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B/Rel Are at the Divergence of CD40-Mediated Proliferation and Survival Pathways
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Division of Immunology, Department of Medicine, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021;
Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan; and
Department of Internal Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel transcription factors is crucial for CD40-mediated
proliferation. Furthermore, our data indicate that PI-3K is
indispensable for CD40-mediated NF-B/Rel activation. This is
achieved via activation of AKT and the degradation of IB
.
Furthermore, we show that PI-3K activity is necessary for the
degradation of cyclin-dependent kinase inhibitor p27kip.
Therefore, both of these events comprise the mechanism by which PI-3K
controls cell proliferation. In contrast to the absolute requirement of
PI-3K and NF-B/Rel for proliferation, these signaling molecules are
only partially responsible for CD40-mediated survival, as blocking of
PI-3K activity did not lead to apoptosis of anti-CD40-treated
cells. However, the PI-3K/NF-B pathway is still required for
CD40-induced Bcl-X gene expression. Taken together, our data indicate
that multiple survival pathways are triggered via this receptor,
whereas NF-B/Rel and PI-3K are crucial for CD40-induced
proliferation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel (2, 3, 4). Known
signaling events occurring upon CD40 stimulation also include
activation of tyrosine kinases (Lyn), activation of
phosphatidylinositol 3-kinase
(PI-3K),3
phosphorylation of phospholipase C
2, and activation of NF-B/Rel
transcription factors (5). Furthermore, CD40 activation
has been linked to the up-regulation of several surface proteins (CD23,
Fas, ICAM-1, and LFA-1; Ref. 6, 7, 8, 9) as well as
anti-apoptotic molecules (Bcl-X, A20; Ref. 10, 11, 12, 13). It
is believed that induction of these apoptosis inhibitors causes the
augmentation of cell survival induced by CD40, although the actual
mechanism accountable for their up-regulation remains elusive.
Experiments presented here are aimed at investigating the events
involved in CD40-mediated proliferation as well as survival
function.
A prominent consequence of CD40 activation is induction of NF-B/Rel
transcription factors. The NF-B/Rel transcription factor family
consists of five members that form homo and heterodimers that
translocate to the nucleus, where they regulate transcription of
various target genes (14). It is believed that, by
activating these genes, NF-B/Rel participates in both proliferation
and survival in B cells. The NF-B/Rel activity is tightly controlled
by IB proteins that serve to sequester transcription factor
complexes in the cytoplasm (14). Data from many
laboratories have established the sequence of biochemical events that
causes the degradation of IB proteins and release of NF-B/Rel
(15). Interestingly, through studies performed in the
knockout (ko) mice, it became clear that individual members of the
NF-B/Rel family have distinct roles in the immune system. We
reported previously that, although the lymphocyte development was
normal in the c-Rel ko mouse, profound functional defects were observed
in both B and T lymphocyte compartments (16, 17). Namely,
c-Rel-deficient B cells were unresponsive to various mitogenic stimuli,
including CD40. Surprisingly, whereas c-Rel ko cells showed clear
defects in CD40-induced proliferation, CD40-mediated survival was still
intact (17). This observation suggested the existence of
at least two signaling pathways stemming from the CD40 receptor,
distinguishable on the basis of c-Rel involvement.
The recent generation of the PI-3K gene-targeted mouse (p85 ko) also
revealed a profoundly impaired B cell phenotype, remarkably similar to
that of c-Rel ko mice (18, 19). It has become clear that
signaling pathways emanating from the mitogenic receptors in B
lymphocytes travel via activation of PI-3K. For example,
PI-3K-deficient cells were unable to mount proliferation in response to
CD40 ligation, as well as other type of mitogenic stimulation
(18). These findings underlie the importance of studying
PI-3K in the context of activation pathways in B lymphocytes. PI-3K
consists of a 110-kDa catalytic subunit and a tightly associated
regulatory subunit encoded by the p85 gene that has splicing
isoforms p55 and p50 (20). Inhibition of PI-3K in
IL-3-dependent cell lines leads to apoptosis. It has been shown that
PI-3K promotes cell survival by activating AKT kinase, which
phosphorylates and inactivates pro-apoptotic molecule, Bad
(21). In addition, recent reports revealed a connection
between AKT and IKKß which is responsible for targeting IB
for degradation and NF-B/Rel activation (22, 23). Based
on these observations, we decided to investigate the role of PI-3K in
CD40-mediated pathways that lead to cell proliferation and/or survival
and to connect these events to the activation of NF-B/Rel
transcription factors by using c-Rel-deficient mouse.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene-targeted c-Rel mice were generated previously
(16) and together with normal counterparts were bred and
maintained at the Weill Graduate School of Medical Sciences of Cornell
University Animal Facility under pathogen-free conditions. Six- to
8-wk-old mice were used for experiments. p85-deficient mice (PI-3K
ko) were generated as described previously (18, 19) and
maintained at Taconic Farms (Germantown, NY).
Reagents
PI-3K inhibitor- LY 294002 (Sigma, St. Louis, MO; Ref.
24) was dissolved in DMSO and further diluted in cell
culture medium before addition to the cell culture at 10 µM final
concentration (final concentration of DMSO did not exceed 0.1%).
Anti-CD40 agonistic Ab (clone 1C10, rat IgG2a) was kindly provided by
Dr. Maureen Howard (Stanford University, Stanford, CA), affinity
purified from hybridoma supernatant, and used at 10µg/ml
concentration. For depleting splenic T cells, anti-Thy1.2 mAb
(clone J1J) in the form of ascites was kindly provided by Dr. Janko
Nikolic-Zugic (Sloan Kettering Institute, New York, NY), and low-tox
rabbit complement was obtained from Cedarlane Laboratory (Ontario,
Canada). The following flow cytometry staining reagents were purchased
from PharMingen (San Diego, CA): PE-conjugated anti-B220,
FITC-conjugated anti-CD3, and FITC-conjugated anti-CD54
(ICAM-1). Propidium iodide for DNA fragmentation analysis was purchased
from Sigma. Abs for immunoblotting were obtained from the following
sources: anti-p27kip, anti-IB,
anti-Bcl-2, and anti-c-Rel were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA); anti-Bcl-X was purchased from
Transduction Laboratories (Lexington, KY); anti-AKT and
anti-pAKT (detects phosphorylation on S-473 residue) were obtained
from New England Biolabs (Beverly, MA); and anti- actin was
obtained from Sigma. For EMSA, anti-p50 and anti-p65 were
affinity purified from rabbit serum.
Purification of B cells and cell culture
B lymphocytes were isolated from total splenocytes by depleting T cells with anti-Thy1.2 mAb and complement and by allowing macrophages to adhere on the plastic surface. This protocol typically yields 95–98% pure population of B cells that are B220+CD3- as determined by flow cytometry. For in vitro cell culture, B cells were plated at 0.5–1 x 106 cells/ml (for some experiments in triplicates) for indicated time periods in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS, essential amino acids, and antibiotics. Cells were pretreated with LY 294002 inhibitor for 1 h before adding anti-CD40 Ab. At the end of culture, cells were collected from individual wells, washed twice with PBS, and further analyzed.
Proliferation assay
For proliferation assay, cells were plated in triplicate at 2 x 105 cells/well in 96-well round-bottom plates (Fisher Scientific, Pittsburgh, PA). After 48 h of stimulation, cells were incubated with 0.5 uCi [3H]thymidine (Am- ersham, Arlington Heights, IL) for 5–8 h. Cells were then harvested and thymidine incorporation into DNA was quantified by scintillation.
Flow cytometry
For two- and single-color flow cytometry, cells were stained with directly conjugated mAb on ice for 30 min and washed in PBS (1% FBS, 0.1% NaN3). Labeled samples were analyzed on a FACScalibur instrument (Becton Dickinson, Mountain View, CA) equipped with CellQuest software. A semiquantitative determination of the DNA content by propidium iodide (PI) staining was used to assess the percentage of cells undergoing DNA fragmentation (25). For PI staining, cultured cells were washed in PBS and resuspended in a hypotonic buffer (0.1% sodium citrate, 0.1% Triton X-100, 20 µg/ml RNaseA) containing 50 µg/ml PI at 1 x 106. Initially, samples were incubated for 30 min at room temperature (to activate RNase); thereafter, samples were kept on ice. For each sample, at least 5 x 103 events were collected and analyzed using a FACScalibur instrument. The percentage of cells undergoing apoptosis is determined as the relative number of cells with DNA content in subdiploid (subG0/1) area, characteristic of apoptotic cells (25). Apoptosis data are presented as: percent of specific DNA fragmentation = (test - spontaneous)/(100 - spontaneous) x 100.
Western blot analysis
After culture, cells were washed with PBS and lysed in RIPA buffer (25 mM Tris (pH 7.4), 75 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.01% SDS, 2.5 mM EDTA) supplemented with the mixture of protease and phosphatase inhibitors (100 µM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate, and 1 mM aprotinin, leupeptin, and PMSF). For AKT immunoblotting, cell lysis buffer was made according to the manufacturer’s instruction. Equal amounts of whole cell lysates, quantified using the commercial Bio-Rad protein assay (equivalent to 106 cells per sample; Richmond, CA), were loaded onto each lane of a 12.5% SDS-PAGE gel. Polyvinylidene difluoride membrane (Amersham) was used to transfer proteins and was blocked with 2% milk in TBST buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. Membranes were further incubated with primary Ab (conditions and concentration of the Ab vary) and, after washing, incubated with appropriate secondary HRP-conjugated secondary Ab and visualized using commercial chemiluminescence detection kit (ECL Plus; Amersham).
EMSA
The nuclear and cytosolic fractionation procedure was performed
as previously described (26). Nuclear proteins were
quantified by the commercial Bio-Rad protein assay, and EMSA was
performed as described (26). Briefly, 10 µg nuclear
extract proteins was incubated with 20 fmol of
32P-labeled B probe dissolved in an
appropriate buffer. The DNA-protein complexes were resolved on a native
6% polyacrylamide gel, dried, and exposed on a film for
autoradiography. For Ab-inhibition experiments, optimal amount of Ab
was determined by using purified proteins. Appropriate Ab was incubated
with a mixture of nuclear extract and poly(dI-dC) in 1x DNA
binding buffer on ice for 30 min before the addition of
32P-labeled B probe. The reaction was
continued for another 15 min before it was loaded on a native 6%
polyacrylamide gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cross-linking of CD40 on the cell surface by agonistic Ab or
ligand binding generates a very strong proliferation signal for normal
B cells. In dissecting the signaling pathway emanating from CD40, we
observed that NF-B/Rel plays a crucial role in CD40-mediated
proliferation. Specifically, c-Rel-deficient cells exhibit very low
levels of proliferation when stimulated with agonistic
anti-CD40 Ab (Ref. 17 and Fig. 1
). While searching for upstream
signaling molecules that are responsible for CD40-mediated
NF-B/Rel activation, we learned that PI-3K-deficient B lymphocytes
were also hyporesponsive to anti-CD40-induced proliferation
(18, 27). These observations thus prompted us to further
investigate the relationship between PI-3K and NF-B/Rel. We took
advantage of the well-characterized PI-3K inhibitor LY 294002 that
specifically blocks the ATP-binding sites of the p110 catalytic subunit
(24). We found that LY 294002 completely abrogates
proliferation induced via CD40 in both wild-type (wt) and c-Rel ko B
cells (Fig. 1). This is not due to the increased rate of cell death,
because CD40 stimulation maintains cell viability following the
treatment with the PI-3K inhibitor, as shown below in Fig. 5.
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B/Rel nuclear translocation
induced via CD40
A critical consequence of CD40 signaling in B lymphocytes involves
high and sustained activation of the NF-B/Rel transcription factors
(Ref. 28 ; S.A. and H.-C.L., unpublished observations).
Because PI-3K inhibition resulted in a serious impairment in
proliferation that was similar to that of c-Rel-deficient B cells, we
investigated whether PI-3K is connected to NF-B/Rel activation in
splenic B lymphocytes. Intriguingly, using the EMSA, we observed that
PI-3K blocking completely prevents nuclear translocation and activation
of NF-B/Rel complexes upon CD40 and anti-IgM stimulation (Fig. 2A). The inhibition was
observed in both wt and c-Rel ko B cells. Note that c-Rel-deficient
cells mobilize NF-B/Rel complexes to a lesser degree than the wt
cells, due to the lack of the predominant c-Rel member. Translocated
complexes in c-Rel ko B cells consist of p65 and p50 proteins as
identified by an Ab inhibition assay shown in Fig. 2B.
Furthermore, we found that the inhibition of NF-B/Rel activity by
the PI-3K antagonist is not due to diminishment of NF-B/Rel
proteins, because their expression level in the cytoplasm (in both wt
and c-Rel ko cells) remained unchanged regardless of the treatment
(Figs. 2C, 3B, and
data not shown). Rather, these results demonstrate that PI-3K
specifically affects nuclear translocation of NF-B/Rel
complexes.
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B/Rel activation
In an effort to exclude possible nonspecific effects of the
chemical compound serving as pharmacological PI-3K inhibitor, we used
PI-3K-deficient mice (p85 ko) (18) to assay for
NF-B/Rel activation. We found that p85 ko B cells stimulated via
CD40 ligation completely lack the induction of nuclear NF-B/Rel
complexes (Fig. 3A), in a manner similar to that observed
with the use of PI-3K inhibitor (Fig. 2
A). Therefore, these
data strongly support our conclusion that PI-3K action is required for
NF-B/Rel activation. Despite the marked absence of the nuclear
NF-B/Rel proteins, cytoplasmic c-Rel is normally present in p85
ko B cells (Fig. 3B), suggesting that PI-3K is crucial for
permitting nuclear translocation of the NF-B/Rel complexes. Previous
studies have demonstrated that IB degradation is required for
NF-B/Rel activation and nuclear translocation (14, 15).
Considering recently published data (22, 23), it was
reasonable to suggest that the diminishment of nuclear NF-B/Rel
caused by PI-3K inhibition is caused by a block in IB
degradation. To test this hypothesis, we performed Western blot
analysis of the IB inhibitor expression in normal and
p85-deficient mice. As shown in Fig. 3C, degradation of
IB occurred upon CD40 treatment in wt mice (lane
2), but was prevented in the B cells that lack PI-3K activity
(lane 4). This result confirms that PI-3K activation
represents the main pathway that conveys activation signals from the
CD40 receptor to the complex machinery that directs IB
degradation via proteosome pathways.
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A recent report on the Kit225 cell line stimulated with IL-2
concluded that PI-3K promotes proliferation via down-regulating cell
cycle inhibitor p27kip (29).
p27kip is a cyclin-dependent kinase (CDK)
inhibitor whose expression level is reduced by an
ubiquitin-proteosome-dependent mechanism during
G1 phase of the cell cycle (30).
Degradation of p27kip CDK inhibitor is necessary
for the activation of CDK kinases and subsequent cell cycle progression
(30). By examining p27kip expression
levels in B lymphocytes, we discovered that PI-3K inhibitor prevents
CD40-induced down-regulation of p27kip over a 24-
to 48-h period (Fig. 4A).
Consistent with the results obtained using the PI-3K inhibitor
LY294002, we confirm that p27kip down-regulation
is also prevented in mice deficient for PI-3K activity (Fig. 4B). Taken together, our data show that PI-3K regulates both
NF-B/Rel activation and p27kip CDK inhibitor
degradation, events that are essential for cellular proliferation.
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Apart from causing cellular activation, CD40 is also crucial for
maintaining the viability of B lymphocytes both in vitro and in vivo
(1, 2). Our studies led us to further test whether PI-3K
and NF-B/Rel contribute to the CD40-mediated survival pathway. In
accordance with our previous finding (17), CD40 treatment
provides the same level of protection from spontaneous apoptosis to
both wt and c-Rel ko B cells (Fig. 5). LY
294002, when applied alone, induces cell death in both wt and c-Rel ko
B lymphocytes (Fig. 5), suggesting that PI-3K is important for
maintaining the viability of resting splenic B cells. Intriguingly,
CD40 stimulation can partially rescue cell death induced via LY 294002,
in both wt and c-Rel-deficient B cells (Fig. 5). We confirmed these
findings by counting the actual number of live cells, where we observed
that in all experiments the number of live cells was comparable in
nontreated control samples and samples treated with combination of
LY294002 and CD40 stimulation (data not shown). These data suggest that
certain aspects of CD40-stimulated apoptosis protection are
operational, even in the absence of both c-Rel and PI-3K activity. To
find effectors of the CD40-mediated survival, we tested known
apoptosis-blocking molecules for their role in this pathway.
PI-3K inhibitor blocks phosphorylation of AKT induced by CD40 stimulation
Previous studies in several cell systems, including B lymphocytes,
demonstrated that AKT kinase is one of the primary targets of PI-3K
(31). Because AKT was shown to prevent apoptosis
(21, 32), we investigated its role in CD40-mediated
survival pathway. As shown in Fig. 6, AKT
phosphorylation occurs within 15 min after anti-CD40 treatment, and
increases up to 1 h poststimulation. However, pretreatment of
cells with LY294002 compound potently blocks phosphorylation, while
having no effect on overall levels of nonphosphorylated AKT in the
cytoplasm (Fig. 6). This result verifies that, in B lymphocytes, CD40
activates AKT exclusively via PI-3K pathway. Nonetheless, the PI-3K/AKT
survival pathway contributes only partially to the CD40-mediated
apoptosis protection in splenic B lymphocytes, because we observed that
lack of PI-3K activation did not significantly alter CD40-mediated
survival function (Fig. 5).
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Bcl-X is recognized to be a major anti-apoptotic protein that
is induced upon CD40 stimulation (10, 12, 13). To test
whether this protein confers CD40-mediated protection from LY
294002-inflicted cell death, we analyzed its expression in wt and c-Rel
ko B cells that were stimulated with CD40 in the presence/absence of
the PI-3K inhibitor (Fig. 7A).
Surprisingly, we observed a complete absence of Bcl-X expression in
c-Rel deficient cells. We further performed experiments to assess Bcl-X
expression in p85 ko B cells. As shown in Fig. 7B,
p85-deficient B cells also lack Bcl-X induction upon CD40
stimulation. This and our other observations (A.O. and H.-C.L.,
unpublished data) thus indicate that Bcl-X is directly regulated via
c-Rel. Bcl-X induction in wt cells was also significantly perturbed by
addition of the PI-3K inhibitor and correlates with the compound’s
ability to inhibit NF-B/Rel activity (Fig. 2A). In
contrast to Bcl-X, Bcl-2 is constitutively present in B lymphocytes and
remained constant and unaffected by c-Rel deficiency or PI-3K
inhibition in all samples tested (Fig. 7A). Taken together,
these data indicate that Bcl-X expression is regulated by both
NF-B/Rel and PI-3K, representing one of the multiple survival
pathways triggered via CD40.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel transcription factor. In splenic B cells, we found that
PI-3K and NF-B/Rel pathway is necessary for CD40-induced
proliferation. In contrast, this pathway is not the only survival
pathways used by CD40, because cells remain viable even though this
pathway is blocked. These data thus suggest the existence of other
survival pathways that are independent of NF-B/Rel and Bcl-X.
One of the intriguing findings of this study is a definitive link
between PI-3K, NF-B/Rel activation, and cell proliferation (Fig. 8). Using PI-3K ko mice or
pharmacological inhibitor LY294002, we showed that PI-3K controls the
nuclear translocation of CD40-induced NF-B/Rel complexes,
through regulation of IB degradation. Together with two
recent reports showing a direct interaction between AKT and
IKK in cell lines (22, 23), we propose that PI-3K may
regulate IB degradation in splenic B lymphocytes via AKT. Indeed,
we showed that AKT phosphorylation and activation is blocked when PI-3K
activity is inhibited. Thus, our study established the necessary
signaling pathway responsible for CD40-mediated proliferation that
involves PI-3K and NF-B/Rel activation.
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B/Rel activation. However, it is most likely that these two
PI-3K-mediated events are independently regulated. First, it is
noticeable that these two events occur at different times upon CD40
ligation. NF-B/Rel nuclear translocation begins 2 h after
stimulation and is sustained for several hours. In contrast,
down-regulation of p27kip-cell cycle inhibitor
appears only upon 24 h of CD40 stimulation. Furthermore, our study
indicates that the down-regulation of p27kip
isindependent from c-Rel, because in c-Rel ko B cells,
p27kip deg- radation still occurs normally upon B
cell receptor
engagement.4
In contrast to the effects PI-3K exerts on proliferation upon CD40
stimulation, apoptosis protection seems to proceed normally even in the
absence of the PI-3K activity. Surprisingly, even though NF-B/Rel
has been implicated in protecting cells from various apoptotic insults,
we found that NF-B/Rel is not the only survival pathway mediated by
CD40 (Fig. 8). This is concluded based on our data that establish that
CD40-mediated apoptosis protection is normal in c-Rel ko mouse and in
cells treated with the PI-3K inhibitor. In dissecting the survival
pathway, we examined several known anti-apoptotic molecules. First,
AKT kinase, known to be a direct target of the PI-3K phosphorylation
and to prevent apoptosis, was not activated when PI-3K action has been
blocked. Still, CD40 prevented apoptosis of these cells, suggesting
that CD40-activated cells can remain viable in the absence of AKT.
Remarkably, we demonstrated that Bcl-X expression is regulated by PI-3K
and NF-B/Rel; and its absence in the c-Rel ko or PI-3K deficient
cells (H.S., T.K., and S.K., unpublished observation) does not affect
the ability of these cells to survive when stimulated via CD40
receptor. In searching for the remaining CD40-mediated survival
pathways or molecules that confer the protection against death mediated
by PI-3K inhibition, we have indirectly excluded the c-IAP1 and c-IAP2
members (inhibitor of apoptosis) of caspase inhibitors (33, 34), as well as transcription factor A-20 (Ref. 11 ;
S. Andjelic, J. Tumang, and H.-Chi Liou, unpublished data),
because these molecules were shown to be dependent on the NF-B/Rel
activity (3, 35), which is blocked in this case. Moreover,
XIAP (X-linked member of the IAP family molecule; Ref.
36) is also not involved according to several experiments
we performed on XIAP ko mice (kindly provided by Dr. Craig Thompson,
University of Pennsylvania Medical Center; data not shown), which
revealed that the CD40 survival pathway was not affected by the absence
of XIAP (that is NF-B/Rel-independent).
Thus, the CD40-mediated survival pathways are more complex than we anticipated, and they are not relying completely on the induction of known anti-apoptotic molecules, such as Bcl-X, A20, and IAPs. Previously, we have reported that CD40 stimulation blocks caspase-3-like activity in normal splenic B lymphocytes (37). Therefore, it is likely that CD40-induced events block caspase action through a direct posttranslational modification of certain signaling molecules that are unknown at present, which in turn inactivate caspases. In addition to searching for these molecules, our future study will be focused on investigating the effect of CD40 stimulation on preventing the apoptotic events occurring in mitochondria during the course of programmed cell death (38).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hsiou-Chi Liou, Division of Immunology, Department of Medicine, Weill Graduate School of Medical Sciences of Cornell University, 1300 York Avenue, LC-929, New York, NY 10021.
3 Abbreviations used in this paper: PI-3K, phosphatidylinositol 3-kinase; CDK, cyclin-dependent kinase; ko, knockout; wt, wild type; PI, propidium iodide.
4 C. Y. Hsia and H-C. Liou. c-Rel regulation of the cell cycle in murine splenic B cells. Submitted for publication.
Received for publication February 17, 2000. Accepted for publication July 6, 2000.
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Y. J. Ha and J. R. Lee Role of TNF Receptor-Associated Factor 3 in the CD40 Signaling by Production of Reactive Oxygen Species through Association with p40phox, a Cytosolic Subunit of Nicotinamide Adenine Dinucleotide Phosphate Oxidase J. Immunol., January 1, 2004; 172(1): 231 - 239. [Abstract] [Full Text] [PDF]
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M. Prendes, Y. Zheng, and A. A. Beg Regulation of Developing B Cell Survival by RelA-Containing NF-{kappa}B Complexes J. Immunol., October 15, 2003; 171(8): 3963 - 3969. [Abstract] [Full Text] [PDF]
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C. W. Dawson, G. Tramountanis, A. G. Eliopoulos, and L. S. Young Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Activates the Phosphatidylinositol 3-Kinase/Akt Pathway to Promote Cell Survival and Induce Actin Filament Remodeling J. Biol. Chem., January 31, 2003; 278(6): 3694 - 3704. [Abstract] [Full Text] [PDF]
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I. Ringshausen, F. Schneller, C. Bogner, S. Hipp, J. Duyster, C. Peschel, and T. Decker Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cdelta Blood, November 15, 2002; 100(10): 3741 - 3748. [Abstract] [Full Text] [PDF]
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 Y. Wang, S. Chan, and B. K. Tsang Involvement of Inhibitory Nuclear Factor-{kappa}B (NF{kappa}B)-Independent NF{kappa}B Activation in the Gonadotropic Regulation of X-Linked Inhibitor of Apoptosis Expression during Ovarian Follicular Development in Vitro Endocrinology, July 1, 2002; 143(7): 2732 - 2740. [Abstract] [Full Text] [PDF]
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K. Kirito, T. Watanabe, K.-i. Sawada, H. Endo, K. Ozawa, and N. Komatsu Thrombopoietin Regulates Bcl-xL Gene Expression through Stat5 and Phosphatidylinositol 3-Kinase Activation Pathways J. Biol. Chem., March 1, 2002; 277(10): 8329 - 8337. [Abstract] [Full Text] [PDF]
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