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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3847-3854
Inhibition of Caspase Cascade by HTLV-I Tax Through Induction of
NF- B Nuclear Translocation
By
Atsushi Kawakami,
Tomoki Nakashima,
Hideaki Sakai,
Satoshi Urayama,
Satoshi Yamasaki,
Ayumi Hida,
Masahiko Tsuboi,
Hideki Nakamura,
Hiroaki Ida,
Kiyoshi Migita,
Yojiro Kawabe, and
Katsumi Eguchi
From The First Department of Internal Medicine, and the Department of
Hospital Pharmacy, Nagasaki University School of Medicine; and the
Department of Pharmacology, Nagasaki University, School of Dentistry,
Nagasaki, Japan.
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ABSTRACT |
NF- B is required for prevention of apoptosis. We examined the
importance of human T-cell leukemia virus-I (HTLV-I) Tax protein to
stimulate NF-B nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study.
Both Jurkat and Tax JPX-9 cells had small amounts of
basal nuclear NF-B activity. The addition of NF-B inhibitors
suppressed NF-B nuclear translocation of the cells, thus inducing
apoptosis. Sequential activation of caspases from caspase-8 to
caspase-3 was shown during this process. NF-B nuclear translocation
in JPX-9 cells was stimulated through Tax expression, and both the
activation of caspases and apoptosis induced by NF-B inhibitors were
significantly suppressed in the Tax+ JPX-9 cells. The
expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat,
Tax JPX-9, and Tax+ JPX-9 cells in the
presence or absence of NF-B inhibitors. X-chromosome-linked inhibitor of apoptosis (XIAP) protein expression in Tax
JPX-9 cells was significantly suppressed by NF-B inhibitors, however, its expression in Tax+ JPX-9 cells was
maintained even by the addition of NF-B inhibitors. Our results
suggest that the activation of NF-B via Tax protein in HTLV-I
infected cells renders the cells resistant to apoptosis. The expression
of anti-apoptotic gene products such as XIAP to suppress caspase
cascade, results in an increase of cytokine production and cell
proliferation; one of the proposed mechanisms that promotes autoimmune
disorders such as Sjögren's syndrome and rheumatoid arthritis
found in HTLV-I seropositive subjects.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INFECTION OF HUMAN host cells with human
T-cell leukemia virus-I (HTLV-I) results in activation of these cells
and induction of various cellular events, such as increased production of various types of cytokines1-3 and augmentation of cell
proliferation.4 The central role of these events is
believed to be initiated by transactivation of expression of host genes
through HTLV-I Tax protein.5 The factors in host cells to
induce gene expression by Tax is thought to be a set of
enhancer-binding proteins.5 One such factor is NF-B,
which is implicated in Tax-mediated transactivation of various genes.
We previously showed that interleukin-6 (IL-6) gene is transactivated
by Tax protein via NF-B binding site using transient cotransfection
by Tax expression plasmid pMax-Neo with IL-6 promotor in COS1 cells,
and JPX-9, in which pMAX-Neo is stably transfected in Jurkat
cells.6 Previous studies suggested that increased
production of cytokines and cell proliferation mediated by activation
of NF-B through Tax protein may have a major pathogenic role in the
induction of autoimmune disorders described in humans such as HTLV-I
associated myelopathy (HAM), Sjögren syndrome (SS), and
rheumatoid arthritis (RA).7-11
A newly described function of NF-B is its inhibitory action against
apoptotic signals.12-19 A variety of stimuli such as
radiation, depletion of growth factors, and signaling through Fas and
tumor necrosis factor receptor (TNFR) induce apoptosis of the cells, whereas the common death pathways of the above stimuli appear to be
mediated through activation of caspases.12-17 Accumulating evidence suggests that the apoptotic process is clearly inhibited by
activation of NF-B, which appears to be mediated through induction of anti-apoptotic gene products.12-19 It is possible to
speculate that NF-B is highly activated by Tax in HTLV-I infected
cells, which increases the resistance against several apoptotic
stimuli, resulting in increased production of cytokines and cell
proliferation. These phenomena may increase the risk for onset of
autoimmune disorders in HTLV-I seropositive subjects.
In the present study, we initially examined whether inhibition of
activation of NF-B induces apoptosis of cells through activation of
the caspase cascade. In the next step, we also investigated whether
HTLV-I Tax protein activates NF-B, which in turn inhibits the
activation of caspases and results in inhibition of apoptosis.
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MATERIALS AND METHODS |
Cell culture.
We used Jurkat (HTLV-I-uninfective T-cell line) and JPX-9 cells in the
following experiments. JPX-9 is a Jurkat subclone generated by the
stable introduction of the Tax expression plasmid vector, pMax-Neo
(kindly provided by Prof Sugamura, Tohoku University, Sendai,
Japan).6,20 Because metallothionein promotor
is included in the plasmid vector, the addition of CdCl2
(30 µmol/L) was necessary to induce Tax expression in JPX-9
cells.6,20 As previously described,6,20 Tax
expression in JPX-9 cells induced by CdCl2 was detected
within 3 hours as determined by reverse transcription-polymerase chain
reaction (RT-PCR) and Western blotting (results of RT-PCR in Fig
1).
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| Fig 1.
Induction of Tax mRNA expression in JPX-9 cells by
CdCl2. JPX-9 cells were treated with 30 µmol/L of
CdCl2 for indicated hours. After incubation, the expression
of Tax or  -actin mRNA in the cells was examined by RT-PCR analysis
on 1% agarose gel electrophoresis. PCR products for Tax mRNA contain
246-bp fragments, whereas that for -actin mRNA contains 236-bp
fragments. Primer pairs used are 5'-AAACAGCCCTGCAGATACAAAGT-3'
(upper primer) and 5'-ACTGTAGAGCTGAGCCGATAACG-3' (lower primer) for
Tax, and 5'-GACGAGGCCCAGAGCAAGAGAG-3' (upper primer) and
5'-ACGTACATGGCTGGGGTGTTG-3' (lower primer) for -actin. Lane A;
CdCl2-untreated JPX-9 cells. Lane B; JPX-9 cells treated
with CdCl2 for 3 hours. Lane C; JPX-9 cells treated with
CdCl2 for 6 hours. Lane D; JPX-9 cells treated with
CdCl2 for 12 hours. Lane E; Jurkat cells (negative
control). Lane F; MT-2 cells (positive control). Lane G; DNA size
marker (Hind III digests of  DNA).
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Induction of apoptosis of Jurkat and JPX-9 cells by
NF-B inhibitors.
Apoptosis of Jurkat and JPX-9 cells was induced by the addition of
pyrrolidine dithiocarbamate (PDTC; Sigma Chemical Co, St Louis, MO),
N-acetylcysteine (NAC, Sigma), or Z-Leu-Leu-Leu-aldehyde (LLL-CHO,
Peptide Institute, Inc, Osaka, Japan), a potent NF-B inhibitor, as
previously described.5,21,22 PDTC and NAC are antioxidants,
which inhibit the action of reactive oxygen intermediates, and are
thought to inhibit nuclear translocation of NF-B in the cells.5 LLL-CHO is reported to inhibit IB degradation
by inhibiting proteasome function. Therefore, dissociation of NF-B from IB is prevented by LLL-CHO, and nuclear NF-B translocation is suppressed.22 In brief, Jurkat or JPX-9 cells,
preincubated with or without CdCl2, were further cultured
with various concentrations of NF-B inhibitors for 24 hours in RPMI
1640 supplemented with 10% fetal bovine serum. After incubation,
apoptotic cell death was quantified by the percentage of cells with
hypodiploid DNA or by the detection of apoptotic nuclei using Hoechst
33258 dye staining (Wako Pure Chemical Industries, Osaka, Japan), as
described previously.23 Briefly, the cells were fixed with
70% ethanol and treated with RNAase (100 µg/mL; Sigma) and then
stained with propidium iodide (100 µg/mL; Sigma) for 30 minutes on
ice. The stained cells were analyzed using a flow cytometer (Epics
Profile-II; Coulter Immunology, Hialeah, FL) to detect the
presence of hypodiploid DNA. We also used Hoechst 33258 dye staining to
detect apoptotic cells. For this purpose, the cells were treated with
or without CdCl2 or NF-B inhibitors, then fixed with 2%
glutaraldehyde solution (Wako) for 10 minutes and stained with 0.2 mmol/L Hoechst 33258 to visualize DNA. Cells were examined under a
fluorescence microscope (AHB-LB; Olympus, Tokyo, Japan) to determine
the number of cells with chromatin condensation and/or nuclear fragmentation.
In some experiments, we added a caspase-1 specific inhibitor,
Ac-Try-Val-Ala-Asp-aldehyde (YVAD-CHO; Peptide Institute), caspase-3 specific inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO; Peptide Institute, Osaka, Japan), or caspase-8 specific inhibitor,
Z-Ile-Glu(OMe)-Thr-Asp(OMe)-FMK (IETD-FMK; Enzyme Systems
Products, Livermore, CA) to the cell culture 3 hours before adding
NF-B inhibitors, and examined their effects on apoptotic cell death.
Determination of nuclear NF-B activity by
electrophoretic mobility shift assay (EMSA).
Determination of nuclear NF-B activity of Jurkat or JPX-9 cells in
EMSA was examined by the use of Promega Gel Shift Assay System (Promega Co, Madison, WI). In brief, binding reactions of 20 µL total volume contained 7.5 µg of nuclear proteins isolated from
Jurkat or JPX-9 cells, 32P-radiolabeled double-stranded
oligonucleotide containing NF-B binding site
(5'-AGTTGAGGGGACTTTCCCAGGC-3'), and 3 µg of poly(dI-dC) in 10 mmol/L
Tris (pH = 7.5), 50 mmol/L NaCl, 0.5 mmol/L EDTA, 1 mmol/L
MgCl2, 0.5 mmol/L DTT, and 4% glycerol. Reactions were incubated for 30 minutes at room temperature and analyzed by 5% polyacrylamide gel electrophoresis. We used nuclear proteins from the
HTLV-I-infected T-cell line MT-2 as a positive control for nuclear NF-B activity in EMSA.
Enzyme activity assay and Western blot analysis of caspase-3 and
caspase-8.
We measured the enzyme activity of caspase-3 in Jurkat and JPX-9 cells
as described previously.24 In brief, treated Jurkat and
JPX-9 cells were washed 3 times with phosphate-buffered saline (PBS),
and cytosolic extracts were lysed with 100 µL extraction buffer (50 mmol/L HEPES, 50 mmol/L KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2,
1 mmol/L DTT, 20 µmol/L cytochalasin B, 2 mmol/L PMSF, 1 µg/mL
leupeptin, 1 µg/mL pepstatin, 10 µg/mL antipain, 3 µg/mL chymostatin, 1% NP-40, 1 µg/mL DNAase) and sonicated. Cell lysates were then diluted with 0.4 mL caspase-3 standard buffer (100 mmol/L HEPES, 10% Sucrose, 0.1% CHAPS, 10 mmol/L DTT, and 0.1 mg/mL
ovalbumin) and incubated at 30°C for 1 hour with 100 µmol/L
fluorescent substrate, Ac-DEVD-MCA (Peptide Institute). The specific
inhibitor for caspase-1 (Ac-YVAD-CHO, Peptide Institute) was added to
the reaction mixture at a concentration of 100 µmol/L to eliminate a
possible cleavage of the substrate by caspase-1. Specific caspase-3
activity was determined by subtracting the values obtained in the
presence of inhibitor. The fluorescence of the cleaved substrates was
determined using a spectrofluorometer set at excitation/emission
wavelengths of 380/460 nm. One unit corresponds to the activity that
cleaves 1 pmol/min of the respective fluorescent substrate at 30°C.
We also examined the expression of pro-caspase-8 and pro-caspase-3 by
Western blotting as previously described.25,26 These forms
are rapidly converted into the active subunits on the course of
activation.25,26 Thus, the decrease of pro-caspase-8/3 on Western blotting means the activation of
pro-caspase-8/3.25,26 In brief, the cells were washed 3 times with PBS, lysed by the addition of extraction buffer, and
sonicated. The protein concentration in cell extracts was determined by
the Bio-Rad (Melville, NY) protein assay kit. An identical amount of
the protein for each lysate (20 µg/well) was subjected to 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins
were transferred to a nitrocellulose filter, and the filter was blocked
for 1 hour using 5% nonfat dried milk in PBS containing 0.1% Tween 20 (PBS-T), washed with PBS-T, and incubated at room temperature for 1 hour in 1:1,000 dilution of mouse anti-human caspase-3 monoclonal
antibody (MoAb; Transduction Laboratories, Lexington, KY) or mouse
anti-human caspase-8 MoAb (MBL; Nagoya, Japan). These antibodies
preferentially recognize 32 kD of pro-caspase-3 or 55/54 kD of
pro-caspase-8, respectively. The filter was washed with PBS-T and
incubated with 1:1,000 dilution of sheep anti-mouse immunoglobulin-G
(IgG), coupled with horseradish peroxidase. The enhanced
chemiluminescence (ECL) system (Amersham) was used for detection. In
some experiments, we added a caspase-8 inhibitor, IETD-FMK, to the
culture, and also examined the expression of pro-caspase-3. We used
MoAb against -actin (Sigma) for an internal control protein in
Western blot analysis.
Expression of Bcl-2 related proteins and inhibitor of apoptosis
protein (IAP) family proteins in Jurkat and JPX-9 cells.
The expression of Bcl-2 related proteins and IAP family of gene
products is important in regulating certain apoptotic
stimuli.17-19,27,28 Thus, we examined the expression of
Bcl-2, Bax, Bcl-x, XIAP, cIAP1, and cIAP2 in Jurkat and JPX-9 cells by
Western blotting. In brief, cells were collected and lysed by the
addition of lysis buffer (50 mmol/L Tris buffer, pH 8, 150 mmol/L NaCl,
0.02% sodium azaide, 0.1% SDS, 100 µg/mL PMSF, 1 µg/mL of
aprotinin, 1% NP-40, 0.5% sodium deoxycholate) for 20 minutes at
4°C, and insoluble material was removed by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was collected and the
protein concentration was determined by the Bio-Rad (Melville, NY)
protein assay kit. An identical amount of protein for each lysate (20 µg/mL) was subjected to SDS-PAGE. Proteins were transferred to a
nitrocellulose filter and the filter was blocked for 1.5 hours using
5% nonfat dried milk in PBS containing 0.1% Tween 20 (PBS-T), washed
with PBS-T and incubated at room temperature for 1 hour in the presence of each antibody (Bcl-2: mouse monoclonal, DAKO Japan, Kyoto; Bax:
rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA; Bcl-x:
rabbit polyclonal, Transduction Laboratories, Lexington, KY; XIAP:
mouse monoclonal, MBL; cIAP1: rabbit polyclonal, Santa Cruz
Biotechnology; cIAP2: rabbit polyclonal, Santa Cruz Biotechnology). The
filter was washed with PBS-T and incubated with 1:1000 dilution of
sheep anti-mouse IgG or donkey anti-rabbit IgG, coupled with horseradish peroxidase. ECL system (Amersham) was used for detection.
Statistical analysis.
Data were expressed as mean ± SD. Differences between groups were
tested for statistical significance using the Student's t-test. A P value less than .05 was considered significant.
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RESULTS |
Induction of apoptosis of Jurkat and JPX-9 cells by
NF-B inhibitors.
We initially examined the apoptotic cell death of Jurkat and JPX-9
cells induced by PDTC. As shown in Table 1,
PDTC induced apoptosis, determined by flow cytometry, of Jurkat and
JPX-9 cells in the absence of CdCl2. The percentage of
apoptotic cells was similar in Jurkat and JPX-9 cells. The maximal
effect was noted at a PDTC concentration of 50 µmol/L. Similar
results were obtained when apoptosis was quantified by the Hoechst
33258 staining method (data not shown). Based on these initial results,
we used 50 µmol/L of PDTC in the remaining experiments. In addition
to PDTC, NAC also clearly induced apoptosis of Jurkat and JPX-9 cells
(Table 2). Furthermore, the addition of 10 µmol/L of LLL-CHO for 24 hours induced apoptosis in both Jurkat and
JPX-9 cells in the absence of CdCl2 (55.9% ± 3.9% in
Jurkat cells and 56.9% ± 4.3% in JPX-9 cells from 4 experiments
determined by flow cytometry). In contrast, when JPX-9 cells were
preincubated with 30 µmol/L of CdCl2 to induce Tax
expression, a significant suppression of apoptosis of the cells induced
by PDTC was observed. This effect was not detected in Jurkat cells
preincubated with or without CdCl2 or JPX-9 cells without
CdCl2 (Fig 2). Inhibition of
apoptosis occurred after preincubation with CdCl2 after 3 hours, and reached a peak level when preincubated for 6 hours (Fig 2).
Similar results were obtained in CdCl2-pretreated JPX-9
cells when NAC or LLL-CHO was used as a NF-B inhibitor. In the
presence of NAC (50 mmol/L), CdCl2 preincubation decreased
hypodiploid DNA+ cells from 44.5% ± 3.2% to
4.9% ± 0.3%, while in the presence of LLL-CHO (10 µmol/L),
hypodiploid DNA+ cells decreased from 56.5% ± 3.9% to
5.9% ± 0.4% with CdCl2 preincubation (results from 4 experiments). As previously described,6,20 Tax expression
in JPX-9 cells induced by preincubation with CdCl2 for 6 hours was determined by RT-PCR (Fig 1) and Western blot analysis.
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| Fig 2.
Induction of apoptosis in Jurkat and JPX-9 cells by PDTC
determined by flow cytometry. Jurkat and JPX-9 cells were preincubated
with 30 µmol/L of CdCl2 for indicated time intervals,
washed with PBS, and further cultured with 50 µmol/L of PDTC for 24 hours. After cultivation, apoptotic cell death was determined by flow
cytometry as described in the text. Numbers in parentheses are
percentages of cells with hypodiploid DNA. The results are
representative examples of 6 similar experiments.
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Activation of caspase-3 during the apoptotic process induced by
NF-B inhibitors.
We next examined whether activation of caspase-3 is involved in the
apoptotic process induced by NF-B inhibitors. During the process of
caspase-3 activation, the expression of pro-caspase-3 is decreased, and
enzyme activity is increased.25 As shown in Fig
3, the expression of pro-caspase-3 was
significantly decreased during PDTC-induced apoptosis of Jurkat cells
with or without preincubation with CdCl2. In contrast,
preincubation of JPX-9 cells with CdCl2 inhibited the
decrease in expression of pro-caspase-3 (Fig 3). Similar results were
obtained in JPX-9 cells preincubated with CdCl2 when NAC or
LLL-CHO was used (Fig 4). The enzyme
activity of caspase-3 was markedly increased in apoptotic JPX-9 cells
incubated with PDTC in the absence of CdCl2. This increase
was inhibited in JPX-9 cells preincubated with CdCl2 (Fig
5). Similar results were obtained when NAC
or LLL-CHO was used in the experiments instead of PDTC (data not
shown). In addition, the peptide DEVD-CHO inhibited the apoptosis of
Jurkat and JPX-9 cells induced by PDTC (Table
3). The inhibitory effect of DEVO-CHO was
also evident in apoptotis induced by NAC or LLL-CHO. (Apoptosis of
JPX-9 cells by NAC, 45.5% ± 3.4%, decreased to 5.8% ± 0.4%,
and that by LLL-CHO, 56.9% ± 4.1%, decreased to 8.7% ± 0.7%
in the presence of DEVD-CHO. The data are mean ± SD from 4 experiments of hypodiploid DNA+ cells). These data suggest
that while caspase-3 is involved in the apoptotic process induced by
NF-B inactivation, the HTLV-I Tax protein inhibits the activation of
caspase-3.
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| Fig 3.
Expression of pro-caspase-3 in Jurkat and JPX-9 cells
determined by Western blotting. Jurkat and JPX-9 cells were
preincubated with or without CdCl2 for 6 hours. After
washing with PBS, the cells were further cultured with or without 50 µmol/L of PDTC for 24 hours. After cultivation, we examined the
expression of pro-caspase-3 as described in the text. Results are
representative examples of 6 similar experiments. Note that the
expression of pro-caspase-3 in Jurkat cells is significantly decreased
by PDTC irrespective of CdCl2 pretreatment, whereas that of
JPX-9 cells in the presence of PDTC is almost suppressed by
preincubation of the cells with CdCl2.
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| Fig 4.
Activation of caspase-3 in JPX-9 cells by NAC or LLL-CHO,
which is inhibited by preincubation of the cells with
CdCl2. JPX-9 cells were preincubated with or without
CdCl2 for 6 hours. After washing with PBS, the cells were
further cultured with or without 50 mmol/L of NAC or 10 µmol/L of
LLL-CHO for 24 hours. After cultivation, the expression of
pro-caspase-3 was determined as described in the text. Results are
representative examples of 4 similar experiments. Note that the
expression of pro-caspase-3 in CdCl2-untreated JPX-9 cells
is significantly decreased by 2 kinds of NF-B inhibitors, whereas
that is almost inhibited by preincubation of the cells with
CdCl2.
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| Fig 5.
Enzyme activity assay of caspase-3 in JPX-9 cells. JPX-9
cells were preincubated with or without CdCl2 for 6 hours,
and further cultured in the presence or absence of 50 µmol/L of PDTC
for 24 hours. After incubation, the enzyme activity of caspase-3 was
determined as described in the text. Results are representative
examples of 5 similar experiments. Note that caspase-3 enzyme activity
is markedly increased in CdCl2-untreated JPX-9 cells by
PDTC, whereas it is significantly suppressed by preincubation of cells
with CdCl2.
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Sequential activation of caspase-8 to caspase-3 is an essential
signal to induce apoptosis during inhibition of NF-B.
Recent reports have suggested that sequential activation of caspases,
initiating from caspase-8, is essential for the activation of caspase-3
during certain apoptotic stimuli.17,26,29 Thus, we next
examined the involvement of caspase-8 and caspase-1 in the apoptotic
process of Jurkat and JPX-9 cells induced by NF-B inactivation.
Blocking experiments using peptide inhibitors indicated the importance
of caspase-8 in PDTC-induced apoptosis because the caspase-8 inhibitor,
IETD-FMK, significantly abrogated PDTC-induced apoptosis, whereas the
caspase-1 inhibitor, YVAD-CHO, did not affect this process (Table
4). Similar results were obtained in NAC-
or LLL-CHO-induced apoptosis (CdCl2-untreated, and
NAC-treated JPX-9 cells without IETD-FMK contained 42.2% ± 2.2%
of hypodiploid DNA+ cells, which was decreased to
6.1% ± 0.4% by the addition of IETD-FMK.
CdCl2-untreated, and LLL-CHO-treated JPX-9 cells without IETD-FMK contained 56.9 ± 4.4% of hypodiploid DNA+
cells, which was decreased to 8.9 ± 0.5% by IETD-FMK. The results are mean ± SD from four experiments). When we added the caspase-8 inhibitor IETD-FMK to cultures, in which the cells did not undergo apoptosis, the expression of pro-caspase-3 was not decreased (Fig 6), indicating that the activation of
caspase-3 was clearly suppressed by the caspase-8 inhibitor. These
results suggest that caspase-8 is upstream of caspase-3 during the
apoptotic signal induced by inactivation of NF-B. In addition, 2 isoforms of pro-caspase-8 determined by Western blotting26
were significantly decreased in CdCl2-untreated JPX-9 cells
after the addition of PDTC. This phenomenon was significantly
suppressed by CdCl2 pretreatment (Fig
7). These data also suggest that the
inhibitory effect of Tax protein on inactivation of caspase-3 is
mediated through inactivation of caspase-8.
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| Fig 6.
Suppression of the activation of caspase-3 by caspase-8
inhibitor in JPX-9 cells during apoptotic process induced by NF-B
inhibitors. CdCl2-untreated JPX-9 cells were cultured with
or without NF-B inhibitors (PDTC, NAC, or LLL-CHO) in the presence
or absence of caspase-8 inhibitor Z-IETD-FMK for 24 hours. After
incubation, the expression of pro-caspase-3 was determined as described
in the text. Note that the decrease of pro-caspase-3 expression, which
indicates caspase-3 activation, in JPX-9 cells induced by NF-B
inhibitors is almost recovered by the addition of Z-IETD-FMK. Results
are representative examples of 4 similar experiments.
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| Fig 7.
Activation of caspase-8 in JPX-9 cells during
PDTC-induced apoptosis. JPX-9 cells were preincubated with or without
CdCl2 for 6 hours, washed with PBS, and further incubated
with or without PDTC (50 µmol/L) for 24 hours. After incubation, the
expression of procaspase-8 was examined as described in the text. Note
that the decrease of pro-caspase-8 expression, which indicates
caspase-8 activation, was almost recovered even in the presence of PDTC
by preincubation of the cells with CdCl2. Results are
representative examples of 5 similar experiments.
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We confirmed the relationship between NF-B inactivation and
activation of caspase cascade by the use of EMSA and kinetic studies of
caspase activation. EMSA showed that small amount of nuclear NF-B
activity was present in CdCl2-untreated JPX-9 cells. The
treatment of these cells with NF-B inhibitors for 3 hours mostly
abrogated NF-B nuclear translocation (Fig
8A). Both flow cytometric analysis and
Hoechst 33258 dye staining showed that these cells (treated with
NF-B inhibitors for 3 hours) did not undergo apoptosis (data not
shown). However, CdCl2-treated JPX-9 cells contained a
significant amount of nuclear NF-B, and substantial NF-B nuclear
activity still remained after the addition of NF-B inhibitors in
these cells (Fig 8B). Nuclear NF-B activity of Jurkat cells was not
increased by CdCl2 (Fig 8C). Kinetic studies of the
expression of pro-caspase-8 or -3 suggested that both activation of the
caspase cascade and induction of apoptosis was operated after the
inhibition of NF-B nuclear translocation because the decrease of
pro-caspase protein expression was not seen until 24 hours after
incubation with PDTC (Fig 9).
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| Fig 8.
Nuclear NF-B activity determined by EMSA. Nuclear
proteins of JPX-9 cells treated with (B; CdCl2 treatment
for 6 hours) or without CdCl2 (A) in the presence or
absence of NF-B inhibitors, was incubated with radiolabeled
oligonucleotide containing NF-B binding site, and nuclear NF-B
activity was examined by EMSA as described in the text. (A) Nuclear
NF-B activity in CdCl2-untreated JPX-9 cells. Lane A;
positive control (MT-2 cells). Lane B; CdCl2-untreated
JPX-9 cells without NF-B inhibitors. Lane C;
CdCl2-untreated JPX-9 cells, and then cultured in 50 µmol/L of PDTC for 3 hours. Lane D; CdCl2-untreated JPX-9
cells, and then cultured in 50 mmol/L of NAC for 3 hours. Lane E;
CdCl2-untreated JPX-9 cells, and then cultured in 10 µmol/L of LLL-CHO for 3 hours. Note that basal nuclear NF-B
activity in CdCl2-untreated JPX-9 cells was clearly suppressed by 3 kinds of NF-B inhibitors. (B) Nuclear NF-B activity in
CdCl2-treated JPX-9 cells. Lane A; control
(CdCl2-untreated JPX-9 cells without NF-B inhibitors).
Lane B; CdCl2-treated JPX-9 cells (without NF-B
inhibitors). Lane C; CdCl2-treated JPX-9 cells, and then
cultured in 50 µmol/L of PDTC for 3 hours. Lane D;
CdCl2-treated JPX-9 cells, and then cultured in 50 mmol/L
of NAC for 3 hours. Lane E; CdCl2-treated JPX-9 cells, and
then cultured in 10 µmol/L of LLL-CHO for 3 hours. Lane F;
CdCl2-treated JPX-9 cells, and then cultured for 3 hours
without NF-B inhibitors. Note that nuclear NF-B in JPX-9 cells
was clearly augmented by CdCl2 treatment, and its activity
is still high in the presence of 3 kinds of NF-B inhibitors. (C)
Nuclear NF-B activity of Jurkat cells was not augmented by
incubation of the cells with CdCl2. Lane A; Jurkat cells
without CdCl2 treatment. Lane B; Jurkat cells treated with
CdCl2 for 6 hours. Results (Fig 8A through C) are
representative of 5 experiments.
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| Fig 9.
Time kinetic study of activation in caspase-8 and
caspase-3 during PDTC-induced apoptosis. CdCl2-untreated
JPX-9 cells were cultured for indicated hours in the presence of 50 µmol/L of PDTC. After incubation, the expression of pro-caspase-8 (A)
and procaspase-3 (B) was examined by Western blotting. Note that the
decrease of pro-caspase protein expression was found at 24 hours
incubation of JPX-9 cells with PDTC, whereas that was not determined at
3 hours incubation of the cells with PDTC in which nuclear NF-B
activity had already been suppressed. Results are representative of 4 experiments.
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Expression of Bcl-2 related proteins and IAP family proteins in
Jurkat and JPX-9 cells.
Bcl-2 related proteins, such as Bcl-2, Bax, and Bcl-x, are involved in
the regulation of apoptotic process.17,27 Therefore, we
investigated changes in the expression of Bcl-2, Bax, and Bcl-x on
Jurkat and JPX-9 cells by Western blot analysis. There was no
significant change of Bcl-2 expression in both Jurkat and JPX-9 cells
with or without of CdCl2 or NF-B inhibitors (Fig
10) although the amount of Bcl-2 in
Jurkat cells was higher than that in JPX-9 cells. In addition, the
expression of Bax and Bcl-x was not different in both Jurkat and JPX-9
cells under these culture conditions (data not shown). Expression of
IAP family proteins was also examined. As shown in Fig
11, XIAP expression in
CdCl2-untreated JPX-9 cells almost disappeared after the
addition of NF-B inhibitors. However, XIAP expression in
CdCl2-treated JPX-9 cells was maintained even after
addition of NF-B inhibitors. cIAP2 expression in JPX-9 cells was not
changed in these culture conditions (Fig 11), and cIAP1 expression was
not detected in Jurkat or JPX-9 cells (data not shown).
View larger version (45K):
[in this window]
[in a new window]
| Fig 10.
Bcl-2 expression in Jurkat and JPX-9 cells. (A) Jurkat
and JPX-9 cells were preincubated with or without CdCl2 for
6 hours, washed with PBS, and further incubated with or without PDTC
(50 µmol/L) for 24 hours. After incubation, the expression of Bcl-2
was examined as described in the text. (B) JPX-9 cells were
preincubated with or without CdCl2 for 6 hours, washed with
PBS, and further incubated with or without NAC (50 mmol/L) or LLL-CHO
(10 µmol/L) for 24 hours. After incubation, the expression of Bcl-2
was examined as described in the text. Note that the expression of
Bcl-2 in Jurkat or JPX-9 was not different in each culture condition.
Results are representative examples of 5 similar experiments.
|
|
View larger version (28K):
[in this window]
[in a new window]
| Fig 11.
Expression of XIAP and cIAP2 in JPX-9 cells. JPX-9
cells were preincubated with or without CdCl2 for 6 hours,
washed with PBS, and further incubated with or without PDTC (50 µmol/L), NAC (50 mmol/L), or LLL-CHO (10 µmol/L) for 24 hours.
After incubation, the expression of XIAP and cIAP2 was examined as
described in the text. Note that XIAP expression in
CdCl2-untreated JPX-9 cells is almost suppressed by NF-B
inhibitors, however, its expression in CdCl2-treated JPX-9
cells is maintained even in the presence of NF-B inhibitors. In
contrast, cIAP2 expression in JPX-9 cells was not changed in each
culture condition. Results are representative examples of 4 similar
experiments.
|
|
| |
DISCUSSION |
The apoptotic process involves a complex machinery regulated by
molecular interactions of various gene products.12-19,22,28 One of the major gene products that induce apoptosis is the caspase family, which is conserved from nematodes to mammals.17 In
humans, more than 10 such proteins are divided in 3 subfamilies.17 Among these, a sequential activation of
caspase-8 to caspase-1, to caspase-3 is one of the major cascades
involved in the induction of apoptotic cell death including signaling
through Fas and TNFR.17,26,29 In contrast, a
major molecule that inhibits apoptosis is the nuclear transcription
factor NF-B.12-19 Binding of TNF to TNFR stimulates both
the caspase cascade and NF-B nuclear translocation. In this model,
TNF-induced cytotoxicity increases when NF-B activation is
inhibited,12-19 suggesting a molecular interaction between
caspases and NF-B. Thus, we initially examined whether inactivation
of NF-B induces sequential activation of the caspase cascade,
resulting in the induction of apoptosis.
Our results showed that both Jurkat and JPX-9 cells underwent apoptosis
in the presence of NF-B inhibitors, without the addition of
CdCl2. As suggested in the recent study that both the
cleavage of poly(ADP-ribose) polymerase (PARP) and induction of
apoptosis in HuT-78 cells was induced by NF-B
inactivation,30 a significant activation of caspase-3 in
the cells was noted as determined by Western blotting and enzyme
activity assay during this process. Furthermore, the addition of
DEVD-CHO, a caspase-3 inhibitor, abrogated apoptotic cell death induced
by NF-B inhibitors, suggesting that caspase-3 is a major effector
caspase to induce apoptosis when NF-B nuclear translocation is
inhibited. We next examined whether caspase-8 and caspase-1, caspases
located upstream of caspase-3, are involved during an apoptotic process
induced by NF-B inhibitors. According to the results using peptide
inhibitors, the involvement of not caspase-1 but caspase-8 is apparent
because the caspase-1 inhibitor YVAD-CHO did not inhibit the apoptotic process. However, the caspase-8 inhibitor IETD-FMK significantly suppressed apoptosis induced by NF-B inhibitors. The evidence that
caspase-1 is not involved in apoptotic process has been reported in
Fas-mediated apoptosis of synovial cells.31 Western blot analysis showed that pro-caspase-8 expression is markedly decreased when we added NF-B inhibitors to the cells, indicating that
activation of caspase-8 is an essential event in the induction of
apoptosis by NF-B inhibition. The caspase-8 inhibitor IETD-FMK
inhibited the activation of caspase-3 in CdCl2-untreated
JPX-9 cells induced by NF-B inhibitors, while caspase-8 activation
in JPX-9 cells by NF-B inhibitors was clearly suppressed in the
cells pretreated with CdCl2. These data strongly suggest
that sequential activation from caspase-8 to caspase-3 is a central
apoptotic pathway in the process of apoptosis induced by inactivation
of NF-B.
What are the molecular mechanisms of HTLV-I Tax protein that inhibit
the activation of caspase cascade? EMSA showed that the treatment of
CdCl2-untreated JPX-9 cells with NF-B inhibitors clearly
suppressed basal nuclear NF-B activity of the cells, whereas nuclear
NF-B activity in JPX-9 cells was significantly augmented by
CdCl2. Furthermore, residual NF-B activity in
CdCl2-treated JPX-9 cells was still high even with the
addition of NF-B inhibitors. These data indicate that nuclear
NF-B activity is a principal factor to suppress the activation of
caspase cascade initiated from caspase-8, and Tax-mediated inhibition
of caspase cascade is mediated through hyperactivation of NF-B in
the cells. However, a kinetic study (Fig 9) suggests that gene products
regulated by NF-B modulate the activation of caspase cascade because
the activation of the caspase cascade was followed by inhibition of NF-B nuclear translocation. Recent reports have suggested that the
expression of IAP family proteins is regulated by NF-B, and their
expression in the cells is important to inhibit the activation of
caspase-3.18,19 We showed in the present study that the expression of XIAP in CdCl2-untreated JPX-9 cells was
clearly suppressed by NF-B inhibitors. In contrast, its expression
in CdCl2-treated JPX-9 cells was maintained even with the
addition of NF-B inhibitors, suggesting an importance of
Tax-mediated NF-B activation in XIAP expression in the cells. cIAP1
expression was not detected, and the expression of cIAP2 was not
changed with or without CdCl2 or NF-B inhibitors.
Furthermore, the expression of Bcl-2, Bax, and Bcl-x was also not
changed in Jurkat and JPX-9 cells with or without CdCl2 or
NF-B inhibitors. Therefore, we speculate that Tax-mediated
anti-apoptotic effect is accomplished, in part, through XIAP
expression, which is regulated by NF-B. Recent experiments suggest
that while XIAP directly inhibits the activation of caspase-3, it does
not prevent the caspase-8-induced proteolytic activation of
caspase-3.28 Because Tax-mediated anti-apoptotic effects
appear to act on caspase-8, anti-apoptotic molecules other than XIAP
such as FLICE-inhibitory protein32 may also be responsible
for this process.
In conclusion, we showed the inhibitory effect of HTLV-I Tax in the
activation of caspase cascade and suggest a role for Tax in the
prevention of apoptosis. The lack of apoptotic cell death in
lpr and in gld mice causes various autoimmune disorders
by the inability to eliminate self-reactive T cells.33,34
Various kinds of viruses encode anti-apoptotic proteins such as an E1B for an adenovirus35 and BHRF1 for an Epstein-Barr
virus,36 and transgenic mice overexpressing Tax protein
develop diseases quite similar to SS and RA.37,38 Although
the precise molecular mechanisms in the process are not fully
understood at present, our present data may imply new insight to why
autoimmune disorders are developed in HTLV-I seropositive subjects.
Perhaps, viral protein-mediated inhibition of apoptosis could also be
involved in autoimmune disorders induced by viruses other than HTLV-I.
| |
ACKNOWLEDGMENT |
We thank Prof Sugamura for providing us Jurkat and JPX-9 cells. We also
thank Y. Matsuo, E. Nogami, and N. Fukuda for their excellent technical
assistance. We also thank Dr F.G. Issa, Sydney, Australia, for the
careful reading and editing of the manuscript.
| |
FOOTNOTES |
Submitted June 6, 1998; accepted July 14, 1999.
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 Atsushi Kawakami, MD, The First Department
of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.
| |
REFERENCES |
1.
Wano Y, Hattori T, Matsuoka M, Takatsuki K, Chua AO, Gunler U, Greene WC:
Interleukin 1 gene expression in adult T-cell leukemia.
J Clin Invest
80:911, 1987
2.
Maruyama M, Shibuya H, Harada H, Hatakeyama M, Seiki M, Fujita T, Inoue J, Yoshida M, Taniguchi T:
Evidence for aberrant activation of the interleukin-2 autocrine loop by HTLV-I-encoded p40x and T3/Ti complex triggering.
Cell
48:343, 1987[Medline]
[Order article via Infotrieve]
3.
Siekevitz M, Feinberg MB, Holbrook N, Wong-Staal F, Greene WC:
Activation of interleukin-2 and interleukin-2 receptor (Tac) promotor expression by the trans-activator (tat) gene product of human T-cell leukemia virus type I.
Proc Natl Acad Sci USA
84:5389, 1987[Abstract/Free Full Text]
4.
Sakai M, Eguchi K, Terada K, Nakashima M, Yamashita I, Ida H, Kawabe Y, Aoyagi T, Takino H, Nakamura T, Nagataki S:
Infection of human synovial cells by human T-cell lymphotropic virus Type I; proliferation and granulocyte/macrophage colony-stimulating factor production by synovial cells.
J Clin Invest
92:1957, 1993
5.
Baeuerle PA, Henkel T:
Function and activation of NF-B in the immune system.
Annu Rev Immunol
12:141, 1994[Medline]
[Order article via Infotrieve]
6.
Yamashita I, Katamine S, Moriuchi R, Nakamura Y, Miyamoto T, Eguchi K, Nagataki S:
Transactivation of the human interleukin-6 gene by human T-lymphotropic virus type 1 Tax protein.
Blood
84:1573, 1994[Abstract/Free Full Text]
7.
Osame M, Matsumoto M, Usuku K, Izumo S, Ijichi N, Amitani H, Tara M, Igata A:
Chronic progressive myelopathy associated with elevated antibodies to human T-lymphotropic virus type I and adult T-cell leukemia-like cells.
Ann Neurol
21:117, 1987[Medline]
[Order article via Infotrieve]
8.
Eguchi K, Matsuoka N, Ida H, Nakashima M, Sakai M, Sakito S, Kawakami A, Terada H, Shimada H, Kawabe Y, Fukuda T, Sawada T, Nagataki S:
Primary Sjögren's syndrome with antibodies to HTLV-I: Clinical and laboratory features.
Ann Rheum Dis
51:769, 1992[Abstract/Free Full Text]
9.
Terada K, Katamine S, Eguchi K, Moriuchi R, Kita M, Shimada H, Yamashita I, Iwata K, Tsuji Y, Nagataki S, Miyamoto T:
Prevalence of serum and salivary antibodies to HTLV-I in Sjögren's syndrome.
Lancet
344:1116, 1994[Medline]
[Order article via Infotrieve]
10.
Eguchi K, Origuchi T, Takashima H, Iwata K, Katamine S, Nagataki S:
High seroprevalence of anti-HTLV-I antibody in rheumatoid arthritis.
Arthritis Rheum
39:463, 1996[Medline]
[Order article via Infotrieve]
11.
Nakamura H, Eguchi K, Nakamura T, Mizokami A, Shirabe S, Kawakami A, Matsuoka N, Migita K, Kawabe Y, Nagataki S:
High prevalence of Sjögren's syndrome in patients with HTLV-I associated myelopathy.
Ann Rheum Dis
56:167, 1997[Abstract/Free Full Text]
12.
Beg AA, Baltimore D:
An essential role for NF-B in preventing TNF--induced cell death.
Science
274:782, 1996[Abstract/Free Full Text]
13.
Liu Z-G, Hsu H, Goeddel D, Karin M:
Dissection of TNF receptor 1 effector function: JNK activation is not linked to apoptosis while NF-B activation prevents cell death.
Cell
87:565, 1996[Medline]
[Order article via Infotrieve]
14.
Wang C-Y, Mayo MW, Balwin Jr AS:
TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-B.
Science
274:784, 1996[Abstract/Free Full Text]
15.
Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM:
Suppression of TNF--induced apoptosis by NF-B.
Science
274:787, 1996[Abstract/Free Full Text]
16.
Baeuerle PA, Baltimore D:
NF-B: Ten years after.
Cell
87:13, 1996[Medline]
[Order article via Infotrieve]
17.
Nagata S:
Apoptosis by death factor.
Cell
88:355, 1997[Medline]
[Order article via Infotrieve]
18.
Wang C-Y, Mayo MW, Korneluk RG, Goeddel DV, Baldwin Jr AS:
NF-B antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.
Science
281:1680, 1998[Abstract/Free Full Text]
19.
Stehlik C, Martin R, Kumabayashi I, Schmid JA, Binder BR, Lipp J:
Nuclear factor (NF)-B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor -induced apoptosis.
J Exp Med
188:211, 1998[Abstract/Free Full Text]
20.
Hiraiwa N, Hiraiwa M, Kannagi R:
Human T-cell leukemia virus-1 encoded Tax protein transactivates 1  3 fucosyltransferase Fuc-T VII, which synthesizes sialyl lewis X, a selectin ligand expressed on adult T-cell leukemia cells.
Biochem Biophys Res Commun
231:183, 1997[Medline]
[Order article via Infotrieve]
21.
Ozaki K, Takeda H, Iwahashi H, Kitano S, Hanazawa S:
NF-B inhibitors stimulate apoptosis of rabbit mature osteoclasts and inhibit bone resorption by these cells.
FEBS Lett
410:297, 1997[Medline]
[Order article via Infotrieve]
22.
Miagkov AV, Kovalenko DV, Brown CE, Didsbury JR, Cogswell JP, Stimpson SA, Baldwin AS, Makarov SS:
NF-B activation provides the potential link between inflammation and hyperplasia in the arthritic joint.
Proc Natl Acad Sci USA
95:13859, 1998[Abstract/Free Full Text]
23.
Kawakami A, Eguchi K, Matsuoka N, Tsuboi M, Koji T, Urayama S, Fujiyama K, Kiriyama T, Nakashima T, Nakane PK, Nagataki S:
Fas and Fas ligand interaction is necessary for human osteoblast apoptosis.
J Bone Miner Res
12:1637, 1997[Medline]
[Order article via Infotrieve]
24.
Enari M, Talanian RV, Wong WW, Nagata S:
Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.
Nature
380:723, 1996[Medline]
[Order article via Infotrieve]
25.
Cui H, Matsui K, Omura S, Schauer SL, Matulka RA, Sonenshein GE, Ju S-T:
Proteasome regulation of activation-induced T cell death.
Proc Natl Acad Sci USA
94:7515, 1997[Abstract/Free Full Text]
26.
Scaffidi C, Medema JP, Krammer PH, Peter ME:
FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b.
J Biol Chem
272:26953, 1997[Abstract/Free Full Text]
27.
Boussiotis VA, Lee BJ, Freeman GJ, Gribben JG, Nadler LM:
Induction of T-cell clonal anergy results in resistance, whereas CD28-mediated costimulation primes for susceptibility to Fas- and Bax-mediated programmed cell death.
J Immunol
159:3156, 1997[Abstract]
28.
Deveraux QL, Roy N, Stennicke HR, Arsdale TV, Zhou Q, Srinivasula SM, Alnemri ES, Salvesen GS, Reed JC:
IAPs block apoptotic events by caspase-8 and cytochrome c by direct inhibition of distinct caspases.
EMBO J
17:2215, 1998[Medline]
[Order article via Infotrieve]
29.
Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH, Peter ME:
FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).
EMBO J
16:2794, 1997[Medline]
[Order article via Infotrieve]
30.
Giri DK, Aggarwal BB:
Constitutive activation of NF-B causes resistance to apoptosis in human cutaneous T-cell lymphoma HuT-78 cells: Autocrine role of tumor necrosis factor and reactive oxygen intermediates.
J Biol Chem
273:14008, 1998[Abstract/Free Full Text]
31.
Sekine C, Yagita H, Kobata T, Hasunuma T, Nishioka K, Okumura K:
Fas-mediated stimulation induces IL-8 secretion by rheumatoid arthritis synoviocytes independently of CPP32-mediated apoptosis.
Biophys Biochem Res Commun
228:14, 1996
32.
Yeh J-H, Hsu S-C, Han S-H, Lai M-Z:
Mitogen-activated protein kinase kinase antagonized Fas-associated death domain protein-mediated apoptosis by induced FLICE-inhibitory protein expression.
J Exp Med
188:1795, 1998[Abstract/Free Full Text]
33.
Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S:
Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.
Nature
356:314, 1992[Medline]
[Order article via Infotrieve]
34.
Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata S:
Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.
Cell
76:969, 1994[Medline]
[Order article via Infotrieve]
35.
White E, Sabbatini P, Debbas M, Wold WS, Kusher DI, Gooding LR:
The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha.
Mol Cell Biol
12:2570, 1992[Abstract/Free Full Text]
36.
Henderson S, Huen D, Rowe M, Dawson C, Johnson G, Rickinson A:
Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death.
Proc Natl Acad Sci USA
90:8479, 1993[Abstract/Free Full Text]
37.
Green JE, Hinrichs SH, Vogel J, Jay G:
Exocrinopathy resembling Sjögren's syndrome in HTLV-I tax transgenic mice.
Nature
341:72, 1989[Medline]
[Order article via Infotrieve]
38.
Iwakura Y, Tosu M, Yoshida E, Takiguchi M, Sato K, Kitajima I, Nishioka K, Yamamoto K, Takeda T, Hatanaka M, Yamamoto H, Sekiguchi T:
Induction of inflammatory arthropathy resembling rheumatoid arthritis in mice transgenic for HTLV-I.
Science
253:1026, 1991[Abstract/Free Full Text]
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TOP
ABSTRACT
INTRODUCTION