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INTRODUCTION |
Fas is a 45-kDa membrane protein that belongs to the tumor
necrosis factor (TNF)1/nerve
growth factor receptor family (1). Engagement of Fas by agonistic
anti-Fas antibodies triggers programmed cell death in a variety of cell
types (2, 3). Its natural ligand, Fas ligand (Fas-L), belongs to the
TNF family (4) and can be found as a 40-kDa membrane-bound or a 26-kDa
soluble cytokine (5). Similarly to agonistic anti-Fas antibodies,
binding of membrane-bound or soluble Fas-L to Fas receptor can induce
apoptosis in Fas-bearing cells (1, 4). The main death pathway initiated
from Fas activation involves a series of death-associated molecules
(6), including FADD (Fas-associated
death domain-containing protein), which is an
adaptor protein that is recruited to Fas receptor upon its engagement
(7-9). FADD then binds to and activates procaspase-8 (also called
FLICE or MACH) (8-10), which is believed to be the first step of a
proteolytic cascade that triggers activation of other caspases such as
caspases-7, -3, and -6 (11). Although other cell death pathways could
be initiated from Fas activation (12, 13), analysis of lymphocytes from
FADD
/
mice has recently demonstrated the prominent role of the
FADD/procaspase-8 pathway in Fas-mediated cell death (14).
Cytotoxic drugs commonly used in cancer therapy can induce tumor cell
death by apoptosis (15-17). These drugs were shown to enhance the
expression of Fas (18) and Fas-L (19, 20) on the surface of certain
malignant cells. It has been proposed that the molecular process
leading from specific cellular damage induced by the drugs to apoptosis
might involve an interaction between Fas ligand and Fas (19). However,
this issue remains controversial because antagonistic anti-Fas
antibodies that block Fas-L-mediated apoptosis do not always inhibit
drug-induced cell death (21, 22). In the present study, we further
addressed the role played by the Fas/FADD pathway in drug-induced cell
death. We show that anticancer drugs can induce Fas receptor clustering
and FADD recruitment to Fas receptor in a Fas ligand-independent
fashion. By modulating FADD expression, we also demonstrate the role of
this adaptor molecule in drug-induced apoptosis.
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MATERIALS AND METHODS |
Cell Lines and Culture Conditions--
The HT29 and HCT116 human
colon carcinoma and the U937 and Jurkat leukemic cell lines were
obtained from the ATCC (Rockville, MD). HT29 and HCT116 were maintained
in Eagle's minimum essential medium complemented with 10% fetal calf
serum and 2 mM L-glutamine (Biowhittaker Co.,
Fontenay sous Bois, France). U937 and Jurkat cells were maintained in
RPMI 1640 (Biowhittaker Co.) complemented as above with
heat-inactivated fetal calf serum.
Soluble Fas-L Production--
sFas-L was collected from Neuro
2A-transfected murine cells supernatant (kindly provided by Dr. A. Fontana, Zurich, Switzerland) (23). This supernatant was obtained as
described previously (18). One arbitrary unit of sFas-L was defined as
1 µl of a 100-fold concentrated supernatant of Neuro 2A cells that
had been confluent for 48 h. An unique pool of sFas-L or mock
supernatant was used throughout the study.
Cytotoxicity and Apoptosis Determination Assays--
Cell
viability was determined by the use of the methylene blue colorimetric
assay (18) 72 h after the beginning of drug exposure, unless
specified. Specific apoptosis was determined after trypsinizing cells
and staining with 1 µg/ml Hoechst for 15 min at 37 °C. The
percentage of apoptotic cells was determined by analyzing 300 cells.
For blocking assays, cells were incubated for 1 h with 2 µg/ml
antagonistic ZB4 monoclonal antibody (Biovalley Co., Rockville, MD) or
2 µg/ml Fas-IgG kindly provided by Dr. S. Nagata (Osaka Bioscience
Institute, Osaka, Japan) prior to treatment with 5 or 10 µM cisplatin (CDDP; Roger Bellon Chemical Co., Neuilly,
France), 10 µM doxorubicin (Sigma Chemical Co., St.
Quentin Fallavier, France), 50 µM etoposide (VP16;
Sigma Chemicals Co.), 10 ng/ml vinblastine (VB; Sigma Chemical Co.), 50 ng/ml anti-Fas CH11 (Biovalley, Rockville, MD) plus 0.8 µg/ml
cycloheximide (Sigma Chemical Co.) or 2 AU/ml recombinant Fas-L
(sFas-L) for 72 h, except U937 cells, which were treated for
4 h with etoposide and 24 h with vinblastine and anti-Fas
receptor CH11 antibody, respectively. During the course of the
treatment cells were co-treated with 2 µg/ml ZB4 antibody or 2 µg/ml Fas-IgG, and apoptosis was determined as described above.
Flow Cytometric Analysis of Fas Ligand Membrane
Expression--
HT29, HCT116, or U937 cells were treated with 10 µM cisplatin, 50 µM etoposide, or 10 ng/ml
vinblastine for 4 h. Jurkat cells were activated with 500 ng/ml
ionomycin and 20 ng/ml phorbol 12-myristate 13-acetate (Sigma Chemical
Co.) for 4 h. Fas-L expression was measured by flow cytometry by
incubating cells for 45 min at +4 °C with the rat IgG2a anti-human
Fas-L clone H11 (Alexis, San Diego, CA) or a rat isotype-matched
control (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted
at 1/500, in PBS containing 0.5% bovine serum albumin and 0.1%
NaN3. After two washes in PBS, cells were incubated for 45 min with a fluorescein isothiocyanate-labeled donkey anti-rat IgG
(Jackson ImmunoResearch Laboratories) and analyzed on a Becton
Dickinson flow cytometer.
Expression Vectors--
pCDNA3 was obtained from Invitrogen
(NV Leek Co., The Netherlands), pCDNA3-FADD was kindly provided by
Dr. V. M. Dixit (University of Michigan Medical School, Ann Arbor,
MI). Antisense construct for FADD was obtained by digestion of the
full-length FADD sequence from pCDNA3-FADD with
BamHI/KpnI and subcloning in the expression vector pBK-CMV (Stratagene, La Jolla, CA) in reverse orientation. PBK-CMV-FADD-AS construct was checked by manual sequencing (data not
shown). PCI-neo was purchased from Promega (Madison, WI), pCI-MC159 and
pCI-E8 were kindly provided by Dr. J. I. Cohen (NIH, Bethesda,
MD). PCI-FADD-DN construct was a kind gift from Dr. C. M. Zacharchuk (NIH, Bethesda, MD).
Western Blotting--
For immunoblotting, cells were washed in
PBS, lyzed in the boiling buffer (1% SDS, 10 mM Tris, pH
7.4) for 10 min at 4 °C, and boiled for 5 min. Proteins (30 µg)
were separated on a polyacrylamide SDS containing gel and transferred
onto polyvinylidene difluoride membranes (Bio-Rad, Ivry sur Seine,
France). After blocking nonspecific binding sites overnight by 5%
nonfat milk in TPBS (PBS with 0.1% Tween 20), the membrane was
incubated for 2 h at room temperature with anti-human FADD
(Transduction Laboratories, Lexington, KY) or the loading control
anti-human Hsp70 (StressGen Biotechnologies, Victoria, Canada). The
membrane was then washed twice with TPBS and incubated for 1 h at
room temperature with horseradish peroxidase-conjugated goat anti-mouse
antibody (Jackson ImmunoResearch Laboratories). The membrane was then
washed twice with TPBS and revealed using an enhanced chemiluminescence
detection kit (Amersham, Les Ulis, France) and autoradiography.
Stable Transfections--
HT29 cells were seeded for 24 h
and transfected with pCDNA3-AU1-FADD containing full-length human
FADD cDNA or an empty vector construct pCDNA3 by the use of the
lipofection reagent Dac-30, according to the manufacturer's
instructions (Eurogentec Co., Seraing, Belgium). Single clones were
picked 2-3 weeks after transfection and selected in medium containing
1 µg/ml geneticin.
Transient Transfections--
HT29, HCT116, and U937 cells were
seeded 24 h before transfection and transfected with 1 µg of
pCI-neo, pCI-MC159, pCI-E8, pBK-CMV, pBK-CMV-FADD-AS, pCI-FADD-DN, or a
combination of different plasmids using LipofectAMINE Plus (Life
Technologies Co., Gaithersburg, MD), except for U937 cells, for which
transfection were performed using Superfect (Qiagen, Courtaboeuf,
France), according to the manufacturer's instructions. HT29 and HCT116
cells were treated or not 16 h after transfection with 10 µM cisplatin or 50 µM VP16 for 72 h.
U937 cells were treated with 50 µM etoposide for 4 h or 10 ng/ml VB for 24 h. Cells were then analyzed for cytotoxicity or apoptosis as described above. Each point is the mean of two to three experiments.
Confocal Laser Scanning Microscopy Analysis of Fas Receptor
Clustering--
HT29 and HCT116 cells were seeded into tissue culture
chambers (Chamber Slide, Life Technologies Co.) 24 h before
treatment with 2 AU/ml sFas-L, 10 µM cisplatin, 50 µM etoposide, or 10 ng/ml TNF-
for 4 h. U937
cells were seeded into 25 cm2 flat bottom flasks the day
prior to treatment with sFas-L, etoposide, cisplatin, 10 ng/ml
vinblastine, or TNF-
, as above for 4 h. Control cells were left
untreated. Cells were fixed in 3% paraformaldehyde (Sigma Chemical
Co.) for 10 min, and washed twice with PBS for 10 min, preincubated
with 1% bovine serum albumin for 15 min, and incubated with a mouse
antibody directed against Fas receptor (ZB4) diluted 1/100 in PBS
containing 1% bovine serum albumin for 2 h at room temperature.
Samples were washed twice in PBS then incubated for 45 min with a Texas
Red-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch
Laboratories) diluted 1/50, and cells were analyzed with a confocal
laser scanning microscope as described (24). For U937 cells, staining
was performed on suspension cells, and the observation was realized
after letting U937 cells attach on parafin-covered glass slides. A
nonrelevant isotype-matching antibody was used as a negative staining
control and showed no staining on HT29, HCT116, or U937 cells (not shown).
Immunoprecipitation--
After treatment, 15 × 106 HT29 cells were washed once in PBS and incubated with
the cleavable cross-linker
3,3'-dithiobis[sulfosuccinimidyl-propionate] (Pierce Chemical Co.,
Rockford, IL) for 10 min at 4 °C. The reaction was stopped by
incubation in PBS containing 10 mM ammonium acetate for 5 min at 4 °C. Cells were detached from dishes by use of a rubber
policeman, washed twice in PBS, and lysed in lysis buffer (30 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, 1% Nonidet P-40,
10% glycerol; all purchased from Sigma Chemicals Co.) for 15 min on
ice. After centrifugation at 14,000 rpm at 4 °C for 15 min, an
anti-human CD95 antibody (APO1-3, Alexis Co., San Diego, CA) was added
and reacted at 4 °C overnight. Immune complexes were precipitated
using protein A-Sepharose (Pharmacia Co., Orsay, France) and washed
three times in lysis buffer. The precipitate was resuspended in a
Laemmli buffer containing 2.5%
-mercaptoethanol and
boiled for 5 min. Samples were resolved on SDS-polyacrylamide gel
electrophoresis and transferred onto polyvinylidene difluoride membrane
(Bio-Rad) for Western blot analysis. FADD and Fas receptor protein
content were analyzed by the use of the anti-human FADD antibody
(Transduction Laboratories) and the rabbit polyclonal anti-human Fas
receptor antibody clone M20 (Santa Cruz Biotechnology, Santa Cruz, CA) as described above.
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RESULTS |
Fas Ligand Is Not Involved in Anticancer Drug-induced
Apoptosis--
We have shown recently that anticancer drugs
could increase Fas receptor membrane expression and sensitize tumor
cells to Fas apoptotic pathway (18). To determine whether drug-induced cell death could involve an interaction between Fas receptor and Fas-L,
we used the antagonistic ZB4 anti-Fas antibody and the Fas-IgG blocking
molecule. HT29 and HCT116 colon carcinoma cells were treated for
72 h with 5 or 10 µM CDDP, 10 µM
doxorubicin, or 2 AU/ml recombinant sFas-L. U937 human leukemic cells
were treated with either 50 µM etoposide for 4 h or
10 ng/ml vinblastine for 24 h or 50 ng/ml CH11 anti-Fas agonistic
antibody in the presence of 0.8 µg/ml cycloheximide for 24 h.
All these treatments were performed in the absence or in the presence
of either 2 µg/ml ZB4 anti-Fas blocking antibody or 2 µg/ml Fas-IgG
molecule added to the culture medium 1 h before drug exposure. In
these conditions, both ZB4 antibody and Fas-IgG reagent inhibited
apoptosis induced by sFas-L and CH11 antibody in colon cancer cell
lines and U937 cells, respectively (Fig.
1). By contrast, neither ZB4 antibody nor
Fas-IgG reagent influenced drug-induced cell death in the tested cell
lines (Fig. 1). These results suggested that Fas-Fas-L interaction
might not play a central role in drug-induced cell death.

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Fig. 1.
Antagonistic ZB4 anti-Fas antibody and
Fas-IgG fail to block anticancer drug-induced apoptosis. HT29 and
HCT116 cells were preincubated (black and shaded
bars) or not (open bars) for 1 h in the presence
of 2 µg/ml ZB4 antagonistic antibody (black bars) or 2 µg/ml Fas-IgG (shaded bars) and then left untreated
(NT) or treated with indicated concentrations of CDDP,
doxorubicin (DXR), and sFas-L for 72 h. U937 cells were
preincubated as above and then treated with VP16 or anti-Fas agonistic
CH11 antibody plus cycloheximide (CHX) or VB for 4, 24, and
24 h, respectively. The percentage of apoptotic cells was
determined by Hoechst 33258 staining. Results are expressed as the
means ± S.D. of three independent experiments.
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Then we used flow cytometry to analyze the expression of Fas-L on the
plasma membrane of the studied cell lines. Untreated Jurkat cells and
Jurkat cells exposed to phorbol 12-myristate 13-acetate + ionomycin
were used as negative and positive controls, respectively. Whereas
Fas-L was expressed on the plasma membrane of untreated U937 cells, it
remained undetectable on the membrane of HT29 and HCT116 cells (Fig.
2). Exposure of the three cell lines to
cytotoxic drugs did not modulate Fas-L expression on their plasma
membrane (Fig. 2). These results further argued against a role for a
Fas-Fas-L interaction in drug-induced cell death.

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Fig. 2.
Flow cytometric analysis of Fas ligand
expression on the plasma membrane of tumor cells. HT29, HCT116,
and U937 cells were treated at doses indicated in the legend to Fig. 1
and analyzed 4 h after the beginning of drug treatment for Fas-L
expression by flow cytometry using the rat anti-human Fas-L antibody
H11. Untreated Jurkat cells and Jurkat cells activated with 500 ng/ml
ionomycin plus 20 ng/ml phorbol 12-myristate 13-acetate were used as
negative and positive controls, respectively. Gray, control
antibody; white, specific antibody. Results are
representative of four independent experiments.
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Anticancer Drugs Induce Fas Receptor Clustering--
We used
confocal laser scanning microscopy and ZB4 anti-Fas antibody to analyze
the effect of anticancer drugs on Fas receptor expression at the
surface of tumor cells. Exposure of tumor cells to recombinant sFas-L,
which induces the clustering of Fas receptor at the surface of tumor
cells, was used as a positive control, whereas cell exposure to 10 ng/ml TNF-
, which does not modify the pattern of Fas expression, was
used as a negative control (Fig. 3). All
the cytotoxic drug/cell line combinations induced the clustering of Fas
receptor on tumor cell plasma membrane as soon as 4 h after the
beginning of cell treatment (Fig. 3).

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Fig. 3.
Anticancer drugs induce Fas receptor
clustering. Cells were either left untreated (NT) or
treated with 10 ng/ml TNF- , 10 µM CDDP, 50 µM VP16, 10 ng/ml VB, or 2 AU/ml sFas-L. 4 h later,
cells were fixed and labeled for confocal laser scanning microscopy as
described under "Materials and Methods." Cells were viewed and
photographed using a confocal microscope with a ×40 objective.
Bar, 10 µM.
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Anticancer Drugs Induce the Recruitment of FADD to Fas
Receptor--
To test the possibility that anticancer drugs could also
induce the recruitment of the adaptor protein FADD to Fas receptor, co-immunoprecipitation studies were performed. HT29 cells were either
left untreated or treated with 10 µM cisplatin, 2 AU/ml sFas-L, or 10 µM doxorubicin for 4 h and analyzed
for co-immunoprecipitation using an antibody directed against Fas
receptor. Western blot analysis using an anti-FADD antibody revealed
that cisplatin and doxorubicin, similar to soluble recombinant Fas-L,
induced FADD recruitment to Fas receptor (Fig.
4). Similar results were obtained with
HCT116 cells after treatment with cisplatin and doxorubicin (not
shown).

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Fig. 4.
Anticancer drug treatment induced
co-immunoprecipitation of FADD with Fas receptor. HT29 cells (15 106) were left untreated (NT) or treated with 10 µM CDDP, 10 µM doxorubicin
(DXR), or 2 AU/ml recombinant sFas-L for 4 h and then
treated with a cross-linker and lyzed. The extract was used to
immunoprecipitate (IP) the Fas receptor as described under
"Materials and Methods." Samples were subsequently analyzed for
FADD and Fas receptor contents after Western blotting with an anti-FADD
and anti-Fas antibodies.
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Overexpression of FADD Sensitizes HT29 Cells to Cisplatin-induced
Cytotoxicity--
To determine whether FADD association to Fas
receptor after clustering could be relevant for anticancer drug-induced
apoptosis, we performed a series of experiments aiming to modulate FADD
expression. HT29 cells were stably transfected with a construct
encoding FADD full-length cDNA in pCDNA3 plasmid. After
geneticin treatment, we selected two clones, namely FD2 and FD5, that
expressed nearly two times more FADD messenger RNA (not shown) and
protein (Fig. 5A,
inset) as compared with the CO4 clone transfected with an empty construct. Both FADD-overexpressing clones were more sensitive to
soluble recombinant Fas-L- (sFas-L) (not shown) and cisplatin-induced cytotoxicity when compared with control mock transfected cells in a
72-h methylene blue colorimetric assay (Fig. 5A). The FD5 clone demonstrated also significantly higher sensitivity to sFas-L- (not shown) and cisplatin-induced apoptosis (Fig. 5B) when
compared with CO4 cells.

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Fig. 5.
Increased sensitivity of HT29 cells
overexpressing FADD toward cisplatin-induced cytotoxicity and
apoptosis. HT29 cells were stably transfected with empty
pCDNA3 plasmid (CO4) or a pCDNA3-FADD construct (FD2 and FD5).
A, CO4 ( ), FD2 ( ), and FD5 ( ) clones were treated
with indicated concentrations of cisplatin for 72 h. Cell
viability was analyzed by the use of a methylene blue colorimetric
assay. Results are expressed as the means ± S.D. of three
independent experiments. Inset, Western blot analysis of
FADD protein expression in transfected clones. An anti-human Hsp70
antibody was used as a loading control. B, percentage of
apoptotic cells, determined by Hoechst staining, in FD5 (black
bars) and in CO4 (open bars) cell clones treated for
72 h with indicated concentrations of cisplatin. Results are
expressed as the means ± S.D. of three independent
experiments.
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Transient Expression of FADD Antisense, FADD-DN, or MC159 and/or E8
Constructs Prevents Cisplatin-induced Cytotoxicity--
To further
confirm the role of FADD in the cytotoxic activity of anticancer drugs,
we transiently transfected HT29 cells with constructs encoding either
antisense FADD or sense FADD-DN, MC159, or E8 proteins. FADD-DN is a
dominant negative construct that is capable of blocking Fas signal
transduction (25). MC159 and E8 are two viral proteins that inhibit
apoptosis at the level of FADD and procaspase-8, respectively (26).
Transient overexpression of both FADD antisense construct (Fig.
6A) and FADD-DN (Fig.
6B) prevented cisplatin-induced cytotoxicity in HT29 cells,
as measured by a methylene blue colorimetric assay. Transient
overexpression of either MC159 and/or E8 proteins demonstrated effects
similar to those of FADD antisense (Fig. 6C) and protected
cells from cisplatin-induced cytotoxicity.

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Fig. 6.
Inhibition of cisplatin-induced cytotoxicity
by FADD antisense, FADD-DN, or viral MC159 and/or E8 constructs.
HT29 cells were transiently transfected for 16 h with indicated
vectors: control vectors (pBK and pCI),
pBK-FADD-AS antisense vector (AS), pCI-FADD-DN
(FADD-DN), pCI-E8 (E8), and/or pCI-MC159
(MC159). Cells were then treated with 10 µM
cisplatin for 72 h, and cell viability was determined by the
methylene blue colorimetric assay. Results are expressed as the
means ± S.D. of three independent experiments.
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Antisense FADD or FADD-DN Prevent Anticancer Drug-induced
Apoptosis--
To determine whether the reduced cytotoxicity observed
after cisplatin treatment in HT29 cells transiently transfected by a
FADD antisense construct was related to a decreased induction of
apoptosis, HT29 cells were analyzed by fluorescence microscopy after
Hoechst staining of nuclear chromatin. These experiments demonstrated
that cisplatin-induced apoptosis was prevented by FADD antisense
construct in HT29 cells (Fig. 7). This
antiapoptotic effect was confirmed in VP16-treated HT29 cells.
Moreover, the ability of FADD antisense to protect from drug-induced
apoptosis was observed in HCT116 cells after treatment with cisplatin
(Fig. 7) and in U937 cells after treatment with etoposide or
vinblastine (Fig. 7). The FADD-DN construct was also capable of
preventing HCT116 cells from cisplatin-induced apoptosis (Fig. 7).

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Fig. 7.
Inhibition of anticancer drug-induced
apoptosis by FADD antisense or FADD-DN constructs. HT29, HCT116,
or U937 cells were transiently transfected for 16 h with indicated
vectors: control vectors (pBK and pCI),
pBK-FADD-AS antisense vector (AS), or pCI-FADD-DN and
treated as indicated with 10 µM CDDP or 50 µM VP16 for 72 h. U937 cells were treated with 50 µM etoposide for 4 h or with 10 ng/ml VB for 24 h. Apoptosis was determined by Hoechst staining. Each point is the mean
of 300 cells counted. Results are expressed as the means ± S.D.
of three independent experiments.
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DISCUSSION |
The molecular mechanisms involved during the course of apoptosis
upon exposure to anticancer drugs are still poorly understood but are
of major importance for the understanding of tumor cell killing.
Induction of apoptosis occurs through multiple pathways and depends on
the stimulus. We have previously shown that anticancer agents could
increase Fas receptor at the tumor cell surface by enhancing Fas gene
expression. The increase in Fas receptor was functional, and
drug-treated tumor cells were rendered more sensitive to Fas-mediated
apoptosis (18). Whether interaction of Fas receptor with its ligand
plays a role in anticancer drug-mediated cytotoxicity remains a
controversial issue (21). Here, we show that the antagonistic anti-Fas
antibody ZB4 and the Fas-IgG molecule, used at a concentration that
inhibits Fas-mediated and radiation-induced apoptosis (27), have no
effect on anticancer drug-induced apoptosis in three different cell
lines. Moreover, we failed to detect any modification of Fas-L
expression on the plasma membrane of tumor cells exposed to cytotoxic
agents (27, 28). In accordance with previously reported observations in
other cell systems (21, 22, 29), these results argue against a role for
a Fas receptor-Fas-L interaction in drug-induced cell death.
Fas signaling pathway has been shown to be triggered by Fas
trimerization in the absence of Fas-L in cells exposed to UV
irradiation (24, 30). In this situation, the adaptor protein FADD is
recruited to Fas receptor through its death domain (7-10), followed by
an interaction of the death receptor domain of FADD with the homologous domain of procaspase-8 leading to the activation of the proteolytic cascade of caspases (11). Confocal laser scanning microscopy and
coimmunoprecipitation studies indicated that anticancer drugs could
induce Fas receptor aggregation and FADD association to Fas.
Analyses of lymphocytes from FADD
/
mice have recently demonstrated
the prominent role of the FADD/procaspase-8 pathway in cell death
triggered by either Fas-L or agonistic anti-Fas antibodies (14). The
influence of FADD on the cell fate depends on the cell system and the
stimulus. FADD exhibits a Fas-L-independent proapoptotic activity in
MCF-7 breast carcinoma cells (9), whereas its overexpression promotes
the growth of lymphoid T cells (31). We show here that FADD plays a
role in drug-induced apoptosis. We obtained two HT29 cell clones that
stably overexpressed FADD, indicating that a limited 2-fold increase in
FADD protein expression was not sufficient to trigger apoptosis in
these cells. These FADD overexpressing clones were more sensitive to
drug-induced apoptosis and cytotoxicity than control cells. A role for
FADD in drug-induced cell death was further demonstrated by the
decreased toxicity of cytotoxic agents in HT29 cells in which FADD
expression was reduced by transient expression of an antisense
construct. Similar results were obtained by inhibiting the
FADD/procaspase-8 pathway at different levels using transient
expression of viral proteins (26) and FADD-DN dominant negative
construct (25). Altogether our data indicate that anticancer drugs can
induce Fas receptor clustering in a Fas-L-independent fashion and
recruit FADD to Fas receptor in the cytotoxic process leading to apoptosis.