Rapamycin-mediated induction of c-globin mRNA accumulationin human erythroid cells

The search for potential therapeutic agents in haematological

diseases, including b-thalassaemia and sickle cell anaemia,

focuses on the pharmacologically mediated regulation of the

expression of human c-globin genes (Fibach et al, 1993a,b;

Perrine et al, 1993; Rodgers et al, 1993; Rochette et al, 1994;

Rodgers & Rachmilewitz, 1995; Steinberg et al, 1997; Olivieri

et al, 1998; Swank & Stamatoyannopoulos, 1998). It is well

established that even a 30% increase in the production of fetal

haemoglobin (HbF, a2c2) leads to a significant improvement

in the clinical status of these patients (Rochette et al, 1994;

Rodgers & Rachmilewitz, 1995; Olivieri et al, 1998). Therefore,

many recently published experiments were designed to find

agents capable of augmenting HbF levels in humans, such as

hormones, cytotoxic agents, haemopoietic cytokines and short

fatty acids (Fibach et al, 1993b; Perrine et al, 1993; Rodgers

et al, 1993; Chiarabelli et al, 2003; Fibach et al, 2003; Lamp-

ronti et al, 2003).

In this respect, rapamycin, a lipophilic macrolide, also called

sirolimus, which was isolated from a strain of Streptomyces

hygroscopicus found in a soil sample from Easter Island (known

by the inhabitants as Rapa Nui) (Sehgal, 2003), could be of

great interest.

This compound was found to effectively induce the differ-

entiation of human myeloid leukaemia HL-60, ML-1, K562

cells (Yamamoto-Yamaguchi et al, 2001) and J2E cells (Jaster

et al, 1996). Rapamycin (as sirolimus) has received approval

from the US Food and Drug Administration for marketing as

an agent for the prevention of acute rejection in renal

transplant recipients. Several studies are available on the

mechanism of action of this compound. Rapamycin shares

Carlo Mischiati,1 Alessia Sereni,1 Ilaria

Lampronti,1 Nicoletta Bianchi,1 Monica

Borgatti,1 Eugenia Prus,2 Eitan Fibach2

and Roberto Gambari1,3

1Department of Biochemistry and Molecular

Biology, University of Ferrara, Ferrara, Italy,2Department of Haematology, Hadassah

University, Jerusalem, Israel, and 3Laboratory for

the Development of Pharmacological and

Pharmacogenomic Therapy of Thalassemia,

Biotechnology Center, Ferrara, Italy

Received 23 February 2004; accepted for

publication 16 May 2004

Correspondence: Professor Roberto Gambari,

Department of Biochemistry and Molecular

Biology, University of Ferrara, Via L. Borsari

n.46, 44100 Ferrara, Italy. E-mail: gam@unife.it

Summary

The present study aimed to determine whether rapamycin could increase the

expression of c-globin genes in human erythroid cells. Rapamycin is a

macrocyclic lactone that possesses immunosuppressive, antifungal and anti-

tumour properties. This molecule is approved as an immunosuppressive

agent for preventing rejection in patients receiving organ transplantation. To

verify the activity of rapamycin, we employed two experimental cell systems,

the human leukaemia K562 cell line and the two-phase liquid culture of

human erythroid progenitors isolated from normal donors and patients with

b-thalassaemia. The results suggested that rapamycin, when compared with

cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of

erythroid differentiation and c-globin mRNA accumulation in human

leukaemia K562 cells. In addition, when normal human erythroid precursors

were cultured in the presence of rapamycin, c-globin mRNA accumulation

and fetal haemoglobin (HbF) production increased to levels that were higher

than those obtained using hydroxyurea. These effects were not associated

with inhibition of cell growth. Furthermore, rapamycin was found to increase

HbF content in erythroid precursor cells from four b-thalassaemia patients.

These results could have practical relevance, because pharmacologically

mediated regulation of the expression of human c-globin genes, leading to

increased HbF, is considered a potential therapeutic approach in

haematological disorders, including b-thalassaemia and sickle cell anaemia.

Keywords: rapamycin, erythroid differentiation, c-globin, fetal haemoglobin,

b-thalassaemia.

research paper

doi:10.1111/j.1365-2141.2004.05083.x ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621

with tacrolimus (FK506) a similar molecular structure and

binding capacity to the cytosolic immunophillin FK506

Binding Protein 12 (FKBP12) (Saunders et al, 2001). However,

despite the similar molecular structure, FK506 and rapamycin

have different mechanisms of action. FK506 acts by inhibiting

the calcineurin phosphatase, whereas rapamycin has no effect

on calcineurin phosphatase, but specifically inhibits the FKBP

and rapamycin-associated protein/mammalian target of rapa-

mycin (FRAP/mTOR) protein in mammalian cells (Gummert

et al, 1999). Rapamycin inhibits FRAP by forming a stable

complex with the immunophilin FK506-binding protein,

which binds to FRAP (Zhang et al, 2000).

The FRAP is a member of the mammalian phosphoinositide

kinase-related kinase (PIKK) family of proteins comprising

ATM, ATR-FRP and DNA-PK. Although the ATM, ATR-FRP

and DNA-PK proteins all respond to DNA damage in the cell,

FRAP/mTOR acts as a checkpoint control protein that

regulates the initiation and elongation of translation, ribosome

biosynthesis, and amino acid transport (Schmelzle & Hall,

2000), which affect the rate of protein synthesis by phosphor-

ylating the proteins p70S6K and 4E-BP1. The phosphorylation

of p70S6K promotes the phosphorylation of the S6 ribosomal

subunit, leading to an increase in translation (Dennis et al,

1996). FRAP also phosphorylates the translation inhibitor

4E-BP1, causing its dissociation from the translation initiation

factor eIF-4E and permitting increased protein translation and

mitogenesis (Gingras et al, 2001).

The aim of our study was to determine whether rapamycin

could induce erythroid differentiation and increase the

expression of c-globin genes in human erythroid cells. For

this purpose, we first used the human leukaemia K562 cells

(Lozzio & Lozzio, 1975; Rutherford et al, 1979; Gambari et al,

1984). The data obtained were further extended to human

erythroid precursors from normal subjects and b-thalassaemia

patients using a two-phase liquid culture procedure (Fibach

et al, 1989, 2003; Pope et al, 2000). This culture system was

very useful for identifying those molecules that were capable of

stimulating HbF production in erythroid precursors derived

from normal subjects as well as patients with thalassaemia and

sickle cell anaemia (Fibach et al, 1989; Pope et al, 2000).

Materials and methods

Materials

Rapamycin, FK506, ascomycin, butyric acid, cytosine arabino-

side, mithramycin and cisplatin were purchased from Sigma/

Aldrich (Milwaukee, WI, USA).

Cell lines and culture conditions

The human leukaemia K562 cells (Lozzio & Lozzio, 1975;

Rutherford et al, 1979; Gambari et al, 1984) were cultured in a

humidified atmosphere of 5% CO2/air in RPMI 1640 medium

(Sigma, St Louis, MO, USA) supplemented with 10% fetal

bovine serum (FBS; Celbio), 50 units/ml penicillin and

50 lg/ml streptomycin (Gambari et al, 1984). Cell growth

was studied by determining the cell number/ml with a ZF

Coulter Counter (Coulter Electronics, Hialeah, FL, USA)

(Bianchi et al, 1999, 2001). Stock solutions of rapamycin

(10 mmol/l) in ethanol were stored at )20�C in the dark and

diluted immediately before use with MeOH/dimethyl sulph-

oxide (DMSO) (1:2). Chemical inducers were added at the

appropriate concentrations at the beginning of the experiment

(cells were usually seeded at 30 000 cells/ml). The medium was

not changed during the induction period. Erythroid differen-

tiation was determined by counting benzidine-positive cells

after suspending the cells in a solution containing 0Æ2%benzidine in 5 mol/l glacial acetic acid, 10% H2O2, as

described elsewhere (Bianchi et al, 1999, 2001).

Human erythroid cell cultures from normal donors andb-thalassaemia patients

The two-phase liquid culture procedure was employed as

previously described (Fibach et al, 1989, 2003; Pope et al,

2000). Mononuclear cells were isolated from peripheral blood

samples of normal donors or b-thalassaemia patients by Ficoll–

Hypaque density gradient centrifugation and seeded in

a-minimal essential medium supplemented with 10% FBS

(both from Biological Industries, Beit-Haemek, Israel),

1 lg/ml ciclosporin A (Sandoz, Basel, Switzerland), and 10%

conditioned medium from the 5637 bladder carcinoma cell

line. The cultures were incubated at 37�C, under an atmo-

sphere of 5% CO2 in air, with extra humidity. After 7-d

incubation in this phase I culture, non-adherent cells were

harvested, washed and cultured in fresh medium, which was

composed of a-medium, 30% FBS, 1% deionized bovine

serum albumin (BSA), 10 lmol/l b-mercaptoethanol,

1Æ5 mmol/l glutamine, 1 lmol/l dexamethasone, and 1 U/ml

human recombinant erythropoietin (EPO; Ortho Pharmaceu-

tical, Raritan, NJ, USA). This stage of the culture is referred to

as phase II (Fibach et al, 2003). Compounds were added on

day 4–5 of phase II and cells were harvested on day 12 of phase

II. The proportion of HbF (% of total Hb) was determined by

high-performance liquid chromatography (HPLC) as des-

cribed elsewhere (Fibach et al, 1989; Pope et al, 2000).

Reverse transcription polymerase chain reaction (RT-PCR)and real-time quantitative RT-PCR

The RT-PCR was performed as described elsewhere using the

PCR primers described in Table I. Quantitative real-time PCR

assay of c-globin, b-globin and a-globin mRNAs were carried

out using gene-specific double-fluorescent-labelled probes in a

ABI Prism 7700 Sequence Detection System version 1.6.3

(Applied Biosystems, Warrington Cheshire, UK) (Fibach et al,

2003) (see Table I for primer sequences). The fluorescent

reporter and the quencher were: 6-carboxyfluorescein (FAM)

and 6-carboxy-N,N,N¢,N¢-tetramethylrhodamine (TAMRA)

Induction of c-globin Expression by Rapamycin

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621 613

respectively. For real-time PCR, the reference gene was human

GAPDH; this probe was fluorescent-labelled with VIC

(Applied Biosystems, Monza, Italy). The primers for b-actinwere 5¢-GTG GGG CGC CCC AGG CAC CA-3¢ (forward) and5¢-CTC CTT AAT GTC ACG CAC GAT TTC-3¢ (reverse); theprimers for GAPDH were 5¢-GAA GGT GAA GGT CGG AGT-

3¢ (forward) and 5¢-GAA GAT GGT GAT GGG ATT TC-3¢(reverse).

Results

Growth and differentiation of K562 cells cultured in thepresence of rapamycin

Figure 1 shows the dose-dependent effects of rapamycin,

FK506 and ascomycin on K562 cell growth and differentiation.

Cells were cultured in the absence or presence of the indicated

concentrations of the drugs and (i) the cell number/ml and

(ii) the proportion of benzidine-positive (haemoglobin-

containing) cells were determined after 6 d of culture. As

positive control, we employed K562 cells treated with

hydroxyurea (HU), a compound known to stimulate HbF

production in adult erythroid precursors (Fibach et al, 1993a)

and already used for the treatment of patients with sickle cell

anaemia and b-thalassaemia (Rodgers et al, 1993; Saxon et al,

1998). The results are given as the mean ± standard deviation

of five experiments performed in triplicate and showed that,

among the used immunophillins, only rapamycin was able to

induce erythroid differentiation at the concentration range

tested. Interestingly, inhibition of cell growth was observed

only when rapamycin was added at 400 nmol/l. Lower

concentrations (10–200 nmol/l) were able to induce erythroid

differentiation in the absence of a significant inhibition of cell

growth. It should be pointed out that, among the immuno-

phillins, rapamycin is a specific inhibitor of FRAP activity.

Figure 2 shows the relationship between the concentration

of rapamycin and its effects on cell growth and erythroid

differentiation when K562 cells were cultured for 5, 6 and 7 d.

As expected, induction of erythroid differentiation increased

with increasing concentrations of rapamycin and, at all the

concentrations used, the proportion of benzidine-positive cells

was the highest after 7 d. Furthermore, differentiation was

obtained by concentrations of rapamycin that did not inhibit

cell growth.

Table I. Sequences of primers and probes used

to analyse the expression of globin genes by

RT-PCR.

Semi-quantitative RT-PCR

c-globinForward primer 5¢-ACTCGCTTCTGGAACGTCTGA-3¢Reverse primer 5¢-GTATCTGGAGGACAGGGCACT-3¢

a-globinForward primer 5¢-CTGGAGAGGATGTTCCTGTCCTTG-3¢Reverse primer 5¢-CAGCTTAACGGTATTTGGAGGTCAT-3¢

b-globinForward primer 5¢-TCCTGAGGAGAAGTCTGCCGTTAT-3¢Reverse primer 5¢-GAAATTGGACAGCAAGAAAGCGGA-3¢

d-globinForward primer 5¢-GCAGATTACTGGTGGTCTACCCTGT-3¢Reverse primer 5¢-GGAAACAGTCCAGGATCTCAATGC-3¢

e-globinForward primer 5¢-TGTGGAGCAAGATGAATGTGGGAA-3¢Reverse primer 5¢-AGGGTCACAGGAAGACCTGCAAAC-3¢

f-globinForward primer 5¢-ACCAAGACTGAGAGGACCATCATTA-3¢Reverse primer 5¢-TCAGGACAGAGGATACGACCGATAC-3¢

Quantitative real-time RT-PCR

c-globinForward primer 5¢-TGGCAAGAAGGTGCTGACTTC-3¢Reverse primer 5¢-TCACTCAGCTGGGCAAAGG-3¢Probe 5¢-FAM-TGGGAGATGCCATAAAGCACCTGG-TAMRA-3¢

b-globinForward primer 5¢-CAAGAAAGTGCTCGGTGCCT-3¢Reverse primer 5¢-GCAAAGGTGCCCTTGAGGT-3¢Probe 5¢-FAM-TAGTGATGGCCTGGCTCACCTGGAC-TAMRA-3¢

a-globinForward primer 5¢-CACGCGCACAAGCTTCG-3¢Reverse primer 5¢-AGGGTCACCAGCAGGCAGT-3¢Probe 5¢-FAM-TGGACCCGGTCAACTTCAAGCTCCT-TAMRA-3¢

C. Mischiati et al

614 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621

Table II shows the rapamycin-induced benzidine positivity

of K562 cells compared with that of other known inducers,

such as cytarabine (ara-C), mithramycin, cisplatin, HU and

butyric acid (Bianchi et al, 1999, 2000, 2001). The results

showed that induction of erythroid differentiation by rapa-

mycin was lower than that of ara-C and mithramycin, was

similar to cisplatin, and was much higher than that of butyric

acid and HU.

RT-PCR analysis of rapamycin-mediated increase ofglobin gene expression in K562 cells

In order to find out whether the rapamycin-induced increase

in the proportion of benzidine-positive K562 cells is associ-

ated with an increase in c-globin mRNA content, we analysed

total cellular mRNA by semi-quantitative RT-PCR (Bianchi

et al, 2001). In the first experiment (Fig 3A), cells were

cultured for 3, 4, 5 and 6 d in the absence or in the presence

of 10 nmol/l rapamycin and total RNA was isolated. After

reverse-transcription, PCR was used to amplify the a-globin,

Fig 1. The effects of various immunophillin-

binding drugs on the proliferation (open

squares) and erythroid differentiation (filled

squares) of K562 cells. Cells were cultured in the

absence or presence of the indicated concen-

trations of drug for 6 d. The numbers of total

cells per ml of culture and the percentage of

benzidine-positive cells were determined. The

values in treated cultures were compared with

untreated control cultures (taken as 100%). The

data represents the mean ± SD of five inde-

pendent experiments.

Fig 2. The effects of rapamycin on the proliferation and erythroid

differentiation of K562 cells. Cells were cultured in the absence or in

the presence of the indicated concentrations of rapamycin. Cell

number/ml was determined at the indicated days (A). On the indicated

days, cells were stained with benzidine and counted, and the per cent of

benzidine-positive cells was determined (B). The data represents the

mean ± SD of five independent experiments.

Table II. Level of K562 erythroid differentiation induced by rapamy-

cin and other known inducers.

Inducer

Differentiation

(% of benzidine-positive cells)

Concentration

used

Rapamycin 45Æ6 ± 5Æ2 20 nmol/l

Ara-C 92Æ5 ± 3Æ6 5 lmol/l

Mithramycin 85Æ4 ± 9Æ1 50 nmol/l

Cisplatin 35Æ8 ± 5Æ2 2 lmol/l

Hydroxyurea 25Æ4 ± 2Æ4 100 lmol/l

Butyric acid 31Æ2 ± 3Æ1 2 mmol/l

Determinations were performed on day 7.

Induction of c-globin Expression by Rapamycin

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621 615

b-globin, c-globin, d-globin, e-globin, and n-globin genes. As

controls, b-actin sequences were also amplified. The results

indicated that, as expected, no expression of b-globin and

d-globin genes was observed. In contrast, the expression of all

the embryo-fetal globin genes was increased. These data were

confirmed by another experiment, in which K562 cells were

cultured for 6 d in the presence of increasing concentrations

of rapamycin (Fig 3B). As can be clearly appreciable, a

concentration of rapamycin as low as 10 nmol/l was sufficient

to induce the highest level of expression of embryo-fetal

globin genes. As expected from the results shown in Fig 1,

ascomycin and FK506 did not increase the expression of

globin genes in K562 cells (Fig 3C). The effects of rapamycin

on the accumulation of c-globin mRNA prompted us to

verify the effects of rapamycin on human erythroid precursor

cells.

Effects of rapamycin on cell growth and differentiation ofnormal human erythroid precursors

The effects of rapamycin on cell growth and differentiation of

erythroid precursor cells was determined by employing the

two-phase liquid culture system as described elsewhere

(Fibach et al, 1989; Pope et al, 2000). In this procedure,

early erythroid-committed progenitors (erythroid burst-form-

ing units) derived from the peripheral blood proliferated and

differentiated during phase I (in the absence of EPO) into late

progenitors (erythroid colony-forming units). In phase II, in

the presence of EPO, the latter cells continue their prolifer-

ation and mature into Hb-containing orthochromatic normo-

blasts.

The effects of rapamycin on erythroid precursor cells

isolated from normal donors are reported in Fig 4.

Rapamycin-mediated effects were compared with those found

in erythroid precursor cells treated with 100 lmol/l HU.

Figure 4A shows representative results regarding the cell

growth of erythroid precursor cells 1, 2 and 4 d after the

addition of 10, 50 and 100 nmol/l rapamycin. Figure 4B

summarizes the dose-dependent effects obtained with pooled

precursors derived from five different donors after 4 d of

treatment. As is clearly evident, only minor inhibitory effects

on cell growth were detectable; in contrast, HU exhibited

antiproliferative effects, confirming previously reported results

(Fibach et al, 2003).

With respect to erythroid differentiation, the proportion of

benzidine-positive cells in the rapamycin-treated cell popula-

tions was found to be even higher than that found in untreated

control cells (Fig 4C, white boxes). This is evident also when

the data are reported as absolute number of benzidine-positive

cells/ml of culture (Fig 4C, black boxes).

These data support the concept that the effects of rapamycin

on the expression of globin genes are not the result of

cytotoxicity of the treatment, at least for the drug concentra-

tions used in this study.

Fig 3. The effects of rapamycin on the expression of globin genes in K562 cells, assayed by semi-quantitative reverse transcription polymerase chain

reaction (RT-PCR). (A) Time-course analysis of the mRNA levels in cells treated with 10 nmol/l rapamycin. (B) Dose-dependent analysis of the

mRNA levels in cells treated with increasing concentration of rapamycin. (C) Comparison of the mRNA levels in cells treated with the immuno-

phillin-binding drugs FK506, ascomycin and rapamycin. In the experiments in (B) and (C), cells were harvested following 6 d of treatment with the

drugs.

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616 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621

Rapamycin-induced increase of c-globin mRNA in normalhuman erythroid precursors

To study the effects of rapamycin on c-globin mRNA produc-

tion in normal human erythroid precursors cells, rapamycin

was added at 10 nmol/l on days 4–5 of phase II, when the cells

started to synthesize Hb. As positive controls, we used cultures

treated with HU (100 lmol/l) and mithramycin (30 nmol/l),

two well-known potent inducers of HbF (Fibach et al, 2003).

The accumulation of c-globin and GAPDH mRNAs was

measured using as template total RNA by quantitative

fluorescence-based RT-PCR (Fibach et al, 2003). The results

(Fig 5A) indicated that the kinetics of the generation of

c-globin RT-PCR products was much faster when using cDNA

from rapamycin treated cells as the substrate (closed symbols)

compared with untreated control cells (open symbols). No

major differences were observed in the cellular content of

mRNA for GAPDH (Fig 5B). The data reported in Fig 5 (A

and B) were analysed using the Sequence Detection Software

System 1.6.3. Relative to untreated cells, rapamycin induced a

12Æ1 ± 3Æ5-fold increase in c-globin mRNA. These results were

consistently reproduced in five independent experiments using

different donors, as shown in Fig 5C, which also enabled a

comparison with the induction of c-globin mRNA following

treatment of erythroid precursors with HU (3Æ5 ± 2Æ8-foldincrease) and mithramycin (5Æ3 ± 2Æ4-fold increase).

Fig 4. The effects of rapamycin on the prolif-

eration of erythroid precursor cells from normal

subjects. (A) Erythroid precursor cells from a

representative normal subject were cultured

without inducers (open circles) or with 10 (open

triangles), 50 (solid triangles) and 100 (solid

squares) nmol/l rapamycin, or with 100 lmol/l

HU (solid circles). On the indicated days, cell

number/ml was determined. (B) A summary of

the results obtained after 4 d of treatment of

erythroid precursor cells from five different

subjects. (C) Effects of rapamycin on differen-

tiation of erythroid precursor cells from normal

subjects. Erythroid precursor cells from five

different donors in five independent experi-

ments were treated with the indicated concen-

trations of rapamycin (nmol/l), with 100 lmol/l

HU or none (control). Following 4 d of treat-

ment, cells were stained with benzidine and

counted, and the percentage and number of

benzidine-positive cells were determined.

(B) and (C) represents the mean ± SD of five

independent experiments.

Induction of c-globin Expression by Rapamycin

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621 617

A second important point we wanted to analyse was the

expression of c-globin genes with respect to the expression of

a-globin and b-globin genes. To this aim, erythroid precursor

cells from five different subjects were cultured for 4 d in the

presence of 10, 50 and 100 nmol/l rapamycin, RNA isolated

and accumulation of GAPDH, a-globin, b-globin and c-globinmRNA determined by real-time quantitative RT-PCR analysis.

The results (Fig 5D) clearly showed that the content of all the

analysed globin mRNAs increased in rapamycin-treated cells.

Interestingly, the increase of c-globin mRNA was found to be

significantly higher than that of b-globin mRNA.

Taken together, these findings strongly suggest that rapa-

mycin treatment could lead to an increase in the amount of

HbF produced by erythroid precursor cells.

Rapamycin-induced increase of HbF in human erythroidprecursor cells from normal subjects and b-thalassaemiapatients

In a first set of experiments, rapamycin, at 10 nmol/l, was

added on days 4–5 of phase II for 7 d. As positive controls, we

used cultures treated with HU (100 lmol/l) and mithramycin

(30 nmol/l). HPLC analyses of the cellular Hb content in these

cultures showed that HbF was increased in rapamycin-treated

cultures (Fig 6A) with respect to untreated cultures. The

proportion of HbF in control cultures was 1Æ4 ± 0Æ6%, it

increased to 4Æ8 ± 0Æ9% and 6Æ6 ± 1Æ1% in HU- and mithra-

mycin-treated cultures respectively, and to 10Æ2 ± 1Æ5%

in rapamycin-treated cultures (average ± SD of six experi-

ments).

In the second set of experiments, erythroid precursor cells

from five different subjects were treated for 4 d with 10, 50 and

100 nmol/l rapamycin and HbF determined, as a percentage of

total Hb and pg/cell, using HPLC as an analytical system and

suitable standards for quantification. The results (Fig 6B)

clearly demonstrated that HbF was significantly increased (at

least 10-fold) in rapamycin-treated cells.

In the third set of experiments, we performed a preliminary

study on the effects of rapamycin on cells derived from

b-thalassaemia patients. The proportion of HbF detected is

summarized in Table III. As expected, the HbF levels in

untreated cultures of these patients were high (from 11Æ3 in

cells from patient 1 to 16Æ5 in cells from patient 2). However,

in cells from all these patients, the percentage of HbF was

always significantly higher (P < 0Æ005) after exposure to

rapamycin. A fourth patient generated cultures with an even

higher starting level of HbF (37Æ1%), and even in this patient,

rapamycin increased HbF up to 52Æ2%.

Discussion

The HbF inducers could be of great interest for the therapy of

b-thalassaemia and sickle cell anaemia (Fibach et al, 1993a,b;

Perrine et al, 1993; Rodgers et al, 1993; Rochette et al, 1994;

Rodgers & Rachmilewitz, 1995; Steinberg et al, 1997; Olivieri

et al, 1998; Swank & Stamatoyannopoulos, 1998), because

Fig 5. c-globin mRNA content in normal erythroid precursor cells treated with rapamycin. Cultures of erythroid precursors were treated from day 4

of phase II in the absence (open squares) or presence of 10 nmol/l rapamycin (filled squares). After 12 d, cells were harvested, total RNA extracted

and then reverse transcribed and 50 ng was used for PCR amplification. For each sample, the kinetics of generation of c-globin (A) and GAPDH (B)

RT-PCR products was determined. D Rn for each mRNA is plotted against the cycle number. (C) The fold increase of c-globin mRNA accumulation

in rapamycin-treated erythroid precursors was compared with the fold increase obtained in hydroxyurea and mithramycin-treated erythroid

precursors. The reported data are the mean ± SD of five independent experiments. (D) Fold increase of c-globin (solid circles), b-globin (open cirles)

and a-globin (open squares) mRNAs in erythroid precursor cells treated for 4 d with 10, 50 and 100 nmol/l rapamycin. The reported data are the

mean ± SD of three independent experiments.

C. Mischiati et al

618 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621

increased HbF concentrations can ameliorate the symptoms of

these diseases.

This study found that rapamycin is a potent inducer of the

erythroid differentiation of K562 cells. Differentiation was

found to be associated with a sharp increase in the production

of c-globin mRNA. Interestingly, this increase was not

observed in cells treated with tacrolimus (FK506) or ascomy-

cin, two immunophillins that display a similar molecular

structure and targets, but unable to modulate FRAP/mTOR

(Saunders et al, 2001). This novel data suggest that FRAP/

mTOR, and not FKBP12, is implicated in the complex pathway

leading to erythroid differentiation.

The K562 cell line has been proposed as a useful in vitro

model for studying the molecular mechanism(s) regulating the

expression of embryonic and fetal human globin genes

(Rutherford et al, 1979), as well as screening for potential

new differentiation-inducing compounds (Bianchi et al, 1999,

2000; Fibach et al, 2003). This cell line, isolated and charac-

terized by Lozzio and Lozzio (1975) from a patient with

chronic myeloid leukaemia in blast crisis, exhibits a low

proportion of Hb-synthesizing cells under standard culture

conditions, but is capable of undergoing erythroid differenti-

ation when treated with a variety of compounds, including

haemin (Rutherford et al, 1979), ara-C (Bianchi et al, 1999),

5-azacytidine (Gambari et al, 1984), chromomycin and

mithramycin (Bianchi et al, 1999), tallimustine (Bianchi et al,

2001; Chiarabelli et al, 2003), cisplatin and cisplatin analogues

(Bianchi et al, 2000). Following the erythroid induction of

K562 cells, Hb Portland (f2c2) and Hb Gower 1 (f2e2)accumulate, because of increases in the expression of human

f-, e- and c-globin genes (Gambari et al, 1984). In vitro studies

demonstrated that known inducers of erythroid differentiation

in K562 cells, such as HU, butyrates and 5-azacytidine, are also

capable of inducing HbF production when administered,

either alone or in combination, to normal erythroid cells

(Fibach et al, 1993b). Butyric acid, HU and 5-azacytidine have

been the subject of reports on the treatment of b-thalassaemia

patients (Lowrey & Nienhuis, 1993; Perrine et al, 1993;

Rodgers et al, 1993; Sher et al, 1995).

Another important conclusion of this study is related to the

evaluation of the effects of rapamycin on the production of

c-globin mRNA in human erythroid precursors grown in the

two-stage liquid culture system. We demonstrated that rapa-

mycin stimulated an increase in c-globin mRNA production.

This increase is higher than that obtained by HU, a potent

inducer of HbF production both in vitro and in vivo (Fibach

et al, 1993a; Saxon et al, 1998; Lavelle et al, 2001).

In full agreement with these results, we found that

rapamycin also induced increased HbF production, when the

data are considered as both % of total haemoglobin produc-

tion and pg/cell. The data obtained by HPLC analysis and

shown in Fig 6 clearly indicate that HbF content is much

higher in rapamycin-treated cells compared with control cells.

The results of this study are also of particular interest for

investigating the biochemical basis of erythroid differentiation.

In fact, it is well known that rapamycin inhibits FRAP/mTOR

by forming a stable complex with the FKBP12 (Zhang et al,

2000), and that FRAP/mTOR acts as a checkpoint control

Fig 6. (A) Increase in fetal haemoglobin (HbF)

content in normal erythroid precursor cells

treated with rapamycin. Cultures of erythroid

precursors were treated from day 4 of phase II in

the absence or presence of 10 nmol/l rapamycin.

At 12 d, cells were harvested, lysed and analysed

for HbF. The fold increase of HbF in rapamycin-

treated cells was compared with the fold increase

obtained in hydroxyurea and mithramycin-

treated cells. The reported data are the

mean ± SD of six independent experiments.

(B) HbF accumulation, expressed as % of total

Hb and pg/cell, in erythroid precursor cells

treated for 4 d with 10, 50 and 100 nmol/l

rapamycin. The reported data are the

mean ± SD of three independent experiments.

Table III. HbF production by erythroid precursor cells from b-tha-lassaemia patients.

Patient

number Untreated

Rapamycin-treated

10 nmol/l 100 nmol/l

1 11Æ3 17 17Æ52 16Æ5 18 20

3 12Æ5 16Æ3 15

Results represent % HbF with respect to total Hb.

Induction of c-globin Expression by Rapamycin

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 612–621 619

protein regulating the rate of protein synthesis (Schmelzle &

Hall, 2000). Further studies, if focused on this pathway, might

identify the biochemical steps and also determine whether

existing, new drugs can control the expression of the

erythroid-specific genes. Furthermore, the recent demonstra-

tion that FRAP/mTOR is a nucleocytoplasmic shuttling

protein that could also have direct targets in the nucleus

(Kim & Chen, 2000) suggest that gene-profiling experiments

could be useful in order to better understand the molecular

basis of erythroid differentiation.

The results of the present study could also have a practical

impact, because it is well known that an increase in c-globinmRNA and HbF production could ameliorate the clinical

status of patients with b-thalassaemia and sickle cell anaemia

(Rochette et al, 1994; Rodgers & Rachmilewitz, 1995; Olivieri

et al, 1998). Interestingly, the pharmacokinetics, adsorption,

route of administration, distribution and metabolism of

rapamycin (as Rapamune or Sirolimus) are well known

(Mahalati & Kahan, 2001). Of great interest is the evidence

that the whole blood concentration of Sirolimus, as measured

by immunoassay or liquid chromatography/mass spectro-

metry/mass spectrometry (LC/MS/MS), is 17Æ3 ± 7Æ4 ng/ml

following the administration of 5 mg/d. The terminal elimin-

ation half-life (t1/2) of Sirolimus after multiple dosages is

estimated to be 62 ± 16 h. Therefore, the concentration of

Sirolimus in the whole blood of patients treated with standard

conditions, could reach steady-state concentrations that are

similar to those found to induce the increased expression of

c-globin mRNA in our experiments.

Our study should encourage the use of Sirolimus in clinical

trials. It should be noted that Sirolimus retains a number of

adverse side-effects, including hypercholesterolaemia, hyperli-

pidaemia and hypertension. Therefore, the first objective

should be to determine the Hb content and HbF/HbA1 ratio

in patients treated with Sirolimus for pathologies other than

thalassaemia. With all these considerations in mind, we believe

that our results indicate that rapamycin warrants further

evaluation as a potential therapeutic drug in b-thalassaemia

and sickle cell anaemia.

Acknowledgements

This work was supported by CNR PF Biotecnologie and by

Associazione Veneta per la Lotta alla Thalassemia (Rovigo).

We thank S.I.T. ULSS 18, Rovigo (Servizio di Immunoemat-

ologia e Trasfusione: Prof. Rocco Potenza, Dr Francesco

Chiavilli, Dr Stefano Modonesi) for clinical samples.

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