From the Department of Hematology and Oncology, Beth
Israel Deaconess Medical Center, Boston, Massachusetts 02215 and
§ St. Elizabeth Medical Center, Boston, Massachusetts
02135
Received for publication, August 21, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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The caspase-8 homologue FLICE-inhibitory protein
(FLIP) functions as a caspase-8 dominant negative, blocking apoptosis
induced by the oligomerization of the adapter protein
FADD/MORT-1. FLIP expression correlates with resistance to apoptosis
induced by various members of the tumor necrosis factor family such as
TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are
largely unknown. In this report, we examined the roles of the MAP
kinase and phosphatidylinositol (PI) 3-kinase signaling pathways
in the regulation of FLIP expression in tumor cells. We observed that
the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor
cell lines tested. In contrast, disruption of the PI 3-kinase pathway
with the specific inhibitor LY294002 reduced Akt (protein kinase
B) phosphorylation and the levels of FLIP protein and mRNA
in all cell lines evaluated. The introduction of a dominant negative
Akt adenoviral construct also consistently reduced FLIP expression as
well as the phosphorylation of the Akt target glycogen synthase
kinase-3. In addition, infection of the same cell lines with a
constitutively active Akt adenovirus increased FLIP expression and the
phosphorylation of GSK-3. These data add FLIP to the growing list of
apoptosis inhibitors in which expression or function is regulated
by the PI 3-kinase-Akt pathway.
The p55 TNF1 receptor,
Fas, LARD (DR3), and the TRAIL receptors DR4 and DR5 are members
of the TNF receptor family that contain a unique cytoplasmic sequence
termed a death domain (DD). Upon engagement by ligand or antibody
cross-linking, these receptors trimerize and recruit an adapter protein
called FADD (Fas-associated death
domain) (1-3), which interacts through its own DD with those of the receptors or other adapters (e.g. TRADD). The
resulting multimolecular complex, referred to as a death-inducing
signaling complex or DISC, recruits an inactive procaspase called FLICE or caspase-8, which is then cleaved to an active form, initiating a
cascade of proteolytic events resulting in the cleavage of numerous proteins essential for DNA repair and the maintenance of cell viability
(4). It has been shown that FADD multimerization is sufficient to kill
some cells even in the absence of TNF family ligands or receptor
cross-linking (5). For example, the apoptosis induced by certain
chemotherapeutic agents is thought to rely on ligand-independent FADD
multimerization, since it can be blocked by a dominant negative FADD
(5). Collectively, these data suggest that FADD plays a critical role
in apoptosis induced by the cross-linking of DD-containing membrane
receptors of the TNF receptor family (e.g. Fas) as
well as that resulting from exposure to cytotoxic drugs.
Many tumors express Fas (6) and TRAIL receptors (7, 8) yet are
resistant to apoptosis induced by exposure to Fas ligand, TRAIL, or
agonist anti-receptor antibodies. It has been proposed that this
resistance is due to the presence of one or more inhibitors of
apoptosis (9). Numerous cellular proteins, including FLIP,
Fas-associated protein (FAP-1) (10), Bcl-2, Bcl-X, Bruton's tyrosine
kinase (11), Toso (12), and SODD (silencer
of death domain) (13, 14) as well as
caspase inhibitors such as cIAP (15) and survivin (16, 17), have been
shown to inhibit the death process, either early on at the level of Fas
(FAP-1) or further downstream. FLIP is a cytoplasmic protein that has
sequence homology to FLICE. FLIP is capable of binding to FADD yet is
unable to be cleaved to an active caspase because of a substitution of
a tyrosine for an active site cysteine, thus preventing the initiation
of the death pathway (18-20). FLIP has been demonstrated to play a
role in the prevention of apoptosis in a number of systems, especially
those involving the immune system. Cells with high levels of FLIP
relative to FLICE are generally resistant to apoptosis (21), whereas
cells with low levels of FLIP relative to FLICE are more sensitive to
apoptosis (21). When naive T cells are initially activated, they
express Fas yet are resistant to Fas-mediated apoptosis (22, 23).
However, when these T cells are rechallenged with antigen, they become sensitive to Fas-mediated apoptosis, even though the expression of Fas
on the surface of these cells is unchanged. This change in
susceptibility to Fas-mediated apoptosis correlated with FLIP levels, which are high during the initial stimulation but significantly reduced upon rechallenge (2, 24, 4). Interleukin-2 has been
shown to markedly enhance the susceptibility of activated T cells to
Fas-mediated apoptosis and at the same time down-modulate FLIP (23),
the loss of which may account for the interleukin-2 effect. Recently,
the protection from Fas-mediated apoptosis in B cells upon
cross-linking of the B cell receptor has been shown to be associated
with an increase in FLIP expression (26). Thus, at least for B and T
lymphocytes, susceptibility to apoptosis induced by death receptor
ligation is governed in part by the level of FLIP.
In addition to the ability of FLIP to act as a competitive inhibitor of
the binding of caspase-8 to FADD, it has also been shown to induce the
activation of NF- Although initially described as a viral product (28), a cellular
analogue of FLIP has since been discovered, and high levels are
commonly found in tumors (18, 29). In one study in melanoma cells, the
levels of FLIP were found to correlate inversely with susceptibility to
apoptosis induced by exposure to TRAIL (30). However, other studies
have failed to identify a linkage between FLIP expression and
sensitivity to Fas or TRAIL (7).
Very little is known about the signaling pathways or transcription
elements that control the expression of FLIP. In activated T cells,
FLIP expression has been shown to be dependent on the ERK/MAP kinase
pathway because the addition of a dominant active MKK1 (MEK-1) induces
FLIP in 293T and Jurkat cells (31). The molecular basis for the high
constitutive levels of FLIP observed in many tumor cell lines is unknown.
PI 3-kinase has been shown to protect cells from apoptosis in a
caspase-dependent manner (32-34). PI 3-kinase catalyzes
the phosphorylation of phosphoinositol-4 phosphate and
phosphoinositol-4,5 phosphosphate at the 3 position. Kinases such
as PDK1 and pkB/Akt bind to these phosphorylated intermediates via
their pleckstrin homology domain. PDK1 in turn phosphorylates and
activates pkB/Akt (35), which then phosphorylates several proteins that
have been implicated in the control of cell survival (32, 34, 36-39). The phosphorylation of procaspase-9 by Akt, for example, inactivates this protease, blocking the propagation of death signals originating in
the mitochondria (39). Likewise, Akt-mediated phosphorylation of
Bad results in the binding of this pro-apoptotic Bcl-2 family member to 14-3-3 proteins and its dissociation from the mitochondria (40-42). Some targets of pkB/Akt, such as NF- The ability of a constitutively active MEK1 to induce FLIP expression
in T cell lines and the multiplicity of anti-apoptotic proteins in
which expression and/or function are regulated through the PI 3-kinase
pathway suggest that the constitutive expression of FLIP by tumor cells
might be attributable to the activation of either the Ras-Raf-MEK-ERK
and/or PI 3-kinase pathways. To test these hypotheses, we carried out a
series of studies with drugs and a dominant negative kinase that
inhibit these pathways. The results of our studies clearly implicate
the PI 3-kinase/Akt pathway as the predominant regulator of FLIP
expression in tumor cells.
Cell Lines and Reagents--
The human colon adenocarcinomas
HT29 and DLD-1, the prostate carcinomas DU145 and PC-3, the breast
carcinoma MCF7, the renal carcinomas 786-0 and 769-P, and the melanoma
G-361 cell lines were purchased from the ATCC. The human colon
carcinoma cell line Clone A was obtained from Dr. Ian Summerhayes of
the Lahey Clinic (Burlington, MA), and the breast carcinomas MDA-MB231
and T47D were obtained from Dr. Hava Abraham of the Beth Israel
Deaconess Medical Center (Boston, MA). The PC-3 cells were maintained
in McCoy's 5A media supplemented with 10% fetal bovine serum,
and the G-361 cells were maintained in Kaign's modification of Ham's F12 media supplemented with 10% fetal bovine serum. All of the remaining cell lines were grown in Dulbecco's modified medium supplemented with glutamine, gentamicin, and 10% fetal bovine serum.
The MEK inhibitor PD98059 and the PI 3-kinase inhibitor LY294002 were
purchased from New England Biolabs (Beverly, MA) and Calbiochem
(Lajolla, CA), respectively. The vinculin antibody was purchased from
Sigma. The hemagglutinin (HA) antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), and the rabbit anti-pERK and
anti-p-GSK3a/b antibodies were obtained from New
England Biolabs. The p-Akt (Ser-473) antibody was purchased from BD/PharMingen. The FLIP antibody was generated in New Zealand White rabbits against a peptide spanning amino acids 2-27
(SAEVIHQVEEALDTDEKEMLLFLCRD) of the human protein (48).
Western Analyses--
Cells were treated with 50 µM PD98059 or LY294002 in medium supplemented with 5%
fetal bovine serum for the indicated times and then lysed with Passive
Lysis Solution (Promega, Madison, WI) supplemented with protease
(phenylmethylsulfonyl fluoride and leupeptin) and phosphatase (NaF,
Na3VO4, glycerophosphate, and sodium
pyrophosphate) inhibitors. Lysates were separated on 12%
SDS-polyacrylamide gels, and the fractionated proteins were transferred
to nitrocellulose. The top portions of the blots were removed and
probed for the cytoskeletal protein vinculin to ensure equivalent
loading of all samples. The bottom portions of the blots were probed
either for FLIP, pERK, HA, or p-GSK3a/b. The blots were
further probed with goat anti-rabbit (FLIP and pERK) or goat anti-mouse
(vinculin and HA) conjugated to horseradish peroxidase and then exposed
to SuperSignal chemiluminescent substrate (Pierce). The blots
were then exposed to Kodak X-Omat Blue XB-1 film. The films were
analyzed by densitometry using a Bio-Rad densitometer and Molecular
Analyst software.
FLIP Expression--
RNA was isolated from the treated tumor
cells using Trizol reagent (Life Technologies, Inc.). RT-PCR was
performed (22) using oligonucleotides specific for human FLIP
(5'-GAAGTTGACTGCCTGCTGGCTTTCT and 5'-TGGGGCAACCAGATTTAGTTTCTCC)
and the ribosomal protein S9 (5'-GATGAGAAGGACCCACGGCGTCTGTTCG and
5'-GAGACAATCCAGCAGCCCAGGAGGGACA). The FLIP
oligonucleotides were selected using DNA STAR software to give an
annealing temperature of at least 60 °C and to amplify FLIP
specifically (i.e. not the homologous caspase-8).
[32P]dCTP was incorporated into the products (550 base
pairs for FLIP and 431 for S9). The radioactive products were resolved
on 4% polyacrylamide gels, dried, and exposed to Kodak XAR-5 film. The
films were analyzed by densitometry using a Bio-Rad densitometer and
Molecular Analyst software.
Infection of DN-Akt--
Cell lines were infected with
adenoviral constructs bearing HA-tagged catalytically inactive (49) or
constitutively active Akt (49) or with the same construct bearing the
gene for FLIP Expression Is Dependent on MEK Activation in Two Colon Cancer
Cell Lines but Not in Other Tumor Cell Lines Examined--
The human
colon carcinoma cell lines HT29, clone A, and DLD-1, the prostate
carcinoma cell lines DU145 and PC-3, the breast cancer cell lines MCF7,
MDA-MB231, and T47D, the renal carcinoma cell lines 786-0 and 769-P,
and the melanoma cell line G-361 all constitutively express FLIP (Fig.
1). Because of prior reports linking FLIP
expression in T cells to the activation of MEK1 (31), we sought to
determine the extent to which the MAP kinase pathway contributed to the
constitutive expression of FLIP in tumor cells. To examine the role of
the Ras-Raf-MEK-ERK pathway in FLIP expression, tumor cells were first
cultured overnight in medium containing 1% FCS and then placed in
medium containing 5% FCS with or without the MEK inhibitor PD98059. As
shown in Fig. 1A, ERK phosphorylation was readily
detectable in both the HT29 and clone A colon carcinoma cell lines
after 24 h of serum starvation (zero time point). The readdition
of serum transiently enhanced ERK phosphorylation in clone A and, to a
lesser extent, in HT29 cells. In both cell lines, the addition of
PD98059 (50 µM) for even 15 min completely eliminated ERK
phosphorylation as determined by Western blot analysis. The FLIP level
declined with time in HT29 but not Clone A cells. Similar studies with
all of the other cell lines listed above except the colon carcinoma
DLD-1 demonstrated that PD98059, at concentrations sufficient to
suppress ERK phosphorylation (50 µM), had no effect on
FLIP levels (Fig. 1B). The DLD-1 cells were similar to the HT29 cells in that their FLIP levels declined, albeit modestly, in
response to PD98059. Thus, of the 11 tumor cell lines studied, HT29 and
DLD-1 were the only cells in which FLIP expression appeared to be
linked to sustained MEK activity.
FLIP Expression Is Dependent on PI 3-Kinase Activity--
To
assess the role of the PI 3-kinase pathway in the regulation of FLIP
levels, four of the tumor cell lines were cultured in fresh medium
containing or lacking the PI 3-kinase inhibitor LY294002. As shown in
Fig. 2, the addition of fresh medium
increased the levels of p-Akt to a variable extent in each cell
line examined, and this increase was blocked by 50 µM
LY294002. In each of the tumor cell lines tested, LY294002
down-modulated FLIP expression. In the case of the HT29 cells, this
suppressive effect was apparent within 6 h (not shown), whereas in
Clone A, MCF7, and DU145 cells, no significant decrement was observed
until 24 h. To determine whether the suppressive effect of
LY294002 on FLIP protein levels was associated with a decrement in the
level of FLIP transcripts, FLIP mRNA levels were analyzed by RT-PCR
at various time points after placing the cells in either control medium
or medium containing 50 µM LY294002. As shown in Fig.
3, the addition of LY294002 resulted in a
decline in the level of FLIP mRNA levels. The extent of inhibition at 6 h ranged from 50% of the control for the DU145 and MCF7
cells to as much as 70 and 85% for the Clone A and HT29 cells,
respectively, as determined by densitometry. In Clone A, MCF7, and
DU145 cells, the FLIP mRNA levels were reduced after only 3 h
of exposure to LY294002.
FLIP Expression Is Dependent upon pkB(Akt) Activity--
To
determine the role of pkB(Akt) in FLIP expression, tumor cells were
infected with an adenoviral construct containing an HA-tagged dominant
negative form of pkB/Akt. A similar adenovirus containing a
The mechanism by which the PI 3-kinase/Akt pathway regulates FLIP
expression is yet to be determined. As mentioned previously, Akt has
been shown to phosphorylate several transcription factors (e.g. CREB, NF-
Our results indicate that sustained MEK activity is not essential for
FLIP expression in most tumor cell lines as it appears to be in
activated T cells. However, FLIP expression is critically dependent
upon PI 3-kinase and Akt activity. These data add FLIP to the growing
list of survival-related proteins regulated by the PI 3-kinase pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B via TNF receptor-1 through TRADD, TRAF-2,
RIP, NIK, and IKK (27), thus providing a second anti-apoptotic
mechanism for FLIP.
B (43), CREB (44, 45),
and Forkhead (46), are transcription factors that regulate cell
survival. In some cells, the anti-apoptotic effect of NF-
B activation is partly attributable to the expression of cIAP, a potent
caspase inhibitor (47). The phosphorylation of Forkhead by Akt blocks
its activity, reducing the levels of expression of pro-apoptotic
proteins such as Fas ligand (46). These data illustrate the diversity
of downstream effectors through which the PI 3-kinase/Akt signaling
pathway affects cell survival.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase at the specified multiplicities of
infection. After overnight incubation, the medium was replaced. Six or
24 h later, the cells were then lysed in preparation for Western
blot analysis.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
A, Western blot of HT29 and Clone A
cells treated either with serum alone or serum with the MEK inhibitor
PD98059 (50 µM). Cells were first serum-starved in 1%
FCS overnight and then exposed to 5% FCS in culture media ± PD98059 for 15 or 30 min or 24 h. The lysates were then probed for
pERK, FLIP, and vinculin as described under "Experimental
Procedures." B, DU145, PC-3, DLD-1, MCF7, MDA-MB231, T47D,
769-P, 786-0, and G-361 cells were similarly treated either with
complete media (5% Y/designated media) alone or complete media and
PD98059 for 24 h. The lysates were probed for pERK, FLIP, and
vinculin.
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Fig. 2.
Western blot of HT29, Clone A, DU145, and
MCF7 cells treated either with serum alone or with the PI 3-kinase
inhibitor LY294002 for 24 h. The lysates were probed for
p-Akt, FLIP, and vinculin.
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Fig. 3.
FLIP and S9 mRNA levels. Cells were
treated either with serum alone or with the PI 3-kinase inhibitor
LY294002 for 3 or 6 h. The lysates were subjected to RT-PCR as
described. Data were analyzed by autoradiography.
-galactosidase insert was used as a negative control. For the HT29,
Clone A, and MCF7 cell lines, infection was carried out with a
multiplicity of 300 and an overnight incubation followed by the
addition of fresh medium and an additional 24 h of incubation. This protocol had no affect on the viability of these cell lines but
proved toxic for the DU145 cells, which were therefore infected at a
lower multiplicity of infection (150) and a shorter incubation time (6 h) to prevent significant cell death. In each case, infection of the
tumor cells was confirmed by Western blot analysis using an HA-specific
antibody to detect the HA-tagged dominant negative pkB/Akt (Fig.
4A). To confirm that this
DN-Akt virus actually inhibited the activation of endogenous Akt, we
first carried out Western blot analyses using commercially available
anti-phospho-Akt antibodies as we had done previously in experiments
with LY294002 (Fig. 2). These studies were largely unsuccessful because
of unexpected cross-reactivity of the antibodies with the DN-Akt
virus. To avoid this confounding artifact, we chose a downstream
Akt target, glycogen synthase kinase-3, as an indicator of Akt activity
in our cell lines (50). As shown in Fig. 4, GSK-3 phosphorylation was
reduced in cells infected with the DN-Akt virus but not in the
uninfected control cells or cells harboring the
-galactisodase
virus. The extent of inhibition was variable, ranging from almost
complete inhibition in the HT29, Clone A, and DU145 cells to 40% for
the MCF7 cells, as determined by densitometry. These data indicate that
the DN-Akt virus effectively blocks the activity of endogenous Akt.
Infection with the DN-Akt virus down-regulated FLIP expression in all
of the tumor cell lines tested (Fig. 4A). This finding corroborates the results of studies with LY294002 implicating the PI
3-kinase/Akt pathway as a key regulator of FLIP expression in tumor
cells. To verify that the down-regulation of FLIP was not a general
effect of adenoviral infection and to further verify the influence of
activated Akt on FLIP expression, the same cell lines were infected
with an adenovirus containing a constitutively active Akt insert. As
shown in Fig. 4B, infection of each cell line with the
constitutively active Akt adenovirus resulted in increased
phosphorylation of GSK-3. FLIP expression in each cell line was also
increased. In addition to confirming a direct link between Akt activity
and FLIP expression, these experiments confirm that the adenovirus was
not responsible for observed changes in FLIP expression.
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Fig. 4.
A, Western blot of HT29, Clone A, DU145,
and MCF7 cells uninfected or infected with either the -galactisodase
control adenovirus or an adenovirus with an insert containing the DN
form of Akt conjugated to HA. The multiplicity of infection was 300 for
clone A, HT29, and MCF7 and 150 for DU145. After overnight infection,
the cells were fed with fresh medium. The lysates were probed
for FLIP, HA, p-GSK3a, and vinculin after an additional 6 (DU145) or 24 h (Clone A, HT29, and MCF7). B, the same
cell lines as in A were infected with a constitutively
active Akt adenovirus conjugated to HA. The multiplicities of infection
and incubation times were the same as in A. The lysates were
probed for FLIP, HA, p-GSK3a, and vinculin.
B, Forkhead), many of which have been
implicated in the expression of genes whose protein products regulate
susceptibility to apoptosis. CREB, for example, plays a major role in T
cell survival (51) and is phosphorylated by Akt at the same site phosphorylated by its structurally relative protein kinase A
(44). The Akt substrate Forkhead is involved in the regulation of Fas ligand expression (46) and NF-
B in the expression of the caspase inhibitor survivin (47). The enhanced expression of survivin is, in
fact, thought to be the dominant factor underlying the protective
effect of NF-
B activation against apoptosis induced by exposure to
tumor necrosis factor (47). It appears likely that one or more factors
may be involved in the regulation of FLIP, and studies to determine the
role played by these factors in FLIP expression are currently under way.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA74401 (to J. W. M.) and AG15051 (to K. W.).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.
¶ To whom correspondence should be addressed: Dept. of Hematology and Oncology, Rm. 309, Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, 4 Blackfan Cr., Boston, MA 02115. Fax: 617-975-8030; E-mail: jmier@caregroup.harvard.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.C000569200
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ABBREVIATIONS |
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The abbreviations used are: TNF, tumor necrosis factor; DD, death domain; FADD, Fas-associated death domain; FLICE, FADD-homologous ICE-like protease; FLIP, FLICE-inhibitory protein; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; p-Akt, phosphorylated Akt; GSK3a/b, glycogen synthase kinase 3a/b; HA, hemagglutinin; MAP, mitogen-activated protein; MEK, MAP/ERK kinase; PI, phosphatidylinositol; CREB, cAMP-response element-binding protein; RT-PCR, reverse transcriptase-polymerase chain reaction; FCS, fetal calf serum; DN-Akt, dominant negative Akt adenoviral construct; cIAP, cellular inhibitor of apoptosis protein; TRAIL, TNF-related apoptosis-inducing ligand.
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