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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3044-3052
Role of NF- B in the Rescue of Multiple Myeloma Cells From
Glucocorticoid-Induced Apoptosis by Bcl-2
By
Rena Feinman,
Jadd Koury,
Michael Thames,
Bart Barlogie,
Joshua Epstein, and
David S. Siegel
From Myeloma and Transplantation Research Center, Arkansas Cancer
Research Center, University of Arkansas for Medical Sciences, Little
Rock, AR.
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ABSTRACT |
The molecular mechanisms by which multiple myeloma (MM) cells evade
glucocorticoid-induced apoptosis have not been delineated. Using a
human IgA MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-B DNA binding and B-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-B complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of
NF-B DNA binding precedes a notable increase in annexin
V binding, thereby indicating that diminished NF-B activity is an
early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1
cells prevents Dex-mediated repression of NF-B activity and
apoptosis. Sustained NF-B DNA binding is also observed in two
previously characterized Dex-resistant MM cell lines (RPMI8226 and
ARH-77) that express moderate levels of endogenous bcl-2 and IB
proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not
prevent the augmentation of IB protein by Dex. We also noted a
possible association between Dex-mediated downregulation of NF-B in
freshly obtained primary myeloma cells and the patients' responsiveness to glucocorticoid-based chemotherapy. Collectively, our
data suggest that the protective effects of bcl-2 in MM cells act
upstream in the NF-B activation-signaling pathway and the potential
use of NF-B as a biomarker in progressive MM.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MYELOMA CELLS are terminally
differentiated neoplastic B cells with a low proliferative index and a
long life span. Glucocorticoids such as dexamethasone (Dex) and
alkylating agents such as melphalan (Mel), all potent inducers of
apoptosis, are frequently used to treat multiple myeloma (MM). Using
standard doses of these agents, this malignancy remains incurable, with a complete remission (CR) rate of 5% and a median survival of 30 to 36 months. Drug resistance increases with prolonged treatment, suggesting
that primary drug resistance is a major underlying factor in the
resilience of myeloma cells to apoptotic signals and therapeutic interventions.
Like in other chronic B-cell malignancies such as chronic lymphocytic
leukemia, chemoresistance has been attributed to bcl-2. Moderate to
high levels of bcl-2 protein have been reported in numerous human MM
cell lines and freshly isolated MM cells.1,2 In diverse
experimental systems, bcl-2 acts as an antioxidant and alters
intracellular calcium levels, plasma membrane asymmetry, mitochondrial
permeability, cytochrome c release, and caspase activation.3 The characterization of bcl-2 and its
antiapoptotic homologs, such as bcl-XL as ion channel proteins and
docking proteins,3,4 indicates that bcl-2 interacts with
proteins that are directly or indirectly involved in the apoptotic
pathway. Several examples are calcineurin,5
Raf1,6 GTPases R-, and H-Ras.7 This cascade of
events ultimately modulates the expression of genes critical for
apoptosis, including the transcription factors (TFs) NF-AT,5,8 p53-BP2,9 and
NF-B.10-14
The NF-B family of dimeric TFs is a critical regulator of genes
expressed during acute phase and inflammatory responses, Ig class
switching, cellular differentiation, and apoptosis. Members of this
family of TFs can form homodimers or heterodimers that bind to a
specific decameric DNA sequence termed the B site.15,16 The activated NF-B prototype is composed of the p50 and p65 (relA) subunits. Other members of this family include relB, c-rel, v-rel, and
p52. With the exception of mature B lymphocytes and plasmacytomas, a
latent form of NF-B is sequestered in the cytoplasm of most cell
types by a family of inhibitor proteins (IB) that mask the nuclear
localization domain of Rel proteins. To date, seven IB proteins
(IB , IB , IB , IB , Bcl3, p100, and p105) have
been described: the roles of only IB and IB have been
investigated in great detail.17 Activators of NF-B, such
as tumor necrosis factor (TNF)- and phorbol esters, cause
site-specific phosphorylation of IB (serines 32 and 36) and
IB (serines 19 and 23), leading to their degradation via the
ubiquitin/proteasome pathway.18,19 The direct consequence
is the release of active NF-B complex, its nuclear translocation,
and transcriptional activation of the B site in promoters of
numerous target genes. Activation of NF-B signaling pathway by
TNF- and interleukin (IL)-1 requires the NF-B-inducing kinase
(NIK), a member of the MAP kinase kinase kinase (MAP3K)
family.20 Although the molecular mechanisms for its
activation are not yet delineated, NIK has been shown to interact with
and activate two downstream targets, IB kinase (IKK) and IKK
that phosphorylate IB and IB,
respectively.21-23
Recent studies implicated NF-B as a critical regulator of apoptosis.
NF-B activation can promote apoptosis or survival, depending on the
cellular context. Because glucocorticoids have been shown to act as
potent inhibitors of NF-B activity24,25 and
susceptibility to glucocorticoid-induced apoptosis is inversely related
to bcl-2 expression, it was of interest to examine the interplay
between Dex-mediated resistance in MM cells, bcl-2, and NF-B
activation. We report that maintenance of NF-B activation is
associated with the ability of both endogenous or enforced bcl-2 to
render MM cell lines resistant to Dex-induced apoptosis. Furthermore,
our findings suggest that NF-B activation is a central factor in the
development of resistance to glucocorticoid therapy and may be useful
as a predictive factor for assessing the efficacy of
glucocorticoid-based therapy in MM patients.
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MATERIALS AND METHODS |
Cell culture and treatments.
The ARP-1 cell line was established from a bone marrow aspirate of a
patient with an IgA/ secreting myeloma.26 The ARH77 and
RPMI 8226 human MM-derived cell lines were obtained from American Type
Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and
100 mg/mL of penicillin/streptomycin. For apoptosis assays, 2.5 × 105 cells/mL (>95% viable cells by trypan blue
exclusion) were treated with and without 0.1 to 10 µmol/L of Dex
(Sigma Chemical Company, St Louis, MO) for different times. For
electrophoretic mobility shift assay (EMSA) and western
blot analysis, 5 to 10 × 106 cells/treatment
were used for the preparation of nuclear extracts (NEs), cytoplasmic
extracts (CytEs), and whole cell extracts (WCEs).
Transfections and luciferase assays.
For transient transfections, ARP-1 cells were electroporated (BTX;
1,300 µF, 175 V) with 10 µg of reporter DNA. The
thymidine-kinase-driven luciferase (TK-Luc) reporter plasmid and the
TK-Luc reporter plasmid containing three copies of the consensus B
site were kindly provided by John L. Cleveland (St Judes Children's
Hospital, Memphis, TN). After transfection, cells were allowed to
recover for 6 to 8 hours and were then treated with Dex (0.1 µmol/L)
for 18 hours. Cells were harvested, lysed, and assayed for luciferase
activity (Luciferase Assay System; Promega, Madison, WI) by using a
Turner TD-20e luminometer (Promega). Luciferase activity was normalized
according to protein content. Protein concentrations of cell lysates
were determined according to the protocol for the Pierce Micro BCA
(Pierce, Rockford, IL). For stable transfections, ARP-1 cells were
transfected with 10 µg of either human bcl-2 pSFFV-neo or pSFFV-neo
empty-containing expression plasmids (kindly provided by Stanley J. Korsemeyer, Washington University School of Medicine, St Louis, MO) by
electroporation at 275 V and 1,180 µF, using a Cell-Porator (Life
Technologies, Grand Island, NY). After 48 hours, bulk selection was
performed in 800 µg/mL G418 sulfate (Life Technologies) for 2 weeks.
Individual clones were then isolated by cell sorting using flow
cytometry. Bcl-2 expression was confirmed by Western blotting analysis
using a mouse anti-bcl-2 monoclonal antibody (Dako Corporation,
Carpinteria, CA).
Nuclear, cytoplasmic, and whole cell extracts.
Cells were collected, centrifuged, and washed twice in ice-cold
phosphate-buffered saline. Crude CytEs and NEs were prepared as
described by Schreiber et al27 with the
following modifications: buffers A and C contain 1 µg/mL of
aprotinin, leupeptin, and pepstatin; 50 mmol/L -glycerophosphate; 10 mmol/L NaF; and 1 mmol/L sodium vanadate. For preparation of WCEs,
cells were lysed in buffer containing 1% (vol/vol) Nonidet P-40; 240 mmol/L NaCl; 50 mmol/L HEPES (pH 7.9); 10% glycerol; 0.1 mmol/L EDTA;
1 mmol/L dithiothreitol; 0.5 mmol/L phenylmethylsulfonyl fluoride; 1 µg/mL of aprotinin, leupeptin, and pepstatin; 50 mmol/L
-glyceraldehyde phosphate; and 1 mmol/L sodium vanadate.
Protein content was determined according to the Bradford method
using the Bio-Rad protein assay dye reagent (Bio-Rad Laboratories,
Hercules, CA).
Electrophoretic mobility shift assay.
Assays were performed as described28 using 5 to 10 µg of
NE or WCE, 0.2 to 0.5 ng of probe, 20 mmol/L Tris (pH 7.5), 50 mmol/L
NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10% glycerol, and 2 µg
poly(dI-dC) (poly(dI-dC); Pharmacia Biotech, Piscataway, NJ). The B
oligonucleotide from the IL-6 promoter
(5'-agctTCAAATGTGGGATTTTCCCATGAG-3', IL-6 B),29 and the
OCT-1 oligonucleotide based on consensus sequence of OCT-1 (Santa Cruz
Biotechnology, Santa Cruz, CA) were end-labeled by T4 polynucleotide
kinase with 32P-ATP (Dupont NEN, Boston, MA). The
binding reaction (25 µL) is incubated at room
temperature for 20 minutes, and then electrophoresis is performed on
4% nondenaturing polyacrylamide gel. In our competition studies, 10 and 50 molar excess of unlabeled oligonucleotide competitor was added
to the binding reaction for 10 minutes before EMSA with radiolabeled
oligonucleotide probe. Competitor oligonucleotides were: IL-6 B,
IL-6 mutant B (5'-agctTCAAATGTTACATTTTCCCATGAG-3'),29 and Ig B (5'-CAGAGGGGACTTTCCGAGA-3'). For supershifts, 5 µg of unrelated or specific antibody to putative binding proteins is added to
the binding reaction for 30 minutes before EMSA with radiolabeled
oligonucleotide probe. Rabbit or goat polyclonal antibodies specific
for the p50 subunit, p65 subunit, c-rel subunit, and unrelated goat
anti-p107 and rabbit anti-USF were obtained from Santa Cruz.
Western blot analysis.
Equal amounts of protein (10 to 50 µg) in CytEs or WCEs were
electrophoresed in a reducing 10% to 12.5%
sodium-dodecyl sulfate polyacrylamide gel. Protein was transferred to
nitrocellulose membranes (Amersham Life Sciences, Cleveland, OH) and
stained with either mouse anti-bcl-2 monoclonal antibody or rabbit
polyclonal IB antibody (Santa Cruz Biotechnology) followed by
goat antimouse or donkey antirabbit IgG horseradish peroxidase
conjugate (Amersham). For detection of proteins, an enhanced
chemiluminescence system (Amersham) was used. Equal protein
loading was confirmed by Fast Green staining of the membrane (Sigma
Chemical Co).
Apoptosis assays.
For in situ end labeling (ISEL), cytospins from untreated and
Dex-treated cells were subsequently fixed in ethanol overnight at
 20°C and air dried. The KLENOW FragEL DNA fragmentation detection assay was used according to the manufacturer's instructions (Oncogene Research Products, Cambridge, MA). For annexin V binding, 0.5 to 1 × 106 cells, before and after treatment, are collected,
washed twice with phosphate-buffered saline (PBS) and resuspended in
0.5 mL of annexin V binding buffer (2.5 mmol/L CaCl2, 20 mmol/L HEPES [pH 7.4], and 140 mmol/L NaCl) containing fluorescein
isothiocyanate (FITC)-conjugated annexin V (Caltag Laboratories,
Burlingame, CA) for 15 minutes in ice. After the addition of propidium
iodide (10 µg/mL), annexin V staining is determined by flow cytometry on a FACScan (Becton-Dickinson, San Jose, CA).
Myeloma cell purification.
Heparinized bone marrow aspirates were obtained from patients with
myeloma during scheduled clinic visits, as prescribed in the
Investigational Review Board (IRB)-approved protocol. Signed informed
consents were obtained and are kept on record. Myeloma plasma cells were purified from bone marrow aspirates as previously described.30 Briefly, light density (=1.077
g/cm3) cells were reacted with monoclonal antibodies to
CD38 (phycoerythrin [PE]-conjugated) and CD45 (FITC-conjugated) and
separated on the FACStar Plus cell sorter (Becton-Dickinson). MM cells
were identified by high CD38 fluorescence, low-intermediate CD45
fluorescence, and by their light scatter properties. MM plasma cells
were then sorted at a flow rate of 4,000 to 6,000 events per second.
The purity of MM cells in the sorted cell preparation was confirmed immunohistochemically by monotypic cytoplasmic immunoglobulin content
and was equal to 97%.
Clinical criteria for patient's response to glucocorticoid-based
therapy.
The definition of clinical responsiveness to the most recent
application of glucocorticoid-based therapy was based on a 50% reduction in the patient's serum or urine paraprotein.
| |
RESULTS |
Ectopic expression of bcl-2 blocks Dex-induced apoptosis in ARP-1
cells.
The human IgA MM cell line, ARP-1, serves as an excellent model to
examine the role of bcl-2 in Dex-induced apoptosis of myeloma cells.
ARP-1 cells undergo extensive apoptosis upon treatment with a
clinically relevant dose of Dex (0.1 µmol/L). In addition, ARP-1
cells express low endogenous levels of bcl-2 protein. We therefore
transfected ARP-1 cells with either bcl-2 or empty parental vectors,
and several stably transfected clones were isolated and characterized.
The degree of bcl-2 expression varied among the clones (data not
shown), and a high bcl-2 expressing clone (B491) confirmed by Western
blot analysis (Fig 1A) was further
characterized. Parental and bcl-2 ARP-1 transfectants were treated for
48 hours with and without Dex, and the percentage of apoptotic cells
was determined by ISEL. The addition of Dex resulted in a sevenfold increase in the percentage of apoptotic cells in our parental ARP-1
transfectants (Fig 1B). The ectopic expression of bcl-2 protected ARP-1
cells from both spontaneous and Dex-induced apoptosis. This result is
consistent with earlier studies that showed an inverse correlation
between glucocorticoid susceptibility and bcl-2
expression.31 It is also noteworthy that induction of apoptosis by Dex and resistance to Dex-induced apoptosis mediated by
bcl-2 were not caused by clonal variation because we obtained similar
results with bulk-selected ARP-1 transfectants.
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| Fig 1.
Overexpression of bcl-2 protects Dex-induced apoptosis in
ARP-1 cells. (A) WCEs prepared from parental and bcl-2 ARP-1
transfectants were Western blotted with antibody specific for bcl-2
protein. (B) Parental and bcl-2 ARP-1 transfectants were treated with
and without Dex (0.1 µmol/L) for 48 hours. Percentage of apoptosis
was measured by ISEL.
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Dex-induced apoptosis in ARP-1 cells is associated with inhibition of
NF-B.
To investigate whether Dex-induced apoptosis correlated with inhibition
of NF-B activity in the ARP-1 cell line, NF-B activity was tested
in NEs prepared from parental and bcl-2 ARP-1 transfectants. Two
constitutive nuclear NF-B complexes were detected. The slower migrating constitutive NF-B complex was dramatically inhibited by a
24-hour exposure to Dex (Fig 2A). In contrast,
overexpression of bcl-2 prevented downregulation of NF-B activity by
Dex. To show that Dex selectively downmodulated NF-B DNA binding,
the same 24-hour NEs used in the B DNA binding assay (Fig 2A) were tested for binding to the consensus binding site for the octamer binding protein (Oct-1). As shown in Fig 2B, Oct-1 DNA binding was not
markedly inhibited by Dex. Collectively, our data indicate that the
selective inhibition of NF-B nucleoprotein complexes that
accompanies Dex-induced apoptosis is prevented by enforced bcl-2
expression.
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| Fig 2.
Enforced bcl-2 prevents the selective downmodulation of
NF-B DNA binding by Dex. Effect of Dex on (A) NF-B and (B)
octamer binding protein (Oct-1) DNA binding activities in parental and
bcl-2 ARP-1 transfectants. (A) NEs from parental and bcl-2 ARP-1
transfectants treated with and without Dex (0.1 µmol/L) for 24 hours
were incubated with a 32P-labeled probe containing the B
binding site of the IL-6 promoter and assayed by EMSA. (B) The same NEs
used in (A) were tested for Oct-1 DNA binding by EMSA using a
radiolabeled Oct-1 probe containing a consensus binding site for Oct
family homeodomain TFs. Arrows indicate two distinct NF-B complexes
in (A) and four Oct-1 complexes in (B).
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The specificity of these two protein-DNA complexes was confirmed by
competition analysis (Fig 3A). Excess unlabeled B
oligonucleotide competed with NF-B complex formation, whereas a
mutated version of the B site failed to compete for NF-B binding.
An EMSA supershift was employed to identify the components of NF-B
subunits bound to the B site. Anti-p50 antibody caused the loss of
both NF-B complexes and yielded a supershifted complex (Fig 3B). The
addition of anti-p65 antibody abrogated complex formation of the slower migrating complex, and a faint supershifted complex was detected. Unrelated and anti-c-Rel antibodies did not react with the NF-B complex. Hence, the slower and faster migrating NF-B complexes contained the classical p50:p65 heterodimer and p50:p50 homodimers, respectively (Fig 3B).
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| Fig 3.
Characterization of constitutive nuclear NF-B
complexes in ARP-1 cells. (A) For competitive EMSA, NEs prepared from
untreated parental ARP-1 transfectants were incubated with either a 10- or 50-fold molar excess of unlabeled wild-type or mutated B
oligonucleotides and assayed for their ability to compete with
radiolabeled wild-type B probe in a standard EMSA. Arrows indicate
two specific NF-B complexes. (B) p50:p50 and p50:p65 dimers bind to
the B site. Supershift EMSA was performed using whole cell extracts
prepared from parental ARP-1 transfectants. When marked, antibodies
specific for p50, p65, c-Rel, and USF and E2F-1 (two control unrelated
antibodies) were incubated in an EMSA reaction. The positions of
p50:p50 and p50:p65 NF-B complexes are indicated by arrows.
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Loss of NF-B DNA binding is an early event in
drug-induced apoptosis.
To test whether the inhibition of NF-B DNA binding was an early
event in the commitment phase of apoptosis or a late event in the
execution phase of Dex-induced apoptosis, we compared the kinetics of
Dex-mediated downregulation of NF-B DNA binding with the kinetics of
annexin V binding, a landmark analysis for early and late apoptosis. As
compared with untreated control, treatment with Dex inhibited NF-B
DNA binding by 5.4% after 1 hour (Fig 4A). At 3 and 6 hours Dex significantly diminished NF-B DNA binding by 30% and
56%, respectively. The percentage of inhibition of NF-B DNA binding
by Dex (relative to untreated control) was quantitated by a
Phospholmager (Molecular Dynamics, Sunnyvale, CA). In comparison, no marked increase in annexin V binding was detected after
1 and 3 hours with Dex with only a minimal increase after 6 hours of Dex treatment (Fig 4B). A significant increase in annexin V binding begins after 12 hours of Dex and further increases with prolonged treatment of Dex, 24 and 48 hours (data not shown). In bcl-2 ARP-1 transfectants, Dex induced only negligible changes in annexin V binding
and in situ end labeling that were associated with lack of Dex-mediated
inhibition of NF-B DNA binding. Thus, the progressive decline of
NF-B DNA binding in parental ARP-1 transfectants after different
exposure times with Dex preceded changes in annexin V binding,
indicating that loss of NF-B DNA binding is an early event in the
commitment of cell death.
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| Fig 4.
Loss of NF-B DNA binding precedes annexin V binding.
(A) For NF-B DNA binding, NEs prepared from parental ARP-1
transfectants treated with and without Dex (0.1 µmol/L) for the
indicated times were examined by EMSA. (B) Parental ARP-1 cells treated
as described above were double stained with annexin-V-FITC and
propidium iodide and analyzed for annexin-V binding by flow cytometry.
Numbers within dot plots represent percentages of cells in early
apoptosis (annexin V+/PI ; lower right) and
in late apoptosis and necrosis (annexin
V+/PI+; upper left). Percentage of
apoptotic cells (next to each lower dot plot) represents the fraction
of cells undergoing apoptosis with Dex.
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Bcl-2 prevents Dex-mediated downregulation of
B-dependent transcription.
The above findings prompted us to determine whether the modulation of
NF-B DNA binding in Dex-treated ARP-1 cells correlated with
B-dependent transcriptional activity in vivo. Parental and bcl-2
ARP-1 transfectants were transfected with either a TK-Luc reporter
construct containing three repeats of the B site (3B) or a
control TK-Luc reporter plasmid, and the effects of Dex on luciferase
activity were examined. Negligible luciferase activity was detected in
both parental and bcl-2 ARP-1 cells transfected with TK-Luc reporter,
and treatment with Dex for 18 hours did not affect TK promoter activity
(Fig 5). The level of B transcriptional activity is indicated as percentage of control (relative light units
[RLU]/µg of protein) and was normalized to protein content. In
untreated parental and bcl-2 ARP-1 cells, transfected 3B reporter displayed 164.8-fold and 94.9-fold higher luciferase activity, respectively, than TK-Luc reporter. The addition of Dex significantly reduced B-dependent luciferase activity in parental ARP-1 cells by
22% (P = 0.019), but only had a marginal effect (6.5%;
P = 0.165) in the bcl-2 overexpressing cells. According to
these functional studies, comparable high levels of constitutive
NF-B activity were detected in both parental and bcl-2 ARP-1 cells.
More importantly, the protective function of bcl-2 in ARP-1 cells seems
to be attributed to the preservation of B-dependent transcription.
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| Fig 5.
Effect of bcl-2 on Dex-mediated downregulation of
B-dependent reporter gene activity. Parental and bcl-2 ARP-1
transfectants were transiently transfected with either a TK-Luc
reporter plasmid or TK-Luc reporter plasmid harboring three B sites
and subsequently cultured in the presence or absence of Dex (0.1 µmol/L) for 20 hours. The level of B transcriptional activity is
indicated as percentage control (relative light units [RLU]/µg of
protein) and normalized to protein content. The average of 13 (parental
ARP-1) and 12 (bcl-2 ARP-1) individual experiments is represented.
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Bcl-2 does not affect Dex-mediated augmentation of
IB protein.
Because transrepression of NF-B activity by glucocorticoids could be
caused, in part by the transcriptional induction of IB
synthesis,25,32 we asked whether enforced bcl-2 expression prevented increased IB expression by Dex. Whole cell extracts were prepared from parental and bcl-2 ARP-1 transfectants with and
without Dex for 0.5, 1, and 3 hours. Steady state levels of IB
protein were analyzed by Western blotting. Dex profoundly increased
IB protein expression after 3 hours, even in the bcl-2 overexpressing ARP-1 cells (Fig 6). Thus,
Dex-mediated upregulation of steady state levels of IB protein is
not sufficient for Dex-induced apoptosis.
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| Fig 6.
Bcl-2 does not prevent upregulation of IB protein
by Dex. Parental and bcl-2 ARP-1 transfectants were treated with and
without Dex (0.1 µmol/L) for different times. WCEs were prepared and
assayed for IB protein expression by Western blotting. WCE from
HeLa cell line was used as a positive control for IB protein
expression. IB and NS, a nonspecific cross-reacting protein, are
indicated by arrows.
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It is also noteworthy that although enforced bcl-2 sustained NF-B
activity, IB degradation was not evident at any of the time
points analyzed. In addition, the relative abundance of IB protein is higher in the bcl-2 ARP-1 transfectants. These observations suggest that the ability of bcl-2 to maintain NF-B activity is independent of IB.
Correlation between Dex-resistance, bcl-2 levels, and constitutive
NF-B activation.
To further show the relationship between persistent NF-B activity,
bcl-2 expression, and resistance to Dex-induced apoptosis, we analyzed
NF-B DNA binding and endogenous bcl-2 in two Dex-resistant human MM
cell lines, RPMI8226 and ARH77. Relative to the low bcl-2 overexpressing parental ARP-1 transfectants, both cell lines expressed moderate endogenous levels of bcl-2 (Fig
7A). In addition, treatment with Dex did
not alter bcl-2 protein levels in the Dex-sensitive (parental ARP-1
transfectants) and the Dex-resistant (bcl-2 ARP-1 transfectants,
RPMI8226 and ARH77) cells. Both RPMI8226 and ARH77 MM cells had
constitutive NF-B DNA binding, and by 20 hours, 10 µmol/L of Dex
did not inhibit NF-B DNA binding (Fig 7B). Thus, resistance to
Dex-induced and spontaneous apoptosis seems to be linked to bcl-2
expression and maintenance of constitutive NF-B activation.
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| Fig 7.
Correlation between Dex-resistance, constitutive NF-B
DNA binding, and endogenous bcl-2 protein levels. RPMI8226, ARH77,
parental, and bcl-2 ARP-1 transfectants were cultured with or without
the indicated concentration of Dex for 20 hours (A) WCEs were prepared
and analyzed for NF-B DNA binding by EMSA with a radiolabeled B
probe (B) and (C) WCEs were blotted with antibody specific for (B)
bcl-2 and (C) I.
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Because IB has been shown to regulate transient and persistent
activation of NF-B, we examined the overall level of IB protein in these three MM cell lines. There was no apparent difference in the levels of IB protein in RPMI8226, ARH77, and parental ARP-1 transfectants (Fig 7C). Exposure to Dex increased the level of
IB protein in parental and bcl-2 ARP-1 transfectants, as well as
in RPMI8226 cells. Dex did not, however, increase IB protein
levels in ARH77 cells. Taken together, our findings suggest that
constitutive NF-B activation in these three MM cell lines cannot be
explained by low or negligible steady state levels of IB.
Dex-mediated downregulation of NF-B DNA binding
activity is associated with patients' clinical response to Dex.
To investigate whether primary myeloma cells respond to Dex in a
similar fashion, we examined NF-B DNA binding in myeloma cells from
five MM patients. WCEs were prepared from sort-purified myeloma plasma
cells ( 95%) from bone marrow aspirates of MM patients. The cells
were cultured in vitro for 24 hours in the presence and absence of Dex,
and NF-B DNA binding was determined by EMSA. Dex reduced DNA binding
in three patient samples. No effect was noticed in the other two (Fig
8). Interestingly, the three patients whose
myeloma cells responded to Dex in vitro, but not those whose myeloma
cells showed no response, were responsive to Dex-based treatment.
Patient's responsiveness to Dex was defined as more than 50% decline
in their serum or urine paraprotein/tumor-specific Ig levels. These
results may suggest an association between Dex-mediated decreased
NF-B activity and favorable clinical response to Dex.
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| Fig 8.
Dex-mediated downregulation of NF-B DNA binding
activity correlates with patients' clinical response to Dex.
FACS-sorted-purified myeloma plasma cells from bone marrow aspirates
of MM patients were treated with and without Dex (0.1 µmol/L) for 24 hours. WCEs were prepared and subjected to EMSA for binding to the B
oligonucleotide probe.
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| |
DISCUSSION |
It is widely accepted that dysregulation of genes that control
apoptosis contributes to the pathogenesis of numerous diseases, including MM. Although bcl-2 has been implicated in primary drug resistance in MM, the molecular mechanisms by which MM cells evade chemotherapy-induced apoptosis remain unclear. In this study, we
present evidence that Dex-induced apoptosis is associated with the
selective downmodulation of NF-B activity in our preclinical myeloma
model (ARP-1 cell line). Enforced bcl-2 expression in ARP-1 cells
prevents Dex-induced apoptosis by maintaining NF-B activity. A
striking association between Dex-mediated resistance, endogenous bcl-2
protein levels, and constitutive nuclear NF-B is observed in two
other MM cell lines that fail to undergo Dex-induced apoptosis.
Moreover, persistent NF-B activation in MM cell lines is not
regulated by differential IB expression. Most intriguing is the
apparent association between Dex-mediated inhibition of NF-B DNA
binding in patients' MM cells and the patients' response to treatment
with glucocorticoid-based therapy.
Persistent NF-B activation is observed in several MM cell lines and
primary patient samples. Earlier studies have shown that mature B cell
lines and plasmacytomas manifest constitutively nuclear NF-B
activity.33 In our model system, the NF-B complex is
composed of a heterodimer of p50 and p65 subunits. Recent studies have
also shown the involvement of the p50:p65 NF-B heterodimer in
promoting survival of Hodgkin/Reed-Sternberg cells,34
estrogen-independent human breast cancer cell lines,35
carcinogen-induced primary rat mammary tumors,36 and
thyroid carcinomas.37 The underlying factors responsible
for constitutive NF-B activation in MM cells are presently unclear.
It is possible that autocrine and paracrine TNF- and IL-1 and
stimulation of CD30 and CD40 ligands in MM cells may be responsible for
constitutive NF-B activation in MM cells. Reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the
presence of TNF- and IL-1 mRNA in ARP-1 cells (data not shown).
Our data clearly show that NF-B plays a critical role in
Dex-mediated resistance in MM cells. We found that Dex-induced
apoptosis in ARP-1 cells is associated with the inhibition of NF-B
DNA binding and B-dependent transcription. These observations are in
agreement with earlier reports showing that activation of NF-B prevents TNF-, ionizing radiation-, and daunorubicin-induced apoptosis.38-40 Similarly, inhibition of p50/65 NF-B
complexes is also involved in IgM-induced apoptosis in the WEHI 231 cell line,41 and overexpression of a super IB
repressor mutant in Hodgkin's cells increased their susceptibility to
stress-induced apoptosis.34
To further explore the role of NF-B as a survival factor in MM
cells, we show in this report a relationship between bcl-2 and NF-B
in conferring resistance to Dex-induced apoptosis in MM cells. Bcl-2 is
able to protect ARP-1 cells from Dex-induced apoptosis, and its
antiapoptotic effects are mediated by NF-B. More specifically,
enforced bcl-2 prevented the downmodulation of NF-B DNA binding and
B-dependent transcription by Dex in ARP-1 cells. Consistent with
this observation was the correlation between resistance to Dex-mediated
apoptosis in two MM cell lines (RPMI8226 and ARH77), endogenous bcl-2
protein levels, and constitutive NF-B DNA binding activity. Several
studies have shown that bcl-2 can either positively or negatively
modulate NF-B activation, depending on the cellular environment.
Restoration of NF-B activation by enforced bcl-2 has been reported
in HeLa10 and Jurkat42 cells that failed to
undergo CD95(fas)-induced apoptosis. Contrary to our
system, bcl-2 has been shown to attenuate the transactivation domain of
p65 during serum-deprived apoptosis in 293 cells11 and
prevent NF-B DNA binding in alphavirus-induced apoptosis in a
prostate carcinoma line.14 With the exception of one report showing that the protective effects of bcl-2 on TNF--mediated cytotoxicity in prostate carcinoma cells are not linked to NF-B signaling,43 the above studies suggest that bcl-2 acts
upstream in the NF-B activation signaling pathway.
A possible mechanism for maintenance of NF-B activation by bcl-2 in
MM cells is caused by the ability of bcl-2 to block proteolysis. A
recent study has shown that inhibition of NF-B activity in Jurkat
cells that undergo apoptosis when triggered with CD95 is a consequence
of the proteolytic cleavage of both p50 and p65 by caspase-3-related
proteases.42 Although we cannot exclude this possibility,
the onset of Dex-mediated inhibition of NF-B DNA binding (3 hours)
in ARP-1 cells was detected before any significant changes in annexin V
binding (6 hours) were seen. Because loss of phospholipid asymmetry
(annexin V binding) is a prominent feature in both early and late
events during apoptosis, the disappearance of NF-B DNA binding in
ARP-1 cells undergoing apoptosis does not seem to reflect proteolysis.
We conclude that inhibition of NF-B may play a critical and
causative role in the execution of apoptosis.
Earlier studies have shown that transcriptional repression domain of
the activated glucocorticoid-receptor is responsible for
glucocorticoid-induced apoptosis of human leukemic cells.44 Two independent mechanisms are involved in the inhibition of NF-B activity by glucocorticoids: induction of IB synthesis and direct interference between the activated glucocorticoid receptor and the
transactivation domain of p65.24,45,46 Our present studies only addressed the effects of Dex and overexpressed bcl-2 on IB protein expression. In other studies, Dex has been shown to markedly increase IB- expression25,32 and facilitate the
nuclear translocation of IB- in which it inhibits NF-B DNA
binding by causing the nuclear export of activated DNA-bound NF-B
complexes. In our system, Dex augmented steady state levels of IB
protein expression in both parental and bcl-2 ARP-1 transfectants
within 3 hours, thereby indicating that bcl-2 does not interfere with
transcriptional induction of IB by Dex. Furthermore, there were
no major differences in the amount of endogenous IB protein in
low (parental ARP-1) or high (RPMI8226, ARH77) bcl-2 expressing MM cell
lines. Recent studies have reported that transrepression of NF-B
activity by glucocorticoids is independent of IB
expression.45,47 In contrast to our findings, low steady
state levels of IB protein have been shown to account for
constitutive NF-B activation in Hodgkin/Reed-Sternberg cells that
express activated p65.48 In a related study, the inability
of CD40 ligand to further activate B-dependent transcription in
three Hodgkin cell lines is possibly caused by aberrant IB
protein expression.49 Although we did not examine inducible
IB degradation in MM cells, our data suggests that continuous
basal IB degradation is not responsible for constitutive NF-B
activation and for the maintenance of NF-B activation by bcl-2 in MM
cells. Nevertheless, from the data herein, we cannot exclude the
possibility that IB protein may be defective in its regulation of
NF-B in MM.
Studies exploring the mechanisms responsible for constitutive NF-B
activity showed that a hypophosphorylated form of IB is found to
shield NF-B complex from IB-mediated
inhibition,50,51 and it is conceivable that IB plays
an important role in the persistent activation of NF-B. The
phosphorylated form of IB has been shown to have a higher
affinity for p50:p65 complexes than for p50-RelB and p50:c-Rel
complexes. A newly identified IB protein has also been recently
characterized to inhibit the late, transient activation of a subset of
genes that are primarily regulated by p65 and c-Rel.52,53
Further studies investigating the roles of IB, IB, and
IB in the regulation of constitutive NF-B activation are warranted.
Although preliminary, the observed relationship between Dex-mediated
inhibition of NF-B DNA binding activity in purified myeloma cells
and the patient's clinical response to glucocorticoid-based treatment,
if confirmed on a larger patient population, is intriguing. It is
tempting to speculate, on the basis of these observations, that the
ability of drugs to inhibit NF-B activity could serve as a
predictive factor for treatment outcome. Indeed, drugs that selectively
inhibit NF-B activity could be used in patients with progressive
disease. The search for new biologically relevant predictive factors,
such as NF-B, to guide therapy and the identification and
characterization of molecular targets involved in the bcl-2 antiapoptotic signaling pathway in MM will hopefully improve the overall median survival of MM patients.
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ACKNOWLEDGMENT |
The authors thank Dr Victoria Richon for comments on the manuscript and
helpful discussions, and Julian A. Terry and Jeff Woodliff for their
expert technical assistance.
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FOOTNOTES |
Submitted July 30, 1998; accepted December 11, 1998.
Supported in part by grant CA-55819 from the National Cancer Institute.
R.F. is a recipient of the 1996 Brian D. Novis Research Grant for
Multiple Myeloma from the International Myeloma Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rena Feinman, PhD, Department of
Surgery MSB G-519, UMDNJ-New Jersey Medical School, 185 South Orange
Ave, Newark, NJ 07103.
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REFERENCES |
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ABSTRACT
INTRODUCTION