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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2658-2663
Apoptotic Signal of Fas Is Not Mediated by Ceramide
By
Shu-Ching Hsu,
Chia-Cheng Wu,
Tien-Yau Luh,
Chen-Kung Chou,
Shau-Hwa Han, and
Ming-Zong Lai
From the Graduate Institute of Microbiology, National Taiwan
University School of Medicine, Taiwan, China; the Institute of
Molecular Biology, Academia Sinica, Taiwan, China; the Graduate
Institute of Microbiology and Immunology, National Yang-Ming
University, Taiwan, China; the Department of Chemistry, National Taiwan
University, Taiwan, China; and the Department of Medical Research,
Veteran General Hospital, Taipei, Taiwan, China.
 |
ABSTRACT |
Ceramide has been suggested as the secondary messenger mediating the
apoptotic signal for Fas engagement. By using different inhibitors, we
demonstrated here that ceramide is unlikely a mediator of Fas-initiated
apoptosis. First, cAMP prevented cell death induced by ceramide but not
by Fas. Second, ceramide-triggered, but not Fas-triggered, apoptosis
was antagonized by the free radical scavenger C60. Third,
the metal chelator pyrrolidinedithiocarbamate suppressed ceramide-initiated DNA fragmentation but had no effect on the Fas-induced cell death. Fourth, the SAPK/ERK kinase dominant negative mutant, which attenuated ceramide-induced cell death, did not prevent
Fas-induced apoptosis. Finally, activation of NF- B inhibited ceramide-induced but not Fas-initiated apoptosis. The fact that many
antagonists of ceramide-induced apoptosis could not suppress Fas-mediated cell death clearly indicates that ceramide is not the
mediator for Fas-initiated apoptotic signal.
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INTRODUCTION |
Fas (APO-1) IS A 45-kD membrane protein
when engaged by anti-Fas antibody or Fas ligand triggers programmed
cell death (for review, see Nagata1). The death pathway
initiated from Fas activation involves a series of death-induced
molecules.1 FADD (Fas-associating protein with death
domain) or MORT1 is recruited to Fas upon its
engagement.2,3 FADD then binds FLICE (FADD-like ICE) or
MACH (MORT-1-associated CED-3 homologue).4,5 The association with the Fas death-inducing signaling complex activates FLICE,6 followed by eventual activation of ICE and
CPP32.7
The activation of acidic sphingomyelinase that leads to the hydrolysis
of sphingomyelin and generation of ceramide has also been suggested as
the apoptotic pathway downstream of Fas ligation.8-12 Ceramide is reported as a common intermediator for stimulation by tumor
necrosis factor (TNF), interleukin-1, nerve growth factor, lipopolysaccharide, ionizing radiation, serum withdrawal, and daunorubicin.12-19 The role of ceramide in the apoptosis
induced by some of these stimuli is illustrated by the ability of
membrane-permeable ceramide to trigger cell death.13,14,19
In addition, defects in the sphingomyelinase/ceramide pathway confers
the resistance to radiation-, TNF- -, and UV-induced
apoptosis,20-22 supporting the role of ceramide in these
types of death induction. Ceramide-induced cell death involves the
activation of c-Jun N-terminal kinase (JNK).23
In this study, we compared the sensitivity of Fas and ceramide to
various regulatory reagents. We showed that ceramide-induced apoptosis
was antagonized by cAMP, C60, and
pyrrolidinedithiocarbamate (PDTC). However, Fas-triggered cell death
proceeded in the presence of these inhibitors. In addition, the
negative mutant of SAPK/ERK kinase (SEK) suppressed ceramide-triggered
death but did not prevent Fas-induced apoptosis. Furthermore,
activation of NF-B antagonized ceramide but not Fas-initiated
apoptosis. These observations are not consistent with the model that
ceramide acts downstream of Fas signaling. Together with recent
observations,24,25 our results argue against a role of
ceramide in Fas-mediated apoptosis.
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MATERIALS AND METHODS |
Reagents and cell lines.
12-O-tetradecanoylphorbol 13-acetate (TPA), A23187,
N6, 2 -O-dibutyryladenosine 3,5-cyclic
monophosphate (Bt2cAMP), forskolin, and PDTC were purchased
from Sigma Chemical Co (St Louis, MO). Anti-Fas antibody
CH-1126 was purchased from Upstate Biotech Inc (Lake
Placid, NY). C2-ceramide and C6-ceramide were
obtained from Biomol (Plymouth Meeting, PA). ICE inhibitor z-VAD-FK was purchased from Kamiya (Thousand Oaks, CA). The two regioisomers with C3
or D3 symmetry of water-soluble carboxylic acid C60
derivatives (carboxylfullerenes) were synthesized as previously
described.27 Both C60 (C3) and C60
(D3) are effective scavengers of oxgen radicals, with complete
elimination of hydroxyl radicals and superoxide radicals at
concentrations of 5 to 50 µmol/L, and are potent inhibitors of
neuronal apoptosis, which is associated with increased intracellular free radical production.27 H-89 and H-85 were purchased
from Seikagaku (Tokyo, Japan). Human T lymphoblastomas CEM (ATCC CCL 119) and human T-cell leukemia Jurkat (ATCC TIB 152) were obtained from
American Type Culture Collection (Rockville, MD). Recombinant human
TNF- was purchased from R&D (Minneapolis, MN).
Plasmids.
CMV-RelA(p65) and B-TATA-CAT28 were kind gifts of Dr
Warren C. Greene (University of California, San Francisco, CA).
HA-JNK129 was obtained from Michael Karin (University of
California, San Diego, CA). SEK-negative mutants SEK1(K R) and
SEK1(AL)30 were obtained from Dr Leonard I. Zon
(Harvard Medical School, Boston, MA). Green fluorescence protein
expression vector pEGFP-N1 was obtained from Clontech (Palo Alto, CA).
Transfection.
T cells (1.6 × 107) were washed once with STBS (25 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 5 mmol/L KCl, 0.6 mmol/L
Na2HPO4, 0.7 mmol/L CaCl2, 0.5 mmol/L MgCl2) and incubated with a total of 10 µg DNA in
1.2 mL STBS containing 0.5 mg/mL diethylaminoethyl (DEAE)-dextran for 20 minutes at room temperature. T cells
were then treated with 15% dimethylsulfoxide for 3 minutes and washed once with STBS.31,32 After 24 to 48 hours, transfected
cells were then stimulated with anti-Fas or ceramide, and the cell
death was quantitated.
JNK activity assay.
The inhibition of JNK by the dominant negative form of SEK was
performed with cotransfection of HA-JNK1. T cells were activated as
indicated, washed twice with phosphate-buffered saline (PBS), and lysed
in ice-cold lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 1% Triton X-100,
10% glycerol, 137 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L
EDTA, 50 mmol/L NaF, 1 mmol/L Na3VO4, and 1 mmol/L phenylmethylsulfonyl fluoride). Detergent-insoluble material was
removed by centrifugation at 14,000g for 10 minutes at 4°C.
For each immuno-precipitation, 200 µg of cell lysate was mixed with
anti-HA 12CA5 (Boehring Mannheim, Mannheim, Germany) and incubated at
4°C for 2 hours. Twenty microliters of protein A-sepharose
(Pharmacia, Uppsala, Sweden) was then added and incubated for an
additional 2 hours at 4°C. Immune complexes were washed three times
with lysis buffer and once with kinase buffer (30 mmol/L Tris-HCl, pH
8.0, 20 mmol/L MgCl2, 2 mmol/L MnCl2). Immune
complexes were then incubated in kinase buffer (30 µL) containing 2 mmol/L ATP, GST-c-Jun(1-79), and 5 µCi of ( -32P)ATP
for 30 minutes at 30°C. Incubations were terminated by adding 15 µL of 3× sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer and boiling for 3 minutes. The
reaction products were resolved on 15% SDS-PAGE followed by
autoradiography and quantitated by Phosphorimager (Molecular Dynamics,
Sunnyvale, CA).33
Cell death measurement.
All cultures (except those treated with ceramide) were performed in
RPMI with 10% fetal calf serum (both from GIBCO, Grand Island, NY), 10 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 × 10 5 mol/L 2-ME.34 For apoptosis
induced with ceramide, serum free-medium were used throughout the
experiment. The extent of apoptosis was determined by propidium iodide
(PI) staining or annexin V staining. Cells were treated with different
inducers and/or inhibitors, washed with PBS, and fixed with
ethanol. DNA content was determined by staining with 20 µg/mL PI and
analyzed by FACScan (Becton Dickinson, Mountain View, CA). The fraction
of cells with sub-G1 DNA content was assessed with the
CELLFIT program (Becton Dickinson). For annexin V staining, the treated
cells were washed, resuspended in annexin V-fluorescein isothiocyanate
(FITC) (1 µg/mL; Clontech), incubated at room temperature for 15 minutes in the dark, and analyzed on FACScan. For the ceramide/Fas
sensitivity of cell transiently transfected with CMV-p65 or
pEBG-SEK(AL), survival was monitored with green fluorescence
protein expression vector pEGFP-N1. Treated cells were examined using a
Nikon Diaphot 2000 fluorescence microscope (Tokyo, Japan)
48 hours after transfection.
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RESULTS AND DISCUSSION |
cAMP prevented ceramide-induced cell death but had no effect on
Fas-initiated apoptosis.
Ceramide is a potent inducer of cell death in T-lymphoma cells Jurkat
and CEM (not shown for CEM). Cell death was assessed by DNA
fragmentation as represented by sub-G1 fraction
(Fig 1B). DNA fragmentation induced by
ceramide never exceeded 45%, despite extensive cell death. A similar
observation was also earlier reported.35 We first
identified a few reagents that could block ceramide-initiated cell
death. cAMP was an effective antagonist of ceramide-induced cell death
(Fig 1C). Forskolin (10 µmol/L) suppressed DNA fragmentation initiated by ceramide by at least 50%. A similar extent of inhibition was observed with dibutyryl cAMP (Bt2cAMP) at 1 mmol/L
(Fig 2A). There was a dose-dependent
inhibition of cAMP on ceramide-triggered cell death. The inhibition of
ceramide-induced cell death could be detected with Bt2cAMP
as low as 100 µmol/L. In contrast, apoptosis triggered by anti-Fas
antibody CH-11 was resistant to cAMP. Treatment with forskolin up to 50 µmol/L or Bt2cAMP up to 2.5 mmol/L did not reduce the
apoptosis triggered by Fas (Fig 2B). cAMP agonists by itself did not
induce any T-cell death at the highest concentrations used here.
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| Fig 1.
The inhibition of ceramide-induced cell DNA
fragmentation. Jurkat T cells were treated with 5 µmol/L of
C2-ceramide (C2-C) in the absence or presence of various
antagonists for 4 hours, washed with PBS, and fixed with ethanol. DNA
content was determined by staining with 20 µg/mL PI and analyzed by
FACScan (Becton Dickinson). Fraction of cells with sub-G1
DNA content (M1 fraction in the diagram) were assessed with CELLFIT
program (Becton Dickinson). CTR, untreated cell control. The inhibitors
used were as follows: forskolin (F), 10 µmol/L; C60 D3
(D3), 100 µmol/L; PDTC, 200 µmol/L; Z-VAD-FK (ICEi), 300 µmol/L.
The sub-G1 fractions were less than 6% for cells treated
with inhibitors only. The exception was PDTC, in which a slightly
elevated background death (8%) was observed.
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| Fig 2.
Inhibitors that blocked ceramide-induced DNA
fragmentation did not prevent Fas-induced DNA fragmentation. (A) Jurkat
cells were treated with ceramide and various inhibitors for 4 hours. The sub-G1 DNA content was quantitated as described in Fig
1. Additional inhibitors used were as follows: dbcAMP, 1 mmol/L; C60 (C3/100), 100 µmol/L; C60 (D3/100), 100 µmol/L; C60 (D3/10), 10 µmol/L. (B) Jurkat cells were
stimulated with anti-Fas antibody CH11 (125 ng/mL) in the absence or
presence of the inhibitors for 14 hours, and the sub-G1 DNA
content was determined. The inhibitors used were as follows: forskolin,
50 µmol/L; dbcAMP, 2.5 mmol/L; C60 (C3), 100 µmol/L;
C60 (D3), 100 µmol/L; PDTC, 200 µmol/L; Z-VAD-FK
(ICEi), 300 µmol/L. None of the inhibitors alone induced DNA
fragmentation at the concentrations used, except PDTC, in which an 1%
to 2% increase over control was observed. The result is the average of
duplicates, with standard deviation shown as an error bar. Those not
shown are too small in scale. Experiments were repeated three times
with the same results.
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The observation that Fas-initiated cell death cannot be prevented by
cAMP is consistent with an earlier report,36 yet is in
direct contrast to the prominent inhibitory effect of cAMP recently
documented.37 Because DNA fragmentation may not well represent actual apoptosis,38,39 we also quantitated cell
death by annexin V staining. Figure 3 shows
that Fas-induced apoptosis was associated with an extensive
phosphatidylserine translocation, which was clearly not inhibited by
cAMP. cAMP is an antagonist for ceramide-induced cell death but not for
Fas-initiated apoptosis.
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| Fig 3.
Inability of cAMP and C60(D3) to prevent Fas-induced
apoptosis as assessed by annexin V binding. Jurkat cells were treated with CH-11 and inhibitors for 14 hours as described in Fig 2. Cells
were washed, resuspended in annexin V-FITC (1 µg/mL; Clontech), and
analyzed on FACScan. M1 is designated as the fraction of apoptotic cells. In data not shown, cells treated with ceramide were similarly assayed. cAMP, (C60)D3, and ICEi were used at the same concentration as
in Fig 2B.
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C60 and PDTC prevented ceramide-induced cell death but
had no effect on Fas-initiated apoptosis.
We have futher observed that two malonic acid derivatives of
C60 (carboxyfullerens)27 were antagonist of
ceramide-induced cell death (Figs 1D and 2A). The pure carbon sphere of
C60 (buckminsterfullerene) is known for its avid reactivity
with free radicals, yet the usage is limited by its water
insolublility. The two regioisoforms with C3 or D3 symmetry of
water-soluble C60 derivatives remain as potent free radical
scavengers and are effective antagonists of apoptotic neronal death
induced by serum deprivation, glutamate receptor agonists, and amyloid
peptide.27 Ceramide-triggered apoptosis was suppressed by
50% in the presence of 10 µmol/L C60 (D3) and was
inhibited by 90% with 100 µmol/L C60 (D3). We have
repeatedly observed that C60 (D3) was more effective than
C60 (C3) in the prevention of ceramide-induced cell death
(Fig 2A). In contrast, C60 (D3) and C60 (C3)
(at 100 µmol/L each) had no preventive effect on Fas-triggered cell
death as analyzed by both sub-G1 fraction and annexin V
binding (Figs 2B and 3D). The same contrast was found with metal
chelator PDTC. High concentration of PDTC antagonized ceramide-induced
apoptosis (Figs 1E and 2A), but did not inhibit Fas-initiated cell
death (Fig 2B). Of all the inhibitors analyzed in this study, PDTC was
the only reagent that by itself increased spontaneous cell death at the
concentration used (1% to 3% over control, data not shown). Because
such increase was minute, the differential effect of PDTC on ceramide-
and Fas-initiated apoptosis was still prominent. As a control, the
potent ICE proteases inhibitor z-VAD-FK completely abolished cell death
induced by either ceramide or Fas (Figs 1, 2, and 3), consistent with
the notion that both ceramide- and Fas-induced cell death are known to
involve caspases such as ICE.7,40
Therefore, we have identified three types of reagents that antagonized
ceramide-induced cell death but did not prevent Fas-induced cell death.
Between 50% and 90% of ceramide-triggered cell death was suppressed
by these inhibitors. If ceramide is the major apoptotic messenger
downstream of Fas, a lesser but still prominent inhibition on
Fas-induced cell death by these inhibitors should be detectable. The
distinct effect on Fas- and ceramide-induced apoptosis suggest that
ceramide is dissociated from Fas-induced death cascade.
SEK dominant negative mutant did not interfere Fas-induced cell
death.
SEK/JNK activation is shown to be essential for ceramide-initiated
apoptosis.23 Despite the fact that JNK is activated after Fas engagement, its role in apoptosis is less certain. Lenczowski et
al41 demonstrated that the activation of SEK/JNK is
downstream of ICE protease and is not required for Fas-induced
apoptosis. On the contrary, the prevention of JNK activation by SEK
dominant negative mutant is shown to block Fas-induced cell death in
L929 and 293 cells.42 We have thus studied the effect of
the dominant negative mutant of SEK30 on the same cell when
treated with ceramide and anti-Fas. A transient transfection analysis
was used to assess the effect on cell death in which SEK mutant was
cotransfected with green fluorescence protein. Expression of the
dominant negative mutants of SEK blocked the activation of the
cotransfected HA-JNK1.32 Ceramide triggered cell death was
suppressed by coexpression of SEK (AL) in Jurkat cells
(Fig 4), but the inhibition was less with
SEK (KR) mutant (data not shown). This is in contrast to the
inability of SEK (AL) to interfere with Fas-induced apoptosis
(Fig 4). In both Jurkat and CEM cells (not shown for CEM), the
inhibition of SEK activation prevented ceramide- but not Fas-induced
apoptosis. JNK activation was not essential for Fas-mediated apoptosis
at least in these two T-lymphoma cells.
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| Fig 4.
Dominant negative mutant of SEK prevented ceramide- but
not Fas-induced apoptosis. Jurkat cells were transfected with 5 µg of
either SEK1(AL) or pEBG vector, together with 5 µg of
pEGFP-N1 (Clontech). Thirty-six hours later, cells were untreated (Ctr) or treated with either C2-ceramide (5 µmol/L) or anti-Fas
antibody CH11 (100 ng/mL) (Induction).
Ceramide-induced cell death was analyzed 6 hours later, whereas
Fas-induced cell death were quantitated 12 hours later. Data are the
average of duplicates.
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Activation of NF-B did not interfere Fas-induced cell death.
In accordance with the recent finding that activation of NF-B
prevents the apoptotic signal of TNF-, we also found that NF-B
activation antagonized ceramide-induced DNA fragmentation (Fig 5). Transient expression of NF-B
p65, which led to activation of B-CAT (not shown), prevented
ceramide-induced apoptosis. On the contrary, Fas-induced cell death was
not affected by the activation of NF-B in Jurkat cells. These
results further showed that ceramide is not a mediator of Fas-induced
apoptosis, for otherwise Fas would be equally sensitive to the
inhibition by NF-B.
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| Fig 5.
Differential sensitivity of ceramide- and Fas-triggered
cell death to activation of NF-B. Jurkat cells were transfected with 5 µg of either CMV-RelA or CMV vector, together with 5 µg of
pEGFP-N1 (Clontech). Thirty-six hours later, cells were untreated or
treated with either C2-ceramide (5 µmol/L) or anti-Fas
antibody CH11 (100 ng/mL). Ceramide-induced cell death was analyzed 6 hours later, whereas Fas-induced cell death were quantitated 14 hours
later. Data are the average of duplicates.
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In summary, we have presented evidence that ceramide is not essential
for Fas-induced cell death. Ceramide-triggered cell death was sensitive
to the inhibition of cAMP, yet its blockage did not prevent
Fas-initiated apoptosis. Two other antagonists, C60 and
PDTC, also demonstrate such selectivity in suppressing ceramide-induced
apoptosis. We have further shown that blocking SEK activation
diminished ceramide-induced cell death in T lymphomas Jurkat and CEM,
yet had no effect on Fas-triggered apoptosis on the same cells (Fig 4).
A similar distinction was also found with NF-B activation (Fig 5).
Therefore, a large fraction of Fas-triggered death pathways must be
ceramide-independent. Even though ceramide has been suggested as the
mediator for Fas-initiated apoptosis, the linkage between ceramide and
Fas-induced cell death is less than confirmative. Recent biochemical
analysis have questioned the role of ceramide in Fas-induced apoptosis.
In one study, the direct measurement of sphingosine-based ceramide
failed to detect induction of ceramide up to 2 hours after Fas
triggering.25 In another study, ceramide increase was found
to be slower than Fas-induced cell death and can be inhibited by
Z-VAD-FMK.24 Our results are fully in support of these
observations and suggest that ceramide is not the mediator of
Fas-initiated apoptosis.
Mixed effect of cAMP and C60 on TNF--induced
apoptosis.
We also examined the effect of cAMP, C60, and PDTC on
TNF--induced apoptosis, which is reported to be mediated by
ceramide.14,16 The study was performed on L929 cells
because TNF- alone does not induce cell death in Jurkat cells. The
effect of these inhibitors on TNF--induced cell death was not as
unequivocal as on Fas-mediated apoptosis. PDTC (5 µmol/L) induced
spontaneous cell death (70%) in L929 cells, and its inhibitory effect
on TNF--induced cell death cannot assessed. TNF--triggered
apoptosis was completely prevented by 50 µmol/L of C60
(C3) and C60 (D3), yet was resistant to treatment with
forskolin and Bt2cAMP (Fig 6).
In contrast to the NF-B-independent Fas-mediated apoptosis (Fig 5),
TNF--induced cell death is also known to be suppressed by the
activation of NF-B.43-45 Some of the discrepancy may be
attributed to the difference in apoptotic signaling between Fas and
TNF- previously reported.36 For example, the different
sensitivity to C60 (Figs 2 and 6) is well correlated with
the involvement of reactive oxgen radicals in TNF--induced, but not
in Fas-induced, cell death.36 It may also be noted that the
mixed effect of the inhibitors on TNF--mediated apoptosis is
consistent with the observation that ceramide contributes to a
fraction, but not all, of TNF--induced cell death.35
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| Fig 6.
Mixed effect of cAMP and C60 on TNF--induced
apoptosis. L929 cells were treated with recombinant TNF- (250 µg/mL) in the absence or presence of the indicated inhibitors for 18 hours. The sub-G1 DNA content was quantitated as described
in Fig 1. The concentration of the inhibitors used were as follows:
forskolin, 10 µmol/L; dbcAMP, 1.0 mmol/L; C60 (C3), 50 µmol/L; C60 (D3), 50 µmol/L; Z-VAD-FK (ICEi), 100 µmol/L.
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The distinction between the Fas-induced apoptotic cascade and
ceramide-induced death pathway shows that death processes induced by
different stimulation can be clearly independent of each other. The
combination of different stimulations may hence be more effective in
the induction of apoptosis, especially in tumor cells for therapeutic purposes, notably, ceramide-mediated cell death initiated by TNF-, daunorubicin, and -irradiation, which are reagents widely used in
tumor treatment.20-22 The dissociation of Fas-induced death signals from ceramide may indicate an additional dimension in manipulation of apoptosis.
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FOOTNOTES |
Submitted October 14, 1997;
accepted December 31, 1997.
Supported by Grant No. DOH86-HR-508 from the Department of Health, by
Grant No. NSC 85-2331-B001-050 M30 from National Science Council, and
by a grant from Academia Sinica, Taiwan, Republic of China.
Address reprint requests to Ming-Zong Lai, PhD, Institute
of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors thank Dr Leonard Zon for SEK(AL) and
SEK(KR), Dr Warren C. Greene for B-TATA-CAT, and Dr
Michael Karin for HA-JNK1. We also thank Douglas Platt for editorial
correction of the manuscript.
| |
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Abstract
Introduction
Abstract