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Tumor Biology |
Tumor Immunology Program, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m) and cytochrome c
release. These events follow activation of caspase-8 and caspase-3 and
induction of DNA fragmentation. In addition, caspase-8-deficient cells
are resistant against TRAIL-induced apoptosis, and inhibition of
caspase-8 but not caspase-9 prevents mitochondrial permeability
transition and apoptosis. In contrast, various Bcl-2- or
Bcl-xL-overexpressing tumor cell lines are sensitive to
TRAIL-induced apoptosis; however, they show a delay in TRAIL-induced
mitochondrial permeability transition compared with control
transfectants. This indicates that TRAIL-induced apoptosis depends on
caspase-8 activation rather than on the disruption of mitochondrial
integrity. Because most chemotherapeutic drugs used in the treatment of
malignancies lead to apoptosis primarily by engagement of the
mitochondrial proapoptotic machinery, we tested whether drug-resistant
tumor cells retain sensitivity for TRAIL-induced apoptosis. Tumor cells
overexpressing Bcl-2 or Bcl-xL become resistant to
apoptosis induced by the chemotherapeutic drug etoposide. However,
these cells are not protected or are only marginally protected against
TRAIL-induced apoptosis. Thus, TRAIL may still kill tumors that have
acquired resistance to chemotherapeutic drugs by overexpression of
Bcl-2 or Bcl-xL. These data will influence future treatment
strategies involving TRAIL. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, CD95L (FasL/APO-1L), and TRAIL/APO-2L (1
, 2)
, have been shown to directly induce apoptosis in sensitive
target cells on binding to those cognate receptors that are capable of
transmitting a caspase-activating signal due to the presence of a so
called cytoplasmic DD (3
, 4)
.
TNF- and CD95L are involved in different physiological and
pathological apoptotic processes such as activation-induced cell death
of T cells, immune privilege, tumor evasion from the immune system, and
autoimmunity (4)
. In addition, TNF- and CD95L kill
various tumor cell lines in vitro. However, the use of these
ligands for anticancer therapy is complicated by their severe toxic
side effects on systemic administration such as shock and acute hepatic
failure (5, 6, 7)
.
Like CD95L and TNF-, TRAIL induces apoptosis in various tumor cell
lines in vitro (8, 9, 10)
. Furthermore, we and
others have recently demonstrated that administration of TRAIL
suppresses the growth of TRAIL-sensitive human tumor cells in severe
combined immunodeficient (SCID) mice or nonhuman primates without
showing any toxicity to normal tissue (11, 12, 13)
. The
tumoricidal activity of TRAIL is also highlighted by recent data
showing that the TRAIL system may be involved in endogenous tumor and
viral surveillance mediated by IFN-
-activated monocytes and
IFN-/ß-activated T cells (14
, 15)
.
TRAIL interacts with five distinct receptors: (a) TRAIL-R1 [DR4/APO-2 (16) ]; (b) TRAIL-R2 [DR5/TRICK/Killer (9 , 17, 18, 19, 20) ]; (c) TRAIL-R3 [DcR1/TRID/LIT (18 , 21) ]; (d) TRAIL-R4 [DcR2/TRUNDD (21 , 22) ]; and (e) osteoprotegerin (23) . Both TRAIL-R1 and -R2 contain the intracellular DD that is essential for the induction of apoptosis on receptor ligation (9 , 16, 17, 18, 19, 20) . In contrast, neither TRAIL-R3 nor TRAIL-R4 can mediate apoptosis due to a complete or partial lack of the intracellular DD, respectively (17 , 19 , 21 , 22 , 24) . Osteoprotegerin is a soluble receptor for TRAIL and thus blocks TRAIL-induced apoptosis (23) .
Although caspase-8 and caspase-3 activation could be detected after triggering of TRAIL-R1 or TRAIL-R2 (10) , thus far the TRAIL-induced signal transduction pathway and the influence of mitochondria-protecting antiapoptotic proteins such as Bcl-2 or Bcl-xL (25) on TRAIL-induced apoptosis remain poorly defined.
Many known chemotherapeutic drugs trigger a receptor-independent
mitochondria-controlled apoptotic pathway that is induced by early loss
of mitochondrial membrane potential (m)
(26, 27, 28, 29, 30)
and the subsequent release of the proapoptotic
proteins cytochrome c; pro-caspase-2, -3, and -9; and
apoptosis-inducing factor from the mitochondria into the cytosol
(31, 32, 33, 34, 35)
. Together with Apaf-1 and dATP as cofactors,
cytosolic cytochrome c facilitates the autoproteolytic
cleavage of caspase-9, leading to subsequent activation of downstream
caspases (36, 37, 38, 39)
. Therefore, we examined to what extent
mitochondria contribute to the pathway leading to TRAIL-induced and
chemotherapeutic drug-induced apoptosis. We were particularly
interested in whether TRAIL can bypass mitochondria and thus also
bypass the antiapoptotic effect of Bcl-2 or Bcl-xL, two
known proto-oncogenes that have been shown to be overexpressed in
several drug-resistant tumors observed in distinct clinical settings
(40, 41, 42, 43, 44)
. We found that during TRAIL-induced apoptosis,
mitochondrial PT and cytochrome c release occur after
caspase-8 and caspase-3 activation and induction of DNA fragmentation.
In addition, caspase-8-deficient Jurkat cells were resistant to
TRAIL-induced apoptosis. In contrast, Bcl-2 overexpression protects
only against early loss of m but not against late
disruption of mitochondrial integrity and apoptosis during
TRAIL-induced apoptosis. These observations suggest that caspase-8 is
necessary and, together with effector caspases, sufficient for
TRAIL-induced apoptosis, whereas disruption of mitochondrial integrity
is a secondary rather than an initial step after triggering of TRAIL-R.
By applying caspase-8-, caspase-9-, and caspase-3-specific inhibitors,
this sequence of events was confirmed. In contrast, overexpression of
Bcl-2- or Bcl-xL led to resistance against the
chemotherapeutic drug etoposide, which induces apoptosis primarily
through the mitochondria-dependent pathway (26, 27, 28, 29, 30)
. Thus,
because overexpression of Bcl-2 or Bcl-xL at levels that
protect cells against chemotherapeutic drugs does not inhibit
TRAIL-induced apoptosis, Bcl-2 and Bcl-xL-overexpressing,
chemoresistant tumors may still respond to treatment with TRAIL.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and Reagents.
mAbs against cytochrome c oxidase/subunit II (12C4-F12),
cytochrome c (7H8.2C12), Bcl-xL, and Bcl-2 were
purchased from Molecular Probes, Inc. (Eugene, OR), PharMingen (San
Diego, CA), Transduction Laboratories (San Diego, CA), and Calbiochem
(La Jolla, CA), respectively. The mouse mAb against PARP (C-II-10) was
a kind gift of Dr. A. Bürkle (German Cancer Research Center,
Heidelberg, Germany). The C15 mAb recognizes the p18 subunit of
caspase-8 (33)
, whereas the anti-caspase-3 mAb recognizes
the p17 subunit of caspase-3, but not the p12 subunit (Transduction
Laboratories, Lexington, Kentucky). LZ-TRAIL and LZ-CD40L are stable
trimers of the respective cytokines, and LZ-TRAIL induces apoptosis
upon binding to TRAIL-sensitive cells (9)
. Both reagents
were kindly provided by Immunex Corp. (Seattle, WA). The horseradish
peroxidase-conjugated goat antimouse IgG1, IgG2a, and IgG2b polyclonal
antibodies were obtained from Southern Biotechnology Associates
(Birmingham, AL). The caspase inhibitors zVAD-fmk and zDEVD-fmk were
purchased from Bachem (Basel, Switzerland), and zIETD-fmk and zLEHD-fmk
were obtained from Calbiochem. All other chemicals used were of
analytical grade and were purchased from Merck (Darmstadt, Germany) or
Sigma. The chemotherapeutic agent etoposide was kindly provided by
Ganti Chemo Supply (Heidelberg, Germany).
Fluorescence-activated Cell-sorting Analysis.
Wild-type or caspase-8-deficient Jurkat cells were incubated with mAbs
M271 (anti-TRAIL-R1), M413 (anti-TRAIL-R2), and M430 (anti-TRAIL-R3),
M444 (anti-TRAIL-R4; Immunex Corp.) or isotype-matched control IgG
followed by phycoerythrin-conjugated secondary goat antimouse
antibodies (Southern Biotechnology Associates). Surface staining was
determined on a FACScan cytometer (Becton Dickinson, San Jose, CA).
Determination of Mitochondrial Membrane Potential and ROS
Production.
Measurement of ROS generation and mitochondrial transmembrane potential
(m) was performed as described previously
(46)
. Briefly, 5 x 104
cells
were incubated in PBS containing 40 nM DiOC6(3)
(Molecular Probes Inc.) and 2 µM dihydroethidin
(Molecular Probes, Inc.) for 15 min at 37°C in the dark, followed by
analysis on a FACScan cytometer (Becton Dickinson).
Measurement of Cell Viability.
To determine the integrity of the cell membrane, cells were incubated
for 5 min in 2.5 µg/ml PI in PBS (Sigma) at 4°C in the dark,
followed by analysis on a FACScan (Becton Dickinson).
Determination of DNA Fragmentation.
As a direct measurement of apoptotic cell death, DNA
fragmentation was quantified essentially as described previously
(47)
. Briefly, 2.5 x 105 cells
were incubated in 24-well plates (Costar, Cambridge, MA) with or
without apoptotic stimuli in 0.5 ml of medium at 37°C. Cells were
collected by centrifugation at 600 x g for
10 min at 4°C, washed twice with PBS, and then resuspended in 100
µl of lysis solution containing 0.1% (v/v) Triton X-100, 0.1% (w/v)
sodium citrate, and 50 µg/ml PI. Apoptosis was quantitatively
determined by flow cytometry after incubation at 4°C in the dark
for at least 24 h as cells containing nuclei with subdiploid DNA
content.
Determination of Phosphatidylserine Translocation to the Outer
Leaflet of the Plasma Membrane by Annexin-V-FITC Surface Staining.
Cells (1 x 105) were incubated in
96-well plates (Costar), with or without apoptotic stimuli, in 200 µl
of medium at 37°C. Cells were collected by centrifugation at
600 x g for 10 min at 25°C, washed twice
in binding buffer [2.5 mM CaCl2, 140
mM NaCl, and 10 mM HEPES (pH 7.4)], and then
resuspended in 50 µl of staining solution [0.2 µg/ml
annexin-V-FITC (R&D Systems, Minneapolis, MN), 4 µg/ml 7-AAD
Molecular Probes), 2.5 mM CaCl2, 140
mM NaCl, and 10 mM HEPES (pH 7.4). After
incubation for 15 min in the dark at 37°C, 150 µl of cold binding
buffer were added, and the degree of single positive
(FITCbright/7-AADdull:early apoptotic
cells) and double-positive cells
(FITCbright/7-AADbright: late apoptotic and
necrotic cells) was determined by flow cytometry.
Preparation of Cell Lysates.
Cells (1 x 107 per sample) were harvested
by centrifugation at 600 x g for 10 min at
4°C and washed twice with PBS, and lysates were prepared by
resuspending the resulting cell pellets in 160 µl of lysis buffer
(PBS and 1% Triton X-100) supplemented with protease inhibitors (2
mg/ml aprotinin, 20 mg/ml amidinophenylmethanesulfanyl fluoride, 200
µg/ml pefabloc, and 40 µg/ml bestatin). After a 30-min incubation
on ice, the lysates were centrifuged once at 15,000 x g at 4°C to remove nuclei. The supernatants were stored at
-20°C or directly subjected to SDS-PAGE.
Subcellular Fractionation.
BL60 cells (1 x 107 per sample) were
harvested by centrifugation at 600 x g for
10 min at 4°C. The cell pellets were washed twice in PBS, resuspended
in 160 µl of isotonic buffer (250 mM sucrose, 20
mM HEPES, 20 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, and 1
mM DTT) supplemented with protease inhibitors (17 µg/ml
phenylmethyl sulfonyl fluoride, 8 µg/ml aprotinin, and 2 µg/ml
leupeptin), and allowed to swell during a 20-min incubation on ice. The
cells were gently broken up using a Dounce homogenizer. Small aliquots
of the lysate were taken and stained with trypan blue to determine the
progression of cell lysis. Homogenization was continued until 
80% of
the cells were broken. The homogenate was centrifuged once at
750 x g at 4°C to remove unbroken cells
and nuclei. The supernatant was centrifuged at 10,000 x g for 25 min at 4°C to collect the mitochondria-enriched
heavy membrane pellet (marked as Mito in the figures). The
resulting supernatant was centrifuged at 100,000 x g for 60 min at 4°C to yield the light membrane pellet and
the soluble cytosolic fraction (marked as Cyto in figures).
Western Blot Analysis.
For Western blot analysis, the resulting postnuclear
supernatants, mitochondrial or cytosolic fractions, were supplemented
with 40 µl of 5-fold concentrated standard reducing sample buffer
(5 x reducing sample buffer), and proteins equivalent
to 106 cells or 20 µg of protein as determined by the BCA
method (Pierce) were separated on 12% SDS-polyacrylamide gels (Novex,
San Diego, CA) by standard electrophoresis. After protein transfer onto
nitrocellulose membranes by electroblotting, membranes were blocked
with 3% BSA and 3% FCS in PBS/Tween overnight, washed with PBS/Tween,
and incubated with primary antibodies against caspase-8 (1:20-diluted
C15 hybridoma supernatant), caspase-3 (1:1,000; Transduction
Laboratories), PARP (mAb C-II-10; 1:20), cytochrome c
(7H8.2C12; 1:500; PharMingen), cytochrome c oxidase/subunit
II (12C4-F12; 1:1,000; Molecular Probes, Inc.), Bcl-2 (1:500;
Calbiochem), or Bcl-xL (1:500; Transduction Laboratories)
for 3 h. After six washes for 5 min each in PBS/Tween, the blots
were incubated with horseradish peroxidase-conjugated
isotype-specific secondary antibody diluted 1:20,000 in PBS/Tween.
After washing six times for 10 min with PBS/Tween, the blots were
developed by enhanced chemiluminescence following the manufacturer’s
protocol (Amersham). For stripping, blots were incubated for 30 min in
a buffer containing 62.5 mM Tris/HCl (pH 6.8), 2% SDS, and
100 mM ß-mercaptoethanol at 60°C. Then blots were
washed six times for 10 min in PBS/Tween and blocked again. To confirm
equal loading and purity of the cytosolic fraction, Western blots were
also developed with an antibody directed against either
extracellular-signal regulated kinase (Santa Cruz Biotechnology,
Santa Cruz, CA; data not shown) or against mitochondrial cytochrome
c oxidase (Molecular Probes, Inc.).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Jurkat cells (Fig. 1B
) with TRAIL. In accordance with
previous reports (48)
, phosphatidylserine translocation to
the outer leaflet of the plasma membrane could be detected as early as
30 min after TRAIL receptor engagement by LZ-TRAIL. In addition, DNA
fragmentation clearly preceded loss of m, suggesting
a mitochondria-independent pathway that leads directly to DNA
fragmentation. Because the degree of DNA fragmentation is identical to
the observed amount of total cell death determined by PI uptake (Fig. 1
), we conclude that TRAIL-induced cell death is exclusively apoptotic
and does not include any additional necrosis in these cell lines.
Although it was previously reported that ROS might initiate
mitochondrial PT in some cellular systems (49)
, we could
not detect an initial or early increase in ROS production during
TRAIL-induced apoptosis. Thus, increased production of ROS seems to be
a secondary effect following mitochondrial PT and does not play a role
at the initiation of TRAIL-induced apoptosis. Stimulation of BL60 or
Jurkat cells with LZ-CD40L, as a control, did not induce any of the
hallmarks of apoptosis induced by LZ-TRAIL (data not shown).
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). This strongly suggests that caspase-8 might be the caspase initiating
TRAIL-induced apoptosis. In accordance with the observed onset of DNA
fragmentation after 1–2 h (Fig. 1
), the active p17 subunit of
caspase-3 (Fig. 2B
) and processing of its substrate PARP
(Fig. 2C
) could already be observed 1 h after
stimulation. In contrast, release of cytochrome c from the
mitochondria to the cytosol was detected after the onset of caspase-3
cleavage and DNA fragmentation (Fig. 2, D and E
)
yet before loss of m (Fig. 1
). Thus, TRAIL-induced
apoptosis involves early activation of caspase-8, which then leads to
cleavage of caspase-3, subsequent caspase-3 substrate cleavage, and DNA
fragmentation. Although the TRAIL-generated apoptotic signal is
also transmitted to the mitochondria and leads to mitochondrial PT and
cytochrome c release, these mitochondrial changes occur late
during TRAIL-induced apoptosis, clearly following caspase-8 activation
and the onset of DNA fragmentation.
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) with TRAIL. Whereas wild-type Jurkat
cells were sensitive to TRAIL-induced apoptosis, caspase-8-deficient
cells were resistant to TRAIL-induced apoptosis (Fig. 3A
). This resistance was due to a deficiency in caspase-8 expression rather
than to reduced expression of caspase-3 (Fig. 3B
) or
TRAIL-R2 (Fig. 3C
, top panels) or, conversely,
increased surface levels of TRAIL-R3 or TRAIL-R4 (Fig. 3C
,
bottom panels). Thus, caspase-8 activation is a necessary
event during TRAIL-mediated apoptosis in Jurkat cells.
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m after TRAIL treatment of
Jurkat cells overexpressing Bcl-2 (Fig. 4A
) compared with control transfectants. Whereas cell death progressed with
similar kinetics in both cell lines tested, the loss of
m was clearly delayed by about 2–4 h in the
Bcl-2-transfected Jurkat cells (Fig. 4, C and D
).
Thus, Bcl-2 protects the mitochondria during the early stages of
TRAIL-induced apoptosis, but not during the late phase. This might be
due to caspase-8- or caspase-3-induced mechanisms that target the
mitochondria later during apoptosis or finally occurring necrotic
processes that cannot be inhibited by Bcl-2 (50)
. These
data provide evidence that Bcl-2 overexpression can indeed inhibit
mitochondrial PT during the early stages of TRAIL-induced apoptosis.
However, independent of the occurrence of mitochondrial PT,
TRAIL-induced apoptosis is initiated and executed with almost equal
efficiency. These data clearly indicate that TRAIL-induced apoptosis
can occur independently of apoptotic mitochondrial alterations.
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m or Cell
Death.
) and increased permeability of the plasma membrane (Fig. 5B
).
We observed that TRAIL-induced reduction of m and
cell death is blocked by high concentrations (100 µM) of
all inhibitors, apparently due to nonspecific broad spectrum caspase
inhibition. However, in contrast to zLEHD-fmk (a caspase-9-specific
inhibitor) and zDEVD-fmk (a caspase-3-specific inhibitor), the
caspase-8-specific inhibitor zIETD-fmk was able to block TRAIL-induced
loss of m even at very low concentrations (1–10
µM; Fig. 5A
, 
). Cell death was inhibited
efficiently by moderate concentrations (Fig. 5B
, ) of
zIETD-fmk, zVAD-fmk, and, to a lesser extent, zDEVD-fmk but was only
marginally inhibited by the caspase-9 inhibitor zLEHD-fmk. Apparently,
the more downstream acting caspase-3 inhibitor zDEVD-fmk is not as
efficient in blocking TRAIL-induced cell death as inhibitors that
directly target caspase-8. This might be due to the caspase-8-mediated
disruption of mitochondrial integrity that occurs even in the absence
of caspase-3 activation. Thus, release of noncaspase apoptotic
mediators from the mitochondria such as cytochrome c and
apoptosis-inducing factor may lead to the observed degree of cell death
with a nonapoptotic pattern of cytolysis (31
, 32
, 35)
.
Because the broad spectrum caspase-inhibitor zVAD-fmk shows an
inhibition similar to that of zIETD-fmk, we conclude that caspases with
target specificity other than IETD are not involved in
the induction of TRAIL-induced loss of m. These data
show that caspase-8 links the apoptotic signal from the activated
apoptosis-inducing TRAIL-R2 both to mitochondria [leading to
dissipation of m (Fig. 5
) and cytochrome c
release (Fig. 2 and E
)] and directly to the
downstream apoptosis-executing caspases (Fig. 2B
).
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-irradiation and
chemotherapeutic drugs (25, 26, 27, 28, 29, 30)
. To investigate the
potential inhibitory effect of these proteins on TRAIL-induced
apoptosis, we compared the apoptosis-inducing potential of TRAIL and
etoposide in cells stably transfected with either Bcl-2,
Bcl-xL, or vector alone as a control (Fig. 4, A and B
). TRAIL-induced apoptosis was slightly reduced by
overexpression of Bcl-2 in both Jurkat and SKW6 cells (Fig. 6C
and Fig. 7C
), whereas Bcl-xL
overexpression in either Jurkat or CEM cells had no inhibitory effect
on TRAIL-induced apoptosis (Fig. 6A
and Fig. 7A
). In contrast, overexpression of Bcl-2 or Bcl-xL in
Jurkat and CEM cells almost completely abrogated etoposide-induced
apoptosis (Fig. 6, A and C
, and Fig. 7A
), and Bcl-2 overexpression in SKW6 cells substantially
reduced their sensitivity to etoposide-induced cell death (Fig. 7C
). These data provide further proof that TRAIL can lead to
apoptosis via a mitochondria-independent pathway, whereas
etoposide-induced apoptosis is at least substantially inhibited by
overexpression of Bcl-2 or Bcl-xL. Thus, although
conventional antitumor treatment failed to kill Bcl-2- and
Bcl-xL-overexpressing tumor cell lines efficiently, these
cells remain highly sensitive to TRAIL-induced apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The bcl-2 gene was first shown to be dysregulated in
the majority of follicular lymphomas in which a t(14:18) chromosomal
translocation is present (40)
. However, Bcl-2 has now been
found to be overexpressed also in the absence of gene rearrangements in
most cases of B-cell chronic lymphocytic leukemia (41)
and
acute lymphoblastic leukemia (42)
. Interestingly, strongly
increased levels of Bcl-2 in B-cell chronic lymphocytic leukemia or
acute lymphoblastic leukemia were particularly pronounced in patients
who had undergone chemotherapy or -irradiation and were subsequently
found to be clinically unresponsive to these treatments (41
, 43
, 44)
. Because most clinically administered chemotherapeutic drugs
mediate apoptosis mainly by inducing mitochondrial dysfunction, tumor
cell resistance is likely to include overexpression of Bcl-2 or
Bcl-xL. This leads to protection of mitochondria and
subsequent cross-resistance to many mitochondria-targeting
chemotherapeutic drugs (25, 26, 27, 28, 29, 30)
. Our data show that TRAIL
is still capable of killing tumor cells that are insensitive to
etoposide-induced apoptosis. Thus, tumor cells that have already
acquired resistance to chemotherapeutic drugs by Bcl-2- or
Bcl-xL overexpression-induced protection of mitochondria
may still remain sensitive to TRAIL-induced apoptosis.
It has previously been shown that coadministration of TRAIL and 5-fluorouracil may have a beneficial effect on tumor eradication in vivo (12) . However, the functional basis for this observation has not been investigated thus far. We provide evidence that TRAIL and chemotherapeutic drugs kill tumors by primarily targeting non-mitochondria-dependent and mitochondria-dependent apoptotic pathways, respectively. Thus, our findings suggest an explanation for the observed enhanced treatment effect of TRAIL together with mitochondria-targeting drugs in vivo (12) . In addition, we show that TRAIL is still capable of inducing apoptosis in Bcl-2- and Bcl-xL-overexpressing tumor cells while these cells are protected against mitochondria-targeting cytotoxic agents.
TRAIL does not exhibit any observable toxicity on systemic treatment of mice or nonhuman primates, and it does not induce apoptosis in many different normal human cells in vitro (11, 12, 13) . Thus, our data suggest that this cytokine may be a promising candidate for the treatment of patients carrying drug-resistant tumors. In addition, because chemotherapy and radiation therapy on the one hand and TRAIL on the other hand trigger tumor cell apoptosis primarily via two distinct pathways, cotreatment of malignancies that still react to chemotherapy or radiation therapy potentially diminishes the pool of tumor cells that may give rise to an outgrowing resistant clonal variant of the parental tumor. Therefore, concomitant treatment of tumor patients with TRAIL and chemotherapeutic drugs or radiation therapy may reduce the relapse rate in malignancies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
|---|
1 H. W. and A. B. contributed equally to
this work. H. W. is supported by the AIDS Stipend Program of the
Bundesministerium für Forschung und Technologie. 
2 To whom requests for reprints should be
addressed, at Tumor Immunology Program, German Cancer Research Center
(DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: h.walczak{at}dkfz-heidelberg.de
3 Present address: Basel Institute for Immunology,
Grenzacherstrasse 487, 4005 Basel, Switzerland.
4 The abbreviations used are: TNF, tumor necrosis
factor; TRAIL, TNF-related apoptosis-inducing ligand; DD, death
domain; PI, propidium iodide; mAb, monoclonal antibody; LZ, leucine
zipper; ROS, reactive oxygen species; 7-AAD, 7-aminoactinomycin D;
PBS/Tween, PBS containing 0.05% Tween-20; PT, permeability transition;
DiOC6(3), 3,3-dihexyloxocarbocyanine iodide; PARP,
poly(ADP-ribose) polymerase.
Received 7/19/99. Accepted 4/ 4/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and protects against TRAIL-induced apoptosis, yet retains an incomplete death domain. Immunity, 7: 813-820, 1997.[Medline]
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F. Dicker, A. P. Kater, T. Fukuda, and T. J. Kipps Fas-ligand (CD178) and TRAIL synergistically induce apoptosis of CD40-activated chronic lymphocytic leukemia B cells Blood, April 15, 2005; 105(8): 3193 - 3198. [Abstract] [Full Text] [PDF]
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 F. A. Sinicrope, R. C. Penington, and X. M. Tang Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis Is Inhibited by Bcl-2 but Restored by the Small Molecule Bcl-2 Inhibitor, HA 14-1, in Human Colon Cancer Cells Clin. Cancer Res., December 15, 2004; 10(24): 8284 - 8292. [Abstract] [Full Text] [PDF]
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 X. Liu, P. Yue, Z. Zhou, F. R. Khuri, and S.-Y. Sun Death Receptor Regulation and Celecoxib-Induced Apoptosis in Human Lung Cancer Cells J Natl Cancer Inst, December 1, 2004; 96(23): 1769 - 1780. [Abstract] [Full Text] [PDF]
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K. Izeradjene, L. Douglas, A. B. Delaney, and J. A. Houghton Casein Kinase I Attenuates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis by Regulating the Recruitment of Fas-Associated Death Domain and Procaspase-8 to the Death-Inducing Signaling Complex Cancer Res., November 1, 2004; 64(21): 8036 - 8044. [Abstract] [Full Text] [PDF]
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K. Izeradjene, L. Douglas, A. Delaney, and J. A. Houghton Influence of Casein Kinase II in Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Rhabdomyosarcoma Cells Clin. Cancer Res., October 1, 2004; 10(19): 6650 - 6660. [Abstract] [Full Text] [PDF]
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J. Sonnemann, V. Gekeler, A. Sagrauske, C. Muller, H.-P. Hofmann, and J. F. Beck Down-regulation of protein kinase C{eta} potentiates the cytotoxic effects of exogenous tumor necrosis factor-related apoptosis-inducing ligand in PC-3 prostate cancer cells Mol. Cancer Ther., July 1, 2004; 3(7): 773 - 781. [Abstract] [Full Text] [PDF]
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A. Ballestrero, A. Nencioni, D. Boy, I. Rocco, A. Garuti, G. S. Mela, L. Van Parijs, P. Brossart, S. Wesselborg, and F. Patrone Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Cooperates with Anticancer Drugs to Overcome Chemoresistance in Antiapoptotic Bcl-2 Family Members Expressing Jurkat Cells Clin. Cancer Res., February 15, 2004; 10(4): 1463 - 1470. [Abstract] [Full Text] [PDF]
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R. L. Hayward, J. S. Macpherson, J. Cummings, B. P. Monia, J. F. Smyth, and D. I. Jodrell Enhanced oxaliplatin-induced apoptosis following antisense Bcl-xl down-regulation is p53 and Bax dependent: Genetic evidence for specificity of the antisense effect Mol. Cancer Ther., February 1, 2004; 3(2): 169 - 178. [Abstract] [Full Text] [PDF]
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V. M. Dirsch, I. M. Muller, S. T. Eichhorst, G. R. Pettit, Y. Kamano, M. Inoue, J.-P. Xu, Y. Ichihara, G. Wanner, and A. M. Vollmar Cephalostatin 1 Selectively Triggers the Release of Smac/DIABLO and Subsequent Apoptosis That Is Characterized by an Increased Density of the Mitochondrial Matrix Cancer Res., December 15, 2003; 63(24): 8869 - 8876. [Abstract] [Full Text] [PDF]
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X. Liao, L. Zhang, J. B. Thrasher, J. Du, and B. Li Glycogen synthase kinase-3{beta} suppression eliminates tumor necrosis factor-related apoptosis-inducing ligand resistance in prostate cancer Mol. Cancer Ther., November 1, 2003; 2(11): 1215 - 1222. [Abstract] [Full Text] [PDF]
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M. Chawla-Sarkar, J. A. Bauer, J. A. Lupica, B. H. Morrison, Z. Tang, R. K. Oates, A. Almasan, J. A. DiDonato, E. C. Borden, and D. J. Lindner Suppression of NF-{kappa}B Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL J. Biol. Chem., October 10, 2003; 278(41): 39461 - 39469. [Abstract] [Full Text] [PDF]
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C. Ruiz-Ruiz, G. Robledo, E. Cano, J. M. Redondo, and A. Lopez-Rivas Characterization of p53-mediated Up-regulation of CD95 Gene Expression upon Genotoxic Treatment in Human Breast Tumor Cells J. Biol. Chem., August 22, 2003; 278(34): 31667 - 31675. [Abstract] [Full Text] [PDF]
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R. L. Hayward, J. S. Macpherson, J. Cummings, B. P. Monia, J. F. Smyth, and D. I. Jodrell Antisense Bcl-xl Down-Regulation Switches the Response to Topoisomerase I Inhibition from Senescence to Apoptosis in Colorectal Cancer Cells, Enhancing Global Cytotoxicity Clin. Cancer Res., July 1, 2003; 9(7): 2856 - 2865. [Abstract] [Full Text] [PDF]
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K. Kandasamy, S. M. Srinivasula, E. S. Alnemri, C. B. Thompson, S. J. Korsmeyer, J. L. Bryant, and R. K. Srivastava Involvement of Proapoptotic Molecules Bax and Bak in Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Mitochondrial Disruption and Apoptosis: Differential Regulation of Cytochrome c and Smac/DIABLO Release Cancer Res., April 1, 2003; 63(7): 1712 - 1721. [Abstract] [Full Text] [PDF]
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 V. Hietakangas, M. Poukkula, K. M. Heiskanen, J. T. Karvinen, L. Sistonen, and J. E. Eriksson Erythroid Differentiation Sensitizes K562 Leukemia Cells to TRAIL-Induced Apoptosis by Downregulation of c-FLIP Mol. Cell. Biol., February 15, 2003; 23(4): 1278 - 1291. [Abstract] [Full Text] [PDF]
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M. Leverkus, M. R. Sprick, T. Wachter, T. Mengling, B. Baumann, E. Serfling, E.-B. Brocker, M. Goebeler, M. Neumann, and H. Walczak Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation Mol. Cell. Biol., February 1, 2003; 23(3): 777 - 790. [Abstract] [Full Text]
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A. B. Werner, E. de Vries, S. W. G. Tait, I. Bontjer, and J. Borst TRAIL Receptor and CD95 Signal to Mitochondria via FADD, Caspase-8/10, Bid, and Bax but Differentially Regulate Events Downstream from Truncated Bid J. Biol. Chem., October 18, 2002; 277(43): 40760 - 40767. [Abstract] [Full Text] [PDF]
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D. Javelaud and F. Besancon Inactivation of p21WAF1Sensitizes Cells to Apoptosis via an Increase of Both p14ARF and p53 Levels and an Alteration of the Bax/Bcl-2 Ratio J. Biol. Chem., September 27, 2002; 277(40): 37949 - 37954. [Abstract] [Full Text] [PDF]
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X. Tang, Y. J. Sun, E. Half, M. T. Kuo, and F. Sinicrope Cyclooxygenase-2 Overexpression Inhibits Death Receptor 5 Expression and Confers Resistance to Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Colon Cancer Cells Cancer Res., September 1, 2002; 62(17): 4903 - 4908. [Abstract] [Full Text] [PDF]
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T. Weber, M. Lu, L. Andera, H. Lahm, N. Gellert, M. W. Fariss, V. Korinek, W. Sattler, D. S. Ucker, A. Terman, et al. Vitamin E Succinate Is a Potent Novel Antineoplastic Agent with High Selectivity and Cooperativity with Tumor Necrosis Factor-related Apoptosis-inducing Ligand (Apo2 Ligand) in Vivo Clin. Cancer Res., March 1, 2002; 8(3): 863 - 869. [Abstract] [Full Text] [PDF]
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P. Secchiero, A. Gonelli, C. Celeghini, P. Mirandola, L. Guidotti, G. Visani, S. Capitani, and G. Zauli Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies Blood, October 1, 2001; 98(7): 2220 - 2228. [Abstract] [Full Text] [PDF]
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V. M. Dirsch, H. Stuppner, and A. M. Vollmar Helenalin Triggers a CD95 Death Receptor-independent Apoptosis That Is Not Affected by Overexpression of Bcl-xL or Bcl-2 Cancer Res., August 1, 2001; 61(15): 5817 - 5823. [Abstract] [Full Text] [PDF]
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 M. L. Jelachich and H. L. Lipton Theiler's Murine Encephalomyelitis Virus Induces Apoptosis in Gamma Interferon-Activated M1 Differentiated Myelomonocytic Cells through a Mechanism Involving Tumor Necrosis Factor Alpha (TNF-{alpha}) and TNF-{alpha}-Related Apoptosis-Inducing Ligand J. Virol., July 1, 2001; 75(13): 5930 - 5938. [Abstract] [Full Text] [PDF]
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N. Özören, K. Kim, T. F. Burns, D. T. Dicker, A. D. Moscioni, and W. S. El-Deiry The Caspase 9 Inhibitor Z-LEHD-FMK Protects Human Liver Cells while Permitting Death of Cancer Cells Exposed to Tumor Necrosis Factor-related Apoptosis-inducing Ligand Cancer Res., November 1, 2000; 60(22): 6259 - 6265. [Abstract] [Full Text]
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D. Bernard, B. Quatannens, B. Vandenbunder, and C. Abbadie Rel/NF-kappa B Transcription Factors Protect against Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis by Up-regulating the TRAIL Decoy Receptor DcR1 J. Biol. Chem., July 13, 2001; 276(29): 27322 - 27328. [Abstract] [Full Text] [PDF]
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T. F. Burns and W. S. El-Deiry Identification of Inhibitors of TRAIL-induced Death (ITIDs) in the TRAIL-sensitive Colon Carcinoma Cell Line SW480 Using a Genetic Approach J. Biol. Chem., October 5, 2001; 276(41): 37879 - 37886. [Abstract] [Full Text] [PDF]
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), DNA fragmentation
(
), loss of 
], and production
of ROS (increased fluorescence at 575 nm by ROS-mediated oxidation of
dihydroethidin to ethidin; •). All data points are the mean of three
independent experiments.
). Cell death was determined 16 h after stimulation by
PI uptake. All data points are the mean of three independent
experiments.