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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3302-3308
An Essential Role for NF- B in Human CD34+ Bone Marrow
Cell Survival
By
David W. Pyatt,
Wayne S. Stillman,
Yanzhu Yang,
Sherilyn Gross,
Jia hua Zheng, and
Richard D. Irons
From The Molecular Toxicology and Environmental Health Sciences
Center, School of Pharmacy, the Department of Pathology, School of
Medicine, and the Cancer Center, University of Colorado Health Sciences
Center, Denver, CO.
 |
ABSTRACT |
The transcription factor, NF- B, is important for T-cell
activation, B-cell maturation, and human immunodeficiency virus
transcription and plays a role in alternatively mediating and
protecting against apoptosis in a variety of cell types. However, a
role for NF-B in human CD34+ bone marrow cells has not
been described. We provide evidence here that virtually all human
CD34+ bone marrow cells express NF-B that can be
activated by exposure to phorbol 12-myristate 13-acetate and a variety
of cytokines, eg, tumor necrosis factor  , interleukin-3, and
granulocyte-macrophage colony-stimulating factor. In addition, we
demonstrate that NF-B may be required for human CD34+
bone marrow cell clonogenic function and survival. These results offer
insight into a new role for NF-B in maintaining survival and
function in hematopoietic stem and progenitor cells and suggest that
proposed strategies involving inhibition of NF-B activation as an
adjunct to cancer chemotherapy should be approached with caution.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
NF-B IS A MEMBER of the mammalian Rel
family of transcriptional activators that plays a central role in the
activation and regulation of immune response.1 The family
consists of 5 discrete DNA binding proteins (p50, p52, p65, c-rel, and
RelB) that share partial homology, including a common DNA binding
domain, dimerization domains, and a nuclear localization
region.2 Rel family members form homodimers or heterodimers
that bind to a specific DNA sequence called the B
motif.3-8 NF-B is a heterodimer consisting of the 50-kD
(p50) and 65-kD (p65) subunits9 that, in its inactive
state, is located in the cytoplasm bound to an inhibitory protein,
IB .10 Upon activation, NF-B disassociates from
IB, translocates to the nucleus11 and binds to DNA to regulate gene expression (for review see Thanos and
Maniatis2 and Siebenlist et al12).
Additionally, complex binding patterns among various members of the Rel
family may account for differential gene activation with or without
interaction with other transcriptional regulatory
proteins.2
NF-B is required for the expression of several gene products
relevant to hematopoiesis including interleukin-1
(IL-1),13 tumor necrosis factor-
(TNF-),14 IL-6,15 macrophage
colony-stimulating factor (M-CSF),16 granulocyte-macrophage
colony-stimulating factor (GM-CSF),17 granulocyte
colony-stimulating factor (G-CSF),18 erythropoietin
(EPO),19 interferon- (INF-),20
c-myc,21 and c-myb.22 In addition, NF-B is
activated by cytokines known to regulate hematopoiesis such as
TNF-,23 IL-1 and IL-1,24 INF-,25 leukemia inhibitory factor,26
M-CSF,27 GM-CSF,27 transforming growth
factor-1 (TGF-1),28 and IL-3.29 The role
of NF-B as a secondary messenger for cytokine response, as well as
the hematopoiesis-specific genes that are responsive to NF-B,
suggest that it may be important in human CD34+ cell
survival and/or differentiation. Studies with disruption of the various
Rel family proteins support this observation. The disruption of the
relA locus (p65) leads to embryonic lethality at 15 to 16 days
of gestation with massive liver degeneration.30 Mice with
homozygous disruption of relB exhibit multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, a reduced number of
thymic dendritic cells, and impaired cellular immunity.31 Mice lacking p50 show no developmental abnormalities, but have impaired
B-cell responses.32 Mice with constitutive NF-B
activation due to the disruption of IB expression show enhanced
granulopoiesis and die within 8 days after birth.33 In
addition, both apoptosis and anti-apoptotic functions can be mediated
via NF-B activation in different cells.34-36
NF-B was originally described as a DNA-binding activity that
recognized a sequence 5'-GGGGACTTTCC-3' in the Ig k light chain gene
enhancer in mature B cells.37 Subsequently, NF-B has
been found in all mature hematopoietic cell lineages (eg, T
cells,10 monocytes,38
granulocytes,39 mast cells,40 and dendritic cells41) and has been recently reported to be involved in
erythropoiesis.42 However, the expression and role of
NF-B in human CD34+ bone marrow cells are currently
unknown. Therefore, we investigated whether NF-B is present in
CD34+ bone marrow cells and whether NF-B is required for
colony formation. The results of these studies demonstrate that
virtually all human CD34+ bone marrow cells contain NF-B
and that NF-B is required for human CD34+ bone marrow
cell clonogenic function and survival.
| |
MATERIALS AND METHODS |
Cell purification.
All protocols were approved by the University of Colorado Health
Sciences Center Internal Review Board, and samples were taken with
informed consent from normal adult volunteers. Human bone marrow and
blood were obtained by aspiration from the posterior iliac crest and
venapuncture, respectively. Mononuclear cells were isolated using
Histopaque-1077 (Sigma, St Louis, MO), and purification
of individual cell subpopulations was achieved using a high magnetic
gradient MiniMACS purification system (Miltenyi, Sunnyvale,
CA). Because NF-B is known to be expressed in B
lymphocytes,43 we used the pan-B-cell antigen CD19 to
isolate CD34+ bone marrow cells devoid of B cells
(CD34+CD19 ) for use in these studies.
CD19+ cells were removed and detected by using a
fluorescein isothiocynate (FITC)-conjugated anti-CD19 monoclonal
antibody followed by anti-FITC MicroBeads (Miltenyi).
CD34+CD19 cells were then obtained by using
the CD34 isolation kit. CD4+ T cells were purified using
CD4 Microbeads. The purity of isolated cells was determined by flow
cytometry analysis (Epics 752; Coulter Electronics, Hialeah, FL).
Electrophoretic mobility shift assay (EMSA).
Human CD34+CD19 bone marrow cells were
either used directly after purification or for selected experiments
cultured at 0.4 × 106 cells/mL for 18 hours at 37°C
with 5% CO2 in complete media (RPMI-1640, 10% fetal
bovine serum, 100 mg/mL streptomycin, 100 U/mL penicillin, and 2 mmol/L
L-glutamine) with and without 25 ng/mL phorbol 12-myristate 13-acetate
(PMA), cytokines, or NF-B nuclear localization-sequence (NLS)
peptides before use in the EMSA. Cytokine concentrations used in these
experiments were identical to those used in the colony-forming assays
and TNF- was used at 50 ng/mL. Initial experiments were performed
with NF-B NLS peptides purchased from Biomol (Plymouth Meeting, PA);
however, quality control issues required us to synthesize these
peptides based on the published sequences.44 The NF-B
NLS peptides were used at 100 µg/mL. Nuclear protein was extracted
using a modified Dignam protocol45 from 0.5 to 1 million
cells per treatment group. Protein concentrations were determined using
a BCA protein kit (Pierce, Rockford, IL) and the
nuclear extracts were frozen at  80°C until used. An NF-B probe
was made and labeled as described.46 For super-shift
samples, cellular extracts were preincubated with appropriate specific antibodies for 1 hour to overnight at 4°C. Antihuman p50, p52, p65,
and rel B antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antihuman c-rel and a second
antihuman p65 antibody were purchased from Rockland (Gilbertsville,
PA). Nuclear protein (4 µg) was incubated on ice for
10 minutes with 1 µg dl-dC, 4 µL binding buffer (20 mmol/L HEPES
[pH 7.9] 40 mmol/L KCl, 10% glycerol, 0.05 mmol/L EDTA, 1.6 mmol/L
MgCl2) 1 mmol/L DTT, and deionized water for a total
volume of 19 µL. One microliter of 32P-labeled probe
(~50,000 cpm) was added, and the binding reaction was continued for
30 minutes at 22°C. After complex formation, 2 µg of loading buffer
(250 mmol/L Tris HCl [pH 7.5], 0.2% bromophenol blue, 0.2% xylene
cyanol, 40% glycerol) was added to the DNA-protein complexes, and the
sample was analyzed by electrophoresis on a prerinsed 6%
polyacrylamide gel. The gels were dried and exposed at 80°C to
Fuji x-ray film (Tokyo, Japan). All reported
experiments were repeated at least 3 times.
Measuring NF-B by flow cytometry.
Cells were exposed to 25 ng/mL PMA and 1 µg/mL ionomycin or buffer
for 1 hour at 37°C in RPMI-1640 containing 1% bovine serum albumin
(BSA). The cells were then washed, followed by permeabilization in 4%
paraformaldehyde in phosphate-buffered saline (PBS), pH 7.2, with 0.1%
saponin and 0.01 mol/L HEPES for 10 minutes at room temperature. The
cells were labeled with 2 µg/mL anti-NF-B antibody
(IgG3; Boerhinger Mannheim, Mannheim,
Germany) or a nonspecific IgG3 antibody
(isotype control). The cells were then labeled with biotin-conjugated
goat antimouse IgG3, washed 2 times, and subsequently labeled with allophyocyanin (APC)-conjugated strepavidin. After the
final wash, the cells were fixed with 1% paraformaldehyde and stored
at 4°C until flow cytometric analysis. Flow cytometry was performed
using a MoFlo system (Cytomation, Fort Collins, CO).
These experiments were repeated at least 3 times.
Colony-forming assays.
These were performed as previously described.47 Briefly,
CD34+ bone marrow cells were purified and plated in 35-mm
culture dishes at a concentration of 3 to 4.5 × 103
cells/mL in 1 mL of modified Iscove's medium containing 10% fetal bovine serum, 100 mg/mL streptomycin, 100 U/mL penicillin, 2 mmol/L L-glutamine, 50 µmol/L 2-mercaptoethanol, 1.2% (wt/vol) methyl cellulose, and recombinant cytokines. Each cytokine was used at concentrations experimentally determined to produce maximal colony formation (eg, 5 ng/mL GM-CSF, 25 ng/mL IL-3, 25 ng/mL G-CSF, 25 ng/mL
M-CSF, 25 ng/mL stem cell factor [SCF], and 5 U/mL EPO). All
chemicals were mixed with the methyl cellulose media before the
addition of cells, which preceded the addition of the cytokines. All
cultures were maintained at 37°C in 5% CO2 and scored on
day 14 of culture. Five plates were scored for each treatment group, and results are expressed as the mean ± 1 standard error of the mean
(SEM). Significant differences (P  .05) between groups were determined using the Student's t-test (Excel 4.0; Microsoft
Corp, Redmond, WA). All reported experiments were
repeated at least 3 times.
Apoptosis assay.
Purified CD34+CD19 bone marrow cells were
exposed to NF-B NLS fusion peptide, control peptide, or buffer in
RPMI-1640 media supplemented with 10% fetal bovine serum in 5%
CO2 at 37°C for 16 hours. Apoptosis was measured by flow
cytometric analysis of deoxynucleotidyl transferase-mediated dUTP nick
end labeling (TUNEL; In Situ Cell Death Detection Kit; Boerhinger
Mannheim) for each sample. All reported experiments were repeated at
least 3 times.
| |
RESULTS |
Induction of NF-B-specific DNA-binding activity in
human CD34+CD19 bone marrow cells.
CD34+ bone marrow cells devoid of B cells
(CD34+CD19) were used in these studies,
because NF-B is known to be expressed in B lymphocytes (see
Materials and Methods).43
CD34+CD19 bone marrow cells were purified
and cultured overnight with and without PMA. EMSA of the nuclear
extracts demonstrate no detectable NF-B specific DNA-binding in the
unstimulated cells; however, PMA exposure induced considerable binding
(Fig 1). Super-shift EMSA using antibodies
specific for the Rel family members demonstrated the presence of p50,
p65, and c-rel by the appearance of shifted bands (Fig 1). Although
antibodies specific for p52 and rel B did not cause a supershift, the
possibility that any of these Rel family members are present cannot be
completely excluded due the heterogeneity of the CD34+
cells. The unreactive antibodies serve as an Ig control demonstrating the specificity of the p50, p65, and c-rel antibodies.
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| Fig 1.
PMA induces NF-B specific DNA-binding in human
CD34+CD19 bone marrow cells. To confirm
specificity and identify which Rel family members may be binding to the
NF-B consensus sequence, super-shift EMSA were performed with
antibodies specific for human p50, p65, p52, rel B, and c-rel. The
CD34+CD19 cells were 96.8% pure for this
representative experiment.
|
|
NF-B distribution in human
CD34+CD19 bone marrow cells.
We evaluated the distribution of activated NF-B in both
PMA-stimulated and unstimulated human
CD34+CD19 bone marrow and CD4+ T
cells by flow cytometry using a monoclonal antibody that recognizes an
epitope within the NF-B nuclear localization region that is exposed
upon activation.48 Using this antibody, we confirmed the
results of the EMSA and determined that activated NF-B is present in
virtually all human CD34+CD19 cells (Fig
2).
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| Fig 2.
Activated NF-B measured by flow cytometry in human
CD4+ peripheral T cells ([A] unstimulated, [B]
stimulated) and CD34+CD19 bone marrow
cells ([C] unstimulated, [D] stimulated). Stimulated cells were
incubated with 25 ng/mL PMA and 1 µg/mL ionomycin for 1 hour at
37°C before NF-B analysis. Activated NF-B is a black line and
the background isotype antibody binding is a gray line for each sample.
The monoclonal antibody used in these experiments has been shown to be
specific for the NF-B nuclear localization region, which is detected
only if NF-B is activated. The CD4+ T cells and
CD34+CD19 bone marrow cells were more than
97% and more than 99% pure, respectively.
|
|
NF-B is required for CD34+ cell
colony formation.
To determine if NF-B is necessary for colony formation, a fusion
peptide containing the NF-B NLS and a membrane-permeable hydrophobic
region that has been previously demonstrated to specifically prevent
NF-B nuclear translocation44 was added to the media in a
methyl cellulose colony-forming assay. After activation of NF-B, its
NLS associates with a specific NLS-receptor, resulting in transport of
NF-B in to the nucleus. This can be blocked by occupying the NF-B
NLS-receptors with peptides possessing the NF-B NLS. NF-B NLS
peptide and control peptide (an inactive NF-B NLS via specific amino
acid substitutions) were used to evaluate the effects of NF-B
inhibition on colony-forming unit (CFU) formation
using human CD34+ bone marrow cells stimulated by either
GM-CSF, IL-3, M-CSF, G-CSF, or a mixture of IL-3, SCF, GM-CSF, and EPO
(Fig 3). These results suggest that NF-B
is either required for cell proliferation and/or required for
clonogenic cell survival.
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| Fig 3.
Blocking NF-B nuclear translocation inhibits human
CD34+ bone marrow cell colony formation. Human
CD34+ bone marrow was purified (>96% pure) and
cultured in methyl cellulose medium containing various cytokines and
(1) 200 µg/mL NF-B NLS peptide, (2) 200 µg/mL control peptide,
and (3) no peptides. Error bars indicate 1 standard error of mean (SEM)
for 5 cultures and are omitted when smaller than the data symbol.
*Significant decrease compared with cultures containing the control
peptide (P .05).
|
|
GM-CSF, IL-3, and TNF-, but not M-CSF and G-CSF,
activate NF-B in human
CD34+CD19 bone marrow cells.
EMSA were performed to determine if CFU formation/inhibition (Fig 3)
correlates with NF-B activation/inhibition, respectively, using the
cytokines and NF-B fusion peptides previously described. As a
positive control, EMSA was also performed using TNF-, because it is
a well-documented activator of NF-B in a variety of cell types. The
results of these experiments demonstrate that GM-CSF, IL-3, and TNF-
activate NF-B and that NF-B nuclear localization is blocked by
the active fusion peptide (Fig 4) for each
stimulus. G-CSF and M-CSF did not activate NF-B in these studies.
These results are consistent with previous findings using other cell types except for M-CSF, which has been shown to activate NF-B in
macrophages.27,28 NF-B activation by cytokines does not correlate with CFU formation, because there are NF-B activators (PMA
and TNF-) that do not induce CFU formation and there are cytokines
(G-CSF and M-CSF) that stimulate CFU formation but do not activate
NF-B. In addition, there were several experiments in which there was
a baseline NF-B activity (Fig 4A, C, and D) suggesting that NF-B
activity may be constitutive. Although NF-B activation does not
correlate with CFU formation, NF-B inhibition does correlate with
CFU inhibition. Taken together, these data suggest that NF-B may be
required for cell survival rather than proliferation.
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| Fig 4.
Cytokine activation of NF-B in
CD34+CD19 bone marrow cells. Human
CD34+CD19 bone marrow was purified and
incubated for 18 hours at 37°C with each cytokine/peptide combination
and EMSA was performed. (A) TNF-, (B) GM-CSF, (C) IL-3, and (E) a
combination of GM-CSF, IL-3, SCF, and EPO activated NF-B and its
nuclear translocation could be blocked by the NF-B NLS peptide. (D)
M-CSF and G-CSF did not activate NF-B. The purity of the
CD34+CD19 cells for experiments (A)
through (E) was 99.1%, 98.7%, 96.8%, 95.6%, and 98.6%,
respectively.
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NF-B is required for human
CD34+CD19 cell survival.
To test whether NF-B is required for human CD34+ cell
survival, apoptosis was measured using the TUNEL assay in human
CD34+CD19 cells incubated for 16 hours with
NF-B NLS fusion active or control peptide. Cells were also exposed
to 50 µmol/L etoposide as a positive control for apoptosis.
Inhibition of NF-B nuclear translocation using 50 µg/mL peptide
resulted in 68.1% of the cells undergoing apoptosis (Fig
5). These results suggest that NF-B
is necessary for human CD34+CD19 bone marrow
cell survival.
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| Fig 5.
(A) Inhibition of NF-B nuclear translocation induced
apoptosis of human CD34+CD19 human bone
marrow cells. The deoxynucleotidyl TUNEL assay was performed on human
CD34+CD19 bone marrow cells (>98% pure)
after (A) 16 or (B) 18 hours of incubation with various
agents.19 The controls were incubated with buffer only
( ) or 50 µmol/L etoposide ( ). (A) NF-B NLS peptide ( );
control mutant peptide ( ). (B) Flow cytogram comparison of apoptosis
induced by 50 µg/mL NF-B NLS peptide (black line) and control
mutant peptide (gray line) shown in (A).
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| |
DISCUSSION |
We demonstrate here that NF-B is present in human
CD34+CD19 bone marrow cells and may play a
role in their survival. Virtually all
CD34+CD19 human bone marrow cells contain
NF-B DNA-binding activity that can be activated by PMA exposure, and
a variety of cytokines (eg, GM-CSF, IL-3, and TNF-) are able to
activate NF-B in these cells. NF-B specificity was verified by
antibody recognition in both the supershift EMSA and flow cytometric
assays and by competitive inhibition of nuclear translocation using a
peptide specific for the NF-B NLS. In addition, inhibition of
NF-B nuclear translocation induced both apoptosis and loss of
clonogenic function in human CD34+ cells. These findings
are consistent with the established role of NF-B as a secondary
messenger for GM-CSF,27 IL-3,29 and TNF-23 signal transduction as measured in other cell
types. Contrary to other studies,27,28 M-CSF did not
activate NF-B in our system. It is possible that the proportion of
the CD34+CD19 bone marrow cells that
expressed M-CSF receptors and are therefore capable of responding to
M-CSF was below the threshold of NF-B detection in the EMSA. Another
explanation is that the M-CSF signal transduction pathway in mature or
peripheral mononuclear phagocytes uses NF-B,27 whereas
CD34+CD19 bone marrow cells used in our
experiments have an NF-B-independent M-CSF signal transduction
pathway. Differences in NF-B activation have been observed for IL-3
signaling, depending on the cell type. Oster et
al27 did not detect NF-B activation
after IL-3 exposure using human peripheral mononuclear phagocytes, but
Besançon et al29 did observe IL-3-induced NF-B
activation in a pro-B IL-3-dependent cell line (Ba/F3). In addition,
these investigators demonstrated that NF-B was required for survival
in these cells. Our results indicate both NF-B activation by IL-3
and a requirement for NF-B in CD34+CD19
bone marrow cell survival.
It has been reported that Rel family members are sequentially expressed
during B-cell development and that ordered expression may help regulate
genes that are involved in any one stage of differentiation.43 Flow cytometric analysis of p65
distribution in CD34+ cells suggests that p65 is present in
virtually all CD34+ cells, suggesting a role for NF-B in
supporting cell survival rather than in the regulation of
differentiation-specific genes. The distribution of c-rel and its
possible role in stage-specific differentiation of human
CD34+ bone marrow cells remains unknown.
A number of different mechanisms may explain the role of NF-B in the
survival of CD34+ cells. Inhibition of NF-B has been
demonstrated to induce apoptosis in a variety of cell types, and in B
lymphocytes, inhibition of NF-B is reported to be associated with a
decline in c-myc expression.29,49 NF-B has also been
shown to transactivate c-myb as well,22 which is also
required for hematopoietic cell survival.50 In addition,
NF-B is a positive regulator for GM-CSF production,51 and GM-CSF has been shown to play an autocrine role in colony formation.52
The inhibition of NF-B by a variety of molecules, such as
glucocorticoids, has been proposed as a means of inducing apoptosis in
target cell populations.34,35 The data presented here
indicate that NF-B activation is necessary for general clonogenic
response and protects against apoptosis in CD34+ bone
marrow cells. Based on these results, proposed strategies involving
inhibition of NF-B activation as an adjunct to anticancer therapeutic paradigms should be approached with caution.
In addition to the potential role for the NF-B in regulating
hematopoiesis, these proteins are also
proto-oncogenes6,53,54 and may play a role in the
transformation process in several types of cancer. The avian homologue
of c-rel, v-rel, causes the tumorigenicity of the avian
reticuloendotheliosis virus (REV-T).55 Human
T-lymphotrophic virus-I (HTLV-I)-mediated T-cell leukemia is
thought to involve constitutive NF-B activation induced by the tax
protein.56 P50/p65 NF-B has also been reported to be
activated by the p210 BCR-ABL fusion protein that mediates
transformation in chronic myelogenous leukemia (CML),57 and
NF-B is reportedly a critical downstream element of Ha-Ras signaling
and may mediate its oncogenic potential.58
In conclusion, our findings demonstrate that NF-B (p50, p65, c-rel)
is present in human CD34+CD19 bone marrow
cells and suggest that it is required for colony formation and cell
survival. These results suggest that NF-B plays an important role in
human CD34+ cell signal transduction, gene expression, and transformation.
| |
ACKNOWLEDGMENT |
The authors thank D. Som and C. Hodge for their technical assistance
and K. Helm for technical assistance with flow cytometry.
| |
FOOTNOTES |
Submitted March 16, 1998; accepted January 7, 1999.
Supported by Grant No. ES06258 from the National Institute of
Environmental Health Sciences, National Institutes of Health (NIH) and
its contents are solely the responsibility of the authors and do not
necessarily represent the official views of the NIEHS, NIH. This
publication was also made possible by the cooperation and support of
University of Colorado Cancer Center Flow Cytometry Core (Grant No. 2 P30 CA 46934-09) and the Clinical Investigation Core.
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 Richard D. Irons, PhD,
D.A.B.T., University of Colorado Health Sciences
Center, 4200 E 9th Ave, Box C238, Denver, CO 80262; e-mail:
Richard.Irons{at}UCHSC.edu.
| |
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ABSTRACT
INTRODUCTION