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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3053-3063
By
From the Department of Internal Medicine I,
Eberhard-Karls-University, Tübingen, Germany.
Proteases of the caspase family are the critical executioners of
apoptosis. Their activation has been mainly studied upon triggering of
death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1,
which recruit caspase-8/FLICE as the most proximal effector to the
receptor complex. Because apoptosis induced by anticancer drugs has
been proposed to involve CD95/CD95 ligand interaction, we investigated
the mechanism of caspase activation by daunorubicin, doxorubicin,
etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs
induced apoptosis and the cleavage of procaspase-8 to its active p18
subunit. However, cells resistant to CD95 were equally susceptible to
anticancer drugs and activated caspase-8 with a similar kinetic and
dose response as CD95-sensitive cells. The broad caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis
and caspase-8 activation in response to CD95 and drug treatment,
whereas a neutralizing CD95 decoy as well as a dominant-negative FADD
construct selectively abrogated CD95, but not drug-induced effects. A
potent activation of caspase-8 was also induced by cycloheximide,
indicating that it was independent of protein synthesis. Our data,
therefore, show that (1) anticancer drug-induced apoptosis does not
require de novo synthesis of death ligands or CD95 interaction, and (2)
that caspase-8 can be activated in the absence of a death receptor signaling.
CHEMOTHERAPEUTIC AGENTS kill cancer cells
by multiple mechanisms, including intercalation into DNA, inhibition of
DNA replication, cell membrane damage, or free radical
generation.1,2 Although the primary intracellular targets
of drug action are rather distinct, it has become evident that
drug-induced cytotoxicity ultimately converges on a common pathway,
causing apoptosis. Cells exposed to anticancer drugs display apoptotic
alterations, such as cell shrinkage, chromatin condensation, and
internucleosomal DNA fragmentation.3 A close link between
apoptosis and the mechanism of drug action has been demonstrated by the
involvement of similar genetic components. Overexpression of Bcl-2
proteins can confer drug resistance in transfected tumor
cells.4-6 A number of investigations exposed a critical
role of the tumor suppressor p53 in apoptosis after drug
treatment.5,7,8 Finally, it has been recently shown that
drug-induced cytotoxicity involves proteases of the caspase family,
because specific inhibitors of caspases prevented cell death after
treatment with different anticancer agents.9-12
Caspases are currently considered as the central executioners of many,
if not all, apoptotic pathways. In mammalian cells, at least 12 different caspases exist, which are cysteine proteases that cleave
their substrates after aspartate residues.13-15 Caspases are synthesized as inactive proenzymes that are proteolytically processed to form an active tetrameric complex composed of two heterodimeric subunits of about 10 and 20 kD. The activation mechanism of caspases is currently unknown and is an area of intense research. Activation may proceed through autoproteolysis of the precursor, through mutual processing, or by still undefined processes. An increasing number of proteins have been found to be cleaved by caspases, and for some of them an apoptotic function has been proposed.13-16 Among different substrates are enzymes
involved in genome function, such as the DNA repair enzyme
poly(adenosine diphosphate-ribose)polymerase (PARP) and
DNA-dependent protein kinase (DNA-PK), or regulators of
the cell cycle, including retinoblastoma protein, the p53 regulator
MDM-2, MEKK, and protein kinase C- It is currently unknown why there are so many different caspases in
mammalian cells. The most intensively studied member is caspase-3,
which is activated by multiple apoptotic stimuli. Depletion of
caspase-3 through homologous recombination results in excessive accumulation of neuronal cells caused by a lack of apoptosis in the
brain, whereas it has no affect in other tissues, indicating that
caspase-3 is redundant in many cell types.18 The
physiologic relevance of the other members of the caspase family is
also not well defined. Cells express more than one of these proteases, but whether all are functionally required for a single apoptotic pathway remains obscure. Current knowledge indicates that individual caspases have distinct substrate specificities, inhibitor profiles, and
abilities to process each other. These findings suggest that caspases
form a hierarchical network which, similar to the complement system,
may function as an amplifier for a given apoptotic stimulus.
One of the best-defined apoptotic pathways is mediated by the surface
receptor CD95 (APO-1/Fas).19-21 Triggering of the receptor by its natural ligand CD95L or agonistic antibodies induces the formation of a death-inducing signaling complex (DISC), which consists
of the adapter protein FADD and FLICE/caspase-8.22-24 Complex formation is initiated through homophilic interaction of the
death domains present in the intracellular part of both CD95 and FADD.
FADD, in addition, contains a second interacting region, called the
death effector domain (DED), which couples to caspase-8 as the most
proximal element in the caspase cascade. Further downstream in the
death pathway, caspase-8 presumably triggers the proteolytic activation
of other caspases and cleavage of cellular substrates.
Although it is evident that different anticancer drugs ultimately
mediate a common apoptotic pathway through the activation of caspases,
the events occuring between primary target action of the drugs and the
activation of apoptotic effectors are unclear. Recently, it has been
proposed that drug-induced apoptosis occurs through the CD95
pathway.25-27 It has been observed that several anticancer
drugs, such as doxorubicin, methotrexate, or bleomycin induce the
upregulation of membrane CD95 and induction of CD95L expression,
followed by the subsequent autocrine or paracrine induction of
CD95-dependent apoptosis. Cell lines resistant to CD95 were found to be
insensitive to anticancer drug-induced apoptosis. Furthermore,
drug-induced apoptosis was prevented by CD95 neutralizing antibodies.
However, there are also reports indicating that antitumor drugs may
induce apoptosis by other pathways.28-32
In the present study, we investigated the mechanism of anticancer
drug-induced apoptosis and the requirement of the CD95 system. We show
that different antineoplastic drugs, such as daunorubicin, doxorubicin,
etoposide, and mitomycin C induce caspase-dependent apoptosis to a
similar extent in both CD95-sensitive and resistant leukemic T cells.
Interestingly, caspase-8 was activated in both cell types by these
drugs and also by the protein synthesis inhibitor cycloheximide. Our
data indicate that (1) anticancer drugs induce caspase-8 activation and
apoptosis independent of CD95L/receptor interaction, and that (2)
caspase-8 can be activated in the absence of receptor signals even on
inhibition of protein synthesis. Thus, these findings show that
caspase-8, which has been previously regarded as the most proximal
caspase in CD95 signaling, can be activated independently of
death-receptor interaction.
Cells and reagents.
The human leukemic T-cell lines Jurkat and CEM were maintained in
RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum
(FCS), 100 U of penicillin per milliliter, 0.1 mg streptomycin per
milliliter, and 10 mmol/L HEPES (all from GIBCO-BRL, Eggenstein, Germany). Cells were grown at 37°C in a 5% CO2
atmosphere and maintained in log phase. The CD95-resistant Jurkat
subline, Jurkat-R, was generated by continuous culture in the presence
of anti-CD95 monoclonal antibody (MoAb) (IgG3, 1 µg/mL;
Cell Diagnostica, Münster, Germany) for 6 months. HeLa cells
stably transfected with a chimeric construct of a dominant-negative
FADD mutant or the vector alone fused to green-fluorescent protein
(GFP) were kindly provided by Dr P. Scheurich and Dr H. Wajant
(University of Stuttgart, Stuttgart, Germany)33 and
cultivated in RPMI-1640 supplemented with 5% FCS, 10 mmol/L HEPES and
antibiotics. The CD95-neutralizing chimeric protein glutathione-S
transferase (GST) CD95, consisting of the extracellular part of CD95
fused to GST, was a gift from Dr E. Gulbins (University of
Tübingen, Tübingen, Germany) and produced in
Escherichia coli. The broad-range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was purchased from Enzyme Systems (Dublin, CA). The chemotherapeutic drugs
daunorubicin, etoposide, and mitomycin C were obtained from the
clinical pharmacy (Medical Clinics, Tübingen, Germany), and doxorubicin was purchased from Sigma (Deisenhofen, Germany).
Daunorubicin and mitomycin C were dissolved in methanol and doxorubicin
and etoposide in ethanol and kept as stock solutions at Measurement of cell death.
For determination of cell death, 3 × 104 cells per well
were seeded in microtiter plates and treated for 24 hours with the indicated concentrations of anti-CD95 or the chemotherapeutic agents.
The leakage of fragmented DNA from apoptotic nuclei was measured by the
method of Nicoletti et al.34 Briefly, apoptotic nuclei were
prepared by lysing cells in a hypotonic lysis buffer (1% sodium
citrate, 0.1% Triton X-100, 50 µg/mL propidium iodide) and
subsequently analyzed by flow cytometry. Nuclei to the left of the 2 N
peak containing hypodiploid DNA were considered apoptotic. Phosphatidylserine externalization of apoptotic cells was visualized by
staining with annexin-V-FITC after the manufacturer's protocol (Boehringer-Mannheim, Mannheim, Germany) and subsequent analysis in a
flow cytometer by using the FSC/FL1 profile. Cell death was assessed by
the uptake of propidium iodide (2 µg/mL; Sigma) in phosphate-buffered
saline (PBS) into nonfixed cells and subsequent flow cytometric
analysis with the FSC/FL2 profile. All flow cytometry analyses were
performed on a FACScalibur (Becton Dickinson, Heidelberg, Germany) by
using CellQuest analysis software. Microscopic analysis of cell
viability was performed with nonfixed GFP-FADD-DN expressing HeLa cells
in an inverse fluorescent microscope.
Immunoblotting.
Cleavage of PARP and caspase-8 was detected by immunoblotting. In
24-well plates 1 × 106 cells were seeded and treated with
the apoptotic stimuli. After the indicated periods, cells were washed
in cold PBS and lysed in 1% Triton X-100, 50 mmol/L Tris, pH 7.6, and
150 mmol/L NaCl containing 3 µg/mL aprotinin, 3 µg/mL leupeptin, 3 µg/mL pepstatin A, and 2 mmol/L phenylmethylsulfonyl fluoride.
Subsequently, proteins were separated under reducing conditions by 8%
to 15% gradient sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and electroblotted to a polyvinylidene difluoride
membrane (Amersham, Braunschweig, Germany). The loading and transfer of
equal amounts of protein was confirmed by staining the membrane with
Ponceau S. Membranes were blocked for 1 hour with 5% nonfat dry milk
powder in Tris-buffered saline (TBS: 10 mmol/L Tris-HCl pH
7.4, 100 mmol/L NaCl) and then immunoblotted for 1 hour with rabbit
anti-PARP polyclonal antibody (1:2,000; Boehringer-Mannheim) or mouse
anti-caspase-8 MoAb (1:10 dilution of a hybridoma supernatant; Cell
Diagnostica). Membranes were washed four times with TBS/0.05% Tween-20
and incubated with peroxidase-conjugated affinity-purified rabbit
antimouse Ig for 1 hour. After extensive washing, the reaction was
developed by enhanced chemiluminescent staining by using ECL reagents (Amersham).
Anticancer drug-induced apoptosis is independent of functional
CD95/CD95L interaction.
The mechanism by which antitumor drugs induce apoptosis remains
controversial. It has been previously demonstrated that antineoplastic agents, such as doxorubicin, bleomycin, and methotrexate induce the
expression of CD95L and elicit cell death by subsequent CD95 interaction.25-27 To investigate whether the inducible
interaction of CD95L with its receptor is a prerequisite for
drug-mediated apoptosis, we used the subclone Jurkat-R that had been
selected for resistance to CD95 signaling. The dose response of
drug-induced apoptosis was assessed by flow cytometric staining of
hypodiploid DNA in apoptotic nuclei. When CD95-sensitive Jurkat and
CD95-resistant Jurkat-R cells were treated with mitomycin C, both cell
lines underwent apoptosis in a similar concentration-dependent manner (Fig 1A). When the CD95 pathway was
stimulated by using agonistic anti-CD95 antibodies, cell death was
induced up to a concentration of as low as 4 ng/mL in Jurkat cells,
whereas virtually no apoptosis was observed in the CD95-resistant cell
line Jurkat-R (Fig 1B). To investigate whether chemotherapeutic drugs
other than mitomycin C induce cell death independently of CD95, both
cell lines were stimulated with daunorubicin, doxorubicin, and
etoposide (Fig 2). Similar as in the
previous experiment, all drugs induced cell death in Jurkat and
Jurkat-R cells with almost the same dose dependency.
Caspases are involved in induction of apoptosis by mitomycin C,
etoposide, daunorubicin, and doxorubicin.
Because caspases are involved in drug-mediated apoptosis, we
investigated whether caspases are also activated by anticancer drugs in
the absence of CD95/CD95L interaction. Therefore, we stimulated
CD95-sensitive and resistant cells under conditions analogous as in Fig
3 and used zVAD-fmk, a broad peptide inhibitor of caspases. Similarly,
as in the previous experiment, all cytotoxic agents induced cell death
in both Jurkat and Jurkat-R cells, whereas CD95 triggering only
resulted in apoptosis in Jurkat cells. Flow cytometric staining of
hypodiploid nuclei revealed that zVAD-fmk strongly attenuated anti-CD95
and drug-induced apoptosis (Fig 4A, upper
panel). Inhibition of caspases was also
able to prevent membrane damage and cell death as determined by
measuring the uptake of propidium iodide into cells (Fig 4A, lower
panel).
Caspase-8/FLICE is activated by anticancer drugs in the absence of
CD95L/receptor interaction.
During CD95-mediated apoptosis, the caspase cascade is initiated by the
recruitment and cleavage of caspase-8 at the DISC. Because anticancer
drugs obviously mediate activation of caspases independently of CD95,
we next investigated whether caspase-8 is activated during drug-induced
cell death. Caspase-8 is synthesized as two isoforms of about 55 kD
(caspase-8a and caspase-8b) which, after formation of intermediate
cleavage products of 43 and 41 kD, are processed to a p18 and p10
heterodimer.37,38 As assessed with an antibody directed
against the p18 subunit, CD95 ligation resulted in the cleavage of
procaspase-8 into its characteristic intermediate fragments and the
active p18 subunit (Fig 5A). Surprisingly, a similar cleavage pattern was obtained after treatment of Jurkat cells
with etoposide (Fig 5A), indicating that caspase-8 was also activated
by a receptor-independent apoptotic pathway.
Inhibition of CD95L/receptor interaction does not affect
drug-mediated caspase-8 activation.
We further analyzed the effect of the neutralizing GST-CD95 fusion
protein on caspase-8 activation after drug treatment. Whereas GST-CD95
potently blocked cleavage of caspase-8 in response to anti-CD95 (Fig
7A) or recombinant CD95L (data not shown),
it had virtually no effect on the processing of caspase-8 accomplished by etoposide or mitomycin C in CD95-sensitive and resistant Jurkat T-cells. GST, which was used as a control, did not affect the processing of caspase-8. In contrast to GST-CD95, the caspase inhibitor
zVAD-fmk blocked not only anti-CD95, but also etoposide and mitomycin
C-induced cleavage of caspase-8. Essentially, the same results were
obtained in leukemic CEM cells, in which both anti-CD95 and anticancer
drugs induced PARP cleavage and the processing of caspase-8 (Fig 7B).
Similar to Jurkat cells, only CD95 but not drug-mediated caspase
activation was inhibited by the neutralizing CD95 decoy construct.
Thus, these data clearly show that mechanisms of cell death and caspase
activation in response to anticancer drugs are not dependent on a
functional interaction of CD95L with its cognate receptor.
Drug-induced activation of caspase-8 and apoptosis is not dependent
on death receptor signaling.
Because caspase-8 is the most proximal caspase involved in
receptor-mediated apoptosis, the previous data implicated other mechanisms responsible for caspase-8 processing. Caspase-8 is not only
recruited to the DISC of CD95, but also to the receptors for tumor
necrosis factor (TNF) and TNF-related apoptosis-inducing ligand
(TRAIL). To further exclude a potential role of death receptors in
drug-mediated activation of caspase-8, we analyzed the effect of
anticancer drugs in HeLa cells, which stably expressed a GFP-tagged dominant-negative mutant of the adapter protein FADD lacking the essential DED region.33 It has been shown that FADD
transduces apoptotic signals triggered by CD95, TNF-R1 and
the TRAIL receptors.22,33,39-43 In accordance, HeLa-FADD-DN
cells are resistant to anti-CD95 and TRAIL and have an intrinsic
resistance to TNF-mediated apoptosis.33 Microscopic
analysis revealed that stimulation with anti-CD95 did not induce any
apoptotic alterations in HeLa-FADD-DN cells, thus confirming the
protective effect of the dominant-negative FADD-mutant (Fig
8A). In contrast, treatment with
staurosporine and mitomycin C caused the appearance of typical
apoptotic features such as cell condensation and membrane blebbing (Fig
8A). The parallel assessment of DNA fragmentation revealed that all
cytotoxic drugs induced apoptosis in both HeLa-FADD-DN and vector
control cells, whereas anti-CD95 induced apoptosis only in HeLa cells transfected with the vector alone (Fig 8B). In accordance,
staurosporine and mitomycin-C activated caspase-8 in both HeLa-vector
and HeLa-FADD-DN cells, whereas anti-CD95 triggered activation of
caspase-8 only in HeLa-vector cells (Fig 8C). These data indicate that
apoptosis by anticancer drugs does not require death receptor signaling and that caspase-8 can be activated in the absence of a FADD-containing receptor signaling complex.
Inhibition of protein synthesis by cycloheximide activates caspase-8.
Because the family of death receptors is steadily growing, it could not
be excluded that the activation of caspase-8 by antitumor drugs is
mediated by yet unknown death receptors that transduce their signals
independently of FADD. Therefore, we asked whether de novo synthesis of
other death ligands, such as TNF or TRAIL, is involved in drug-induced
caspase-8 activation. To this end, cells were treated with
cycloheximide, an inhibitor of protein synthesis. In some cell types,
the sole inhibition of translation by cycloheximide induces apoptosis.
This apoptosis is independent on CD95 signaling and occurred to a
similar extent in both Jurkat and Jurkat-R cells (Fig
9A). Interestingly, treatment with
cycloheximide also induced processing of caspase-8 in the absence of
any additional stimuli in CD95 sensitive and resistant cells (Fig 9B).
These data finally exclude that inducible expression of CD95L or other death ligands is an indispensable prerequisite for the activation of
caspase-8 and subsequent apoptosis.
In the present study, we investigated the mechanism of cell death
induced by a number of anticancer agents. We show that daunorubicin, doxorubicin, mitomycin C as well as etoposide induce caspase-dependent apoptosis in drug-sensitive target cells. This suggests that
drug-induced cytotoxicity is mediated by mechanisms that take place
downstream of primary intracellular target action. These data are in
line with previous results by us and others showing that effector
caspases, such as caspase-3, -6, and -7 are critically involved in
anticancer drug-mediated cell death.9-12
We thank Dr E. Gulbins for kindly providing us with the GST-CD95
construct and Dr P. Scheurich and Dr H. Wajant for HeLa cells stably
transfected with GFP-FADD-DN.
Submitted June 18, 1998; accepted December 28, 1998.
Supported in part by grants from the Deutsche Forschungsgemeinschaft
(SFB 364/A7) and the European Union Biomed-2 program. S.W. acknowledges
a fellowship from the Bundesministerium für Bildung und Forschung.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Klaus Schulze-Osthoff, PhD,
Department of Internal Medicine I, Eberhard-Karls-University,
Otfried-Müller-Str. 10, D-72076 Tübingen, Germany; e-mail:
schulze-osthoff{at}uni-tuebingen.de.
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TOP
ABSTRACT
INTRODUCTION
. Substrates of the nucleus and
cytoskeleton include lamins, Gas2, gelsolin, and fodrin. Furthermore,
it has been found that DNA cleavage is triggered upon caspase-mediated
degradation of the inhibitory subunit of a novel endonuclease,
designated caspase-activated DNase.
70°C.
) or resistant cells (Jurkat-R; -----, 
) were
stimulated with (A) mitomycin C or (B) agonistic anti-CD95 antibodies
at the indicated concentrations for 24 hours. Cells in the absence of
apoptotic stimuli were treated with the diluent of the respective
highest drug concentration. Induction of apoptosis was assessed by
propidium iodide staining of hypodiploid apoptotic nuclei and
subsequent flow cytometry.
) or Jurkat-R (
) cells
(3 × 104 per well) were preincubated with (A) medium,
(B) GST-CD95 (100 µg/mL), or (C) GST (100 µg/mL) for 1 hour, and
then treated with either anti-CD95 (20 ng/mL), daunorubicin (Dauno; 5 µg/mL), doxorubicin (Doxo; 1 µg/mL), etoposide (Etopo; 25 µg/mL),
or mitomycin C (Mito; 25 µg/mL). Apoptosis was assessed after 24 hours by measuring phosphatidylserine exposure by
annexin-V-FITC-staining and flow cytometry.
and 
)
or 100 µmol/L zVAD-fmk (
) indicate the uncleaved p116 and open arrowheads (
)
indicate the cleaved p89 form of PARP.
). Ig light chain of stimulatory anti-CD95 antibody is indicated
with an asterisk. (B) Jurkat or Jurkat-R cells
(1 × 106) were treated for the indicated time with
anti-CD95 (1 µg/mL), doxorubicin (Doxo; 1 µg/mL), or (C) with
daunorubicin (Dauno; 5 µg/mL), etoposide (Etopo; 20 µg/mL), or
mitomycin C (Mito; 25 µg/mL). Cellular proteins were immunoblotted
with anti-caspase-8 as described in (A). Only a section of the
immunoblots indicating the cleaved intermediate forms (p43 and p41) of
caspase-8a and caspase-8b is shown.
-converting enzyme (ICE) family protease CPP-32 during chemotherapeutic agent-induced apoptosis in ovarian carcinoma cells.
Cancer Res
56:5224, 1996