surface of 95% of CD40L L cells and 40%
of phytohemagglutinin (PHA)-activated T-cell line Jurkatt
used as control. CDw32 expressing L cells were also used in these
experiments alone or in the presence of an anti-CD40 agonist antibody
(monoclonal antibody [MoAb] 89) at 0.5 µg/mL to mimic the effect of
CD40L. In some experiments, various concentrations of MoAb 89 were
used, ranging from 0.05 µg/mL to 2 µg/mL.
DOX was added at the initiation of the culture during 24 hours (0.5 or
1 µg/mL) or 30 hours (0.1 µg/mL). A 24-hour exposure was chosen
because of the long cellular half-life of this compound in vivo in B
NHL cells; a prolonged exposure to DOX may be more relevant to the in
vivo situation, as previously reported.17,34 After 24 hours, the medium was then removed and replaced with the same culture
medium without DOX for 24 additional hours of culture. Irradiated (75 Gy) L-cell lines (ie, CD40L or CDw32 expressing L cells) were added at
the initiation of DOX exposure at 105 cells/mL. Cytokines
(IL-10, IL-6, IL-2, and TNF) were added at the initiation of the
culture with or without DOX. Apoptosis and caspase 3 activation were
tested 48 hours after the initiation of culture.
Drugs, cytokines, and antibodies.
DOX (Pharmacia, Paris, France) was dissolved in sterile distilled water
before each experiment. Recombinant human (rh) IL-10 and MoAb 89 (anti-CD40 antibody) were provided by Dr F. Rousset and Dr J. Banchereau (Schering-Plough, Dardilly, France). The monoclonal
anti-CD40L antibody was purchased from Immunotech (Marseilles, France).
rhIL-10 was used at 100 ng/mL. rhIL-2 (Eurocetus, Amsterdam, the
Netherlands) was used at 100 U/mL; rhIL-6 (Sandoz, Basel, Switzerland)
was used at 40 ng/mL; rhTNF (Eurocetus) was used at 10 ng/mL. The
caspase 3 (Yama/CPP-32/apopain)-specific Asp-Glu-Val-Asp (DEVD) tetrapeptide labeled with a fluorogenic substrate
(7 amino-4-trifluoro methyl coumarin, AFC), DEVD-AFC, and the
inhibitor (DEVD-CHO) (Tebu, Le Perray-en-Yvelines, France) were
used at 50 µmol/L.24 A cell permeable inhibitor of
caspase 3 (Tebu, Le Perray-en-Yvelines, France) was used at 100 µmol/L and added at the initiation of cell culture.
Determination of apoptosis.
NHL cells were removed from 24-wells microtiter plates using gentle
aspiration to avoid the removal of adherent CD40L L cells. The
phenotype of the recovered cell population was consistently analyzed.
The mean size of L cells was fourfold to fivefold higher than the mean
size of the B NHL cell lines. In all experiments, the size and the
level of CD20 expression was similar in noncocultured B NHL cell lines
and in the recovered cell population, indicating that the contamination
of recovered NHL cells by irradiated L cells is minimal, if it exists.
The percentages of apoptotic NHL cell lines after removal from the
CD40L or CD32 L cells layer ranged from 2% for the BL70 cell line to
23% for the Daudi cell line and were not found significantly different
from that of cell lines cultured alone. For the quantification of
apoptosis in the recovered cell population, DNA breaks characteristic
of apoptotic cells were assessed by terminal deoxytransferase
(TdT)-mediated deoxyuridine triphosphate (d-UTP) nick end
labeling (TUNEL) assay (Boehringer Mannheim Corp, Indianapolis, IN).
NHL cell lines were fixed with 1% paraformaldehyde, permeabilized with
0.1% Triton X100 in 0.1% sodium citrate and washed extensively.
Incubation with TdT and fluorescein-labeled d-UTP provided
visualization of DNA strand breaks by flow cytometry on a FACScan
instrument (Becton Dickinson, Pont de Claix, France).
In each sample, 2,000 cells were analyzed for their content in
fluorescein-labeled DNA strand breaks. The intensity of fluorescence was proportional to the number of fluorescein-labeled DNA strand breaks
within the cell. The threshold level of fluorescence intensity beyond
which cells were considered to be apoptotic was 101.
Proliferation assay.
NHL cell lines (3.5 × 104 cells in 200 µL) were cultured in 96-well flat-bottomed microtiter
plates. DOX was added at the initiation of the culture during 24 hours
(0.5 or 1 µg/mL) or 30 hours (0.1 µg/mL). After 24 hours, the
medium was removed and replaced with the same culture medium without
DOX for 24 additional hours of culture. Irradiated (75 Gy) L cell lines
(ie, CD40L or CDw32 expressing L cells) were added at the initiation of
DOX exposure at 2.5 × 104 cells/mL. After 72 hours (or
more) of culture, cells were pulsed with 1 mCi/well of
[3H]TdR (25 Ci/mmol, Amersham, Les Ulis, France) for 20 hours. [3H]TdR incorporation was measured by tritium
detector using standard liquid scintillation counting techniques on a
 counter (Packard, Rungis, France).
Assay for caspase 3 (Yama/CPP32/apopain) activity.
After 48 hours of culture, cells were harvested, washed in
phosphate-buffered saline (PBS), and then resuspended in the lysis buffer (5× buffer Cold Spring Harbor [CSH], Triton
0.01%, orthovanadate 1×, protease inhibitor 1×) at 4°C
for 30 minutes and finally centrifuged at 4°C for 15 minutes at
13,000g. For each cell lysate, three measures were performed.
The negative control: cell lysates were preincubated at
30°C for 3 hours with DEVD-CHO in the reaction buffer (50 mmol/L
HEPES, 10% sucrose, 0.1% CHAPS [pH, 7.5]) and 10 mmol/L dithiothreitol (dTT) before addition of the
DEVD-AFC substrate. The blank group: DEVD-AFC substrate
incubated in the reaction buffer only at 30°C for 1 hour. The
assay group: cell lysates were directly incubated 1 hour at
30°C with DEVD-AFC substrate in the reaction buffer and dTT.
Fluorescence measurements were performed after 1 (t0) and 2 hours (t60)
of incubation. Caspase 3 activity was detected by AFC release monitored
on a spectrofluorometer (Kontron Analytical SFM 25, Velizy,
France), using an excitation wavelength of 400 nm and an
emission wavelength of 505 nm. Caspase 3 activity was calculated with
the following formula: 1 unit = (dFU/min) × (calibration curve
slope) 1 × (1 U/1 × 106 mmol AFC/min), where dFU is the difference of
fluorescence units: (FU of the assay group at t60  FU of the
blank group at t60) (FU of the assay group at t0 FU of
the blank group at t0).
Statistics.
Statistical analyses were performed using paired Student's
t-test.
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RESULTS |
Modulation of DOX-induced apoptosis by CD40L.
The capacity of CD40L expressed on L cells to modulate the apoptosis
induced by DOX in B-lymphoma cell lines (BJAB, Daudi, BL70, BL36, Raji)
was investigated. Incubation with DOX during 24 hours at
therapeutic34 concentrations (0.1 to 1 µg/mL) induced a
dose-dependent increase of apoptosis in the five cell lines tested
(Fig 1). With a shorter duration (3 hours)
of incubation, DOX at 1 to 2 µg/mL also induced the apoptosis of
these cell lines, while lower concentrations (0.1 to 0.5 µg/mL) of
DOX were not cytotoxic (not shown).
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| Fig 1.
Dose-dependent induction of apoptosis by DOX in five
lymphoma cell lines. Lymphoma cells were incubated with DOX for 24 hours (0.5 or 1 µg/mL) or 30 hours (0.1 µg/mL), then washed, and
cultured without DOX, but otherwise in the same conditions during 24 additional hours, before quantification of the percentage of apoptotic
cells. DOX concentrations: 0 µg/mL (1,  ), 0.1 µg/mL (2,  ),
0.5 µg/mL (3,  ), 1 µg/mL (4,  ). These results
are the mean (and standard deviation [SD]) of five
different experiments.
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All five NHL cell lines tested express CD40 at their cell surface
(>95% of positive cells, not shown). Coculture of lymphoma cell
lines in the presence of irradiated (75 Gy) L cells expressing CD40L
(CD40L L cells) resulted in a decrease of the percentage of lymphoma
cell undergoing apoptosis (Fig 2A), as
compared with lymphoma cells cultured with DOX alone or with L cells
expressing CDw32, ie, the FcRII receptor for IgG (Fig 2B). Coculture
with CD40L L cells significantly (Student's t-test, P < .05) reduced the intensity of TdT signal as well as the percentage
(33% to 89%) of apoptotic cells after treatment with DOX as compared
with no CD40L L cells or with CDw32 L cells (Fig 2B,
Table 1), in all five cell lines at
concentrations of 0.1 to 0.5 µg/mL. With 1 µg/mL of DOX, the
inhibition of apoptosis was significant only for the Daudi, Raji, and
BL36 cell lines (Fig 2C). Coculture with CDw32 L cells (without
anti-CD40 Ab) did not significantly affect the apoptosis of the five
NHL cell lines incubated with a 0.5-µg/mL concentration of DOX (Table
1), or with 0.1 or 1 µg/mL (not shown). However, the addition of the
agonist IgG anti-CD40 antibody (MoAb 89, 0.5 µg/mL) to the coculture
of NHL cells with CDw32 expressing L cells, thus mimicking the effect
of CD40L expressed at the cell surface, reduced the percentage of
apoptotic NHL cells similarly to CD40L L cells (Table 1). Of note, a
similar protective effect was observed with lower concentrations of
anti-CD40 (0.05 µg/mL): for instance, in the BL70 cell line, the
percentage of apoptotic cells was reduced from 57% ± 2% to 21% ± 1% with a 2-µg/mL concentration of MoAb 89 and to 19% ± 1% with a 0.05-µg/mL concentration of MoAb 89 in a representative
experiment. The apoptosis induced by a shorter (3 hours) exposure to
DOX (2 µg/mL) was inhibited similarly on coculture with CD40L L
cells, with a reduction of the percentage of apoptotic cells ranging
from 30% ± 2% for BL70 to 55% ± 2.5% for Daudi (not shown).
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| Fig 2.
Inhibition of DOX-induced apoptosis by CD40L L cells in
lymphoma cell lines. Lymphoma cells were incubated with DOX for 24 hours (0.5 or 1 µg/mL) or 30 hours (0.1 µg/mL) with or without
irradiated (75 Gy) L cells expressing CD40L or CDw32, then washed, and
cultured without DOX, but otherwise in the same conditions during 24 additional hours and tested for apoptosis using the TUNEL assay as
indicated in Materials and Methods. (A and B) Inhibition of DOX-induced
apoptosis of the BL36 cell line by CD40L. The intensity of fluorescence
is proportional to the number of fluorescein-labeled DNA strand breaks
within lymphoma cells. The threshold level of fluorescence intensity
beyond which cells were considered to be in apoptosis was
101; 5% of lymphoma cells not treated with DOX were above
this level. BL36 cells were cultured 48 hours without DOX (negative
control) (5% ± 0.4% of apoptotic cells) or with 0.5 µg/mL of DOX
during the first 24 hours (65% ± 1% of apoptotic cells) with
irradiated CD40L L cells (40% ± 1% of apoptotic cells) (A) or CDw32
L cells (65% ± 1% of apoptotic cells) (B). (C) Inhibition of
DOX-induced apoptosis by CD40L L cells in lymphoma cell lines. Cell
lines were exposed to various concentrations of DOX during the first 24 hours: 0 µg/mL (1), 0.1 µg/mL (2) and (3), 0.5 µg/mL (4) and (5),
1 µg/mL (6) and (7) and cocultured in the presence of irradiated L
cells expressing CD40L (3), (5), and (7). The SD of the percentages is
under 2% in all conditions. This experiment is representative of seven
different experiments.
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IL-10, IL-2, IL-6, and TNF were tested for their capacity to modulate
CD40L-mediated inhibition of apoptosis. The percentage of apoptotic
cells induced by DOX (0.5 µg/mL) was significantly reduced when NHL
cells were incubated with IL-10 alone in four cell lines (Table 1).
IL-2 and IL-6 also marginally reduced DOX-induced apoptosis in the BJAB
cell line only, while TNF alone had no effect (not shown). No additive
protective effect was observed between CD40L and IL-10 in any cell line
tested (Table 1) or between CD40L and the other cytokines tested.
Activation of caspase 3 by doxorubicin: Modulation by CD40L.
Caspase 3 (Yama/CPP32/apopain) is a key enzyme involved in the cleavage
of poly (ADP-ribose) polymerase (PARP) at the onset of
apoptosis.32,33 DOX was found to induce a dose-dependent increase of caspase 3 activity in the five NHL cell lines
(Table 2). This increase of caspase 3 activity was completely inhibited when cell lysates were preincubated
with the caspase 3 inhibitor DEVD-CHO32 (Table 2). In all
five cell lines, coculture with CD40L L cells, but not with CDw32 L
cells, inhibited DOX-induced (0.1 or 0.5 µg/mL) caspase 3 activity by
38% to 100% (Table 2). With a 1-µg/mL concentration of DOX in
contrast, coculture with CD40L L cells significantly reduced caspase 3 activity only in the BJAB and BL70 cell lines (Table 2).
IL-10, IL-2, IL-6, and TNF alone also significantly inhibited
DOX-induced caspase 3 activity in three cell lines, respectively (Table 3). IL-10 or IL-6 exerted a
significant additive inhibitory effect on DOX-induced caspase 3 activity in the Daudi cell line only (Table 3).
CD40L increases the proliferation of doxorubicin-treated NHL cells.
DOX induced a dose-dependent inhibition of tritiated thymidine uptake
by the five lymphoma cell lines at 72 hours of culture (Fig 3) and also after 96, 120, 144, and
240 hours of culture (not shown). When cocultured with CD40L L cells,
but not with CDw32 L cells, the proliferation of the five cell lines
treated with 0.1 to 0.5 µg/mL of DOX was partially restored at 72 hours (Fig 3) and also at 96, 120, 144, and 240 hours of culture (not shown). In contrast, CD40L L cells reversed the antiproliferative effect of a 1-µg/mL concentration of DOX in only two of the five cell
lines (Daudi, BL70) (Fig 3). Thymidine incorporation by NHL cells
cultured with DOX alone or with CDw32 L cells was similar (not shown).
Cytokines (IL-10, IL-2, IL-6, or TNF) either alone or added to the
coculture of lymphoma cells with CD40L L cells did not further increase
the proliferative potential of these cell lines after exposure to DOX
(not shown).
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| Fig 3.
CD40L L cells stimulate the proliferation of DOX-treated
lymphoma cells. Cells were incubated with DOX 0 µg/mL (1), 0.1 µg/mL (2) and (3), 0.5 µg/mL (4) and (5), 1 µg/mL (6) and (7) for
24 hours (0.5 or 1 µg/mL) or 30 hours (0.1 µg/mL) and with (3),
(5), and (7) or without (1), (2), (4), and (6) irradiated CD40L L
cells, then washed and cultured without DOX, but otherwise in the same
conditions during 48 additional hours before measurement of
[3H]TdR incorporation. [3H]TdR
incorporation of all five lymphoma cell lines treated with DOX 0.1 µg/mL (2 v 3), 0.5 µg/mL (4 v 5) was significantly
(Student's t-test, P < .05) increased on coculture
with irradiated CD40L L cells as compared with no L cells or L cells
expressing CDw32 (not shown); with 1 µg/mL of DOX (6 v 7),
the increase was significant only for the Daudi and BL70 cell lines.
These results are the mean and SD of five different experiments.
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DISCUSSION |
The results presented here show that drug resistance to a major
cytotoxic agent can be induced by a ligand expressed on adjacent nontumoral cells. Coculture of lymphoma cells with CD40L-expressing L
cells (or with L cells expressing CDw32, ie, FcRII, in the presence
of an IgG anti-CD40) was found capable (1) to partially inhibit the
apoptosis of lymphoma cell lines induced by therapeutic concentrations
of DOX, (2) to inhibit the activation of caspase 3 induced by DOX, and
(3) to increase the proliferative potential of these cell lines after
the end of exposure to DOX. An external signal provided by adjacent
cells, L cells in the present case, is therefore capable of inducing
the resistance of lymphoma cells to the proapoptotic and
antiproliferative effects of cytotoxic drugs. Of note, the addition of
the same concentrations of the anti-CD40 Ab alone without CDw32 L cells
did not significantly protect these cell lines against DOX-induced
apoptosis (not shown). CD40 ligand mRNA has been found detectable in
lymphoma tumor samples by reverse transcriptase-polymerase chain
reaction (RT-PCR).31 Recently, the presence of CD40L
protein in NHL tumor samples has been reported in aggressive NHL and
follicular NHL tumor samples.35,36 Conceivably, CD40L
expressed on nontumoral infiltrating T cells may play a similar role in
vivo, protecting tumor cells against the cytotoxic activity of
anticancer drugs. However, the protective role of CD40L in vivo still
remains to be demonstrated. It must indeed be noted that the level of
CD40L expression at the surface of CD40L L cells is superior to that of
T cells; for instance, CD40L was detectable at the surface of 95% of
CD40L L cells as compared with 40% of PHA activated T-cell line
Jurkatt. Therefore, we cannot exclude that the lower levels of CD40L
expression in vivo may induce a less potent protective effect than
CD40L L cells. However, it must be noted that the protective effect of
CDw32 L cells with anti-CD40 Ab on DOX-induced apoptosis was similar when all five NHL cell lines were incubated with a 2-µg/mL of anti-CD40 Ab or 40-fold lower concentrations (0.05 µg/mL), suggesting that even low levels of expression of CD40L or CD40 agonist Ab are
capable of protecting B NHL cells against drug-induced apoptosis.
Interestingly, at the highest (1 µg/mL) concentration of DOX tested,
CD40L failed to protect some of the NHL cell lines against the
apoptosis and antiproliferative effect of DOX, thereby providing a
biological basis for the use of a high dose of cytotoxic agents to
overcome this mechanism of drug resistance.2 Of note, the level of CD40 expression was high in all five cell lines and no correlation between the level of CD40 expression and the protective response to CD40L was observed (not shown), suggesting that the differential protective effect of CD40L in these cell lines may result
from differences in the intracellular signaling pathways of CD40 in
these cell lines.
The protective effect of CD40L L cells on DOX-induced apoptosis was not
enhanced by the addition of IL-10, IL-2, IL-6, or TNF. Interestingly,
however, IL-10 alone blocked the induction of apoptosis by 0.1 to 0.5 µg/mL concentrations of DOX in four lymphoma cell lines, whereas IL-6
and IL-2 exerted this effect in only one cell line. The magnitude of
the protective effect of cytokines on DOX-induced apoptosis was less
important than that of CD40L and the cytokines tested did not synergize
with CD40L. Some of the NHL cell lines tested in this study produce IL-10 (BJAB) or IL-6 (BL70 and RAJI). However, the level of IL-10 and
IL-6 are in the range of 100 to 300 pg/mL/106cells/24 hours
(not shown), and therefore 100-fold to 200-fold lower than those used
in the experiments. In addition, no correlation between cytokine
production by NHL cell lines and the modulation of drug-induced
apoptosis by the same cytokines was observed. These observations
suggest that the endogenous production of IL-10 or IL-6 is unlikely to
affect significantly the results presented in this study. Although
IL-10 and IL-6 are frequently produced locally in NHL tumors in
vivo,31 other biological phenomena may account for the
negative prognostic value of the overproduction of these cytokines in
vivo in lymphoma patients.37-39
Coculture with L cells expressing CD40L and/or in the presence
of cytokines inhibited the activation of caspase 3 induced by DOX. The
cytokines tested were found to inhibit caspase 3 activation alone and
additively to CD40L for IL-6 and IL-10. The inhibition of caspase 3 activity has been reported to protect cells against the apoptosis
induced by camptothecin and etoposide.32,40 The coexistence
of the inhibitory effects of CD40L L cells on DOX-induced apoptosis and
on caspase 3 activation suggests that caspase 3 inhibition is involved
in the antiapoptotic effect of CD40L. However, the results shown here
indicate that the cytotoxic activity of DOX is likely to involve other
pathways because (1) the inhibition of caspase 3 activation was not
consistently associated with a protective effect on apoptosis in these
experiments, (2) a low (0.1 µg/mL) concentration of DOX induces the
apoptosis in all five cell lines, but increases caspase 3 activity in
only three of the five cell lines, (3) finally, the modulation of
caspase 3 activity is not correlated with the protection from apoptosis by CD40L, in particular at a 1-µg/mL concentration of DOX.
The intracellular pathways involved in the protective effects of CD40L
and cytokines on drug-induced apoptosis are currently under
investigation. Several studies have shown that the apoptosis induced by
TNF and cytotoxic agents is suppressed by NF- B
induction.41-43 CD40 induces the activation of NF-B
through a TNF associated factor-2 (TRAF2)-mediated
mechanism suggesting a possible role of NF-B in the antiapoptotic
effect of CD40L.44 IL-10, IL-2, and IL-6 have also been
reported to inhibit the apoptosis of B-lymphoid cells and other
hematopoietic cell lines,23-27 by modulating the expression
of genes of the bcl-2 family.28-30 Preliminary results indicate that bcl-2 expression is not affected by CD40L or cytokines in
any of the five cell lines studied (Voorzanger et al, unpublished results). However, a recent report suggests that CD40L may upregulate bcl-xL expression in lymphoma cell lines suggesting a possible role of
this protein in the above-described phenomenon.45
In conclusion, these results show a new mechanism of drug resistance of
lymphoma cells triggered by CD40L expressed on adjacent nontumoral
cells. The expression of CD40L by nontumoral cells in tumor
microenvironment could play a role in drug resistance of lymphoma cells
in vivo.
| |
FOOTNOTES |
Submitted October 9, 1997;
accepted June 22, 1998.
Supported by La Ligue contre le Cancer (Comités
Départementaux du Rhône, de la Saône et Loire, de la
Drôme, et de l'Ardèche), L'Association pour la Recherche
sur le Cancer.
Address reprint requests to Jean-Yves Blay, MD, PhD,
Unité Cytokine et Cancer, Unité INSERM U453, Centre
Léon Bérard, 28, rue Laennec, 69008 Lyon, France; e-mail:
blay{at}lyon.fnclcc.fr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract