From the Division of Medical Oncology,
¶ Division of Pulmonary Medicine, and
Department of
Pathology, University of Colorado Health Sciences Center, Denver,
Colorado 80262 and ** Aventis Pharmaceuticals,
65926 Frankfurt, Germany
Received for publication, June 15, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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We find that the prostate cancer cell lines
ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by
TRAIL (tumor-necrosis factor-related
apoptosis-inducing ligand), while
the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the
LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase
PTEN, a negative regulator of the phosphatidylinositol (PI)
3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity.
Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed
constitutive Akt activity and sensitized LNCaP cells to TRAIL.
Treatment of LNCaP cells with TRAIL alone induced cleavage of the
caspase 8 and XIAP proteins. However, processing of BID, mitochondrial
release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either
wortmannin, LY-294002, or cycloheximide. Blocking cytochrome
c release by Bcl-2 overexpression rendered LNCaP cells
resistant to TRAIL plus wortmannin treatment but did not affect caspase
8 or BID processing. This indicates that in these cells mitochondria
are required for the propagation rather than the initiation of the
apoptotic cascade. Infection of LNCaP cells with an adenovirus
expressing a constitutively active Akt reversed the ability of
wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI
3-kinase-dependent blockage of TRAIL-induced apoptosis in
LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.
TRAIL (tumor-necrosis factor-related
apoptosis-inducing ligand) (1) also
known as Apo-2 ligand (2) is a proapoptotic cytokine that together with
three related proteins (tumor necrosis factor- TRAIL is capable of inducing apoptosis in a wide variety of cancer
cells in culture and in tumor implants in mice, including cancers of
the colon, breast, lung, kidney, central nervous system, blood, and
skin (1, 6, 8-11). At the same time, unlike tumor necrosis factor- Despite the ubiquitous expression of TRAIL receptors, a significant
proportion of cell lines originating from various cancer types
demonstrate either partial or complete resistance to the proapoptotic
effects of TRAIL. These findings suggest either defects in apoptotic
pathways or the presence of inhibitors of TRAIL-induced apoptosis. The
latter possibility appears to be more likely, since the resistance of
many types of cancer cells to TRAIL can be reversed by treatment with
protein synthesis inhibitors (15-19) or chemotherapeutic agents (9,
11). Some normal human cells can also be sensitized to TRAIL by the
inhibition of protein synthesis (20). The elucidation of mechanisms
that control sensitivity to TRAIL may lead to better understanding of
death receptor-mediated signaling and help to develop TRAIL-based
approaches to cancer treatment.
Activation of death receptors leads to the formation of the
death-inducing signaling complex
(DISC)1 (21), which includes
the receptor itself, and caspase 8 (22). The recruitment of caspase 8 to TRAIL receptors DR4 and DR5 is thought to be mediated by the adaptor
protein FADD (23-25). The formation of the DISC triggers
autoprocessing and activation of caspase 8 (22) that in turn results in
the cleavage and activation of the effector caspase 3 or 7 (26, 27),
leading to apoptosis. Activated caspase 8 may also cleave a
proapoptotic protein BID, whose cleavage product triggers cytochrome
c release from mitochondria (28, 29). In some but not all
cell types, the mitochondrial step may be required to amplify the
apoptotic signal and fully activate caspase 8 (30). Since the
TRAIL-induced apoptotic signal is a multistep process, inhibition of
this cascade may occur at several stages. For example, at the
ligand-receptor level, TRAIL signaling could be inhibited by the
overexpression of nonfunctional TRAIL receptors DcR1 or DcR2 (31) or by
proteins that induce rapid internalization of TRAIL receptors (similar
to Fas inhibition the adenoviral protein E3) (32). At the DISC, the
apoptotic pathway may be inhibited by cFLIP protein that is capable of
blocking processing and activation of caspase 8 (33, 34). Downstream of
DISC, IAP proteins may specifically inhibit the executor caspases 3 and
7 (35). In those cells that require mitochondria to stimulate apoptosis, the signal may be inhibited by Bcl-2/Bcl-XL
types of proteins that prevent the release of proapoptotic factors from the mitochondria (30).
In the present study, we tested the cytotoxic effects of TRAIL on six
human prostate cancer cell lines, demonstrating variable responses,
with some cell lines being extremely sensitive and others highly
resistant. The highly resistant cell line LNCaP was further
investigated to examine mechanisms that protect it from TRAIL-mediated
apoptosis. We find that the TRAIL-induced death signal in LNCaP cells
is negatively regulated by a high constitutive activity of protein
kinase Akt. Furthermore, the antiapoptotic block occurs downstream of
caspase 8 activation at the level of BID protein cleavage. This study
is the first demonstration that the PI 3-kinase/Akt pathway may
interfere with an apoptotic signal by inhibiting processing of BID.
Antibodies--
Antibodies were obtained from the following
sources: anti-phospho-Akt (New England Biolabs, Beverly, MA);
anti-cytochrome c and anti-BID (Zymed
Laboratories Inc.); anti-Akt and anti-XIAP (Transduction
Laboratories, Lexington, KY); anti-HA1 tag (Babco, Richmond, CA);
anti-caspase 8 (Upstate Biotechnology, Inc., Lake Placid, NY);
anti-caspase 7 (PharMingen, San Diego, CA); anti-caspase 9 (Oncogene
Research Products, Boston, MA); anti-FLIPL (Affinity BioReagents, Golden, CO); anti-FLIP Cell Culture--
Prostate cancer cell lines LNCaP, PC-3, DU
145, TSU-Pr1, JCA-1, and ALVA-31 were passaged in RPMI 1640 with 10%
fetal calf serum, 50 units/ml penicillin, and 50 units/ml streptomycin.
The sources for these cell lines, their characterization, and use in
our laboratories have been described previously (36). LNCaP cells
overexpressing Bcl-2 (37) were kindly provided by Dr. R. Buttyan
(Columbia Presbyterian Medical Center, New York, NY) and grown in
medium supplemented with 400 µg/ml of G418.
Expression of Recombinant TRAIL in Yeast Pichia pastoris--
A
cDNA encoding for soluble human TRAIL (residues 114-281) was
amplified by polymerase chain reaction from the expressed sequence tag
clone 117926 (GenBankTM accession number T90422) in frame
with the N-terminal hexahistidine tag using oligonucleotides
5'-AGTCATGAATTCCATCACCATCACCATCACGTGAGAGAAAGAGGTCCTCAGAGAGTAG-3' and
5'-AGTCATGGTACCTTAGCCAACTAAAAAGGCCCCGAAAAA-3'. This cDNA was then
cloned into the EcoRI/KpnI sites of pPICZ Cytotoxicity Assays--
Cell viability was determined
spectrophotometrically using an Aqueous One tetrazolium-based
assay (Promega, Madison, WI). Absorbance was measured at 490 nm, and
data from duplicate determinations were plotted as percentage of
untreated control cells. Quantitative analysis of DNA fragmentation was
done using a Cell Death Detection ELISAplus kit (Roche
Diagnostics Corp., Indianapolis, IN) by measuring relative amounts of
DNA-histone complexes released into the cytoplasm. Data from triplicate
determinations were plotted as percentage of control of untreated
cells. A TUNEL assay was performed using the FragELTM DNA
fragmentation detection kit (Oncogene Research Products, Cambridge, MA).
Measurement of Cytochrome c Release from
Mitochondria--
Cytosolic extracts from LNCaP cells were prepared by
the hypotonic lysis procedure originally described by Bossy-Wetzel
et al. (39) and modified by Carson et al. (40).
LNCaP cells grown on 15-cm plates to 50% confluence were placed on ice
and then scraped directly into growth medium and centrifuged for
2 min at 200 × g. Cell pellets were then washed once
with ice-cold phosphate-buffered saline and resuspended in 300 µl of
hypotonic lysis buffer (220 mM mannitol, 68 mM
sucrose, 50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 5 mM EDTA, 2 mM MgCl2, 1 mM dithiothreitol) containing protease inhibitors,
including Complete Mixture (Roche Molecular Biochemicals, Germany), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
2 µg/ml aprotinin. Cells were incubated on ice for 45 min and
homogenized by pipetting (10 passes up and down). Supernatants were
cleared by 10-min centrifugation at 1000 × g, followed
by 30 min at 100,000 × g and analyzed by Western
blotting with the anti-cytochrome c antibody.
Construction of Adenoviral Vectors Expressing myr-Akt--
The
full-length coding sequence of human Akt1 was fused in frame with the
myristoylation signal from the human Src protein in the N
terminus and HA tag in the C terminus (myr-Akt). Kinase-dead construct
was created by mutating lysine 179 for alanine, destroying in that way
an ATP-binding site (myr-Akt(K Effect of Soluble TRAIL on Six Prostate Cancer Cell
Lines--
Recombinant human TRAIL (residues 114-281) was produced in
methylotrophic yeast P. pastoris as a fusion protein
containing an N-terminal hexahistidine tag and a cleavable secretion
signal from yeast
To investigate the mechanisms controlling the resistance of LNCaP cells
to the cytotoxic effect of TRAIL, a series of Western and Northern blot
experiments were done to compare the expression of various components
of the TRAIL signaling pathway among the six prostate cancer cell
lines. However, no correlation was found between the sensitivity of
cells to TRAIL and the expression of TRAIL receptors DR4 and DR5, decoy
receptors for TRAIL DcR1 and DcR2, initiator caspase 8, and apoptosis
inhibitory protein cFLIP (data not shown). LNCaP cells contain a
deactivating frameshift mutation in the gene encoding the tumor
suppresser PTEN (42). This dual specificity phosphatase cleaves D3
phosphate of second messenger lipid phosphatidylinositol (PI)
3,4,5-trisphosphate (43). PI 3,4,5-trisphosphate produced by PI
3-kinase activates protein kinase Akt, and therefore, the lack of
negative regulation by PTEN results in the constitutive activation of
Akt in LNCaP cells (40). Immunoblot analysis with an antibody that
specifically recognizes the phosphorylated/activated form of Akt
(Ser473) demonstrates that LNCaP cells possess the highest
Akt activity among the six prostate cancer cell lines (Fig.
2A). Treating cells with the
inhibitor of PI 3-kinase, wortmannin (200 nM), for 6 h
reverses the high constitutive activity of Akt (Fig.
2B).
Inhibition of PI 3-Kinase Activity or Protein Synthesis Renders
LNCaP Cells Sensitive to TRAIL--
To test whether the high
constitutive activity of Akt in LNCaP cells results in their resistance
to TRAIL, we first examined how PI 3-kinase inhibitors wortmannin (200 nM) and LY-294002 (20 µM) effect TRAIL
cytotoxicity. Wortmannin acts at nanomolar concentrations by covalently
modifying PI 3-kinase (44) but is unstable in aqueous solutions (45),
making it possible that some PI 3-kinase activity can be restored by
de novo synthesis in the course of the experiment. LY-294002
does not bind the enzyme covalently and has an IC50 value
for PI 3-kinase about 500-fold higher than that of wortmannin (46) but
is much more stable in culture medium. We have found that both
substances significantly enhanced the proapoptotic activity of TRAIL in
LNCaP cells as judged by apoptotic morphology (Fig.
3A) and DNA fragmentation
(Fig. 3B), quantitated by measuring the relative amounts of
DNA-histone complexes released into cytoplasm. Since wortmannin and
LY-294002 inhibit PI 3-kinase by different mechanisms, this result
confirms that sensitization of cells to TRAIL occurs through the
inhibition of the PI 3-kinase pathway. Inhibition of protein synthesis
with cycloheximide also sensitized LNCaP cells to TRAIL (Fig. 3,
A and B). The DNA fragmentation induced by TRAIL
in combination with wortmannin, LY-294002, or cycloheximide was greater
than that triggered by the potassium ionophore valinomycin (Fig.
3B), a potent inducer of apoptosis (47). Thus, the
resistance of LNCaP cells to TRAIL results from the blockage of the
TRAIL-induced apoptotic signal transduction cascade rather than the
defects in apoptotic machinery. These data demonstrate that the
inhibition of TRAIL-mediated apoptosis in LNCaP cells requires PI
3-kinase activity and involves some short lived protein
component(s).
TRAIL-mediated Cytochrome c Release Is Blocked in LNCaP
Cells--
Depending on the cell type, apoptotic signaling mediated by
CD95/Fas may or may not require the release of proapoptotic factors (cytochrome c and apoptosis-inducing factor) from
mitochondria. In type II, but not in type I cells, inhibition of
mitochondrial apoptogenic activities by overexpression of Bcl-2 protein
blocks Fas-mediated apoptosis (30). To examine whether the apoptogenic activity of mitochondria is required for the transduction of the TRAIL-induced death signal in LNCaP cells, the cytotoxic effects of
TRAIL alone or in combination with wortmannin were studied in an LNCaP
cell line overexpressing Bcl-2 (37). Quantitation of apoptotic nuclei
by the TUNEL technique clearly demonstrates that Bcl-2 overexpression
impairs the cytotoxic effect of TRAIL (Fig.
4A), indicating that
mitochondria play an important role in TRAIL-induced apoptosis of LNCaP
cells. If the resistance of LNCaP cells to TRAIL results from the high
constitutive activity of Akt, this enzyme may block apoptosis either
upstream (48, 49) or downstream (50) of mitochondrial cytochrome
c release. To discriminate between these two possibilities,
experiments were done to examine whether TRAIL-induced cytochrome
c release is inhibited in LNCaP cells. LNCaP cells were
incubated for 6 h with TRAIL alone or TRAIL in combination with
cycloheximide or wortmannin. Cytosolic extracts were then prepared
under conditions that keep mitochondria intact (39), and cytochrome
c released to the cytosolic fraction was then detected by
immunoblotting (Fig. 4B). This experiment demonstrated that
in LNCaP cells TRAIL alone does not trigger the release of cytochrome
c from the mitochondria, but it does so in combination with
wortmannin and, to a lesser extent, cycloheximide. Thus, TRAIL-induced
apoptotic signaling in LNCaP cells is blocked upstream of the
mitochondria.
TRAIL-induced Apoptotic Signaling in LNCaP Cells Is Blocked at the
Level of BID Cleavage--
To understand at what biochemical step the
TRAIL-mediated apoptotic cascade is blocked in LNCaP cells, a series of
immunoblotting experiments were carried out using antibodies to
proteins involved in this cascade. Our results demonstrate that
processing of initiator caspase 8 is induced by TRAIL alone as
efficiently as when TRAIL is combined with cycloheximide and wortmannin
(Fig. 5A). Similarly, these
two compounds did not enhance TRAIL-induced cleavage of the apoptosis
inhibitory protein XIAP, a substrate for several caspases including
caspase 8 (51). These results suggest that the antiapoptotic block in
LNCaP occurs downstream of caspase 8 activation. In contrast,
proteolytic cleavage of the caspase 8 substrate BID was not detected in
TRAIL-treated cells unless TRAIL was administered in combination with
cycloheximide or wortmannin. Caspase 8-mediated cleavage of BID
generates a proteolytic fragment, tBID, that is capable of inducing
mitochondrial cytochrome c release and providing a
functional link between death receptors and the mitochondria (28, 29).
The lack of BID cleavage is thus consistent with the observation that
TRAIL alone is not capable of inducing cytochrome c release.
TRAIL-mediated processing of cytochrome c-dependent caspase 9 and effector caspase 7 were also detected only if TRAIL was combined with wortmannin or
cycloheximide. The involvement of PI 3-kinase in the blockage of
TRAIL-induced BID cleavage was further confirmed by the experiment with
another PI 3-kinase inhibitor, LY-294002. Fig. 5B
demonstrates that treatment of LNCaP cells with LY-294002 in
combination with TRAIL results in the decreasing of cellular BID level.
Thus, the PI 3-kinase- and protein synthesis-dependent
antiapoptotic block in LNCaP cells occurs downstream of caspase 8, at
the level of BID cleavage.
Alternatively, it is possible that the lack of BID cleavage may result
from an inhibition of mitochondrial function. By analogy with the
CD95/Fas system, LNCaP cells may be classified as type II cells, since
mitochondrial function appears to be necessary for apoptosis. In type
II cells, mitochondrial cytochrome c release serves as an
amplification loop that potentiates the activation of caspase 8. If a
similar mitochondria-dependent amplification loop is
involved in TRAIL signaling in LNCaP cells, its disruption may affect
caspase 8-mediated BID cleavage. To test whether or not cleavage of BID
in LNCaP cells depends on mitochondrial function, the processing of BID
in Bcl-2 overexpressor LNCaP cells versus parental cells was
examined. Immunoblot analysis (Fig. 5C) demonstrates that
after 6 h of treatment with TRAIL plus wortmannin or TRAIL plus
cycloheximide, BID is processed equally well in parental and
Bcl-2-overexpressing LNCaP cells. In addition, caspase 8 was processed
efficiently in both cell lines as judged by the TRAIL-induced appearance of a cleavage product that corresponds to the 20-kDa active
subunit of caspase 8. Thus, apoptogenic activity of mitochondria is not
required for TRAIL-induced cleavage of BID and caspase 8.
Our results demonstrate that the blockage of TRAIL-induced apoptosis at
the level of BID cleavage can be removed by cycloheximide treatment,
suggesting the possibility that this inhibition may be mediated by a
short lived protein. It has been hypothesized that inhibition of
protein synthesis sensitizes cells to death-inducing ligands by
down-regulating antiapoptotic cFLIP proteins (15, 19, 52). To determine
whether this is the case for LNCaP cells, cell lysates from a previous
experiment (Fig. 5A) were immunoblotted with antibodies that
recognize different splice variants of cFLIP proteins:
FLIPL, FLIP Constitutively Active Akt Blocks TRAIL/Wortmannin-induced BID
Cleavage--
The potentiating effect of wortmannin on TRAIL-induced
BID cleavage suggests that Akt may be involved in the inhibition of TRAIL signaling in LNCaP cells. To confirm this hypothesis, a constitutively active Akt, constructed by fusing Akt to the
myristoylation signal of Src protein (myr-Akt) was introduced into
LNCaP cells by adenovirus-mediated gene transfer. If Akt is the sole
target of the wortmannin effect, then this infection would be expected to counteract the ability of wortmannin to sensitize LNCaP cells to
TRAIL-induced BID cleavage. As a control, an adenovirus containing kinase-inactive Akt (myr-Akt(K
We next tested whether activated Akt can also inhibit cleavage of BID
induced by TRAIL plus cycloheximide treatment. However, no rescue was
observed even when the adenovirus titer was 16 times higher than that
sufficient to inhibit proapoptotic effects of TRAIL plus wortmannin
treatment (Fig. 6B). These results suggest that the
protective effects of Akt on BID cleavage may require Akt-induced
protein synthesis.
Our results (Figs. 1B and 2A) indicate the
existence of TRAIL-sensitive cell lines that possess an elevated
Akt activity, albeit at a much lower level than that found in LNCaP
cells. This result raises the question of whether the protective effect
of Akt is cell type-specific or it occurs only when the level of Akt
activity is above a certain threshold. To examine these possibilities, we overexpressed myristoylated Akt in various TRAIL-sensitive cell
lines: DU 145 and ALVA-31 prostate cancer cells, A498 renal cancer
cells, and HeLa cervical cancer cells. Of them, only ALVA-31 cells
acquired significant resistance to TRAIL upon myr-Akt overexpression (Fig. 6C). Thus, the protective effect of Akt appears to be
cell type-specific.
We have developed a novel approach to obtaining preparative
amounts of proapoptotic ligand TRAIL and tested the effects of this
reagent on a panel of six prostate cancer cell lines. Soluble TRAIL was
produced by a methylotrophic yeast P. pastoris, secreted into the medium, and then purified to homogeneity by one-step chromatography on a nickel-chelate column. Cytotoxicity assays demonstrated that three cell lines, ALVA-31, DU 145, and PC-3, were
very sensitive to TRAIL, while in comparison JCA-1 and TSU-Pr1 revealed
moderate sensitivity, and LNCaP cells were resistant to as high as 4 µg/ml TRAIL. Comparing these results with the data published on Fas
ligand-induced apoptosis indicates that prostate cancer cells differ in
their responses to these two apoptotic stimuli. Whereas cells believed
to be derived from primary prostate cancer tumors (ALVA-31 and JCA-1)
were reported to be sensitive to Fas ligand-induced apoptosis, cells
originating from distant metastasis (DU 145, PC-3, TSU-Pr1, and LNCaP)
appeared to be Fas-resistant despite the expression of Fas antigen on
the cell surface (36, 54). In contrast, among the above listed cell
lines, only LNCaP cells were resistant to TRAIL-induced apoptosis,
indicating that TRAIL has a greater potential as an agent to treat
metastatic prostate cancer. These data also suggest that despite the
similarity of CD95/Fas and TRAIL receptors, TRAIL and Fas
ligand-mediated apoptosis may employ different signal transduction
pathways or be negatively regulated by different mechanisms in these
prostate cancer cells.
We found that among six prostate cancer cell lines examined, the LNCaP
cells, which are the most highly resistant to TRAIL-induced apoptosis,
have the highest constitutive activity of the Akt protein kinase. This
result is consistent with the lack of the functional tumor suppressor
PTEN, a negative regulator of the PI 3-kinase/Akt pathway in these
cells (42). Because the Akt protein kinase is known to block apoptosis
(55), we tested whether inhibition of this pathway affects the
sensitivity of LNCaP cells to TRAIL. We found that treatment with the
PI 3-kinase inhibitors wortmannin and LY-294002 or the protein
synthesis inhibitor cycloheximide renders them sensitive to
TRAIL-induced apoptosis. Thus, the resistance of LNCaP cells to TRAIL
results not from defects in apoptotic machinery, but from PI
3-kinase-dependent inhibition of the TRAIL-mediated apoptotic signaling pathway.
It has been reported that apoptosis induced by triggering of CD95/Fas
(56, 57) is counteracted by the PI 3-kinase/Akt pathway, but the
molecular mechanisms that cause apoptosis resistance remain unclear. To
identify which step of the TRAIL-mediated apoptotic pathway is blocked
in LNCaP cells, we first tested whether the release of proapoptotic
factors from mitochondria is essential for TRAIL-induced death of these
cells. The involvement of mitochondria in apoptosis induced by death
receptors remains controversial. Scaffidi et al. (30) have
proposed that two types of cells exist that differ with respect to
their requirement for mitochondria during Fas-mediated apoptosis. In
type I cells, caspase 8 is activated without involvement of
mitochondria to a level sufficient to process the effector caspase 3. In contrast, in type II cells a mitochondria-dependent amplification loop is required to fully activate caspase 8 and transduce an apoptotic signal. This model has recently been questioned by Huang et al. (58), who argue that the difference between type I and type II cells is an artifact of using agonistic anti-Fas antibodies to trigger Fas signaling instead of Fas ligand. To clarify
the role of mitochondria in TRAIL-induced apoptosis in LNCaP cells, we
used Bcl-2-overexpressing LNCaP cells, which were shown to exhibit an
impaired cytochrome c release in response to various
apoptotic stimuli (37). Our results demonstrate that these cells are
much more resistant to TRAIL plus wortmannin-induced apoptosis compared
with the parental cells. In these experiments, apoptosis was triggered
by soluble death receptor ligand and not agonistic antibody, supporting
the notion that in some cells mitochondrial function is indeed
essential for death receptor-mediated apoptosis.
Using a cell fractionation approach, we have found that TRAIL-induced
cytochrome c release was blocked in LNCaP cells, but both
wortmannin and cycloheximide are capable of overcoming this block.
Release of mitochondrial cytochrome c by death receptors is
triggered by a multistep mechanism. The formation of the DISC results
in autoprocessing and activation of the initiator caspase 8 followed by
cleavage of the proapoptotic protein BID (28, 29). A proteolytic
fragment of BID translocates to the mitochondria as an integral
membrane protein and triggers the release of mitochondrial cytochrome
c (59). Using immunoblot analysis, we found that cleavage of
caspase 8 and one of its substrates, the antiapoptotic protein XIAP
(51) were induced by TRAIL alone as efficiently as when TRAIL was
combined with either wortmannin or cycloheximide. This important result
indicates that DISC formation or caspase 8 activation was not blocked
in LNCaP cells. In contrast, wortmannin and cycloheximide were required
for TRAIL-induced cleavage of BID, the release of cytochrome
c, and processing of caspases 9 and 7. Thus, the PI
3-kinase-dependent block of TRAIL-induced apoptosis in
LNCaP cells occurs at the level of BID cleavage.
The requirement for mitochondrial apoptogenic activity in TRAIL-induced
death suggests that LNCaP cells are similar to type II cells. If so,
the lack of BID cleavage could, in principle, be explained by the
disruption of a mitochondria-dependent amplification loop,
resulting in only partial activation of caspase 8. To see whether this
hypothesis could be true, we compared the cleavage of BID and caspase 8 in Bcl-2-overexpressing versus parental LNCaP cells and
found that these proteins are processed equally well in both cell
lines. These results demonstrate that although mitochondrial function
is important for TRAIL-induced apoptosis in LNCaP cells, unlike
"typical" type II cells mitochondria are required not to amplify
caspase 8 activation but to transduce apoptotic signal downstream of
the initiator caspase. Therefore, it may be possible to classify LNCaP
as type III cells where mitochondria are involved in the propagation
rather than the initiation of the apoptotic cascade.
Involvement of PI 3-kinase in the block of apoptosis suggests that Akt
could mediate resistance of LNCaP cells to TRAIL. To confirm this
hypothesis, we tested whether overexpression of constitutively active
Akt could inhibit the proapoptotic effect of TRAIL plus wortmannin
treatment. For this purpose, we used a myristoylated derivative of Akt,
which exhibits kinase activity independently of PI 3-kinase (60). Both
apoptosis (data not shown) and BID cleavage induced by treatment of
LNCaP cells with TRAIL plus wortmannin were inhibited by overexpression
of myristoylated Akt, indicating that resistance of LNCaP cells to
TRAIL is, at least in part, mediated by Akt.
It has been documented that Akt may inhibit a variety of apoptotic
stimuli in multiple ways (55). These include direct phosphorylation and
modulation of proapoptotic proteins BAD (48) and caspase 9 (50),
activation of antiapoptotic NF- Although it remains unclear how the PI 3-kinase/Akt pathway mediates
inhibition of BID cleavage, our data suggest an indirect mechanism.
First, inhibition of protein synthesis by cycloheximide affected the
same step of TRAIL apoptotic cascade as the inhibition of PI 3-kinase.
However, even very high levels of constitutively active Akt did not
rescue BID from cleavage when TRAIL was combined with cycloheximide
rather than wortmannin. These results suggest that a short lived
protein is involved in the PI 3-kinase/Akt-mediated blockage of BID
cleavage, and the synthesis of this hypothetical protein may be
triggered by Akt. Second, the effect of myristoylated Akt appears to be
cell type-specific, since its overexpression did not rescue HeLa,
DU-145, or A498 cells from TRAIL-induced apoptosis (data not shown) but
did rescue LNCaP and ALVA-31 cells. This could reflect either the
difference in apoptotic pathways employed by different cell types or
the absence of certain factors required for the protective effect of
Akt. In particular, human prostate cancer cell lines have scores of
chromosomal deletions and rearrangements (64), so that LNCaP and PC-3
differ in much more than Akt levels.
It has been reported that short term (3-7-h) treatment of human
keratinocytes (52), HeLa and Kym-1 cells (19) with cycloheximide significantly reduces the level of cellular cFLIP protein. Since upon
overexpression cFLIP is capable of inhibiting Fas-mediated apoptosis
(33, 34), it has been suggested that protein synthesis inhibitors
sensitize cells to TRAIL by down-regulating cFLIP. To examine this
hypothesis, we tested the level of various splice variants of cFLIP
(FLIPL, FLIP
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, CD95L/FasL, and
TWEAK/Apo3L) constitutes a family of ligands that transduce death
signals through death domain-containing receptors (3-5). TRAIL is a
type II transmembrane protein that functions by binding to two closely
related receptors, DR4 and DR5 (6). Both TRAIL and its receptors are
ubiquitously expressed (7), suggesting the existence of mechanisms that
protect normal tissues from TRAIL-induced apoptosis.
and Fas ligand, whose use for cancer therapy has been hampered by their
severe toxicity (12, 13), TRAIL has no toxic effects when systemically
administered in rodents (10) and nonhuman primates (9). Although the
majority of normal human cells tested so far appear to be
TRAIL-resistant, recent experiments have demonstrated that cultured
human liver cells may be sensitive to TRAIL (14), suggesting that
additional studies are required to investigate what determines
resistance or sensitivity to this agent.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
(Calbiochem).
A
vector (Invitrogen, Carlsbad, CA) in frame with the cleavable secretion
signal from yeast
factor. All manipulations of yeast were performed
in general as outlined in the Invitrogen manual. Briefly, the
expression vector was linearized and transformed by electroporation
into P. pastoris strain SMD1168 (38). Transformants were
selected on 500 µg/ml of Zeocin, and secretion of TRAIL was tested by
Western blotting. For large scale production, yeast were grown for
24 h in 10 liters of complex medium containing glycerol and
antifoam 289 (Sigma, St. Louis, MO) and buffered with 100 mM potassium phosphate buffer, pH 6.0, at constant aeration
and mixing to A600 of 15. To induce TRAIL
production, cells were pelleted by centrifugation, resuspended in
complex medium containing 0.5% methanol, and grown for 24 h. The supernatant was concentrated using tangential flow Prep/Scale-TFF cartridge (Millipore Corp., Bedford, MA) and
recombinant TRAIL purified by nickel-chelate chromatography on a
Ni2+-nitrilotriacetic acid-agarose column (Qiagen,
Valencia, CA). This procedure yielded about 2 mg of pure protein from 1 liter of yeast supernatant.
)). Recombinant adenoviruses were
constructed by the method described by Crouzet et al. (41). Briefly, cDNAs of interest were subcloned into the expression cassette in plasmid vector pXL2996 under the control of the
cytomegalovirus promoter. Each expression cassette was subcloned into
the shuttle vector pXL3474. The resulting shuttle plasmids were
introduced into Escherichia coli JM83 cells by
electroporation. After double homologous recombinations, plasmid DNA
for recombinant virus was purified by CsCl density gradient
centrifugation. This DNA was linearized and transfected into 293 cells.
2-3 weeks after transfections, recombinant adenovirus was harvested
from the conditioned medium and amplified in 293 cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor. These features allowed quick one-step
purification of secreted 20-kDa TRAIL by nickel-chelate chromatography
from yeast supernatant yielding ~2 mg of pure protein from
each liter of yeast culture medium (Fig.
1A). The cytotoxic effects of
TRAIL were tested on a panel of six prostate cancer cell lines (Fig. 1B). Cell viability assays demonstrated that three of these
cell lines, ALVA-31, DU 145, and PC-3 were very sensitive to TRAIL, JCA-1, and TSU-Pr1 revealed moderate sensitivity, whereas LNCaP cells
were resistant to as high as 4 µg/ml of TRAIL. Internuclosomal fragmentation (DNA laddering) confirmed that cell death occurred by
apoptosis (data not shown).
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Fig. 1.
Sensitivity of human prostate cancer cell
lines to soluble human TRAIL. A, purification of
recombinant TRAIL from P. pastoris supernatant by
nickel-chelate chromatography. B, relative viability of six
prostate cancer cell lines treated for 24 h with TRAIL, as
measured by the tetrazolium conversion assay. Data are expressed
as the means for duplicate determinations.
View larger version (45K):
[in a new window]
Fig. 2.
Constitutive activity of Akt in prostate
cancer cells determined by immunoblot with anti-phospho-Akt antibody
(Ser473). A, cell lysates prepared from six
prostate cancer cell lines were probed by immunoblotting with
anti-phospho-Akt antibody (top panel) or anti-Akt
antibody (bottom panel). B, LNCaP
cells were treated with wortmannin (200 nM) or
cycloheximide (10 µM) for 6 h, and cell lysates were
immunoblotted with anti-phospho-Akt antibody (top
panel) or anti-Akt/PKB antibody (bottom
panel).
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Fig. 3.
Inhibitors of PI 3-kinase or protein
synthesis potentiate the cytotoxic activity of TRAIL.
A, LNCaP cells were treated for 24 h with 1 µg/ml
TRAIL, 200 nM wortmannin, 20 µM LY-294002, or
10 µM cycloheximide alone or in combinations. The cells
were visualized by light microscopy. B, LNCaP cells were
treated for 6 h with 1 µg/ml TRAIL, 200 nM
wortmannin (WM), 20 µM LY-294002, 10 µM cycloheximide (CHX), or 100 µM valinomycin alone or in combinations. DNA
fragmentation was quantitated by measuring the relative amounts of
DNA-histone complexes released into the cytoplasm using a Cell Death
Detection ELISAplus kit.
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Fig. 4.
The role of mitochondrial cytochrome
c release for TRAIL-induced apoptosis in LNCaP
cells. A, parental LNCaP cells or LNCaP cells
overexpressing Bcl-2 were treated as described in the legend to Fig.
3B, and apoptotic nuclei were scored by TUNEL staining.
Several randomly chosen microscopic fields were visualized, and both
normal and TUNEL-positive cells were counted. The numbers of
TUNEL-positive versus total numbers of counted cells are
represented as ratios above the bar
graphs. B, LNCaP cells were treated with TRAIL,
wortmannin, or cycloheximide as described above. Cells were lysed in
hypotonic buffer, and cytochrome c in the cytosolic fraction
was measured by immunoblotting with cytochrome c-specific
antibodies.
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[in a new window]
Fig. 5.
Block of TRAIL-mediated apoptotic
signal in LNCaP cells occurs at the level of BID cleavage.
A, LNCaP cells were treated for 6 or 16 h with 1 µg/ml TRAIL, 200 nM wortmannin (WM), or 10 µM cycloheximide (CHX) alone or in
combinations. Cell lysates were electrophoresed and consecutively
immunoblotted with antibodies specific to caspase 8, XIAP, BID, caspase
9, and caspase 7. The arrows on the left indicate
cleavage products. B, LNCaP cells were treated for 6 h
with 1 µg/ml TRAIL or 20 µM LY-294002 alone or in
combinations. Cell lysates were electrophoresed and consecutively
immunoblotted with antibodies specific to BID or the phosphorylated
form of Akt (Ser473). C, parental LNCaP cells
and LNCaP cells overexpressing Bcl-2 were treated for 6 h with 1 µg/ml TRAIL and 200 nM wortmannin alone or in
combination. Cleavage of caspase 8 and BID was analyzed by
immunoblotting with the corresponding antibodies. Blots were processed
by ECL, and two different exposures were taken to visualize holocaspase
8 (short exposure) and its 20-kDa proteolytic fragment (long exposure).
The arrow indicates caspase 8 cleavage product.
D, cell lysates from the experiment described for
A were immunoblotted with antibodies that specifically
recognize different splice variants of cFLIP protein:
FLIPL, FLIP , and FLIP
.
, and FLIP
(53). In contrast to published data, treatment of LNCaP cells for up to 16 h with cycloheximide or wortmannin had no effect on the level of cFLIP proteins (Fig. 5C), suggesting that they are unlikely to be involved in the
inhibition of TRAIL signaling in LNCaP cells.
) was used. LNCaP cells infected with
adenoviral constructs 16 h prior to the experiment were treated for an additional 6 h with TRAIL or TRAIL plus wortmannin, and BID
cleavage was examined by immunoblotting. Our results demonstrate (Fig.
6A) that the infection of
LNCaP cells with myr-Akt, but not with the kinase-inactive Akt,
inhibits processing of BID induced by TRAIL plus wortmannin treatment.
TRAIL-mediated cell death was also inhibited in myr-Akt-infected cells
as judged by cell morphology (data not shown). Thus, activated Akt is
capable of rescuing LNCaP cells from the apoptogenic action of TRAIL
plus wortmannin treatment, supporting the hypothesis that the
resistance of LNCaP cells to TRAIL results from high constitutive
activity of Akt.
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Fig. 6.
Constitutively active Akt inhibits
proapoptotic effects of TRAIL. A, LNCaP cells were
infected with adenoviral constructs expressing myristoylated Akt
(Adeno-myr-Akt) or kinase-inactive myristoylated Akt
(Adeno-myr-Akt(K )) at a titer of 3 × 106 TCID50/ml. Control cells were not infected
with adenoviruses. 16 h postinfection, the cells were treated for
6 h with 1 µg/ml TRAIL and 200 nM wortmannin alone
or in combination. Cell lysates were consecutively probed with
BID-specific antibody and anti-HA1 antibody that recognizes
hemagglutinin-tagged myr-Akt. B, LNCaP cells were infected
where indicated with adenoviral constructs expressing myristoylated Akt
(Adeno-myr-Akt) at a titer increasing from 6 to 48 × 106 TCID50/ml. Control cells were not infected
with adenovirus. 16 h after infection, the cells were treated for
6 h with 1 µg/ml of TRAIL and 10 µM cycloheximide
alone or in combination. Cell lysates were probed as outlined for
A. C, ALVA-31 cells were transiently
cotransfected with an expression plasmid encoding the E. coli lacZ gene plus an expression plasmid for
myristoylated Akt (myr-Akt) (60) or empty expression vector
(Mock). 24 h after transfection, the cells were
incubated with or without 0.1 µg/ml TRAIL and scored for apoptosis
24 h later. Cells positive for
-galactosidase activity were
checked for morphological changes characteristic of apoptosis, and the
percentage of live cells was quantitated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-mediated transcriptional pathways
(61, 62), or phosphorylation of the Forkhead family of transcription
factors, preventing them from inducing the transcription of
proapoptotic genes (63). Inhibition of BID cleavage has not been
previously reported as a mechanism through which PI 3-kinase and Akt
block apoptotic signals.
, and FLIP
) in LNCaP cells and found that
neither cycloheximide nor wortmannin treatment affected cFLIP levels
after as long as 16 h of treatment. These data are consistent with
our observation on renal carcinoma cells (65) and published results on
Kaposi's sarcoma cells (17) in which that inhibition of protein
synthesis sensitized cells to TRAIL without affecting the expression of
cFLIP proteins. Thus, mediators of the PI
3-kinase-dependent blockage of TRAIL-induced BID cleavage and apoptosis in LNCaP cells still await identification and characterization.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Ralph Buttyan (Columbia Presbyterian Medical Center, New York, NY) for Bcl-2-overexpressing LNCaP cells, and we thank Dr. Richard A. Roth (Stanford University School of Medicine, Stanford, CA), Dr. Joseph Biggs, and other members of Kraft laboratory for helpful discussions. We appreciate the excellent technical assistance of Sarah Winbourn.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA 78631 (to A. S. K.) and United States Public Health Service Grant CA 53520 (to G. J. M.).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 may be addressed: Division of Medical Oncology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-7691; Fax: 303-315-5502; E-mail: Alexander.Nesterov@UCHSC.edu.
To whom correspondence may be addressed: Division of Medical
Oncology, University of Colorado Health Sciences Center, 4200 E. Ninth
Ave., Denver, CO 80262. Tel.: 303-315-8802; E-mail:
Andrew.Kraft@UCHSC.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M005196200
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ABBREVIATIONS |
---|
The abbreviations used are: DISC, death-inducing signaling complex; PI, phosphatidylinositol; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
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REFERENCES |
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