|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 241-251
Interleukin-6-Induced Inhibition of Multiple Myeloma Cell
Apoptosis: Support for the Hypothesis That Protection Is
Mediated Via Inhibition of the JNK/SAPK Pathway
By
Feng-hao Xu,
Sanjesh Sharma,
Agnes Gardner,
Yiping Tu,
Arthur Raitano,
Charles Sawyers, and
Alan Lichtenstein
From the Department of Medicine, West LA VA Medical Center, and
Jonsson Comprehensive Cancer Center, Los Angeles; and the Department of
Medicine, UCLA Medical Center, Los Angeles, CA.
 |
ABSTRACT |
The mechanism by which interleukin-6 (IL-6) protects multiple
myeloma (MM) plasma cells from apoptosis induced by
anti-fas antibodies and dexamethasone was studied.
Anti-apoptotic concentrations of IL-6 had no effect on cell-cycle
distribution or activation of RAF-1 or ERK in dexamethasone- or
anti-fas-treated 8226 and UCLA #1 MM cell lines. However,
IL-6-dependent protection of viability correlated with an inhibition
of dexamethasone- and anti-fas-induced activation of
jun kinase (JNK) and AP-1 transactivation. To test the
hypothesis that cytokine-induced protection was mediated through inhibition of JNK/c-jun, we also inhibited c-jun
function in 8226 cells via introduction of a mutant dominant negative
c-jun construct. Mutant c-jun-containing MM cells were
also resistant to anti-fas-induced apoptosis but were
significantly more sensitive to dexamethasone-induced apoptosis. These
results support the notion that IL-6 protects MM cells against
anti-fas through its inhibitory effects on JNK/c-jun but indicate protection against dexamethasone occurs through separate, yet unknown pathways.
| |
INTRODUCTION |
INTERLEUKIN-6 (IL-6) is a well-known
growth factor for multiple myeloma (MM) plasma cells. This cytokine is
capable of inhibiting plasma cell apoptosis1,2 as well as
stimulating some MM cell types to proliferate.3,4 Some
experimental evidence indicates these two cytokine-dependent effects
are dissociable and, thus, likely mediated by distinct
mechanisms.2 Because the predominant effect of IL-6 in
nontransformed B-lineage cells is that of inducing differentiation,5 a process which normally results in
apoptosis of the terminally differentiated plasma cell, there must be
inherent differences in IL-6 signaling in malignant plasma cells which result in continued cell growth and viability. Elucidation of these
signaling pathways and differences may, thus, provide clues for future
therapeutic manipulations that could be relatively selective for the
malignant plasma cell clone while sparing normal B cells.
We have been actively studying the potential signal transduction
pathways used by IL-6 to inhibit apoptosis in MM plasma cells. After
receptor binding to IL-6 and homodimerization of gp 130, the
signal-transducing molecule of this cytokine, there are two major
pathways activated6: (1) Activation of JAK and TYK kinases
followed by tyrosine phosphorylation of STAT 3 and, to a lesser extent,
STAT 1 transcription factors; and (2) activation of ras by
exchange factors such as SOS with subsequent activation of kinase
cascades involving mitogen-activated protein kinases (MAPKs). Of these
two major pathways, the latter MAPK cascades have been implicated as
important regulatory pathways in several other in vitro models of
apoptosis.7-11
One of these MAPK pathways is mediated by sequential activation of
RAS/RAF/MEK/ and ERK.12 The ERKS (1 and 2) are then capable of activating transcription factors such as ELK-1/SAP-1.13
More recently, a second MAPK pathway has been detected that involves JUN N-terminal kinase (JNK) or stress-activated protein kinase (SAPK).14,15 Activation of JNK/SAPK in this pathway is also dependent on prior ras activation14,16 and the
signaling proceeds through a kinase cascade involving MEKK and SEK1 in
an analagous fashion to the RAF/MEK/ERK cascade.17 Through
phosphorylation, JNK activates the transcription factors c-jun
and ATF-2.18,19 Dimerization of c-jun with other
jun family members or with c-fos leads to formation of
the AP-1 transcriptional activating complex. Thus, subsequent to
receptor triggering, activated ras appears to direct signals
into one or both kinase pathways, either RAF/MEK/ERK or MEKK/SEK1/JNK.
The relative activation of each pathway versus the other may ultimately
influence the outcome of receptor triggering. For example, the balance
between ERK and JNK/SAPK pathway signaling is critical for determining
whether neuronal cells survive or undergo apoptosis.7
Recent work by Chauhan et al20 showed an activation of SAPK
by anti-fas during apoptosis of MM plasma cells. The ability of
IL-6 to protect against anti-fas-induced apoptosis correlated with its ability to inhibit anti-fas-induced SAPK activation. Because experiments with neuronal cells7 indicate
anti-apoptotic influences could be exerted by activation of the ERK
pathway and/or inhibition of the JNK pathway, these latter
studies with MM cells suggested that differential activation/inhibition
signals through the two parallel MAPK cascades might regulate
IL-6-induced protection of MM plasma cells against apoptosis. However,
while showing a correlation, the study of Chauhan et al20
did not prove a causal relationship between IL-6-dependent protection
and inhibition of the JNK pathway. Thus, to further investigate this
issue, we also studied the effects of IL-6 on both MAPK pathways in MM
cells that were protected against apoptosis induced by dexamethasone and anti-fas antibody. In similar fashion to the work of
Chauhan et al,20 we also found that JNK activity and
subsequent c-jun transactivation induced during MM cell
apoptosis was inhibited by protective concentrations of IL-6. To test
whether these specific inhibitory effects of IL-6 were crucial to
protection of MM cell viability, we introduced a dominant negative
c-jun into MM cells, which resulted in inhibition of
jun activity induced by anti-fas or dexamethasone which
was comparable to the inhibitory effects of IL-6. These dominant
negative c-jun containing MM cells were resistant to apoptosis
induced by anti-fas but not by dexamethasone. These data
support the hypothesis that IL-6 protects MM cells from
anti-fas through its inhibition of c-jun activity but
also indicate that protection against dexamethasone proceeds along separate pathways.
| |
MATERIALS AND METHODS |
Cell lines.
The myeloma cell line 8226 was a kind gift from J. Epstein (Little
Rock, AR). AF-10 MM cells were a kind gift of James Berenson (UCLA, Los
Angeles, CA). UCLA #1 MM cells is a cell line started from the
peripheral blood of a patient with plasma cell leukemia. It has the
morphology of plasma cells, and expresses high amounts of CD38 as well
as the identical Ig isotype of the patient's M protein. Both lines
were maintained in RPMI media, supplemented with 10% fetal bovine
serum, L-glutamine, nonessential amino acids, sodium pyruvate, and
antibiotics.
Reagents.
Human recombinant IL-6 was from R&D Labs (Minneapolis, MN). Anti-MAPK
and anti-RAF-1 antibodies were purchased from Santa Cruz Biotech Inc
(Santa Cruz, CA). Antiphosphotyrosine antibody was obtained from UBI
(Lake Placid, NY). Dexamethasone and myeline basic protein (MBP) was
purchased from Sigma (St Louis, MO).  -32P-ATP was
obtained from Amersham Labs (Arlington Heights, IL). Anti-fas
antibody was obtained from Kamiya Inc (Thousand Oaks, CA). All other
chemicals were obtained from Sigma Labs.
Induction of apoptosis.
Cells were treated with anti-fas antibody or a control antibody
of the same isotype. When dexamethasone was used to induce apoptosis,
controls contained identical concentrations of alcohol (always
<0.1%).
Immunoblotting.
MM cells were stimulated with or without IL-6 or with PMA for 5 to 15 minutes. Cells were then lysed in lysis buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% NP40, 1 mmol/L
Na3VO4, 10 mmol/L NaF, 2 mmol/L
phenylmethylsulfonyl fluoride [PMSF], 0.5 mmol/L EDTA, 10 µg/mL
leupeptin, and 10 µg/mL aprotinin). One milligram of protein of each
sample was then precipitated with 5 µg of antiphosphotyrosine antibody at 4°C with constant shaking for 1 hour, followed by addition of 35 µL of protein A-Sepharose and protein G-Sepharose. After another 1-hour reaction, the immunocomplexes were washed once
with lysis buffer, twice with washing buffer (same as lysis buffer
except NP40 was decreased to 0.1% and EDTA was removed), and the
samples were boiled with 30 µL of sodium dodecyl sulfate (SDS)-sample
buffer and resolved on 10% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). The separated samples were transferred onto membranes and
blotted with anti-MAPK or antiphosphotyrosine antibodies. The bands
were detected by an enhanced chemiluminescence (ECL)
system.
In vitro kinase assay for MAP kinase activity.
The assay was performed as previously described.21 Briefly,
cells were treated for 5 to 15 minutes, were washed twice, and lysed in
lysis buffer (20 mmol/L Tris, pH 7.4, 2 mmol/L MgCl2, 10 mmol/L  -glycerophosphate, 10 mmol/L p-nitrophenylphosphate, 1 mmol/L
EGTA, 0.1 mmol/L Na3VO4, 10 mmol/L NaF, 0.5%
Triton X-100, and 10 µg/mL aprotinin). Five micrograms of each lysate
was incubated with 5 micrograms of MBP in kinase buffer (20 mmol/L
HEPES, pH 7.6, 20 mmol/L MgCl2, 10 mmol/L
-glycerophosphate, 20 mmol/L p-nitrophosphate, 0.5 mmol/L
Na3VO4, 2 mmol/L dithiothreitol [DTT], 50 µmol/L ATP0 plus 20 µCi of -32P-ATP) for 30 minutes
at room temperature. SDS-sample buffer was then added and the samples
boiled for 3 minutes. Samples were separated on SDS-PAGE. After
separation, the gel was dried and exposed on film at  70°C
overnight.
In vitro kinase for jun kinase activity.
The GST-jun vector was a kind gift of M. Karin (San Diego, CA).
The GST-jun substrate was expressed in bacteria and then
purified with glutathione (GSH)-beads. The purity and
semi-quantitation of the substrate was monitored by SDS-PAGE. Cells
were pretreated with IL-6 for increasing durations and then stimulated
with anti-fas or dexamethasone. They were then lysed in JEB
buffer (25 mmol/L HEPES, pH 7.7, 300 mmol/L NaCl, 1.5 mmol/L
MgCl2, 0.1 mmol/L EDTA, 0.1% Triton X-100, 20 mmol/L -glycerophosphate, 0.1 mmol/L Na3VO4, 0.5 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin), the
lysates were centrifuged at 14,000 rpm for 10 minutes, and the
supernatants saved. Twenty-five microliters of GST-jun-agarose was then added, followed by rocking at 4°C for 2 hours. The
mixtures were washed twice in HBIB buffer (20 mmol/L HEPES, pH 7.7, 50 mmol/L NaCl, 0.1 mmol/L EDTA, 2.5 mmol/L MgCl2, and 0.05%
Triton X-100). Kinase buffer was then added (25 µL of 0.5 µCi
32P-ATP, 20 µmol/L ATP, 20 mmol/L MgCl2,
20 mmol/L HEPES, pH 7.6, 20 mmol/L -glycerophosphate, 20 mmol/L
PNPP, 0.1 mmol/L Na3VO4, and 2 mmol/L DTT) and
the mixtures were incubated at 30°C for 30 minutes. The mixtures
were then resolved on an 8% SDS-PAGE, and the gel was dried and
exposed on film.
In some experiments, lysates were first incubated with specific
antibody to SAPK/JNK (Santa Cruz Biotechnology) for 2 hours at 4°C
before adding protein A-Sepharose for 1 hour. Immune complexes were
then washed with JEB buffer, followed by kinase buffer and then
resuspended in kinase buffer containing GST-jun and
32P-ATP.
Immune complex kinase assay for RAF-1 activity.
The assay was performed as previously described.22 Briefly,
cells were lysed in 25 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.1%
SDS, 0.5% Na-deoxycholate, 1% NP40, 10% glycerol, 2 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L PMSF, 20 µmol/L
leupeptin, and 5 µg/mL aprotinin. Raf-1 was then immunoprecipitated
using protein A-Sepharose preadsorbed with anti-RAF-1 antibody and the
complex was washed twice in 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton-X-100, 10% glycerol, 2 mmol/L EDTA, 1 mmol/L
Na3VO4, and protease inhibitors. A final wash
in kinase buffer (25 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 25 mmol/L
glycerol phosphate, 1 mmol/L DTT, 5 mmol/L MgCl2) was
performed before incubation in 30 µL of kinase buffer containing
purified recombinant MEK protein (generated from GST-MEK vector), 10 µmol/L ATP, and 20 µCi 32P-ATP for 30 minutes at
room temperature. Samples were centrifuged at 16,000g for 1 minute and the supernatant containing MEK and the pellet containing
RAF-1 immune complexes were separated on SDS-PAGE. Phosphorylated MEK
was detected by autoradiography in the dried gel and RAF-1 was
transferred to nitrocellulose and detected by immunoblotting.
Detection of apoptosis.
DNA electrophoresis was performed as previously described.2
After extraction in PCI, 5 to 10 µg of DNA per lane was
electrophoresed in a 1% agarose gel for 2 hours at 45 V and the gels
were visualized with ethidium bromide. Viability was determined by dye
exclusion assays using trypan blue. Briefly, cells were seeded at a
concentration of 2 to 4 × 105/mL in six-well tissue
culture plates and drugs were added at the designated time points. At
specified incubation times, cell viability was determined by trypan
blue staining and percent viability was determined in at least 300 cells. Triplicate wells were run for each group and the standard
deviation of the groups was always less than 5% of the mean viability.
The percent apoptotic nuclei was determined by
4 ,6-diamidine-2-phenylindole dihydrochloride (DAPI)
staining. Cells were first fixed with 3.7% formaldehyde in
phosphate-buffered saline (PBS) at room temperature for 10 minutes and
then washed with PBS. Fixed cells were then stained with 1 µg/mL DAPI
in PBS at room temperature for 15 minutes. After washing three times,
cells were resuspended in glycerol:PBS (10:1) and were mounted onto
glass slides and covered with a coverslip. The slide was examined under
400× magnification using a fluorescent microscope with a 340/380
nm excitation filter and LP 430-nm barrier filter. At least 300 nuclei
were examined per group. The diphenylamine DNA fragmentation assay was
performed by first lysing cells in 0.5 mL of buffer containing 0.5%
Triton X-100, 25 mmol/L Tris (pH 8.0), 10 mmol/L EGTA, and 10 mmol/L
EDTA for 15 minutes on ice. Samples were then centrifuged for 20 minutes at 13,000g to separate fragmented (supernatant) from
intact chromatin (pellet). DNA content of each fraction was determined
using the diphenylamine reagent and results are expressed as
percentages of DNA in each sample that resisted sedimentation at
13,000g.
AP-1-dependent transcription assay.
As previously described,23 cells were cotransfected with
equal amounts of cytomegalovirus (CMV) gal and the
reporter containing three copies of the tetradecanoyl phorbol acetate
response element (TRE) present in the collagenase promoter fused to the
chloramphenicol acetyltransferase gene (TRE-CAT).
Transfection was by the DEAE-dextran method. Forty-eight hours
posttransfection, cells were treated with the appropriate drugs for 1 hour. Cells were then washed with Tris HCl (pH 7.5) and incubated in
fresh media for 24 hours. Cells were then obtained and the CAT was
extracted. The colorimetric -galactosidase assay provided a rough
indication of transfection efficiency, from which we determined how
much cell extract to use in the CAT assay. CAT activity was measured
with (14C)chloramphenicol as substrate. The percentage
converted to acetylated forms was quantified with a PhosphorImager
(Molecular Dynamics, Chicago, IL).
Transduction of 8226 cells with mutant c-jun.
The cDNA encoding mutant jun was originally obtained from I. Verma (Salk Institute, La Jolla, CA). It was subcloned into the retroviral vector pSR MSVtkNeo. Retrovirus stocks were
prepared by transient transfection of 293 T cells with the ecotropic
 -packaging plasmid. Indicator lines were generated by infection with
the appropriate retrovirus stock and by selection for 2 to 3 weeks in
G418. Naïve 293 T cells were then newly infected with viral supernatant from the indicator lines containing the empty retroviral vector (neo control) or vector containing the mutant
c-jun. Forty-eight hours later, high-titer viral supernatant
was collected and used to transduce 8226 MM cells. 8226 cells were
plated in complete media containing 5% fetal bovine serum (FBS) at 5 × 106 cells/plate and incubated at 37°C
overnight. Media was aspirated and 1 mL of viral supernatant containing
1 µg/µL polybrene was added to each plate. Cells were incubated
with the viral supernatant for 1 hour at 37°C, after which 10 mL of
RPMI media was added and culture was continued. Forty-eight hours
later, selection in 0.5 mg/mL G418 was initiated.
Cell-cycle analysis.
Cells were stained with hypotonic propidium iodide (50 µg/mL in 0.1%
sodium citrate and 0.1% Triton X-100) for 1 hour at 4°C. They were
kept in the dark at 4°C before analysis. Cell-cycle distribution
was then determined by analyzing 10,000 events on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). The DNA data
were fitted to a cell-cycle distribution analysis by use of the MODFIT
program for MAC V2.0.
Statistics.
The t-test was used to determine significance of differences
between groups.
| |
RESULTS |
IL-6 protects MM cells from dexamethasone and anti-fas-induced
apoptosis.
We have used the 8226 and UCLA #1 MM cell lines for the following
mechanistic studies because, though expressing competent IL-6
receptors, exogenous IL-6 only protects against apoptosis but does not
stimulate proliferation. Thus, we can isolate cytokine-induced effects
on apoptosis without potential obfuscating effects on proliferation.
These two MM cell lines undergo apoptotic death within 72 hours of
incubation with 10 6 mol/L dexamethasone and within
20 hours of incubation in 0.5 µg/mL of anti-fas.
Co-incubation with IL-6, at 1,000 U/mL, significantly inhibited both
dexamethasone- and anti-fas-induced apoptosis. Figure 1 shows the ability of IL-6 to block
endonucleosomal fragmentation induced by anti-fas or
dexamethasone in both 8226 and UCLA #1 target cells.
Figure 2 shows that IL-6-dependent
protection against anti-fas-induced apoptosis is also
demonstrated by scoring apoptotic nuclei on DAPI-stained cytospins and
viability by dye exclusion assays (mean percent viability shown above
each bar). Pretreatment with IL-6 for 60 or 120 minutes is more
effective than shorter treatments for subsequent survival of
anti-fas-challenged MM targets (Fig 2). Dye exclusion and DAPI
staining of 8226 cells challenged with dexamethasone also showed
significant protection afforded by IL-6 (63% viability and 35%
apoptosis induced by dexamethasone [106 mol/L]
v 81% and 9% when IL-6 is present [1,000 U/mL]). Similar results were seen with UCLA #1 cells treated with
106 mol/L dexamethasone (59% viability and 32%
apoptosis v 79% viability and 12% apoptosis when IL-6 is
present).
View larger version (118K):
[in this window]
[in a new window]
| Fig 1.
IL-6 protects against anti-fas and dexamethasone. 8226 (lanes A through F) and UCLA #1 cells (lanes G through K) were cultured in media alone for 48 hours (lanes A and G), IL-6 alone for 48 hours
(1,000 U/mL, lane B), anti-fas for 20 hours (0.5 µg/mL, lanes
C and H), dexamethasone for 48 hours (106 mol/L, lanes E
and J) or the combination of anti-fas + IL-6 (lanes D and I)
or dexamethasone + IL-6 (lanes F and K). In the anti-fas + IL-6 combination, IL-6 was present for 1 hour before addition of
anti-fas. DNA was then extracted and electrophoresed. Culture of both cell lines with a control antibody of identical isotype to the
anti-fas antibody resulted in normal, intact,
high-molecular-weight DNA (identical to that shown in lanes A and G
[not shown]).
|
|
View larger version (44K):
[in this window]
[in a new window]
| Fig 2.
IL-6 protects against anti-fas. 8226 cells ( )
or UCLA #1 cells ( ) cultured in anti-fas (AF, 0.5 µg/mL)
alone for 20 hours, or anti-fas + IL-6 (1,000 U/mL). As shown
in the figure, the IL-6 was present for either 15, 30, 60, or 120 minutes before addition of anti-fas. Results are the percent
apoptosis from DAPI-stained cytospins, mean ± SD of three separate
experiments. 8226 cells cultured in media alone for 20 hours showed
only 3.5% apoptosis in this assay. Above each bar is shown the mean
percent viability (from dye exclusion assays of the three experiments).
8226 cells cultured in media alone for 20 hours showed a viability of
88%. Only the presence of IL-6 for 60 or 120 minutes significantly (P < .05) decreased the % apoptosis and increased the
percent viability relative to the cells cultured in anti-fas
alone.
|
|
IL-6-induced protection is not caused by alteration of cell-cycle
distribution.
In M1 myeloid leukemia blasts, IL-6 protects against p53-induced
apoptosis and complements the antiproliferative effect of p53 resulting
in cell-cycle exit with as high as 95% of cells in a quiescent G0
state.24 It is possible that this extreme blockage of
target cells in a dormant state may actually protect them from
p53-induced apoptotic death. In similar fashion, the ability of IL-6 to
protect cells against tumor necrosis factor (TNF)
cytotoxicity correlates with the cytokine's ability to arrest cells in
G1.25 Although IL-6 stimulation of unchallenged 8226 and
UCLA #1 cells neither stimulates nor inhibits in vitro growth, we
considered the possibility that IL-6 could be interacting with dexamethasone or anti-fas for an enhanced antiproliferative
effect similar to the combination of p53 and IL-6 or TNF and IL-6 with resulting protection against apoptosis. However, cell-cycle analysis (Table 1) showed that, in contrast to M1
leukemia cells, IL-6 did not complement dexamethasone-induced cell
cycle exit in these cells. As expected, at both 48 and 72 hours,
dexamethasone alone decreased the percentage of cells in S/G2M by
approximately 50% with a concurrent increase in distribution of cells
in G0/G1. This inhibition of cell-cycle transit through S and G2M
induced by dexamethasone was not enhanced by concurrent IL-6 treatment. Thus, a synergistic increase in cell-cycle exit is not present and
cannot, therefore, explain the ability of IL-6 to protect against
apoptosis. The results in Table 1 also show that the inhibition of
cell-cycle transit through S and G2M induced by dexamethasone was not
prevented by concurrent IL-6 exposure. Thus, the palliative effect of
IL-6 in these cultures is specific for apoptosis as the cytokine could
not protect against dexamethasone-induced cytostasis. This lack of
protection against cytostasis is similar to results we previously
obtained when viable cell recoveries were determined.2
Exposure of these cells to anti-fas for 20 hours did not
significantly affect cell-cycle distribution (not shown).
IL-6 protects against apoptosis in the absence of activating effects
on RAF-1 and ERKs.
We first tested effects of anti-apoptotic concentrations of IL-6 on the
ras-dependent RAF-1/MEK/ERK pathway. RAF-1 activation was
investigated by use of an in vitro kinase assay using MEK as a
substrate. We used AF-10 MM cells as a positive control because prior
studies26,27 confirmed the ability of IL-6 to activate the
RAS/RAF/MEK/ERK pathway in these cells. Activation of the ERK pathway
may mediate IL-6-dependent stimulation of proliferation in AF-10
cells.26,27 AF-10 and 8226 target cells were treated with
or without IL-6 (1,000 U/mL) for 5 minutes. RAF-1 was then immunoprecipitated from cell lysates and tested for its enzymatic activity against GST-MEK. As shown in Fig
3, IL-6 was capable of activating RAF-1 in AF-10 cells but could not
activate RAF-1 activity in 8226 target cells. Coomassie blue staining
confirmed equal loading of protein in each individual lane (Fig 3,
lower band). Furthermore, in Western blot analyses not shown,
reblotting immunoprecipitated RAF-1 with an anti-RAF-1 antibody
confirmed that equal amounts of RAF-1 protein were immunoprecipitated
from cell extracts. In addition, when cells were treated with phorbol myristate acetate (PMA) (500 nmol/L for 5 minutes), RAF-1 activation was clearly detected in 8226 cells (Fig 3). These latter control experiments show that 8226 cells contain RAF-1 that is capable of being
activated by appropriate stimuli but is not susceptible to activation
by IL-6 when used in concentrations that protect these same MM cells
from apoptosis. Repeated in vitro kinase assays testing longer
exposures of cells to IL-6 (10 or 15 minutes, not shown) consistently
showed no IL-6-dependent RAF-1 activation in 8226 cells, although
positive activation was seen in AF-10 cells.
View larger version (41K):
[in this window]
[in a new window]
| Fig 3.
Protective concentrations of IL-6 do not activate RAF-1.
8226 and AF-10 MM cells were cultured with or without IL-6 (1,000 U/mL)
or PMA (500 nmol/L) for 5 minutes. RAF-1 was then immunoprecipitated and tested for enzymatic activity against the substrate GST-MEK in an
in vitro kinase assay. Similar results, ie, a lack of IL-6-dependent activation of RAF-1 in UCLA #1 cells was also shown (not shown). Bottom
panel shows Coomassie blue staining of the gel.
|
|
Activation of ERKs was tested in two ways. First, the three MM cell
lines (8226, UCLA #1, or AF-10) were treated with or without IL-6
(1,000 U/mL for 15 minutes), and tyrosine phosphorylated proteins were
immunoprecipitated from extracts with an antiphosphotyrosine antibody.
We then immunoblotted with anti-MAPK1 (ERK 1; p44) and anti-MAPK2 (ERK
2; p42) antibodies. As shown in Fig 4, both
ERK 1 and ERK 2 were constitutively tyrosine phosphorylated in 8226, UCLA #1, and AF-10 target cells. However, no further stimulation of ERK
activation was demonstrated upon stimulation of 8226 or UCLA #1 cells
with IL-6. In contrast, IL-6 efficiently activated ERK 2 (p42) in AF-10
target cells. Immunoblotting with antiphosphotyrosine antibodies (Fig
4) confirmed equal amounts of phosphotyrosine proteins were applied to
the lanes. This assay was repeated twice with similar results.
View larger version (50K):
[in this window]
[in a new window]
| Fig 4.
Protective concentrations of IL-6 do not induce tyrosine
phosphorylation of ERK-1 or ERK-2. 8226, UCLA #1, and AF-10 cells were
treated with or without IL-6 (1,000 U/mL) for 15 minutes and extracts
were immunoprecipitated with an antiphosphotyrosine antibody
(IP:PTY). Immunoprecipitates were then blotted with anti-ERK
antibodies (anti-MAPK, upper blot) and with an antiphosphotyrosine antibody (PTY, lower blot).
|
|
The second method used for testing ERK activity was by in vitro kinase
assays using MBP as a substrate (Fig 5).
The results obtained (panel A) were consistent with the immunoblot
analyses described above. As shown, constitutive ERK activity was
detected in UCLA #1, 8226, and AF 10 cells. However, only in AF 10 cells was an IL-6-dependent increase in ERK activity demonstrated.
Also shown in Fig 5 (panel B) is the finding that PMA is capable of significantly stimulating ERK activity above the constitutively expressed level in 8226 cells. Thus, these experiments collectively argue against the RAF/MEK/ERK pathway as playing any role in mediating IL-6-induced protection against apoptosis in 8226 and UCLA #1 cells
because anti-apoptotic concentrations of the cytokine failed to
activate RAF-1 or ERK above baseline levels in these cells, although
these signaling proteins were sensitive to activation by PMA and IL-6
was successful in activating them in AF-10 cells.
View larger version (36K):
[in this window]
[in a new window]
| Fig 5.
Protective concentrations of IL-6 do not activate ERK
function. 8226, AF-10, and UCLA #1 MM cells cultured with and without IL-6 (1,000 U/mL, upper panel) or with and without PMA (500 nmol/L, lower panel). Whole-cell lysates were then tested for enzymatic activity against the substrate MBP in an in vitro kinase assay.
|
|
Effects on jun kinase activation.
Initial experiments used in vitro kinase assays with c-jun as
substrate. As shown in Fig 6A,
anti-fas antibody and dexamethasone clearly activate JNK
activity in 8226 cells. By densitometric analysis, dexamethasone
induced a 5-fold and anti-fas induced a 3.5-fold activation in
JNK activity. In a time-course experiment not shown, 10 minutes was the
optimal incubation duration with anti-fas or dexamethasone for
JNK activation. When cells are co-incubated with IL-6, the
anti-fas and dexamethasone-induced enhancement of JNK activity
is inhibited. Pretreatment with IL-6 for 30 or 60 minutes was more
effective than only 10 minutes on subsequent anti-fas-induced
JNK activity. Figure 6B shows equal loading of protein in individual
lanes by Coomassie blue staining of the gel.
Figure 7A, top band, shows a similar
activation of JNK activity in UCLA #1 cells when exposed to
anti-fas (3.3-fold increase in activity by densitometry) or
dexamethasone (2.8-fold increase in activity) and a similar inhibition
of activity by IL-6 when IL-6 was present 60 minutes before addition of
anti-fas or dexamethasone. Because p38 MAPK can also
phosphorylate c-jun, we also performed the in vitro kinase
assay on UCLA #1 cells using anti-JNK antibody to specifically
precipitate cellular JNK. The bottom portion of Fig 7A shows
jun phosphorylation induced by immunoprecipitated JNK in cells
either treated with dexamethasone (lane B, 106
mol/L, for 10 minutes) or anti-fas (lane C, 0.5 µg/mL for 10 minutes). Figure 7B shows the Coomassie blue-stained gel confirming equal protein loading of Fig 7A's (top panel) experiment.
View larger version (41K):
[in this window]
[in a new window]
View larger version (23K):
[in this window]
[in a new window]
| Fig 6.
(A) Anti-fas and dexamethasone activate and IL-6
inhibits jun kinase in 8226 cells. 8226 MM cells incubated in
media (control) or dexamethasone alone (106 mol/L), IL-6
(1,000 U/mL) alone, dexamethasone + IL-6 (IL-6 present for 60 minutes
followed by dexamethasone for 10 minutes, anti-fas alone (0.5 µg/mL for 10 minutes), or anti-fas + IL-6 where IL-6 is
present for 10, 30, or 60 minutes before addition of anti-fas. Whole-cell lysates were then tested for enzymatic activity against GST-jun in an in vitro kinase assay. (B) Coomassie blue
staining of gel shown in (A) to confirm equal protein loading.
|
|
View larger version (36K):
[in this window]
[in a new window]
View larger version (26K):
[in this window]
[in a new window]
| Fig 7.
(A) Dexamethasone and anti-fas activate and IL-6
inhibits jun kinase activity in UCLA #1 cells. Top band: UCLA
#1 cells were incubated in dexamethasone alone (lane A,
106 mol/L, for 10 minutes), anti-fas alone (lane
B, 0.5 µg/mL for 10 minutess), media alone (control,
lane C), dexamethasone (106 mol/L) + IL-6 (lane D,
IL-6 at 1,000 U/mL present for 60 minutes before addition of
dexamethasone), anti-fas + IL-6 (lane E, IL-6 present for 10 minutes before anti-fas), or anti-fas + IL-6 (lane F,
IL-6 present for 60 minutes before anti-fas). In vitro kinase assay performed for jun kinase activity as shown in Fig 6.
Bottom band: UCLA #1 cells incubated in media (lane A), dexamethasone (106 mol/L, lane B), or anti-fas (0.5 µg/mL,
lane C) for 10 minutes. Jun kinase then immunoprecipitated from
protein lysates and tested against GST-jun in in vitro kinase
assay. (B) Coomassie blue staining of gel shown in upper band of (A).
|
|
We next tested whether anti-fas and dexamethasone also activate
c-jun transcriptional activity downstream of JNK activation and
whether IL-6 prevents such activation. After phosphorylation by JNK,
jun/fos heterodimers bind to DNA at TRE, the binding site in
the jun promoter.28 Thus, we exploited a reporter
gene assay which uses a promoter containing three TRE sites fused to
the CAT gene (TRE-CAT). As shown in Fig 8,
anti-fas and dexamethasone treatment activated TRE-CAT 7.5-and
5.5-fold in 8226 and 5- and 3.5-fold, respectively, in UCLA #1 cells.
Thus, these apoptosis-inducing drugs lead to an increase in AP-1
activity in the same cells that demonstrate activated JNK activity.
Concurrent exposure to IL-6 significantly inhibited this activation of
AP-1 activity induced by both agents in both cell types (Fig 8).
View larger version (25K):
[in this window]
[in a new window]
| Fig 8.
Dexamethasone and anti-fas induce and IL-6
inhibits TRE-CAT activity. 8226 cells () or UCLA #1 cells () were
transfected with the AP-1-dependent transcriptional reporter 3×
TRE-CAT and treated with anti-fas (0.5 µg/mL), dexamethasone
(106 mol/L), IL-6 alone (1,000 U/mL), or the
combinations of dexamethasone + IL-6 or anti-fas + IL-6.
Treatments were for 60 minutes and cells were lysed after 24 hours; CAT
activity was measured with (14C) chloramphenicol as
substrate. Data represent means ± SE of three independent
trasnfections and are expressed as fold increase in activity compared
with control cells that received the TRE-CAT vector but no drug
treatment.
|
|
A dominant negative mutant that inhibits c-jun prevents
anti-fas-induced MM cell apoptosis.
Several prior studies have provided evidence that JNK activation and
subsequent c-jun activity play a role in induction of apoptosis. Thus, we considered the possibility that IL-6-dependent protection of MM cells was mediated through the cytokine's inhibitory effects on anti-fas and dexamethasone-induced stimulation of
JNK and c-jun. To further test this hypothesis, we examined the
effect of a well-characterized jun mutant, Jun
In282,23 on apoptosis: Jun In282 is a DNA binding mutant
caused by an insertion in the basic region and acts as a
dominant negative construct by competing with endogenous c-jun
as a substrate for JNK activity. We reasoned that if IL-6-dependent
protection against apoptosis was mediated via inhibition of JNK
activity with subsequent prevention of c-jun phosphorylation
and c-jun transactivation, then disruption of c-jun
function by use of the dominant negative construct should also protect
against apoptosis. Thus, 8226 cells were transduced to express the
jun mutant or neo alone by retroviral infection. After
selection in G418, the two polyclonal cell populations were tested for
the ability of anti-fas or dexamethasone to stimulate the
activation of TRE-CAT. As shown in Fig 9,
the c-jun dominant negative line was prohibited in TRE-CAT
activation by both apoptosis-inducing agents. The dominant negative
c-jun had no significant effect on fas expression or
viability of the continuously cultured MM cells (not shown). However,
when c-jun mutant-containing MM cells were exposed to
anti-fas, they were protected against apoptosis. This was
confirmed by testing viability with dye exclusion assays (Table 2), testing DNA fragmentation by the
diphenylamine assay (Table 2), and by electrophoresis of extracted DNA
(Fig 10). In contrast, the apoptotic
response of mutant c-jun-containing MM cells to dexamethasone
was, in fact, significantly increased (Table 2 and Fig 10). We also
cloned the mutant c-jun-containing MM cells by limiting
dilution and generated four clones. After confirming an inhibition of
TRE-CAT activation by dexamethasone and anti-fas in these
clones, we tested them for sensitivity to apoptosis. The results were
comparable to the transduced polyclonal population of cells. Although
neo control cells exposed to anti-fas (0.5 µg/mL) for 20 hours or dexamethasone (106
mol/L) for 48 hours resulted in a significant loss of viability (from
87% to 69% for dexamethasone and 83% to 47% for anti-fas) and increase in DNA fragmentation (from 4% to 26% for
anti-fas and 3% to 23% for dexamethasone), the clones were
relatively resistant to anti-fas (viability, 73% to 89%
v control of 87%; DNA fragmentation, 2% to 5% v
control of 4%) and showed a modest increase in sensitivity to
dexamethasone (viability, 49% to 60% v control of 90%; DNA fragmentation, 30% to 37% v control of 4.7%). Thus, these
data provide supportive evidence that IL-6 protects MM cells against anti-fas-induced apoptosis via its inhibitory effects on
jun kinase activity and subsequent c-jun function but
also indicate that protection against dexamethasone proceeds along
different pathways.
View larger version (17K):
[in this window]
[in a new window]
| Fig 9.
Mutant c-jun-containing 8226 cells have a
blunted TRE-CAT response to anti-fas and dexamethasone.
Neo control or dominant negative c-jun-transduced 8226 cells were transfected with TRE-CAT and treated without or with
anti-fas (0.5 µg/mL) or dexamethasone (106
mol/L) for 10 minutes. TRE-CAT activity is mean ± SE of three separate experiments.
|
|
View larger version (101K):
[in this window]
[in a new window]
| Fig 10.
Mutant c-jun-containing 8226 cells are
protected from anti-fas-induced DNA laddering but not from
dexamethasone. Neo control (lanes A through D and I through K)
and mutant c-jun-transduced 8226 cells (lanes E through H and
L through N) were cultured in media for 20 hours (lanes A and E) or 48 hours (lanes I and L) or with increasing concentrations of
anti-fas (0.1, 0.25, 0.5 µg/mL, lanes B through D and F
through H) or with dexamethasone (106 mol/L and 2 × 106 mol/L, lanes J and K and M and N, respectively). DNA
was then extracted and electrophoresed.
|
|
| |
DISCUSSION |
The results of this study indicate that IL-6-dependent protection
against anti-fas- and dexamethasone-induced apoptosis of malignant plasma cells is not mediated via alterations in cell-cycle distribution or the RAF-1/MEK/ERK pathway. However, IL-6 prevented anti-fas and dexamethasone-induced activation of jun
kinase in these cells. Our experiments with mutant
c-jun-containing MM cells provide support that these
inhibitory effects on jun kinase mediate IL-6-induced
protection against death at least for MM cells challenged with
anti-fas. Protection against dexamethasone-induced MM cell death appears to be mediated by other pathways.
These results confirm and extend the work of Chauhan et
al.20 These investigators also detected a correlation
between IL-6-dependent protection against anti-fas-induced
apoptosis and the cytokine's ability to inhibit
anti-fas-stimulated JNK activity. Furthermore, IL-6 inhibited
the downstream events of JNK activation, namely c-jun
transactivation of the reporter TRE-CAT. Although these latter data and
the study of Chauhan et al20 suggested a similar mechanism
whereby IL-6 protects against apoptosis and prevents JNK activation,
they did not prove a causal relationship. It was certainly possible
that JNK/c-jun activation were either epiphenomena or even
occurred downstream of the apoptosis machinery, which has been
suggested for TNF29 and anti-fas-induced apoptosis
of Jurkat cells.30 Thus, we hypothesized that, if
IL-6-induced protection was mediated through inhibition of JNK
activation and subsequent c-jun activity, a comparable
inhibition of c-jun function, achieved by different means,
should also protect against apoptosis. The resistance of dominant
negative c-jun-containing MM cells to
anti-fas-induced apoptosis thus provides support for this
hypothesis. We did not test the effects of IL-6 on p38MAPK in this
study because the previous work of Chauhan et al20 clearly
showed that anti-apoptotic concentrations of IL-6 had no effect on the
activation of p38MAPK induced by anti-fas.
The role of the MEKK/SEK1/JNK pathway in the regulation of apoptosis is
controversial. The best evidence that JNK activation can initiate or
sustain apoptosis comes from experiments like ours where selective
disruption of the pathway by introduction of mutant
genes7,31-34 or by use of antisense
oligonucleotides35,36 inhibited the apoptotic response
induced by TNF,31 ceramide,31,35 growth factor
withdrawal,7,36 or anti-fas.32
Specifically for myeloma cells and IL-6, two other previous studies
support a role for JNK activation and AP-1 activity in apoptosis.
One37 showed that cAMP-stimulated AP-1 activity might be
involved in inhibiting MM cell growth, and a second36
showed that anti-sense oligonucleotides directed to c-jun
prevented apoptosis of MM cells induced by IL-6 deprivation. However, a
critical role for JNK activation and c-jun function implies
that gene transactivation would be required for MM cell apoptosis and
most models of cellular apoptosis do not require protein
synthesis.38,39 We have not been able to test the
requirements for protein synthesis in MM cell apoptosis because all our
MM cell lines are exquisitely sensitive to the apoptotic effects of
cycloheximde, emetine, and actinomycin-D when used alone (not shown).
It is also possible that the protection conferred by mutant
c-jun is mediated through yet unknown pathways that do not
involve its well-known role as a transcription factor.
It is not clear whether all activators of JNK/c-jun will induce
MM cell apoptosis. For example, CD40 triggering induce marked JNK
activation in other cell types, although previous
studies40,41 did not detect apoptosis in CD40-stimulated MM
cells. However, these latter cells may also be stimulated to secrete
IL-6,40, 41 which might protect them from a CD40-initiated
apoptosis program. In fact, in MM cells that are not triggered for IL-6
secretion by CD40, we have detected an induction of apoptosis.
Nevertheless, our own data suggest that, though activation of
JNK/c-jun is critical, it may not be sufficient for apoptosis.
Thus, although dexamethasone activates JNK in these cells, this
activation does not mediate apoptosis as shown by experiments with the
c-jun mutant. Another possible example of this may be IL-1,
which induces marked JNK activation, but is not known to be an
apoptotic stimulus, and, in fact, may even activate MM cells. It is
possible that the cellular context within which JNK and c-jun
become activated is important for determining outcome. For example, the
duration of JNK activation42 or the presence of other
activated pathways working in concert with the JNK cascade, may be
critical in determining whether target cells undergo activation or
apoptosis.
Chauhan et al,20 who also studied 8226 MM cells, were
unable to detect activation of ERK-1 or -2 by IL-6 in plasma cells protected against apoptosis. In similar fashion, we also were unable to
detect IL-6-dependent activation of the RAF/MEK/ERK cascade in
protected 8226 as well as UCLA #1 MM cells, although PMA successfully
activated both proteins. In contrast, IL-6 successfully activated both
RAF-1 and ERK in AF-10 plasma cells. This differential effect on
RAF/MEK/ERK in these different MM targets may reflect the finding that
IL-6 can stimulate proliferation in AF-10 cells but only has
anti-apoptotic function in 8226 and UCLA #1 cells without effects on
proliferation. This is consistent with previous work26,27
that suggested IL-6 signaling through RAF/MEK/ERK was crucial for
stimulation of MM cell proliferation. Recent work by Ogata et
al43 indicates that lack of SOS phosphorylation and
activation in 8226 cells exposed to IL-6 explains the absence of
RAF/MEK/ERK activation and loss of proliferative responsiveness in
these cells. However, we detected an IL-6-independent constitutive activation of ERK in 8226 and UCLA #1 cells as determined by in vitro
kinase assays (Fig 5) and by immunoblotting with antiphosphotyrosine antibodies (Fig 4). Such constitutive activation may be due to the
presence of an activating mutation of ras in these tumor cells. Activating ras mutations are relatively common in
MM44 and would allow downstream activation of ERKs in the
absence of SOS phosphorylation.
Our results that argue against ras-dependent MAPK signaling
events being important in IL-6-induced protection against MM cell apoptosis appear inconsistent with results of Billadeau et
al.45 These latter investigators showed that activating
mutations of ras suppress apoptosis in MM cells when they are
deprived of IL-6. The most obvious difference between Billadeau's
model and ours is that he studied an IL-6-dependent MM line and we
studied MM cell lines that are not IL-6 dependent. Antibodies to IL-6
have no effect on the in vitro growth of our 8226 and UCLA #1 cell lines2 and, furthermore, exogenous IL-6 does not stimulate any proliferation of these two cell lines. The inconsistency between the studies of Billadeau et al and our own could be reconciled by the
presence of two separate IL-6-dependent anti-apoptotic pathways in MM
cells, only one of which (that studied by Billadeau et
al45) being intimately integrated into IL-6-dependent
signals that result in proliferation as well.
Although our results provide some insight into the mechanism by which
IL-6 protects MM cells against anti-fas, the pathways involved
in protection against dexamethasone remain unclear. Although we
demonstrated a dexamethasone-induced activation of JNK/SAPK and TRE-CAT
reporter gene expression, mutant c-jun did not protect against
dexamethasone and, in fact, significantly sensitized MM cells to
enhanced apoptosis induced by dexamethasone. This suggests that JNK
activation during dexamethasone exposure is a protective response to
dexamethasone and that enhanced AP-1 transactivation induces expression
of protective proteins. This is consistent with a recent
study46 where the immunosuppressant drug rapamycin was
found to inhibit jun kinase activity and markedly potentiate dexamethasone-induced apoptosis of lymphoblastoid cells. It should be
mentioned that other investigators could not detect SAPK/JNK activation
in 8226 cells challenged with dexamethasone.47 The reason
for this discrepancy is not readily obvious to us. It could be caused
by the need for costimulatory molecules selectively present in our
culture media. Other investigators have documented the ability of
dexamethasone to induce JNK activation in different cell
models.48-50
The results of our cell-cycle analysis confirm that protection against
dexamethasone-induced apoptosis by IL-6, at least in 8226 cells, is not
accompanied by protection against dexamethasone-induced cytostasis.
This shows that IL-6-induced protection against dexamethasone-induced apoptosis is not likely due to inhibition of very proximal events such
as dexamethasone receptor expression or binding of dexamethasone to its
receptor, but is selective for dexamethasone-induced downstream events
that are more specific for apoptosis. However, cell-cycle analysis also
showed that, unlike the interaction between IL-6 and activated p53 in
M1 cells,24 IL-6 was not protecting MM cells by
complementing the antiproliferative effect of dexamethasone and
blocking cells in a dormant state. We have also been unable to
correlate IL-6-induced protection against dexamethasone-induced MM
cell death with any alteration in expression of BCL-2 or BAX proteins.2 In a previous study,51 we detected
an IL-6-dependent upregulation of BCL-XL in UCLA #1 cells,
which is similar to previous findings in other MM
lines.52,53 However, repeated studies with 8226 targets
exposed to IL-6 have failed to demonstrate any alteration of
BCL-XL expression.51 We have also assessed the ability of IL-6 to phosphorylate BAD protein in protected MM cells because recent work54 suggests BAD phosphorylation is
crucial for mediating IL-3-induced protection against apoptosis.
However, we detected very little BAD protein in these human MM cells
and IL-6 was incapable of phosphorylating BAD. Thus, although there are
many more members of the BCL protein family that have not been
examined, work to date has not detected consistent alterations of this
family of proteins that could conceivably account for IL-6-induced
protection.
In summary, this study indicates that inhibition of c-jun
function protects MM cells against apoptosis induced by
anti-fas. The results support the hypothesis that IL-6-induced
protection against anti-fas is mediated through its inhibition
of the JNK/c-jun pathway. Furthermore, it indicates that the
ability of IL-6 to protect MM cells against dexamethasone must be
mediated by yet unknown mechanisms.
| |
FOOTNOTES |
Submitted October 6, 1997;
accepted February 25, 1998.
Supported by research funds from the Veteran's Administration.
Address reprint requests to Alan Lichtenstein, MD, Hematology-Oncology,
VA West LA Hospital, 691/W111H, 11301 Wilshire Blvd, Los Angeles, CA
90073.
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.
| |
REFERENCES |
1.
Hardin J,
MacLeod S,
Grigorieva I,
Chang R,
Barlogie B,
Xiao H,
Epstein J:
Interleukin-6 prevents dexamethasone-induced myeloma cell death.
Blood
84:3063,
1994[Abstract/Free Full Text]
2.
Lichtenstein A,
Tu Y,
Fady C,
Vescio R,
Berenson J:
Interleukin-6 inhibits apoptosis of malignant plasma cells.
Cell Immunol
162:248,
1995[Medline]
[Order article via Infotrieve]
3.
Anderson K,
Jones RM,
Morimoto C,
Leavitt P,
Barut BA:
Response patterns of purified myeloma cells to hematopoietic growth factors.
Blood
73:1915,
1989[Abstract/Free Full Text]
4.
Barut BA,
Zon LI,
Cochran MK,
Paul SR,
Chauhan D,
Mohrbacher A,
Fingeroth J,
Anderson K:
Role of interleukin 6 in the growth of myeloma-derived cell lines.
Leukemia Res
16:951,
1992[Medline]
[Order article via Infotrieve]
5.
Muraguchi A,
Hirano T,
Tang B,
Matsuda T,
Horii Y,
Nakajima K,
Kishimoto T:
The essential role of B cell stimulating factor 2 for the terminal differentian of B cells.
J Exp Med
167:332,
1988[Abstract/Free Full Text]
6.
Kishimoto T,
Akira S,
Narazaki M,
Taga T:
Interleukin-6 family of cytokines and gp 130.
Blood
86:1243,
1995[Free Full Text]
7.
Xia Z,
Dickens M,
Raingeaud J,
Davis RJ,
Greenberg ME:
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:1326,
1995[Abstract/Free Full Text]
8.
Graves JD,
Draves KE,
Craxton A,
Saklatvala J,
Krebs EG,
Clark EA:
Involvement of stress activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes.
Proc Natl Acad Sci USA
93:13814,
1996[Abstract/Free Full Text]
9.
Ichijo H,
Nishida E,
Irie K,
ten Dijke P,
Saitoh M,
Moriguchi T,
Takagi M,
Matsumoto K,
Miyazono K,
Gotoh Y:
Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.
Science
275:90,
1997[Abstract/Free Full Text]
10.
Johnson N,
Gardner AM,
Diener KM,
Lange-Carter CA,
Gleavy J,
Jarpe MB,
Minden A,
Karin M,
Zon LI,
Johnson GL:
Signal transduction pathways regulated by mitogen activated extracellular response kinase kinase kinase induce cell death.
J Biol Chem
271:3229,
1996[Abstract/Free Full Text]
11.
Wilson DJ,
Fortner KA,
Lynch DH,
Mattingly RR,
Macara IG,
Posada JA,
Budd RC:
JNK, but not MAPK, activation is associated with fas-mediated apoptosis in human T cells.
Eur J Immunol
26:989,
1996[Medline]
[Order article via Infotrieve]
12.
Marshall CJ:
Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation.
Cell
80:179,
1995[Medline]
[Order article via Infotrieve]
13.
Hill CS,
Treisman R:
Transcriptional regulation by extracellular signals: Mechanisms and specificity.
Cell
80:199,
1995[Medline]
[Order article via Infotrieve]
14.
Derijard B,
Hibi M,
Wu I-H,
Barrett T,
Su B,
Karin M,
Davis R:
JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.
Cell
76:11025,
1994
15.
Kyriakis JM,
Benerjee P,
Nikolakaki P,
Dai T,
Rubie EA,
Ahmad MF,
Avruch J,
Woodgett JR:
The stress-activated protein kinase subfamily of c-jun kinases.
Nature
369:156,
1994[Medline]
[Order article via Infotrieve]
16.
Minden A,
Lin A,
McMahon M,
Lange-carter C,
Derijard B,
Davis RJ,
Johgnson G,
Karin M:
Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.
Science
266:1719,
1994[Abstract/Free Full Text]
17.
Lange-Carter CA,
Johnson GL:
Ras-dependent growth factor regulation of MEK kinase in PC12 cells.
Science
265:1458,
1994[Abstract/Free Full Text]
18.
Gupta S,
Campbell D,
Derijard B,
Davis RJ:
Transcription factor ATF2 regulation by the JNK signal transduction pathway.
Science
267:389,
1995[Abstract/Free Full Text]
19.
Hibi M,
Lin A,
Smeal T,
Minden A,
Karin M:
Identification of an oncoprotein and UV responsive protein kinase that binds and potentiatesthe c-jun activation domain.
Genes Dev
7:2135,
1993[Abstract/Free Full Text]
20.
Chauhan D,
Kharbanda S,
Ogata A,
Urashima M,
Teoh G,
Robertson M,
Kufe DW,
Anderson KC:
Interleukin-6 inhibits fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells.
Blood
89:227,
1997[Abstract/Free Full Text]
21.
Minden A,
Lin A,
Smeal T,
Derijard B,
Cobb M,
Davis R,
Karin M:
cjun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.
Mol Cell Biol
14:6683,
1994[Abstract/Free Full Text]
22.
Wang H-G,
Millan JA,
Cox AD,
Der CJ,
Rapp UR,
Beck T,
Zha H,
Reed JC:
R-ras promotes apoptosis caused by growth factor deprivation via a BCL-2 suppressible mechanism.
J Cell Biol
129:1103,
1995[Abstract/Free Full Text]
23.
Raitano A,
Halpern JR,
Hambuch TM,
Sawyers CL:
The Bcr-Abl leukemia oncogene activates jun kinase and requires jun for transformation.
Proc Natl Acad Sci USA
92:11746,
1995[Abstract/Free Full Text]
24.
Levy N,
Yonish-Rouach E,
Oren M,
Kimchi A:
Complementation by wild type p53 of interleukin-6 effects on M1 cells: Induction of cell cycle exit and cooperativity with c-myc suppression.
Mol Cell Biol
13:7942,
1993[Abstract/Free Full Text]
25.
Belizario JE,
Dinarello CA:
Interleukin 1, interleukin 6, tumor necrosis factor and transforming growth factor-beta increase cell resistance to tumor necrosis factor cytotoxicity by growth arrest in the G1 phase of the cell cycle.
Cancer Res
51:2379,
1991[Abstract/Free Full Text]
26.
Daeipour M,
Kumar G,
Amaral MC,
Nel AE:
Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line AF-10.
J Immunol
150:4743,
1993[Abstract]
27.
Kumar G,
Gupta S,
Wang S,
Nel AE:
Involvement of janus kinases, p52shc, Raf-1 and MEK-1 in the IL-6-induced mitogen-activated protein kinase cascade of a growth-responsive B cell line.
J Immunol
153:4436,
1994[Abstract]
28.
Angel P,
Imagawa M,
Chiu R,
Stein B,
Imbra RJ,
Rahmsdorf HJ,
Jonat C,
Herrlich P,
Karin M:
Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor.
Cell
49:729,
1987[Medline]
[Order article via Infotrieve]
29.
Liu Z,
Hsu H,
Goeddel DV,
Karin M:
Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kB activation prevents cell death.
Cell
87:565,
1996[Medline]
[Order article via Infotrieve]
30.
Lenczowski JM,
Dominguez L,
Eder AM,
King LB,
Zacharchuk CM,
Ashwell JD:
Lack of a role for jun kinase and AP 1 in fas-induced apoptosis.
Mol Cell Biol
17:170,
1997[Abstract]
31.
Verheij M,
Bose R,
Lin XH,
Yao B,
Jarvis WD,
Grant S,
Birrer MJ,
Szabo E,
Zon LI,
Kyriakis JM,
Haimovitz-Friedman A,
Fuks Z,
Kolesnick RN:
Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.
Nature
380:75,
1996[Medline]
[Order article via Infotrieve]
32.
Goillot E,
Raingeaud J,
Ranger A,
Tepper RI,
Davis RJ,
Harlow E,
Sanchez I:
Mitogen-activated protein kinase-mediated fas apoptotic signaling pathway.
Proc Natl Acad Sci USA
94:3302,
1997[Abstract/Free Full Text]
33.
Estus S,
Zaks WJ,
Freeman RS,
Gruda M,
Bravo R,
Johnson EM:
Altered gene expression in neurons during programmed cell death:Identification of c-jun as necessary for neuronal apoptosis.
J Cell Biol
127:1717,
1994[Abstract/Free Full Text]
34.
Ham J,
Babji C,
Whitfield J,
Pfarr CM,
Lallemand D,
Yaniv M,
Rubin LL:
A c-jun dominant negative mutant protects sympathetic neurons against programmed cell death.
Neuron
14:927,
1995[Medline]
[Order article via Infotrieve]
35.
Sawai H,
Okazaki T,
Yamamoto H,
Okano H,
Takeda Y,
Tashima M,
Sawada H,
Okuma M,
Ishikura H,
Umehara H,
Domae N:
Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells.
J Biol Chem
270:27326,
1995[Abstract/Free Full Text]
36.
Collata F,
Polentarutti N,
Sironi M,
Mantovani A:
Expression and involvement of c-fos and c-jun proto-oncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines.
J Biol Chem
267:18278,
1992[Abstract/Free Full Text]
37.
Rezzonico R,
Loubat A,
Lallemand D,
Pfarr CM,
Far DF,
Proudfoot A,
Rossi B,
Ponzio G:
Cyclic AMP stimulates a junD/Fra-2 AP-1 complex and inhibits the proliferation of IL-6-dependent cell lines.
Oncogene
11:1069,
1995[Medline]
[Order article via Infotrieve]
38.
Martin SJ:
Apoptosis: Suicide, execution or murder?
Trends Cell Biol
3:141,
1993
39.
Nagata S,
Golstein P:
The fas death factor.
Science
267:1449,
1995[Abstract/Free Full Text]
40.
Westendorf JJ,
Ahmann GJ,
Armitage RJ,
Spriggs MK,
Lust JA,
Greipp PR,
Katzmann JA,
Jelinek DF:
CD40 expression in malignant plasma cells.
J Immunol
152:117,
1994[Abstract]
41.
Urashima M,
Chauhan D,
Uchiyama H,
Freeman GJ,
Anderson KC:
CD40 ligand triggered IL-6 secretion in multiple myeloma.
Blood
85:1903,
1995[Abstract/Free Full Text]
42.
Chen Y-R,
Wang X,
Templeton D,
Davis RJ,
Tan T-H:
The role of c-jun N-terminal kinase in apoptosis induced by ultraviolet C and gamma radiation.
J Biol Chem
271:31929,
1996[Abstract/Free Full Text]
43.
Ogata A,
Chauhan D,
Urashima M,
Teoh G,
Treon SP,
Anderson K:
Blockade of mitogen-activated protein kinase cascade signaling in IL-6-independent multiple myeloma cells.
Clin Canc Res
3:1017,
1997[Abstract]
44.
Liu P,
Leong T,
Quam L,
Billadeau D,
Kay NE,
Greipp P,
Kyle RA,
Oken MM,
Van Ness B:
Activating mutations of N- and K-ras in multiple myeloma show different clinical associations: Analysis of the Eastern Cooperative Oncology group phase III trial.
Blood
88:2699,
1996[Abstract/Free Full Text]
45.
Billadeau D,
Liu P,
Jelinek D,
Shah N,
LeBien T,
Van Ness B:
Activating mutations in the N- and K-ras oncogenes differentially affect the growth properties of the IL-6-dependent myeloma cell line ANBL6.
Cancer Res
57:2268,
1997[Abstract/Free Full Text]
46.
Ishizuka T,
Sakata N,
Johnson GL,
Gelfand EW,
Terada N:
Rapamycin potentiates dexamethasone-induced apoptosis and inhibits JNK activity in lymphoblastoid cells.
Biochem Biophys Res Comm
230:386,
1997[Medline]
[Order article via Infotrieve]
47. Anderson K: Interleuklin-6 related regulation of growth and
apoptosis of myeloma cells. Proceedings of the VI International workshop on multiple myeloma. Boston, MA, June 1997
48.
Kanatani Y,
Kasukabe T,
Okabe-Kado J,
Hayashi S,
Yamamoto-Yamaguchi Y,
Motoyoshi K,
Nagata N,
Honma Y:
Transforming growth factor beta and dexamethasone cooperatively enhance c-jun gene expression and inhibit the growth of monocytoid leukemia cells.
Cell Growth Diff
7:187,
1996[Abstract]
49.
Sikora E,
Grassilli E,
Radziszewska E,
Bellesia E,
Barbieri D,
Franceschi C:
Transcription factors DNA-binding activity in rat thymocytes undergoing apoptosis after heat-shock or dexamethasone treatment.
Biochem Biophys Res Comm
197:709,
1993[Medline]
[Order article via Infotrieve]
50.
Goldstone SD,
Lavin MF:
Prolonged expression of c-jun and associated activity of the transcription factor AP-1, during apoptosis in a human leukemic cell line.
Oncogene
9:2305,
1994[Medline]
[Order article via Infotrieve]
51.
Tu Y,
Renner S,
Xu F-H,
Fleishman A,
Taylor J,
Weisz J,
Vescio R,
Rettig M,
Berenson J,
Krajewski S,
Reed JC,
Lichtenstein A:
BCL-X expression in multiple myeloma: Possible indicator of chemoresistance.
Cancer Res
58:256,
1998[Abstract/Free Full Text]
52.
Schwarze MMK,
Hawley RG:
Prevention of myeloma cell apoptosis by ectopic BCL-expression or interleukin-6 mediated upregulation of BCL-X-L.
Cancer Res
55:2262,
1995[Abstract/Free Full Text]
53.
Lotem J,
Sachs L:
Regulation of BCL-2, BCL-X and BAX in the control of apoptosis by hematopoietic cytokines and dexamethasone.
Cell Growth Differ
6:647,
1995[Abstract]
54.
Zha J,
Harada H,
Yang E,
Jockel J,
Korsmeyer SJ:
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL.
Cell
87:619,
1996[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
P. Frost, Y. Shi, B. Hoang, J. Gera, and A. Lichtenstein
Regulation of D-cyclin translation inhibition in myeloma cells treated with mammalian target of rapamycin inhibitors: rationale for combined treatment with extracellular signal-regulated kinase inhibitors and rapamycin
Mol. Cancer Ther.,
January 1, 2009;
8(1):
83 - 93.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Shi, P. J. Frost, B. Q. Hoang, A. Benavides, S. Sharma, J. F. Gera, and A. K. Lichtenstein
IL-6-Induced Stimulation of c-Myc Translation in Multiple Myeloma Cells Is Mediated by Myc Internal Ribosome Entry Site Function and the RNA-Binding Protein, hnRNP A1
Cancer Res.,
December 15, 2008;
68(24):
10215 - 10222.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Sharma and A. Lichtenstein
Dexamethasone-induced apoptotic mechanisms in myeloma cells investigated by analysis of mutant glucocorticoid receptors
Blood,
August 15, 2008;
112(4):
1338 - 1345.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kiziltepe, T. Hideshima, K. Ishitsuka, E. M. Ocio, N. Raje, L. Catley, C.-Q. Li, L. J. Trudel, H. Yasui, S. Vallet, et al.
JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells
Blood,
July 15, 2007;
110(2):
709 - 718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. R. Hodge, B. Peng, J. C. Cherry, E. M. Hurt, S. D. Fox, J. A. Kelley, D. J. Munroe, and W. L. Farrar
Interleukin 6 Supports the Maintenance of p53 Tumor Suppressor Gene Promoter Methylation
Cancer Res.,
June 1, 2005;
65(11):
4673 - 4682.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Tassone, P. Neri, R. Burger, R. Savino, M. Shammas, L. Catley, K. Podar, D. Chauhan, S. Masciari, A. Gozzini, et al.
Combination Therapy with Interleukin-6 Receptor Superantagonist Sant7 and Dexamethasone Induces Antitumor Effects in a Novel SCID-hu In vivo Model of Human Multiple Myeloma
Clin. Cancer Res.,
June 1, 2005;
11(11):
4251 - 4258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. F. Gera, I. K. Mellinghoff, Y. Shi, M. B. Rettig, C. Tran, J.-h. Hsu, C. L. Sawyers, and A. K. Lichtenstein
AKT Activity Determines Sensitivity to Mammalian Target of Rapamycin (mTOR) Inhibitors by Regulating Cyclin D1 and c-myc Expression
J. Biol. Chem.,
January 23, 2004;
279(4):
2737 - 2746.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Brocke-Heidrich, A. K. Kretzschmar, G. Pfeifer, C. Henze, D. Loffler, D. Koczan, H.-J. Thiesen, R. Burger, M. Gramatzki, and F. Horn
Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation
Blood,
January 1, 2004;
103(1):
242 - 251.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Hideshima, D. Chauhan, T. Hayashi, K. Podar, M. Akiyama, C. Mitsiades, N. MItsiades, B. Gong, L. Bonham, P. de Vries, et al.
Antitumor Activity of Lysophosphatidic Acid Acyltransferase-{beta} Inhibitors, a Novel Class of Agents, in Multiple Myeloma
Cancer Res.,
December 1, 2003;
63(23):
8428 - 8436.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Greil, G. Anether, K. Johrer, and I. Tinhofer
Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools
J. Leukoc. Biol.,
September 1, 2003;
74(3):
311 - 330.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. C. Platanias
Map kinase signaling pathways and hematologic malignancies
Blood,
June 15, 2003;
101(12):
4667 - 4679.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. H. Underhill, D. George, E. G. Bremer, and G. S. Kansas
Gene expression profiling reveals a highly specialized genetic program of plasma cells
Blood,
May 15, 2003;
101(10):
4013 - 4021.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Shi, J.-h. Hsu, L. Hu, J. Gera, and A. Lichtenstein
Signal Pathways Involved in Activation of p70S6K and Phosphorylation of 4E-BP1 following Exposure of Multiple Myeloma Tumor Cells to Interleukin-6
J. Biol. Chem.,
May 3, 2002;
277(18):
15712 - 15720.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Grad, N. J. Bahlis, I. Reis, M. M. Oshiro, W. S. Dalton, and L. H. Boise
Ascorbic acid enhances arsenic trioxide-induced cytotoxicity in multiple myeloma cells
Blood,
August 1, 2001;
98(3):
805 - 813.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Tu, A. Gardner, and A. Lichtenstein
The Phosphatidylinositol 3-Kinase/AKT Kinase Pathway in Multiple Myeloma Plasma Cells: Roles in Cytokine-dependent Survival and Proliferative Responses
Cancer Res.,
December 1, 2000;
60(23):
6763 - 6770.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Renner, J. Weisz, S. Krajewski, M. Krajewska, J. C. Reed, and A. Lichtenstein
Expression of BAX in Plasma Cell Dyscrasias
Clin. Cancer Res.,
June 1, 2000;
6(6):
2371 - 2380.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
E. E. Plowright, Z. Li, P. L. Bergsagel, M. Chesi, D. L. Barber, D. R. Branch, R. G. Hawley, and A. K. Stewart
Ectopic expression of fibroblast growth factor receptor 3 promotes myeloma cell proliferation and prevents apoptosis
Blood,
February 1, 2000;
95(3):
992 - 998.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Tinhofer, I. Marschitz, T. Henn, A. Egle, and R. Greil
Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma
Blood,
January 15, 2000;
95(2):
610 - 618.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R.-H. Chen, M.-C. Chang, Y.-H. Su, Y.-T. Tsai, and M.-L. Kuo
Interleukin-6 Inhibits Transforming Growth Factor-beta -induced Apoptosis through the Phosphatidylinositol 3-Kinase/Akt and Signal Transducers and Activators of Transcription 3 Pathways
J. Biol. Chem.,
August 13, 1999;
274(33):
23013 - 23019.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Y. Chang, X. Yang, and D. Baltimore
Dissecting Fas signaling with an altered-specificity death-domain mutant: Requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation
PNAS,
February 16, 1999;
96(4):
1252 - 1256.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Chauhan, P. Pandey, T. Hideshima, S. Treon, N. Raje, F. E. Davies, Y. Shima, Y.-T. Tai, S. Rosen, S. Avraham, et al.
SHP2 Mediates the Protective Effect of Interleukin-6 against Dexamethasone-induced Apoptosis in Multiple Myeloma Cells
J. Biol. Chem.,
September 1, 2000;
275(36):
27845 - 27850.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. H. Wang, X. Y. Yang, K. Mihalic, W. Xiao, D. Li, and W. L. Farrar
Activation of Estrogen Receptor Blocks Interleukin-6-inducible Cell Growth of Human Multiple Myeloma Involving Molecular Cross-talk between Estrogen Receptor and STAT3 Mediated by Co-regulator PIAS3
J. Biol. Chem.,
August 17, 2001;
276(34):
31839 - 31844.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract