Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 863-869
HEMATOPOIESIS
Quantitative PCR analysis of HbF inducers in primary human
adult erythroid cells
Reginald D. Smith,
Jin Li,
Constance T. Noguchi, and
Alan N. Schechter
From the Laboratory of Chemical Biology, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MD 20892.
 |
Abstract |
The development and evaluation of drugs to elevate fetal hemoglobin
in the treatment of the genetic diseases of hemoglobin would be
facilitated by the availability of reliable cell assays. We have used
real-time, quantitative polymerase chain reaction (PCR) analyses of
globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to
modulate levels of adult (
) and fetal (
) globin mRNA.
Complementary DNA synthesized from total RNA extracted at timed
intervals from aliquots of cells were assayed throughout the period
that the culture was studied. -globin mRNA levels were found to be
much lower (less than 1%) than -globin mRNA levels. At
concentrations of agents chosen for minimal effect on cell division, we
find that the 3 drugs studied, 5-azacytidine
(5µmol/L), hydroxyurea (40µmol/L),
and butyric acid (0.5mmol/L), significantly increase -globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on -globin mRNA levels, while butyric acid caused
a twofold inhibition of -globin mRNA levels, and 5-azacytidine had
little effect on -globin mRNA levels. The net result of all 3 drugs
was to increase the /( + ) mRNA ratios by threefold to
fivefold. These data suggest that the mechanism is distinct for each
drug. The profile of butyric-acid-induced changes on globin gene
expression is also quite distinct from changes produced by trichostatin
A, a known histone deacetylase inhibitor. Quantitative PCR analyses of
human erythroid cells should prove useful for studying the mechanism(s)
of action of known inducers of -globin and identifying new drug candidates.
(Blood. 2000;95:863-869)
© 2000 by The American Society of Hematology.
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Introduction |
Expression of the globin genes is developmentally
regulated during vertebrate ontogeny. Two major switches characterize
hemoglobin production in humans1: embryonic hemoglobins
switch to fetal hemoglobins (HbF) after the first 2 months of
gestation, and these switch at birth to production of the adult
hemoglobins (HbA and HbA2). These changes are believed to
be due primarily to changes in the transcriptional control of the
individual globin chain messenger RNAs (mRNAs).
An increase in HbF ameliorates the clinical symptoms in both sickle
cell anemia and -thalassemia.2 In sickle cell anemia, HbF-containing red blood cells have lower concentrations of sickle hemoglobin (HbS), and HbF itself inhibits HbS polymerization, which
decreases the potential for intracellular polymerization in such
cells.3 In -thalassemia, increased levels of -globin chains decrease the chain imbalance due to decreased -globin protein
levels and the resultant destruction of cells during erythropoiesis. Such compounds as 5-azacytidine4-6 and
hydroxyurea7,8 as well as, more recently, butyric acid and
its analogues9,10 have been studied in great detail. (See
reviews by Rodgers and Rachmilewitz,2 Saleh and
Hillen,11 and Swank and Stamatoyannopoulos.12) The detailed molecular mechanism(s) by which these pharmacologic agents
produce increases in HbF is as yet unclear, but obviously such
information would greatly assist in the design of new drugs that might
have a lower toxicity as well as greater efficacy in stimulating HbF.
In general, it is also assumed that the major effects of these drugs
will be on transcription rates, although effects on mRNA processing,
stability, or other posttranscriptional mechanisms cannot be excluded.
The recent introduction of real time, fluorescence-based quantitative
polymerase chain reaction (PCR) as a rapid, sensitive technique for
precise quantitation of nucleic acid template13,14 now
allows for detailed monitoring of gene expression. This approach is
based on the 5' nuclease activity of Thermus aquaticus
(Taq) polymerase15 to hydrolyze a
dual fluorescently labeled oligonucleotide probe.16
Emission of a reporter dye at the 5' end of the intact sequence-specific probe is quenched by resonance energy transfer to a
second fluorophore at the 3' end. Probe hydrolysis by Taq polymerase during primer extension releases the 5' reporter,
which is detected in real time as an increase in fluorescence intensity within the tube. Quantitation is possible over a considerable range of
starting template concentrations since the fluorescence intensity is
directly proportional to the quantity of amplified copies. Absolute
quantitation is accomplished with the use of a standard curve with an
identical target of known quantity.
We report the combined use of a primary erythroid culture
system17,18 with the fluorescence-based, real-time
quantitative PCR technique to determine the effects of these 3 drugs,
5-azacytidine, hydroxyurea, and butyric acid, on fetal () and adult
() globin gene expression in human erythroid cells. The ex vivo
culture of primary adult erythroid cells provides a convenient source of immature progenitors and is divided into 2 phases. In the first, an
erythropoietin-independent phase, peripheral blood cells are cultured
in the presence of a combination of growth factors. In this phase,
early erythroid-committed progenitors (burst-forming units) proliferate
and differentiate into colony-forming unit-like progenitors. In the second phase, the cells are cultured
in an erythropoietin-supplemented medium, where they continue to
proliferate and differentiate into more mature erythroid progenitors,
such as orthochromatic normoblasts.19 This procedure
provides an experimental tool for studying various aspects of erythroid
cell development as well as modulation of Hb production by
pharmacological agents. It is anticipated that this combination of
culture and assay system will provide a convenient, yet powerful method
of screening potential drug candidates that affect gene expression as
well as of investigating the underlying molecular mechanisms by which
such drugs work.
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Materials and methods |
Two-phase liquid cultures of erythroid progenitor cells
from normal donors were established as previously
described.17,18 Mononuclear cells from at least 3 (and as
many as 6) normal donors were pooled in culture for these analyses in
order to minimize the influence of individual variation in response.
Briefly, peripheral blood mononuclear cells were isolated by
centrifugation on a cushion of Lymphocyte Separation Medium (ICN,
Aurora, OH), washed twice with Dulbecco's phosphate buffered saline
(Life Technologies, Grand Island, NY), and resuspended in 
-minimum
essential medium (Sigma, St. Louis, MO) supplemented with 10% fetal
calf serum (FCS) (Intergen, Purchase, NY), 1 µg/mL cyclosporin A
(Sandoz, Basel, Switzerland), 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Biofluids,
Gaithersburg, MD), and 10% conditioned medium obtained from cultures
of the 5637 bladder carcinoma cell line.20 Cultures were
incubated at 37°C in an atmosphere of 5% CO2 in air
with extra humidity. The nonadherent cells were harvested from this
phase I culture by centrifugation, washed twice in -medium (without
supplements), and resuspended in fresh -medium with 30% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL
streptomycin, 10% deionized bovine serum albumin,
10
5 mol/L -mercaptoethanol, 106
mol/L dexamethasone, 33 µg/mL holo-transferrin (Sigma, St. Louis, MO), and 1 U/mL human recombinant erythropoietin (Ortho Pharmaceutical, Raritan, NJ). Hydroxyurea, 5-azacytidine, and butyric acid were obtained from Sigma. Viable cell counts were performed with the use of
the trypan-blue-exclusion technique. The number of Hb-containing cells
was determined by means of the benzidine-HCl procedure.21 Total RNA was isolated with the use of the RNeasy mini-kit (Qiagen, Santa Clarita, CA). Superscript II reverse transcriptase (Life Technologies Inc) was used to synthesize complementary DNA (cDNA) after
priming with oligodeoxythymidine.
Quantitative real-time PCR assay of transcripts was carried out with
the use of gene-specific double fluorescently labeled probes in a 7700 Sequence Detector (PE Applied Biosystems, Norwalk, CT). We used
6-carboxy fluorescein (FAM) as the 5' fluorescent reporter while
we added tetramethylrhodamine (TAMRA) to the 3' end as quencher.
The following primer and probe sequences were used: -globin forward
primer, 5'-CTCATGGCAAGAAAGTGCTCG-3'; -globin reverse
primer, 5'-AATTCTTTGCCAAAGTGATGGG-3'; -globin probe, 5'-FAM-CGTGGATCCTGAGAACTTCAGGCTCCT-TAMRA-3'; -globin
forward primer, 5'-GGCAACCTGTCCTCTGCCTC-3'; -globin
reverse primer, 5'-GAAATGGATTGCCAAAAC-GG-3'; -globin
probe, 5'-FAM-CAAGCTCCTGGGAAATGTGCTGGTG-TAMRA-3'. All probes are designed to span exon junctions in the fully processed message in order to prevent reporting of amplification of any possible
contaminating genomic DNA. All primers and probes were made with the
use of reagents from Glen Research (Chantilly, VA) on an ABI 394 synthesizer (PE Applied Biosystems).
Double fluorescently labeled probes were high performance liquid
chromatography (HPLC)-purified on a 250 mm × 10 mm Biovantage C8
reverse phase column (Thomson, Chantilly, VA) fitted to a Gilson HPLC
system (Gilson Inc., Middleton, WI) with the use of a 10% to 35%
acetonitrile gradient in 0.1 mol/L triethylamine acetate, pH 7, with
the detector set to monitor column eluate at 260 nm. Products were
lyophilized and characterized by absorption spectroscopy as well as
DNAse I treatment to confirm a more than twofold increase in
fluorescence. All oligonucleotide primers and probes were quantitated by absorbance at 260 nm. Specific transcripts within cDNA reaction products were quantitated in a reaction mix consisting of 10 mmol/L Tris, pH 8.3; 50 mmol/L KCl; 4 mmol/L MgCl2; 1 mmol/L
ethylenediaminetetraacetic acid; 200 µmol/L
deoxynucleotide triphosphate; and 0.025 U/µL Platinum Taq polymerase (Life Technologies Inc).
Standard curves were constructed with the use of dilutions of an
accurately determined plasmid containing the cDNA of interest as
template. A dynamic range of 5 log orders of concentration or greater
was routinely achieved for each transcript of interest. Data are
presented as fold change relative to values immediately prior to the
addition of test compounds.
To perform HPLC quantitation of hemoglobin, we separated hemoglobins by
cation-exchange HPLC of supernatants from cell lysates as previously
described.18,22 Briefly, cells were pelleted and then
suspended and lysed in sterile distilled water. After the debris was
pelleted, the supernatant was chromatographed on a Synchropak CM 300, 250 mm × 4.6 mm column (Synchron Inc, Lafayette, IN) fitted to a
Maxima 820 (Waters, Milford, MA) developed with a sodium acetate
gradient in 30 mmol/L Bis
Tris buffer (pH 6.3-pH 6.15).
The area under the fetal (HbF) and adult (HbA) globin peaks were
integrated with the use of the system software.
| |
Results |
The effect of the 3 compounds 5-azacytidine,
hydroxyurea, and butyric acid on the number of benzidine-positive cells
present in the erythroid cultures 96 hours after their introduction is shown in panels A to C of Figure 1. We
added the compounds to the final concentrations shown on the fourth day
after adding erythropoietin to the cultured cells. In the range of
concentrations tested, hydroxyurea (Figure 1B) does not show a
consistent and reproducible impact on cell number.
5-Azacytidine (Figure 1A) and butyric acid (Figure 1C) addition both
resulted in reduced cell counts. Hydroxyurea has previously been shown
to reduce benzidine-positive cell counts in primary erythroid
cultures18 at concentrations 4 times the maximum used in
these experiments. There was also a time dependence of this decrease in
benzidine-positive cells: addition of the drug 4 days after
erythropoietin yielded a greater impact than addition at 7 or 10 days
(data not shown).
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| Fig 1.
Effect of 5-azacytidine (A), hydroxyurea (B), and butyric
acid (C) on primary adult erythroid cell proliferation.
Each bar represents the number of benzidine-positive cells present 96 hours after addition of the compound or vehicle.
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We also examined the effects of the 3 compounds on the percentage of
HbF, the total Hb, and the amount of Hb per cell. 5-Azacytidine, hydroxyurea, and butyric acid all produced dose-dependent increases in
the HbF fraction 96 hours after their addition to primary cultures on
the fourth day after erythropoietin addition (Figure
2A-C); hydroxyurea and butyric acid yielded
the largest effects. Linear correlation coefficients are as follows:
r2 = 0.788 for 5-azacytidine (Figure 2A);
r2 = 0.915 for hydroxyurea (Figure 2B); and
r2 = 0.996 for butyric acid (Figure 2C). Similar results
have previously been reported for hydroxyurea.18 However,
the total hemoglobin level (Figure 2D-F) was decreased in a
dose-dependent manner for both hydroxyurea and butyric acid while
5-azacytidine produced no consistent effect. In order to
estimate the effect of these compounds on the hemoglobin content per
cell, we used the cell counts after 96 hours' exposure. The results
are shown in Figure 2G-I. There was no effect on the hemoglobin content
per cell for the lowest concentration of 5-azacytidine used, but higher
concentrations resulted in a slightly increased total hemoglobin
content per cell. Hydroxyurea tended to decrease levels of hemoglobin
per cell, and butyric acid showed a strong dose-dependent reduction in
hemoglobin content per cell.
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| Fig 2.
Effect of varying concentrations of 5-azacytidine,
hydroxyurea, and butyric acid in erythroid cultures.
(A-C) Effect on fractional HbF content in erythroid cultures. (D-F)
Effect on total Hb. (G-I) Effect on the amount of HbF per cell.
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The interpretation of changes in absolute globin mRNA levels in these
primary human erythroid cells is complex for 2 reasons. First, in
general, -globin mRNA is less than 1% of -globin mRNA levels.
Second, during the course of culture, -globin mRNA levels gradually
decrease while -globin mRNA levels simultaneously increase. We
compare the effects of the various agents to that of control cultured
cells in which these changes are occurring in the absence of drugs.
Little net effect of 5-azacytidine is in evidence on
-globin mRNA levels in 24-hour, 48-hour, and 96-hour samples of
cells treated with 5 µmol/L 5-azacytidine as compared
with control cells (Figure 3A). Expression
of -globin message, on the other hand, showed a marked increase from
5-azacytidine, and the effect continued to increase with days in
culture (Figure 3B) in comparison with the untreated controls. The net
effect was a threefold to fourfold increase in the fraction of
-globin mRNA compared with -globin message (Figure 3C). This
suggests that the mechanism of pharmacologic stimulation of HbF
synthesis by 5-azacytidine reported in primates23 and in
patients with thalassemia or sickle cell disease4 does indeed involve a specific increase in -globin gene transcription.
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| Fig 3.
Time course of 5-azacytidine effects on the absolute
levels of globin mRNA in primary human adult erythroid cultures.
Hatched bars show the effect of 5 µmol/L 5-azacytidine
on (A) the levels of -globin message, (B) the levels of -globin
message, and (C) fractional -globin message content.
Untreated controls are shown as solid bars.
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In comparison with the untreated control cells, there is a small
increase in -globin mRNA expression evident 48 to 72 hours after
treatment with 40 µmol/L hydroxyurea (Figure
4A). In contrast, -globin mRNA
expression is increased by approximately 20-fold to 50-fold over the
equivalent controls (Figure 4B), correlating with the increased HbF
expression previously reported for in vitro erythroid cell
culture18 and sickle cell patients taking the drug.7,24 In the absence of the drug, -globin mRNA
levels declined over the period of observation. The resultant effect on
the fractional -globin mRNA content (/[ + ]) is a
significant, time-independent increase over the equivalent controls, as
expected, in the fourfold to fivefold range (Figure 4C).
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| Fig 4.
Hydroxyurea effects on absolute globin mRNA levels in
primary human adult erythroid cultures.
The effect of 40 µmol/L hydroxyurea on (A) the levels of
-globin message, (B) the levels of -globin message, and (C)
fractional -globin message content are shown. Untreated controls are
shown as solid bars, while hydroxyurea-treated samples are shown as
hatched bars.
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Although there was a monotonic increase of -globin mRNA expression
both in cells treated with 0.5 mmol/L butyric acid and in
the untreated controls (Figure 5A), the
level of expression was significantly lower in treated cells
(P < .05, paired t test), indicating a relative
inhibition of -globin gene expression. In contrast, -globin
message was expressed at considerably higher levels in treated cells in
a time-dependent fashion (Figure 5B). Consequently, fractional
-globin message expression is increased twofold to threefold in
butyric-acid-treated cells (Figure 5C). While these observations are
in agreement with previous reports that butyric acid increases
fractional and absolute HbF,25 simultaneous lowering of
-globin expression has not been reported.
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| Fig 5.
Butyric-acid effects on absolute globin mRNA levels in
primary human adult erythroid cultures.
The effect of 0.5 mmol/L butyric acid on (A) the levels
of -globin message, (B) the levels of -globin message, and (C)
fractional -globin message content are shown. Untreated controls are
shown as solid bars while butyric-acid-treated samples are shown as
hatched bars.
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Inhibition of histone deacetylase activity26 has been
reported and has recently been proposed as being directly involved in
increased -globin gene expression by butyric acid.27
Trichostatin A, a reversible inhibitor of histone deacetylase activity
both in vitro and in vivo,28 was used to treat primary
erythroid cells, and the response profile it induced in primary cell
cultures was found to be different from that induced by butyric acid.
Despite having a relatively small impact on the benzidine-positive cell count (data not shown), hemoglobin protein product was not detectable for cells treated with trichostatin A over a concentration range covering 62.5 nmol/L to 1 µmol/L. A profound decrease
in both - and -globin message expression was observed in primary
erythroid cells treated with 125 nm trichostatin A as
early as 24 hours after addition (samples were normalized to controls
at equivalent time points instead of there being one sample taken at
time 0) (Figure 6). Despite this, a small
relative increase in the fraction of -globin mRNA to -globin mRNA
was found.
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| Fig 6.
Effect of trichostatin A on globin expression in primary
human adult erythroid cultures.
The fold change in (A) -globin message levels, (B) -globin
message levels, and (C) fractional -globin message content are
shown. Controls are represented by filled bars while trichostatin A
treated samples are shown as hatched bars. Fold changes are expressed
relative to equivalent controls.
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| |
Discussion |
During growth and differentiation, gene expression undergoes
selective activation and repression/silencing.1 Silenced
genes can, however, be reactivated and expressed. This is the basis of
therapies for certain monogenic genetic diseases, such as Duchenne muscular dystrophy,29,30 and holds promise for the
development of therapies for certain other genetic diseases.
Reactivation of -globin synthesis to increase fetal hemoglobin with
the use of 5-azacytidine, hydroxyurea, or butyric acid (or its
derivatives) in the treatment of sickle cell disease and thalassemia
syndromes is an example of this approach.
The dose-dependent cytotoxicity of benzidine-positive cells observed in
this study with butyric acid has not been published previously although
similar results were reported for sodium phenylacetate.31 Treatment of erythroid cultures with 5-azacytidine, hydroxyurea, or
butyric acid resulted in both a 30-fold-to-120-fold increase in
expression of -globin mRNA and approximately 5-fold
increase in /( + ) fractional mRNA levels. In contrast to
the uniform increase in -globin mRNA by all 3 compounds, hydroxyurea
treatment produced a small stimulatory effect on -globin mRNA
expression. This observation could explain, at least in part,
previous reports of increased total Hb expression both in human
erythroid cells in culture18 and in patients32
treated with hydroxyurea.
In contrast to the increase in -globin mRNA observed for
hydroxyurea, butyric-acid treatment resulted in a twofold decrease in
-globin mRNA levels. This finding invites speculation on the possibility that the butyrate-induced increase in -globin may be
due, at least in part, to a compensatory mechanism related to the
concomitant decrease in -globin mRNA levels. The molecular mechanisms underlying the pharmacologic regulation of -globin gene
expression have been proposed as involving histone
deacetylation27 and/or a specific butyrate response
element.33 While butyrate response elements have been
described in the promoters of several butyrate-inducible genes, they
have been only partially defined in the globin genes.34
Butyrate was discovered to be an inducer of HbF as a result of an
investigation into the reasons behind the delayed
fetal-to-adult-hemoglobin switch in infants born to diabetic
mothers.35 Although the histone deacetylase inhibitory activity of butyrate is well known and is proposed as responsible for
the HbF-induction activity, it is yet to be demonstrated that alteration of acetylation levels of histones packaging the globin locus
affects globin gene expression specifically. Interestingly, although
treatment of cultures with trichostatin A, a widely used and
well-characterized histone deacetylase inhibitor,28 has minimal effect on the viable cell count, it yielded drastic decreases in both -globin and -globin transcripts. In addition, there was
no detectable hemoglobin protein product, suggesting the possibility of
incomplete (or no) overlap of their cellular targets. This raises the
further intriguing possibility that different classses of histone
deacetylases, possibly restricted in their locus of activity, act as
specific targets for various inhibitors, such as butyric acid and
trichostatin A. Alternatively, the difference in activities may reflect
a more potent inhibitory effect of trichostatin A on histone
deacetylases in primary human erythroid progenitors. In addition to
affecting mRNA levels, treatment of cultures with any of the 3 therapeutic compounds also resulted in increases in HbF/(HbF + HbA)
parallel to that observed for /( + ) fractional mRNA values.
Despite the use of butyrate for the treatment of some patients with
thalassemia and sickle cell disease and the reports of striking
inductions of HbF for some of these
patients,9,36 possible in vivo correlates of
this cytotoxicity should be investigated along with its exact clinical effectiveness.
The proposed mechanisms of action for the 3 therapeutic compounds
tested include alteration of the kinetics of erythropoiesis by
hydroxyurea,37 direct induction of -globin gene
transcription by demethylation of regulatory sequences by
5-azacytidine,4 and alteration of the acetylation status of
histones packaging the globin genes by inhibition of histone
deacetylase activity in the case of butyric acid.27 That
perturbations of such widely varying mechanisms can lead to increases
in HbF in peripheral blood (as well as in ex vivo cultures of erythroid
progenitors from normal donors) suggests that the overall regulation of
globin gene expression is likely to comprise many parts and be quite complex. The high risk of carcinogenicity with
5-azacytidine has limited its use to severe cases of homozygous
-thalassemia.38 Hydroxyurea, on the other hand, is
believed to present a low risk of carcinogenicity, is administered
orally, and is well tolerated by the patient.39 The effect
of treatment with hydroxyurea, however, as with the other drugs, is
quite transient. When administration of the drug stops, the production
of HbF-containing erythrocytes ceases, and HbF in the patient's blood
declines. Therefore, the accumulation and maintenance of therapeutic
levels of fetal hemoglobin in the blood require continuous treatment
with hydroxyurea. Butyrate action appears to allow use of a pulse
dosing regimen.10 In addition, it is known that not all
patients respond to the now standard hydroxyurea therapy. Among those
who do, there is significant variation in the level of HbF induction;
some show drug-induced HbF levels near 40% while others show only
minor changes.40 The reasons for this variation remain unknown.
An in vitro system that can be used for prediction of treatment
outcomes (measured as HbF and/or -globin mRNA response) would be of
value as a prognostic tool and also as a platform for testing novel
potential pharmacologic modulators of hemoglobin production. The
biphasic erythroid culture system employed in this study is a potential
candidate for such a tool. The quantitative PCR assay provides a
sensitive, reproducible, and specific assay of DNA templates and
provides a convenient assay of changes in gene expression under various
conditions and at varying time points. In combination with the
erythroid culture system, the quantitative PCR assay could serve as the
basis for development of such a system. Before any prognostic value
could be realized, a correlation would have to be established between
the response of cells in cultures derived from donors and the response
in the donors themselves.
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Footnotes |
Submitted July 21, 1999; accepted October 1, 1999.
Reprints: Alan N. Schechter, Laboratory of Chemical Biology,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Building 10, Room 9N307, 10 Center
Drive, MSC 1822, Bethesda, MD 20892-1822; e-mail:
aschecht{at}helix.nih.gov.
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.
Baron MH.
Developmental regulation of the vertebrate globin multigene family.
Gene Expr.
1996;6:129[Medline]
[Order article via Infotrieve].
2.
Rodgers GP, Rachmilewitz EA.
Novel treatment options in the severe beta-globin disorders.
Br J Haematol.
1995;91:263[Medline]
[Order article via Infotrieve].
3.
Schechter AN, Noguchi CT, Rodgers GP.
Sickle Cell Anemia. Philadelphia, PA: Saunders; 1987.
4.
Ley TJ, DeSimone J, Anagnou NP, et al.
5-Azacytidine selectively increases gamma-globin synthesis in a patient with beta+ thalassemia.
N Engl J Med.
1982;307:1469[Abstract].
5.
Charache S, Dover G, Smith K, Talbot CC Jr, Moyer M, Boyer S.
Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the gamma-delta-beta-globin gene complex.
Proc Natl Acad Sci U S A.
1983;80:4842[Abstract/Free Full Text].
6.
Ley TJ, DeSimone J, Noguchi CT, et al.
5-Azacytidine increases gamma-globin synthesis and reduces the proportion of dense cells in patients with sickle cell anemia.
Blood.
1983;62:370[Abstract/Free Full Text].
7.
Charache S, Dover GJ, Moore RD, et al.
Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia [see comments].
Blood.
1992;79:2555[Abstract/Free Full Text].
8.
Rodgers GP, Dover GJ, Noguchi CT, Schechter AN, Nienhuis AW.
Hematologic responses of patients with sickle cell disease to treatment with hydroxyurea.
N Engl J Med.
1990;322:1037[Abstract].
9.
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 [see comments].
N Engl J Med.
1993;328:81[Abstract/Free Full Text].
10.
Atweh GF, Sutton M, Nassif I, et al.
Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease [see comments].
Blood.
1999;93:1790[Abstract/Free Full Text].
11.
Saleh AW, Hillen HF.
Pharmacological induction of foetal haemoglobin synthesis in sickle-cell disease.
Neth J Med.
1997;51:169[Medline]
[Order article via Infotrieve].
12.
Swank RA, Stamatoyannopoulos G.
Fetal gene reactivation.
Curr Opin Genet Dev.
1998;8:366[Medline]
[Order article via Infotrieve].
13.
Heid CA, Stevens J, Livak KJ, Williams PM.
Real time quantitative PCR.
Genome Res.
1996;6:986[Abstract/Free Full Text].
14.
Gibson UE, Heid CA, Williams PM.
A novel method for real time quantitative RT-PCR.
Genome Res.
1996;6:995[Abstract/Free Full Text].
15.
Holland PM, Abramson RD, Watson R, Gelfand DH.
Detection of specific polymerase chain reaction product by utilizing the 5'3' exonuclease activity of Thermus aquaticus DNA polymerase.
Proc Natl Acad Sci U S A.
1991;88:7276[Abstract/Free Full Text].
16.
Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K.
Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.
PCR Methods Appl.
1995;4:357[Medline]
[Order article via Infotrieve].
17.
Fibach E, Manor D, Oppenheim A, Rachmilewitz EA.
Proliferation and maturation of human erythroid progenitors in liquid culture.
Blood.
1989;73:100[Abstract/Free Full Text].
18.
Fibach E, Burke KP, Schechter AN, Noguchi CT, Rodgers GP.
Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or beta-thalassemia.
Blood.
1993;81:1630[Abstract/Free Full Text].
19.
Fibach E.
Techniques for studying stimulation of fetal hemoglobin production in human erythroid cultures.
Hemoglobin.
1998;22:445[Medline]
[Order article via Infotrieve].
20.
Myers CD, Katz FE, Joshi G, Millar JL.
A cell line secreting stimulating factors for CFU-GEMM culture.
Blood.
1984;64:152[Abstract/Free Full Text].
21.
Orkin SH, Harosi FI, Leder P.
Differentiation in erythroleukemic cells and their somatic hybrids.
Proc Natl Acad Sci U S A.
1975;72:98[Abstract/Free Full Text].
22.
Wilson JB, Headlee ME, Huisman TH.
A new high-performance liquid chromatographic procedure for the separation and quantitation of various hemoglobin variants in adults and newborn babies.
J Lab Clin Med.
1983;102:174[Medline]
[Order article via Infotrieve].
23.
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].
24.
Zeng YT, Huang SZ, Ren ZR, et al.
Hydroxyurea therapy in beta-thalassaemia intermedia: improvement in haematological parameters due to enhanced beta-globin synthesis.
Br J Haematol.
1995;90:557[Medline]
[Order article via Infotrieve].
25.
Blau CA, Constantoulakis P, Shaw CM, Stamatoyannopoulos G.
Fetal hemoglobin induction with butyric acid: efficacy and toxicity.
Blood.
1993;81:529[Abstract/Free Full Text].
26.
Sealy L, Chalkley R.
The effect of sodium butyrate on histone modification.
Cell.
1978;14:115[Medline]
[Order article via Infotrieve].
27.
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].
28.
Yoshida M, Kijima M, Akita M, Beppu T.
Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A.
J Biol Chem.
1990;265:17,174[Abstract/Free Full Text].
29.
Deconinck N, Tinsley J, De Backer F, et al.
Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice.
Nat Med.
1997;3:1216[Medline]
[Order article via Infotrieve].
30.
Tinsley J, Deconinck N, Fisher R, et al.
Expression of full-length utrophin prevents muscular dystrophy in mdx mice.
Nat Med.
1998;4:1441[Medline]
[Order article via Infotrieve].
31.
Fibach E, Prasanna P, Rodgers GP, Samid D.
Enhanced fetal hemoglobin production by phenylacetate and 4-phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia.
Blood.
1993;82:2203[Abstract/Free Full Text].
32.
Fucharoen S, Siritanaratkul N, Winichagoon P, et al.
Hydroxyurea increases hemoglobin F levels and improves the effectiveness of erythropoiesis in beta-thalassemia/hemoglobin E disease.
Blood.
1996;87:887[Abstract/Free Full Text].
33.
Glauber JG, Wandersee NJ, Little JA, Ginder GD.
5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.
Mol Cell Biol.
1991;11:4690[Abstract/Free Full Text].
34.
Pace BS, Li Q, Stamatoyannopoulos G.
In vivo search for butyrate responsive sequences using transgenic mice carrying A gamma gene promoter mutants.
Blood.
1996;88:1079[Abstract/Free Full Text].
35.
Perrine SP, Greene MF, Faller DV.
Delay in the fetal globin switch in infants of diabetic mothers.
N Engl J Med.
1985;312:334[Abstract].
36.
Sher GD, Ginder GD, Little J, Yang S, Dover GJ, Olivieri NF.
Extended therapy with intravenous arginine butyrate in patients with beta-hemoglobinopathies [see comments].
N Engl J Med.
1995;332:1606[Abstract/Free Full Text].
37.
Torrealba-de Ron AT, Papayannopoulou T, Knapp MS, Fu MF, Knitter G, Stamatoyannopoulos G.
Perturbations in the erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine.
Blood.
1984;63:201[Abstract/Free Full Text].
38.
Lowrey CH, Nienhuis AW.
Brief report: treatment with azacitidine of patients with end-stage beta-thalassemia [see comments].
N Engl J Med.
1993;329:845[Free Full Text].
39.
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 [see comments].
N Engl J Med.
1995;332:1317[Abstract/Free Full Text].
40.
Steinberg MH, Lu ZH, Barton FB, Terrin ML, Charache S, Dover GJ.
Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Multicenter Study of Hydroxyurea.
Blood.
1997;89:1078[Abstract/Free Full Text].
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Abstract
Introduction
