Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-06-1720.
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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1324-1328
HEMATOPOIESIS
Stem cell factor increases the expression of FLIP that inhibits
IFN -induced apoptosis in human erythroid progenitor cells
Ik-Joo Chung,
Chunhua Dai, and
Sanford B. Krantz
From the Department of Veterans Affairs Medical
Service, Department of Medicine, Division of Hematology/Oncology; and
the Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville,
TN.
 |
Abstract |
Interferon  (IFN) acts on human erythroid colony-forming
cells (ECFCs) to up-regulate Fas, without a demonstrable change of Fas
ligand (FasL) or Fas-associated DD-containing protein (FADD) expression
and activates caspase-8 plus caspase-3, which produce apoptosis. Our
previous data showed that stem cell factor (SCF) reduced the inhibitory
effect of IFN on human ECFCs when both factors were present in the
cultures. However, the mechanism by which SCF prevents IFN-induced
apoptosis in ECFCs is unclear. In this study we used highly purified
human ECFCs to investigate the mechanism of the effect of SCF on
IFN-induced apoptosis. Because the binding of FasL to Fas is the
first step of the apoptosis cascade and IFN strongly up-regulates
Fas expression, we added FasL (50 ng/mL) to the cultures with IFN to
accentuate the IFN-induced activation of caspase-8 and caspase-3
plus subsequent apoptosis. SCF (100 ng/mL) clearly inhibited the
activation of caspase-8 and caspase-3 induced by IFN and/or FasL,
and it also reduced apoptosis as measured by the terminal dUTP nick-end
labeling (TUNEL) assay. SCF did not decrease the surface expression of
Fas on the ECFCs. FADD-like interleukin 1  (IL-1)-converting
enzyme (FLICE)-inhibitory protein (FLIP) has been reported to interact
with FADD and/or caspase-8 at the death-inducing signaling complex
(DISC) level following Fas stimulation and acts as a dominant-negative
caspase-8. SCF increased FLIP mRNA and protein expression, concomitant
with reduced apoptosis, whereas IFN and/or FasL did not change FLIP expression. Reduction of FLIP expression with antisense
oligonucleotides decreased the capacity of SCF to inhibit
IFN-induced apoptosis, demonstrating a definite role for FLIP in the
SCF-induced protection of ECFCs from IFN-initiated apoptosis.
(Blood. 2003;101:1324-1328)
© 2003 by The American Society of Hematology.
| |
Introduction |
Fas (CD95/APO-1) is a prominent member of the death
receptor family. Its cardinal death-signaling function is ensured by
the presence of a cytoplasmic protein-protein interaction motif called the death domain (DD).1 Clustering of Fas induces
association of the cytoplasmic adaptor protein Fas-associated
DD-containing protein (FADD) with the oligomerized DD of the
receptor.2,3 FADD in turn recruits the zymogen form of the
initiator caspase-8/FADD-like interleukin 1 (IL-1)-converting enzyme (FLICE),4-6 thus leading to
the formation of the death-inducing signaling complex (DISC) that is
the most receptor-proximal element of signal transduction by
Fas.7 Recruitment of the zymogen form of caspase-8 to the DISC leads to its autoproteolytic cleavage and the release of its
active enzyme form in the cytosol, which initiates the cascade of
caspase activation and apoptosis. One mechanism for inhibition of
Fas-mediated apoptosis occurs through the action of FLICE-inhibitory protein (FLIP), a novel Fas pathway inhibitory protein, which acts as a
dominant-negative caspase-8.8,9
The inhibitory effects of interferon (IFN) on murine and human
granulocyte-macrophage colony-forming units (CFU-GMs), burst-forming units-erythroid (BFU-Es), and colony-forming units-erythroid (CFU-Es) in vitro have been reported by many investigators.10-17
Experiments in our laboratory have shown that IFN reduced erythroid
colony formation, cell proliferation, and differentiation of highly
purified human day  3 to day 6 BFU-Es in a dose-dependent manner and
produced profound erythroblast apoptosis.17 We also have
shown that IFN markedly increased the percentage of cells expressing
Fas on the surface of human erythroid colony-forming cells (ECFCs) as
well as the intensity of Fas expression on these cells and induced the
up-regulation and activation of caspase-8 and caspase-3 to produce
apoptosis in human ECFCs.18,19
Stem cell factor (SCF) has an essential role in the development of
erythroid cells and affects intracellular signaling associated with
proliferation, differentiation, and survival of erythroid progenitor
cells.20-24 Several studies have shown that SCF reduces IFN-induced inhibition of ECFC development17 and
Fas-mediated apoptosis in hematopoietic progenitor cells. Nishio et
al25 reported that SCF inhibits the activation of
caspase-8 and caspase-3 without down-regulation of the surface
expression of Fas and prevents Fas-mediated apoptosis of human ECFCs
with Src-family kinase dependency. Recently Endo et al26
demonstrated that SCF induces phosphorylation of Akt at Ser473 in human
erythroid cells and suggested that c-kit-mediated Src kinase
activation is involved in Akt activation and cell survival. Despite
those developments, the precise mechanisms by which SCF prevents IFN
and/or Fas-induced apoptosis in ECFCs have remained unknown. In this
study we investigated the effect of SCF on IFN and/or Fas ligand
(FasL)-induced apoptosis in human ECFCs.
| |
Materials and methods |
Generation of ECFCs
This method has been previously described.27 In
brief, 400 mL of blood was obtained from healthy donors who signed
consent forms approved by the Vanderbilt Committee for the Protection of Human Subjects and the Nashville Department of Veterans Affairs Research and Development Committee. BFU-Es (day-0 cells) were purified
by sequential density gradient centrifugation, depletion of platelets
and lymphocytes, and removal of adherent cells after overnight culture.
A further negative selection and removal of contaminant cells with CD2,
CD11b, CD16, and CD45 monoclonal antibodies was performed as previously
described.18,27 The day 1 BFU-Es were suspended in
Iscove modified Dulbecco medium (IMDM) containing 20% heat-inactivated
fetal calf serum (FCS), 5% heat-inactivated, pooled, human AB serum,
1% deionized bovine serum albumin (BSA), 5 × 10 5 M
2-mercaptoethanol, 10 µg/mL insulin (Sigma, St Louis, MO), 2 U/mL
erythropoietin (Amgen, Thousand Oaks, CA), 50 U/mL IL-3 (R&D Systems,
Minneapolis, MN), 100 ng/mL SCF (Amgen), and streptomycin plus
penicillin to generate ECFCs. After 5 days of culture the average
purity of day 6 ECFCs was 60% or higher as measured by the plasma
clot assay. The day-6 cells were then collected and further incubated
in the above medium lacking IL-3, with or without SCF, IFN
1× 107 U/mg (R&D Systems), and FasL (R&D Systems) in
liquid suspension. In some experiments, BFU-Es were incubated for
additional days as indicated.
Western blot analysis
Whole cell lysates were prepared in lysis buffer (1% Triton
X-100, 20 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, pH
7.5, 140 mM NaCl, 100 mM sodium fluoride, 10 mM EDTA
(ethylenediaminetetraacetic acid), 2 mM vanadate, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.15 U/mL aprotinin). Equivalent
amounts of total cellular protein were electrophoresed on 10% or 12%
sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to
nitrocellulose membranes (BIO-RAD, Hercules, CA). The membranes were
blocked in 5% dry milk in 0.05% Tween 20-Tris-buffered saline (TBST)
for 2 hours at room temperature. Incubation with primary antibodies was
done at 4°C overnight, and incubation with secondary horseradish
peroxidase (HRP)-linked antibodies (Amersham Pharmacia Biotech,
Piscataway, NJ) was performed for 1 hour at room temperature. After
washing the membranes extensively in TBST, antibody binding was
detected by using enhanced chemiluminescence (Amersham Pharmacia
Biotech). Antibodies used in this study are as follows: polyclonal
rabbit anti-FLIP antibody (kindly provided by Dr Donald Nicholson and
Merck Frosst Canada, Quebec, Canada), polyclonal anticaspase-3 antibody
(BD Pharmingen, San Diego, CA), monoclonal anticaspase-8 antibody (Cell
Signaling Technology, Beverly, MA), and monoclonal antiactin antibody
(Santa Cruz Biotechnology, Santa Cruz, CA).
RNA preparation and Northern analysis
Total RNA was prepared from ECFCs treated with or without SCF
for 48 and 96 hours using ULTRASPEC (BIOTECX Laboratories, Houston, TX). The full sequence of FLIP was used as DNA probe. Quantification of
RNA, formaldehyde gel electrophoresis, blotting onto nylon membranes,
and hybridization with 32P-labeled FLIP probe were
performed as previously described.18 To verify sample
loading variation, the same blots were reprobed with human G3PDH cDNA
control probes that were purchased from Clontech (Palo Alto, CA).
Transfection with FLIP antisense oligonucleotides
We used FLIP antisense oligonucleotides (ASOs) and nonsense
oligonucleotides (NSOs) as reported by Bannerman et al28
with a little modification. The
2'-O-methyl/2'-deoxynucleotide chimeric oligonucleotides28,29 used in all experiments were
synthesized by Operon Technologies (Alameda, CA). Chimeric
oligonucleotides were used to support an RNase H-dependent mechanism of
action, which results in a selective loss of target
mRNA.30 The FLIP ASOs contained 8 mismatches, as compared
with the control NSOs. The sequence of the ASOs to FLIP is
5'-ACUUGTCCCTGCTCCUUGAA-3' and the sequence of the NSOs is
5'-UCUAGCCTCTCCTCGUAGUA-3'. The first 5 and last 5 bases represent
2'-O-methyl-modified nucleotides. The middle 10 bases
represent 2'-deoxynucleotides. Oligonucleotides are further modified
with phosphorothioate linkages. Day 5 ECFCs were incubated with or
without SCF (30 ng/mL) for 48 hours. FLIP ASOs or NSOs were added to
day 7 ECFCs at 1 µM final concentrations and were incubated in the
presence of Oligofectamine (Gibco BRL, Gaithersburg, MD) in 5% serum
medium for 12 hours. IFN (400 U/mL) and FasL (50 ng/mL) were then
added to the ECFCs for an additional 36 hours of incubation at 37°C.
Down-regulation of the relative protein levels was evaluated by
immunoblotting, and apoptosis was evaluated by terminal uridine dUTP
nick-end labeling (TUNEL) assay at 48 hours after ASO and NSO incubation.
TUNEL assay for quantitation of apoptotic cells
Apoptotic cells were identified by the TUNEL method using an in
situ cell death detection kit (Roche Diagnostics, Indianapolis, IN)
according to the manufacturer's instructions. Briefly, ECFCs were
harvested and washed twice in phosphate-buffered saline (PBS). After centrifugation, cell pellets were resuspended in 200 µL PBS
containing 4% paraformaldehyde for 1 hour at 26°C and were washed
twice again. Permeabilization was conducted by using 100 µL 0.1%
Triton X-100 and 0.1% sodium citrate in PBS. After washing, the cells
were resuspended in TUNEL reaction mixture containing fluorescein
isothiocyanate (FITC)-dUTP and
terminal-deoxy-nucleotidyl-transferase (TdT). Control cells were
suspended in the TUNEL reaction mixture containing FITC-dUTP without
TdT, and incubations were performed for 1 hour at 37°C before washing
the cells twice. Fluorescein labels incorporated into DNA strand breaks
were detected by flow cytometry.
Statistics
Student t test was used to determine statistical
significance. The minimal level of significance was
P = .05.
| |
Results |
Addition of FasL augments caspase-8 and caspase-3 activation
induced by IFN
IFN increases the percentage of cells expressing Fas on the
surface of the human ECFCs as well as the intensity of Fas expression and induces the activation of caspase-8 and caspase-3 in human ECFCs.16,17 We added the FasL with IFN to augment
caspase-8 and caspase-3 activation and apoptosis. Human day 6 ECFCs
were treated with IFN (400 U/mL) and/or FasL (10, 25, or 50 ng/mL) for 48 hours at 37°C, and cell lysates were prepared and probed with
the anticaspase-8 and anticaspase-3 antibodies. The addition of FasL
augments caspase-8 and caspase-3 activation induced by IFN in a
dose-dependent manner (data not shown). When day 6 ECFCs were
cultured with medium with or without IFN (400 U/mL) and FasL (50 ng/mL) for 24 to 96 hours, the cleavage of caspase-8 and caspase-3 was
increased during 24 hours of incubation with IFN/FasL, and the
activation of caspase-8 and caspase-3 persisted or further increased
over 96 hours (Figure 1).
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| Figure 1.
Caspase-8 and caspase-3 are activated by IFN plus
FasL in a time-dependent manner.
Day 6 ECFCs were cultured in medium with or without IFN (400 U/mL), FasL (50 ng/mL) at 37°C for 24 to 96 hours. Cell protein
lysates were prepared, and immunoblot analyses were performed with
anticaspase-8 and anticaspase-3 antibodies.
|
|
SCF inhibits caspase-8 and caspase-3 activation and apoptosis
induced by IFN
To investigate the effect of SCF on IFN and/or FasL-induced
caspase-8 and caspase-3 activation and apoptosis, day 6 ECFCs were
cultured in medium with or without IFN (400 U/mL), FasL (50 ng/mL),
or SCF (100 ng/mL). After 72 hours of incubation at 37°C, cell
lysates were prepared, and immunoblot analysis was performed with
anticaspase-8 and caspase-3 antibodies. As shown in Figure
2, the addition of FasL (50 ng/mL)
augmented the activation of caspase-8 and caspase-3 induced by IFN,
and SCF clearly reduced the activation of caspase-8 and caspase-3
induced by IFN and FasL. TUNEL assay showed that the percentage of
apoptotic cells was 27% in the IFN-treated group and 50% in the
IFN/FasL-treated group after 120 hours of incubation at 37°C in
5% serum medium (Figure 3). The addition
of SCF decreased the percentage of apoptotic cells to 6% in the
IFN-treated group and to 18% in the IFN/FasL-treated group. The
same pattern was present in 6 experiments using slightly different time
intervals. These experiments showed that SCF inhibits apoptosis induced
by IFN and/or FasL. Consistent with reported data,25
SCF did not decrease the surface expression of Fas on the ECFCs (data
not shown), which suggests that SCF acts on a pathway downstream of Fas
and upstream of the caspase cascade to inhibit apoptosis.
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| Figure 2.
SCF inhibits caspase-8 and caspase-3 activation induced
by IFN and/or FasL.
Day 6 ECFCs were incubated in medium with or without IFN (400 U/mL), FasL(50 ng/mL), or SCF (100 ng/mL) for 72 hours. Cell protein
lysates were prepared, and immunoblot analyses were performed with
anticaspase-8 and anticaspase-3 antibodies.
|
|
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| Figure 3.
SCF inhibits apoptosis induced by IFN and/or FasL.
Day 6 ECFCs were incubated in medium with or without IFN (400 U/mL), FasL (50 ng/mL), or SCF (100 ng/mL) at 37°C for 120 hours in
5% serum medium, and apoptosis was determined by TUNEL
assay.
|
|
SCF increases the expression of FLIP protein and mRNA
We evaluated the effect of SCF on FLIP expression because FLIP has
been reported to interact with FADD and/or caspase-8 at the DISC level
after Fas stimulation and to act as a dominant-negative caspase-8. To
identify the potential involvement of FLIP in the SCF protection
pathway, we incubated day 6 ECFCs for up to 72 hours with SCF (100 ng/mL) and incubated day 6 ECFCs with increasing concentrations of
SCF for 48 hours. Because day 1 BFU-Es were incubated in medium
containing SCF (100 ng/mL) and SCF is present in serum at a
concentration of approximately 3 ng/mL, in our initial experiments the
day 5 ECFCs were washed twice with PBS and then were
incubated in low (5%) serum medium at 37°C for the duration of the
experiment in an attempt to reduce baseline stimulation by SCF. As
shown in Figure 4A, the increase of FLIP
protein was detectable after 36 hours, reaching a maximum by 72 hours
of incubation with SCF. An increase of FLIP protein was evident at an
SCF concentration of 25 ng/mL and continued to increase at 50 to 100 ng/mL when day 6 ECFCs were incubated with SCF for 48 hours (Figure
4B). In subsequent experiments the prior incubation in 5% serum medium was omitted. We then incubated day 6 ECFCs in regular medium with or
without SCF and assayed FLIP protein expression and FLIP mRNA levels.
Immunoblot experiments showed that FLIPL expression level
was much higher in the group treated with SCF (100 ng/mL) compared with
the control group after 72 hours of incubation (Figure 4C). IFN or
FasL did not change FLIP expression. When we added SCF together with
IFN or IFN/FasL, the FLIP expression level was higher than when
IFN or FasL was added alone but was less than when SCF was added
alone. The FLIP mRNA expression level was also higher in the
SCF-treated group than in the control group after 48 and 96 hours of
incubation (Figure 4D). These findings suggest that the increase in the
FLIP protein is most likely controlled by the level of mRNA and that
SCF may be acting through transcriptional up-regulation.
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| Figure 4.
SCF enhances expression of FLIP and mRNA.
(A,B) Day 5 ECFCs were washed twice with PBS and incubated in 5%
serum medium for 24 hours. Day 6 ECFCs were then incubated with SCF
(100 ng/mL) for up to 72 hours (A) or with SCF at increasing
concentrations for 48 hours (B), and cell protein lysates were prepared
at each indicated time. FLIPL (long form of FLIP) protein
was determined by immunoblot analysis. (C) Without a prior incubation
in 5% serum medium, day 6 ECFCs were cultured at 37°C in regular
medium with or without IFN (400 U/mL), FasL (50 ng/mL), or SCF (100 ng/mL) for 72 hours. FLIPL protein was determined by
immunoblot analysis. (D) Without a prior incubation in 5% serum
medium, day 6 ECFCs were cultured in regular medium with or without
SCF (100 ng/mL) for 48 and 96 hours. FLIP mRNA was determined by
Northern blot analysis.
|
|
FLIP ASO reduces FLIP expression and enhances
IFN/FasLinduced apoptosis that is not reduced by SCF in the
presence of ASO
To establish a causal connection between the antiapoptotic effect
of SCF and increased expression of FLIP by SCF, ASOs were designed to
specifically reduce the expression of FLIP. We exposed SCF (30 ng/mL)-treated or untreated ECFCs, transfected with FLIP ASO or NSO,
to IFN and FasL and assayed for FLIP protein expression levels and
apoptosis. Immunoblot analysis of ECFCs transfected with FLIP ASOs
revealed a significant decrease in the expression of FLIPL
compared with ECFCs transfected with NSOs in both the SCF-treated and
-untreated groups (Figure 5A). Reduction
of FLIP expression with ASOs enhanced the IFN/FasL-induced apoptosis of the ECFCs. ECFCs treated with SCF and NSO had reduced apoptosis compared with cells treated with NSO without SCF, but this effect of
SCF was not seen in ASO-treated cells, demonstrating a definitive role
for FLIP in the protection of ECFCs from IFN and FasL-induced apoptosis (Figure 5B). These experiments showed that antiapoptotic effect of SCF against IFN and FasL in human ECFCs is due at least partly to the increased expression of FLIP by SCF.
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| Figure 5.
FLIP ASOs reduce FLIP expression and enhance
IFN/FasL-induced apoptosis that is not reduced by SCF in the
presence of ASOs.
Day 5 ECFCs were incubated at 37°C with or without SCF (30 ng/mL)
for 48 hours, and then the resulting day 7 ECFCs were transfected
with FLIP ASOs or control NSOs over 12 hours before incubation with
IFN (400 U/mL) and FasL (50 ng/mL) over 48 hours. (A) Cell lysates
were prepared, and immunoblot analyses were performed with anti-FLIP
antibody. (B) Apoptosis was determined by TUNEL assay, and the
percentages of apoptotic cells were expressed as the mean ± SD
from 4 independent experiments. *P < .05, ASO-treated
group versus NSO-treated group (without SCF preincubation);
**P < .05, ASO-treated group versus NSO-treated group
(with SCF preincubation); ***P < .05, NSO-treated group
without SCF preincubation versus NSO-treated group with SCF
preincubation; ****P = .21, ASO-treated group without SCF
preincubation versus ASO-treated group with SCF preincubation.
|
|
| |
Discussion |
Other investigators have reported that SCF inhibits apoptosis of
ECFCs induced by CH11, a Fas ligand-mimetic antibody,25,26 but the mechanism by which SCF mediates this process is not known. To
investigate the mechanism by which SCF reduces IFN-induced apoptosis
in ECFCs, we first examined the effect of SCF on the Jak/STAT1 (janus
kinase signal transducer and activator of transcription 1) pathway
activated by IFN. Biologic responses to IFN are mediated mainly
by the regulation of gene expression, and it has been established that
most of the pleiotropic effects of IFN are mediated by several gene
products that are regulated by the Jak-STAT1 pathway.31 Our experiments showed that IFN definitively induced the
phosphorylation of STAT1 and expression of IRF1 (IFN-regulatory factor
1) in the ECFCs, but SCF did not affect the phosphorylation of STAT1
and the expression of IRF1 induced by IFN (I.-J.C., C.D., and
S.B.K., unpublished data, 2001). Although the lack of an
apparent SCF effect on STAT1 could be due to an activation of
STAT1 by SCF that counterbalanced a change in the IFN activation or
could be due to activation of a STAT1-independent pathway, we decided to examine an effect on later events in the traditional IFN
transduction pathway.
We evaluated the effect of SCF on the Fas pathway and the caspase
cascade because our previous data showed that IFN markedly increased
the percentage of cells expressing Fas on the surface of the human
ECFCs as well as the intensity of Fas expression and induced the
activation of caspase-8 and caspase-3 to produce apoptosis in human
ECFCs.18,19 This was greatly reduced by both NOK-2
antihuman Fas ligand and soluble Fas ligand receptor, Fas-Fc.18 As shown in Figures 2 and 3, SCF inhibited the
activation of caspase-8 and caspase-3 along with the apoptosis induced
by IFN and/or FasL. Consistent with reported data,25 we
also found that SCF did not decrease the surface expression of Fas on
the ECFCs (unpublished data, 2001). These observations suggested that SCF acts on the pathway downstream of Fas and upstream of the caspase
cascade to inhibit apoptosis.
FLIP, which structurally resembles caspase-8, was identified as a
cellular homologue of viral FLIPs, except that it lacks proteolytic
activity.8,9,32 FLIP is recruited to the Fas DISC through
the adaptor molecule FADD similar to caspase-8, thereby preventing the
recruitment of caspase-8 into the complex and subsequent caspase-8
activation.33 Cellular FLIP (c-FLIP) is found mainly with
2 splice variants, a long form (FLIPL) and a short
form (FLIPS).33 So far, most of the studies
concerning FLIP have focused on the long form, FLIPL, most
likely because it is generally more abundant in cells. Both
c-FLIPL and c-FLIPS block procaspase-8
activation at the DISC even though the 2 splice variants produce this
effect in a distinctly different way.34
The signaling pathways by which FLIP expression is modulated are not
well understood. In the present study, we found that increased
expression of FLIP is clearly induced by SCF in human ECFCs (Figure 4).
Reduction of FLIP expression with ASO-sensitized ECFCs to
IFN/FasL-induced apoptosis (Figure 5), demonstrating a definitive
role for FLIP in the protection of ECFCs from IFN/FasL-induced apoptosis. These experiments also showed that the antiapoptotic effect
of SCF against IFN/FasL in human ECFCs was greatly reduced by the
ASOs. The fact that SCF increases FLIP and reduces apoptosis, coupled
with the fact that ASOs to FLIP prevent the increase in FLIP and at the
same time prevent the decrease in apoptosis produced by SCF, lead us to
the conclusion that SCF inhibits IFN/FasL-induced apoptosis through
increased FLIP expression.
| |
Acknowledgments |
We thank Dr Donald Nicholson and Merck Frosst Canada, Inc, for
their gift of polyclonal rabbit antibody to FLIP
(Usurpin35).
| |
Footnotes |
Submitted June 11, 2002; accepted September 16, 2002.
Prepublished
online as Blood First Edition Paper, October 10, 2002; DOI
10.1182/blood-2002-06-1720.
Supported by a Veterans Health Administration Merit Review Grant
(S.B.K.) and by grants ROI DK-15555 and 2 T32-DK-07186 (S.B.K.) and
CA-68485 (Vanderbilt-Ingram Cancer Center) from the National Institutes
of Health.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Sanford B. Krantz, Department of
Medicine-Hematology/Oncology, Vanderbilt University, 777 PRB, 2220 Pierce Ave, Nashville, TN 37232-6307; e-mail:
sanford.krantz{at}med.va.gov.
| |
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Abstract
Introduction