| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
University Children’s Hospital, D-89075 Ulm, Germany
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-induced apoptosis. Because blockade of death receptor
triggering by FLIP overexpression has recently been implicated in
tumorigenesis and treatment resistance in vivo,
strategies to inhibit FLIP expression, e.g., by
metabolic inhibitors, may prove to be a useful complementary tool for
the treatment of cancer. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To avoid inappropriate cell death, death receptor signals must be tightly controlled (1) . The CD95 signaling pathway can be inhibited at different checkpoints: at the receptor level [by mutations or down-regulation of the receptor, e.g., by oncogenic Ras (9) , decoy receptors, or soluble ligands (10) ], during signal transduction (by FLIP and Bcl-2-related proteins), or during the effector phase (by IAPs). c-FLIP, which structurally resembles caspase-8, was identified recently as a cellular homologue of viral FLIPs, except that it lacks proteolytic activity (11–13) . FLIP is recruited to the CD95 DISC through the adaptor molecule FADD similar to caspase-8, thereby preventing the recruitment of caspase-8 into the complex and subsequent caspase-8 activation (14) . At the mitochondrial level, apoptosis can be inhibited by antiapoptotic members of the Bcl-2 family, such as Bcl-2 or Bcl-XL, which inhibit relocalization of cytochrome c or apoptosis-inducing factor from mitochondria into the cytosol or binding of cytochrome c to Apaf-1 (15–22) . The effector phase of apoptosis can be blocked by IAPs that interfere with activation of effector caspases, e.g., caspase-3 and caspase-7 (23, 24) .
Recently, induction of tumor cell death by triggering of death
receptors was found to constitute a more prominent mechanism for tumor
clearance in vivo than has been thought previously, and
blockade of CD95-induced apoptosis has been implicated in tumorigenesis
and treatment resistance (25–28)
. CD95 is expressed on a
variety of different cell types, yet many CD95-expressing cells are
resistant to CD95-triggered apoptosis (1)
. Maintaining a
state of resistance to apoptosis often requires de novo
protein or RNA synthesis (24, 29–31)
. Thus, treatment
with protein synthesis inhibitors, such as CHX, or RNA synthesis
inhibitors, such as ActD, is usually required for TNF--mediated
apoptosis (29)
. Likewise, treatment with CHX or ActD may
render many resistant cells sensitive to CD95 triggering (24, 29, 30)
. This sensitizing effect is thought to result from
inhibition of the synthesis of short-lived regulatory proteins
(24, 29, 30)
. However, it is not exactly known at which
stage(s) of the signaling pathway CHX or ActD interferes, which hampers
the potential therapeutic application of metabolic inhibitors as
sensitizers for death receptor-induced apoptosis. Therefore, to
elucidate the mechanism(s) of CHX or ActD-mediated sensitization, we
selected SHEP neuroblastoma cells as a prototype cell line susceptible
to CHX- or ActD-mediated sensitization. In addition to the parental
cell line, we used derivative cells in which receptor-associated
signaling events were blocked by FADD-DN or in which the mitochondrial
pathway was inhibited by overexpression of Bcl-2.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Determination of Apoptosis.
Cells were incubated for indicated times with 1 µg/ml mouse
anti-APO-1 IgG3 monoclonal antibody (2)
and 5 ng/ml
protein A (Sigma, Deisenhofen, Germany), 10 ng/ml TRAIL (Pepro Tech,
Inc., Rocky Hill, NJ), 30 ng/ml TNF- (Calbiochem, Bad Soden,
Germany), or 1 µM staurosporine (Sigma) in the presence
or absence of 1 µg/ml CHX (Sigma) or 0.1 µg/ml ActD (Sigma). The
broad spectrum tripeptide inhibitor of caspases zVAD.fmk (Enzyme
Systems Products, Dublin, CA) was used at a concentration of 50
µM. Quantification of DNA fragmentation was performed by
FACS analysis of propidium iodide-stained nuclei as described
previously (33)
using CELLQuest software (Becton
Dickinson, Heidelberg, Germany).
Metabolic Labeling Studies.
Protein synthesis of untreated or CHX-treated cells (0.01–3 µg/ml
CHX) was determined by pulse-chase labeling of cells with
[3H]leucine (31)
. Cells were
washed with leucine-free RPMI 1640 (Life Technologies, Inc.) and
incubated for 1 h at 37°C in leucine-free RPMI 1640 supplemented
with 5% dialyzed FCS to deplete cellular leucine. Cells were pulsed
for 30 min with [3H]leucine (0.2 mCi/ml;
Amersham Pharmacia, Freiburg, Germany) and incubated for 2 h at
37°C in RPMI 1640 containing excess leucine (15 mg/l; Sigma). Protein
precipitation was performed using trichloroacetic acid (Roth,
Karlsruhe, Germany). RNA synthesis of cells, treated with 0.001–0.3
µg/ml ActD or left untreated, was determined by incorporation of
5,6-[3H]uridine (5 µCi/ml; Amersham
Pharmacia) into cells. Radioactivity was quantified by scintillation
counting (liquid scintillation analyzer 1500 TR; Packard, Frankfurt,
Germany). Results are expressed as percentage of inhibition of protein
or RNA synthesis as compared with untreated cells.
Western Blot Analysis.
Cells were lysed for 30 min at 4°C in PBS with 0.5% Triton
X-100 (Serva, Heidelberg, Germany) and 1 mM PMSF (Sigma,)
followed by high-speed centrifugation. Protein concentration was
assayed using bicinchoninic acid (Pierce, Rockford, IL). Forty µg of
protein/lane was separated by 12% SDS-PAGE and electroblotted onto
nitrocellulose (Amersham Pharmacia). Equal protein loading was
controlled by Ponceau red staining of membranes. After blocking for
1 h in PBS supplemented with 2% BSA (Sigma) and 0.1% Tween 20
(Sigma), immunodetection of caspase-8, caspase-3, FADD, FLIP, PARP,
RIP, XIAP, or ß-actin was done using mouse anti-caspase-8 monoclonal
antibody C15 (1:10 dilution of hybridoma supernatant; Ref.
34
), mouse anti-caspase-3 monoclonal antibody (1:1000;
Transduction Laboratories, Lexington, KY), mouse anti-FADD monoclonal
antibody (1:500; Transduction Laboratories), mouse anti-CD95 monoclonal
antibody (1:1000; Transduction Laboratories), mouse anti-FLIP
monoclonal antibody NF6 (1:10 dilution of hybridoma supernatant; Ref.
14
), rabbit anti-PARP polyclonal antibody (1:10000; Enzyme
Systems Products), mouse anti-RIP monoclonal antibody (1:1000;
Transduction Laboratories), mouse anti-XIAP monoclonal antibody (clone
#H62120; 1:1000; Transduction Laboratories), or mouse anti-ß-actin
monoclonal antibody (1:5000; Sigma), followed by goat anti-mouse IgG or
goat anti-rabbit IgG (1:50;0, Santa Cruz Biotechnology, Santa Cruz,
CA). Enhanced chemiluminescence (ECL; Amersham Pharmacia) was used for
detection.
Immunoprecipitation.
Immunoprecipitation of the CD95 DISC was performed as described
previously (35)
. Briefly, cells were treated with 1
µg/ml anti-APO-1 IgG3 monoclonal antibody (2)
for 15 min
and lysed for protein extraction. Immunoprecipitation of the CD95 DISC
was performed using mouse anti-APO-1 IgG3 monoclonal antibody and
protein A-Sepharose (Sigma). Beads were washed three times in lysis
buffer and proteins were separated on 12% SDS-PAGE. Westerns blot for
FADD, FLIP, or caspase-8 protein was performed as described above.
Down-Regulation of FLIP Protein Expression by FLIP Antisense
Oligonucleotides.
To inhibit FLIP expression phosphorothiorate antisense
oligodeoxynucleotides to inhibit the FLIP initiation codon, control
sense and nonsense oligodeoxynucleotides with the following published
sequences were used (36)
: FLIP antisense,
5'-gacttcagcagacatcctac-3'; FLIP sense, 5'-catcctacagacgacttcag-3'; and
FLIP nonsense, 5'-tggatccgaacatgtcaga-3'. The uptake of
FITC-conjugated oligonucleotides was measured by flow cytometry after
24-h incubation with oligonucleotides.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sensitization to Anti-APO-1-induced Apoptosis.
Apoptosis triggered by an agonistic anti-APO-1 antibody was strongly
increased in the presence of CHX or ActD (Fig. 1
). Treatment with CHX or ActD alone at the concentrations required for
sensitization (1 µg/ml CHX or 0.1 µg/ml ActD) was devoid of any
significant toxicity (data not shown). Overexpression of FADD-DN or the
caspase inhibitor zVAD.fmk almost completely blocked
anti-APO-1-triggered apoptosis or CHX- or ActD-mediated sensitization
(Fig. 1, A and B)
. Overexpression of Bcl-2
initially (24 h) provided protection against CD95-induced apoptosis in
the presence or absence of CHX or ActD (Fig. 1C
). However,
upon prolonged treatment for 48–72 h, overexpression of Bcl-2 only
partially inhibited CD95-triggered apoptosis and only poorly blocked
CHX-or ActD-mediated sensitization (Fig. 1C
). In control
experiments, Bcl-2 was overexpressed in the mitochondrial fraction and
blocked loss of mitochondrial 
m (data not
shown), indicating that CHX or ActD may affect a step upstream of
mitochondria that eventually leads to cell death, even in the absence
of functional mitochondria.
|
). Caspase-8 cleavage was followed by cleavage of the
effector caspase-3 and PARP detectable at 6 h, indicating that
caspase-8 was the apical caspase in the cascade upon CD95 stimulation
(Fig. 2A
). In cells overexpressing Bcl-2, increased cleavage
of caspase-8 in the presence of CHX or ActD was found at 2 h after
CD95 triggering similar to vector control cells, whereas cleavage of
caspase-3 and PARP was markedly delayed to 24 h (Fig. 2B
). Treatment with CHX or ActD alone did not induce caspase
cleavage at any time point tested (data not shown). Thus, caspase-8
cleavage was increased by CHX or ActD independently of mitochondrial
functions, suggesting that CHX or ActD act upstream of caspase-8
activation, possibly at the receptor complex.
|
). In addition, expression of XIAP protein decreased after
12 h of treatment with CHX or ActD (Fig. 3A
). In
contrast, no changes in protein expression levels of Bcl-2,
Bcl-XL, Bax, CD95, or CD95L and no alterations in
CD95 aggregation were detected (data not shown).
|
), despite any signs of
cell death (data not shown). In contrast, no change in caspase-8 or
FADD expression was seen (Fig. 3B
), indicating that the
intracellular concentration of FLIP relative to other components of the
CD95 DISC, in particular caspase-8, may determine the susceptibility to
CD95 triggering.
Increased DISC Formation by Reduction of DISC-bound FLIP.
FLIP has been reported to interact with FADD or caspase-8 at the DISC
level upon CD95 stimulation, and no constitutive association of
endogenous FLIP, FADD, or caspase-8 was found in the cytoplasm
(14)
. Upon CD95 triggering, FLIP is recruited to the CD95
DISC through the adaptor molecule FADD, similar to caspase-8
(14)
. Therefore, to test how FLIP levels could influence
the susceptibility to CD95-mediated apoptosis, we immunoprecipitated
the CD95 receptor and tested for the presence of CD95-associated FLIP
by Western Blot analysis. FLIP was recruited to the CD95 DISC after
receptor triggering (Fig. 4A
). However, in CHX- or ActD-treated cells, in which FLIP
protein levels were strongly reduced (Fig. 3)
, no recruitment of FLIP
could be detected in response to CD95 triggering (Fig. 4A
).
Reciprocally, the recruitment of caspase-8 to CD95 upon receptor
triggering was strongly increased in the presence of CHX or ActD (Fig. 4)
. Thus, treatment with CHX or ActD reduces the amount of DISC-bound
FLIP, thereby facilitating the recruitment of caspase-8 to the DISC and
subsequent caspase-8 activation. Interestingly, in addition to
caspase-8, the recruitment of FADD to the CD95 receptor was also
enhanced upon treatment with CHX or ActD (Fig. 4)
. This indicates that
other labile repressors in addition to FLIP, such as RIP, may modulate
DISC formation at the interaction of FADD and CD95. However, although
RIP protein expression was down-regulated by CHX or ActD, we could not
detect any CD95-RIP interaction (data not shown), consistent with a
previous report (37)
, suggesting that RIP may indirectly
modulate the recruitment of FADD to CD95. Similarly, the recruitment of
FADD-DN to CD95 was increased by treatment with CHX or ActD in cells
overexpressing FADD-DN (Fig. 4B
). However, overexpression of
FADD-DN completely blocked binding of caspase-8 to the DISC (Fig. 4B
). In contrast, the presence of zVAD.fmk or overexpression
of Bcl-2 did not affect DISC formation (Fig. 4B
), consistent
with unaltered cleavage of caspase-8 found in Bcl-2-overexpressing
cells compared with vector control cells (Fig. 2)
. Altered recruitment
of caspase-8, FADD, or FADD-DN to CD95 occurred without any change in
protein expression levels (Fig. 4B
). These results indicate
that CHX or ActD can modulate DISC formation at least at two different
levels, at the interaction between CD95 and FADD and at the interaction
between FADD and caspase-8.
|
). In these cells, FADD and
caspase-8 were recruited to CD95 upon receptor stimulation and
recruitment of FADD, and caspase-8 was further increased in the
presence of CHX or ActD (Fig. 5B
). Similar to SHEP
neuroblastoma cells, treatment with CHX or ActD strongly reduced FLIP
and RIP expression, decreased XIAP expression, and increased activation
of caspases (Fig. 5C
). In contrast, no sensitization for
CD95-induced cell death upon treatment with CHX or ActD was seen in
type II Jurkat or CEM cells (35)
or in GG62 and CADO
cells, in which no recruitment of FADD and caspase-8 was found upon
CD95 stimulation, even in the presence of CHX or ActD (Fig. 5, A and B)
. However, FLIP expression similarly
decreased upon addition of CHX or ActD in type I and type II cells, and
no differences in constitutive expression of FADD, caspase-8, or FLIP
proteins were found between both cell types (Fig. 5D
;
compare Fig. 3B
), suggesting that down-regulation of FLIP
only results in increased DISC formation and caspase-8 activation in
type I cells that form a DISC upon CD95 triggering, whereas
down-regulation of FLIP was of no consequence in type II cells that do
not form a DISC detectable by the method used in our analysis. In
addition, these data demonstrate that our findings are relevant for
sensitization mechanisms of tumor cells of different origin.
|
). Western blot analysis of antisense
oligonucleotide-treated SHEP cells showed a strong suppression of
FLIPlong and FLIPshort
protein expression compared with medium (mock) or control sense or
nonsense oligonucleotide-treated cells (Fig. 6B
). Treatment
with FLIP antisense oligonucleotides sensitized SHEP cells for
CD95-induced apoptosis to a similar degree compared with treatment with
CHX, whereas control sense or nonsense oligonucleotides had no effect
on CD95-induced apoptosis (Fig. 6C
). Sensitization for
CD95-triggered apoptosis by FLIP antisense oligonucleotides was also
found in other cell types, such as NT68 Ewing’s sarcoma cells, whereas
no effect was seen in Jurkat cells (Fig. 6C
). These data
show that selective down-regulation of FLIP expression can sensitize
for CD95-induced apoptosis and suggest that this mechanism might be
especially important in certain cell types, e.g., in cells
that do form a CD95 DISC upon CD95 triggering.
|
). After preincubation with CHX or ActD for 12 h, apoptosis levels
after treatment with anti-APO-1 antibody alone were close to those
found after simultaneous treatment with anti-APO-1 antibody and CHX or
ActD for 12 h (Fig. 7)
. These results provide additional support
for the premise that inhibition of synthesis of short-lived proteins
sensitizes cells for CD95-mediated apoptosis.
|
). These results indicate that the synergistic effect of CHX or ActD on
CD95-mediated apoptosis was the result of inhibition of novel synthesis
of labile repressors.
|
-induced Apoptosis. in SHEP neuroblastoma cells. In the
presence of CHX or ActD, TRAIL- or TNF--induced apoptosis was
strongly increased (Fig. 9
,
A and B). In addition, pretreatment with CHX or
ActD resulted in enhanced apoptosis compared with cells treated with
TRAIL or TNF without CHX or ActD pretreatment (data not shown). In
contrast, CHX or ActD had no effect on apoptosis induced by the protein
kinase C inhibitor staurosporine (Fig. 9C
). Because
staurosporine is reported to trigger apoptosis independent of death
receptor signaling (38)
, these results further indicate
that sensitization by CHX or ActD involves death receptor-associated
signaling events.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Here, we report for the first time that metabolic inhibitors such as
CHX or ActD regulate the susceptibility to CD95-induced apoptosis
through down-regulation of FLIP expression at the CD95 DISC. This
conclusion is based on a number of independent pieces of evidence:
first, FLIP levels rapidly decreased upon addition of CHX or ActD at
concentrations that blocked protein or RNA synthesis, indicating that
FLIP has a high turnover rate. Expression of FADD and caspase-8
remained unchanged upon treatment with CHX or ActD, thereby increasing
the ratio of caspase-8 to FLIP. Thus, the intracellular concentration
of FLIP relative to the other components of the DISC, especially
caspase-8, may determine the susceptibility of cells to CD95
triggering; second, selective down-regulation of FLIP expression by
FLIP antisense oligonucleotides sensitized for CD95-induced apoptosis.
Furthermore, down-regulation of intracellular FLIP levels resulted
in undetectable levels of DISC-bound FLIP, thereby enhancing the
recruitment of caspase-8 to the DISC and caspase-8 activation. In the
CD95 pathway, FLIP was reported to act as an inhibitor of the CD95 DISC
activity, because most of the intracellular pool of FLIP was found at
the DISC upon CD95 stimulation, thereby preventing the recruitment of
caspase-8 into the complex, whereas no association of FLIP and
caspase-8 was detected in the cytoplasm (14)
. In addition,
the relevance of reduced FLIP levels at the CD95 DISC was further
supported by our findings in different cell types. Treatment with CHX
or ActD only sensitized type I cells, in which a DISC was formed upon
CD95 triggering, but not type II cells, in which no DISC formation was
detected. Because FLIP levels similarly decreased upon addition of CHX
or ActD in both cell types and because no differences in constitutive
expression of FADD, caspase-8, or FLIP proteins were found between type
I and type II cells (Figs. 3B
and 5B), these
results suggest that down-regulation of FLIP levels at the CD95 DISC
results in increased caspase-8 activation and apoptosis predominantly
in cells that form a CD95 DISC upon CD95 triggering (type I cells). In
contrast, down-regulation of FLIP levels by CHX or ActD, or more
specifically by antisense FLIP oligonucleotides, could not further
sensitize the CD95-sensitive prototype type II Jurkat or CEM cells, in
which no DISC is found upon CD95 stimulation. However, because some
CD95-resistant type II cells, such as the pre-B leukemia cell line
BoeR, can be sensitized by CHX for CD95-triggered apoptosis at a
level downstream of DISC formation
(39)
,4
CHX may affect additional cellular functions that regulate sensitivity
for CD95 triggering in different cell types. Moreover, pretreatment
with CHX or ActD, which rapidly reduced FLIP levels sensitized for
subsequent CD95 stimulation in a time- and concentration-dependent
manner. In addition to CD95, CHX or ActD also sensitized for TRAIL- or
TNF--induced apoptosis, implying that modulation of FLIP levels
might be a common mechanism of CHX or ActD to regulate the
susceptibility to death receptor signals. Thus, the data clearly
demonstrate that FLIP is one of the important labile repressors that is
down-regulated by CHX or ActD treatment, which in turn leads to
increased sensitivity to death receptor-induced apoptosis.
Although the exact function of FLIP has to be completely elucidated, FLIPlong has been shown to render cells resistant to apoptotic signals transmitted by CD95 and all other death receptors known to date by inhibition of caspase-8 recruitment to the death receptor complex, whereas the role of FLIPshort in apoptosis signaling is not exactly known (13) . FLIP expression has been implicated to regulate sensitivity to CD95-mediated cell death in T cells upon TCR stimulation (40, 41) or to modulate TRAIL-induced apoptosis in melanoma cells (42, 43) . Moreover, inhibition of death receptor signaling by FLIP has recently been implied as a novel mechanism for immune escape of tumors from T-cell immunity in vivo (25–27) . In Burkitt’s lymphoma, the tumorigenic potential of EBV was reported to involve the development of resistance to CD95-induced apoptosis through antagonistic regulation of caspase-8 and FLIP expression (28) . In addition, in vivo expression of FLIP has been shown to act as a tumor progression factor by promoting tumor establishment and growth through prevention of death receptor-induced apoptosis mediated by T cells (26, 27) . FLIP was also found to be involved in the development of resistance in vivo, because resistant tumor cells were selected for elevated FLIP expression (26) . Up-regulation of FLIP expression may be involved in the pathogenesis of tumors such as melanomas, because FLIP was found to be overexpressed in malignant melanoma, whereas no FLIP expression was detected in normal melanocytes (11) . Thus, FLIP may play an important role in tumorigenesis and in modulation of sensitivity or resistance toward therapy.
Interestingly, in addition to FLIP, treatment with CHX or ActD strongly
reduced the expression of RIP. RIP has been identified as a death
domain kinase in TNF-RI signaling mediating activation of nuclear
factor-
B, which in turn may exert an antiapoptotic function
(44)
. Recently, RIP has been reported to be cleaved in
TNF-induced apoptosis, promoting the interaction between TRADD
and FADD and increasing the sensitivity to death receptor triggering
(45)
. Reduction of RIP expression by CHX or ActD may
similarly enhance the recruitment of FADD to CD95 to facilitate DISC
formation upon CD95 stimulation. In line with this, increased FADD
recruitment to the CD95 DISC was found in CHX-treated cells. However,
we could not detect any CD95-RIP interaction (data not shown),
consistent with a previous report (37)
suggesting that RIP
may indirectly modulate the recruitment of FADD to CD95. Treatment with
CHX or ActD also decreased expression of XIAP, which has been shown to
suppress the effector phase of apoptosis by directly interacting with
and inhibiting caspase-3, caspase-7, and caspase-9, respectively
(23, 46)
. Moreover, other yet undefined short-lived
repressors, e.g., SODD-related proteins, may regulate the
interaction between the death domain of CD95 and FADD. SODD has been
identified recently as a negative regulatory protein that is associated
with the death domain of TNF-RI, inhibiting the intrinsic
self-aggregation properties of the death domain (47)
. SODD
association with death domains of death receptors may represent a
general mechanism for preventing spontaneous signaling by death
domain-containing receptors, and SODD-related proteins may play a
similar role in preventing spontaneous signaling by CD95. Thus, CHX or
ActD may regulate formation of the DISC at two different levels, at the
interaction between CD95 and FADD, e.g., through
down-regulation of RIP or SODD-like proteins, and at the interaction
between FADD and caspase-8 through down-regulation of FLIP.
The synergistic effect of CHX or ActD in CD95-induced apoptosis has been explained as a consequence of inhibition of protein or mRNA synthesis (24, 29, 30) . Consistent with this concept, we found a clear correlation between CHX or ActD concentrations necessary to facilitate apoptosis induction and those required for inhibition of protein or RNA synthesis. In addition, FLIP and RIP levels rapidly decreased upon addition of CHX or ActD, and pretreatment of cells with CHX or ActD enhanced CD95-induced apoptosis, supporting that inhibition of short-lived repressors may account for the synergistic effect of CHX or ActD. Alternatively, CHX or ActD have also been reported to modulate apoptosis by activating intracellular signaling pathways (30) . Some cell types such as GG62 or CADO Ewing’s sarcoma cells, however, remained completely resistant to anti-CD95-triggered apoptosis, even in the presence of CHX or ActD. In these cells, only minimal constitutive expression of caspase-8 was detected (data not shown), suggesting that in addition to labile repressors, defects in apoptosis pathways, such as an absence of apoptosis signaling molecules, may account for a resistant phenotype in some cells.
Thus, by demonstrating that metabolic inhibitors such as CHX or ActD can sensitize tumor cells to apoptosis after death receptor ligation through down-regulation of FLIP expression, our findings provide new insights into mechanisms that regulate the susceptibility to death receptor signals. Blockade of the CD95 pathway by overexpression of FLIP was shown to offer significant protection against the in vivo tumor response through immune escape from T-cell immunity (25–28) . In addition, overexpression of FLIP also promoted the development of resistance in vivo (26) . In light of these recent data, our findings that metabolic inhibitors sensitize tumor cells to death receptor-induced apoptosis through reduction of FLIP expression may have important implications for cancer therapy. Current attempts to improve cancer survival depend on strategies to target tumor cell resistance and on the development of new treatment modalities, e.g., gene therapy using the herpes simplex thymidine kinase/ganciclovir system or immunotherapy. Because the potential success of these approaches largely depends on intact death receptor pathways in target cells (48) , strategies to inhibit FLIP expression, e.g., by metabolic inhibitors, may prove to be a useful complementary tool to the treatment of cancer.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was partially supported by grants from
the Deutsche Forschungsgemeinschaft; the Bundesministerium für
Forschung and Technologie, Bonn; the Tumor Center Heidelberg/Mannheim;
and the Deutsche Leukämieforschungshilfe (to K. M. D.). 
2 To whom requests for reprints should be
addressed, at University Children’s Hospital, Prittwitzstrasse 43,
D-89075 Ulm, Germany. Phone: 49-731-502-7700; Fax: 49-731-502-6681;
E-mail: klaus-michael. debatin{at}medizin.uni-ulm.de
3 The abbreviations used are: TNF, tumor necrosis
factor; TNF-RI, TNF receptor type I; FADD, Fas-associated death domain;
FADD-DN, dominant negative mutant of FADD; DISC, death-inducing
signaling complex; IAP, inhibitor of apoptosis protein; ActD,
actinomycin D; CHX, cycloheximide; FACS, fluorescence-activated
cell-sorting; FLIP, FLICE-inhibitory protein; PARP,
poly(ADP-ribose)polymerase; zVAD.fmk,
benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; SODD, silencer of
death domains.
4 S. Fulda and K-M. Debatin, unpublished
results.
Received 11/17/99. Accepted 5/16/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B but not Fas/APO-1-initiated apoptosis. EMBO J, 15: 6189-6196, 1996.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. Chalaris, B. Rabe, K. Paliga, H. Lange, T. Laskay, C. A. Fielding, S. A. Jones, S. Rose-John, and J. Scheller Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils Blood, September 15, 2007; 110(6): 1748 - 1755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Wilson, K. M. McLaughlin, M. McEwan, H. Sakai, K. M.A. Rogers, K. M. Redmond, P. G. Johnston, and D. B. Longley c-FLIP: A Key Regulator of Colorectal Cancer Cell Death Cancer Res., June 15, 2007; 67(12): 5754 - 5762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M.A. Rogers, M. Thomas, L. Galligan, T. R. Wilson, W. L. Allen, H. Sakai, P. G. Johnston, and D. B. Longley Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells Mol. Cancer Ther., May 1, 2007; 6(5): 1544 - 1551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Bai and A. I. Cederbaum Cycloheximide Protects HepG2 Cells from Serum Withdrawal-Induced Apoptosis by Decreasing p53 and Phosphorylated p53 Levels J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1435 - 1443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Palacios, R. Yerbes, and A. Lopez-Rivas Flavopiridol Induces Cellular FLICE-Inhibitory Protein Degradation by the Proteasome and Promotes TRAIL-Induced Early Signaling and Apoptosis in Breast Tumor Cells. Cancer Res., September 1, 2006; 66(17): 8858 - 8869. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Golks, D. Brenner, P. H. Krammer, and I. N. Lavrik The c-FLIP-NH2 terminus (p22-FLIP) induces NF-{kappa}B activation J. Exp. Med., May 15, 2006; 203(5): 1295 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Poukkula, A. Kaunisto, V. Hietakangas, K. Denessiouk, T. Katajamaki, M. S. Johnson, L. Sistonen, and J. E. Eriksson Rapid Turnover of c-FLIPshort Is Determined by Its Unique C-terminal Tail J. Biol. Chem., July 22, 2005; 280(29): 27345 - 27355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Golks, D. Brenner, C. Fritsch, P. H. Krammer, and I. N. Lavrik c-FLIPR, a New Regulator of Death Receptor-induced Apoptosis J. Biol. Chem., April 15, 2005; 280(15): 14507 - 14513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Terrasson, S. Allart, H. Martin, J. Lule, H. Haddada, D. Caput, and C. Davrinche p73-Dependent Apoptosis through Death Receptor: Impairment by Human Cytomegalovirus Infection Cancer Res., April 1, 2005; 65(7): 2787 - 2794. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-L. Sun and C. C.-K. Chao Cross-Resistance to Death Ligand-Induced Apoptosis in Cisplatin-Selected HeLa Cells Associated with Overexpression of DDB2 and Subsequent Induction of cFLIP Mol. Pharmacol., April 1, 2005; 67(4): 1307 - 1314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Poulaki, C. S. Mitsiades, C. McMullan, G. Fanourakis, J. Negri, A. Goudopoulou, I. X. Halikias, G. Voutsinas, S. Tseleni-Balafouta, J. W. Miller, et al. Human Retinoblastoma Cells Are Resistant to Apoptosis Induced by Death Receptors: Role of Caspase-8 Gene Silencing Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 358 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A E Greijer and E van der Wall The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis J. Clin. Pathol., October 1, 2004; 57(10): 1009 - 1014. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gilbert, A. Loranger, and N. Marceau Keratins Modulate c-Flip/Extracellular Signal-Regulated Kinase 1 and 2 Antiapoptotic Signaling in Simple Epithelial Cells Mol. Cell. Biol., August 15, 2004; 24(16): 7072 - 7081. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kreuz, D. Siegmund, J.-J. Rumpf, D. Samel, M. Leverkus, O. Janssen, G. Hacker, O. Dittrich-Breiholz, M. Kracht, P. Scheurich, et al. NF{kappa}B activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP J. Cell Biol., August 2, 2004; 166(3): 369 - 380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mezzanzanica, E. Balladore, F. Turatti, E. Luison, P. Alberti, M. Bagnoli, M. Figini, A. Mazzoni, F. Raspagliesi, M. Oggionni, et al. CD95-Mediated Apoptosis Is Impaired at Receptor Level by Cellular FLICE-Inhibitory Protein (Long Form) in Wild-Type p53 Human Ovarian Carcinoma Clin. Cancer Res., August 1, 2004; 10(15): 5202 - 5214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Schmitz, H. Weyd, A. Krueger, S. Baumann, S. C. Fas, P. H. Krammer, and S. Kirchhoff Resistance of Short Term Activated T Cells to CD95-Mediated Apoptosis Correlates with De Novo Protein Synthesis of c-FLIPshort J. Immunol., February 15, 2004; 172(4): 2194 - 2200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Davies, J. Mason, M. J. O. Wakelam, L. S. Young, and A. G. Eliopoulos Inhibition of Phosphatidylinositol 3-Kinase- and ERK MAPK-regulated Protein Synthesis Reveals the Pro-apoptotic Properties of CD40 Ligation in Carcinoma Cells J. Biol. Chem., January 9, 2004; 279(2): 1010 - 1019. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. R. Rosato, J. A. Almenara, Y. Dai, and S. Grant Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells Mol. Cancer Ther., December 1, 2003; 2(12): 1273 - 1284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Kuenzi, P. Schneider, and D. A. E. Dobbelaere Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/Fas Ligand-Induced Apoptosis J. Immunol., August 1, 2003; 171(3): 1224 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Munoz-Pinedo, C. Ruiz-Ruiz, C. Ruiz de Almodovar, C. Palacios, and A. Lopez-Rivas Inhibition of Glucose Metabolism Sensitizes Tumor Cells to Death Receptor-triggered Apoptosis through Enhancement of Death-inducing Signaling Complex Formation and Apical Procaspase-8 Processing J. Biol. Chem., April 4, 2003; 278(15): 12759 - 12768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Kudo, P. Rayman, C. Horton, M. K. Cathcart, R. M. Bukowski, M. Thornton, C. Tannenbaum, and J. H. Finke Gangliosides Expressed by the Renal Cell Carcinoma Cell Line SK-RC-45 Are Involved in Tumor-induced Apoptosis of T Cells Cancer Res., April 1, 2003; 63(7): 1676 - 1683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Fanzo, C.-M. Hu, S. Y. Jang, and A. B. Pernis Regulation of Lymphocyte Apoptosis by Interferon Regulatory Factor 4 (IRF-4) J. Exp. Med., February 3, 2003; 197(3): 303 - 314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Langelier, S. Bergeron, S. Chabaud, J. Lippens, C. Guilbault, A. M.-J. Sasseville, S. Denis, D. D. Mosser, and B. Massie The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation J. Gen. Virol., November 1, 2002; 83(11): 2779 - 2789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Erwert, R. K. Winn, J. M. Harlan, and D. D. Bannerman Shiga-like Toxin Inhibition of FLICE-like Inhibitory Protein Expression Sensitizes Endothelial Cells to Bacterial Lipopolysaccharide-induced Apoptosis J. Biol. Chem., October 18, 2002; 277(43): 40567 - 40574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. M. Kim, Y. Wang, A. Gambotto, Y.-M. Kim, R. Weller, B. S. Zuckerbraun, Y. Hua, S. C. Watkins, and T. R. Billiar Hepatocyte Fas-associating Death Domain Protein/Mediator of Receptor-induced Toxicity (FADD/MORT1) Levels Increase in Response to Pro-apoptotic Stimuli J. Biol. Chem., October 4, 2002; 277(41): 38855 - 38862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Li, M. S. Kluger, L. A. Madge, L. Zheng, A. L. M. Bothwell, and J. S. Pober Interferon-{gamma} Augments CD95(APO-1/Fas) and Pro-Caspase-8 Expression and Sensitizes Human Vascular Endothelial Cells to CD95-Mediated Apoptosis Am. J. Pathol., October 1, 2002; 161(4): 1485 - 1495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. McNees, C. T. Garnett, and L. R. Gooding The Adenovirus E3 RID Complex Protects Some Cultured Human T and B Lymphocytes from Fas-Induced Apoptosis J. Virol., August 28, 2002; 76(19): 9716 - 9723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kim, N. Suh, M. Sporn, and J. C. Reed An Inducible Pathway for Degradation of FLIP Protein Sensitizes Tumor Cells to TRAIL-induced Apoptosis J. Biol. Chem., June 14, 2002; 277(25): 22320 - 22329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Thomas, A. Kallenborn, C. Wickenhauser, J. L. Schultze, A. Draube, M. Vockerodt, D. Re, V. Diehl, and J. Wolf Constitutive Expression of c-FLIP in Hodgkin and Reed-Sternberg Cells Am. J. Pathol., April 1, 2002; 160(4): 1521 - 1528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Krueger, S. Baumann, P. H. Krammer, and S. Kirchhoff FLICE-Inhibitory Proteins: Regulators of Death Receptor-Mediated Apoptosis Mol. Cell. Biol., December 15, 2001; 21(24): 8247 - 8254. [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Ahn, S.-M. Park, H.-S. Cho, M.-S. Lee, J.-B. Yoon, J. Vilcek, and T. H. Lee Non-apoptotic Signaling Pathways Activated by Soluble Fas Ligand in Serum-starved Human Fibroblasts. MITOGEN-ACTIVATED PROTEIN KINASES AND NF-kappa B-DEPENDENT GENE EXPRESSION J. Biol. Chem., December 7, 2001; 276(50): 47100 - 47106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Saelens, M. Kalai, and P. Vandenabeele Translation Inhibition in Apoptosis. CASPASE-DEPENDENT PKR ACTIVATION AND eIF2-alpha PHOSPHORYLATION J. Biol. Chem., November 2, 2001; 276(45): 41620 - 41628. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Chatterjee, I. Schmitz, A. Krueger, K. Yeung, S. Kirchhoff, P. H. Krammer, M. E. Peter, J. H. Wyche, and P. Pantazis Induction of Apoptosis in 9-Nitrocamptothecin-treated DU145 Human Prostate Carcinoma Cells Correlates with de Novo Synthesis of CD95 and CD95 Ligand and Down-Regulation of c-FLIPshort Cancer Res., October 1, 2001; 61(19): 7148 - 7154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. D. Zhang, X. Y. Zhang, C. P. Gray, T. Nguyen, and P. Hersey Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis of Human Melanoma Is Regulated by Smac/DIABLO Release from Mitochondria Cancer Res., October 1, 2001; 61(19): 7339 - 7348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Schwieger, L. Bauer, J. Hanusch, C. Sers, R. Schafer, and G. Bauer ras oncogene expression determines sensitivity for intercellular induction of apoptosis Carcinogenesis, September 1, 2001; 22(9): 1385 - 1392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gilbert, A. Loranger, N. Daigle, and N. Marceau Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation J. Cell Biol., August 20, 2001; 154(4): 763 - 774. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, 
) or presence (•, 
) of 1 µg/ml CHX or
0.1 µg/ml ActD (
, 
). Apoptosis was determined by FACS analysis
of propidium iodide-stained DNA content. The percentage of specific
apoptosis was calculated as follows: 100 x [experimental apoptosis (%) - spontaneous apoptosis
(%)/100% - spontaneous apoptosis (%)]. Spontaneous
apoptosis was <1%. Data are the mean of triplicates; similar results
were obtained in three separate experiments; bars,
SD.
18,000, the active fragment of caspase-3 at
Mr 
).
Apoptosis was determined by FACS analysis of propidium iodide-stained
DNA content as described above. Data are the means of triplicates;
similar results were obtained in three separate experiments; bars,
SD. B, increased DISC formation by CHX or ActD in
type I cells. Type I BJAB, SKW6.4, HepG2, or NT68 cells (upper
panel) or type II Jurkat and CEM cells, GG62, or CADO cells
(lower panel) were treated with 1 µg/ml anti-APO-1
monoclonal antibody for 2 h in the presence or absence of 1
µg/ml CHX or 0.1 µg/ml ActD. Immunoprecipitation was performed as
described above. C, down-regulation of FLIP, RIP, and
XIAP expression and enhanced caspase activation by CHX or ActD in type
I cells. HepG2 cells were treated for the indicated periods with 1
µg/ml anti-APO-1 monoclonal antibody in the presence or absence of 1
µg/ml CHX or 0.1 µg/ml ActD. Western blot analysis for FLIP, RIP,
XIAP, caspase-8, PARP, or ß-actin protein was performed as described
above. D, down-regulation of FLIP expression by CHX in
type II cells. Jurkat cells were treated for the indicated periods with
1 µg/ml CHX. Western blot analysis for FLIP, RIP, caspase-8, FADD,
and ß-actin protein was performed as described above.
