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RED CELLS
Zileuton induces hemoglobin F synthesis in erythroid progenitors: role of the
L-arginine–nitric oxide signaling pathway
Johnson Haynes Jr, B. Surendra Baliga, Boniface Obiako, Solomon Ofori-Acquah, and Betty Pace
Induction of fetal hemoglobin (Hb F) is
an important therapeutic tool in ameliorating complications of sickle cell disease. Nitric oxide has been implicated in
the mechanism of Hb F synthesis induced by hydroxyurea (HU). This study
examined whether zileuton (ZL), a structural analog of hydroxyurea, possessed
Hb F–inducing properties and the potential role nitric oxide plays. ZL caused a
dose-dependent increase in ␥-globin expression in K562 cells. This effect was
confirmed by a dose-dependent increase in Hb F synthesis in erythroid
progenitors from individuals with sickle
cell anemia and normal hemoglobin genotypes. L-arginine had no effect on Hb
F production; however, it dose-dependently inhibited ZL’s ability to induce Hb
F. The nitric oxide synthase inhibitor
NG-monomethyl–L-arginine (L-NMMA) inhibited L-arginine’s effect and restored
ZL-mediated increase in Hb F synthesis.
In addition, 8-PCPT–cGMP (8-(4-chlorophenylthio)guanosine 3ⴕ, 5ⴕ-cyclic monophosphate) inhibited ZL-mediated induction of Hb F synthesis. When comparing
L-NMMA effects alone on ZL and HU, a
partial reversal of increased Hb F synthesis was seen only with HU. Neither
L-arginine alone nor L-arginine in combination with L-NMMA effected hydroxyurea-mediated induction of Hb F synthesis. This study demonstrates that ZL
induces Hb F through a mechanism that
involves L-arginine/nitric oxide/cGMP in
a manner distinctly different from HU.
(Blood. 2004;103:3945-3950)
© 2004 by The American Society of Hematology
Introduction
Zileuton (ZL; (N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea)
is a specific inhibitor of 5-lipoxygenase (5-LO), which results in
decreased formation of leukotrienes (LTB4, LTC4, LTD4, and
LTE4).1 Structurally, ZL contains N-hydroxyurea (HU).2 This
study investigated whether or not ZL can stimulate fetal
hemoglobin (Hb F) synthesis in erythroid progenitors, as has
been observed with HU. Currently HU is the only Federal Drug
Administration–approved agent used to stimulate Hb F synthesis with proven efficacy in the treatment of sickle cell disease.
HU has been documented to reduce the severity and frequency
of painful vasoocclusive crisis, the incidence of acute chest
syndrome,3,4 and mortality5 in sickle cell anemia (HbSS).
Despite its use in HbSS, the mechanism of action by which HU
induces Hb F remains uncertain. HU is cytotoxic and inhibits
growth of burst forming unit-erythroid (BFU-E) colonies in a
dose-dependent manner, while Hb F levels increase. The
decrease in BFU-E colony growth observed with HU treatment
is related to interruption of DNA synthesis in rapidly dividing
late erythroid progenitors by inhibition of the enzyme, ribonucleotide reductase. This leads to a transient arrest of hematopoiesis.
Suppression of BFU-E colony growth has been demonstrated to
enhance premature commitment of earlier progenitor cells,
accelerate erythropoiesis, and increase survival of sickle red
blood cells containing Hb F.6-8
Nitric oxide has been implicated in the regulation of Hb F
induction by HU, which was recently shown to react with heme
proteins to generate nitric oxide.9-11 Ikuta et al12 demonstrated
that an intracellular pathway, which includes soluble guanylate
cyclase and cyclic guanosine monophosphate (cGMP)–dependent protein kinase, plays a role in the induction of ␥-globin
gene expression in 2 independent K562 erythroleukemic cell
lines. Most recently, Cokic et al13 demonstrated that HU induces
Hb F by the nitric oxide–dependent activation of soluble
guanylate cyclase in K562 cells and human erythroid progenitors.
While, structurally, ZL contains HU, there are no published
data that document whether ZL stimulates Hb F synthesis in
primary erythroid progenitors.6-8 In the present study, we
demonstrate ZL is a potent inducer of ␥-globin mRNA in normal
and sickle erythroid progenitors and in K562 cells. This study
further demonstrates that ZL decreases BFU-E colonies in cell
culture and stimulates Hb F synthesis similarly to that observed
with HU. We postulated that ZL induced Hb F through a nitric
oxide–dependent pathway, as previously suggested with HU.
We found that ZL induction of Hb F synthesis occurs through a
mechanism that does not involve the nitric oxide/cGMP pathway as demonstrated for HU. Furthermore, we observed that the
nitric oxide/cGMP pathway does play a negative regulatory role
in ZL induction of Hb F in contrast to HU.
From the Departments of Medicine, Pediatrics, Cell Biology, and Neuroscience,
University of South Alabama Comprehensive Sickle Cell Center, Mobile, AL;
and the Department of Molecular and Cell Biology, University of Texas at
Dallas, Richardson, TX.
Development (contract grant no. P60 HL-38639).
Submitted September 2, 2003; accepted January 23, 2004. Prepublished
online as Blood First Edition Paper, February 5, 2004; DOI 10.1182/blood2003-08-2969.
Supported by Comprehensive Sickle Cell Program from the National Heart,
Lung and Blood Institute and the Florence Foundation Research Career
BLOOD, 15 MAY 2004 䡠 VOLUME 103, NUMBER 10
Reprints: Johnson Haynes Jr, University of South Alabama Medical Center,
10th Floor, Suite H, 2451 Fillingim St, Mobile, AL 36617; e-mail:
[email protected].
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.
© 2004 by The American Society of Hematology
3945
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3946
BLOOD, 15 MAY 2004 䡠 VOLUME 103, NUMBER 10
HAYNES et al
Patients, materials, and methods
1 for Hb F determination. Fetal hemoglobin was measured as a percent of
total hemoglobin, normalized to total protein by alkaline denaturation as
published previously.6,14
Materials
Statistics
Bovine serum albumin (BSA, 66 000 molecular weight [mol wt]), and
L-arginine were purchased from Sigma (St Louis, MO). ZL and HU were
purchased from the University of South Alabama Pharmacy (Mobile, AL).
NG-monomethyl–L-arginine (L-NMMA) was purchased from RBI (Natick,
MA). Phorbol myristate acetate (PMA) and A23187 were dissolved in
dimethyl-sulfoxide (DMSO). ZL was dissolved in 95% ethanol. HU,
L-arginine, and L-NMMA were dissolved in sterile water. The 8-PCPT–
cGMP (8-(4-chlorophenylthio)guanosine 3⬘,5⬘-cyclic monophosphate) was
purchased from BIOLOG (Bremen, Germany) and dissolved in phosphatebuffered saline (PBS).
All results are presented as means ⫾ SEM. Statistical analyses were
performed using the unpaired Student t test and one-way analysis of
variance (ANOVA). The Tukey test and Dunnett test15 were used for
multiple comparisons when ANOVA indicated statistically significant
differences between or within groups. Differences were considered to be
significant when P is less than .05.
Results
Blood collection
ZL augments ␥-gene mRNA levels in K562 cells
All participants gave informed consent and the University of South
Alabama Institutional Review Board approved the protocol. During steadystate, blood samples (40 mL) were obtained from individuals with
homozygous sickle cell anemia in the sickle cell clinics at the University of
South Alabama. Whole blood samples were collected in syringes containing
heparin and used within 48 hours of collection. Individuals who received a
transfusion within 6 weeks of sample procurement were excluded from this
study. Sickle cell anemia was documented by hemoglobin electrophoresis
on each subject.
Several pharmacologic Hb F inducers such as HU, 5-azacytidine,16
erythropoietin,17 didox,18 and butyrate19 alter ␥-globin gene expression in culture. Although the correlation between in vitro response
and in vivo Hb F production is not necessarily direct, the induction
of ␥-globin gene activity in culture is a good indicator of potential
clinical usefulness. Therefore, we analyzed the ability of ZL to
induce ␥-globin gene activity at the mRNA level. K562 cells were
cultured in ZL (0 to 100 ␮M) or HU (50 ␮M to 100 ␮M) for 48
hours. Total cellular RNA was analyzed by RPA for ␥-globin
mRNA levels. We observed an increase in ␥-globin mRNA for ZL
Cell culture
Human K562 erythroleukemic cells were grown in suspension cultures in
RPMI-1640 containing 10% fetal bovine serum, penicillin (100 U/mL), and
streptomycin (0.1 mg/mL) in a humidified incubator at 37°C, 5%CO2/95%
air atmosphere. The following experimental conditions were analyzed in
triplicate with 0.3 ⫻ 106 cells per well: an untreated control; ZL dissolved
in 0.1% DMSO at final concentrations of 20, 40, 50, 75, or 100 ␮M; HU at
50, 75, or 100 ␮M; and 2 positive controls, hemin (50 ␮M; Sigma) and
sodium butyrate (NB; Sigma, 2 mM). The K562 cells were induced for 48
hours then RNA isolated for further analysis. Cell viability was determined
by 2% trypan blue exclusion at times 0 and 48 hours.
RNase protection analysis
RNA was isolated using RNA Stat-60 (TEL-TEST “B,” Inc, Friendswood,
TX) according to the manufacturer’s instructions. In brief, cell pellets were
suspended in RNA Stat-60 (1 mL) and the RNA was precipitated with
isopropanol. The mRNA levels were quantitated by RNase protection assay
(RPA) with probes designed to yield protected fragments for human
␥-globin (Hu␥) and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPD) as previously described.14 Hu␥ mRNA levels were
normalized to that of GAPD and compared with untreated K562 cells.
BFU-E colony growth
Mononuclear cells were isolated from the peripheral blood of each
participant studied by density gradient centrifugation using Histopaque1077 (Sigma). Erythroid progenitors were incubated in methylcellulose
culture medium as previously described.14 ZL was added on day 0 at 25, 50,
75, or 100 ␮M concentrations for 14 days, at which time the number of
BFU-E colonies were counted on an inverted microscope and harvested for
hemoglobin determinations. We previously published similar experiments
for HU to analyze Hb F inducibility in erythroid progenitors.6 Informed
consent was obtained from all participants following the University of
South Alabama Institutional Review Board guidelines.
Hemoglobin F determination
After 14 days of incubation, erythroid progenitors were collected, washed
in PBS, and then lysed at room temperature in 200 ␮L of water. The
supernatant was split into 2 tubes, 1 for total hemoglobin determination and
Figure 1. ␥-Globin gene induction by ZL in K562 cells. K562 cells were treated
with ZL at 20 to 100 ␮M concentration or hydroxyurea (HU) 50 to 100 ␮M
concentrations for comparison. ␥-Globin mRNA levels were quantitated by RNase
protection assay (RPA) with human ␥ (Hu␥) and internal control gylcearaldehyde-3phosphate dehydrogenase (GAPD) probes to yield protected fragments of 170 bp
and 316 bp, respectively. (A) A representative RPA gel showing the mRNA bands for
ZL-treated cells on the left and HU-treated cells in the middle. (B) Quantitative values
for Hu␥ and GAPD mRNA production by phosphorimager analysis. The untreated
K562 samples (0 ␮M) were normalized to one (f). The total increase in ␥ mRNA was
calculated as a ratio of GAPD to control for variations in sample loading for ZL- (䡺) or
) and hemin (HE,
) were used
HU-treated (p) K562 cells. Sodium butyrate (NB,
as positive control for ␥ gene induction. *2 ⫽ NB at a 2 mM concentration. Error bars
indicate SEM (n ⫽ 4 for each concentration/groups reported); each study done in
triplicate.
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BLOOD, 15 MAY 2004 䡠 VOLUME 103, NUMBER 10
ZILEUTON INDUCES HEMOGLOBIN F
3947
Table 1. Effects of zileuton on BFU-E colony growth and fetal hemoglobin production in primary erythroid cultures
Zileuton concentrations, ␮M
0
25
50
75
100
Patient
no.
BFU-E
1
87
2
61
4
47
7
27
11
12
2
135
7
91
11
72
14
43
18
7
21
3
148
2
137
3
62
6
23
11
13
13
4
93
3
86
5
66
7
34
Mean
116
3.5
93.8
5.8
61.8
8.5
31.8
Hb F, %
BFU-E
Hb F, %
BFU-E
Hb F, %
BFU-E
Hb F, %
9
12.3
BFU-E
Hb F, %
14
21
16
13.3
16.0
SEM
15.2
1.2
15.8
1.8
5.3
1.8
4.4
2.0
2.9
1.8
P
—
—
⬎ .05
⬎ .05
⬍ .01
⬎ .05
⬍ .01
⬍ .01
⬍ .01
⬍ .01
P value represent a comparison of BFU-E colonies and Hb F in the absence of zileuton (0 ␮M) versus 25 to 100 ␮M concentrations. BFU-E indicates burst-forming
unit-erythroid; Hb F, fetal hemoglobin; and —, not applicable.
at 20 ␮M with a maximal increase at 75 ␮M (Figure 1A). The level
of ␥-globin gene mRNA synthesis was quantitated relative to the
internal control, GAPD. As shown in Figure 1B, we observed a
4.1-fold increase in ␥-globin mRNA at a 75 ␮M ZL concentration
compared with a 2.7-fold increase for HU at 100 ␮M (P ⬍ .05). Of
note is the observation that ZL increased ␥-globin mRNA 2.6-fold
at the 20 ␮M concentration demonstrating comparable induction to
100 ␮M HU (Figure 1B).
K562 cell viability was decreased 10% when measured by
trypan blue exclusion at 75 ␮M and 100 ␮M concentrations for
both ZL and HU. The level of ␥-globin gene induction was
3.9-fold and 10-fold for 2 ␮M sodium butyrate and 50 ␮M
hemin, respectively.
ZL induces fetal hemoglobin in primary erythroid progenitors
The parent compound HU is known to induce Hb F production in
vivo in sickle cell patients treated for an extended period and in
primary erythroid cell culture. Therefore, we analyzed the capability of ZL to induce Hb F in HbSS erythroid progenitors, grown
from peripheral blood mononuclear cells (MNCs). Each experiment was performed in triplicate. BFU-E colonies were grown in
methylcellulose media supplemented with erythropoietin, interleukin-3, granulocyte-macrophage colony-stimulating factor, stem
cell factor, and ZL at concentrations from 0 to 100 ␮M. The mean
BFU-E colony number was 116.0 ⫾ 15.2 without ZL compared
with 13.3 ⫾ 2.9 at 100 ␮M concentrations (P ⬍ .01; Table 1).
Despite the decline in BFU-E colonies, Hb F levels increased
from 3.5% ⫾ 1.2% to 16.0% ⫾ 1.8% (P ⬍ .01), thus demonstrating the ability of ZL to induce Hb F production in primary erythroid
progenitors. Pair-wise comparisons for BFU-E at 0 ␮M and 50, 75,
and 100 ␮M were significant at P less than .01. Pair-wise
comparisons for Hb F at 0 ␮M and 75 to 100 ␮M were significant
at P less than .01. The correlation coefficient between the number
of BFU-E colonies or percent Hb F versus the different ZL
concentrations was ⫺0.90 and ⫹0.79, respectively (P ⬍ .05).
These results are comparable to data we previously published for
HU.6 A dose-dependent 8.7-fold decrease in BFU-E colony number
(P ⬍ .05) was observed at 100 ␮M ZL compared with control.
Similar studies performed with HU (0 and 100 ␮M) resulted in a
21.3-fold decrease for HU in BFU-E colonies grown from sickle
cell anemia patients (P ⬍ .05). Despite less change in the number
of BFU-E growth with ZL, we observed a 4.6-fold increase in Hb F
compared with a 3.8-fold increase (5.1 ⫾ 1.0 to 19.4 ⫾ 1.5) for
HU, suggesting comparable Hb F induction for both drugs.
Next, the effect of ZL on ␥-globin gene expression was assessed
in erythroid progenitors obtained from healthy donors. Using the
same protocol described in “Patients, materials, and methods,”
BFU-E colonies from healthy donors were measured in the
presence of ZL at 0 to 100 ␮M concentrations. In normal erythroid
progenitors, we observed a decrease in BFU-E colony number from
94.0 ⫾ 30.6 to 10.1 ⫾ 2.4 (Figure 2A) and an increase in Hb F
from 1.4% ⫾ 0.05% to 6.0% ⫾ 0.38% (Figure 2B). This pattern
was similar to that obtained for sickle cell progenitors. Of note is
the lower average BFU-E colony number for normal progenitors.
This represents a 9.3-fold decrease and 4.3-fold increase in BFU-E
and Hb F, respectively. Pair-wise comparisons of BFU-E colony
growth for normal samples revealed significant decreases between
ZL (0 ␮M) and the 50, 75, and 100 ␮M concentrations (P ⬍ .05).
Similarly, Hb F levels were significantly increased at ZL concentrations of 50, 75, and 100 ␮M (P ⬍ .05).
L-arginine
dose-dependently reverses the effect of ZL in sickle
primary erythroid progenitors
Based on data previously published with HU, the nitric oxide–
dependent activation of soluble guanylate cyclase–protein kinase G
pathway in primary erythroblast constitutes a mechanism that
regulates expression of the ␥-globin gene.12 In this study, the
effect(s) of the nitric oxide donor, L-arginine,20,21 on ZL induction
of Hb F in primary sickle erythroid progenitors in culture was
assessed. L-arginine (0-100 ␮M) dose-dependently reversed ZL (50
␮M) suppression of BFU-E colony growth and stimulation of Hb F
synthesis (Figure 3). In contrast, L-arginine (100 ␮M) did not
reverse the suppression of BFU-E colony growth and stimulation
of Hb F synthesis seen with HU, 100 ␮M controls (Figure 4A-B).
When sickle erythroid progenitors were treated with L-arginine
(100 ␮M) alone, there was a 31% increase in BFU-E colony growth
compared with the BFU-E control (P ⬍ .01; Figure 4). L-arginine
Figure 2. BFU-E colony growth and fetal hemoglobin production in the
presence of zileuton for sickle cell and healthy individuals in methylcellulose
culture. Peripheral blood mononuclear cells were cultured as described in “Patients,
materials, and methods.” The results are expressed as the number of BFU-E colonies
per 3 ⫻ 105 mononuclear cells (MNCs). 䡺 indicates sickle cell samples; and u,
normal samples. Panel A demonstrates the BFU-E colonies on day 14 in the
presence of zileuton at 0 to 100 ␮M concentrations. Panel B represents Hb F for
sickle cell (䡺) and normal samples (u). Note Hb F induction for both groups tested.
Error bars represent SEM (n ⫽ 4 for each concentration/groups reported); each study
done in triplicate.
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3948
BLOOD, 15 MAY 2004 䡠 VOLUME 103, NUMBER 10
HAYNES et al
8-PCPT–cGMP (10 ␮M) and compared with ZL treatment and
control (no treatment) groups. The 8-PCPT–cGMP reverses ZL
(50 ␮M) suppression of BFU-E colony growth and stimulation
of Hb F synthesis, similar to that observed with L-arginine
(Figure 5A-B).
Nitric oxide synthase inhibition abrogates the L-arginine
inhibitory effect on zileuton
Figure 3. Effects of increasing concentrations of L-arginine on ZL-treated
erythroid progenitors isolated from patients with homozygous sickle cell
anemia. Cells were cultured on methylcellulose culture media and treated with ZL
(100 ␮M) as described in “Patients, materials, and methods.” L-arginine (10-100 ␮M)
was added at the beginning of the incubation period. The number of BFU-E colonies
(f) and levels of hemoglobin F (Hb F; ) were measured after 14 days of incubation.
Data are expressed as mean ⫾ SEM of 4 experiments done in triplicate. *P is less
than .001 compared with baseline (BL); BL indicates the number of BFU-E colonies or
percent Hb F measured in cultured cells not treated with ZL. The 0 ␮M point indicates
the number of BFU-E colonies or percent Hb F measured in cultured cells treated with
ZL alone. ␪ indicates that P is less than .01 c/w L-arginine (0 ␮M); ⌿, P is less than .05
c/w L-arginine (0 ␮M); and ␾, P is less than .001 c/w L-arginine (0 ␮M).
had no effect on Hb F synthesis in the absence of ZL treatment of
sickle erythroid progenitors in culture.
The 8-PCPT–cGMP reverses the effect of ZL in sickle primary
erythroid progenitors
Since the L-arginine/nitric oxide synthase product, nitric oxide,
activates soluble guanylate cyclase, which results in the accumulation of cGMP, we postulated that the cell-permeable cGMP analog,
8-PCPT–cGMP,22 would mimic L-arginine inhibition of ZL induction of Hb F and decrease of BFU-E colony growth. Using the same
protocol described with L-arginine, sickle erythroid progenitors
were treated with ZL (50 ␮M) in the absence or presence of
Nitric oxide synthase produces nitric oxide by converting Larginine and molecular oxygen into citrulline and nitric oxide.20,21
The previous data suggested that L-arginine plays a negative
regulatory role in ZL-mediated induction of Hb F. To test this, the
nitric oxide synthase inhibitor, L-NMMA (100 ␮M), was used to
determine if the inhibitory effect of L-arginine on ZL-induced Hb F
synthesis was mediated by nitric oxide production. This dose of
L-NMMA was chosen based on previous studies demonstrating
nitric oxide synthase inhibition.23 Studies (n ⫽ 4, in triplicate) with
L-NMMA alone were performed initially using sickle erythroid
progenitors compared with controls (no L-NMMA). L-NMMA did
not affect BFU-E colony growth or Hb F synthesis relative to
control. Subsequent studies were performed comparing ZL or HU
alone to ZL ⫹ L-NMMA and HU ⫹ L-NMMA. Interestingly,
L-NMMA did not effect ZL-mediated inhibition of BFU-E colony
growth or induction of Hb F synthesis. In contrast to ZL, L-NMMA
did partially reverse the HU effects on BFU-E colony growth and
Hb F synthesis (Table 2). Figure 6A-B demonstrates that L-NMMA
reverses the L-arginine effect on ZL in sickle erythroid progenitors
resulting in restoration of decreased numbers of BFU-E colonies
and stimulation of Hb F synthesis.
These data, cumulatively, support the hypothesis that nitric
oxide plays an inhibitory role in Hb F induction by ZL and supports
previously reported studies that nitric oxide at least in part
facilitates the synthesis of Hb F synthesis by HU.
Discussion
ZL is a selective inhibitor of 5-LO and structural analog of HU1,2
that is currently being used as an anti-inflammatory agent in the
treatment of asthma.24-29 To date there are no published studies that
address the potential significance of ZL as a therapeutic agent in the
treatment of sickle hemoglobinopathies. HU is the only drug
currently approved for the treatment of sickle cell disease (SCD)
that is known to ameliorate its most common symptom, pain,3 and
Figure 4. Effects of L-arginine and L-NMMA on HU-treated erythroid progenitors
isolated from patients with homozygous sickle cell anemia. The experimental
conditions were the same as in Figure 3. L-arginine (ARG; 100 ␮M) effects on
hydroxyurea (HU)–treated (100 ␮M) erythroid progenitors in the absence and
presence of the nitric oxide synthase inhibitor, NG-monomethyl–L-arginine (L-NMMA;
100 ␮M) were assessed. The number of BFU-E colonies (A) and percent Hb F (B)
were measured as described in “Patients, materials, and methods.” Data are
expressed as mean ⫾ SEM. n ⫽ 4 in each group. Each experiment was done in
triplicate. *P is less than .001 compared with control (CON).
Figure 5. Effects of 8-PCPT–cGMP on ZL-treated erythroid progenitors isolated
from patients with homozygous sickle cell anemia. The experimental conditions
were the same as in Figure 3. The effect of 8-(4-chlorophenylthio)guanosine
3⬘,5⬘-cyclic monophosphate (8-PCPT–cGMP; 10 ␮M) on BFU-E colony growth (A)
and Hb F synthesis (B) in zileuton-treated (ZL; 50 ␮M) sickle erythroid progenitors
was assessed. Data are expressed as the mean ⫾ SEM. n ⫽ 7 in control (CON) and
ZL treatment groups; n ⫽ 4 in the ZL ⫹ 8-PCPT–cGMP treatment group. Each
experiment was done in triplicate. *P is less than .001 compared with the CON and
ZL ⫹ 8-PCPT–cGMP treatment groups.
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BLOOD, 15 MAY 2004 䡠 VOLUME 103, NUMBER 10
ZILEUTON INDUCES HEMOGLOBIN F
3949
Table 2. Effects of NG-monomethyl-L-arginine on zileuton-and hydroxyurea-mediated BFU-E colony growth and fetal hemoglobin
production in erythroid progenitors
ZLⴙLNMMA, 100
n
CON
ZL, 50 ␮M
␮M
HU, 100 ␮M
HUⴙLNMMA, 100 ␮M
BFU-E
4
124.4 ⫾ 1.3
24.3 ⫾ 2.3
27.5 ⫾ 2.3
8.2 ⫾ 0.9
20.8 ⫾ 1.7*
Hb F, %
4
1.8 ⫾ 0.2
11.5 ⫾ 0.6
9.4 ⫾ 0.7
19.7 ⫾ 0.6
14.8 ⫾ 1.1*
BFU-E indicates burst-forming unit-erythroid; Hb F, fetal hemoglobin; CON, control; ZL, zileuton; LNMMA,
*P ⬍ .01 compared with HU alone.
common complications associated with death, including the acute
chest syndrome.30,31 The amelioration of symptoms by HU occurs
at least in part through its ability to stimulate Hb F synthesis.
Exactly how HU stimulates Hb F synthesis is not known.
In the present study, an erythroid cell culture system and K562
cells were used to study the effects of ZL on BFU-E colony growth,
Hb F synthesis, and ␥-globin mRNA synthesis. In addition, studies
were performed that analyzed the role of the nitric oxide/cGMP
pathway in regulating Hb F induction in primary sickle erythroid
progenitors. In this study the major findings were as follows: (1) ZL
induced ␥-globin mRNA synthesis in K562 cells; (2) ZL induced
Hb F synthesis in normal and sickle primary erythroid progenitors;
(3) the nitric oxide donor, L-arginine, and cGMP analog, 8-PCPT–
cGMP, blocked ZL-induced Hb F synthesis; and (4) the nitric oxide
synthase inhibitor, L-NMMA, blocks the L-arginine inhibitory
effect on ZL. These observations strongly support that the in vitro
effects of ZL are at least in part negatively regulated through a
nitric oxide–dependent pathway. Unlike ZL, L-arginine did not
affect HU induction of Hb F. Furthermore, L-NMMA had no effect
on ZL induction of Hb F. In contrast, L-NMMA partially abrogates
the decrease in number of BFU-E colonies and increase in Hb F
synthesis seen with HU. These observations strongly suggest that
the role of nitric oxide signaling in regulating ZL and HU induction
of Hb F synthesis occur through different mechanisms.
Previously, we and others demonstrated the ability of HU to
induce ␥-globin mRNA production at the transcriptional level.6,32
Thus the experimental data supports a multilevel mechanism for
Hb F induction and the antisickling effects of HU therapy. In this
study, a dose-response curve for ZL versus BFU-E colony number
was established similar to that previously shown for HU.6 These
data suggest cytotoxicity plays a role in the mechanism of action
for Hb F synthesis by ZL as well. Our data clearly demonstrated a
comparable ability of ZL to induce ␥-globin gene expression at the
transcriptional level and suggest ZL may have clinical utility
similar to that reported with HU in the treatment of sickle
hemoglobinopathies. Furthermore, the concentrations of ZL used
in this study are pharmacologically achievable with standard
dosing previously reported in patients with asthma.33
The nitric oxide–dependent activation of soluble guanylate
cyclase–protein kinase G pathway in primary erythroblast consti-
Figure 6. Effects of L-arginine and L-NMMA on zileuton-treated
erythroid progenitors isolated from patients with homozygous sickle
cell anemia. The experimental conditions were the same as in Figure 3.
L-arginine (ARG; 100 ␮M) effects on zileuton-treated erythroid progenitors
in the absence and presence of the nitric oxide synthase inhibitor LNMMA
(100 ␮M) were assessed. The number of BFU-E colonies (A) and percent
Hb F (B) were measured as described in “Patients, materials, and
methods.” Data are expressed as mean ⫾ SEM. n ⫽ 7, control (CON);
n ⫽ 7, zileuton (ZL)–treated; n ⫽ 5, ZL ⫹ L-arginine (ARG); and n ⫽ 4,
ZL ⫹ ARG ⫹ LNMMA–treated erythroid progenitors. Each experiment was
done in triplicate. *P is less than .001 compared with CON and ZL ⫹ ARG.
NG-monomethyl-L-arginine;
and HU, hydroxyurea.
tutes a mechanism that regulates expression of the ␥-globin gene12
by HU. In this study, the effect(s) of the nitric oxide donor,
L-arginine,20,21 on ZL induction of Hb F in primary sickle erythroid
progenitors in culture was assessed. L-arginine dose-dependently
blocked ZL-mediated induction of Hb F and caused the BFU-E
colony number and Hb F percentage to return to control values.
This suggested that ZL mediated increased Hb F synthesis through
a cytotoxic pathway that is nitric oxide dependent. L-arginine and
molecular oxygen in the presence of nitric oxide synthase produce
citrulline and nitric oxide, which activate soluble guanylate cyclase
and increase intracellular cGMP accumulation.20 Administration of
the cell-permeant cGMP analog, 8-PCPT–cGMP, mimicked Larginine effects, suggesting that the upstream activation of nitric
oxide is required to achieve ␥-globin inhibition. The role of
L-arginine as a nitric oxide donor is supported by results obtained
with the nitric oxide synthase inhibitor, L-NMMA. L-NMMA in the
presence of ZL and L-arginine resulted in the restoration of Hb F
induction and a decrease in the number of BFU-E colonies by ZL.
These findings cumulatively support that ZL increases Hb F
synthesis and decreases the number of BFU-E colonies through a
pathway that is at least in part negatively regulated by nitric oxide.
In addition, the ZL dose-response data suggest cytotoxicity plays a
role in the mechanism of action for Hb F synthesis by ZL and HU.
In contrast to ZL, L-arginine did not have any effect on sickle
erythroid progenitors treated with HU. However, L-NMMA partially antagonized HU induction of Hb F and the decrease in
BFU-E colony number. These data demonstrate that nitric oxide
plays a negative role in Hb F synthesis induced by ZL in contrast to
a small but significant positive regulatory role in HU-mediated
induction of Hb F synthesis.
Ikuta et al12 recently demonstrated that soluble guanylate
cyclase activators or analogs increased ␥-globin in K562 cells and
human erythroid progenitors. In addition, hemin- and butyratemediated ␥-globin induction was prevented by inhibiting soluble
guanylate cyclase or cGMP-dependent protein kinase.12 Subsequently, Cokic et al13 demonstrated that S-nitrosocysteine, a nitric
oxide donor, induced ␥-globin mRNA and Hb F protein in K562
cells and human erythroid progenitors. Both S-nitrosocysteine and
HU increased cGMP levels, and guanylate cyclase inhibitors
abolished S-nitrosocysteine and HU-induced ␥-globin expression.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
3950
BLOOD, 15 MAY 2004 䡠 VOLUME 103, NUMBER 10
HAYNES et al
In this study, L-arginine (100 ␮M), a precursor to nitric oxide, did
not stimulate Hb F synthesis over a 14-day period in cultured sickle
erythroid progenitors. Whether or not L-arginine induces ␥-globin
mRNA in sickle erythroid progenitors was not assessed. Although
L-arginine is a precursor to nitric oxide, studies performed using
HU and L-arginine did not mirror the findings of a nitric oxide–
dependent pathway reported by Cokic et al13 with the nitric oxide
donors, S-nitrosocysteine and HU, on Hb F synthesis. However, the
nitric oxide synthase inhibitor, L-NMMA, did partially antagonize
the HU effects. The lack of an L-arginine effect in the presence of
L-NMMA suggests that the basal level of nitric oxide may be more
significant in defining the physiologic significance of nitric oxide in
HU induction of Hb F synthesis. While Cokic et al13 suggest that
nitric oxide releasing or potentiating agents may have potential
therapeutic benefit, L-arginine is not supported by our study to be
such an agent. Our data does not rule out the possibility that
specific nitric oxide donors directly stimulate the ␥-globin promoter, however, the data highlight the importance of evaluating
potential ␥-globin inducers in the context of L-arginine, the major
dietary source of nitric oxide.
Acknowledgments
Many thanks to Marilyn Chancellor and Kathy Billingsley for the
preparation of this manuscript.
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2004 103: 3945-3950
doi:10.1182/blood-2003-08-2969 originally published online
February 5, 2004
Zileuton induces hemoglobin F synthesis in erythroid progenitors: role
of the l-arginine−nitric oxide signaling pathway
Johnson Haynes, Jr, B. Surendra Baliga, Boniface Obiako, Solomon Ofori-Acquah and Betty Pace
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