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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3555-3561
RED CELLS
Hemoglobin switching in unicellular erythroid culture of sibling
erythroid burst-forming units: kit ligand induces a dose-dependent
fetal hemoglobin reactivation potentiated by sodium butyrate
Marco Gabbianelli,
Ugo Testa,
Adriana Massa,
Elvira Pelosi,
Nadia Maria Sposi,
Roberta Riccioni,
Luisella Luchetti, and
Cesare Peschle
From the Department of Hematology and Oncology, Istituto Superiore
di Sanità, Rome, Italy, and T. Jefferson University, Kimmel
Cancer Center, Philadelphia PA.
 |
Abstract |
Mechanisms underlying fetal hemoglobin (HbF) reactivation in adult
life have not been elucidated; particularly, the role of growth factors
(GFs) is controversial. Interestingly, histone deacetylase (HD)
inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF.
We developed a novel model system to investigate HbF reactivation: (1)
single hematopoietic progenitor cells (HPCs) were seeded in serum-free
unilineage erythroid culture; (2) the 4 daughter cells (erythroid
burst-forming units, [BFU-Es]), endowed with equivalent
proliferation/differentiation and HbF synthesis potential, were seeded
in 4 unicellular erythroid cultures differentially treated with graded
dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated
in terminal erythroblasts by assay of F cells and  -globin content
(control levels, 2.4% and 1.8%, respectively, were close to
physiologic values). HbF was moderately enhanced by interleukin-3
(IL-3) and granulocyte-macrophage colony-stimulating factor treatment
(up to 5%-8% -globin content), while sharply reactivated in a
dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The
stimulatory effects of KL on HbF production and erythroid cell
proliferation were strictly correlated. A striking increase of HbF was
induced by combined addition of KL and NaB or TSA (40%-43%). This
positive interaction is seemingly mediated via different mechanisms:
NaB and TSA may modify the chromatin structure of the  -globin gene
cluster; KL may activate the -globin promoter via up-modulation of
tal-1 and possibly FLKF transcription factors. These studies indicate
that KL plays a key role in HbF reactivation in adult life.
Furthermore, combined KL and NaB administration may be considered for
sickle cell anemia and -thalassemia therapy.
(Blood. 2000;95:3555-3561)
© 2000 by The American Society of Hematology.
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Introduction |
In humans, the embryonic, fetal, and adult -globin
cluster genes are developmentally regulated. In the perinatal switch, fetal hemoglobin (HbF) is almost completely replaced by adult Hb
(HbA).1 The HbF present in adult life (less than 1% of
total Hb) is restricted to F cells, which represent less than 5% (mean values, 2.7% ± 1.4%) of the red blood cells
(RBCs).2,3
In diverse postnatal conditions, particularly rapid marrow regeneration
(stress erythropoiesis), HbF synthesis may be reactivated up to 10% to
20% relative -globin content.4,5 HbF reactivation provides an interesting model of partial reverse of the HbF  HbA perinatal switch. Furthermore, pharmacologic reactivation of
-globin expression may provide an effective treatment for patients
affected by -globin disorders, as sickle cell anemia or
-thalassemia. In sickle cell anemia, HbF interferes with sickling by
inhibiting the polymerization of sickle hemoglobin; subjects with
elevated HbF levels appear to have a milder disease.6,7 In
thalassemia, a significant increase in -globin production could
partially replace the defective -globin, thus preventing precipitation of unpaired  -globin chains.8
In humans and primates, HbF reactivation has been induced by diverse
agents. Cytostatic drugs such as 5-azacytidine, bromo-deoxyuridine or
hydroxyurea (HU) are effective in vitro.9-11 HU has been
successfully used for treatment of sickle cell anemia12,13;
however, myelotoxicity, potential long-term carcinogenicity, and only
moderate therapeutic effects limited this clinical use. Histone
deacetylase (HD) inhibitors (ie, trapoxin [TPX], trichostatin A
[TSA], arginine butyrate [ArgB], sodium butyrate, and sodium
phenylbutyrate [NaB and NaPB]) increase HbF levels in human
cultures.14 NaB and NaPB are currently evaluated in
clinical trials15,16; intermittent therapy with ArgB
sustainedly induced HbF in sickle cell anemia.17
The role of hematopoietic growth factors (HGF) in HbF reactivation is
controversial. Large dosages of erythropoietin (Epo), while stimulating
HbF production in primates,18,19 induced modest and
variable reactivation of HbF in clinical trials.20-22 In
the serum-free culture of adult hematopoietic progenitor cells (HPCs),
granulocyte-macrophage colony-stimulating factor (GM-CSF)23 and interleukin-3 (IL-3)24 induced a significant rise of
HbF synthesis, but these results were not confirmed25;
furthermore, the c-kit ligand (KL) markedly stimulated HbF production
in sickle cell anemia26 and normal27 HPC
culture. However, these bulk culture studies involved methodology
limitations (see "Discussion"), which hampered elucidation of the
role of HGFs in HbF reactivation.
We have investigated HbF reactivation by a novel in vitro approach.
Purified adult HPCs were grown in a single-cell unilineage erythroid
culture. The generated sibling erythroid burst- forming units (BFU-Es)
were then reseeded in unicellular erythroid cultures differentially treated with diverse HGFs (IL-3, GM-CSF, KL), and/or HD
inhibitors (TSA, NaB).
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Materials and methods |
Hematopoietic growth factors and chemical inducers
Recombinant human IL-3 and GM-CSF were supplied by Behring
(Behringwerke AG, Marburg, Germany) and Sandoz (Basel, Switzerland), respectively. Recombinant human KL was provided by R & D Systems (Minneapolis, MN), whereas recombinant human Epo was supplied by Amgen
(Thousand Oaks, CA). TSA and NaB were provided by Sigma Chemical, (St
Louis, MO).
Peripheral blood
Adult peripheral blood (PB) was obtained from healthy adult male
donors after informed consent. Blood (450 mL) was collected in
preservative-free citrate/phosphate/dextrose/adenine (CPDA-1) anticoagulant. A buffy coat was obtained by centrifugation (Beckman J6M/E, 1400 rpm per 20 minutes at room temperature; Beckman
Instruments, Fullerton, CA).
Hematopoietic progenitor cell purification
Adult PB HPCs were purified according to a
modification28 of a method previously
reported,29 which consists of 3 steps. These purified cells
are more than 90% CD34+ and comprise 90% to 95% of early
hemopoietic progenitor cells, HPCs (including colony-forming units
mixed, CFU-Mix, BFU-E, CFU-GM, and CFU-MK), 5% to 10% of primitive
HPCs (high proliferative potential colony-forming cells, HPP-CFCs and
colony-forming units-blast, CFU-B), and 0.5% to 2% of putative
hematopoietic stem cells (HSCs) (8-week long-term culture
initiating cells, LTC-ICs).30
Unilineage erythroid differentiation cultures
Unicellular cultures.
Unicellular cultures were performed in flat-bottomed 96-microwell
plates in 0.1 mL of 5% fetal calf serum (FCS)-medium supplemented with
all the ingredients used in FCS culture as
previously reported.31,32
In the sibling cell assay, to avoid 2 cells in the same well, the cells
were plated at 0.5 cell per well.
To selectively stimulate erythroid differentiation, low dosages of IL-3
and GM-CSF (0.01 units and 0.001 ng/mL, respectively) and saturating
levels of Ep (3 U/mL) were added to the cultures (this culture
condition was defined in the text as erythroid medium).
The sibling cells were picked up by a micromanipulator from 4 cell
clones scored at day 3 and day 4 and seeded in 4 different wells
containing 0.1 mL of the same medium supplemented or not with graded
amounts of IL-3 (1, 10, or 100 U/mL) or GM-CSF (1, 10, or 100 ng/mL) or
KL (1, 10, or 100 ng/mL) or KL + TSA (100 ng and 10 ng/mL,
respectively). Cultures were incubated in a fully humidified atmosphere
of 5% CO2/5% O2/90%
N2 and colonies were analyzed on day 14 to day 18.
Mini-bulk cultures.
Purified HPCs grown in FCS liquid culture
(5.104 cells per milliliter per well in IMDM, supplemented
as reported by Valtieri et al32) were induced to specific
erythroid differentiation by an appropriate HGF cocktail (Epo and
IL-3/GM-CSF as indicated above) supplemented or not with KL (100 ng/mL), TSA (10 ng/mL), NaB (0.5 mmol/L), or their combination, in a
fully humidified atmosphere as indicated above.
Morphologic analysis.
Cells were harvested from day 14 to day 21, smeared on glass slides by
cytospin centrifugation and stained with standard
May-Grünwald-Giemsa. For single sibling colony
analysis, specific polylysinated glass slides were used.
F cells
The percentage of erythroblasts containing HbF was evaluated by
indirect fluorescence as previously described.3
Briefly, cells from pooled or single bursts33 were
cytocentrifuged on a glass slide, fixed for 5 minutes at room
temperature in acetone-methanol (9:1, vol/vol), washed 3 times with
phosphate-buffered saline (PBS), once with PBS containing 2 mg/mL
bovine serum albumin (BSA), and incubated for 40 minutes at 37°C
with a 1:40 dilution of an antihuman HbF MoAb (Caltag Laboratories,
Burlingame, CA). The slides were washed twice with PBS, once with
PBS/BSA, incubated for 30 minutes at room temperature with a 1:20
dilution of fluorescein isothiocyanate (FITC)-conjugated
F(ab1)2 antimouse IgGs (Dakopatts, Copenhagen,
Denmark), and extensively washed in PBS. The slides were then mounted
in PBS/glycerol (50:50, vol/vol) and observed under an Axiophot Zeiss
microscope (Zeiss, Jena, Germany) equipped for
fluorescence. As a negative control, cells were incubated with normal
mouse IgG instead of anti-HbF and processed as above.
-Chain content
High-performance liquid chromatographic (HPLC) separation of globin
chains was performed according to Leone et al.34 Briefly, cell lysates from bulk culture and/or pooled sibling erythroid colonies
were separated on chromatographic columns (Merck LiChrospher 100 CH8/2,5 µm; E. Merck, Darmstadt, Germany) using as eluents a linear
gradient of acetonitrile/methanol/0.155 mol/L sodium chloride (pH 2.7, 68:4:28 vol/vol/vol) (eluent A), and acetonitrile/methanol/0.077 mol/L
sodium chloride (pH2.7, 26:33:41 vol/vol/vol) (eluent B). Gradient was
from 20% to 60% eluent A in 60 minutes at a flow rate of 0.8 mL/min.
The optimal absorbance of the different globin was evaluated at 214 nm,
because the absorbance coefficients of the different chains are
identical at this wavelength.34
Tal-1 and FKLF expression
Tal-1 expression was investigated at the messenger RNA (mRNA) level
by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
and at the protein level by Western blotting on erythroid bulk
cultures, according to experimental procedures previously described.35 In the same cultures, FKLF mRNA expression was investigated by RT-PCR using primers and amplification conditions as
recently reported.36
| |
Results |
We have analyzed reactivation of human HbF synthesis in erythroid
colonies generated by sibling adult BFU-Es in unicellular culture.
Several studies also involved minibulk cultures of purified BFU-Es.
In the first series of experiments, the effect of IL-3, GM-CSF, and KL
was analyzed in sibling BFU-Es (Figure 1).
The purified HPCs were grown in unicellular erythroid liquid suspension
culture (ie, in medium supplemented with saturating amounts of Epo,
combined with very low concentrations of IL-3 and GM-CSF to allow
erythroid differentiation and to stimulate cell proliferation up to
terminal maturation, Sposi et al37). After 2 cell
divisions, the 4 generated BFU-Es were subdivided by micromanipulation
and individually reseeded in secondary erythroid culture. Three
siblings were grown in erythroid culture supplemented with graded
concentrations of IL-3, GM-CSF, or KL, whereas the fourth control
sibling was grown in standard erythroid culture. Finally, we evaluated
HbF levels in the mature erythroblasts of terminal erythroid colonies;
it is noteworthy that we analyzed single colonies and/or pools of
corresponding sibling colonies.
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| Fig 1.
A model system for analysis of sibling BFU-Es, generated
in unicellular erythroid culture (0.5 cell per well) supplemented with
low dosages of IL-3 and GM-CSF and saturating levels of Epo.
The 4 sibling BFU-Es were subdivided and individually reseeded in
unicellular erythroid culture: 3 siblings were treated with graded
amounts of IL-3, GM-CSF, or KL ± HD inhibitor (NaB or TSA); the
fourth control sibling was untreated. The sibling clones were analyzed
on day 14 through day 18 as single or pooled mature erythroblast
colonies.
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Both IL-3 and GM-CSF reactivated HbF synthesis, as evaluated in terms
of -chain content (up to 5% and 8% at 10 units and 1 ng,
respectively, mean values) and F cells (Figure
2A and 2B, and data not shown).
Comparison with control erythroid cultures indicates that the HbF
reactivation was unrelated to maturation stage and cell
proliferation, ie, percentage of late erythroblasts was unmodified
and cell number per colony showed only a borderline increase
in cultures treated with graded GM-CSF or IL-3 dosages (Figure 2A).
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| Fig 2.
HbF reactivation in sibling BFU-E colonies.
(A) HbF reactivation in sibling BFU-Es grown in the presence of graded
concentrations of GM-CSF or IL-3. HbF synthesis was evaluated in terms
of -chain content in pooled sibling erythroid colonies. A
representative experiment is shown. (B) Globin chain HPLC scans and
percentage of F cells from pooled sibling BFU-E colonies grown in
unilineage erythroid culture in the presence or absence of GM-CSF (1 ng/mL) (representative results).
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The effects of KL were evaluated in either pooled or single erythroid
colonies generated by sibling BFU-Es (Figure
3). At pooled colony level (Figure 3A), KL
induced a dose-dependent increase in percentage of F cells (from 2.8%
up to 58%) and -chain content (from 0.8% up to 22.5%). HbF
reactivation was paralleled by an increase of colony size (from
2 × 103 up to 105 cells per colony).
The correlation between cell numbers per colony and percentage values
of F cells or HbF content was highly significant (P < .001,
legend of Figure 3A). Although erythroid maturation was delayed in
KL-treated culture, we consistently analyzed erythroid clones composed
of more than 90% mature orthochromatic erythroblasts. At the single
colony level (Figure 3B), KL induced a comparable dose-related HbF
reactivation (from 2.5% up to 55% F cells) (top); percentage of F
cell values and cell numbers per colonies were strictly correlated
(P < .001) (bottom).
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| Fig 3.
HbF reactivation in sibling BFU-E colonies.
(A) HbF reactivation in pooled sibling BFU-E colonies grown in
erythroid culture in presence of graded amounts of KL. Clonogenetic
parameters are shown in top panels. In the bottom part, HbF synthesis
is monitored in terms of percentage of F cells and -chain content.
Mean ± SEM values from 3 separate experiments. The correlation
between cell numbers per colony and percentage values of F cells
(r = 0.8, P = .0002) or HbF content
(r = 0.91, P < .0001) is highly significant. (B)
HbF reactivation in single sibling BFU-E colonies treated with graded
amounts of KL, as evaluated in terms of F cells (% values) in each
colony. Top panel, results from 8 groups of sibling colonies (mean
values are shown). Bottom, direct correlation between F cell number (% values) and cell number × 103 per colony in the 32 sibling clones (see top panel).
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To comparatively evaluate the HbF synthesis potential of sibling
BFU-Es, we analyzed 9 experimental groups, each composed of 4 daughter
progenitors reseeded in a unicellular erythroid culture. At variance
with the standard protocol (Figure 1), all sibling BFU-Es within each
group were treated with 0, 1, or 100 ng KL; specifically, 3 groups were
treated with each KL concentration. In all groups, the 4 sibling
colonies consistently showed equivalent size, maturation stage, and
percentage of F cells (Figure 4 and results
not shown). This indicates that sibling BFU-Es have a similar
potential, in terms of proliferation and differentiation,38 as well as HbF synthesis.
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| Fig 4.
Variation of HbF synthesis among sibling BFU-E clones in
9 experimental groups.
At variance with the standard protocol (Figure 1), the 4 sibling
colonies within each group were identically treated, ie, the 4 sibling
wells were supplemented with 0, 1, or 100 ng KL. Correlation between
percentage values of F cells and cell number × 103
per colony: r = 0.8603, P < .0001.
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In a second series of experiments, we have analyzed the effects of the
HD inhibitors NaB and TSA in minibulk erythroid culture. Treatment with
TSA alone blocks erythroid differentiation at an early stage, thus
rendering a difficult analysis of HbF content in mature erythroblasts.
Nevertheless, the analysis of F cells in minibulk cultures revealed an
increase of F erythroblasts (Figure 5A). In
a dose-response experiment, NaB markedly reactivated HbF with a peak
effect at 0.5 mmol/L, as evaluated in terms of F cells (up to 26%) and
-globin content (up to 24%) (Table 1
and Figure 5A and 5B).
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| Fig 5.
Minibulk HPC erythroid cultures.
(A) F cells from minibulk HPC erythroid cultures supplemented or not
with KL (100 ng/mL) ± TSA (10 ng/mL) ± NaB (0.5 mmol/L). F
cells were evaluated by indirect immunofluorescence using an antihuman
HbF mAb as a primary antibody and an FITC-conjugated goat antimouse IgG
as a secondary antibody. Original magnification × 630.
Representative results are shown. (B) Globin chain HPLC scans from
mature erythroblasts obtained in minibulk HPC erythroid cultures
supplemented or not with KL, TSA, NaB, or combinations thereof
(representative results).
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Table 1.
Representative experiment showing HbF reactivation,
evaluated in terms of F cells and -chain content, in minibulk
erythroid HPC cultures supplemented or not with graded NaB dosages ± saturating KL level
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The third series of experiments was performed by combined addition of
plateau levels of KL (100 ng/mL) and NaB or TSA (0.5 mmol/L and 10 ng/mL, respectively) in both minibulk and sibling BFU-E erythroid
cultures (Figure 6A and 6B). Interestingly,
the combined addition of KL and NaB or TSA induced a striking increase of HbF levels in terms of F cells and -globin content, up to 71% or
73% and 40% or 43%, respectively (Figure 6A). Although colonies
treated with NaB alone were markedly small, KL restored a virtually
normal proliferation in NaB-treated cultures, as well as in
TSA-supplemented ones (Figure 6B). Furthermore, both NaB and TSA
delayed differentiation/maturation along the erythroid pathway (more
than 90% of mature erythroblasts were observed at day 20 through
day 25 in KL + NaB or TSA culture, ie, 5 days later than in the
KL-treated group) (Figure 6A and 6B).
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| Fig 6.
HbF reactivation and synthesis.
(A) HbF reactivation in minibulk HPC erythroid cultures supplemented or
not with KL (100 ng/mL) ± NaB (0.5 mmol/L) or ± TSA (10 ng/mL).
Clonogenetic parameters are shown in top panels. On the bottom, Hb
synthesis is shown as F cells and -chain content percentage.
Mean ± SEM values from 3 separate experiments. Variance analysis
was performed to evaluate the difference between groups and expressed
as Bonferroni P values. *P < .05 and
***P < .001 when compared with the indicated group. (B) HbF
synthesis, evaluated in terms of F cells and -chain content, in
pooled sibling BFU-E colonies grown in unilineage erythroid culture
supplemented or not with KL ± NaB or ± TSA. Mean ± SEM
values from 3 separate experiments. *P < .05 and
***P < .001 when compared with the indicated group.
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Finally, to explore the molecular mechanisms underlying -globin
modulation induced by KL and HD inhibitors, we evaluated the expression
of Tal-1 and FKLF in minibulk erythroid cultures grown under standard
conditions (control) or in the presence of saturating levels of
KL ± NaB (Figure 7). Semiquantitative
RT-PCR showed that KL caused a significant increase in Tal-1 mRNA
expression (top left panel), which was paralleled by a marked increase
of Tal-1 protein levels (top right). Conversely, NaB caused a drop of
Tal-1 mRNA and protein, which was rescued by combined NaB + KL
treatment (top). Evaluation of fetal Krüppel-like factor (FKLF) mRNA levels showed a moderate increase in KL-supplemented cultures, in
contrast to the marked drop induced by NaB, which was rescued by
combined NaB + KL treatment (bottom panel).
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| Fig 7.
Tal-1 and FKLF expression in bulk HPC erythroid cultures
supplemented or not with KL (100 ng/mL) ± NaB (0.5 mmol/L).
Tal-1 expression was evaluated in terms of RT-PCR (right top panel) and
Western blotting (left top panel). FKLF expression was analyzed only by
RT-PCR (bottom panel).
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Discussion |
Human HbF reactivation has been consistently observed in bulk
cultures of normal adult erythroid progenitors.39-41
However, elucidation of the underlying mechanisms was hampered by major methodology limitations, namely, (1) the variable effects of different FCS batches, (2) the rarity and heterogeneity of the HPCs, and (3) the
large number of coplated accessory cells releasing endogenous cytokines. HbF reactivation was almost completely abolished in HPC
FCS-free cultures,23,25,42 indicating that FCS directly and/or indirectly enhances HbF production. Thereafter, HbF reactivation was analyzed in bulk cultures of 90% to 95% purified HPCs, thus eliminating the accessory cell bias.27 Despite these
advances, the heterogeneity of progenitors still represented a
significant hurdle, particularly with respect to result interpretation
(see below). In this regard, we developed a system for unilineage
erythroid culture in liquid suspension FCS-free medium.37
In this model, purified HPCs undergo a gradual and homogeneous wave of
differentiation/maturation along the erythroid lineage up to terminal
cells (ie, at day 14 to day 16,  97% cells are orthochromatic
normoblasts). Thereafter, we developed a system for unicellular
culture, whereby single sibling BFU-Es are grown in parallel in
erythroid-specific medium and then comparatively analyzed at the
level of their progeny.38 Specifically, while a small
fraction (less than 20%) of the 4 sibling clones undergo
apoptosis in this culture system, the other sibling
progenitors generate pureerythroblast colonies and show an equivalent
proliferative/ differentiative capacity.38
In the current studies, we used the sibling BFU-E approach to
investigate the effects of IL-3, GM-CSF, and particularly KL in the
reactivation of the HbF synthesis program. Four sibling BFU-Es,
generated by a single progenitor and endowed with an equivalent HbF
synthesis potential (Figure 4) were grown in a single-cell culture, and
the resulting erythroid colonies were comparatively analyzed.
Specifically, the control sibling BFU-E was untreated, whereas the
other 3 siblings were treated with KL ± HD inhibitor at 3 graded
concentrations; this allowed to perform a rigorous dose
response with an internal negative control. Furthermore, the progeny of
each sibling BFU-E was analyzed at the stage of terminal erythroblast,
thus excluding that the positive effects on HbF synthesis were mediated
by insufficient maturation of the erythroblasts. It is also noteworthy
that GF treatment only slightly diminished the fraction of clones
undergoing apoptosis (ie, from less than 20% to less than 10%).
Although previous studies on IL-3 and GM-CSF provided controversial
results,25 the current observations unequivocally
demonstrate that both multilineage HGFs exert a mild, but significant,
stimulatory effect on HbF reactivation, as originally indicated in bulk
BFU-E cultures.23,24
The stimulatory action of KL on HbF production in the bulk BFU-E
culture is of uncertain significance,27 ie, it may be
mediated by an effect on the BFU-E HbF synthesis program or
the recruitment of BFU-Es with elevated HbF production potential.
The latter mechanism, however, is excluded by the present observations,
which demonstrate a direct, dose-related stimulatory effect of KL on
HbF reactivation in sibling BFU-Es. It is well established that KL
potentiates the erythroid proliferation induced by Epo.30
Interestingly, the KL-induced rise of erythroid cell proliferation is
strictly correlated with the reactivation of HbF. In human development, the erythroid proliferation rate gradually declines from the fetal through the perinatal and adult period.43 In this regard,
fetal liver HPCs are particularly sensitive to KL,44 whose
concentration is more elevated in cord than adult blood.45
On this basis, it is hypothesized that the perinatal Hb switch may be,
at least in part, mediated via a decline of KL activity and hence,
erythroid proliferation activity.
Chemical compounds reactivating HbF9-12,14-16,46 may act
via different mechanisms: TPX, TSA, and probably butyrate, and
derivatives act via HD inhibition,47 whereas HU neither
affects bulk histone acetylation, nor activates -globin promoter
constructs.14 On the other hand, KL induces
tal-148 and possibly other transcription factors such as
FKLF,36 which may up-modulate -globin seemingly via
promoter activation.36,48 Tal-1, in cooperation with other transcription factors, interacts with a conserved E-box in the -globin control region enhancer to stimulate -globin gene
transcription.49,50 FKLF is preferentially expressed in
fetal rather than in adult erythroid tissue, whereas it stimulates
-globin gene expression in the K562 cell line.36 Our
experiments, performed on HPCs grown in erythroid culture in the
presence of KL ± NaB, provided evidence that KL, but not NaB,
exerted a marked stimulatory effect on Tal-1 expression, and a moderate
increase of FLKF mRNA. This suggests that the stimulatory effect of KL
on -globin gene expression may, at least in part, be mediated
through enhanced Tal-1 and possibly FKLF expression. On the other hand,
NaB seemingly exerts its stimulatory effect on HbF reactivation by a
different molecular mechanism, ie, modification of chromatin structure
of the -globin gene cluster, resulting in increased transcriptional
output of the -globin gene.51
Agents reactivating HbF synthesis via different molecular mechanisms
may act additively or synergistically.52-54 The current studies demonstrate that the HD inhibitors TSA and NaB significantly potentiate the KL-induced reactivation of HbF in human adult BFU-Es. TSA also caused a partial block of BFU-E proliferation/differentiation, which was restored by combined treatment with KL. Similarly, the drop
of Tal-1 and FLKF expression induced by NaB was restored by combined
treatment with KL. We suggest that the additive effect exerted by KL
and the HD inhibitors on HbF reactivation may be mediated via
complementary mechanisms, as indicated above.
Finally, it is noteworthy that, in the clinical setting, combined
treatment with HD inhibitors and KL may be considered for HbF
reactivation in patients with -globin disorders, as sickle cell
anemia or -thalassemia.
| |
Footnotes |
Submitted June 23, 1999; accepted January 21, 2000.
Reprints: C. Peschle, T. Jefferson University, Kimmel Cancer
Center, Bluemle Life Science Bldg, 233 S 10th St, Philadelphia PA
19107-5541.
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.
| |
References |
1.
Stamatoyannopoulos G, Nienhuis AW.
Hemoglobin switching. In:
Stamatoyannopoulos G,Nienhuis AW,Majerus PW,Varmus H, eds.
The Molecular Basis of Blood Disease. Philadelphia, PA: WB Saunders; 1994:107.
2.
Boyer SH, Belding TK, Margolet L, Noyes AN.
Fetal hemoglobin restriction to a few erythrocytes (F cells) in normal human adults.
Science.
1975;188:361[Abstract/Free Full Text].
3.
Wood WG, Stamatoyannopoulos G, Lim G, Nute PE.
F-cells in the adult: normal values and levels in individuals with hereditary and acquired elevations of HbF.
Blood.
1975;46:671[Abstract/Free Full Text].
4.
Peschle C, Migliaccio AR, Covelli A, Giuliani A, Mavilio F, Mastroberardino G.
Hemoglobin switching in humans. In:
Dunn CDR, ed.
Current Concepts in Erythropoiesis. London, UK: John Wiley; 1983.
5.
Alter BP, Rappeport JM, Huisman TH, Schroeder WA, Nathan DG.
Fetal erythropoiesis following bone marrow transplantation.
Blood.
1976;48:843[Abstract/Free Full Text].
6.
Noguchi CT, Rodgers GP, Serjeant G, Schechter AN.
Levels of fetal hemoglobin necessary for treatment of sickle cell disease.
N Engl J Med.
1988;318:96[Medline]
[Order article via Infotrieve].
7.
Wood WG, Pembrey ME, Serjeant GR, Perrine RP, Weatherall DJ.
HbF synthesis in sickle cell anaemia: a comparison of Saudi Arab cases with those of African origin.
Br J Haematol.
1980;45:431[Medline]
[Order article via Infotrieve].
8.
Stamatoyannopoulos G, Nienhuis AW.
Therapeutic approaches to hemoglobin switching in treatment of hemoglobinopathies.
Ann Rev Med.
1992;43:497[Medline]
[Order article via Infotrieve].
9.
DeSimone J, Heller P, Hall L, Zwiers D.
5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons.
Proc Natl Acad Sci U S A.
1982;79:4428[Abstract/Free Full Text].
10.
Letvin NL, Linch DC, Beardsley GP, McIntyre KW, Nathan DG.
Augmentation of fetal-hemoglobin production in anemic monkeys by hydroxyurea.
N Engl J Med.
1984;310:869[Abstract].
11.
Comi P, Ottolenghi S, Giglioni B, et al.
Bromodeoxyuridine treatment of normal adult erythroid colonies: an in vitro model for reactivation of human fetal globin genes.
Blood.
1986;68:1036[Abstract/Free Full Text].
12.
Charache S, Terrin ML, Moore RD, et al.
Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia.
N Engl J Med.
1995;332:1317[Abstract/Free Full Text].
13.
Golberg MA, Brugnara C, Dover GJ, Schapiza L, Charache S, Bunn HF.
Treatment of sickle cell anemia with hydroxyurea and erythropoietin.
N Engl J Med.
1990;323:366[Abstract].
14.
McCaffrey PG, Newsome DA, Fibach E, Yoshida M, Su MS.
Induction of gamma-globin by histone deacetylase inhibitors.
Blood.
1997;90:2075[Abstract/Free Full Text].
15.
Sher GD, Ginder GD, Little J, Yang S, Dover GJ, Olivieri NF.
Extended therapy with intravenous arginine butyrate in patients with beta-hemoglobinopathies.
N Engl J Med.
1995;332:1606-1610[Abstract/Free Full Text].
16.
Collins AF, Pearson HA, Giardina P, McDonagh KT, Brusilow SW, Dover GJ.
Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial.
Blood.
1995;85:43-49[Abstract/Free Full Text].
17.
Atwech GF, Sutton M, Nassif I, et al.
Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease.
Blood.
1999;93:1790[Abstract/Free Full Text].
18.
Al-Khatti A, Veith RW, Papayannopoulou T, Fritsch EF, Goldwasser E, Stamatoyannopoulos G.
Stimulation of fetal hemoglobin synthesis by erythropoietin in baboons.
N Engl J Med.
1987;317:415[Abstract].
19.
Umemura T, al-Khatti A, Donahue RE, Papayannopoulou T, Stamatoyannopoulos G.
Effects of interleukin-3 and erythropoietin on in vivo erythropoiesis and F-cell formation in primates.
Blood.
1989;74:1571-1576[Abstract/Free Full Text].
20.
Rachmilewitz EA, Goldfarb A, Dover G.
Administration of erythropoietin to patients with thalassemia intermedia: a preliminary trial.
Blood.
1991;78:1145[Free Full Text].
21.
Olivieri NF, Sheridan B, Freedman M, Dover G, Perrine S, Nagel RS.
Trial of recombinant human erythropoietin in thalassemia intermedia.
Blood.
1992;80:3258[Free Full Text].
22.
Rachmilewitz EA, Aker M.
The role of recombinant human erythropoietin in the treatment of thalassemia.
Ann N Y Acad Sci.
1998;30:129.
23.
Gabbianelli M, Pelosi E, Bassano E, et al.
Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults.
Blood.
1989;74:2657[Abstract/Free Full Text].
24.
Gabbianelli M, Pelosi E, Labbaye C, Valtieri M, Testa U, Peschle C.
Reactivation of HbF synthesis in normal adult erythroid bursts by IL-3.
Br J Haematol.
1990;74:114[Medline]
[Order article via Infotrieve].
25.
Migliaccio AR, Migliaccio G, Brice M, Constantoulakis P, Stamatoyannopoulos G, Papayannopoulou T.
Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe.
Blood.
1990;76:1150[Abstract/Free Full Text].
26.
Miller BA, Perrine SP, Bernstein A, et al.
Influence of steel factor on hemoglobin synthesis in sickle cell disease.
Blood.
1992;79:1861-1868[Abstract/Free Full Text].
27.
Peschle C, Gabbianelli M, Testa U, et al.
c-kit ligand reactivates fetal hemoglobin synthesis in serum-free culture of stringently purified normal adult burst-forming unit-erythroid.
Blood.
1993;81:328[Abstract/Free Full Text].
28.
Labbaye C, Valtieri M, Barberi T, et al.
Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis.
J Clin Invest.
1995;95:2346.
29.
Gabbianelli M, Sargiacomo M, Pelosi E, Testa U, Isacchi G, Peschle C.
"Pure" human hematopoietic progenitors: permissive action of basic fibroblast growth factor.
Science.
1990;249:1561[Abstract/Free Full Text].
30.
Gabbianelli M, Pelosi E, Montesoro E, et al.
Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors.
Blood.
1995;86:1661[Abstract/Free Full Text].
31.
Guerriero R, Testa U, Gabbianelli M, et al.
Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture.
Blood.
1995;86:3725[Abstract/Free Full Text].
32.
Valtieri M, Gabbianelli M, Pelosi E, et al.
Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture.
Blood.
1989;74:460[Abstract/Free Full Text].
33.
Comi P, Giglioni B, Ottolenghi S, et al.
Globin chains synthesis in single erythroid bursts from cord blood: studies on - and G-A-switches.
Proc Natl Acad Sci U S A.
1980;77:362[Abstract/Free Full Text].
34.
Leone L, Monteleone M, Gabutti V, Amione C.
Reversed phase high-performance liquid chromatography of human haemo-globin chains.
J Chromatogr.
1985;321:407[Medline]
[Order article via Infotrieve].
35.
Condorelli G, Vitelli L, Valtieri M, et al.
Coordinate expression and developmental role of Id2 protein and TAL1/E2A heterodimer in erythroid progenitor differentiation.
Blood.
1995;86:164[Abstract/Free Full Text].
36.
Asano H, Li XS, Stamatoyannopoulos G.
FKLF, a novel kruppel-like factor that activates human embryonic and fetal beta-like globin genes.
Mol Cell Biol.
1999;19:3571[Abstract/Free Full Text].
37.
Sposi NM, Zon LI, Care A, et al.
Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors.
Proc Natl Acad Sci U S A.
1992;89:6353[Abstract/Free Full Text].
38.
Ziegler BL, Müller R, Valtieri M, et al.
Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level.
Blood.
1999;93:3355[Abstract/Free Full Text].
39.
Papayannopoulou T, Brice M, Stamatoyannopoulos G.
Hemoglobin F synthesis in vitro: evidence for control at the level of primitive erythroid stem cells.
Proc Natl Acad Sci U S A.
1977;74:2923[Abstract/Free Full Text].
40.
Kidoguchi K, Ogawa M, Karam JD.
Hemoglobin biosynthesis in individual erythropoietic bursts in culture: studies of adult peripheral blood.
J Clin Invest.
1979;63:804.
41.
Peschle C, Migliaccio G, Covelli A, et al.
Hemoglobin synthesis in individual bursts from normal adult blood: all bursts and subcolonies synthesize G gamma-and A gamma-globin chains.
Blood.
1980;56:218[Abstract/Free Full Text].
42.
Fujimori Y, Ogawa M, Clark SC, Dover GJ.
Serum-free culture of enriched hematopoietic progenitors reflects physiologic levels of fetal hemoglobin biosynthesis.
Blood.
1990;75:1718-1722[Abstract/Free Full Text].
43.
Peschle C, Migliaccio AR, Migliaccio G, et al.
Identification and characterization of three classes of erythroid progenitors in human fetal liver.
Blood.
1981;58:565[Free Full Text].
44.
Zandstra PW, Conneally E, Piret JM, Eaves CJ.
Ontogeny-associated changes in the cytokine responses of primitive human haemopoietic cells.
Br J Haematol.
1998;101:770[Medline]
[Order article via Infotrieve].
45.
Yamaguchi H, Ishii E, Saito S, et al.
Umbilical vein endothelial cells are an important source of c-kit and stem cell factor which regulate the proliferation of haemopoietic progenitor cells.
Br J Haematol.
1996;94:606[Medline]
[Order article via Infotrieve].
46.
Perrine SP, Ginder GD, Faller DV, et al.
A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders.
N Engl J Med.
1993;328:81[Abstract/Free Full Text].
47.
Hassig CA, Schreiber SL.
Nuclear histone acetylases and deacetylases and transcriptional regulation.
Curr Opin Chem Biol.
1997;1:300[Medline]
[Order article via Infotrieve].
48.
Miller BA, Floros J, Cheung JY, et al.
Steel factor affects SCL expression during normal erythroid differentiation.
Blood.
1994;84:2971[Abstract/Free Full Text].
49.
Elnitski L, Miller W, Hardison R.
Conserved E boxes function as part of the enhancer in hypersensitive site 2 of the beta-globin locus control region: role of basic helix-loop-helix proteins.
J Biol Chem.
1997;272:369[Abstract/Free Full Text].
50.
Holmes ML, Haley JD, Cerruti L, et al.
Identification of Id2 as a globin regulatory protein by representational difference analysis of K562 cells induced to express gamma-globin with a fungal compound.
Mol Cell Biol.
1999;19:4182[Abstract/Free Full Text].
51.
Struhl K.
Histone acetylation and transcriptional regulatory mechanisms.
Genes Dev.
1998;12:599[Free Full Text].
52.
McDonagh KT, Dover GJ, Donahue RE, et al.
Hydroxyurea-induced HbF production in anemic primates: augmentation by erythropoietin, hematopoietic growth factors, and sodium butyrate.
Exp Hematol.
1992;20:1156[Medline]
[Order article via Infotrieve].
53.
Lavelle D, DeSimone J, Heller P.
Fetal hemoglobin reactivation in baboon and man: a short perspective.
Am J Hematol.
1993;42:91-95[Medline]
[Order article via Infotrieve].
54.
Constantoulakis P, Knitter G, Stamatoyannopoulos G.
On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC.
Blood.
1989;74:1963[Abstract/Free Full Text].
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Abstract
Introduction