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INTRODUCTION |
Apoptotic cell death plays a central role in immune system
development, in normal lymphocyte function, and in the progression of
lymphoid neoplasia (1). B-lymphocytes have a propensity to undergo
apoptosis as evidenced by their rapid death following the loss of
cell-cell contacts, as well as their sensitivity to apoptosis induction
following exposure to drugs or chemicals. Importantly, the role of
appropriate signals within lymphoid organs in controlling the survival
and apoptotic responses of B-lymphocytes has become increasingly
apparent, and indicate a contribution of
mitochondrial-dependent and independent pathways in B-cell apoptosis (2-9).
Cell lines derived from human Burkitt's lymphoma
(BL)1 have proven to be
useful models of germinal center (GC) B-lymphocytes for investigating
proliferative and apoptotic signals transduced by surface receptors,
including the B-cell antigen-receptor (BCR), CD40, and CD95/Fas (9-19)
as well as mechanisms of chemically induced cytotoxicity (18, 20-25).
We previously identified a panel of BL cell lines that vary
dramatically in their sensitivity to apoptosis induced by arsenite and
to mitochondrial inhibitors (22, 23). The BL cell line ST486 shows
sensitivity to BCR-mediated as well as chemically induced apoptosis,
and it was selected here as a model for studying signaling pathways
involved in regulation of antigen receptor and stress-induced cell
death pathways.
Arsenite is a potent inducer of apoptosis in lymphoid cells. It is a
ubiquitous environmental contaminant originating from geological and
industrial sources with immunotoxic potential (26, 27). Furthermore,
arsenite, in the form of arsenic trioxide, has emerged as a promising
therapeutic agent for the treatment of acute promyelocytic leukemia
(28). Arsenite is a prototypic chemical inducer of apoptosis that
activates the c-Jun amino-terminal kinase (JNK) and the p38 pathways
(23, 29, 30, 31) and acts via mitochondrial effectors of apoptosis
(32). The differential sensitivity of BL cell lines to apoptosis
induction by arsenite and other chemical agents can be attributed, at
least in part, to expression of Bcl-2 proteins (22-25). This family of
proteins is highly regulated throughout B-cell development (33), but the signaling pathways that control the expression and activity of
Bcl-2 and related proteins in B-cells are not completely known. Such
information is important, because these proteins play a key role in
regulating mitochondrial homeostasis and B-cell responses to
stress-associated as well as BCR-mediated signals.
Depending on the stage of development, activation of the B-cell antigen
receptor (BCR) has different cellular outcomes, including proliferation, growth arrest, apoptosis, and tolerance. BL cell lines
serve as in vitro models for such BCR-mediated responses. Activation of the BCR by cross-linking of surface IgM leads to the
induction of cell cycle arrest and apoptosis, whereas co-stimulation with CD40 and/or interleukin-4 results in rescue from IgM-mediated apoptosis. These processes parallel negative and positive selection of
B-lymphocytes in vivo (9-19, 34-36). Although the ultimate
outcome of BCR activation in BL cells is induction of apoptosis, the
immediate signals are associated with survival/proliferative responses
and include activation of the Ras/Raf/MEK/ERK and phosphoinositide 3-kinase (PI3K)/3-phosphoinositide-directed kinase-1 (PDK1)/protein kinase B/AKT pathways (AKT) (34, 37-42).
Among the downstream targets activated in response to surface IgM
cross-linking is p90S6 ribosomal kinase (RSK) (38).
Although first identified as a substrate of ERK, recent studies have
shown that activation of RSK is dependent not only on MEK/ERK, but
rather on the coordinated activity of MEK/ERK and PI3K/PDK1. ERK and
PDK1 phosphorylate RSK at discrete sites, thereby activating the
carboxyl- and amino-terminal domains (CTD and NTD), respectively
(43-45). In addition, at least some of the downstream responses to IgM
receptor activation involve members of the Bcl-2 family of proteins.
Surface IgM engagement has been reported to induce cell death agonists
Bax and Bik, whereas CD40 activation induces cell death antagonists
BclXL and A1 (10, 11, 13-15). The pro-apoptotic protein,
Bad is a substrate of both AKT and RSK, which phosphorylate it at
serine 136 and 112, respectively, subsequently blocking its
pro-apoptotic activity (46-51). Phosphorylation of Bad has been
reported following exposure of cells to cytokines and growth factors,
but an involvement in IgM-mediated responses has yet to be described.
In the present study we tested the hypothesis that the rapid,
coordinated activation of ERK and PI3K pathways following IgM cross-linking provides an initial survival signal that confers protection against the rapid induction of apoptosis by a broad range of
stress factors. We found that surface IgM cross-linking in the ST486
cell line provides a window of protection against apoptosis induced by
arsenite, multiple drugs that differ in their initial molecular targets
in cells, and hyperthermia. This protection involves an inhibition of
mitochondrial depolarization as well as caspase-9 cleavage, key points
of regulation in chemically induced apoptotic pathways. Importantly,
our study shows that the IgM-mediated protection requires activation of
the ERK and the PI3K pathways. Moreover, these two signaling pathways
converge upon a common downstream target, RSK, resulting in
phosphorylation of the pro-apoptotic Bcl-2 family member, Bad.
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EXPERIMENTAL PROCEDURES |
Chemicals and Antibodies--
Sodium arsenite
(NaAsO2), carbonyl cyanide
m-chlorophenylhydrazone (mClCCP), etoposide, and
camptothecin were obtained from Sigma Chemical Co. U0126, LY294002, and
rapamycin were also obtained from Sigma. C6-ceramide
(N-hexanoylsphingosine) was obtained from BIOMOL. The AKT
inhibitor, 1L-6-hydroxymethyl-chiro-inositol
2-[(R)-2-O-methyl]-3-O-octadecylcarbonate (AKTi5) was obtained from Calbiochem. It is a phosphoinositol ether
analog that binds the pleckstrin homology domain of AKT thereby
preventing its phosphorylation and activation (compound 5 (52);
IC50 = 5.0 µM). The dye
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolycarbocyanine iodide (JC-1) was obtained from Molecular Probes, Inc. (Eugene, OR).
Sodium arsenite was dissolved in water, mClCCP in methanol, and all
other chemicals in dimethyl sulfoxide.
Antibodies specific for total and/or phosphorylated p44/42 ERK, AKT
threonine 308 and serine 378, RSK serine 380, Bad serine 112, Bad
serine 136, PDK1 serine 241, and caspase-9 were purchased from Cell
Signaling Inc. The antibody for poly(ADP-ribose) polymerase (PARP,
AAP-250) was purchased from Stressgen, Inc. and for RSK serine 227 from
Santa Cruz Biotechnology. Goat affinity-purified F(ab')2
fragment to human IgM was obtained from ICN/Cappel.
Cell Lines and Culture Conditions and Chemical
Treatment--
The BL cell lines ST486 and CA46 were obtained from
ATCC, Rockville, MD. Both of the cell lines grow optimally and
similarly in the same medium formulation, have doubling times of ~24
h, and are Epstein-Barr virus-negative. The cell lines were cultured in
medium RPMI 1640 (Invitrogen) supplemented with 15% fetal calf serum,
penicillin-streptomycin, and L-glutamine. Cells were grown at 37 °C, in 5% CO2 and 95% humidity.
For all experiments, cultures were set up at a density of 0.4 × 106 cells/ml and allowed to grow for 24 h. (For
experiments with C6-ceremide, cells were transferred to medium
containing 1% serum.) After appropriate pretreatment (i.e.
5 µg/ml anti-IgM or glycerol control) cells were plated into six-well
plates, at 3 ml/well, and sodium arsenite or other chemicals were added
at the indicated concentrations. At the designated times, cells were
harvested for protein immunoblotting or for detection of morphological
apoptosis as described below.
Cytological Detection of Apoptosis and Necrosis with the Hoechst
33342/Propidium Iodide Assay--
The induction of
apoptosis was analyzed using a double-fluorescence staining technique
(53-55). The procedure allows simultaneous detection of plasma
membrane integrity by dye exclusion and apoptotic phenotypes by
observing condensed, segregated chromatin in "live" cells. Briefly,
cells were stained in 20 µg/ml propidium iodide (emitting red
fluorescence) and 100 µg/ml Hoechst 33342 (emitting blue
fluorescence) for 15 min, at 37 °C in the dark. The double fluorescence was detected with a Leitz Aristoplan microscope equipped with an epifluorescence system and a long-pass filter cube A. Dead
cells emit red and live cells blue fluorescence. Apoptotic cells have a
characteristic phenotype of condensed, segregated chromatin in intact
but shrunken cells (fluorescing blue in early stages and red later on).
The apoptotic phenotype was easy to detect and discriminate from
necrotic cells, which were swollen, had irregular/damaged membranes,
and were propidium iodide-positive. The chromatin was minimally
condensed with some accumulation near the nuclear membrane. Typically,
200 cells were scored for each sample and classified as either
necrotic, apoptotic, or normal/viable, and data were subjected to
statistical analysis as described below.
Protein Immunoblotting--
Following chemical exposure for the
specified period of time, cells (1 ml of culture) were collected,
washed in phosphate-buffered saline, and solubilized in 50 µl of 1×
Laemmli sample buffer (65.2 mM Tris-Cl, pH 6.8, 25%
glycerol, 2% SDS, 0.01% bromphenol blue, and 5%
-mercaptoethanol). 10 µl of lysate (~4 × 105
cells/sample) was subjected to SDS-PAGE in a 4 to 15% gradient gel.
Gels were electrophoretically transferred to nitrocellulose membrane
(Bio-Rad) in 25 mM Tris, pH 8.3, 192 mM
glycine, 20% MeOH. For detection of phosphorylated kinases, membranes
were first probed with antibodies specific for the phosphorylated forms then subsequently re-probed using antibodies that recognize the proteins independent of phosphorylation status as loading controls. Membranes were washed in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) then blocked for 1 h in TBS
containing 5% dried milk. Filters were then washed in TBS containing
0.1% Tween 20 then incubated overnight at 4 °C with primary
antibody diluted appropriately in TBS containing 5% bovine serum
albumin. Filters were washed again and incubated with the second
antibody-horseradish peroxidase conjugate. Detection was then performed
using an enhanced chemiluminescent (ECL) system. Quantitation of the
signals was performed using an Alpha Imager 2000 documentation and
analysis system, equipped with AlphaEase version 3.2 software (Alpha
Innotech Corp.).
Detection of Mitochondrial Toxicant-induced Membrane
Depolarization--
Mitochondrial toxicant-induced loss in

m was monitored at the single cell level using JC-1,
a membrane potential-sensitive probe. JC-1 is taken up selectively by
energized mitochondria and forms J-aggregates that emit red
fluorescence upon excitation at 490 nm (55). Mitochondria with low

m (depolarized) take up little JC-1 and emit a low
level of green fluorescence, indicative of the monomeric form of JC-1.
A major advantage of the JC-1 assay is that the energy status of
mitochondria can be observed in individual cells and a diagnosis made
to determine whether 1) a majority of mitochondria are energized (red),
2) most mitochondria are depolarized (green), or 3) there is a
mixture of energized and depolarized mitochondria in the same cell
(partial depolarization) (56).
Cultures were seeded as described above, incubated for 24 h, and
then exposed to graded concentrations of arsenite or mClCCP at the
indicated concentrations. Cell cultures were sampled after 2, 4, and
8 h of chemical exposure to study the kinetics of loss of

m. At each of the respective time points, 5 µl of
JC-1 stain (from a 400 µM stock in Me2SO) was
added to 200 µl of cell suspension (10 µM final
concentration) taken directly out of the culture flasks (22, 54). Cells
were then incubated with the JC-1 dye for 20 min at 37 °C in the
dark. Then, 20 µl of cell suspension was pipetted onto a slide that
was gently mounted with a coverslip and examined using a Leitz
Aristoplan microscope equipped for epifluorescence. The red
versus green mitochondrial-specific fluorescence from JC-1
was detected using a Leitz I3 filter cube. The JC-1 fluorescence status
in each of the 200 cells was determined for each treatment sample in
the study. Cells were classified as energized (red), depolarized
(green), or partially depolarized (mixture of red and green
mitochondria in same cell). Mitochondria in control cultures fluoresced
bright red after the 20-min incubation indicative of J-aggregate
formation at high membrane potential (56). Also, cell viability
remained unchanged during the 20-min labeling period. Additional
toxicity tests showed no measurable deleterious effects of 10 µM JC-1 on J-aggregate-linked red fluorescence and cell viability over 4 h of continuous exposure. Mitochondria in control cultures fluoresced bright red after the 20-min incubation period in
JC-1, indicating uptake and retention of JC-1 molecules in mitochondria
with high membrane potential. In fact, the J-aggregate-mediated red
fluorescence in mitochondria has been shown to be dependent upon the
maintenance of high membrane potential (56). Also, cell viability
remained unchanged during the 20-min incubation period.
Statistical Analysis of the Data--
Statistical evaluations of
all data sets were performed using the statistical program NCSS 6.0 (Kaysville, UT). Percentage data were transformed by arc sine prior to
statistical analysis to normalize the data. The data were analyzed by
analysis of variance followed by post-hoc testing using Fisher's least
significant difference test to determine all possible differences among
control and treatment groups. All statistical evaluations were
performed at a significance level of p < 0.05.
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RESULTS |
Activation of the Surface IgM Receptor Protects ST486 Cells from
Arsenite-induced Apoptosis during an 8-h Period--
The ST486 cell
line is a model system for time-dependent IgM-mediated
apoptosis (17-19) as well as apoptosis induction by the arsenite and
other chemicals (22, 23). However, the kinetics of apoptosis induction
by arsenite treatment compared with surface IgM receptor activation
differed markedly. Sodium arsenite induced apoptosis in ST486 cells as
early as 4 h following exposure as evidenced by levels of PARP
cleavage of over 50% at this time point (Fig.
1,
anti-IgM). In contrast,
no PARP cleavage was detected at 4 h following cross-linking of
surface IgM (Fig. 1, +anti-IgM, 0 µM
AS). Similar to PARP cleavage, morphological changes
characteristic of apoptosis were detected as early as 4 h
following exposure to 20 µM arsenite (Fig.
2A, shaded bars),
whereas apoptosis induction following surface IgM cross-linking was
detected only at 24 h (Fig. 2A, black
bars).

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Fig. 1.
Cross-linking of surface IgM inhibits the
rapid induction of arsenite-induced PARP cleavage in ST486 cells.
Cultures of ST486 cells were established at a density of 4 × 105 cells/ml and grown for 24 h. IgM antibody at 5 µg/ml (+anti-IgM) or glycerol ( anti-IgM) was
added 20 min prior to the addition of arsenite, at the indicated
concentrations. At 4 h following chemical addition, aliquots were
taken, and cells were lysed and subjected to SDS-polyacrylamide gel
electrophoresis and immunoblotting using PARP antibody that recognizes
the 113-kDa uncleaved protein and the 85-kDa cleavage fragment. The
percentage of cleaved PARP, as determined by densitometry, is indicated
below each lane. The results are representative
of a minimum of three independent experiments.
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Fig. 2.
Surface IgM cross-linking provides transient
protection against the induction of morphological apoptosis, as well as
PARP and caspase-9 cleavage in ST486 cells that is independent of the
order of addition of antibody or arsenite. A, cultures
of ST486 cells were treated with IgM antibody (IgM,
solid bars), 20 µM sodium arsenite
(AS, shaded bars), IgM antibody plus arsenite
(IgM + AS, striped bars), or left untreated
(control, open bars). In those cultures subjected to IgM
cross-linking, antibody was added 20 min prior to the addition of
arsenite. After incubation for the times indicated, aliquots were taken
and morphological apoptosis was quantified using Hoechst
33342/propidium iodide staining as described under "Experimental
Procedures." The mean values and S.E. were established from three
independent experiments. Statistically significant
differences (p < 0.05) between samples treated with
arsenite alone and those treated with IgM antibody prior to arsenite
addition were found at 4, 6, and 8 h. Cultures treated with
arsenite alone were significantly different from control cultures for
all time points, whereas cultures treated with anti-IgM plus arsenite
were significantly different from controls at 6, 8, and 24 h.
B, cultures of ST486 cells were treated with 20 µM sodium arsenite (AS) or IgM antibody plus
arsenite (anti-IgM + AS) as described above and sampled at
the indicated times. Cells were lysed and subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotting using PARP
antibody or an antibody to caspase-9 (CSP 9), which detects
the 47-kDa parental protein, plus 37- and 35-kDa cleavage products. The
percentage of cleaved PARP and caspase-9, as determined by
densitometry, is indicated below the lane.
C, cultures of ST486 cells were treated with 20 µM arsenite for a total of 4 h. IgM antibody was
added as follows: no antibody ( IgM), antibody 20 min prior
to chemical addition ( 20m), or 30 min, 1 h, 2 h,
or 3 h after chemical addition (+30m, +1h,
+2h, +3h, respectively). After a total of 4 h of chemical exposure, lysates were made and subjected to protein
immunoblotting with PARP antibody.
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Despite the rapid induction of apoptosis by arsenite, cross-linking of
surface IgM prior to addition of arsenite almost completely suppressed
arsenite-induced PARP cleavage at 4 h (Fig. 1,
anti-IgM compared with +anti-IgM). IgM-mediated
suppression of arsenite-induced apoptosis was confirmed by the
morphological assay (Fig. 2A). Significantly fewer apoptotic
cells were detected in cultures treated with IgM-antibody plus arsenite
(striped bars) compared with those exposed to arsenite alone
(shaded bars) at 4 h, and reduced levels of apoptosis
due to IgM cross-linking were sustained for a period of at least 8 h. Consistent with morphological apoptosis, reduced levels of PARP
cleavage were detected over several hours, with only a modest increase
appearing at the later time points of 6 and 8 h (Fig.
2B, PARP, AS compared with AS + anti-IgM).
Caspase-9 is a proximal caspase in the apoptotic pathway. It is
activated upon release of cytochrome c from mitochondria, an
event that is central to the execution of the apoptotic program and
common to a broad spectrum of apoptotic stimuli. The activation of
procaspase-9 involves the autocatalytic cleavage of the full-length 47-kDa protein to generate fragments of 35 and 17 kDa. Among the substrates cleaved by activated caspase-9 is caspase-3. In addition to
autocatalytic cleavage, procaspase-9 can be cleaved by
mitochondrial-associated/activated caspase-3 to generate a 37-kDa
fragment, which contributes to the amplification of caspase-9
processing. IgM cross-linking delayed the generation of both the 35- and 37-kDa cleavage products of procaspase-9 in a manner that
paralleled the delay in PARP cleavage over the 8-h period (Fig.
2B).
In the experiments described above, IgM antibody was added 20 min prior
to the addition of arsenite. However, IgM cross-linking also blocked
PARP cleavage when added to cultures up to 2 h after the chemical
(Fig. 2C). Arsenite itself is a potent inducer of stress
kinases, including p38 and JNK in many cell types, including those
derived from BL. We have previously shown that activation of stress
kinases by arsenite occurs rapidly in ST486 cells, reaching maximal
levels within 1 h of chemical addition (23). Thus, IgM cross-linking blocks arsenite-induced apoptosis after the immediate signaling events induced by arsenite, such as stress-kinase activation, but prior to the cleavage of pro-caspase-9. Together, these data suggest the possibility that mitochondria may be a target of
IgM-mediated protection.
Cross-linking of the Surface IgM Receptor Inhibits Mitochondrial
Depolarization Induced by Arsenite--
Loss of 
m is
an important marker of mitochondrial involvement in the apoptotic
pathway. This event is often accompanied by permeability transition and
release of cytochrome c leading to caspase-9
cleavage/activation. We used the mitochondrial membrane-sensitive fluorochrome JC-1 to measure 
m and to
determine if IgM cross-linking inhibited the depolarization of
mitochondria by arsenite compared with mClCCP (Fig.
3A). This latter agent
directly affects mitochondrial function, causing a rapid and extensive
uncoupling of oxidative phosphorylation leading to collapse of

m (53, 54).

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Fig. 3.
Cross-linking of surface IgM delays the loss
of  m following
exposure to arsenite but not to mClCCP and fails to protect against
mClCCP-induced apoptosis. A, cultures of ST486 cells
were treated as follows: IgM antibody alone (IgM), 20 µM arsenite (AS), IgM antibody plus 20 µM arsenite (IgM + AS), 5 µM
mClCCP (CCP), or IgM antibody plus 5 µM mClCCP
(CCP + IgM). Aliquots of cultures were taken at the
indicated times, and loss of  m was determined using
the membrane potential-sensitive JC-1 fluorochrome as described under
"Experimental Procedures." The graph shows the
percentage of cells remaining polarized (normal membrane potential).
The mean values and S.E. were established from three independent
experiments. The values for cultures treated with IgM plus arsenite
were significantly higher than those for cultures treated with arsenite
alone at 2 and 4 h, whereas the values for cultures treated with
anti-IgM plus mClCCP were significantly lower than those for cultures
treated with mClCCP alone at 2, 4, and 8 h (p < 0.05). B, cultures of ST486 cells were treated
with IgM antibody (+anti-IgM) or glycerol
( anti-IgM) for 20 min prior to the addition of 2, 5, or 10 µM mClCCP (CCP) or 20 µM
arsenite (AS). At 4 h following chemical addition,
aliquots were taken, and cells were lysed and subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotting using PARP
antibody that recognizes the 113-kDa uncleaved protein and the 85-kDa
cleavage fragment. The percentage of cleaved PARP and caspase-9, as
determined by densitometry, is indicated below each
lane. C, cultures of ST486 cells were treated as
described for B, and aliquots were collected and subjected
to analysis of morphological apoptosis at the indicated times. The
treatments are: untreated (control, open bars), IgM antibody
(IgM, solid bars), 5 µM mClCCP
(CCP, shaded bars), IgM antibody plus mClCCP
(IgM + CCP, striped bars). The mean values and
S.E. were established from three independent experiments. Statistically
significant differences (p < 0.05) between samples
treated with mClCCP alone and those treated with anti-IgM plus mClCCP
were found at 6 and 8 h, with no statistical difference revealed
at 4 h.
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A progressive decline in 
m was detected in
arsenite-treated cells (AS) at 2-8 h of chemical exposure. Prior
cross-linking of surface IgM (AS plus IgM) inhibited the
loss of 
m over a 4-h period in a manner that
paralleled the delay of caspase-9 and PARP cleavage and morphological
apoptosis. In contrast, IgM cross-linking failed to protect against,
and even augmented, loss of 
m induced by 5 µM mClCCP (CCP compared with CCP plus IgM). Further
analysis revealed that IgM cross-linking provided only minimal
protection against mClCCP-induced PARP or caspase-9 cleavage (Fig.
3B) that was limited to the lowest concentration of 2 µM. No protection was observed for 5 or 10 µM mClCCP, concentrations that gave levels of PARP
cleavage similar to 20 µM arsenite. Analysis of
morphological apoptosis induced by 5 µM mClCCP over a
period of 8 h revealed that IgM cross-linking did not protect, but
actually potentiated, apoptosis induced by this toxicant (Fig.
3C).
The ability of IgM cross-linking to transiently inhibit mitochondrial
depolarization by arsenite, but not by mClCCP, suggests that protection
may be associated with a specific mitochondrial target involved in
apoptosis, as opposed to a general stabilization of mitochondrial
function. However, such an effect should not be limited to arsenite but
should extend broadly to other agents that activate a mitochondrial
pathway for apoptosis induction. Thus, we investigated whether IgM
cross-linking conferred protection to apoptosis induced by other
chemicals that differ in their initial molecular targets in cells as
well by potential endogenous effectors of apoptosis (Fig.
4). Arsenite interacts with sulfhydryl
groups in proteins and can generate reactive oxygen species. Other
chemicals used were camptothecin, an inhibitor of DNA topoisomerase I;
etoposide, an inhibitor of DNA topoisomerase II; and the protein
synthesis inhibitor, cycloheximide (Fig. 4A). We also
examined the effect of anti-IgM pretreatment on apoptosis induced by
hyperthermia (Fig. 4, B and D) and by C6-ceramide
(Fig. 4, C and D). Ceramide is generated by the
hydrolysis of complex sphingolipids and has emerged as an important
coordinator of cellular stress responses in a number of cell types,
including immune cells (reviewed in Ref. 57). It is considered a second
messenger in apoptotic pathways, and it is induced by a variety of
exogenous and endogenous stresses, including chemicals, bacterial
endotoxin, hyperthermia, and hypoxia (57-59). All of the above
treatments induced apoptosis in ST486 cells within 4-6 h of exposure.
Prior cross-linking of surface IgM substantially inhibited PARP
cleavage and apoptosis induction.

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Fig. 4.
IgM-mediated protection extends to a variety
of chemical toxicants with different molecular targets in cells.
Cultures of ST486 cells were treated either without ( ) or with (+)
IgM antibody for 20 min prior to addition of: A,
camptothecin (CPT, 1 µM), etoposide
(ETP, 10 µM), cycloheximide
(CHX, 5 µg/ml), arsenite (AS, 20 µM); B, hyperthermia for 30 min at 43 °C;
C, C6-ceramide at the indicated concentrations. After 4 h (for A) or 6 h (for B and C) of
incubation at 37 °C, lysates were made and subjected to protein
immunoblotting for PARP or for caspase-9 (CSP9) (cleavage
products are indicated by arrows). D,
morphological apoptosis for cells treated as in B and C. Values represent averages of two independent experiments ± S.E.
Statistical differences (p < 0.05) were found for
samples exposed to hyperthermia (hs) or ceramide
(cer) alone compared with those pretreated with anti-IgM
(ST+IgM) for all treatments.
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Contribution of the MEK/ERK and
PI3K/PDK1/AKT Pathways to IgM-mediated Protection
against Apoptosis--
A number of signaling pathways are activated as
a consequence of engagement of the surface IgM receptor, including
MEK/ERK and PI3K/PDK1/AKT. However, their role in regulating cellular responses to stress is unclear. We examined the potential contribution of these pathways in IgM-mediated protection against apoptosis. Consistent with published studies, we found that IgM cross-linking rapidly induced the phosphorylation of AKT and ERK in ST486 cells (Fig.
5A). Although phosphorylation
was sustained over the first 4 h of treatment, the levels of
phosphorylated kinases declined somewhat from 4 to 8 h (Fig.
5B), particularly for AKT. Exposure of cells to arsenite
accelerated the loss in signal for both kinases. It should be noted
that, although the two isoforms of ERK, p42 and p44, are not completely
resolved here, they showed similar activation profiles in all
experiments. In addition, although the results shown for AKT were
obtained using an antibody specific for phosphorylation at threonine
308, similar results were obtained using a serine 478-specific
antibody.

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Fig. 5.
IgM cross-linking induces transient
phosphorylation of ERK and AKT. A, ST486 cells were
exposed to 0, 5, or 10 µg/ml anti-IgM for 20 min. Lysates were made
and subjected to immunoblotting with antibodies specific for dual
phosphorylated ERK (p-ERK) or AKT phosphorylated at
threonine 308 (p-AKT), then re-probed with antibodies that
recognize each kinase regardless of phosphorylation state.
B, anti-IgM was added to ST486 cells at 5 µg/ml. After 20 min of exposure, the parent culture was divided into three aliquots, to
which arsenite was added to yield concentrations of 0 ( AS), 2, or 20 µM. Samples were taken from
each culture at the times indicated after arsenite addition, and cell
lysates were made and subjected to immunoblotting for p-AKT and p-ERK
as in A. Numbers beneath each lane
indicate percentage of signal compared with parent culture treated with
anti-IgM only for 20 min (0 h).
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Both of these signaling pathways are well documented in their ability
to promote proliferative and/or survival responses in cells. Thus, we
assessed the potential contribution of each pathway to IgM
receptor-mediated protection using specific chemical inhibitors (Fig.
6). The inhibitors used were: LY294002
(LY), an inhibitor of PI3K that is upstream of AKT; U0126, an inhibitor
of MEK1/2, the kinase immediately upstream of ERK1/2; AKTi5, a
phosphatidylinositol ether analog that binds the pleckstrin homology
domain of AKT and inhibits its phosphorylation (52); or rapamycin, an
inhibitor of the mTOR, which is a downstream substrate of AKT.
Each of these agents effectively blocked IgM-mediated phosphorylation
of the expected target kinases the concentrations used (Figs.
6C and 8A).

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Fig. 6.
IgM-mediated protection against
arsenite-induced apoptosis is blocked by the MEKK1/2 inhibitor
U0126 and the PI3K inhibitor LY294002. Cultures of ST486 cells
were treated with (A) LY or U0126 or (B) AKTi5 or
rapamycin (RAP) at the indicated concentrations for 1 h. After that time, either anti-IgM (+anti-IgM) or glycerol
( anti-IgM) was added to the appropriate cultures for an
additional 20 min. Cultures were then exposed to 20 µM
arsenite ( AS or +AS), as indicated. At 4 h
following addition of arsenite, lysates were made and subjected to
immunoblotting with PARP antibody. The percentage of cleaved PARP, as
determined by densitometry, is indicated below each
lane. C, cultures of ST486 cells were treated
with 0, 10, 20, or 40 µM AKTi5 for 1 h prior to the
addition of anti-IgM as indicated. After an additional 1-h incubation,
protein lysates were made and immunoblotted with antibodies that
recognize AKT and phospho-AKT. The percentage of signal for phospho-AKT
in anti-IgM plus AKTi5-treated cultures compared with the culture
treated with anti-IgM alone is indicated below each
lane.
|
|
The inhibitors did not induce apoptosis induction or PARP cleavage when
added alone to cells (Fig. 6,
AS/
IgM) or when
added to cultures treated with either IgM antibody
(
AS/+ anti-IgM) or arsenite
(+AS/
anti-IgM) alone for the duration of the
experimental period (i.e. a total of 6 h of treatment).
However, it should be noted that long term exposure (i.e.
24 h) to the high concentration of LY, U0126, or AKTi5 did induce
some apoptosis.
As shown previously, IgM cross-linking effectively blocked
arsenite-induced PARP cleavage at 4 h (Fig. 6,
+AS/
anti-IgM compared with
+AS/+anti-IgM for 0 µM inhibitor).
Prior exposure of cells to graded concentrations of LY or U0126 (Fig.
6A) resulted in a partial reversal of the protective effect
of IgM cross-linking, whereas AKTi5 and rapamycin did not (Fig.
6B). The later two inhibitors failed to reverse IgM-mediated
protection at inhibitor concentrations as high as 40 µM
and 40 nM, respectively (data not shown). In addition, wortmannin, another PI3K inhibitor that is structurally unrelated to LY, acted in a similar manner (data not shown). We were
unable to determine the effects of simultaneously blocking both the ERK
and PI3K pathways, because the combined treatment of ST486 with U0126
plus LY (or wortmannin) resulted in substantial levels of apoptosis
within the few hours required for the experiment. However, treatment of
cells with phorbol ester, a potent activator of ERK, also resulted in
protection against arsenite-induced apoptosis that was inhibitable by
U0126 (data not shown). Together, these data suggest the independent
contributions of the MEK/ERK pathway, as well as components of the PI3K
pathway, separate from AKT or mTOR, to cell survival signals that are
initially activated by surface IgM cross-linking.
MEKK/ERK and PI3K-mediated Signals Converge on
Phosphorylation of Bad--
Activation of the MEK/ERK and PI3K/AKT
pathways have several possible outcomes in cells, including modulating
of mitochondrial resistance to apoptotic stimuli. Therefore, we
examined the effect of IgM cross-linking on Bad, a pro-apoptotic member
of the Bcl-2 family of proteins that promotes the release of cytochrome
c/caspase-9 activation and a potential downstream target of
both of these signaling pathways.
The pro-apoptotic activity of Bad is mediated by its ability to
interact with, and inhibit, the anti-apoptotic activity of Bcl-2 and
Bcl-XL. Phosphorylation of Bad at serine 112 and/or serine
136 results in its release from these proteins, thereby facilitating
their anti-apoptotic function. Serine 112 is a known, although
indirect, substrate of ERK, whereas serine 136 is a substrate of AKT
(46-51). We found that cross-linking of surface IgM in ST486 cells
rapidly induced phosphorylation of Bad at serine 112 (Fig. 7A). In contrast, we were
unable to detect phosphorylation of Bad at serine 136 (data not shown).
Similar results were found for a second BL cell line, CA46.
Importantly, we found that phosphorylation of Bad serine 112 was
blocked by inhibition of ERK activation with U0126, as expected, but
also with the PI3K inhibitor LY (Fig. 7B), whereas rapamycin
had no effect. Thus, the data suggest that, in addition to the MEK/ERK
pathway, a downstream target of PI3K, but not AKT/mTOR, contributes to
IgM-mediated protection against apoptosis as well as to Bad
phosphorylation at serine 112.

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Fig. 7.
Anti-IgM treatment induces phosphorylation of
Bad at serine 112 that can be inhibited by U0126 and LY294002.
A, cultures of ST486 and CA46 cells were treated with
anti-IgM, or left untreated, as indicated for 1 h. Lysates were
made and immunoblotted using antibodies specific for Bad phosphorylated
at serine 112 or with antibody that recognizes Bad regardless of
phosphorylation state. B, cultures of ST486 cells were
treated with LY, U0126, or rapamycin (RAP) at the indicated
concentrations for 1 h prior to the addition of anti-IgM. Lysates
were made 1 h after antibody addition and subjected to
immunoblotting with an antibody specific for Bad serine 112.
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RSK is a substrate of ERK responsible for phosphorylation of numerous
protein targets, including Bad at serine 112 (50, 60). In addition,
recent studies show that MEKK/ERK and PI3K/PDK1 together contribute to
the activation of RSK1 and 2 by a series of independent
phosphorylations (43-45). The regulation of RSK is complex and is
described in more detail under "Discussion." Briefly,
ERK-dependent phosphorylations occur at sites in its CTD
and linker region, including serines 380/386, whereas PDK1 phosphorylates serines 221/227 in the NTD. These multiple
phosphorylations confer full activity to the amino-terminal kinase.
Thus, we predicted that U0126 and LY would inhibit phosphorylation of
the critical sites on RSK that are dependent upon the activity of
MEK/ERK (i.e. serines 380/386) and PI3K/PDK1 (serines
221/227), respectively, thereby blocking the phosphorylation of Bad at
serine 112. (It should be noted that the phospho-specific antibodies
used in these experiments recognize both isoforms of RSK, 1 and 2.)
Inhibition of the MEK/ERK pathway using U0126 blocked the
phosphorylation of RSK serine 380/386 following IgM cross-linking (Fig.
8A). Consistent with published
studies on other cell types, PDK1 was constitutively phosphorylated in
ST486 cells, as was its target in RSK, serine 221/227, although we
detected a small, but consistent increase in the intensity of
phospho-serine 221/227 following IgM cross-linking. Importantly,
treatment with LY effectively blocked the phosphorylation of this site,
in control and in antibody-treated cultures, indicating a requirement
for PI3K. Rapamycin had no effect on phosphorylation of either site,
consistent with its failure to inhibit Bad phosphorylation or reverse
IgM-mediated protection against apoptosis (Fig. 8B).

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Fig. 8.
The MEKK1/2 inhibitor U0126 and the PI3K
inhibitor LY block anti-IgM-mediated phosphorylation of serine 380/386
and serine 221/227 of RSK, respectively. A, cultures of
ST486 cells were treated with U0126 (20 µM), LY (25 µM), or no inhibitor ( ) as indicated, for 1 h.
Anti-IgM was then added to cultures as indicated (+). After an
additional 1 h, lysates were made and subjected to immunoblotting
with antibodies that recognize phospho-AKT, phospho-ERK,
phospho-Bad/serine 112 (lower band), phospho-PDK1, RSK
phosphorylated at serine 221/227 (lower band), or RSK
phosphorylated at serine 380/386. B, cultures were treated
with 25 µM LY or 20 nM rapamycin
(RP) for 1 h then treated with anti-IgM, and lysates
were subjected to immunoblotting as in A.
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DISCUSSION |
Our study shows, for the first time, that cross-linking of surface
IgM initially confers a survival signal that protects ST486 cells
against the induction of apoptosis for a period of several hours
following BCR engagement. This protective effect extends to a variety
of chemicals with different molecular targets, including inhibitors of
transcription and protein synthesis, as well as to hyperthermia and
ceramide. Furthermore, BCR-mediated protection targets mitochondrial
effectors of apoptosis, blocking the loss of 
m
and cleavage of caspase-9.
GC B-lymphocytes rapidly undergo apoptosis when deprived of appropriate
cell-cell contact or when exposed to chemical/physical stress. Loss of
signals derived primarily from follicular dendritic cells results in
the rapid degradation of the Fas-associated death domain
(FADD)-inhibitory protein, FLIP, and induction of apoptosis by
activation of the FAS/FADD/caspase-8 pathway (2-5). Association of GC
B-cells with follicular dendritic cells inhibits this response by
preventing the loss of FLIP. Importantly, this is a type I Fas pathway,
independent of mitochondrial effectors of apoptosis, and is therefore
unaffected by the expression of members of the mitochondrial-associated
Bcl-2 family of proteins.
However, GC B-cells are also vulnerable to many pro-apoptotic
signals that exert effects through a mitochondrial route. Apoptosis induced by engagement of antigen receptor during the process of negative selection against self-reactive B-cells requires mitochondrial signals (61-65). In addition, conditions that may alter the GC microenvironment, including endogenous stresses that occur during infection (i.e. hyperthermia, anoxia, and septic shock) as
well as exogenous stresses resulting from drug/chemical exposure
(66-68), also induce apoptosis in B-cells. In
contrast to the Fas pathway described above, stress-associated stimuli
activate mitochondrial effectors and that can be modulated by Bcl-2 and
its related proteins (22-25).
Survival factors within the GC, such as CD40, can protect mitochondria
by up-regulation of Bcl-2-related proteins, A1 and BclX, via the
transcriptional trans-activator NF
B (13, 14). Because these
responses involve new protein synthesis, they are postulated to
contribute to long term B-cell survival. Our results suggest an
additional, but presently overlooked, role for the BCR that
specifically modulates the sensitivity of B-cells to apoptotic stimuli.
Although at present we cannot exclude an interaction between
BCR-mediated signals and the Fas pathway, we have clear evidence that
activation of surface IgM can influence survival of ST486 cells by
stabilizing mitochondria. The BCR-mediated response is immediate and,
unlike CD40 protection, does not require new protein synthesis, as
evidenced by the surface IgM-mediated protection against cycloheximide.
Thus, the transient protection afforded by BCR engagement may ensure a
window of protection during which a decision can be made for either
selection/proliferation or apoptosis of B-cells after encountering antigen.
We found that the protective effect of IgM receptor activation against
chemically induced apoptosis was mediated by PI3K-associated signals
and by the MEK/ERK pathway. Pharmacological inhibition of each
pathway using LY or U0126, respectively, partially reversed IgM-mediated suppression of arsenite-induced apoptosis. Our data also
showed a gradual reduction in the phosphorylation of the target kinases
activated by surface IgM cross-linking that was accelerated in the
presence of arsenite and paralleled the decay in suppression of
apoptosis. However, we found that inhibiting AKT, or downstream target
mTOR, failed to block IgM-mediated protection against apoptosis, thus
suggesting that the required PI3K-mediated signals are independent of
AKT and/or mTOR.
The importance of PI3K activation has clearly been demonstrated in
B-lymphocyte development and functions in both in vivo and
in vitro systems. PI3K-knockout mice are defective in B-cell responses and fail to develop germinal centers. Surface IgM
cross-linking of B-lymphoma cell lines induced a transient activation
of p70S6 kinase, a downstream target of AKT/mTOR, as well
as an initial increase in c-Myc protein (40), both of which are
associated with cell growth and survival. Inhibition of PI3K with LY,
or transfection of cells with dominant-negative PI3K, acted
synergistically with IgM cross-linking to enhance growth inhibition and
apoptosis induction, whereas transfection with constitutively active
PI3K abrogated the cytotoxic effects of IgM cross-linking. Similarly, AKT has been shown to be essential for proliferation of lymphoid cells
(70). However, consequences of PI3K activation in B-cells in all of
these cases are attributed to the PI3K/AKT pathway and subsequent
downstream targets of AKT that effect gene expression. We have
identified a target of PI3K that is activated by engagement of surface
IgM but independent of AKT, specifically, RSK/Bad, which influences
B-cell survival by inducing a rapid and complete protection of mitochondria.
Mitochondrial changes, in particular loss of 
m,
leading to release of cytochrome c and subsequent caspase-9
cleavage, are associated with apoptosis induced by a variety of
stimuli. We found that IgM cross-linking transiently inhibited the loss
of 
m in arsenite-treated cells with kinetics that
paralleled the delay in caspase-9 and PARP cleavage. Such specific
effects suggest the regulation of mitochondrial sensitivity by IgM
cross-linking, possibly mediated by members of the Bcl-2 family of proteins.
ST486 cells have relatively low levels of the anti-apoptotic Bcl-2
protein and relatively high levels of the pro-apoptotic protein Bax
(22, 24). Studies show that IgM receptor- and chemically induced
apoptosis may be mediated, in part, by changes in the levels or ratio
of Bcl-2 to Bax. We did not observe a change in either of these
proteins upon arsenite exposure or IgM receptor cross-linking (data not
shown). However, we found that IgM cross-linking did induce a rapid
phosphorylation of Bad, a pro-apoptotic Bcl-family member. Bad forms
heterodimers with Bcl-2 and Bcl-XL via its conserved BH3
domain, thus inhibiting their anti-apoptotic activities (46, 50, 71,
72). Phosphorylation of Bad at serine 112 or 136 disrupts this
association and results in its sequestration in the cytosol by 14-3-3 protein. Much of the published literature demonstrates that the MEK/ERK
pathway mediates the phosphorylation of serine 112, whereas the
phosphorylation of serine 136 is mediated by the PI3K/AKT pathway,
either directly by AKT, or by the downstream target p70S6
kinase (46-51). However, our results are most consistent with a model
in which both pathways independently contribute to the phosphorylation
of Bad at serine 112 and invoke RSK as a point of convergence.
RSK is a major target activated by the MEK/ERK pathway following
exposure of cells to growth factors, insulin, and other stimuli (73)
and by engagement of the surface IgM receptor (38). It has been invoked
as the kinase primarily responsible for phosphorylation of Bad at
serine 112 in response to ERK activation (48, 49, 60). The regulation
of RSK is complex, but data suggest the involvement of both MEK/ERK as
well as PI3K/PDK1 (43-45). Current models indicate the requirement for
phosphorylation of at least two sites by ERK located in the
carboxyl-terminal domain and in the linker region. These
phosphorylations subsequently activate the carboxyl-terminal
kinase leading to autophosphorylation of serine 380 or 386 in
the linker region of RSK 1/2. This event generates a docking site for
PDK1, which phosphorylates serine 221/227 and activates the
amino-terminal kinase of RSK. Full activity of RSK is dependent
on all of these phosphorylation events. Our data are consistent with
this model in that inhibition of MEK/ERK with U0126 and inhibition of
PI3K with LY specifically blocked phosphorylation of serine 380/386 and
serine 221/227, respectively, and, consequently, the ability of RSK to
phosphorylate Bad serine 112 following surface IgM cross-linking.
Our result showing phosphorylation of Bad as an early response to BCR
activation is consistent with a central role for members of this
protein family in regulating apoptotic responses of B-cells. Studies
show that other components of the germinal center microenvironment, such as CD40 ligation or association with follicular dendritic cells, not only modulate normal B-cell responses but also can protect them from apoptosis induced by antineoplastic drugs (5, 74). Our study suggests not only a role for BCR in modulation of normal
B-cell responses described above but also the potential, along with
co-stimulatory signals, to influence the outcome of antineoplastic
treatment or other forms of immunotherapy.