Leukemia Research 30 (2006) 561–568

Transcriptional and post-transcriptional regulation of glutathioneS-transferase P1 expression during butyric acid-induced

differentiation of K562 cells

Michael Schnekenburger a, Franck Morceau a, Estelle Henry a, Romain Blasius a,Mario Dicato a, Chantal Trentesaux b, Marc Diederich a,∗

a Laboratoire de Biologie Moleculaire et Cellulaire du Cancer, Hopital Kirchberg, 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourgb Laboratoire d’Onco-Pharmacologie, JE2428, IFR 53, UFR de Pharmacie, 51 rue Cognacq-Jay, F-51096 Reims, France

Received 4 November 2004; accepted 26 August 2005Available online 4 October 2005

Abstract

Over-expression of glutathione S-transferase P1 is related to chemotherapeutic drug resistance as well as to differentiation of humanecGdaf©

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rythroleukemia cells. In opposition to previously described differentiating inducers which enhance the GST-resistance phenotype, time- andoncentration-dependent activation of both erythroid and megakaryocytic differentiation pathways by butyric acid progressively diminishedSTP1 mRNA expression. GSTP1 mRNA expression decreased by 25% (p < 0.01) and 64% (p < 0.01) in 1 mM and 2 mM butyric acid-ifferentiated K562 cells, respectively. These results were associated to both a reduction of GATA-1 binding activity to the GSTP1 promoternd to a posttranscriptional destabilization of GSTP1 mRNA in a concentration dependent manner. Indeed, GSTP1 mRNA half-life decreasedrom 43.8 to 36.2 h and 12.6 h in 1 mM- and 2 mM-treated cells, respectively.

2005 Elsevier Ltd. All rights reserved.

eywords: Erythroid and megakaryocytic differentiation; Butyric acid; GSTP1; GATA-1; K562

. Introduction

The glutathione S-transferase (GST, EC 2.5.1.18) familyf multifunctional enzymes plays a particularly importantole in cellular detoxification [1]. Over-expression of theSTP1 isoform characterizes many human tumor cell lineserived from stomach, brain and colon as well as leukemia2]. In the clinic, this over-expression is a bad prognostic foratient survival [3]. In addition, GSTP1 is involved in resis-ance against antineoplastic drugs [4] and against apoptosisy binding to Jun N-terminal kinase (JNK) [5], whereas inhi-ition of GSTP1 leads to apoptosis in K562 leukemia cells6].

Transcriptional regulation by SP-1 [7], AP-1 [8] and NF-B [9] was described to be part of the molecular mechanisms

∗ Corresponding author. Tel.: +352 2468 4040; fax: +352 2468 4060.E-mail address: marc.diederich@lbmcc.lu (M. Diederich).

controlling GSTP1 gene expression. GSTP1 down-regulationwas also described by methylation of CpG residues withinthe core promoter in various pathologies, including prostatecarcinoma [10] and leukemia [11]. In addition, we recentlydemonstrated that the erythroid differentiation-specific tran-scription factor GATA-1 binds to a GATA sequence withinthe GSTP1 gene promoter with similar variations of GATA-1binding activity and GSTP1 expression during both inducederythroid- and megakaryocytic K562 cell differentiation [12].

Differentiation therapy aims to induce the reversal of themalignant phenotype by using non toxic doses of differen-tiating agents and appears to become an alternative strat-egy to conventional anti-cancer therapies [13,14]. However,we recently demonstrated that this clinical approach mightincrease cancer cell resistance by enhanced detoxificationpotential via over-expressed GSTP1 [12,15].

The aim of this paper was to study the effect of butyricacid as an inducer of megakaryocytic and erythroid differ-

145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2005.08.023

562 M. Schnekenburger et al. / Leukemia Research 30 (2006) 561–568

entiation on GSTP1 expression. We report here butyric acidto act as an interesting inducer of differentiation leading to areduction of GSTP1 mRNA expression levels linked to a dropin GATA-1 binding as well as to a concentration-dependentdestabilization of the GSTP1 mRNA.

2. Materials and methods

2.1. Cell culture and reagents

The human chronic myelogenous leukemia K562 cell linewas purchased from the Deutsche Sammlung von Mikroor-ganismen und Zellkulturen (DSMZ) and cultured in RPMI1640 medium supplemented with 10% (v/v) heat-inactivatedfetal calf serum (FCS) and 1% (v/v) antibiotic-antimycotic(all Bio-Whittaker) in 5% (w/w) CO2 humidified atmo-

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sphere at 37 ◦C. Induction of K562 differentiation was car-ried out at exponential growth phase by supplementingcultures with 20 nM aclarubicin (Sigma), 40 nM doxoru-bicin (Sigma), 30 �M hemin (MP-Biomedicals), 1 mM or2 mM butyric acid (Merck). Cell growth and viability wereassessed by trypan blue dye exclusion test. The percent-age of cell growth inhibition was calculated as follows:[(Cn − C0) − (Tn − T0)] × 100/(Cn − C0) where Cn, C0, Tn

and T0 represent the number of control (C) or treated (T)cells/ml at days 0 and n, respectively.

2.2. Assessment of differentiation

Erythroid differentiation was scored by the benzidinestaining method for the determination of the percentageof hemoglobin-positive cells upon induction as previouslydescribed [16]. Briefly, 3.105 cells in 100 �L of NaCl 0.9%were incubated for 20 min at room temperature in the darkwith 50 �L of benzidine solution (20 �L of H2O2 30% in1 ml of benzidine 0.2% in acetic acid 0.5 M). Cellspresenting blue coloration were scored independently two

ig. 1. Northern blot analysis of GSTP1 mRNA expression in differentiating562 cells. K562 cells were grown in medium alone or treated for 48 hith various inducers of differentiation as indicated. (A) Total mRNA from

ontrol and treated K562 cells were extracted and expression was analyzed byorthern blot assays probed sequentially with human GSTP1 and ubiquitinDNAs. Ethidium bromide-stained gel is shown as a loading control. (B)orthern blots were quantified by phosphorimaging and data were expressed

s a ratio between GSTP1 and ubiquitin hybridization signals. Results areresented as the mean of a fold change relative to the control. Error barsepresent the standard deviation from three independent experiments. (*)ndicate p < 0.01 compared to control using paired t-test.

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ig. 2. Time course of differentiation of butyric acid-treated K562 cells.562 cells were grown in medium alone or treated for various times withutyric acid at 1 mM (A) or 2 mM (B). Erythroid and megakaryocyticifferentiation of cells were detected for each time point indicated by deter-ination of the percentage of benzidine-positive cells and CD61 surfacearker expression, respectively. Effects of butyric acid-induced differentia-

ion in vitro on growth inhibition and cellular viability are indicated. Theseata are the mean (±S.D.) of three independent experiments.

M. Schnekenburger et al. / Leukemia Research 30 (2006) 561–568 563

times by two investigators. Determination of megakaryocyticdifferentiation was realized by using a FACSCalibur (Becton-Dickinson) flow cytometer with a mouse monoclonalantibody fluorescein isothiocyanate (FITC)-conjugatedanti-CD61 (Dako) or isotype-matched immunoglobulin(IgG1-FITC). Background was measured using FITCconjugated preimmune isotype antibody and detection limitwas set accordingly. The mean of fluorescence intensityand the percentage of positive cells were analyzed by usingCellquest software (Becton-Dickinson).

2.3. RNA extraction

Total RNA was extracted from treated or control K562cells using a Nucleospin RNA II kit (Macherey-Nagel)according to the manufacturer’s protocol. To study mRNAstability, cells were treated for 48 h with butyric acid at 1 mMor 2 mM and total RNA was extracted after incubation for

0, 8, 24, 32 and 48 h with actinomycin D (5 �g/ml) (MP-Biomedicals).

2.4. Analysis of GSTP1 gene expression by northern blotanalysis

Northern blot analysis was performed by using a 720-bp fragment of the human GSTP1 cDNA (American TypeCulture Collection, ATCC, USA) as a probe and a fragmentof human ubiquitin cDNA (Becton-Dickinson) as a control.Probes were labeled with [�-32P] dCTP using a randomprimer labeling system (RediprimeTMII, Amersham Pharma-cia Biotech). Ethidium bromide-stained gel was used as aloading control. Results were acquired on a Cyclone phos-phorimager (Perkin-Elmer) and quantified by Kodak 1D soft-ware (Perkin-Elmer). Quantification of blots was expressedas a ratio between GSTP1 and ubiquitin hybridizationsignals.

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ig. 3. Effect of butyric acid-induced erythroid (1 mM) and megakaryocytic differelone or treated for 4 days with 1 mM or 2 mM butyric acid. (A) Changes of GSTas extracted and its expression was analyzed by northern blot experiments probed

ontrol. (B) Northern blots were quantified by phosphorimaging and data were exprere presented as the mean of a fold change relative to the control. Error bars represndicate p < 0.05 and p < 0.01 compared to control, respectively. (#) Correspondinreated with 1 and 2 mM butyric acid, using paired t-test.

ntiation (2 mM) on GSTP1 expression. K562 cells were grown in mediumP1 mRNA expression. Total mRNA from control and treated K562 cellssequentially with human GSTP1 cDNA and ubiquitin cDNA as a loadingssed as a ratio between GSTP1 and ubiquitin hybridization signals. Resultsent the standard deviation from three independent experiments. (* and **)

g to p < 0.05 indicate a significant difference between the values for cells

564 M. Schnekenburger et al. / Leukemia Research 30 (2006) 561–568

2.5. Analysis of GATA-1 gene expression by PCR

cDNA synthesis was realized using the SuperScriptTM

First-strand Synthesis System for RT-PCR (Invitrogen). Fivemicrograms of RNA were submitted to reverse transcrip-tion (RT) using oligo(dT) primers. The resulting RT productswere used as templates for PCR amplification according tothe manufacturer’s instructions using the Platinium® TaqDNA Polymerase High Fidelity and gene-specific primers ofGATA-1 (sense: 5′-TCAATTCAGCAGCCTATTCC-3′; anti-sense: 5′-TTCGAGTCTGAATACCATCC-3′). The amountof cDNA synthesized was evaluated by amplification ofthe S14 gene (sense: 5′-GGCAGACGAGATGAATCCTC-3′; antisense: 5′-CAGGTCCAGGGGTCTTGGTCC-3′) as astandard. Amplification was performed at 94 ◦C for 2 min,

60 ◦C for 1 min and 68 ◦C for 2 min for 25 cycles. PCR prod-ucts were separated on a 2% agarose gel.

2.6. Electrophoretic mobility shift assay (EMSA)

After treatment, K562 cells were harvested and nuclearextracts were prepared according to Muller et al. [17].Protein content was determined for each sample usingthe Bradford assay (Bio-Rad). The oligonucleotide probe(Eurogentec) used in EMSA harbors the GATA-1 sitelocated within the GSTP1 promoter (nucleotides −1219to −1200 relative to the transcriptional start site, 5′-AGCTAAGGGATACTGGGCTT-3′). Gel shift analysis wasrealized as previously described [8]. For supershift experi-ments, nuclear proteins were preincubated with anti-GATA-1

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ig. 4. Effect of butyric acid-induced erythroid (1 mM) and megakaryocytic (2 mM562 cells were grown in medium alone or treated for 4 days with 1 mM or 2 mM

ncubated with 32P-labeled GATA probe. (B) Nuclear extracts from control K5622P-labeled GATA probe. (C) Total RNA from control and treated K562 cells was exT products were used as templates for PCR amplification (25 cycles) using gene-sp

he amount of cDNA used in each reaction. Amplification products were separatedD) PCR results were expressed as a ratio between GATA-1 and S14 amplificationnd values are reported as the mean (±S.D.) of a fold change relative to the control#) Corresponding to p < 0.05 indicate a significant difference between the values fo

) differentiation on GATA-1 binding activity and mRNA expression. (A)butyric acid. Nuclear extracts from control and treated K562 cells were

cells were incubated with or without anti GATA-1 antibody and then withtracted and submitted to reverse transcriptase for each time point indicated.ecific sense and antisense primers for GATA-1. S14 was amplified to controlby electrophoresis onto a 2% agarose gel stained with ethidium bromide.

signals. Results shown are representative of three independent experiments. (* and **) indicate p < 0.05 and p < 0.01 compared to control, respectively.r cells treated with 1 and 2 mM butyric acid, using paired t-test.

M. Schnekenburger et al. / Leukemia Research 30 (2006) 561–568 565

antibody (active motif). Results were acquired on a Cyclonephosphorimager (Perkin-Elmer).

2.7. Statistics

Results were expressed as the mean ± S.D. of n exper-iments. Significance was assessed with two tailed, pairedStudent’s t-tests. p-values below 0.05 were considered as sta-tistically significant.

3. Results

3.1. GSTP1 expression during butyric acid-induceddifferentiation

As GSTP1 is differentially regulated during erythroid andmegakaryocytic differentiation [12,15], we investigated herethe effect of 1 mM and 2 mM butyric acid as erythroid andmegakaryocytic lineage inducer of K562 cells. We first com-pared the effect of butyric acid and known chemical inducersof differentiation on GSTP1 mRNA expression levels. Ourresults show that the anthracyclins doxorubicin and aclaru-bicin as well as hemin, all inducers of the erythroid lineage,enhance the GSTP1 mRNA expression (Fig. 1A). Interest-icwetntm

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dependent decrease of GSTP1 mRNA expression that reaches30% (p < 0.01) and 54% (p < 0.01) after 4 days of inductionby 1 and 2 mM butyric acid, respectively (Fig. 3B).

3.2. GATA-1 DNA binding activity in butyricacid-induced differentiation of K562 cells

In order to determine the molecular mechanisms whichexplain butyric acid-induced GSTP1 mRNA decrease, weanalyzed GSTP1 promoter/GATA-1 binding activity. Weused nuclear factors from K562 cells treated with or with-out butyric acid (1 mM or 2 mM) for 1–4 days. Results showa time-dependent decrease of GATA-1 binding activity after1–4 days of butyric acid treatments (Fig. 4A). However, thedecrease of GATA-1 binding activity with 1 mM butyric acidis less important and occurs later as compared to the con-centration of 2 mM. Supershift experiments confirmed the

Fig. 5. Effect of butyric acid-induced K562 erythroid or megakaryocyticdifferentiation on GSTP1 mRNA stability. K562 cells were grown in mediumalone or in the presence of 1 mM or 2 mM butyric acid for 48 h. (A) Total RNAwas isolated 0, 8, 24, 36 and 48 h after actinomycin D addition (5 �g/ml)and submitted to northern blot analysis probed with human GSTP1 cDNA.Ethidium bromide stained gel is shown as a loading control. (B) Northernblots were quantified by phosphorimaging and results were expressed as themeans of three independent experiments. S.D. are indicated.

ngly, butyric acid induces erythroid differentiation at a con-entration of 1 mM and decreases GSTP1 mRNA expressionhereas the same compound at 2 mM reduces GSTP1 mRNA

xpression stronger while differentiating K562 cells towardshe megakaryocytic lineage (Fig. 1B). These variations areot due to an unspecific regulation of RNA synthesis, sincereatments did not significantly modify the level of ubiquitin

RNA expression.Butyric acid-induced differentiation was studied at the cel-

ular level by measuring CD61 expression which is increasedn K562 cells in a time-dependent manner and reached 26.2%fter 4 days of treatment with butyric acid at 2 mM (Fig. 2).egacaryocytic differentiation was associated with marked

rowth inhibition (57.2%) and weak cellular cytotoxicity6.3%) (Fig. 2). On the other hand, butyric acid at 1 mMnduced expression of hemoglobin with 36.8% of benzidineositive cells after 4 days of treatment (Fig. 2). Erythroid dif-erentiation was associated with a growth inhibition of 43.1%nd weak cellular cytotoxicity (4.3%).

Northern blot analysis of RNA extracted from K562 cellsreated with or without butyric acid (1 mM or 2 mM) for 1–4ays is shown in Fig. 3A. Results display a significant time-

able 1STP1 mRNA half-life

Half-life of GSTP1 mRNA (h)

ontrol 43.8 ± 1.8utyric acid 1 mM 36.2 ± 2.1utyric acid 2 mM 12.6 ± 1.1**

ata are mean ± S.D. of three independent experiments.** p < 0.01 compared to control.

566 M. Schnekenburger et al. / Leukemia Research 30 (2006) 561–568

presence of GATA-1 within the retarded complex (Fig. 4B).Results were ascertained by RT-PCR analysis showing a time-dependent decrease of GATA-1 mRNA expression (Fig. 4Cand D) after butyric acid-induced differentiation of K562cells towards erythroid and megakaryocytic pathways.

3.3. Half-life of the GSTP1 mRNA in butyric acidtreated K562 cells

As the decrease of GSTP1 mRNA synthesis appearedstronger in 2 mM versus 1 mM butyric acid-treated K562cells (Fig. 3A and B), we assessed whether butyric acidconcentration-dependently modified the stability of theGSTP1 mRNA. In order to assess the half-life of the GSTP1mRNA during differentiation, treated and untreated K562cells were co-treated with actinomycin D. mRNA expres-sion was analyzed by northern blot (Fig. 5A) and resultswere quantified (Fig. 5B). Half-lives of the GSTP1 mRNAare 43.8 h, 36.2 h and 12.6 h for untreated cells, 1 and 2 mMbutyric acid treated cells, respectively (Table 1).

4. Discussion and conclusion

This report compares the effect of different concentra-tions of butyric acid on the K562 cell line which has thecodtoowmbei

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[23]. Caco-2 cells, which are closely related to normal humanenterocytes, if differentiated by butyric acid, express higherGSTP1 levels, contributing to enhanced detoxification capac-ity in the gut [24].

The reduction of GSTP1 expression described in thisreport can nevertheless be explained by the cell signalingpathways by which butyric acid specifically induces differ-entiation of K562 cells: ERK and JNK are dephosphorylatedand thus inhibited whereas p38 kinases are phosphorylatedfollowing butyric acid exposure [20]. According to the lit-erature, neither ERK nor p38 were described to be involvedin GSTP1 expression, but we previously published that JNKinhibitors including curcumin down-regulate GSTP1 mRNAexpression as well as the essential AP-1 binding [6]. More-over, chemical inducers of differentiation, like doxorubicin,induce GSTP1 mRNA synthesis [12] while activating JNKand leading to increased binding of AP-1 to a site locatedat −73 relative to the GSTP1 transcriptional start site [6].Therefore, inhibition of GSTP1 via JNK by butyric acid is inaccordance with previously published data and contributes toexplain the decrease of GSTP1 mRNA synthesis. Addition-ally, butyric acid markedly suppresses tumor necrosis factor(TNF) �-induced activation of NF-�B [25]. We previouslypublished that NF-�B homo- and heterodimers positivelyregulate GSTP1 expression as well as the TNF� → TNFreceptor 1 → TNF receptor associated factor (TRAF) → NF-�SiGGtitsees

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apacity to differentiate along the erythroid or megakary-cytic lineage [18]. Compared to other chemical inducers ofifferentiation exhibiting lineage specificity, butyric acid hashe property to induce differentiation differently, dependingn the concentration used: low butyric acid concentrationsf 0.5–1 mM activate the erythroid differentiation processhereas concentrations above 2 mM differentiate towards theegakaryocytic lineage. Our results are confirmed by a num-

er of individual reports where butyric acid is used as anrythroid differentiating agent [19,20] or as a megakaryocyticnducer [21].

In order to elucidate molecular mechanisms relatedo chemoresistance during chemically induced differentia-ion of leukemia cells, we previously showed that 12-O-etradecanoyl phorbol 13-acetate (TPA), a megakaryocyticathway inducer, leads to decreased GSTP1 expression atoth mRNA and protein levels whereas induction of ery-hroid differentiation by aclarubicin and doxorubicin gen-rates increased GSTP1 gene expression [12] as does theanonical erythroid inducer hemin [15]. Here, our resultshow a decrease in GSTP1 mRNA expression after butyriccid treatment and this independently of the differentiationineage.

These results were unexpected as earlier reports docu-ented an increase in GSTP1 expression after butyric acid-

nduced differentiation of a rat liver non parenchymal epithe-ial cell line [22]. Similarly, in HT-29 colon cancer cells,ifferentiation by butyric acid activates the extracellular reg-lated kinase (ERK) 1/2 cell signaling pathway leading toncreased GSTP1 expression and enhanced chemoprotection

B inducing kinase (NIK) → I�B kinase (IKK) pathway [9].uppression of the TNF� signaling pathway by butyric acid

s therefore likely to contribute to the down-regulation ofSTP1 mRNA synthesis in human leukemia cells. Finally,ATA-1 transcription factor was previously described to con-

ribute to GSTP1 up-regulation by chemical inducers includ-ng hemin and aclarubicin [12,15]. A reduced binding ontohe GSTP1 GATA-1 site located at −1208 relative to the tran-criptional initiation site contributes to a reduction of GSTP1xpression. Published data confirm these results as Busfieldt al. [26] previously reported a depletion of GATA-1 tran-cripts in butyric acid-treated J2E erythroid cells.

Our results show furthermore, that higher concentrationsf butyric acid additionally affect GSTP1 mRNA stabil-ty leading to a strongly reduced half-life. Concentration-ependent effects of butyric acid on RNA stability and cellignaling pathways involved in the regulation of GSTP1xpression remain to be elucidated but our results are con-rmed by a number of reports where butyric acid reduces

he half-life of selected mRNA including decay-acceleratingactor (DAF) mRNA expressed in colon cancer cells [27],he cholesterol acyltransferase mRNA expressed in HepG2ells [28] and the surfactant protein (SP) A, B and C mRNAxpressed in fetal rat lung [29]. Decreased mRNA stabil-ty explains the even stronger reduction of GSTP1 transcriptevel after treatment at 2 mM butyric acid versus 1 mM.

In future, it will be interesting to investigate the effectf chemical derivatives of butyrate on GSTP1 expressions the clinical use of butyric acid salt derivatives is limitedy an extremely short half-life due to a rapid metabolism

M. Schnekenburger et al. / Leukemia Research 30 (2006) 561–568 567

[30]. The long-term goal of this research aims to determinethe molecular mechanisms of leukemia cell resistance toanticancer drugs during differentiation and to identify com-pounds, which do not trigger resistance mechanisms. Weconclude that butyric acid-induced megakaryocytic differen-tiation prevents adverse effects like GSTP1 up-regulation thatcould be observed with hemin and anthracyclines during ery-throid differentiation. Nevertheless, the rate of differentiationwith butyric acid remains low compared to hemin and it isnoteworthy to mention that butyric acid does not generate ahomogenous population of differentiated cells at a given con-centration, which should not lower its therapeutic potential.

Present data demonstrate that GSTP1 expression dependsnot only on the differentiation pathway, but essentially on thenature and concentration of the chemical inducer used as wellas the signal transduction pathways activated or inhibited. Atthe molecular level, butyric acid decreases the expressionand the binding of the transcription factor GATA-1 leadingto a reduction of transcriptional regulatory steps. Addition-ally, butyric acid acts at the post-transcriptional level. Wesuggest that butyric acid could destabilize GSTP1 mRNA byactivating specific endonucleases or by inhibiting the interac-tion with stabilizing mRNA binding proteins. In conclusion,butyric acid appears to be a promising chemical inducer ofdifferentiation as it prevents adverse effects like GSTP1 up-regulation, which would eventually lead to chemoresistancem

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[5] Adler V, Yin Z, Fuchs SY, Benezra M, Rosario L, TewKD, et al. Regulation of JNK signaling by GSTp. EMBO J1999;18(5):1321–34.

[6] Duvoix A, Morceau F, Delhalle S, Schmitz M, Schnekenburger M,Galteau MM, et al. Induction of apoptosis by curcumin: media-tion by glutathione S-transferase P1-1 inhibition. Biochem Pharmacol2003;66(8):1475–83.

[7] Moffat GJ, McLaren AW, Wolf CR. Sp1-mediated transcriptionalactivation of the human Pi class glutathione S-transferase promoter.J Biol Chem 1996;271(2):1054–60.

[8] Borde-Chiche P, Diederich M, Morceau F, Wellman M, Dicato M.Phorbol ester responsiveness of the glutathione S-transferase P1gene promoter involves an inducible c-jun binding in human K562leukemia cells. Leuk Res 2001;25(3):241–7.

[9] Morceau F, Duvoix A, Delhalle S, Schnekenburger M, Dicato M,Diederich M. Regulation of glutathione S-transferase P1-1 geneexpression by NF-kappaB in tumor necrosis factor alpha-treatedK562 leukemia cells. Biochem Pharmacol 2004;67(7):1227–38.

[10] Millar DS, Paul CL, Molloy PL, Clark SJ. A distinct sequence(ATAAA)n separates methylated and unmethylated domains at the5′-end of the GSTP1 CpG island. J Biol Chem 2000;275(32):24893–9.

[11] Borde-Chiche P, Diederich M, Morceau F, Puga A, Wellman M,Dicato M. Regulation of transcription of the glutathione S-transferaseP1 gene by methylation of the minimal promoter in human leukemiacells. Biochem Pharmacol 2001;61(5):605–12.

[12] Schnekenburger M, Morceau F, Duvoix A, Delhalle S, Trentesaux C,Dicato M, et al. Expression of glutathione S-transferase P1-1 in dif-ferentiating K562: role of GATA-1. Biochem Biophys Res Commun2003;311(4):815–21.

[13] Waxman S. Differentiation therapy in acute myelogenous leukemia

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echanisms in differentiated leukemia cells.

cknowledgements

This work was supported by “Fondation de Rechercheancer et Sang” and “Recherches Scientifiques Luxem-ourg” association. MS was supported by a fellowship fromhe Government of Luxembourg, EH and RB are supported byelevie grants. RB is a “Legs Kanning” recipient. The authors

hank Myriam Weinacht (Hematology Laboratory, Centreospitalier du Luxembourg). “Een Haerz fir kriibskrank Kan-er” association is also thanked for generous support.

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