* Department of Genetics and Department of Anatomy, Osaka University Medical School, Yamada-oka, Suita, Osaka
565-0871, Japan; and § Osaka Bioscience Institute, Furuedai, Suita, Osaka 565-0874, Japan
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Abstract |
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The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.
Key words: apoptosis; caspase; Fas; Fas-associated protein with death domain; necrosis ![]() |
Introduction |
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TWO categories of cell death have been defined: apoptosis and necrosis (Kerr et al., 1972). The apoptotic
process is accompanied by condensation and fragmentation of cells and nuclei, as well as by degradation of
the chromosomal DNA into nucleosomal units (Wyllie
1980
; Wyllie et al., 1980
). The necrotic process is characterized by swelling and disintegration of the cells and nuclei. Most apoptotic processes are mediated by a family of
cysteine proteases called caspases (Cohen, 1997
; Nicholson and Thornberry, 1997
). Caspases are activated by various apoptotic stimuli and cleave cellular substrates such as
poly(ADP-ribose) polymerase (PARP),1 lamin, actin, p27,
and inhibitor of caspase-activated DNase, which is responsible to cause morphological changes of nuclei, and degradation of chromosomal DNA. The molecular mechanism
of the necrotic cell death process, however, is not well understood.
Fas ligand (FasL) is a member of the tumor necrosis factor (TNF) family. By binding to Fas, its receptor, FasL induces apoptosis (Baker and Reddy, 1996; Nagata, 1997
).
This apoptotic process proceeds by the binding of an adaptor molecule, Fas-associated protein with death domain
(FADD), to the death domain of the Fas cytoplasmic region. This binding causes recruitment of pro-caspase 8 to
the Fas receptor to establish the death-inducing signaling complex (DISC) (Kischkel et al., 1995
). Pro-caspase 8 is
processed at the DISC to the mature active enzyme, and
activates other caspases, such as caspase 3, in the downstream of the caspase cascade. In this report, we describe a
Jurkat-derived cell line that does not express caspase 8. This cell line was resistant to Fas-induced cell death. However, multimerization of FADD in this cell line killed the
cells. This killing process proceeded without caspase activation, and was accompanied neither by apoptotic morphological changes of the cells nor by DNA fragmentation. An analysis of the dying cells under the electron
microscopy indicated that this process was accompanied
by necrotic morphological changes.
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Materials and Methods |
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Establishment of Stable Transformants
Plasmid MF2FE carries a DNA fragment coding for the FK506 binding
protein (FKBP)-Fas chimeric molecule that is consisting of the cytoplasmic region of Fas, two tandem repeats of FKBP12, the myristilation-targeting peptide, and the influenza hemagglutinin (HA) epitope (see Fig. 2
A). The coding region of FKBP-Fas was excised from pMF2FE (Spencer et
al., 1996) (provided by Dr. M. Gilman in ARIAD, Cambridge, MA), and
inserted in pEF-BOS expression vector to generate pEF-FKBP-Fas. The
expression vector for FKBP-FADD (pEF-FKBP-FADD) was constructed
by replacing the Fas cytoplasmic region of pEF-FKBP-Fas by the entire
coding region of FADD/MORT1 (Boldin et al., 1995
; Chinnaiyan et al.,
1995
). To construct the expression vector for human caspase 8, the entire
coding region of human caspase 8 (Boldin et al., 1996
; Muzio et al., 1996
)
was fused in frame with the sequence of FLAG epitope, and inserted in
pEF-BOS vector (Mizushima and Nagata, 1990
) to generate pEF-CAS8.
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Jurkat is a human leukemic T lymphoma cell line, and JB-6 is a Jurkat-derived cell line that overexpresses human Bcl-2. To establish stable transformants expressing FKBP-Fas, FKBP-FADD, or human caspase 8, Jurkat and JB-6 cells (107) were cotransfected with 50 µg of the expression
vectors (pEF-FKBP-Fas, pEF-FKBP-FADD, or pEF-CAS8) with 1 µg of
the hygromycin-resistant gene (pMiwhyg) by electroporation using a
Gene Pulser (Bio-Rad Laboratories, Hercules, CA) as described previously (Suda et al., 1993). The hygromycin-resistant clones were selected
by culturing cells in the presence of 800 µg/ml of hygromycin. The transformant clones expressing FKBP-Fas, FKBP-FADD, or caspase 8 were
then identified by Western blotting using anti-HA, anti-FADD antibody, or anti-FLAG antibody, respectively. JF and JM clones were Jurkat-derived cells expressing FKBP-Fas and FKBP-FADD, respectively.
Whereas, BC, BF, and BM clones were JB-6-derived cells expressing human caspase 8, FKBP-Fas, and FKBP-FADD, respectively.
Cell Viability Assay
The cell viability was determined by the WST-1 assay (Kawahara et al.,
1998b) or 3H-thymidine incorporation. In brief, 5 × 104 cells were stimulated with various concentrations of FK1012 for 6 h in 100 µl. The WST-1
reagent, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2[H] tetrazolium monosodium salt (Dojin Laboratories, Kumamoto, Japan), and
1-methoxy-5-methylphenazinium methylsulfate were added to the cells at
the final concentrations of 5 and 0.2 mM, respectively, and incubated for
1 h. The cell viability was determined by measuring the difference between the absorbance at 450 and 620 nm using an ELISA autoreader. For
3H-thymidine incorporation assay, 3 × 104 cells were incubated with 0.5 µM of FK1012 for 20 h in 100 µl, and then pulsed with 1 µCi of 3H-thymidine (6.7 Ci/mmol; Dupont-NEN, Boston, MA) for 4 h before harvest.
Assay for Caspase Activity
The caspase activity in the cell lysates was determined as described (Enari
et al., 1996). In brief, the cell lysates (S-30 fraction, 10 µg) were incubated
for 30 min at 30°C in 0.5 ml of the assay buffer (100 mM HEPES-KOH,
pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM DTT, and 0.1 mg/ml ovalbumin) containing 1 µM MCA-DEVDAPK(dnp) (Peptide Institute, Osaka,
Japan). The fluorescence was measured at an excitation wavelength of 325 nm and an emission wavelength of 395 nm using a spectrofluorometer
(RF-1500; Shimadzu, Tokyo, Japan).
Western Blotting and DNA Fragmentation
Cells were incubated on ice for 30 min in the lysis buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM p-amidinophenyl methanesulfonyl fluoride hydrochloride, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10% glycerol). After removing insoluble materials by centrifugation, the supernatant was recovered as cytosolic extracts. In some cases, the crude lysates were prepared by heating the cells at 85°C for 30 min in 100 µl of lysis buffer (2.5% SDS, 120 mM Tris-HCl, pH 6.8, 200 µM DTT, 20% glycerol). The lysate was then mixed with 100 µl of 2× Laemmli's sample buffer.
Western blotting was performed as previously described (Kawahara et al.,
1998a). Anti-human caspase 8 (clone 5F7) antibody was purchased from
Medical and Biological Laboratories (Nagoya, Japan). Anti-Lamin B1
and anti-
-tubulin antibodies were from Oncogene Science (Cambridge,
MA), and anti-human FADD and anti-human caspase 3 antibodies were
from Transduction Laboratories (Lexington, KY). Anti-HA and anti-
human Bcl-2 antibodies were from Boehringer Mannheim Biochemicals
(Indianapolis, IN) and from PharMingen (San Diego, CA), respectively.
Anti-human PARP antibody (clone C-2-10) was provided by Dr. G.G.
Poirier (Centre Hospitalier de l'Université Laval Research Center, Quebec, Canada). Proteins recognized by the primary antibody were visualized using the enhanced chemiluminescence system (Renaissance; Dupont-NEN) after reacting with the horseradish peroxidase-conjugated
anti-mouse IgG (DAKO Corp., Santa Barbara, CA). DNA fragmentation assay was performed as described previously (Enari et al., 1995
).
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Results |
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Requirement of Caspase 8 for Fas-induced Apoptosis
During establishment of Jurkat cell transformants overexpressing Bcl-2, we obtained a clone (JB-6) that was completely resistant to Fas-induced cell death (Fig. 1 A). This
cell line, JB-6, expressed Fas as abundantly as the parental
Jurkat cells (Fig. 1 B); however, it expressed very little, if
any, caspase 8 (Fig. 1 B). Other signaling molecules, such
as FADD and caspase 3, were expressed in JB-6 cells as
abundantly as in Jurkat cells. The expression level of
caspase 10 (Mch 4, FLICE 2), which is another caspase containing the death effector domain (Fernandes-Alnemri
et al., 1996; Vincenz and Dixit, 1997
), was also comparable
between Jurkat and JB-6 cells (data not shown). These results suggested that the resistance of this cell line to Fas-induced apoptosis was likely due to the lack of caspase 8. Accordingly, when an expression vector for human
caspase 8 was introduced into the JB-6 cell line, the transformants (BC clones) regained the sensitivity to Fas-induced apoptosis (Fig. 1, A and B).
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Oligomerization of FADD Kills the Cells Lacking Caspase 8
It is assumed that the engagement of Fas causes oligomerization of FADD, which leads to activation of caspase 8 (Boldin et al., 1996; Muzio et al., 1996
). To examine
whether the oligomerization of FADD causes cell death,
two copies of FKBP (FK506-binding protein) were joined
to the death domain of human Fas (Itoh and Nagata, 1993
)
or to full-length human FADD (Boldin et al., 1995
; Chinnaiyan et al., 1995
; Fig. 2 A), and introduced into Jurkat
cells or JB-6 cells. As shown in Fig. 2 B, the Jurkat-derived
transformant clones JF-1 and -19, and the JB-6-derived
BF-2 and -4 clones all expressed FKBP-Fas of 44 kD,
which was recognized by the anti-HA antibody. In addition, the Jurkat-derived JM-84 and -94 clones, as well as
the JB-6-derived BM-5 and -79 clones, expressed a FKBP-FADD of 50 kD, detected by an anti-human FADD antibody. The expression level of the FKBP-FADD was comparable with that of the endogenous FADD, which is 32 kD
(Fig. 2 B). As found in the parental JB-6 cells, all transformant clones derived from JB-6 were deficient in expressing caspase 8. However, the expression levels of the endogenous Fas, FADD, and caspase 3 were comparable
among all transformants (Fig. 2 B, and data not shown).
The transformants were then treated for 6 h with increasing concentrations of FK1012, which should induce
FKBP dimerization (Spencer et al., 1993, 1996
), and thus
oligomerization of FKBP-Fas or FKBP-FADD. As shown
in Fig. 2 C, FK1012 did not show any toxicity to the parental Jurkat or JB-6 cells at a concentration of 0.5 µM. When
Jurkat cell transformants expressing FKBP-Fas or FKBP-FADD were treated with FK1012, viability (as assayed by
the WST-1 method) was lost, indicating that the oligomerized FKBP-Fas or FKBP-FADD could kill the cells. The
JB-6 transformants expressing FKBP-Fas were resistant to
the FK1012-induced cell death, and >65% of the cells
were still alive after treatment with 0.5 µM of FK1012 for 6 h. These results were consistent with the observation
that JB-6 cells were resistant to the anti-Fas antibody-induced cell death as described above (Fig. 1 A). On the
other hand, the JB-6 transformants expressing FKBP-FADD were efficiently killed by FK1012 (Fig. 2 C). The
sensitivity of these cells to FK1012 was comparable to that
found with Jurkat cell transformants expressing FKBP-FADD. The cell viability was also assayed by 3H-thymidine uptake. As shown in Fig. 2 D, the cells were incubated for 20 h with 0.5 µM of FK1012, and then pulsed for
4 h with 3H-thymidine. The Jurkat cell transformants (JF
and JM clones) expressing FKBP-Fas or FKBP-FADD
lost the ability to incorporate thymidine. The JB-6 transformants expressing FKBP-Fas (BF clones) survived after
treatment with FK1012, while the BM clones expressing FKBP-FADD were efficiently killed by the same treatment.
Necrotic Death Caused by FADD
The above results indicated that the killing of cells by oligomerization of Fas requires caspase 8. However, FADD, which mediates the Fas signal to caspase 8, can kill cells without caspase 8. To examine the molecular mechanism behind the FADD-induced cell death, activation of caspase 3 and DNA fragmentation were analyzed in Jurkat and JB-6 transformant clones. As shown in Fig. 3, A and D, when the Jurkat cell transformants were treated with FK1012, the proforms of caspases 8 and 3 disappeared within 6 h. This processing of caspases was accompanied by a strong activation of caspase 3-like protease, the cleavage of PARP and lamin B1, and the degradation of chromosomal DNA (Fig. 3). On the other hand, the JB-6 cell transformants expressing FKBP-Fas did not show any proteolysis of the nuclear proteins lamin B1 and PARP, nor any DNA fragmentation, as predicted by their unresponsiveness to FK1012 treatment. The transformants expressing FKBP-FADD also showed neither caspase 3 activation nor DNA degradation by the treatment with FK1012, despite massive cell death. These results indicated that oligomerization of FADD can kill the cells without activating caspases or causing DNA fragmentation. The dying cells were then examined using transmission electron microscopy. As shown in Fig. 4, death of Jurkat cells induced by oligomerization of FKBP-Fas or FKBP-FADD was accompanied by apoptotic morphological changes such as chromatin condensation, cell shrinkage, and apoptotic bodies. On the other hand, the caspase 8-deficient BM cells dying as result of FADD oligomerization showed neither condensation of cells nor fragmentation of chromatin. Rather, the cells swelled without forming apoptotic bodies. These results indicated that FADD can kill cells without inducing apoptosis in BM cells.
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We then examined the effect of a broad caspase inhibitor, Z-VAD-fmk, on the cell death in Jurkat cells. As shown in Fig. 5, A and B, Z-VAD-fmk completely blocked the proteolysis of nuclear proteins and DNA fragmentation that occur during FKBP-Fas-induced cell death. Accordingly, the cell death induced by FKBP-Fas was largely prevented by Z-VAD-fmk (Fig. 5 C). In contrast, Z-VAD-fmk did not inhibit the FKBP-FADD-mediated cell death, although it inhibited proteolysis of nuclear proteins and DNA degradation. The dying Jurkat cells under electron microscope showed a necrotic morphological change (Fig. 4 E). These results suggested that FADD can kill the Jurkat cells via two pathways. One is an apoptotic pathway in which activation of a caspase cascade leads to fragmentation of chromosomal DNA, and the other is a nonapoptotic pathway that does not involve caspases, but is accompanied by necrotic morphological changes of the cells.
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Discussion |
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In this report, we have shown that a derivative of human
Jurkat cells that does not express caspase 8 was resistant to
the apoptosis induced by the activation of endogenous Fas
or oligomerization of the Fas death domain. These results
indicate that caspase 8 is indispensable for the Fas-mediated apoptotic cell death process, which agrees with the recent results that embryonal fibroblasts from caspase 8 null
mice are resistant to the Fas-induced apoptosis (Varfolomeev et al., 1998). On the other hand, oligomerization of
FADD, an adaptor molecule that normally mediates the
Fas signal to caspase 8, could kill cells that were deficient
in caspase 8. This pathway does not use caspases to kill the
cells, and seems to operate even in the cells (the parental
Jurkat cells) that express caspase 8, because a broad
caspase inhibitor could not prevent the cell death induced
by oligomerization of FADD.
The Fas-induced cell death is so far thought to transduce
only apoptotic signal. On the other hand, TNF was shown
to transduce not only an apoptotic signal, but also a necrotic signal. In particular, treatment of mouse L929 cells
with TNF produces oxidants and kills the cells by inducing
necrotic morphological changes of the cells (Vercammen
et al., 1998a). This process was shown to be enhanced by caspase inhibitors. More recently, Vercammen et al.
(1998b)
established mouse L929 cells overexpressing human Fas. When these cells were treated with the agonistic
anti-Fas antibody, they underwent apoptosis. But, caspase
inhibitors could not prevent the cell death process, and the
cells died by necrosis. This result is apparently in contrast
to our observation that the Fas engagement alone cannot
kill the caspase 8-deficient human Jurkat cells. However,
the sensitivity of the cells to necrosis may depend on the
strength of the death signal evoked by Fas or FADD, the
expression level of the downstream signal transducer for
necrosis, and/or the balance between apoptotic and necrotic signals. In L929 cells, a downstream molecule(s)
leading to necrosis is more abundant than in Jurkat cells,
and the necrotic signal can be easily seen with the weak
signal from the Fas receptor in the presence of caspase inhibitors, while its strong activation by direct oligomerization of FADD may be necessary to activate the necrotic
pathway in Jurkat cells. FADD can also be activated by
the TNF-TNF type I receptor system through an adaptor
molecule called TRADD (TNFR1-associated death domain protein; Chinnaiyan et al., 1996
; Hsu et al., 1996
). It
will be interesting to determine whether the TNF-induced
necrosis in L929 cells is mediated by FADD or not. In addition, the TNF-induced necrosis in L929 cells can be inhibited by butylated hydroxyanisole, an anti-oxidant, suggesting an involvement of oxidants in this cell death
process (Vercammen et al., 1998a
). Whether or not a similar oxidant(s) is activated during necrosis by oligomerization of FADD in Jurkat cells remains to be determined.
The FADD-mediated apoptosis occurs through recruitment of pro-caspase 8 to the FADD death effector domain, which leads to processing of caspase 8 and the activation of the downstream caspases, such as caspase 3, and
a DNase (CAD; Nagata, 1997; Enari et al., 1998
; Sakahira
et al., 1998
). It would be interesting to examine whether or
not the same region of FADD is responsible for the necrotic signal transduction and what kinds of molecules are
activated by FADD. The JB-6 cells abundantly express
Bcl-2, yet oligomerization of FADD killed the JB-6 cells,
suggesting that the FADD-induced necrotic cell death
cannot be inhibited by Bcl-2. This agrees with no inhibitory effect of Bcl-2 on the TNF-induced cytotoxicity in
mouse L929 cells (Vanhaesebroeck et al., 1993
). In any
case, the establishment of a system for studying necrosis in
the absence of caspase activation will contribute to our understanding of the molecular mechanisms underlying necrosis.
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Footnotes |
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Received for publication 31 August 1998 and in revised form 13 October 1998.
Dr. Kawahara's present address is Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892.
Address all correspondence to Shigekazu Nagata, Osaka University
Medical School, B-3, Department of Genetics, 2-2 Yamada-oka, Suita,
Osaka 565-0871, Japan. Tel.: 81-6-879-3310. Fax: 81-6-879-3319. E-mail:
nagata{at}genetic.med.osaka-u.ac.jp
We thank M. Gilman and H. Berger for the plasmid carrying FKBP and FK1012, V.M. Dixit for human caspase 8 (FLICE) cDNA, and S. Kumagai for secretarial assistance.
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture in Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government. A. Kawahara was supported by a postdoctoral fellowship in Cancer Research of the Japan Society for the Promotion of Science.
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Abbreviations used in this paper |
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FADD, Fas-associated protein with death domain; FKBP, FK506 binding protein; HA, hemagglutinin; PARP, poly(ADP-ribose) polymerase; TNF, tumor necrosis factor.
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