From the Department of Laboratory Medicine and
Pathology and ¶ Oncology, University of Alberta, Edmonton, Alberta
T6G 2S2, Canada and
Tumorimmunology Program, German Cancer
Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
Received for publication, November 4, 2002
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ABSTRACT |
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Fas, upon cross-linking with Fas ligand (FasL) or
Fas agonistic antibody, transduces apoptotic yet also proliferative
signals, which have been implicated in tumor pathogenesis. In this
study, we investigated the molecular mechanisms that control
Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11,
induced apoptosis in sensitive glioma cells through caspase-8
recruitment to the Fas-mediated death-inducing signaling complex (DISC)
where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell
growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like
interleukin-1 Fas (CD95 or APO-1) induces apoptosis upon stimulation with
FasL1 (CD95L or APO-1L) or an
agonistic Fas antibody such as CH-11 (1-4). Fas is a type I
transmembrane protein that has multiple cysteine-rich repeats in the
extracellular domain and an intracellular motif termed a death domain
(DD). Binding of FasL or CH-11 to the Fas extracellular domain induces
trimerization of Fas, resulting in the recruitment of the intracellular
adapter FADD (5, 6). FADD has a carboxyl-terminal DD and an
amino-terminal death effector domain (DED). Through its DED, FADD
recruits the DED-containing apoptosis-initiating proteases caspase-8
(7, 8) and caspase-10 (9-11) to the Fas receptor to assemble a DISC
(12). In the DISC, caspase-8 is cleaved through autoproteolysis
of caspase-8 molecules in close proximity (13). Active caspase-8
subunits are released into the cytoplasm to cleave downstream effector
caspases such as caspase-3 (14), which subsequently cleaves its
substrates such as DNA fragmentation factor 45 (DFF45) (15), to execute programmed cell death.
Recently, the recruitment of other DED-containing proteins to the
Fas-mediated DISC, which has been described, modulates DISC functions. These include a family of virus-encoded proteins referred to
as v-FLIP (16, 17). v-FLIP contains two DEDs that can bind to the
Fas/FADD complex to inhibit Fas-mediated apoptosis by interfering with
the recruitment of caspase-8 to the DISC. A mammalian cellular homolog
of v-FLIP is termed c-FLIP (18), CASH (19), CASPER (20), CLARP (21),
FLAME1 (22), I-FLICE (23), MRIT (24), and Usurpin (25). These studies,
however, have generated controversy as to the functions of c-FLIP in
apoptosis. Some groups have described it as pro-apoptotic (19-21, 24),
whereas others as anti-apoptotic (18, 22, 25). Recent analysis of
Fas-mediated DISC in c-FLIP-transfected BJAB cells has shown that
c-FLIP proteins are recruited to the Fas-mediated DISC to inhibit
caspase-8 cleavage (26, 27), which supports the role of c-FLIP as an
anti-apoptotic molecule.
The c-FLIP gene is composed of 13 exons that are clustered within
~200 kilobases within the caspase-8 and caspase-10 genes on human
chromosome 2q33 to 34 (25, 28). c-FLIP is expressed as four main
mRNA splice variants but only two forms of protein in human tissues
(18, 20). The short form protein (c-FLIPS,
Mr ~28) contains two DEDs and is structurally
related to v-FLIP; the longer form (c-FLIPL,
Mr ~55) is structurally similar to caspase-8 and contains two DEDs and a caspase-like domain that lacks catalytic activity (18). c-FLIPL is expressed in many tissues, but
c-FLIPS is found mainly in lymphatic tissue (18).
Expression of c-FLIP mRNA and proteins is regulated by
mitogen-activated protein kinase kinase in T lymphocytes (29) and by
phosphatidylinositol 3-kinase in tumor cells (30). Here we show
calcium/calmodulin-dependent protein kinase II (CaMK II)
regulates c-FLIP expression and phosphorylation in malignant glioma cells.
Many tumor cells express Fas yet are resistant to Fas-mediated
apoptosis (31, 32). An earlier observation, that c-FLIP is
overexpressed in human melanomas, suggests that c-FLIP may inhibit
Fas-mediated apoptosis in these tumors (33). Recent studies in
vivo have shown that high c-FLIP expression in transfected tumor
cells promotes tumor growth and facilitates tumor immune escape (34,
35), which further strengthens the concept that c-FLIP up-regulation is
implicated in tumor pathogenesis. To investigate the molecular
mechanisms in c-FLIP-mediated regulation of Fas signaling, we examined
Fas-mediated DISC and its modulation in glioma cells and showed that
c-FLIP regulates Fas signaling through its recruitment to the
Fas-mediated DISC. Furthermore, we have demonstrated that CaMK II
regulates c-FLIP expression and phosphorylation, thus modulating
Fas-medicated signaling in malignant glioma cells.
Human Glioma Cell Lines, Antibodies, and Reagents--
The human
malignant glioma cell lines LN-18, LN-215, LN-464, and LN-443 (gifts
from N. De Tribolet, Lausanne, Switzerland) and U343MG and T98G
(American Type Culture Collection) were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(Invitrogen). Primary monoclonal antibodies used in the study included
anti-human Fas clone 13, FADD, and CaMK II (Transduction Laboratories,
Lexington, KY), Fas CH-11, caspase-8, and caspase-10 (Medical & Biological Laboratories, Nagoya, Japan), DFF45 (StressGen, Victoria,
BC, Canada), and c-FLIP NF6 clone (26). Primary polyclonal rabbit
antibodies included anti-human caspase-3, c-FLIP, and ERK1/2
(StressGen, Victoria, BC, Canada), phosphothreonine (Research
Diagnostics, Inc. Flanders, NJ), and PED serum (36). Secondary
antibodies included HRP-conjugated goat anti-mouse IgG2b and IgG1
antibodies (Southern Biotech, Birmingham, AL) and HRP-conjugated goat
anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove,
PA). [32P]Orthophosphate (Amersham Biosciences),
phosphate-free Dulbecco's modified Eagle's medium (Invitrogen), the
CaMK II activity assay kit (Upstate Biotechnology, Lake Placid, NY),
and [ Cell Death Assay and Cleavage of Caspases, DFF45, and
c-FLIP--
For cell death, cells were seeded in 96-well plates at
2×104 cells/well and treated at 37 °C for 24 h
with 1 µg/ml anti-Fas CH-11 or 100 µM KN-93 alone or in
combination. Cell death was determined by crystal violet assay (37).
For cleavage of caspases, DFF45, and c-FLIP, subconfluent cells were
treated with 1 µg/ml CH-11 either in the presence or absence of 100 µM KN-93 at 37 °C for the times indicated. At each
time point, cells were lysed in lysis buffer (1%Triton X-100, 150 mM NaCl, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mixture). After centrifugation at 16,000 × g for 15 min at 4 °C, supernatants were
subjected to Western blotting.
DISC Analysis by Immunoprecipitation--
Immunoprecipitation
for DISC analysis was performed according to a modified protocol as we
previously reported (38). 5 ×107 cells were stimulated
with 1 µg/ml CH-11 (mouse IgM) for 30 min at 37 °C and then lysed
for 30 min on ice with lysis buffer. In unstimulated controls, the
cells were lysed, and 1 µg of CH-11 was added to 1 ml of cell lysates
to immunoprecipitate non-stimulated Fas receptors. The soluble fraction
was immunoprecipitated with 20 µl of goat anti-mouse
IgM-agarose overnight at 4 °C and analyzed by Western blotting and
two-dimensional PAGE immunoblotting.
Two-dimensional-PAGE, 32P-Labeling, and
Dephosphorylation--
For each sample, 107 cells were
either treated with 100 µM KN-93 for 24 h at
37 °C or left untreated and lysed in 40 mM Tris-HCl, pH
8.0, 1% Triton X-100, 65 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, complete protease
inhibitor mixture. After centrifugation, the supernatants were
precipitated with acetone. The pellets were dissolved in 9.8 M urea, 2% CHAPS, 0.5% pH 3-10 non-linear IPG buffer (Amersham Biosciences) 65 mM dithiothreitol, applied
by rehydration in 13-cm DryStrips (pH 3-10 non-linear), and
electrofocused with IPGphorTM System following the
manufacturer's protocol (Amersham Biosciences). The strips were
equilibrated with 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 10 mg/ml dithiothreitol and subjected to
SDS-PAGE followed by transfer to a nitrocellulose membrane (Bio-Rad)
and immunoblotting. For labeling of cellular phosphoproteins, cells
were incubated for 1 h in phosphate-free Dulbecco's modified Eagle's medium (DMEM) and then labeled for 4 h in phosphate-free DMEM supplemented with 0.5 mCi/ml 32P. Cells were lysed,
and cell extracts were subjected to two-dimensional-PAGE followed by
autoradiography and immunoblotting, as described above. For
dephosphorylation of cellular phosphoproteins, cell lysates obtained
from cells were treated with 0.2 units/ml acid phosphatase for 3 h
at 37 °C and subsequently subjected to two-dimensional PAGE immunoblotting.
Western Blot and Two-dimensional PAGE Immunoblot--
For
Western blot, cell extracts and immunoprecipitated DISC samples were
separated through SDS-PAGE and transferred to nitrocellulose membranes.
The membranes were blocked with blocking buffer (5% nonfat dry milk
(for phosphoprotein detection, dry milk was substituted with 3% bovine
serum albumin), Tris-buffered saline (20 mM Tris-HCl, pH
7.5, 500 mM NaCl), 0.5% Tween 20)) for 2 h at room
temperature and then incubated overnight at 4 °C with the following
primary antibodies diluted in blocking buffer: anti-caspase-8,
anti-DFF45 and anti-c-FLIP (1:1000), anti-caspase-3 and anti-PED
(1:5000), anti-caspase-10, anti-Fas clone 13, anti-CaMK II, and
anti-ERK1/2 (1:500), anti-phosphothreonine (1:300), anti-FADD (1:250),
and anti-c-FLIP NF6 (1:10). The membranes were washed in Tris-buffered saline, 1% Tween 20 and incubated for 2 h at room temperature with the following secondary antibodies diluted in Tris-buffered saline, 1% Tween 20: anti-mouse IgG2b-HRP, anti-mouse IgG1-HRP (1:20,000), and anti-rabbit IgG-HRP (1:5000). The blots were washed and
developed by chemiluminescence (Amersham Biosciences).
CaMK II Activity Assay and CaMK II cDNA
Transfection--
CaMK II activity was assayed in cell lysates using
CaMK II assay kits following the manufacturer's protocol (Upstate
Biotechnology). For transfection, CaMK II cDNA (Stratagene, La
Jolla, CA) was subcloned into pcDNA3.1 expression vector using
EcoR I and HindIII restriction sites.
Transfection of the pcDNA3.1 expression vector containing CaMK II
cDNA into glioma cells was accomplished using the LipofectAMINE
method following the manufacturer's protocol (Invitrogen). After
transfected for 48 h, the cells were subjected to cell death assay
and Western blot analysis for cleavage of caspases and DFF45.
Caspase-8 Is Recruited to the Fas-mediated DISC to Initiate
Apoptosis--
Malignant glioma cells express cell surface Fas and are
susceptible to agonistic Fas antibody-induced apoptosis (31). To illustrate the signal events in Fas-mediated apoptosis, we first analyzed the Fas-mediated DISC in sensitive glioma cells. Three glioma
cell lines (U343MG, LN-18, T98G) were selected in the study for their
expression of cell surface Fas, as shown by flow cytometry, and
sensitivity to agonistic Fas antibody CH-11, as determined by crystal
violet assay (data not shown). We stimulated these cells with 1 µg/ml
CH-11 (mouse IgM) for 30 min at 37 °C, and the Fas-mediated DISC was
immunoprecipitated using goat anti-mouse IgM-agarose and examined by
Western blotting to identify the proteins that are incorporated
into the Fas-mediated DISC. In unstimulated controls, cells were lysed,
and CH-11 was then added to the lysates to immunoprecipitate
unstimulated Fas. Total cell protein extracts from unstimulated cells
were also included in the Western blot analysis to examine endogenous
expression of the proteins.
Western blots detected one p45 protein band of Fas, one p25 protein
band of FADD, four protein bands of caspase-8, and four protein bands
of caspase-10 in the Fas-mediated DISC in CH-11 stimulated cells (Fig.
1A). Two caspase-8 precursor
proteins (p55 and p53) were detected in both the cell extracts and the
DISC, but two caspase-8 first-step cleavage products (p43 and p41) were detected only in the DISC. Similarly, two caspase-10 precursor proteins
(p59 and p54) and two caspase-10 first-step cleavage products (p47 and
p43) were detected in the DISC. Next we examined the Fas-medicated DISC
to look for the other two DED-containing proteins; c-FLIP and
phosphoprotein enriched in diabetes/phosphoprotein enriched in
astrocytes-Mr 15 (PED/PEA-15) (36). The long
form p55 c-FLIPL and the short form p25 c-FLIPS
were endogenously expressed in the cells as shown by Western blot
analysis of the cell extracts (Fig. 1A), but they were not
detected in the Fas-mediated DISC in these CH-11-sensitive cells (Fig.
1A). PED/PEA-15 was reported to inhibit Fas-mediated
apoptosis (39, 40). Western blots failed to detect PED/PEA-15
in the Fas-mediated DISC in these sensitive glioma cells (Fig.
1A).
Caspase-8 cleavage occurs in two consecutive steps in Fas-mediated
DISC, first-step cleavage to produce p12, p43, and p41 subunits and
second-step cleavage to generate prodomain and active p18 and p10
subunits (41) to cleave downstream caspase-3 p32 precursors into large
p20 and p17 and small p10 subunits (42). To examine this
caspase-8-initiated cascade, we analyzed cell extracts
collected from the cells exposed to 1 µg/ml CH-11 to look for
cleavage products. Western blots detected caspase-8 first-step cleavage
p43 and p41 products within 30 min and second-step cleavage p18
proteins within 60 min after CH-11 stimulation (Fig. 1B). Western blots also detected caspase-3 cleavage p20 and p17 subunits after CH-11 stimulation (Fig. 1B). Finally, we examined the
cell extracts for cleavage products of DFF45, a caspase-3 downstream substrate (15, 43). Two forms of DFF45, the long form DFF45 and
the short form DFF35, were endogenously expressed in the glioma cells and proteolytically cleaved upon CH-11 stimulation (Fig. 1B).
c-FLIP Proteins Are Recruited to the Fas-medicated DISC in
Resistant Glioma Cells--
Many glioma cell lines express cell
surface Fas yet are resistant to CH-11 and soluble FasL (31, 37). To
investigate molecular mechanisms of this resistance, we analyzed
Fas-mediated DISC in three glioma cell lines (LN-215, LN-443, LN-464)
that express Fas but are resistant to CH-11 (data not shown). In the
cells stimulated with 1 µg/ml CH-11, Western blots detected one p45 Fas band, one p25 FADD band, and four caspase-8 bands, p55 and p53
caspase-8 precursors and p43 and p41 caspase-8 first-step cleavage
products (Fig. 2A). These
findings indicated that FADD and caspase-8 are recruited to the
Fas-medicated DISC, where caspase-8 completes its first-step cleavage.
However, Western blot analysis failed to show caspase-8 second-step
cleavage products in these resistant cells treated with CH-11 (Fig.
2B), indicating that caspase-8 second-step cleavage is
inhibited in the DISC. Western blots also failed to detect caspase-3
and DFF45 cleavage products (Fig. 2B), which indicated that
caspase-8-initiated cascade is inhibited as well in the resistant
cells. Neither caspase-10 precursors nor its cleavage products was
detected in the Fas-mediated DISC (data not shown) due to the lower
levels of caspase-10 expression in these cells, as we previously
reported (38).
We further examined the Fas-medicated DISC to look for c-FLIP and
PED/PEA-15. The long form p55 c-FLIPL and the short form p25 c-FLIPS were endogenously expressed in these glioma
cells (Fig. 2A). However, Western blot analysis of the DISC
revealed three forms of c-FLIP proteins, a very weak band of p55
c-FLIPL, a new band of p43 c-FLIP, and a band of p25
c-FLIPS (Fig. 2A). These findings indicated that
c-FLIPL is recruited to the DISC and cleaved into an
intermediate p43 form, as previously reported in c-FLIP transfectants
(18, 26, 27). Caspase-8 first-step cleavage occurred in both
CH-11-sensitive and -resistant glioma cells (Figs. 1A and
2A), but it appeared that only resistant cells showed
simultaneous recruitment of caspase-8 and c-FLIPL to the DISC, resulting in c-FLIPL cleavage into a p43 intermediate
form (Fig. 2A). To further confirm this, we examined
c-FLIPL cleavage in the sensitive and resistant cell lines.
Indeed, Western blots detected p43 intermediate products in the
resistant cells (LN-215, LN-443, LN-464) but not in the sensitive cells
(U343MG, LN-18, T98G) after exposure to 1 µg/ml CH-11 (Fig.
2C). Western blots also detected PED/PEA-15 in the
Fas-mediated DISC (Fig. 2A). Simultaneous detection of
caspase-8 first-step cleavage products, c-FLIP, and PED/PEA-15
suggested that both c-FLIP and PED/PEA-15 proteins may be responsible
for inhibition of caspase-8 second-step cleavage in the Fas-mediated
DISC in the CH-11-resistant glioma cells.
c-FLIP and CaMK II Are Up-regulated in Resistant Glioma
Cells--
The differential recruitment of c-FLIP and PED/PEA-15 to
the Fas-mediated DISC suggests that c-FLIP and PED/PEA-15 proteins may
be up-regulated in the resistant glioma cells. Indeed, our earlier work
has shown that CaMK II up-regulates PED/PEA-15 expression and
phosphorylation and modulates TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in glioma cells (38). We
speculated that a similar mechanism might exist in the glioma cells,
which regulates c-FLIP expression and function. To test this
hypothesis, we first investigated whether treatment of the resistant
cells with KN-93, a CaMK II inhibitor, might restore CH-11 sensitivity.
LN-215, LN-443, and LN-464 cell lines were treated with 1 µg/ml CH-11
alone or in the presence of 100 µM KN-93 for 24 h.
Crystal violet analysis showed a consistent 20% cell growth in the
CH-11-stimulated cells (Fig.
3A). These resistant cell
lines, however, became sensitive to CH-11 in the presence of KN-93
(Fig. 3A). Kinetic analysis on Western blots showed
caspase-8 first-step and second-step cleavage products in the cells
exposed to CH-11 and KN-93 but not to KN-93 or CH-11 alone (Fig.
3B). Western blots also detected caspase-3 and DFF45
cleavage products (Fig. 3, C and D). These
results indicated that the CaMK II inhibitor KN-93 rescues CH-11
sensitivity in resistant glioma cells and that CH-11-induced cell death
in the resistant cells occurs through a caspase-8-initiated caspase
cascade, similar to the apoptotic signal pathway observed in the
sensitive glioma cells (Fig. 1).
Given that caspase-8 is able to complete two-step cleavage in resistant
cells after KN-93 treatment, we speculated that CaMK II might
up-regulate c-FLIP expression and, thus, modulate Fas-mediated apoptosis in the resistant cells. To test this, we first compared the
c-FLIP protein expression levels in the CH-11-sensitive and resistant
glioma cell lines. Western blot analysis showed that both
c-FLIPL and c-FLIPS were expressed at higher
levels in the resistant (LN-215, LN-443, LN-464) than in the sensitive
cell lines (U343MG, LN-18, T98G) (Fig.
4A) and that the
c-FLIPL and c-FLIPS expression levels were
markedly reduced in resistant cells after treatment with 100 µM KN-93 for 24 h (Fig. 4A). Next, we examined CaMK II protein expression in the glioma cells and showed that
CaMK II protein was highly expressed in CH-11-resistant cells as
compared with the sensitive cells (Fig. 4A). Finally, we
examined CaMK II activity to determine whether overexpressed CaMK II is biologically functional in glioma cells. CaMK II activity was measured
in a total of 10 µg proteins from each of the cell extracts using a
specific kinase substrate and a mixture of kinase inhibitors to block
the activity of other kinases. CaMK II activity was significantly higher in the resistant cell lines LN-215, LN-443, and LN-464 than in
the sensitive cell lines U343MG, LN-18, and T98G (Fig. 4B).
Treatment of the resistant cells with 100 µM KN-93 for
24 h markedly reduced CaMK II activity in the resistant cells
(Fig. 4B).
CaMK II Regulates c-FLIPL Phosphorylation and
Recruitment to the DISC--
CaMK II regulates c-FLIP expression to
modulate Fas-mediated apoptosis in glioma cells. Here, we further
proposed that CaMK II might regulate c-FLIP phosphorylation and
recruitment to the Fas-mediated DISC. To test this, we first subjected
the cell extracts to two-dimensional PAGE immunoblots to identify
isoforms of the c-FLIP proteins. Two-dimensional PAGE immunoblots
detected one c-FLIPS spot in either the sensitive or
resistant tumor cells (Fig.
5A), indicating that there is
only one isoform of c-FLIPS endogenously expressed in
glioma cells. In contrast, however, two-dimensional PAGE blots revealed
three isoforms of c-FLIPL, indicated as
c-FLIPLa, c-FLIPLb, and c-FLIPLc
(Fig. 5A). Two of the isoforms, c-FLIPLa and
c-FLIPLb, were present in both sensitive (U343MG, LN-18)
and resistant cell lines (LN-215, LN-443), but isoform
c-FLIPLc was detected only in the resistant cell lines (Fig. 5A).
Treatment of the resistant cells with 100 µM KN-93 for
24 h inhibited CaMK II activity (Fig. 4B) and
eliminated the expression of the isoform c-FLIPLc (Fig.
5A); the results suggested that c-FLIPLc might
be a phosphorylated protein. To test this hypothesis, we first treated
cell extracts of resistant cell lines LN-215 and LN-443 with acid
phosphatase for 3 h at 37 °C and then subjected the cell
extracts to two-dimensional PAGE immunoblots. The results showed that
acid phosphatase treatment eliminated the c-FLIPLc isoform
in the resistant cells (Fig. 5A). Next, we labeled the resistant cell lines LN-215 and LN-443 with
[32P]orthophosphate and subjected the cell extracts to
two-dimensional PAGE that was examined first by autoradiography and
then by immunoblotting with anti-c-FLIP antibody. Autoradiography of
the two-dimensional PAGE membrane revealed one 32P-labeled
spot among the three c-FLIPL isoforms (Fig. 5B)
and subsequent immunoblot analysis of the same membrane showed that the
32P-labeled spot was identical to c-FLIPLc
(Fig. 5B). In contrast, similar analysis of autoradiography
and immunoblots of two-dimensional PAGE failed to show any
phosphorylated spot of the c-FLIPS form (Fig.
5B).
CaMK II is a S/T protein kinase (44). To further confirm
c-FLIPLc phosphorylation, we examined cell extracts from
the sensitive U343MG and resistant LN-215 cells on two-dimensional PAGE
immunoblots using anti-phosphothreonine antibody. The same membranes
were then stripped of primary and secondary antibodies and analyzed using c-FLIP antibody. The c-FLIPLc spot, detected using
c-FLIP antibody, was also recognized by anti-phosphothreonine antibody (Fig. 6). Similar analysis showed an
absence of c-FLIPLc in CH-11-sensitive U343MG cells (Fig.
6). Taken together, these studies indicated that c-FLIPLc
is a phosphoprotein and is highly expressed in the resistant glioma
cells.
We showed that c-FLIPL was recruited to the Fas-mediated
DISC only in the resistant cells (Fig. 2A), and of three
isoforms of c-FLIPL, only c-FLIPLc was
expressed in the resistant cells (Figs. 5 and 6). These results
suggested that c-FLIPLc, but neither c-FLIPLa
nor c-FLIPLb, is recruited to the Fas-mediated DISC. Western blot analysis showed that immediately upon recruitment, c-FLIPL proteins were cleaved into p43 intermediate forms
in the DISC (Fig. 2A), and thus, we were not surprised by
the fact that two-dimensional PAGE immunoblots using anti-c-FLIP
antibody detected p43 intermediate form protein but not
c-FLIPL in the CH-11-induced DISC in LN-215 cells (Fig. 6).
The same membrane was then analyzed using anti-phosphothreonine
antibody, and the results showed that p43 intermediate form protein was
phosphorylated (Fig. 6); the results suggested that p43 intermediate
forms are generated from the phosphoprotein c-FLIPLc but
not from c-FLIPLa or c-FLIPLb.
CaMK II cDNA Transfection in Sensitive Glioma Cells Resulting
in Cell Resistance--
To further define the function of CaMK II in
modulation of Fas-mediated apoptosis in tumor cells, we transiently
transfected CaMK II cDNA in CH-11-sensitive U343MG and LN-18 cell
lines. Crystal violet analysis showed a near 100% cell death (Fig.
7A) in untransfected or vector
transfected U343MG and LN-18 cell lines upon stimulation with 1 µg/ml
CH-11 for 24 h. Western analysis revealed cleavage of caspase-8,
caspase-3, and DFF45 in these untransfected or vector-transfected cell
lines upon stimulation with 1 µg/ml CH-11 for 3 h (Fig.
7B). In contrast, transfection of these CH-11-sensitive
cells with CaMK II cDNA resulted in significantly increased
expression of CaMK II proteins, as demonstrated on Western blots (Fig.
7A), and remarkably decreased cell death, as shown by
crystal violet analysis (Fig. 7A). Western blot analysis
further showed a significantly decreased cleavage of caspase-8,
caspase-3, and DFF45 (Fig. 7B). CaMK II cDNA-transfected
cells were further exposed to 1 µg/ml CH-11 in the presence of 100 µM KN-93 to inhibit CaMK II activity. KN-93 treatment
restored the sensitivity of CaMK II cDNA-transfected cells to
CH-11-induced cell death (Fig. 7A) and cleavage of
caspase-8, caspase-3, and DFF45 (Fig. 7B). Collectively, the
results indicate that CaMK II plays a crucial regulatory role in
Fas-mediated apoptosis in tumor cells.
The Fas/FasL-signaling pathway is an essential process in the
regulation of programmed cell death and has been implicated in the
pathogenesis of immune system diseases and various malignancies (45).
Recent studies have provided several lines of evidence to support the
role of the Fas-signaling pathway in tumorigenesis of malignant
gliomas. Gliomas express both Fas and FasL, and glioma-derived FasL
induces apoptosis of Fas-positive T cells that infiltrate tumors, thus
facilitating tumor escape from the host immune system (46). FasL
stimulates gliomas to produce chemokines that modulate immune responses
(47). FasL or the Fas agonistic antibody CH-11 triggers apoptosis in
some glioma cells (48) but transduces proliferative signals in others
(49). In the present study, we analyzed the Fas-mediated DISC and
elucidated the molecular mechanisms that regulate the biological
functions of Fas-signaling pathways in human gliomas.
The signal transduction of Fas-mediated apoptosis has been well
characterized in transfectants and lymphocytes (45) but remains to be
established in non-transfected solid tumor cells. Here, we showed that
Fas-mediated apoptosis in sensitive glioma cells occurs through
recruitment of apoptosis-initiating caspase-8 and caspase-10 to the
Fas-mediated DISC. Caspase-8 completes its consecutive two-step
cleavage in the DISC, resulting in release of its active subunits into
the cytoplasm that initiate programmed cell death by subsequent
cleavage of downstream effector caspases such as caspase-3 (14, 42) and
caspase-3 substrates such as DFF45 (15, 43). However, many glioma cells
are resistant to Fas-mediated apoptosis. Analysis of the Fas-mediated
DISC provides a new molecular model for Fas-mediated resistance in
tumor cells. Apoptosis-initiating caspase-8 is recruited to the
Fas-mediated DISC and completes its first-step cleavage, but its
second-step cleavage is inhibited in the DISC. Detection of c-FLIP and
PED/PEA-15 molecules in the DISC suggests that these DED-containing
proteins may inhibit the second-step cleavage of caspase-8 and, thus,
prevent Fas-mediated apoptosis in tumor cells.
Recent studies of c-FLIP overexpression have provided two models for
c-FLIP modulation of the Fas-mediated DISC (26, 27). In the first
model, caspase-8 and c-FLIPL are simultaneously recruited to the DISC, where caspase-8 completes its first-step cleavage, whereas
c-FLIPL is cleaved into a p43 intermediate form. The p43 intermediate c-FLIPL remains in the DISC to interrupt
caspase-8 second-step cleavage, and, thus inhibit caspase-8-initiated
apoptosis. In contrast in another model, c-FLIPS is
recruited to the DISC to inhibit first-step cleavage of caspase-8.
Here, we demonstrated the p43 intermediate form in the Fas-mediated
DISC, which supports the c-FLIPL-mediated model of
regulation of Fas-mediated signaling. However, simultaneous detection
of c-FLIPS and caspase-8 first-step cleavage in the DISC
raises a question about the role of c-FLIPS in Fas
signaling in glioma cells. It also remains to be clarified why both
c-FLIP and PED are simultaneously recruited to the Fas-mediated DISC in
glioma cells.
There is now increasing evidence that Fas transduces proliferative
signals, but the signaling pathways remain poorly defined (50).
Overexpression of c-FLIPL has been reported to activate nuclear factor kappa B (NF- We have further identified three isoforms of c-FLIPL in
glioma cells. The three isoforms were separated by the difference in
their molecular weights and isoelectric points on two-dimensional PAGE.
A combined two-dimensional PAGE 32P autoradiography and
immunoblot analysis further revealed that one of the three isoforms was
phosphorylated. The phosphorylated c-FLIPL was expressed
only in resistant cells, and inhibition of CaMK II activity in the
resistant cells eliminated this phosphorylated isoform of
c-FLIPL and rendered the cells sensitive to CH-11-induced apoptosis. Furthermore, we showed that the p43 intermediate form of
c-FLIPL was phosphorylated, which suggested that the
phosphorylated c-FLIPL isoform is recruited to and cleaved
into a p43 intermediate form in the Fas-mediated DISC. The p43 form of
c-FLIPL remains bound in the DISC to inhibit
caspase-8-initiated apoptosis and, thus, switch the glioma cells from
apoptosis to proliferation.
CaMK II has recently been implicated in the modulation of
microcystin-induced apoptosis (56). Our earlier studies showed that
CaMK II regulates PED/PEA-15 phosphorylation and, thus, modulates TRAIL
(tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in glioma cells (38). We now provide several lines of
evidence that CaMK II up-regulates c-FLIP expression and
phosphorylation to modulate Fas-mediated signaling in the same tumor
cells. Both c-FLIPL and c-FLIPS were highly
expressed in resistant glioma cells, and treatment of these cells with
the CaMK II inhibitor KN-93 reduced the expression of both long and
short forms of c-FLIP and rescued the cell sensitivity to Fas-mediated
apoptosis. CaMK II protein and biological activity were up-regulated,
and KN-93 treatment eliminated the phosphorylated c-FLIPL
in the resistant cells, suggesting that CaMK II may regulate
c-FLIPL phosphorylation. Other kinases such as
mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase,
and protein kinase C have also been reported to regulate c-FLIP and
PED/PEA-15 to modulate apoptosis (29, 30, 37). Therefore, further
studies of these kinase-mediated signaling pathways will shed some
light on the complex regulatory mechanisms that control tumor cell
death and proliferation.
-converting enzyme (FLICE)-inhibitory protein) to the
Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in
glioma cells, but only the phosphorylated isoform was recruited to and
cleaved into a p43 intermediate form in the Fas-mediated DISC in
resistant cells. Calcium/calmodulin-dependent protein
kinase II (CaMK II) activity was up-regulated in resistant cells.
Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited
CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP
phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II
cDNA in sensitive cells rendered them resistant to CH-11. These
results indicated that CaMK II regulates c-FLIP expression and
phosphorylation, thus modulating Fas-mediated signaling in glioma cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol) (PerkinElmer Life
Sciences) were purchased from the commercial sources. Goat anti-mouse
IgM-agarose, KN-93, acid phosphatase, complete protease inhibitor
mixture, Triton X-100, CHAPS, and all other chemicals of analytical
grade were purchased from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (59K):
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Fig. 1.
DISC analysis of CH-11-sensitive glioma
cells. Each column represents one cell line as
indicated above the panels. The proteins detected
are indicated to the left, and Western blot (WB)
antibodies are indicated to the right. A, DISC
analysis. Anti-Fas CH-11 (1 µg/ml) was added to the cells either
after cell lysis in unstimulated cells ( ) or for 30 min before cell
lysis in stimulated cells (+). The samples were immunoprecipitated with
goat anti-mouse IgM-agarose and analyzed by WB. Cell extracts
(EX) were also included in the Western blot analysis for the
presence of the endogenous proteins in the cells. B,
cleavage of caspases and DFF45. The cells were treated with 1 µg/ml
CH-11 for the times indicated, and cell lysates were examined by
Western blot using anti-caspase-8, caspase-3, and DFF45 antibody,
respectively.
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[in a new window]
Fig. 2.
Fas-mediated DISC in CH-11-resistant glioma
cells. A, DISC analysis. The cell lines are indicated
above the panels. Cell extracts (EX)
and immunoprecipitated samples collected from unstimulated ( ) and
CH-11-stimulated (+) cells were analyzed as described in Fig.
1A. B, cleavage of caspases and DFF45. Each
column represents one cell line as indicated above in Fig.
2A. The cells were treated as described above in Fig.
1B and examined on Western blot (WB) for cleavage
products of caspase-8, casapase-3, and DFF45. C, kinetic
analysis of c-FLIP cleavage. The cell lines are indicated
under the panels. The CH-11-sensitive (U343MG, LN-18, T98G)
and CH-11-resistant (LN-215, LN-443, LN-464) cell lines were treated
with 1 µg/ml CH-11 for the times indicated, and c-FLIP proteins were
examined on Western blot using anti-c-FLIP NF6 antibody.
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Fig. 3.
Effects of CaMK II inhibitor KN-93 on
CH-11-resistant glioma cells. A, cell growth and death
analysis. CH-11-resistant LN-215, LN-443, and LN-464 cells were treated
with 100 µM KN-93 or 1 µg/ml CH-11 alone or in
combination for 24 h. The cell growth and death was determined by
crystal violet assay. Each value represents the mean ± S.D. of
triplicate samples, and experiments were repeated three times.
B, C, and D, cleavage of caspase-8,
caspase-3, and DFF45. CH-11-resistant LN-215 cells were treated with 1 µg/ml CH-11 in the presence of 100 µM KN-93 for the
times indicated or with KN-93 alone for 360 min. Cell lysates were
examined by Western blot (WB) using anti-caspase-8
(B), caspase-3 (C), and DFF45 (D)
antibodies.
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Fig. 4.
c-FLIP and CaMK II protein expression and
CaMK II activity. A, c-FLIP and CaMK II protein
expression in glioma cell lines. The cell lines are indicated
above the panels. CH-11-sensitive (U343MG, LN-18, T98G) and
CH-11-resistant (LN-215, LN-443, LN-464) cell lines were analyzed by
Western blot (WB) using anti-c-FLIP NF6 and CaMK II antibody
in which ERK1/2 was used as a loading control. CH-11-resistant cells
were also treated with 100 µM KN-93 for 24 h and
analyzed on the WB. B, CaMK II activity in glioma cell
lines. Cell extracts were obtained from glioma cells either untreated
or treated with 100 µM KN-93 for 24 h, and 10 µg
of cell extract from each cell line were analyzed for CaMK II
activity following the manufacturer's protocol. CaMK II activity
was expressed as pmol/min/mg of protein. Data represent the means ± S.E. of four experiments from three batches of cells and analyzed by
Student's t test (*, p < 0.001 between the
resistant and sensitive cells; #, p < 0.001 between
the untreated and KN-93-treated resistant cells).
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Fig. 5.
Two-dimensional PAGE analysis of c-FLIP
isoforms. Isoelectric point (PI) ranges are
indicated above the panels. A, two-dimensional
PAGE immunoblots. Cell lines and treatments are indicated to the
left. Cell extracts from CH-11-sensitive cell lines (U343MG,
LN-18) and CH-11-resistant cell lines (LN-215, LN-443) that were
untreated or treated with either 100 µM KN-93 for 24 h or 0.2 units/ml acid phosphatase for 3 h were subjected to
two-dimensional PAGE immunoblotting using anti-c-FLIP polyclonal
antibody. The isoform of c-FLIPLa, c-FLIPLb, or
c-FLIPLc was indicated as a, b, or
c, and c-FLIPL and c-FLIPS are
indicated under the panels. IEF, isoelectric
focusing. B, two-dimensional PAGE autoradiography and
immunoblots. 32P-Labeled cell lines are indicated
above the panels. The cells were labeled with
[32P]orthophosphate for 4 h and then subjected to
two-dimensional PAGE autoradiography. The arrows indicate
the locations of phosphorylated protein spots c-FLIPL (p55)
and c-FLIPS (p25). After two-dimensional (2D)
PAGE autoradiography, the same membranes were probed with anti-c-FLIP
antibody to identify the c-FLIP proteins. The arrows
indicate the same spots identified by 32P autoradiography
and anti-c-FLIP antibody immunoblotting. WB, Western
blot.
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Fig. 6.
Analysis of phosphorylation of
c-FLIPL and its p43 intermediate form.
Isoelectric point ranges are indicated above the
panels. Cell extracts from U343MG (top panel) and LN-215
(middle panel) and the DISC sample from CH-11-stimulated
LN-215 (bottom panel) were subjected to two-dimensional PAGE
immunoblotting. The membranes were probed with anti-phosphothreonine
(anti-pT) antibody (left panels). The same
membranes were then stripped of primary and secondary antibodies and
probed with polyclonal anti-c-FLIP antibody (right panels).
c-FLIPL and its p43 form were indicated as p55 and p43, and
the arrows indicate the same spots identified on the same
membranes. WB, Western blot. IEF, isoelectric focusing.
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Fig. 7.
CaMK II cDNA transfection in
CH-11-sensitive glioma cells. A, CaMK II cDNA
transfection. CH-11-sensitive U343MG and LN-18 cells were transiently
transfected with CaMK II cDNA or with the expression vector
pcDNA3.1 alone as indicated. The cells were incubated further with
1 µg/ml CH-11 in the presence or absence of 100 µM
KN-93, and cell death was determined by crystal violet assay. Some of
the cells were lysed and then analyzed on Western blots with anti-CaMK
II antibody. B, cleavage of caspases and DFF45. The
transfected cells were treated with 1 µg/ml CH-11 in the presence or
absence of 100 µM KN-93 for 3 h, and cell lysates
were examined on Western blot (WB) using anti-caspase-8,
caspase-3, and DFF45 antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B) through unknown signal pathways (51).
On the other hand, c-FLIPS transfection up-regulates the proto-oncogene c-Fos in FADD- and caspase-8-dependent
manner (52); the results suggest that c-FLIPS-mediated cell
proliferation occurs through the DISC. Here, we showed that glioma
cells proliferate in response to CH-11 stimulation. Further detection
of c-FLIPL and c-FLIPS in the Fas-mediated DISC
enhances the concept that c-FLIP proteins are recruited to the DISC to
activate its downstream proliferative signals. PED/PEA-15 has also been
shown to interfere with FADD and caspase-8 interactions and, thus,
prevents DISC formation and subsequent apoptosis induced by FasL (39,
40). PED/PEA-15 is recently reported to regulate Ras-initiated
mitogen-activated protein kinase pathways through its DED (53) and,
thus, modulate integrin-mediated cell growth, migration, and tumor
metastasis (54, 55), but the molecular events that connect the
PED/PEA-15-mediated apoptotic signaling pathway to the Ras-initiated
mitogen-activated protein kinase pathway remain elusive.
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ACKNOWLEDGEMENTS |
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We thank Dr. Alex S. Easton, University of Alberta, for critical reading of the manuscript and Dr. Francesco Beguinot, Federico II University of Naples, Italy for kindly providing anti-PED/PEA-15 serum.
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FOOTNOTES |
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* This work was supported by grants from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research (to C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to the work.
** An Alberta Heritage Foundation for Medical Research Clinical Investigator. To whom correspondence should be addressed: Dept. of Laboratory Medicine and Pathology, University of Alberta Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-4985; Fax: 780-492-9249; E-mail: chao@ualberta.ca.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211278200
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ABBREVIATIONS |
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The abbreviations used are:
FasL, Fas ligand;
DD, death domain;
DED, death effector domain;
FADD, Fas-associated
death domain;
DISC, death-inducing signaling complex;
DFF45, DNA
fragmentation factor 45;
CaMK, calcium/calmodulin-dependent
protein kinase;
ERK, extracellular signal-regulated kinase;
HRP, horseradish peroxidase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
FLIP, FADD-like interleukin-1-converting enzyme.
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