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From the
Received for publication, September 4, 2002, and in revised form, September 23, 2002
B cells in the germinal center are known to
undergo apoptosis after B cell receptor (BCR) ligation, a process
relevant to immunological tolerance. Human CD27 is a B cell
co-stimulatory molecule. The aim of this study was to compare the
effects of CD27 and CD40 signals on BCR-mediated apoptosis of B cells.
BCR ligation activated mitochondrial apoptotic pathways including down-regulation of Bcl-XL, dissipation of
mitochondrial transmembrane potential, release of cytochrome
c, and activation of caspase-9. Each of these effects was
significantly inhibited by CD27 and CD40. Bik expression was weakly but
significantly down-regulated by CD27 but up-regulated by CD40. BCR
ligation resulted in p53 activation including its phosphorylation at
Ser15, nuclear translocation, and target gene
p53AIP1 induction. CD27 and CD40 clearly suppressed these
processes. Analyses that used dominant-negative p53 variants revealed a
low but still substantial level of BCR-mediated apoptosis and intact
mitochondria-mediated apoptotic pathway. These pathways were further
inhibited by CD27 and CD40, although the cells showed no p53
phosphorylation or p53AIP1 expression. Our results
suggested that, at the mitochondrial level, CD27 and CD40
co-stimulatory signals regulated the p53-amplified apoptotic pathway in
B cells through the inhibition of p53-independent apoptotic pathway
primarily induced by BCR ligation.
B cell fate is controlled by signals through the B cell receptor
(BCR)1 on the cell surface.
BCR signaling initiates receptor editing, apoptosis, anergy, and immune
response (1). The nature of the antigen (Ag)-induced response depends
on the developmental stage of the responding cell, the avidity of the
Ag-BCR interaction, and the availability of co-stimulatory signals from
helper T cells. This differential responsiveness to Ag plays a critical
role in the elimination of autoreactive B cells from the functional
repertoire and results in immunological tolerance (2). The human
Burkitt lymphoma cell line, Ramos, expresses surface IgM as BCR, and
engagement of this BCR with an anti-IgM antibody (Ab) induces apoptotic
cell death. Because of these features, Ramos cells have been used
extensively as a model of clonal deletion of B cells in the germinal
center, and Ramos cells facilitate the dissection of molecular and
biochemical pathways that lead to cell death (3, 4).
Accumulating evidence suggests that BCR ligation activates the
mitochondria-mediated apoptotic pathway (5-8). General agreement exists that alterations in the ratio of proapoptotic to antiapoptotic members of the Bcl-2 family can protect against the initiation or
progression of apoptosis by modulating mitochondrial membrane permeability (9). Previous studies have reported that overexpression of
antiapoptotic protein Bcl-XL prevents BCR-mediated
apoptosis (10, 11) and that BCR ligation induces proapoptotic protein Bik (12).
On the other hand, a growing number of gene products have been
identified as components of the machinery that leads to cell death.
Among these, the tumor suppressor gene p53 is of particular interest.
The p53 protein plays important roles in the control of cell death as
well as cell-cycle inhibition (13, 14). The precise molecular
mechanism(s) through which p53 exerts these effects is not clear but
seems to depend on the ability of p53 protein to act as a transcription
factor. Recently one of the important p53 transcriptional target genes
was found to be p53-regulated apoptosis-inducing protein 1 (p53AIP1) (15). p53AIP1 is localized within the mitochondria
and plays a direct role in the mediation of p53-dependent
apoptosis. Although it was reported that in the murine B lymphoma cell
line WEHI-231, p53 and the p53 target gene, cyclin-dependent kinase inhibitor p21WAF1/CIP1,
are involved in BCR-mediated apoptosis (16), whether BCR-mediated signaling primarily activates p53 remains unresolved.
Among a number of cell surface molecules involved in T-B cell
cooperation and subsequent B cell fate, the B cell molecule CD40, a
member of the TNFR family, mediates a co-stimulatory signal and plays a
pivotal role in B cell proliferation, memory B cell differentiation,
and B cell survival (3, 17, 18). Previous studies have shown that Bcl-2
and Bcl-XL are involved in CD40-mediated rescue from
BCR-mediated apoptosis (17-20). CD27 is also a member of the TNFR
family, and we have previously reported that this molecule mediates a
co-stimulatory signal for B cell proliferation and plasma cell
differentiation in humans (21, 22). Although CD27 signaling, in
addition to CD40 signaling, induces activation of nuclear factor Antibodies--
The following Abs were used in this study: Bax
(Medical & Biological Laboratories, Nagoya, Japan), Bcl-XL,
caspase-3, cytochrome c, poly(ADP-ribose) polymerase (PARP),
p53 (BD Pharmingen, San Diego, CA); caspase-9, phospho-p53
(Ser15) (Cell Signaling Technology, Beverly, MA); NBK
(Bik), Bcl-2, histone H4 (Santa Cruz Biotechnology, Santa Cruz, CA);
cytochrome oxidase type IV (COV IV; Molecular Probes, Eugene, OR);
Cell Cultures and Treatment Conditions--
The Epstein-Barr
virus-negative Burkitt lymphoma Ramos cells (sIgM+
CD27+ CD40+) were purchased from Human Science
Research Resources Bank (Osaka, Japan) and grown in RPMI 1640 medium
supplemented with 25 mM Hepes, 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium
pyruvate, 5.5 × 10 Detection of Apoptotic Cells by Using Fluorescein
Isothiocyanate-labeled Annexin V--
To detect apoptotic cells,
phosphatidylserine exposure was measured by using an annexin
V-fluorescein isothiocyanate apoptosis detection kit according to the
instructions provided by the manufacturer (BD Biosciences).
Propidium iodide was added just before analysis on a FACSCalibur
(BD Biosciences).
Subcellular Fractionation and Immunoblot Analysis--
Nuclear
protein extraction was performed as described previously (15) except
for the use of digitonin instead of Nonidet P-40. Briefly, cells washed
in phosphate-buffered saline were resuspended in cold
TKM10-10 Mitochondrial Transmembrane Potential ( Real-time PCR--
The expression of p53AIP1 (16) was
determined by a method for real-time quantitative reverse
transcription-PCR with use of the GeneAmp 5700 sequence
detection system (PE Applied Biosystems) (24). The sense and antisense
primers for p53AIP1 were 5'-GATCTTCCTCTGAGGCGAGCT-3' and 5'-AAACCCAGCCAGGTGTGTGT-3', respectively. The sense and antisense primers for Construction of Transfected Cell Lines--
The p53
dominant-negative construct, in which cysteine at position 135 in the
p53-binding site is mutated to tyrosine (C135Y), abrogates the
endogenous p53 wild-type function (25). To construct a p53DN-expression
vector, a cDNA that encoded full-length p53DN protein was subcloned
from pCMV-p53mt135 vector (Clontech, Palo Alto, CA)
into pBluescript SK (+) (Stratagene, La Jolla, CA) and finally into the
pBCMGSneo expression vector (26) at the XhoI and
NotI sites, generating a clone termed pBCMGS-p53DN.
pBCMGS-p53WT was also constructed. Ramos cells were electroporated with
pBCMGS-p53DN, pBCMGS-p53WT, or pBCMGSneo alone. The human
CD70 and CD154 and the vector alone (mock)-transfected cells in the
murine pre-B 300-19 cell line have been described previously (22).
Statistical Analysis--
The paired Student's t
test was used to determine the statistical significance of the data.
Values of p < 0.05 were considered significant.
CD27 and CD40 Inhibit the Mitochondria-mediated Apoptotic Pathway
Activated by BCR Ligation--
BCRs on Ramos cells ligated with
anti-IgM Ab displayed their apoptotic figures as annexin V-positive
cells. Apoptotic cells started to appear at 6 h. These cells
constituted approximately 40% of the cells at 24 h and
approximately 50% at 48 h (Fig.
1A). We then compared the
effects of CD27 and CD40 signals on BCR-mediated apoptosis. Ramos cells
were mixed with increasing numbers of transfected cells that expressed
CD70 (CD27L) or CD154 (CD40L), both of which are expressed by activated
T cells, in the presence of anti-IgM Ab. CD27 ligation as well as CD40
ligation inhibited BCR-mediated apoptosis in a
dose-dependent manner (Fig. 1B). When the ratio of transfectant cells to Ramos cells was 20%, CD70- and
CD154-transfected cells exhibited a significant inhibition of apoptosis
of Ramos cells stimulated with anti-IgM Ab compared with
mock-transfected cells (70%, p < 0.05 and 75%,
p < 0.01, respectively). The extent of inhibitory
effect by CD27 ligation was almost comparable to that by CD40 ligation
(no significant difference). These inhibitory effects were specifically
abrogated by the addition of anti-CD70 or anti-CD154 Ab (data not
shown). These results suggest that both CD27 and CD40 signals are
involved in antiapoptotic effects for B cell survival.
In agreement with previous reports that BCR ligation activates the
mitochondria-mediated apoptotic pathway (5-8), the initial cleavage of
caspase-9, caspase-3 and PARP started to appear at 12 h and was
evident at 24 h (Fig.
2A). In addition, Bik was
strongly induced by 6 h, whereas Bcl-XL was gradually
down-regulated to a barely detectable level at 48 h (Fig.
2B). The expression of Bcl-2 was absent in unstimulated
Ramos cells and was induced weakly at a relatively late phase. CD27
ligation suppressed the activation of caspase-9, caspase-3, and the
cleavage of PARP with the same efficacy as that observed for CD40
ligation (Fig. 2A). As previously reported (17-20), CD40
ligation up-regulated Bcl-2 and Bcl-XL as much as 9.7- (p < 0.01) and 6.8-fold (p < 0.01),
respectively (Fig. 2, B and C). Under similar
conditions, CD27 ligation also up-regulated Bcl-2 and
Bcl-XL as much as 5.0- (p < 0.01) and
3.9-fold (p < 0.01), respectively. Interestingly,
proapoptotic Bik was weakly down-regulated (1.3-fold decrease by CD27
ligation, p < 0.05), whereas CD40 ligation resulted in
a 1.6-fold increase in Bik expression (p < 0.01) (Fig.
2, B and C).
Cytochrome c release from the intramitochondrial space into
the cytosol was detectable even after 6 h and increased by 48 h (Fig. 3A). BCR ligation
altered the mitochondrial CD27 and CD40 Inhibit p53 Activation--
p53 can directly engage
apoptotic pathways in the cell and stimulate mitochondrial
perturbations including cytochrome c release (13, 14). To
assess the functional activation of p53 in response to BCR ligation, we
examined p53 phosphorylation at Ser15. p53 phosphorylation
at Ser15 represents a response to cellular stress such as
DNA damage (27). As shown in Fig.
4A, phospho-p53 was identified
at 24 h after BCR ligation, and a steady increase in
phosphorylation level was observed. Phosphorylation of p53 at
Ser6, Ser9, Ser20, or
Ser392 was not detected (data not shown). Recent studies
have demonstrated that p53 phosphorylation at Ser15
decreased its nuclear export activity and resulted in accumulation of
p53 in the nucleus (28). On the basis of this finding, we next examined
the location of p53 after BCR ligation. The intracellular distribution
of p53 was found to be biased toward the nucleus (Fig. 4C).
Recent studies demonstrated that, among the identified target genes of
p53, p53AIP1, an indispensable mediator of the p53-dependent apoptotic pathway, was induced directly by
p53 in response to DNA damage and that p53AIP1 was responsive to
functional p53 by phosphorylation at Ser15 and
Ser46 residues (15). In the present study, the
p53AIP1 gene was clearly induced in Ramos cells at
24 h after BCR ligation compared with unstimulated cells (Fig.
4E). Compared with a mock control at 24 h after BCR
ligation, CD27 and CD40 ligation inhibited p53 phosphorylation at
Ser15 (p < 0.01) (Fig. 4, A and
B), its translocation into the nucleus (p < 0.05 and p < 0.01, respectively) (Fig. 4, C
and D), and p53AIP1 expression induced by BCR
ligation (p < 0.05) (Fig. 4E). These results suggest that CD27 and CD40 signals inhibit p53 activation.
p53 Activation Occurs Downstream of Mitochondria and Is Inhibited
by CD27 and CD40 Signals--
It is assumed that
BCRmediated signaling primarily activates p53 downstream,
which in turn executes the apoptotic process. However, the above
finding of p53 phosphorylation at Ser15 and
p53AIP1 induction on BCR ligation could also be interpreted to mean that p53 activation was secondarily induced in response to
cellular stress such as DNA damage. To examine the direct involvement of p53 activation in BCR-mediated apoptosis, stably transfected Ramos
cells with p53DN or p53WT were prepared. Western blot analysis showed
high levels of both p53DN and p53WT expression, and
fluorescence-activated cell sorter analysis showed no difference in
surface IgM expression between these transfectants (data not shown). As
shown in Fig. 5, A and
B, overexpression of p53WT caused Ramos cells to become hypersensitive against BCR ligation, demonstrating rapid cell death by
6 h (p < 0.01) and magnified p53AIP1
gene expression (p < 0.01) compared with transfected
Ramos cells with vector alone. On the other hand, p53DN still showed a
substantial level of BCR-mediated apoptosis after 24 h incubation
compared with vector control in the presence of anti-IgM Ab
(p < 0.05) despite the fact that p53AIP1 gene expression at 12 h after BCR ligation was almost completely suppressed by p53DN to a basal level compared with p53DN in the presence of control Ab. After BCR ligation, p53DN only slightly suppressed phosphorylation of p53 at Ser15, the
cleavage of PARP, and cytochrome c release compared with vector control (no significance except for PARP cleavage
(p < 0.05), whereas p53WT strongly amplified these
(p < 0.01) (Fig. 5, C and D, and
Fig 6, A and B). Interestingly, although no
significant difference was found in Bcl-XL expression among
these transfectants, up-regulation of Bax and Bik was observed in p53WT
compared with vector control in the presence of anti-IgM Ab
(p < 0.01) (Fig. 6,
C and D). Consistent with the above results,
whereas p53WT strongly magnified the alteration of mitochondrial
Finally, we examined the effects of CD27 and CD40 signals on
BCR-mediated apoptosis of p53DN cells to clarify whether their signaling inhibits p53-independent apoptotic pathway. Compared with
vector control after BCR ligation, CD27 and CD40 signals significantly
inhibited apoptosis of p53DN cells (p < 0.05) (Fig. 7A), up-regulated
Bcl-XL and Bcl-2 expression (p < 0.01)
(Fig. 7, B and C), and suppressed cytochrome
c release into the cytosol (p < 0.01) (Fig.
7, D and E). Thus, these results suggest that CD27 and CD40 signals regulate the p53-independent apoptotic pathway at
the mitochondrial level and consequently regulate the p53-amplified pathway.
In this report we compared the effects of CD70/CD27 and CD154/CD40
interactions that belong to the TNF/TNFR family on human T
cell-dependent B cell survival after Ag stimulation. For
this purpose, we used Ramos cells as a model for germinal center B cells and mimicked provision of T cell help through the selective use
of transfectant cells that express the ligands CD70 and CD154 for the
receptors CD27 and CD40 on B cells, respectively. The potential
physiologic relevance of these TNF/TNFR family molecules in B cell
activation, differentiation, and survival has yet to be fully proved.
However, it is likely that multiple surface molecules are involved in
the fine tuning of T cell-dependent B cell responses and
fate and that different molecules are functionally implicated in each
maturation stage of B cells. In addition, to evaluate the role of p53
in BCR-mediated apoptosis, we developed and used p53DN and p53WT Ramos cells.
First we found that both CD27 ligation and CD40 ligation each clearly
inhibited BCR-mediated apoptosis (Fig. 1B). Of note, these
results are in contrast to those of Prasad et al. (29) who
reported that CD27 signaling itself induced apoptosis of Ramos cells.
The reason for the opposite results is that CD27 might provide
different intracellular signals to B cells under different culture
conditions, for instance the presence or absence of Ag stimulation as
observed with other TNF receptors. For example, Fas (CD95) provides
co-stimulatory signals to resting T cells but triggers apoptosis of
activated T cells (30). Thus, CD27 could provide proapoptotic signals
for B cells in the absence of Ag stimulation or low-affinity Ag
recognition but could provide survival and/or co-stimulatory signals
for activated B cells with high-affinity Ag stimulation.
Our results also showed that both CD27 and CD40 ligation altered the
expression of the Bcl-2 family molecules, mitochondrial transmembrane
potential, cytochrome c release, p53 phosphorylation at
Ser15, nuclear translocation of p53, and p53AIP1
induction (Figs. 2-4). These findings clearly indicate that CD27 and
CD40 signals exhibit their regulatory effects on BCR-mediated apoptosis
at least at the mitochondrial level. It should be noted that Bik was
weakly down-regulated by CD27 ligation, whereas it was up-regulated by CD40 ligation (Fig. 2, B and C). Thus, it is
possible that CD27 signals regulate BCR-mediated apoptosis at the
mitochondrial level in a manner different from CD40 signals through the
alteration of the balance among Bcl-2 family members to regulate
mitochondrial permeability. In addition to Bax, one of the target
proteins for p53, Bik might be involved in the
p53-dependent apoptosis pathway. Because previous studies
have demonstrated that BCR ligation increased the amount of Bik
associated with Bcl-XL during apoptosis (12), Bik may
contribute at least in part to the high apoptotic sensitivity of p53WT
together with Bax to antagonize the function of Bcl-XL.
We also examined the role of p53 in BCR-mediated apoptosis and the role
of CD27 and CD40 signals on this mechanism. Previous studies indicated
that DNA damage could induce p53 phosphorylation at Ser15
as well as p53AIP1 expression (15, 17). In addition, p53 activation seems to occur at a relatively late phase after BCR ligation
when compared with the appearance of annexin V-positive cells and
cleavage of caspases and PARP (Figs. 1A, 2A, and
4A). After BCR ligation, p53DN still revealed a detectable
level of cytochrome c release, cleavage of PARP, p53
phosphorylation, down-regulation of Bcl-XL, and
up-regulation of Bik to levels almost comparable to those observed in
vector control and showed a substantial level of apoptosis despite the
failure of induction of p53AIP1 (Figs. 5 and 6). In
addition, although the exact time of activation of the executioner,
caspase-3, after BCR ligation was not clear, we were able to detect DNA
fragmentation even after 6 h (data not shown). When we used
z-VAD-fmk, a broad-spectrum caspase inhibitor, we observed a complete
inhibition of both DNA fragmentation and p53 phosphorylation at
Ser15 during a 24-h culture period after BCR ligation (data
not shown). Taken together, these results strongly suggest that 1) BCR
ligation initiated a p53-independent apoptotic pathway that resulted in DNA damage, 2) DNA damage in turn activated a p53-dependent
pathway, and 3) both the p53-independent and p53-dependent
apoptotic pathways are located in the mitochondria, and their
activation resulted in amplification of the apoptotic process. This
conclusion is supported by the results of previous studies
demonstrating that splenic B cells from p53-null mice were not more
resistant to apoptosis than normal mice (31), and that BCR ligation
activated a p53-independent pathway of c-Myc-induced apoptosis in
murine WEHI-231 cells (32). To our knowledge, however, no report has described the relationship between p53-dependent and
p53-independent pathways or the relationship between CD27/CD40
signaling and p53 activation in BCR-mediated apoptosis in humans. This
issue is important in the consideration of the potential biological
role of p53 in human B cell fate and responses. It should
be noted that murine B cells showed only a marginal expression of CD27 (33-35) and that a normal B cell response to antigen challenge was
observed in CD27-deficient mice (36). CD27 and CD40 signals primarily
inhibited p53-independent pathway in human BCR-mediated apoptosis at
the mitochondrial level (Fig. 7).
It is possible that the findings obtained in the present study are
specific for Ramos cells. To rule out this possibility, we performed
the same series of experiments using another human B cell line,
P32/ISH. P32/ISH cells express IgM, CD40, and CD27, and are susceptible
to BCR-mediated apoptosis (37). Our results in P32/ISH cells were
similar to those described in Ramos cells (data not shown), thus
indicating that the aforementioned findings were not specific to Ramos
cells but rather were common to B cells.
In conclusion, we have demonstrated in the present study that at the
mitochondrial level CD27 and CD40 co-stimulatory signals suppressed the
p53-mediated apoptotic pathway in human B cells by inhibiting
p53-independent apoptotic pathway induced by BCR ligation. In humans,
CD40 signals induce proliferation of B cells and generation of memory B
cells, and CD27 signals induce marginal proliferation of B cells and
generation of plasma cells, resulting in Ig production (3, 7, 18, 21,
22, 38). Therefore, in humans CD27 signals might rescue the
differentiation of germinal center B cells, especially memory B cells
(35, 39, 40), into plasma cells from apoptosis in the secondary immune
response, whereas CD40 signals might rescue the clonal expansion and
differentiation of germinal center B cells into memory B cells.
We thank Drs. Mitsufumi Mayumi and
Yoshinobu Matsuo for helpful discussion and Yoshie Nitta for
excellent secretarial assistance.
*
This work was supported in part by grants from the Welfide
Medicinal Research Foundation, the Naito Foundation, the Takeda Science
Foundation, the Mochida Memorial Foundation for Medical and
Pharmaceutical Research, AstraZeneca, and the Promotion and Mutual Aid
Corporation for Private Schools of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 24, 2002, DOI 10.1074/jbc.M209050200
1
The abbreviations used are: BCR, B cell
receptor; Ag, antigen; Ab, antibody; TNFR, tumor necrosis factor
receptor; PARP, poly(ADP-ribose) polymerase; p53AIP1,
p53-regulated apoptosis-inducing protein; DN, dominant negative; WT,
wild type.
CD27 and CD40 Inhibit p53-independent Mitochondrial Pathways in
Apoptosis of B Cells Induced by B Cell Receptor Ligation*
,,,
Division of Immunology, Institute for
Medical Science, Dokkyo University School of Medicine, Tochigi
321-0293, Japan, § Division of Clinical Immunology,
Advanced Clinical Research Center, Institute of Medical Science,
University of Tokyo, Tokyo 108-8639, Japan, and
¶ Department of Immunology, Juntendo University School of
Medicine, Tokyo 113-8421, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-B) (18, 23), the role of CD27 signaling in BCR-mediated
apoptosis and p53 activation is still obscure. In the present study we
offer evidence for the crucial roles of both CD27 and CD40 signals in
human BCR-mediated apoptosis. Our results showed that p53 is
secondarily activated and amplifies the apoptotic cascade that is
regulated by both CD27 and CD40 signals at the mitochondrial level.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (Sigma).
2 mM
2-mercaptoethanol, 100 units/ml penicillin, and 100 units/ml streptomycin (all from Invitrogen). Cells were treated with anti-IgM Ab
(Sigma) or control Ab (goat IgG, Sigma) to a final concentration of 10 µg/ml. For co-culture with cells transfected with human CD154 gene,
CD70 gene, or vector alone (22), 1 × 105 Ramos cells
were preincubated with varying amounts of transfectants treated with
mitomycin C (Sigma) in 96-well, round-bottomed plates. After
incubation for 30 min, anti-IgM Ab was added to the culture and cells
were harvested at 24 h. The caspase inhibitors zVAD-fmk and
zLEHD-fmk were obtained from Calbiochem. For treatment with caspase
inhibitors, 1 × 105 Ramos cells were preincubated
with serial dilution of zVAD-fmk or zLEHD-fmk in 96-well,
round-bottomed plates. After the incubation for 30 min, anti-IgM
Ab was added to the culture and cells were harvested at 24 h.
5 (10 mM Tris-HCl, pH 7.6, 10 mM KCl, and 5 mM MgCl2) with
protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride,
20 µg/ml aprotinin, 10 µg/ml pepstatin A, and 10 µg/ml
leupeptin). After incubation on ice for 10 min, cells were lysed by
digitonin (Sigma) to 0.1 mM (final concentration). Nuclei
were removed by centrifugation, and nuclear protein was extracted with
radioimmune precipitation assay buffer (10 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium
deoxycholate, and 1 mM dithiothreitol) supplemented with
protease inhibitors. For cytochrome c release assay, the
cells washed in phosphate-buffered saline were lysed in isotonic buffer
A (10 mM Hepes, 0.3 M mannitol, and 0.1%
bovine serum albumin) plus 0.1 mM digitonin with protease
inhibitors. After incubation on ice for 5 min, cytosol fraction was
collected by centrifugation, and insoluble fraction was disrupted by
sonication with a sonication buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 2 mM EDTA, and 0.5% Tween
20) supplemented with protease inhibitors. For whole cell extractions,
the cell pellets washed in phosphate-buffered saline were resuspended
with 0.5% SDS solution and boiled for 5 min. Proteins (4-8 µg) were
separated on an SDS-PAGE, transferred to an Immobilon-P membrane
(Millipore, Bedford, MA), blocked with 5% skim milk, and immunoblotted
with arbitrary Ab- and horseradish peroxidase-labeled secondary Ab. The
blotting was developed by using the enhanced chemiluminescent substrate
(SuperSignal West Pico, Pierce) and visualized with a LumiVision
analyzer (Taitec Co., Tokyo, Japan). The densitometric analysis was
performed by using a LumiVision analyzer (Taitec Co.).
m)--
Changes in
m were tested by MitoLight apoptosis detection kit according to
the instructions provided by the manufacturer (Chemicon International,
Inc., Temecula, CA). Labeled cells were analyzed on a FACSCalibur.
-actin were 5'-tcacccacactgtgcccatctacga-3' and
5'-CAGCGGAACCGCTCATTGCCAATGG-3', respectively. The reaction master mix
that contained a target hybridization probe labeled with
6-carboxyfluorescein at the 5' end was used. The p53AIP1
probe was 5'-CAGATCTGCTCAAGCTTCCTGCAGTGG-3'. -Actin was
5'-ATGCCCCCCCCATGCCATCCTGCGT-3'. The thermal cycling conditions
included 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min for denaturing and anneal-extension, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Effects of CD27 and CD40 ligation on
BCR-mediated apoptosis of Ramos cells. A, Ramos cells
(1 × 105/well) were stimulated with anti-IgM Ab or a
control Ab (10 µg/ml) for the indicated time periods followed by
determination of annexin V-positive cells as described under
"Experimental Procedures." B, Ramos cells (1 × 105/well) were cultured with increasing numbers of
mitomycin C-treated transfected cells (5 × 103 (5%),
1 × 104 (10%), and 2 × 104/well
(20%)) in the presence of anti-IgM Ab or a control Ab (10 µg/ml),
and the proportion of cells positive for annexin V was measured at
24 h. Data shown are representative of three different experiments
and are presented as mean ± S.D.; *, p < 0.05;
**, p < 0.01.
View larger version (47K):
[in a new window]
Fig. 2.
Effects of CD27 and CD40 ligation on
activation of caspases and expression of Bcl-2 family molecules in
BCR-mediated apoptosis of Ramos cells. Ramos cells (1 × 105/well) were cultured with or without mitomycin
C-treated transfected cells (2 × 104/well) in the
presence or absence of anti-IgM Ab (10 µg/ml) as indicated in Fig. 1.
After the treatment, cells were lysed, followed by Western blotting to
detect the activation of caspases (A) or the expression
level of Bcl-2 family members (B). The blotted membrane was
visualized after development by the enhanced chemiluminescent substrate
as described under "Experimental Procedures." -Actin was
detected as a loading control. C, histogram represents
relative expression of the indicated proteins analyzed in A
and B. Band density quantified as a percentage of the
maximum level in each combination of Ramos cells co-cultured with 20%
transfectants. Data shown are representative of three
different experiments and are presented as mean ± S.D.; *,
p < 0.05; **, p < 0.01).
m (Fig. 3C). These results
confirm that BCR ligation activates the mitochondria-mediated apoptotic
pathway. As expected, CD27 ligation strongly reduced cytochrome
c release after BCR ligation, in which the inhibitory level
was almost the same as CD40 ligation (Fig. 3B). Likewise, CD27 ligation significantly inhibited the dissipation of mitochondrial m, similar to CD40 ligation (Fig. 3C). These results
clearly indicate that CD27 and CD40 signals regulate BCR-mediated
apoptosis at the mitochondrial level through the alteration of the
balance among Bcl-2 family members to regulate mitochondrial
permeability.
View larger version (43K):
[in a new window]
Fig. 3.
Effects of CD27 and CD40 ligation on
mitochondrial permeability in BCR-mediated apoptosis of Ramos
cells. Ramos cells (1 × 105/well) were cultured
with or without mitomycin C-treated transfected cells (2 × 104/well) in the presence or absence of anti-IgM Ab (10 µg/ml) as indicated in Fig. 1. A and B, after
the treatment cytoplasmic fraction was prepared and subjected to
Western blotting to detect cytochrome c release as described
under "Experimental Procedures." Cytochrome oxidase type IV
(COX IV) and -actin were detected as an extraction and
loading control, respectively. C, twenty-four h after the
treatment, cells were harvested and prepared for analysis of
mitochondrial m as described under "Experimental Procedures."
mhigh and
mlow correspond to the emission and
extinction wavelengths, respectively. Data shown are representative of
three different experiments.
View larger version (32K):
[in a new window]
Fig. 4.
Effects of CD27 and CD40 ligation on
functional transactivation of p53 in BCR-mediated apoptosis of Ramos
cells. After culture of Ramos cells (1 × 105/well) with or without mitomycin C-treated transfected
cells (2 × 104/well) in the presence or absence of
anti-IgM Ab (10 µg/ml) as indicated in Fig. 1. A, whole
cell lysates were analyzed by Western blotting with use of Ab for
phosphorylated p53 at Ser15, and its expression level is
presented in the histogram in B. Unphosphorylated p53 was
detected as a loading control. C, both cytoplasm and nuclear
subcellular fractions were prepared and p53 distribution was determined
by using anti-p53 Ab. Histone H4 was detected as a nuclear marker.
D, relative expression of p53 is represented as a ratio of
p53 to histone H4 expression quantified from the band density indicated
in C. E, after co-culture, total RNA of Ramos cells was
extracted, reverse-transcribed into cDNA, and amplified by
real-time quantitative PCR for p53AIP1. The amount of p53AIP1 was
normalized to the level of -actin and designated as a relative
quantity as described under "Experimental Procedures." Data shown
are representative of three different experiments and are presented as
mean ± S.D.; *, p < 0.05; **, p < 0.01.
m, p53DN still showed a substantial level of mitochondrial
m dissipation compared with vector control after BCR ligation
(Fig. 6E). These results suggest that p53-independent and
mitochondrial apoptotic pathways may operate in BCR-mediated apoptosis.
In addition, p53 may be activated secondarily and subsequently drive
apoptosis at the mitochondrial level through p53AIP1, because p53AIP1
is located in the mitochondria and its overexpression causes
dissipation of mitochondrial m (15).
View larger version (36K):
[in a new window]
Fig. 5.
Effects of ectopic expression of p53 on p53
activation and PARP cleavage on BCR-mediated apoptosis of Ramos
cells. Stable transfected Ramos cells with pBCMGSneo
vector that encoded p53WT, p53DN, or vector alone were established as
described under "Experimental Procedures." A,
transfected Ramos cells (1 × 105/well) were
stimulated with anti-IgM Ab (10 µg/ml) for the indicated time periods
followed by determination of annexin V-positive cells. B,
after treatment with control Ab or anti-IgM Ab for 12 h, total RNA
was extracted and subjected to real-time quantitative reaction
temperature-PCR for p53AIP1 as described in Fig. 4E.
C, after treatment for the indicated time intervals,
cytoplasmic fractions or whole cell lysates were prepared for Western
blotting to detect the indicated proteins, and their expression levels
are presented in the histogram in D. -Actin was detected
as a loading control. Data shown are representative of three different
experiments and are presented as mean ± S.D.; *,
p < 0.05; **, p < 0.01.
View larger version (43K):
[in a new window]
Fig. 6.
Effects of ectopic expression of p53 on
mitochondrial permeability in BCR-mediated apoptosis of Ramos
cells. After treatment as described in Fig. 5, cytoplasmic
fraction (A) or whole cell lysates (C) were
prepared for Western blotting to detect the indicated proteins, and
their expression levels are represented in the histograms shown in
B and D, respectively. E, twenty-four
h after the treatment, mitochondrial m was assessed as described
in Fig. 3 (C). m high and m
low corresponded to the emission and extinction
wavelengths, respectively. Data shown are representative of three
different experiments and are presented as mean ± S.D.; *,
p < 0.05; **, p < 0.01.
View larger version (32K):
[in a new window]
Fig. 7.
Effects of CD27 and CD40 ligation on
BCR-mediated apoptosis of Ramos cells that express dominant-negative
p53 protein. Stable transfected Ramos cells (1 × 105/well) with p53DN gene or vector alone were
co-cultured with mitomycin C-treated transfected cells (2 × 104/well) in the presence of control Ab or anti-IgM Ab (10 µg/ml). Twenty-four h after the treatment, annexin V-positive cells
were determined by fluorescence-activated cell sorter analysis
(A), or whole cell lysates (B) or cytoplasmic
fraction (D) were prepared for Western blotting to detect
the indicated proteins. Their expression levels were presented in the
histograms shown in C and E, respectively. Data
shown are representative of three different experiments, and are
presented as mean ± S.D.; *, p < 0.05; **,
p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Division of
Immunology, Institute for Medical Science, Dokkyo University School of
Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan. Tel.:
81-282-87-2401; Fax: 81-282-86-2075; E-mail:
tkobata@dokkyomed.ac.jp.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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