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NEOPLASIA
From INSERM E9910, Institut Claudius Regaud, Toulouse;
and INSERM U517/EPHE, Faculté de Pharmacie, Dijon, France.
Leukemic CD34+ immature acute myeloid leukemia (AML)
cells express Fas receptor but are frequently resistant to Fas
agonistic reagents. Fas plays an important role in T-cell-mediated
cytotoxicity, and recently it has been suggested that altered Fas
signaling may contribute to drug resistance. Therefore, Fas resistance
could be one of the mechanisms by which AML progenitors escape
chemotherapy or T-cell-based immune intervention. However, the
molecular mechanism of Fas resistance in AML cells has not been
identified. Fas signaling can be interrupted at 3 mains levels: Fas
clustering, alteration of death-inducing-signaling-complex (DISC)
formation, and effector caspase inhibition of downstream
caspase-8. This study shows that in the Fas-resistant
CD34+CD38 Fas (APO-1/CD95) is a 45-kd membrane protein that
belongs to the tumor necrosis factor (TNF)-nerve growth factor
receptor family, a group of type 1 transmembrane
receptors.1 Mutational analysis of Fas and the human TNF
receptor (TNFR-1) proteins demonstrates that the cytoplasmic domains
share a homologous region necessary to transduce the apoptotic signal.
This conserved region of approximately 70 amino acids was, therefore,
designated as the death domain (DD). The only known physiological
ligand of Fas, Fas-L (CD95L), belongs to the family of TNF-related
cytokines.2 Fas-L is synthesized as a transmembrane
molecule, and soluble Fas-L trimers can be generated through processing
by a metalloprotease.3,4 Engagement of Fas by agonistic
anti-Fas antibodies or by Fas-L triggers apoptosis in a variety of cell
types. However, only membrane-bound or multimerized Fas-L induces cell
death.3,4 Moreover, ligand-dependent activation of Fas
death pathway requires the oligomerization of Fas receptor, but
ligand-independent activation can occur on Fas aggregation induced by
Fas overexpression or treatment with anticancer drugs or
radiation.5-9 Clustering of Fas recruits Fas-associated
death domain (FADD)-containing protein, which is a bipartite molecule with a death effector domain (DED) at the amino terminus and a DD at
the carboxyl terminus. FADD binds to Fas through a DD-DD interaction
and recruits the DED-containing procaspase-8 through a DED-DED
interaction. The formation of this death-inducing signaling complex
(DISC) results in caspase-8 activation, believed to be the first step
of a proteolytic cascade that triggers the activation of other caspases
such as caspase-3, -7, and -6.10,11 Although other cell
death pathways could be initiated from Fas
activation,12-14 analysis of lymphocytes from
FADD Normal CD34+ hematopoietic cells, including the most
immature CD34+CD38 CD34+CD38 Cell cultures
Reagents
Fas clustering and confocal analysis For the detection of Fas monomer aggregation, KG1a and U937 cells were treated or not treated with 0.5 µg/mL Fas-L for 4 hours, fixed for 10 minutes in 3% paraformaldehyde, and washed twice with phosphate-buffered saline (PBS) for 10 minutes. After 15-minute pre-incubation with 2% bovine serum albumin, cells were incubated for 2 hours at room temperature with or without anti-Fas monoclonal antibody (IgG1 clone ZB4, 1/100) diluted in PBS containing 1% bovine serum albumin. Nonimmune mouse IgG1 was used as a negative control. Samples were then washed in PBS and incubated for 45 minutes with FITC-conjugated goat anti-mouse monoclonal antibody. Subsequently, cells were fixed in paraffin on slides and were examined with a confocal imaging system (Zeiss, Oberkochen, Germany) scanning assembly incorporating argon and helium-neon lasers coupled to a Zeiss Axiovert 100 fluorescence microscope.Western blot analysis Exponentially growing cells were pre-incubated in the presence or absence of inhibitors and then treated by CH11 monoclonal antibody for different time periods. Cells were washed twice in serum-free medium, centrifuged, and lysed in RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA, 1 mM dithiothreitol [DTT], 2 µg/mL leupeptin, 2 µg/mL aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]) for 20 minutes on ice, followed by centrifugation at 10 000g for 15 minutes. Protein concentration in the supernatants was determined as previously described.36 For each lysate, 40 µg total protein was boiled for 5 minutes at 95°C in the presence of 3% -mercaptoethanol. Proteins were separated on
12.5% (wt/vol) SDS-polyacrylamide gel electrophoresis (PAGE) and were
transferred electrophoretically onto nylon membranes (Hybond-C extra;
Amersham Life Science, Cergy-Pontoise, France). Nonspecific binding
sites were blocked in 10 mM Tris-buffered saline containing 0.1%
Tween-20 and 10% nonfat milk. Membranes were then incubated overnight
at 4°C with specific primary monoclonal antibody diluted at an
appropriate concentration in 10 mM Tris-buffered saline containing
0.1% Tween-20 and 1% nonfat milk. Membranes were then washed 5 times
at room temperature, and bound immunoglobulin was detected with
anti-isotype monoclonal antibody coupled to horseradish peroxidase
(Beckman-Coulter). The signal was visualized by enhanced
chemiluminescence (Amersham, Buckinghamshire, United Kingdom) and autoradiography.
DISC formation analysis Exponentially growing cells (100 × 106) were incubated with 1 µg/mL Fas-L-FLAG (Alexis, San Diego, CA) and 1 µg anti-FLAG monoclonal antibody (Sigma, Saint-Quentin-Fallavier, France) for 15 minutes or 1 hour. Cells were then centrifuged and lysed in lysis buffer (0.2% NP-40, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM sodium vanadate, 10% glycerol, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 0.1 mM PMSF) before protein A-Sepharose was added. Immunoprecipitates were washed 3 times in lysis buffer without protease inhibitors before SDS-PAGE and Western blot analysis.Caspase-8 activity assay Caspase-8 colorimetric activity assay (R&D Systems, Abingdon, United Kingdom) was performed according to the manufacturer's recommendations. Briefly, exponentially growing cells treated by CH11 monoclonal antibody (2 µg/mL) for 4 hours were collected by centrifugation. Lysis buffer was added on the cell pellet, incubated on ice for 10 minutes, and centrifuged at 10 000g for 1 minute. For each lysate, 100 µg total protein was incubated with caspase-8 colorimetric substrate for 2 hours at 37°C. Cleavage of the substrate by caspase-8 was quantified spectrophotometrically at a wavelength of 405 nm.PKC  . Briefly, exponentially growing cells were cultured with sense or
antisense oligonucleotides 48 hours before CH11 monoclonal antibody (2 µg/mL) was added. After treatment, viability assay (Trypan blue
exclusion) and Western blot analysis or caspase-8 activity assay were
performed as described above.
Cytochemical staining Changes in cellular nuclear chromatin were evaluated by DAPI staining. Briefly, after CH11 treatment (2 µg/mL for 5 hours), cells were cytocentrifuged and fixed in 4% paraformaldehyde. Slides were then stained with 1 µg/mL DAPI and analyzed by fluorescence microscopy.Immunoprecipitation Cell lysates (5 × 106) were prepared in RIPA lysis buffer for 30 minutes on ice, sonicated, and centrifuged (15 minutes, 10 000g at 4°C). Supernatants were normalized for protein concentration, and each sample (1 mg protein) was immunoprecipitated with anti-PKC monoclonal antibody (4 µg) or
anti-FADD monoclonal antibody (2 µg) and collected by absorption to
protein G-Sepharose. Immunoprecipitates were washed 3 times in RIPA
buffer without protease inhibitors before analysis by SDS-PAGE and
Western blotting.
In vitro PKC monoclonal antibody and collected by absorption to
protein G-Sepharose. Immunocomplexes bound to protein G-Sepharose
were washed in lysis buffer without PMSF and subsequently were
resuspended in reaction buffer (20 mM HEPES, 1 mM DTT, 10 mM
MgCl2, 4 µg/mL phosphatidylserine, 20 µM cold ATP). For
each sample, 10 µCi [ -32P] ATP (6000 Ci/mmol
[222 TBq/mmol]; ICN, Orsay, France) and 1 µg FADD agarose or 3 µg
histone H1 were added. Samples were then incubated for 5 minutes at
32°C. The reaction was terminated by the addition of protein loading
buffer. Proteins were separated on 10% SDS-PAGE, and the gel was
subjected to autoradiography. In parallel, an aliquot of each sample
was analyzed by Western blot using anti-PKC monoclonal antibody to
quantify immunoprecipitated proteins.
Par-4 transfection in KG1a cells Exponentially growing cells were transfected by a plasmid containing full-length Par-4 cDNA sequence (kindly gift from M. T. Diaz-Meco, Madrid, Spain) using Effectene transfection reagent (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. Clones were further selected for Par-4, CD34+, CD38 expression by Western blot analysis or flow cytometry.
Statistics Quantitative experiments were analyzed using Student t test. All P values resulted from the use of 2-sided tests.
Fas expression and function in KG1a cells KG1a, U937, and Jurkat cells were treated with increasing concentrations of CH11 monoclonal antibody for 30 minutes in cold medium, then were stained by an indirect immunofluorescence technique using phycoerythrin-labeled goat-anti-mouse immunoglobulin (Ig)M. Fluorescence was evaluated by flow cytometry for each cell line. Saturating concentrations ranged between 0.5 and 2 µg/mL, depending on the cellular model. At the saturating dose of 2 µg/mL, KG1a cells displayed mean fluorescence intensity similar to, if not higher than, that of Fas-sensitive Jurkat or U937 cells (data not shown).KG1a cells were then treated with various doses of CH11 (2-10 µg/mL)
in supplemented Iscoves modified Dulbecco medium culture for 24, 48, and 72 hours. Cell viability was measured by Trypan blue dye exclusion
assay. CH11 monoclonal antibody, at a 2 µg/mL dose, induced only a
modest, though significant, growth inhibitory effect on KG1a cells
compared to IgM-isotypic control-treated cells (Figure
1A). Higher doses (up to 10 µg/mL) were
also inefficient for inducing KG1a cell death (data not shown).
However, CH11-treated Jurkat and U937 cells rapidly died, the former
more sensitive than the latter (Figure 1B). Morphologic examination
after DAPI staining showed typical features of apoptosis in
CH11-treated Jurkat and U937 cells, whereas CH11-treated KG1a cells
displayed no morphologic changes (data not shown). Similar results were obtained with recombinant human Fas-L used at various doses (0.2-2 µg/mL) (data not shown). These results confirmed that despite high
level of Fas expression, KG1a cells were resistant to Fas-induced apoptosis.
Fas clustering in KG1a cells We addressed whether Fas ligation could induce Fas receptor aggregation in KG1a cells. To resolve this question, we used an immunofluorescence technique coupled to confocal microscopy analysis as reported elsewhere.8 In these experiments, KG1a cells were or were not stimulated with Fas-L (0.5 µg/mL) for 4 hours, fixed with 4% paraformaldehyde, incubated with ZB4 murine anti-Fas monoclonal antibody or nonimmune mouse IgG1 (data not shown), and stained by FITC-labeled goat-anti-mouse IgG. For this study, ZB4 monoclonal antibody was preferred to CH11 because, unlike CH11, this antibody recognized a Fas epitope distinct from the Fas-L binding site. Jurkat (data not shown) and U937 cells were used as controls. Confocal laser microscopy showed that though untreated KG1a cells exhibited diffuse staining of Fas (Figure 2A), stimulation of cells with Fas-L resulted in Fas aggregation, enabling a dense, patchy staining that was primarily membrane localized (Figure 2C). Similar findings were found in the Fas-sensitive cell lines, as shown in Figure 2B and D, for U937 cells. Because Fas oligomerization appeared to be functional in Fas-activated KG1a cells, we hypothesized that the interruption of Fas signaling was situated immediately downstream of the Fas receptor and that in these cells, for example, some regulators interfered with DISC formation and caspase-8 activation.
Caspase-8 activation in KG1a cells In preliminary experiments, whole-cell lysates were examined by immunoblot analysis and demonstrated that the adapter protein FADD (27-28 kd), procaspase-8 (53-55 kd), and procaspase-3 (32 kd) were present in KG1a cells at a level similar to that of Jurkat cells (Figure 3A). These results showed that the proteins that potentially constitute DISC ie, Fas, FADD, and
caspase-8are present in KG1a cells.
We next determined whether Fas ligation could lead to the formation of a functional DISC. As shown on Figure 3B, DISC formation was well detected in Fas-L-treated Jurkat cells at 15 minutes. In contrast, incomplete DISC formation was detected in Fas-L-treated KG1a cells because only FADD was observed in the complex. This result suggested that in Fas-L-treated KG1a cells, the defect of DISC formation was caused by the absence of procaspase-8 recruitment. Finally, we examined the generation of procaspase-8 cleavage products in KG1a cells treated with CH11 monoclonal antibody. Therefore, KG1a cells were treated with CH11 (2 µg/mL) for 24 and 48 hours, after which whole-cell lysates were subjected to immunoblotting with a mixture of antibodies directed against procaspase-8 and its p20 and p10 cleavage products. As shown in Figure 3, whereas exposure to CH11 monoclonal antibody resulted in procaspase-8 proteolysis in Fas-sensitive Jurkat cells (Figure 3C), there was no generation of cleavage products in CH11-treated KG1a cells (Figure 3D). The lack of caspase-8 activation may explain why in KG1a cells, Fas ligation was unable to generate caspase-3 cleavage intermediates (Figure 3F). Together these results suggested that in KG1a cells, the lack of Fas-induced apoptosis was related to the presence of negative regulators that interfere with DISC formation and subsequent inhibition of caspase-8 activation. Among different parameters, we speculated that in these cells, PKC activity might play an important role in regulating the formation of functional DISC and Fas-mediated cell death. This was investigated by evaluating the capacity of chelerythrin or calphostin C, 2 known PKC inhibitors, to restore Fas-induced cytotoxicity. Effect of PKC inhibitors on Fas-mediated cytotoxicity in KG1a cells KG1a cells were pretreated with either chelerythrin (20 µM) or calphostin C (50 nM) for 1 hour, then were incubated in the presence of CH11 monoclonal antibody for 4 hours. Cell viability was measured by Trypan blue exclusion assay. Under these conditions, neither chelerythrin, calphostin C, nor CH11 monoclonal antibody used alone influenced KG1a cell viability (data not shown). As shown in Figure 4A, CH11 monoclonal antibody induced a rapid loss of viability with 50% of residual viable cells at 4 hours in the chelerythrin-pretreated population. In addition, in chelerythrin-pretreated cells, CH11 treatment restored caspase 8 activity (Figure 4B). However, cotreatment with calphostin C and CH11 for 4 hours did not affect KG1a cell viability (Figure 4A) or caspase-8 activity (Figure 4B). Chelerythrin and calphostin C are known to target distinct sites of PKC. Indeed, the former interferes with the catalytic site, which is present in all PKC isoforms, whereas the latter acts at the regulatory site of classical and novel PKC isoforms.37,38 Therefore, the fact that chelerytherin, but not calphostin C, could overcome Fas resistance in KG1a cells suggested that atypical or  PKC isoforms, but not classical ( , ,
), or novel ( ,  ,  ,  , µ) isozymes, interfere with Fas signaling. Based on previous studies that have extensively documented the role of PKC as a potent negative regulator of apoptosis, including TNF-induced apoptosis, in different cellular
models,39,40 we have speculated that this PKC isoform
might play an important role in Fas resistance of KG1a cells.
Effect of PKC in Fas resistance, KG1a cells were
exposed to the action of antisense oligonucleotides directed against
PKC and then were treated or not treated by CH11 monoclonal antibody. At first, 48-hour oligonucleotide pretreatment had no effect
on cell viability as observed by Trypan blue exclusion assay (data not
shown). However, antisense, but not sense, oligonucleotide dramatically
decreased PKC expression (Figure 5A)
and, in parallel, facilitated CH11-induced cytotoxicity. Indeed, though
CH11 was unable to induce significant cytotoxicity in KG1a cells
treated with sense oligonucleotide, this antibody induced a cytotoxic effect in PKC antisense oligonucleotide-treated cells (Figure 5B).
Moreover, in these conditions, CH11 induced caspase-8 activity (Figure
5C) and apoptosis (Figure 5E). These findings suggested that decreased
PKC expression resulted in the restoration of functional DISC and
activation of downstream Fas death pathway.
PKC in Fas-resistance of KG1a
cells, we investigated the influence of Par-4 (prostate apoptosis response-4), a known specific regulator of PKC.41,42
Western blot analysis revealed that KG1a cells expressed no detectable Par-4 protein, whereas Jurkat cells displayed high Par-4 level (Figure
6A). Hence, KG1a cells were stably
transfected by a plasmid containing the full-length Par-4 cDNA
sequence. Ten clones were obtained, for which only 2 (clones KG1a/G8
and KG1a/G9) had an immature phenotype (CD34+,
CD38) such as the parental KG1a cell line. Par-4
overexpression in the KG1a/G8 subclone (Figure 6A) resulted in a
noticeable reduction of PKC activity compared to KG1a cells (Figure
6B), whereas it did not influence PKC expression (Figure 6C).
Moreover, in KG1a/G8 cells, CH11 induced the activation and the
cleavage of caspase-8 and, thus, cytotoxicity and apoptosis (Figure
7). These results suggested that in KG1a
cells, low Par-4 expression level and subsequent PKC overactivity
played an important role in the lack of DISC formation.
Interaction between PKC inhibition restored Fas-induced caspase-8
activation in KG1a cells suggested that PKC might interact with DISC
components. To test this hypothesis, whole-cell extracts were subjected
to immunoprecipitation with anti-FADD antibody, and immunoprecipitates
were blotted with anti-PKC monoclonal antibody. PKC was found to
interact with FADD (Figure 8A); in parallel, PKC immunoextracts prepared from wild-type KG1a cells were
able to phosphorylate FADD-agarose complexes (Figure 8B). Furthermore,
this phosphorylation is dramatically reduced in Par-4 overexpressed
KG1a-G8 cellular extracts (Figure 8B).
Our study shows that in KG1a cells, Fas activation results in Fas
aggregation, incomplete DISC formation leading to a defect in caspase-8
activation. However, pretreatment with chelerythrin, an inhibitor of
all types of PKC isozymes, and, more specifically PKC The mechanism by which PKC Oncogenic Ras and growth factors including platelet-derived growth
factor or nerve growth factor (TrkA/NGF) may enhance PKC In this study we also showed that in KG1a cells, the lack of Par-4
expression plays an important role in Fas resistance. Par-4 interacts
with the regulatory domain of PKC To conclude, our study shows that in myeloid leukemic cells, PKC
We thank Dr M. T. Diaz-Meco and Dr J. Moscat (Madrid, Spain) for the kind gift of Par-4 cDNA, and Dr C. Bezombes-Cagnac for helpful discussions.
Submitted April 17, 2001; accepted August 9, 2001.
Supported by the Association pour la Recherche contre le Cancer (grants 5526 and 5968). A.d.T. is the recipient of a grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur, et de la Recherche.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anne Quillet-Mary, INSERM E9910, Institut Claudius Regaud, 20 rue du Pont St Pierre, 31052 Toulouse, France; e-mail: quillet_mary{at}icr.fnclcc.fr.
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
isoform in Fas resistance of
immature myeloid KG1a leukemic cells
Top
Abstract
Introduction
) or IgM (
). (B)
Jurkat cells treated by CH11 (
) or IgM (
), U937 cells treated by
CH11 (
) or IgM (
). Results are the mean ± standard
deviation of 3 independent experiments.
) or IgM (
), KG1a/G8 cell viability
was evaluated by Trypan blue exclusion assay. Results are the mean ± SD of 3 independent experiments. *P < .05. (D, E)
Morphology of CH11-treated cells was analyzed by fluorescence
microscopy after DAPI staining. (D) KG1a cells. (E) KG1a/G8
cells.
) fetal liver cells.
Exp Hematol.
1999;27:1428-1439