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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 990-995
Triggering of CD40 Antigen Inhibits Fludarabine-Induced Apoptosis
in B Chronic Lymphocytic Leukemia Cells
By
Maria Fiammetta Romano,
Annalisa Lamberti,
Pierfrancesco Tassone,
Fiorella Alfinito,
Silvia Costantini,
Federico Chiurazzi,
Thierry Defrance,
Patrizio Bonelli,
Franca Tuccillo,
Maria Caterina Turco, and
Salvatore Venuta
From the Dipartimento di Biochimica e Biotecnologie Mediche,
Università Federico II, Napoli, Italia; Dipartimento di Oncologia
Sperimentale, Istituto Nazionale Tumori Napoli, Italia; Dipartimento di
Medicina Sperimentale e Clinica, Università of Reggio Calabria,
Italia; DACM area of Haematology, Università Federico II, Napoli,
Italia; and INSERM, Lyon, France.
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ABSTRACT |
We analyzed the effect of CD40 triggering on the
fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL)
cells. Peripheral blood samples obtained from 15 patients were
incubated with fludarabine in the absence or the presence of the
anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a
significant proportion of apoptotic cells, ranging from 22% to 38%
(mean ± SE: 28.5 ± 1.6), were detected after 3 days of culture.
In 9 of these samples, the addition of G28-5 reduced apoptosis by at
least 30.1% and by 57.1% ± 7.8% on average
(P = .0077). Because the CD40 antigen activates NF- B/Rel
transcription factors in B cells, and NF-B/Rel complexes can inhibit
cell apoptosis, we investigated whether the antiapoptotic effect of
G28-5, in our system, could be related to modulation of NF-B/Rel
activity. As expected, B-CLL cells displayed significant levels of
nuclear NF-B/Rel activity; p50, RelA, and c-Rel components of the
NF-B/Rel protein family were identified in these complexes. After
exposure to fludarabine, NF-B/Rel complexes were decreased in the
nuclei. The addition of G28-5 upregulated the NF-B/Rel levels. To
determine the involvement of NF-B/Rel activity in the
G28-5-mediated inhibition of apoptosis, we blocked the transcription
factor with a decoy oligonucleotide, corresponding to the NF-B/Rel
consensus sequence. Cells incubated with the anti-CD40 MoAb in the
presence of the decoy oligonucleotide but not a control
oligonucleotide displayed a complete impairment of the
G28-5 antiapoptotic effect, indicating that NF-B/Rel activity was
required for the inhibition of apoptosis. These results suggest that
CD40 triggering in vivo could counteract the apoptotic effect of
fludarabine on B-CLL cells and that its neutralization, or the use of
NF-B/Rel inhibitors, could improve the therapeutic effect of
fludarabine.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE ABILITY TO induce apoptosis is a
relevant property of several chemotherapeutic drugs.1
Particularly, neoplasias that have a low growth fraction, such as
chronic lymphocytic leukemia (CLL), might be more vulnerable to
apoptotic stimuli than to agents that attack dividing cells. In these
pathologies, the extent of drug-induced apoptosis correlates with the
response to chemotherapy in vivo.2 Mechanisms that affect
cell sensitivity to apoptosis, including mutations and/or
altered expression of apoptosis-regulating genes such as
p53,3,4 Rb,5 and bcl-2,6 are
believed to play roles in CLL neoplastic expansion and resistance to
chemotherapy. Furthermore, physiological modifiers of cell
apoptosis interferon (IFN)- 7 and - ,8
interleukin-4 (IL-4),9 basic fibroblast growth factor,10 and IL-1311can protect CLL
cells from death. The analysis of the complex arrays of factors that
influence CLL apoptosis is needed for the understanding of the disease
pathogenesis and the improvement of therapies.
Among the intracellular modulators of apoptosis, there are NF-B/Rel
transcription factors. These are dimers of proteins (p50/p105 or
NF-B1, p52/p100 or NF-B2, p65 or RelA, c-Rel and RelB)
that have approximately 300 aminoacid Rel regions. The NF-B/Rel
complexes are either found in cell nuclei or retained in the
cytoplasm by inhibitors of the IB (- ) family; these
latter are proteolyzed on cell stimulation by a number of agents,
allowing NF-B/Rel dimers to reach the nucleus. The
NF-B/Rel factors control the expression of a wide range of
genes, such as those encoding immunoglobulins, cytokines, chemokines,
interferons, major histocompatibility complex proteins, growth factors,
and cell adhesion molecules.12 Furthermore, the apoptotic
response of some normal and neoplastic cell types to tumor
necrosis factor (TNF) or daunorubicin13-16 can be
downregulated by the NF-B/Rel nuclear activity. In some cells,
including B lymphocytes, the NF-B/Rel complexes stimulate the
expression of the zinc finger protein-coding gene A20 that inhibits
apoptosis17; other apoptosis-suppressing,
NF-B/Rel-regulated genes will presumably be
identified.
The induction of NF-B/Rel activities by apoptogenic agents such as
TNF- is believed to constitute a pathway by which the apoptotic
stimulus limits or terminates its own effect.13-16
Independent mechanisms that result in NF-B/Rel stimulation might be
expected to inhibit apoptosis as well. In B cells, a possible link
between NF-B/Rel induction and the rescue from apoptosis might be
recognized in the activity of CD40 antigen. This is a 45- to 50-kD
transmembrane glycoprotein18-20 that belongs to the TNF
receptor superfamily.21 The interaction between CD40 and
its ligand CD40-L (CD154), expressed on activated T
cells,19-23 can inhibit the normal and neoplastic B-cell
apoptosis induced by engagement of the B-cell receptor (BcR) or serum
deprivation.24-26 Because of its antiapoptotic properties, the CD40 molecule is believed to exert profound influences on the
growth of B lymphocytes and to direct the T-lymphocyte-mediated rescue
of germinal center B cells.19,20,27 Furthermore, the CD40
molecule appears functional in B-lineage acute lymphoblastic leukemia
cells that can be stimulated to proliferate and maturate via CD40
triggering.28 In mouse spleen,29 human
tonsilla, Daudi cells,30 and immature B
cells,31 the triggering of CD40 induces the nuclear
activity of NF-B/Rel complexes. Although CD40 antigen interacts with
the NF-B/Rel intracellular inducer TRAF2,32 the
NF-B/Rel activation was found to be independent of the association
with this molecule in B cells.33
We investigated whether CD40 antigen could influence the
apoptosis of B-CLL cells induced by fludarabine, and we also
investigated the involvement of NF-B/Rel factors in this regulation.
The aim of this work was to contribute to the understanding of the
mechanisms that regulate B-CLL apoptosis, and to the improvement of
therapies active on slowly dividing malignancies.
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MATERIALS AND METHODS |
Patients.
We studied 15 patients affected by B-cell CLL. Fourteen patients had a
CD5+ CD23+ phenotype, whereas 1 patient was
CD5+ CD23 . These patients had a clinical Rai
stage34 of 0 to 4 and had not received any therapy for at
least 3 years before blood collection.
Cells.
Peripheral blood mononuclear cells were isolated from the heparinized
blood of healthy donors by differential centrifugation through a
Ficoll-Hypaque density gradient (ICN Flow, Opera, Italy) at
400g for 30 minutes. Cells were washed with phosphate-buffered saline (PBS) and resuspended in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal calf serum (FCS; ICN Flow).
Fludarabine, monoclonal antibodies (MoAbs), and oligonucleotides.
Fludarabine (9- -D-arabinosyl-2-fluoroadenine-monophosphate) was
obtained from Schering (SpA, Milan, Italy). The anti-CD40 MoAb-producing hybridoma (G28-5) was obtained from American Type Culture Collection. An anti-HLA-A1 MoAb, HO5, kindly provided by Dr
Soldano Ferrone (New York Medical College, NY), was used as control.
The phosphorothioate oligonucleotides, corresponding to the B
consensus sequence
(5 -CCTTGAAGGGATTTCCCTCC-3)
or its mutated ("scrambled") form
(5-CCTTGAATTTATTTAAATCC-3)35-36
were purchased from Primm Srl (Milan, Italy).
Analysis of apoptosis.
Apoptosis was measured by flow cytometry as described.37
Briefly, the cells (5 × 105) were washed in PBS and
resuspended in 1 mL of a solution containing 0.1% sodium citrate,
0.1% Triton X-100, and 50 µg/mL propidium iodide (Sigma Chemical Co,
Gallarate, Italy). After incubation at 4°C for 30 minutes in the dark, cell nuclei were analyzed with a Becton Dickinson
FACScan (Sunnyvale, CA) flow-cytometer using the Lysis 1 program.
Cellular debris was excluded from analysis by raising the forward
scatter threshold, and the DNA content of the nuclei was registered on
a logarithmic scale. The percentage of cells in the hypodiploid region
was calculated.
Immunofluorescence.
The cells (5 × 105/100 µL) were incubated with CD19
fluorescein isothiocyanate (FITC) or CD69 phycoerythrin (PE)-conjugated antibodies (Becton Dickinson) in 10% FCS/RPMI 1640 medium. After a
30-minute incubation at 4°C, the cells were washed in PBS and analyzed with the FACScan.
Nuclear extracts.
Cell nuclear extracts were prepared38 from 20 × 106 cells by cell pellet homogenization in two volumes of
10 mmol/L Hepes, pH 7.9; 10 mmol/L KCl; 1.5 mmol/L MgCl2; 1 mmol/L EDTA; 0.5 mmol/L dithiothreitol (DTT); 0.5 mmol/L
phenylmethylsulfonyl fluoride; and 10% glycerol. Nuclei were
centrifuged at 1,000g for 5 minutes, and washed and resuspended
in two volumes of the above specified solution. KCl (3 mol/L) was added
to reach 0.39 mol/L KCl. Nuclei were extracted at 4°C for 1 hour and
centrifuged at 10,000g for 30 minutes. The supernatants were
clarified by centrifugation and stored at  80°C. Protein
concentration was determined by using the Bradford method.
Electrophoretic mobility shift assays (EMSAs).
The double-stranded B oligonucleotide (Promega, Madison, WI)
corresponded to the IL-2 receptor NF-B consensus sequence 5-CAACGGCAGGGGAATCTCCCTCTCCTT-3.39 The SP1
oligonucleotide was purchased from CEINGE (Naples, Italy).
The oligonucleotides were end-labeled with [-32P] ATP
(Amersham International Plc, Milan, Italy) using a polynucleotide kinase (Boehringer Mannheim, Milan, Italy) to a specific activity of 2 to 5 × 104 cpm/mL. The AP1 oligonucleotide was purchased
from Promega. EMSAs were performed as described.40 Briefly,
end-labeled DNA fragments (2 × 104) cpm were incubated
at room temperature for 20 minutes with 5 µg of nuclear protein in
the presence of 1 µg poly(dI-dC) in 20 µL of a buffer consisting of
10 mmol/L Tris-HCl, pH 7.5; 50 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L
DTT; and 5% glycerol. Protein-DNA complexes were separated from a free
probe on a 5.5% polyacrilamide gel and run in 0.25× Tris borate
buffer at 200 V for 3 hours at room temperature. The gels were dried
and exposed to X-ray film (Kodak AR, Milan, Italy). The intensity of
the bands, expressed as integrated optical density (O.D.),
was measured by using NIH Image 1.61 for PowerMacintosh.
The values were normalized by comparison with SP1 complex as an
internal control.
Anti-p50, -p65 (Rel A), and -c Rel antibodies.
The rabbit antibodies against the C-terminal peptide (529-551) of human
p65 and against the N-terminal peptide (1-21) of p105/p50 were kindly
provided by Dr Warner C. Greene (Gladstone Institute of Virology and
Immunology, San Francisco, CA). The rabbit antibodies against the
C-terminal peptide (537-587) of c Rel were a kind gift of Dr Nancy Rice
(Frederick Cancer Research and Development Center, Frederick, MD).
Statistical analysis.
Wilcoxon's test was used to perform statistical analysis of the
results.
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RESULTS |
Effect of CD40 triggering on the fludarabine-induced apoptosis of
B-CLL.
We incubated the cells of 15 patients with fludarabine (0.8 µg/mL) in
the presence or absence of MoAb G28-5 and measured the percentage of
apoptotic elements after 3 days of culture. In 3 samples very low
levels of apoptosis (<10%) were detected in the presence of the
drug. Twelve patients displayed apoptosis, with values ranging from
22% to 38% (mean ± SE: 28.5% ± 1.6%). In 9 of these samples
(Fig 1A), the addition of MoAb G28-5 to the
cultures reduced apoptosis by at least 30.1% and by 57.1% ± 7.8% on average. The inhibition of apoptosis was significant
(P = .0077), because the apoptosis percentages were reduced
from 28.3% ± 1.6% to 12.5% ± 2.6% in the presence of
the anti-CD40 MoAb. In the remaining 3 cases, CD40 triggering did not
reduce or slightly ( 10%) reduced the fludarabine-induced apoptosis
(Fig 1B). A dose-response curve of the MoAb G28-5 effect is shown in
Fig 2.
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| Fig 1.
(A) Inhibition of fludarabine-induced apoptosis by
the anti-CD40 MoAb G28-5. (B) Samples with no response or only low
response to anti-CD40 MoAb. Cells
(1 × 106/mL) from patients with B-CLL were incubated in
10% FCS-RPMI 1640 medium with fludarabine (0.8 µg/mL) and in the
absence or presence of MoAb G28-5 (ascites, 1:1000). After 3 days of
cell culture the percentages of apoptosis were calculated. Any
experimental point has been performed in duplicate; standard deviations
of the mean apoptosis values were less than 10%. The levels of
apoptosis in cells incubated without fludarabine, either in the
presence or the absence of anti-CD40 MoAb, were less than 10%.
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| Fig 2.
Dose-response curve of MoAb G28-5 antiapoptotic effect.
B-CLL cells (1 × 106/mL) were incubated in 10%
FCS-RPMI 1640 medium with fludarabine (0.8 µg/mL) in the presence of
the indicated concentrations of MoAb G28-5 or control (HO5) antibody.
After 3 days of cell culture, the apoptosis percentages were
calculated.
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Analysis of NF-B/Rel activity in B-CLL cells.
In cells obtained from 13 of the 15 patients studied, we could
analyze the nuclear levels of NF-B/Rel complexes by EMSA. Twelve of
the 13 samples showed a migration pattern in which four bands could be
recognized. A representative result is shown in Fig
3. The bands indicated by arrows 1 through
3 (a) appeared to correspond to specific NF-B/Rel complexes because
they were not affected by the incubation of the extract with unlabeled
AP1 oligo, whereas (b) disappeared in the presence of a competing, unlabeled B oligonucleotide (c). Band 4 was probably caused by the
not specific binding of the labeled oligonucleotide to an abundant protein present in the nuclear extracts.41 To
analyze the composition of the NF-B/Rel complexes, the extract was
incubated in the presence of rabbit anti-p50 (d), -RelA (e), -c-Rel
(f) or preimmune (g) serum. The band indicated by arrow 1 was
supershifted by both anti-p50 and -Rel A antibodies, whereas the band
indicated by arrow 2 was supershifted by both anti-p50 and -c-Rel and
band 3 only by anti-p50. Therefore, p50/RelA, p50/c-Rel, and p50/p50 dimers were likely to be present in the complexes corresponding to,
respectively, bands 1, 2, and 3.
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| Fig 3.
Analyses of NF-B/Rel nuclear complexes in B-CLL cells.
A nuclear extract was obtained from B-CLL cells and incubated with a
32P-labeled B oligonucleotide, in the absence (a) or
presence of a 50× molar excess of unlabeled AP1 oligonucleotide (b),
a 50× molar excess of unlabeled B oligonucleotide (c), anti-p50
(d), -RelA (e), -c-Rel (f), or preimmune (g) serum.
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In 9 of the 12 samples, fludarabine induced both apoptosis and a
marked reduction of NF-B/Rel levels; in the remaining 3 samples, the
drug induced very low levels (<10%) of apoptosis and apparently did
not modulate the NF-B/Rel levels. Of the 9 fludarabine-responsive
samples, 2 could not be rescued from drug-induced apoptosis by the
anti-CD40 MoAb; consistently, the MoAb did not upregulate the
NF-B/Rel activity. The remaining 7 samples displayed both inhibition
of apoptosis and induction of NF-B/Rel complexes by MoAb G28-5. A
representative result is shown in Fig 4.
Compared with cells incubated in control medium (A, a), cells incubated with fludarabine (A, b) displayed a reduction in the intensities of the
bands, corresponding to NF-B/Rel-DNA complexes (arrows 1 through 3).
The intensities of these bands were markedly higher in cells incubated
with MoAb G28-5, either alone (A, c) or with fludarabine (A, d).
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| Fig 4.
Effect of fludarabine and anti-CD40 MoAb on NF-B/Rel
nuclear complexes. B-CLL cells (1 × 106/mL) were
incubated in 10% FCS-RPMI 1640 medium, in the absence (a) or presence
of fludarabine (0.8 µg/mL) (b), MoAb G28-5 (10 µg/mL) (c), or MoAb
G28-5 + fludarabine (d). After 20 hours, nuclear extracts were
obtained and incubated with a 32P-labeled B (A) or SP-1
(B) oligonucleotide. The DNA-protein complexes were analyzed by EMSA.
The SP1-normalized integrated O.D. (×102) (see
Materials and Methods) of the bands 1 + 2 were: a, 1.1; b, 0.2; c,
3.9; d, 2.7.
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Effect of NF-B/Rel inhibition on the antiapoptotic
activity of MoAb G28-5.
To determine whether the NF-B/Rel activity was required for the
antiapoptotic effect of the MoAb G28-5, we incubated the cells with a
decoy oligonucleotide, corresponding to the B consensus sequence.35 The oligonucleotide subtracts NF-B/Rel
complexes, resulting in a decrease of their nuclear levels and
impairment of target genes expression.35,36,42,43 We
verified that in cells triggered via CD40, the increase of the
NF-B/Rel-controlled CD69 antigen44 was inhibited by the
decoy, but not by a mutated form ("scrambled") oligonucleotide
(Fig 5A). Furthermore, the levels of
nuclear NF-B/Rel complexes, stimulated by CD40 (Fig 5B, lane b),
were reduced in cultures incubated with the decoy (lane c) but not the
scrambled (lane d) oligonucleotide. In five different experiments, we
compared the effects of the MoAb G28-5 on the apoptotic responses of
the cells incubated with fludarabine in the absence or presence of
decoy or scrambled oligonucleotides. The results are shown in Fig
6. The presence of the decoy
oligonucleotide slightly modified the apoptosis percentages of cells
incubated with or without fludarabine. Therefore, the inhibition of
NF-B/Rel complexes was not sufficient to induce or significantly
enhance cell apoptosis. On the other hand, the rescuing effect of MoAb G28-5 was specifically abolished by cell incubation with the decoy oligonucleotide.
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| Fig 5.
Effect of a B decoy oligonucleotide on CD69 expression
(A) and the levels of NF-B/Rel nuclear complexes (B) in
CD40-stimulated B-CLL cells. (A) B-CLL cells
(1 × 106/mL) were stimulated with anti-CD40 MoAb (10 µg/mL) in the absence or the presence of the 5-µmol/L B decoy or
scrambled oligonucleotide. After 3 days the cells were collected,
washed, incubated with CD69 PE and CD19 FITC, and analyzed by FACScan.
The proportion of the CD19+ cells in the different
cultures was 74.8 ± 2.4. ( ), Medium; ( ), anti-CD40 MoAb. (B)
B-CLL cells (1 × 106/mL) were incubated in 10%
FCS-RPMI 1640 medium (a), and with anti-CD40 MoAb (10 µg/mL) in the
absence (b) or presence of the 5-µmol/L B decoy (c) or scrambled
(d) oligonucleotide. After 20 hours, nuclear extracts were obtained and
incubated with the 32P-labeled B oligonucleotide. The
DNA-protein complexes were analyzed by EMSA.
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| Fig 6.
Effect of the B decoy oligonucleotide on the levels of
B-CLL cell apoptosis. B-CLL cells (1 × 106/mL) were
incubated in 10% FCS-RPMI 1640 medium, in the presence of the
indicated reagents. After a 3-day incubation, cell apoptosis was
analyzed. ( ), RPMI; (), decoy 5 µmol/L; (), scrambled 5 µmol/L.
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DISCUSSION |
These results show that CD40 antigen modulates the fludarabine-induced
apoptosis in B-CLL lymphocytes. The CD40 effects were analyzed in
unfractionated mononucleate cell populations, which, when cultured,
displayed a low proportion of apoptotic elements (<10%), compared
with the spontaneous apoptosis percentages observed in purified B
leukemic cells.11,45-48 However, in three cases, in which
the 15%, 41%, and 73% of spontaneous apoptosis were detected after 5 days of culture, the presence of anti-CD40 MoAb did not reduce such
percentages, in accord with results reported by other authors49 (results not shown). On the other hand, in
fludarabine-treated cultures, the CD40 triggering rescued the cells
from apoptosis. Therefore, in addition to inhibiting the spontaneous or
BcR-induced apoptosis of normal B cells and B-cell
lines,18,19,24-27 CD40 appears to regulate also the
chemotherapeutic drug-induced apoptosis in cells from patients with
B-CLL.
A constitutive activity of NF-B/Rel activity was found in B-CLL. The
complexes comprised p50 homodimers, p50/c-Rel, and p50-RelA heterodimers. This pattern is similar to those described in immature and mature B-cell lines.12,50 Cell incubation with
fludarabine resulted in the disappearance of the nuclear NF-B/Rel
activity. This might rely on a reduced production of NF-B/Rel
proteins, due to the fludarabine-caused hampering of RNA synthesis.
Alternatively, the drop in NF-B/Rel nuclear activity might be
induced during the apoptosis programconsistently, a decline in
NF-B/Rel nuclear levels had been observed during the BcR-induced
apoptosis of murine B cells.51 However, although being part
of the fludarabine activity, the downmodulation of NF-B/Rel nuclear
factors is not likely to be sufficient for the triggering of apoptosis.
Indeed, the block of NF-B/Rel complexes, obtained by incubating the
cells with the decoy oligonucleotide alone, did not result in inducing apoptosis.
On CD40 triggering, the nuclear levels of NF-B/Rel complexes were
increased. When the NF-B/Rel complexes were blocked by the presence
of a decoy oligonucleotide, the antiapoptotic activity of CD40 was also
abrogated. This strongly argues for a role of NF-B/Rel complexes in
the antiapoptotic activity of CD40. Whether A2051
and/or other NF-B/Rel-controlled genes induced by CD40
triggering are responsible for the antiapoptotic mechanism remains to
be determined.
These results suggest that the CD40-mediated survival pathway,
triggered by CD40-L-expressing T lymphocytes,19-23 could
counteract the therapeutic effect of fludarabine, and possibly other
apoptogenic drugs in vivo. Furthermore, the CD40-mediated regulation of
fludarabine-induced apoptosis in B-CLL cells can represent a model of
NF-B/Rel-controlled response to chemotherapy in a lymphoid tumor.
This example supports the possible use of NF-B/Rel inhibitors in
antitumour therapies.52 Reagents, like anti-CD40-L
antibodies, that neutralize NF-B/Rel-stimulating molecules of the
cell's environment could as well potentiate the therapeutic effect of
the drug.
Finally, the question arises whether the CD40-mediated antiapoptotic
pathway, active in B-CLL cells, can contribute to the prolonged life
span and accumulation of these leukemic cells53 as a part
of the pathogenetic mechanism of the disease. The role of CD40/CD40-L
system in cell resistance to apoptotic drugs should, as well, be
investigated.
| |
FOOTNOTES |
Submitted July 14, 1997;
accepted March 23, 1998.
Supported by AIRC, FSN 96, and Regione Campania.
M.C.T. and S.V. equally contributed to this work.
Address reprint requests to M. Caterina Turco, MD,
Dipartimento di Biochimica e Biotecnologie Mediche, University of
Napoli, Federico II, Via S Pansini, 5, 80131 Napoli, Italia; e-mail:
turco{at}dbbm.unina.it.
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.
| |
ACKNOWLEDGMENT |
We thank Dr Antonella De Pascale (Schering SpA, Milan, Italy) and Dr
Felicetto Ferrara (Cardarelli Hospital, Naples, Italy) for providing us
with the fludarabine. We also thank Carmine Del Gaudio for his
excellent technical help.
| |
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Abstract
Introduction
Abstract