Flow Cytometric Analysis of Hydroxyurea Effects on Fetal Hemoglobin Production in Cultures of...

ORIGINAL ARTICLE

Flow Cytometric Analysis of Hydroxyurea Effectson Fetal Hemoglobin Production in Cultures of

b-Thalassemia Erythroid Precursors

Ilana Amoyal, Ada Goldfarb, and Eitan Fibach*

Department of Hematology, Hadassah University Hospital, Jerusalem, Israel

ABSTRACT

An increase in fetal hemoglobin (Hb F) ameliorates the clinical symptoms of the

underlying disease in the b hemoglobinopathies—sickle cell anemia and b-thalassemia

(thal). Hydroxyurea (HU) can elevate Hb F production in erythroid cells and is the

agent currently in clinical use for patients with sickle cell anemia; it is presently being

tested in clinical trials for thalassemia. We have developed a two-phase liquid culture

system that mimics the in vivo hematological changes that are observed in patients

treated with HU. Adding HU during the second phase of the culture increases the

proportion of Hb F, increases the levels of total hemoglobin (Hb) content per cell and

increases cell size, but it decreases the numbers of cells and the total amount of

Hb produced. In the present study we developed and utilized a double labeling

procedure for flow cytometric analysis of the cellular distribution of Hb F. Cells

exposed to various concentrations of HU on day 6 of the second phase of the culture

were harvested on day 12, and stained simultaneously with fluorochrome-conjugated

monoclonal antibodies specific for human glycophorin A, an erythroid specific marker,

and human Hb F. Both the percentage of the Hb F-containing cells and their intensity of

fluorescence were recorded. The latter value gives a semi-quantitative estimation of the

mean cellular Hb F content. The results indicated that cultures derived from different

b-thalassemic patients differ in their response to HU. In most patients, low doses of HU

decreased the percentage of Hb F-cells as well as their Hb F content. At high doses,

*Correspondence: Dr. Eitan Fibach, Department of Hematology, Hadassah University Hospital, Ein-

Kerem, P.O. Box 12,000, Jerusalem 91120, Israel; Fax: 972-2-6423067; E-mail: [email protected].

HEMOGLOBIN

Vol. 27, No. 2, pp. 77–87, 2003

DOI: 10.1081=HEM-120021539 0363-0269 (Print); 1532-432X (Online)

Copyright # 2003 by Marcel Dekker, Inc. www.dekker.com

77

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some patients showed an increase in both parameters, while others showed an increase

in the percentage of Hb F-cells with minimal increase in their mean Hb F content, while

still other patients showed little effect at all. In all patients, high doses of HU caused

a decrease in cell numbers. These results suggest that HU has mixed effects on

erythroid precursors. Both the two-phase liquid culture and the flow cytometric

analysis procedures described herein provide the experimental tools for screening of

Hb F-inducing drugs and for evaluating patients’ cell response prior to treatment.

Key Words: Flow cytrometric analysis; Hydroxyurea (HU); Fetal hemoglobin (Hb F);

Thalassemia (thal); Erythroid precursors.

INTRODUCTION

Fetal hemoglobin (Hb F) is frequently increased in the b hemoglobinopathies—sickle

cell anemia and b-thalassemia (thal) (1). Epidemiological studies have indicated that an

increase in Hb F ameliorates the clinical symptoms of the underlying disease (2,3). In

sickle cell anemia not only do Hb F-containing cells have a lower concentration of sickle

hemoglobin (Hb), but Hb F inhibits polymerization of sickle Hb directly, accounting for

the lower propensity of such cells to undergo sickling (4–6). In b-thal, elevated Hb F

should compensate partially for the deficiency in b-globin chains.

Several pharmacological agents have been used to stimulate Hb F synthesis in vitro

and in vivo. The initial studies involved treatment of baboons (7) or patients with either

b-thal (8) or sickle cell anemia (9), with 5-azacytidine. The treatment resulted in a

considerable stimulation of g chain synthesis. Since this drug has been implicated as a

carcinogen (10), hydroxyurea (HU) is the agent currently in clinical use for patients with

sickle cell anemia and is being tested in clinical trials for thalassemia (11–14). The

response to HU treatment is variable; about 30% of the patients do not respond at all, and

the patients who do respond do it to various degrees.

Various cell culture systems have been used to better characterize the molecular

mechanisms by which HU mediates these effects (15). We developed a two-phase liquid

culture procedure that mimics the hematological changes that are observed in patients

treated with HU (16). In phase I, peripheral blood mononuclear cells are cultured in the

presence of a combination of growth factors, but in the absence of erythropoietin (Epo).

During this stage, early erythroid committed progenitors (BFU-E) proliferate and differ-

entiate into erythroid colony-forming unit (CFU-E)-like progenitors. In phase II, the latter

cells, in response to Epo, continue to proliferate and mature into orthochromatic

normoblasts (17,18). This system yields large and relatively pure populations of erythroid

cells that are semi-synchronous in maturity (19). Cells exposed to high concentrations

of HU during the second part of phase II were found to have an increased proportion of

Hb F (%Hb F), increased levels of total Hb content per cell (MCH), and increased cell

size (MCV), but the numbers of cells and the total amount of Hb produced were

decreased (16,20).

The analysis of the Hb content of these cultures was carried out by high performance

liquid chromatography (HPLC) (16) that measures the percentage of Hb F produced, and

by reverse transcriptase polymerase chain reaction (RT–PCR) that measures the content of

mRNA for the various globin genes (21). However, the results obtained by these

78 Amoyal, Goldfarb, and Fibach

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techniques provide an average data of the entire cell population studied, and do not allow

analysis at the single cell basis. Such an analysis is possible by flow cytometry.

In the present study we developed and utilized a double labeling procedure for a flow

cytometric analysis. Cells were stained simultaneously with monoclonal antibodies

specific for human glycophorin A (GPA) and human Hb F. Glycophorin A is an erythroid

specific marker that appears on the surface of erythroid precursors in parallel with the

intracellular accumulation Hb. The GPA positive cells were gated and analyzed for their

distribution with respect to Hb F. Both the percentage of Hb F-containing cells (%Hb

F-cells) and their intensity of fluorescence, expressed as the mean fluorescence channel

(MFC), were recorded. The MFC provides a comparative, semi-quantitative estimation of

the cellular Hb F content. The results indicated that cultures derived from different


resulted in a decrease in both the %Hb F-cells as well as in their Hb F content. At high

doses, some patients showed an increase in both parameters; other patients showed an

increase in the %Hb F-cells with no increase in Hb F content; while still other patients

showed no effect at all. High doses of HU caused a decrease in cell numbers in all patients.

MATERIALS AND METHODS

Patients

Twelve b-thal patients were studied. Table 1A summarizes some clinical data of

these patients.

Culture of b-Thalassemic Erythroid Cells

After informed consent was obtained, 10 mL blood was withdrawn into a heparin-

containing tube. In the case of transfused patients, blood was obtained just prior to

transfusion. Mononuclear cells were isolated by centrifugation over Ficoll-Hypaque

(1.077 g=mL; Sigma, St. Louis, MO, USA). The cells were cultured as previously

described (17,18,22). Briefly, the cells were first cultured in a-minimal essential medium

containing 10% pre-selected fetal bovine serum (FBS) (both from Biological Industries,

Beit-HaEmek, Israel), 1 mg=mL of cyclosporine A (Sandoz, Basel, Switzerland), and 10%

conditioned medium collected from cultures of a human bladder carcinoma 5637 cell line

[American Type Culture Collection (ATCC), Manassas, VA, USA]. After 5–7 day

incubation in this phase I culture, non adherent cells were harvested, washed and

resuspended in phase II medium composed of a medium, 30% FBS, 1% deionized bovine

serum albumin (BSA; Sigma) supplemented with 10�6 mol=L dexamethasone (Elkin-Sinn,

Cherry Hill, NJ, USA), 10�5 mol=L b-mercaptoethanol (Sigma), 0.3 mg=mL of human

holo-transferrin (Sigma), and 1 U=mL of human recombinant Epo (Ortho Pharmaceuticals,

Raritan, NJ, USA). After exposure to Epo, erythroid progenitors proliferated and matured

after 12 days into Hb-containing orthochromatic normoblasts. Various concentrations of

HU (Sigma) were added to the cultures on day 6 of phase II. On day 12, all cultures were

terminated, and the cells were harvested and analyzed. The Hb-containing cells were

Flow Cytometry Analysis of HU Effect on Hb F 79

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Ta

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var

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amo

ng

the

pat

ien

ts.


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counted following staining with a mixture of benzidine dihydrochloride and hydrogen

peroxide, as described previously (18).

Flow Cytometry

For immuno-phenotyping, cells were harvested and washed with phosphate buffered

saline containing 1% BSA. The cells were fixed in 1 mL of 4% paraformaldehyde (Sigma)

for 15 min at room temperature. The cells were then centrifuged and the pellet permea-

bilized by resuspending the cells in 1 mL mixture of ethanol : acetone (1 : 4, v=v) for 1 min

at room temperature. The cells were washed twice, the pellet was resuspended in 0.1 mL

buffer and the cells stained by incubation (at room temperature for 60 min) with

fluorescein isothiocyanate (FITC)-conjugated anti-human GPA and=or phycoerythrin

(PE)-conjugated anti-human Hb F (both from Immunoquality Products, Groningen,

The Netherlands). The cells were then washed with the same buffer and analyzed, using

a FACS-Calibur1 flow cytometer (Becton-Dickinson, Immunofluorometry Systems,

Mountain View, CA, USA). Cells were passed at a rate of about 1000 cells=second,

using saline as the sheath fluid. A 488 nm argon laser beam served as the light source for

excitation. Emission of 10,000 cells was measured using logarithmic amplification for

fluorescence and linear amplification for forward light scattering and side scatter. FL-1

measured FITC-derived fluorescence and FL-2 measured PE-derived fluorescence. In both

cases geometrical amplification was used. Background noise was determined using isotype

control stained cells. The percentage of Hb F-containing cells (%Hb F-cells) out of the

total population or out of the GPA-positive (erythroid) population was calculated using the

CELLQuest1 (Becton-Dickinson) software. The geometrical MFC of the entire cell

population or of the Hb F-cells, as indicated in the text, were also calculated.

RESULTS

Peripheral blood mononuclear cells were cultured according to the two-phase liquid

culture protocol. On day 6 of phase II, cells were exposed to different concentrations of

HU. On day 12, the cultures were harvested; an aliquot was counted for Hb-containing

cells by benzidine staining, and another aliquot was double labeled for GPA and Hb F and

analyzed by flow cytometry. Figure 1 shows the data as dot plots of a representative

experiment with cultures derived from a thalassemic patient. Figure 1(A), (B) show the

controls: cells stained with PE-conjugated anti-Hb F antibodies had minimal (0.44%) FL-1

fluorescence [Fig. 1(A)], and cells stained with FITC-conjugated anti-GPA antibodies

had minimal (0.73%) FL-2 fluorescence [Fig. 1(B)]. These results indicate that under the

flow conditions used, there was no spillover of fluorescence of cells stained for FL-2 (Hb F)

into the FL-1 detector, and likewise, no FL-2 fluorescence by cells stained for FL-1 (GPA).

Figure 1(C) shows the FL-1 and FL-2 fluorescence of cells double stained for both

GPA and Hb F. The results of this particular experiment show that 8.0% of the entire

population had no fluorescence at all; these (GPA�Hb F�) cells were probably non

erythroid cells (lymphocytes, myeloid precursors, and some macrophages) or very early

erythroid precursors prior to accumulation of either Hb or GPA; 77.2% of the cells were

GPAþHb F�, erythroid cells not containing appreciable amount of Hb F; 12.6% of the cells


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were GPAþHb Fþ, Hb F-containing erythroid cells; and 2.2% GPA�Hb Fþ cells, probably

very immature erythroid precursors that do not yet contain GPA but already contain Hb F.

Fetal hemoglobin has been shown to start accumulating at early stages of the erythroid

maturation (23), probably prior to GPA expression.

A gate was set on the GPAþ cells, and cells within this gate were analyzed for Hb F.

The results show 14.5% Hb Fþ cells within this population. The MFC was calculated to be

36. Figure 1(D) shows the FL-1=FL-2 dot plot of cells from the same patient treated with

150 mM HU. The results show that within the total population 24% were GPAþHb Fþ.

Within the GPAþ cells 29.4% were Hb Fþ with an MFC of 50.3. Thus, in this particular

experiment, HU increased the percentage of erythroid cells with Hb F from 14.5 to 29.4%

(a 2.0-fold increase) and the MFC from 36 to 50.3 (a 1.4-fold increase).

We next studied cultures derived from 12 b-thalassemic patients treated with different

concentrations of HU. The maximal fold increase by HU of the %Hb F cells and their MFCs

are summarized in Table 1 (column B). The results show a variable degree of response. Some

patients (patients 2 and 9) showed increase in both the %Hb F cells and their MFCs. Other

Figure 1. Flow cytometric analyses of Hb F content in thalassemic erythroid precursors. Cultures

derived from a b-thal patient, untreated (A–C) or treated with 150mM HU (D), were stained with

PE-conjugated antibodies for Hb F (A), with FITC-conjugated antibodies for GPA (B), or both

(C–D). The percentages of cells out of the entire population in each quadrant are shown.


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patients (e.g., patient 11) showed and increase in the %Hb F cells with minimal increase in

MFC, while still other patients (e.g., patients 7 and 12) showed little effect at all. In all

patients, low doses of HU resulted in a decrease in both the %Hb F-cells as well as their MFC,

whereas in all patients high doses of HU caused a decrease in cell numbers. Figure 2 depicts

the results of the %Hb F cells and MFC obtained in cultures derived from three

representative patients. Patient 9 [see Table 1 (column B)] showed an HU dose-response

increase of either parameter, patient 11 showed an increase in %Hb F cells but no change

in MFC, and patient 5 showed no change in either parameter.

Figure 2. Effect of different doses of HU on Hb F in cultured thalassemic cells. Cells derived from

three representative patients were cultured from day 6 of phase II in the presence of the indicated

doses of HU. On day 12, the cells were harvested and stained for Hb F and GPA. The variations in

%Hb F cells (A) and MFC (B) are shown.


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DISCUSSION

The two-phase liquid culture procedure is a valuable tool for screening of new Hb

F-stimulating drugs and for studying their mechanism of action. Two broad hypotheses

have been explored for the drug effect. One is based on drug-induced modifications of

DNA, such as hypo-methylation (9) or activation of responsive regions of the globin

promoter (24). The other mechanism involves modification of the rate of differentiation or

maturation of erythroid progenitors, such as rapid recruitment of erythroblasts directly

from progenitors with higher Hb F programs due to the cytoreduction effect of drugs such

as HU (25), or direct induction of down-stream maturation of progenitor cells (26,27).

Observations of elevated Hb F following accelerated erythropoiesis strongly suggest that

the rate of erythroid maturation is involved in the modulation of Hb F production (28).

This was supported by our experiments with hemin (29) and Epo (30).

In order to study the mechanism of action of HU, we analyzed the cultured cell

distribution with respect to Hb F content. In normal adults, most Hb F is restricted to a few

‘‘F cells.’’ Both the number and the Hb F content of these F cells may be increased in

various acquired and genetic conditions (31,32). In the present study, two parameters were

studied: the percentage of Hb F-containing cells and their Hb F content. For this purpose,

we developed a double staining flow cytometric procedure. In order to exclude from

analysis some non erythroid cells, such as lymphocytes, myeloid precursors, and macro-

phages (33) or mature red blood cells (RBC) derived from the original blood that may

contaminate the culture, the cells were labeled for GPA. Glycophorin A is a specific

erythroid marker that is present on intermediate erythroid precursors, but not on non

erythroid cells or mature RBC. By gating on GPAþ cells, only erythroid precursors were

included in the analysis. The cellular Hb F content was inferred from the MFC. Although a

direct relationship between MFC and Hb F content is not available, the MFC provides a

comparative, semi-quantitative estimation of Hb F content. Thus, MFC of cord blood RBC

is 6-fold higher than thalassemic Hb F-containing RBC (F-cells) and 10-fold higher than

normal F-cells. Similar results were obtained when cultured erythroid precursors from cord

blood, thalassemic blood, and normal blood were compared (data not shown).

Increase in %Hb F-cells without a change in Hb F content would suggest that HU

works through its cytoreduction effect; e.g., selective survival of Hb F-containing cells or a

selective maturation of Hb F-committed progenitors. In contrast, increased cellular Hb F

content would suggest that HU induces the potential of cells to produce Hb F, probably

through direct action at the DNA level. Molecular studies reported that HU affects the

proportion of Hb F, g-globin chain, and g-globin mRNA produced (21). But these effects

fail to suggest a mode of action; both selection or induction mechanism would cause an

elevation of these parameters.

The results of flow cytometry analyses indicated that cultures derived from different


resulted in a decrease in both the %Hb F-cells as well as their Hb F content. These results

are in agreement with a recent report showing that HU exerts a bimodal, dose-dependent,

effect on erythropoiesis in culture; low doses increase the cellularity and total Hb content,

but not the Hb F level. High doses, decreased the cellularity and total Hb content, but

increased the proportion of Hb F (20). The present study substantiates these results with

thalassemic patients, and further demonstrates that while some patients respond by an

increase in both the %Hb F-cells and Hb F content, others show an increase in %Hb F-cells


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with minimal increase in Hb F content, while still other patients show little effect at all.

These results, albeit based on a relatively small group of patients, indicated no correlation

between the pattern of response to HU and the genetic (the mutations involved) and

phenotype (clinical manifestations and mode of treatment, e.g., blood transfusion, chelation

therapy by desferral or deferiprone, etc.) of the patients. These results suggest that HU has

mixed effects on erythroid precursors. The relationship of changes in these two parameters

of Hb F to the clinical outcome of the treatment is a subject of further study.

Most of the patients have been treated by regular or occasional blood transfusions.

However, in the culture procedure used, the transfused blood did not affect the Hb

produced, since the transfused normal donor progenitors do not develop in culture (data

not shown). This is due to: (a) thalassemic blood contains elevated number of erythroid

progenitors compared to normal blood; (b) progenitors are removed from the donor blood

in the process of separation of the RBC fraction (by removal of the ‘‘buffy-coat’’)

following blood collection in the blood bank; (c) progenitors do not survive the storing

conditions of the blood bank.

The unresponsiveness of a significant proportion of patients to HU suggests that in vitro

predictive assay prior to treatment is highly desirable. Both the two-phase liquid culture and

the flow cytometric analysis described herein provide the experimental tools for such an

assay. In vivo studies have shown that continuous infusion of a therapeutic dose of HU at

20 mg=kg will maintain a serum concentration at 100 mM (34). The HU doses used in the

two-phase culture closely resembled the in vivo pharmacokinetics of HU treatment. In

addition, these cultures have been demonstrated to recapitulate many aspects of the in vivo

hematological changes following treatment with HU [reviewed in Ref. (15)]. By using the

flow cytometric method less than one million cells are required for analysis; a cell number

that can be obtained by culturing mononuclear cells derived from 1 mL of peripheral blood.

Flow cytometers are standard equipment in most hematological laboratories (for diagnostic

immunophenotyping of immunological and hematological diseases), thus, the procedure

provides an affordable, objective, accurate, and quantitative assay for determining Hb F

production in culture. However, further studies are required to conclude that results

obtained in culture could be extrapolated to patient’s response to HU and other drugs.

ACKNOWLEDGMENTS

We wish to thank Ms. A. Sinberger, Ms. A. Treves, and Dr. E. Prus (Department of

Hematology, Hadassah University Hospital, Jerusalem, Israel) for their help throughout the

study.

REFERENCES

1. Bunn HF, Forget BG. Hemoglobins A2, F, and A1c and other human hemoglobin

components. In: Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia:

W.B. Saunders Company, 1986:73–74.

2. Al-Awamy BH, Niazi GA, el-Mouzan MI, Altorki MT, Naeem MA. Relationship of

Haemoglobin F and a thalassaemia to severity of sickle-cell anaemia in the Eastern

Province of Saudi Arabia. Ann Trop Paediatr 1986; 6:261–265.


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y.

3. Haghshenass M, Ismail-Beigi F, Clegg JB, Weatherall DJ. Mild sickle-cell anaemia in

Iran associated with high levels of fetal haemoglobin. J Med Genet 1977; 14:168–171.

4. Benesch RE, Edalji R, Benesch R, Kwong S. Solubilization of Hemoglobin S by other

hemoglobins. Proc Natl Acad Sci USA 1980; 77:5130–5134.

5. Noguchi CT, Rodgers GP, Serjeant G, Schechter AN. Levels of fetal hemoglobin

necessary for treatment of sickle cell disease. N Engl J Med 1988; 318:96–99.

6. Eaton WA, Hofrichter J. The biophysics of sickle cell hydroxyurea therapy. Science

1995; 268:1142–1143.

7. DeSimone J, Heller P, Hall L, Zwiers D. 5-Azacytidine stimulates fetal hemoglobin

synthesis in anemic baboons. Proc Natl Acad Sci USA 1982; 79:4428–4431.

8. Ley TJ, DeSimone J, Anagnou NP, Keller GH, Humphries RK, Turner PH, Young NS,

Keller P, Nienhuis AW. 5-Azacytidine selectively increases g-globin synthesis in a

patient with bþ thalassemia. N Engl J Med 1982; 307:1469–1475.

9. 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 g-d-b-globin

gene complex. Proc Natl Acad Sci USA 1983; 80:4842–4846.

10. Carr BI, Reilly JG, Smith SS, Winberg C, Riggs A. The tumorigenicity of

5-azacytidine in the male Fischer rat. Carcinogenesis 1984; 5:1583–1590.

11. 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; 322:1037–1045.

12. Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB, Eckert SV, McMahon RP,

Bonds DR. 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; 332:1317–1322.

13. Rodgers GP, Rachmilewitz EA. Novel treatment options in the severe b-globin

disorders. Br J Haematol 1995; 91:263–268.

14. Olivieri NF, Rees DC, Ginder GD, Thein SL, Waye JS, Chang L, Brittenham GM,

Weatherall DJ. Elimination of transfusions through induction of fetal hemoglobin

synthesis in Cooley’s anemia. Ann NY Acad Sci 1998; 850:100–109.

15. Fibach E. Cell culture and animal models to screen for promising fetal hemoglobin-

stimulating compounds. Semin Hematol 2001; 38:374–381.

16. Fibach E, Burke LP, 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 b-thalassemia. Blood 1993; 81:1630–1635.

17. Fibach E, Rachmilewitz EA. Proliferation and differentiation of erythroid progenitors

in liquid culture: analysis of progenitors derived from patients with polycythemia vera.

Am J Hematol 1990; 35:151–156.

18. Fibach E. Techniques for studying stimulation of fetal hemoglobin production in

human erythroid cultures. Hemoglobin 1998; 22(5&6):445–458.

19. Fibach E, Rachmilewitz EA. The two-step liquid culture: a novel procedure for

studying maturation of human normal and pathological erythroid precursors. Stem

Cells 1993; 11(suppl 1):36–41.

20. Wang M, Tang DC, Liu W, Chin K, Zhu J, Fibach E, Rodgers GP. Hydroxyurea exerts

bi-modal dose-dependent effects on erythropoiesis in human cultured erythroid cells.

Br J Haematol 2002; 119:1–8.


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C B

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ley

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/14

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onal

use

onl

y.

21. Pope SH, Fibach E, Sun J, Chin K, Rodgers GP. Two-phase liquid culture system

models normal human adult erythropoiesis at the molecular level. Eur J Haematol

2000; 64:292–303.

22. Fibach E, Manor D, Treves A, Rachmilewitz EA. Growth of human normal erythroid

progenitors in liquid culture: a comparison with colony growth in semisolid culture.

Int J Cell Cloning 1991; 9:57–64.

23. Dalyot N, Fibach E, Rachmilewitz EA, Oppenheim A. Adult and neonatal patterns of

human globin gene expression are recapitulated in liquid cultures. Exp Hematol 1992;

20:1141–1145.

24. Hudgins WR, Fibach E, Safaya S, Rieder RF, Miller AC, Samid D. Transcriptional

upregulation of g-globin by phenylbutyrate and analogous aromatic fatty acids.

Biochem Pharmacol 1996; 52:1227–1233.

25. Letvin NL, Linch DC, Beardsley GP, McIntyre KW, Miller BA, Nathan DG. Influence

of cell cycle phase-specific agents on simian fetal hemoglobin synthesis. J Clin Invest

1985; 75:1999–2005.

26. Torrealba-de Ron AT, Papayannopoulou Th, Knapp MS, Fu MF, Knitter G,

Stamatoyannopoulos G. Perturbations in the erythroid marrow progenitor cell

pools may play a role in the augmentation of Hb F by 5-azacytidine. Blood 1984;

63:201–210.

27. Humphries RK, Dover G, Young NS, Moore JG, Charache S, Ley T, Nienhuis AW.

5-Azacytidine acts directly on both erythroid precursors and progenitors to increase

production of fetal hemoglobin. J Clin Invest 1985; 75:547–557.

28. Alter BP. Fetal erythropoiesis in stress hematopoiesis. Exp Hematol 1979; 7:200–209.

29. Fibach E, Kollia P, Schechter AN, Noguchi CT, Rodgers GP. Hemin-induced

acceleration of hemoglobin production in immature cultured erythroid cells:

preferential enhancement of fetal hemoglobin. Blood 1995; 85:2967–2974.

30. Fibach E, Schechter AN, Noguchi CT, Rodgers GP. Reducing erythropoietin in

cultures of human erythroid precursors elevates the proportion of fetal haemoglobin.

Br J Haematol 1994; 88:39–45.

31. Dover GJ, Boyer SH, Pembrey ME. F-cell production in sickle cell anemia: regulation

by genes linked to b-hemoglobin locus. Science 1981; 211:1441–1444.

32. Garner C, Tatu T, Reittie JE, Littlewood T, Darley J, Cervino S, Farrall M, Kelly P,

Spector TD, Thein SL. Genetic influences on F cells and other hematologic variables:

a twin heritability study. Blood 2000; 95:342–346.

33. Miller JL, Njoroge JM, Gubin AN, Rodgers GP. Prospective identification of erythroid

elements in cultured peripheral blood. Exp Hematol 1999; 27:624–629.

34. Fabricius E, Rajewsky F. Determination of hydroxyurea in mammalian tissues and

blood. Rev Eur Etud Clin Biol 1971; 16:679–683.

Received January 6, 2003

Accepted January 28, 2003


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