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Pathophysiology 16 (2009) 297–303

Hypoxia-HIF-1�-C/EBP�/Runx1 signaling in leukemic celldifferentiation

Jing Zhang, Guo-Qiang Chen ∗Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education,and Institute of Health Science, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences-Shanghai

Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China

bstract

Acute myeloid leukemia (AML), a class of prevalent hematopoietic malignancies, is caused by the acquisition of gene mutations that confereregulated proliferation, impaired differentiation and a survival advantage of hematopoietic progenitors. More recently, we reported thatobalt chloride (CoCl2)/iron chelator desferrioxamine (DFO)-mimicked hypoxia or moderate hypoxia (2% and 3% O2) can directly triggerifferentiation of many subtypes of AML cells. Also, intermittent hypoxia significantly prolongs the survival of the transplanted leukemicice with differentiation induction of leukemic cells. Additionally, these hypoxia-simulating agents selectively stimulate differentiation in

cute promyelocytic leukemic cells induced by arsenic trioxide, an effective second-line drug for this unique type of leukemia. Based onhis interesting evidence in vitro and in vivo, the ongoing investigations showed the role of hypoxia-inducible factor-1alpha (HIF-1�) proteinhrough its non-transcriptional activity in myeloid cell differentiation, as evidenced by chemical interference, the conditional HIF-1� induction,he specific short hairpin RNAs (shRNAs) against HIF-1� and HIF-1�, an essential partner for transcription activity of HIF-1. Furthermore,IF-1� and two hematopoietic transcription factors CCAAT/enhancer binding protein alpha (C/EBP�) and Runx1/AML1 interact directlyith each other. Such interactions increase the transcriptional activities of C/EBP� and Runx1/AML1, while C/EBP� competes with HIF-1�

or direct binding to HIF-1� protein, and significantly inhibits the DNA-binding ability of HIF-1. As a protein is rapidly responsive to all-transetinoic acid (ATRA), a classical clinical differentiation-inducing drug for AML, HIF-1� also plays a role in ATRA-induced differentiationf leukemic cells.

2009 Elsevier Ireland Ltd. All rights reserved.

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eywords: Leukemia; Hypoxia; Hypoxia-inducible factor-1� (HIF-1�); Di

. Introduction

Hematopoiesis is a delicately regulated process, duringhich hematopoietic stem cells (HSC) in the bone marrowive rise to committed progenitor cells, which in turn dif-erentiate into various functional mature blood cells. Acuteyeloid leukemia (AML), a class of prevalent hematopoi-

tic malignancies, is characterized by the block of normal

ifferentiation pathway at a stage where the cells continueo proliferate and do not move on to terminal differen-iation, mainly due to specific gene rearrangements and

∗ Corresponding author at: Department of Pathophysiology, Shanghaiiao-Tong University School of Medicine, No. 280, Chong-Qing South Rd,hanghai 200025, China. Tel.: +86 21 64154900; fax: +86 21 64154900.

E-mail address: chengq@shsmu.edu.cn (G.-Q. Chen).

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928-4680/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.pathophys.2009.02.005

ation

utations targeting a variety of transcription factors and sig-aling molecules. For example, cells of acute promyelocyticeukemia (APL), a unique subtype of AML, are blocked athe stage of the promyelocytes due to the expression of chro-

osome translocation t(15;17), yielding the specific fusionrotein PML-RAR� (for promyelocytic leukemia–retinoiccid receptor �), which interacts with transcriptional co-epressors such as the nuclear co-repressor (N-CoR)-histoneeacetylase complex and exerts dominant-negative effects onunctions of the wild type PML and RAR� proteins [1–3].

Based on the tacit assumption that AML cells exhibiteversible defects in their differentiation process, a poten-

ially less cytotoxic therapeutic strategy for cancer knowns ‘differentiation therapy’ is being developed. It employsrugs to induce cancer cells to undergo terminal differenti-tion, thus preventing their further proliferation. As early as

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he 1960–1970s, control of normal cell differentiation andhe phenotypic reversion of malignancy in myeloid leukemiaere reported and differentiation of myeloid leukemia cellsas put forward as new possibilities for therapy. Until 1980s,

his differentiation therapy comes true from hypothesis forhe first time, concomitant with the successful differentiationnduction of all-trans retinoic acid (ATRA) in APL patientsy Chinese doctors [4,5]. From then on, ATRA has alwayseen the first-line drug for the APL patient in clinic and theechanisms by which it induces differentiation with growth

rrest as well as apoptosis have been provoking extensivetudies in the past decades [6–8]. Despite earlier optimism,he concept of “differentiation therapy” has not yet beensefully extended to other subtypes of leukemia. Thus, itecomes even more urgent to explore practicable differen-iation induction therapy in other leukemia subtypes besidesPL.Along with the world-wide use of ATRA in clinic, at the

ame time, some problems also emerge such as drug resis-ance in APL patients. To this end, arsenic trioxide (As2O3,TO, commercial name TrisenoxTM) is employed by Chi-ese doctors to treat relapsed/refractory APL patients [9], andhe mechanisms through which it works have been gainingtremendous amount of insight. A series of studies showed

hat clinical concentration of As2O3 (1–2 �mol/L) triggerspoptosis not only in APL with the down-regulation of bcl-2ene expression and the modulation of PML-RAR� pro-ein [9,10], but also in an impressive array of other cancerells, in which various kinds of mechanisms have been docu-ented [11–13]. However, the clinical effectiveness of As2O3

ppears to be restricted to APL, which suggests that somether effects besides the apoptosis induction contribute to thefficacy of As2O3 in clinic. Further investigation indicatedhat after 2–3 weeks of continuous in vivo As2O3 treatment,

yelocyte-like cells and degenerative cells in bone marrownd peripheral blood increase when leukemic promyelo-ytes decrease, which is probably as a result of partial inivo differentiation [14,15]. This incomplete differentiationffect of As2O3 was confirmed by other clinical trials and inransplanted APL mice. On the other hand, in vitro, As2O3as dose-dependent dual effects on APL cell lines: induc-ng apoptosis preferentially at relatively high concentrations0.5–2 �mol/L) and inducing partial differentiation at lowoncentrations (0.1–0.5 �mol/L) [14,16], the latter, however,s not so obvious as that of As2O3 in vivo. Based on thesebservations, it is reasonable to assume that the partial differ-ntiation induction of As2O3 also plays a role in its impressivelinical efficacy, and some factors in the bone marrow (BM)icroenvironments could modulate the in vivo activity ofs2O3.When we set to figure out the difference existing in vivo

nd in vitro, oxygen concentration comes into our consid-

ration. As it is well known that in vitro leukemic cells areultured in ambient O2 concentration around 21%, while inivo, tissues are exposed physiologically to lower O2 tension,anging from 16% in pulmonary alveoli and artery blood to

iU

iology 16 (2009) 297–303

ess than 5% in distal tissues. The O2 tension of bone marrown leukemic patients might be much lower due to the rapid anderrible proliferation of the leukemic cells [17]. Furthermore,ncreasing vascular endothelial growth factor (VEGF) levels,hich is associated with angiogenesis, growth, dissemina-

ion, metastasis and poor outcome in solid tumors, is reportedo be related to shorter survival as the independent predictor ofhe outcome in diagnosed AML [18,19]. According to these,e begin to broaden our view about the regulation of oxygenetabolism in the organism and to investigate whether oxy-

en concentration influences As2O3-induced leukemic cellifferentiation.

Cellular and systemic O2 homeostasis, consisting of2 delivery and consumption, is essential for the sur-ival of higher eukaryotes. As a consequence, delicateechanisms are utilized to regulate O2 delivery, amonghich, hypoxia-inducible factor-1 (HIF-1) plays an essen-

ial role [20]. HIF-1 is a heterodimeric transcriptionalactor composed of the constitutively expressed HIF-1�also named aryl-hydrocarbon receptor nuclear transporter,RNT) subunit and the highly regulated HIF-1� subunit

21]. Under normoxia, the HIF-1� subunit is hydroxylatedy oxygen-activated HIF prolyl hydroxylases (PHDs) in thexygen-dependent degradation domain (ODD) and rapidlyegraded by the von Hippel-Lindau (VHL) tumor suppres-or protein-mediated ubiquitin–proteasome pathway. On theontrary, under hypoxic or hypoxic-mimic condition, theIF-1� subunit accumulates due to the significantly reduced

nzymatic activities of PHDs. The stabilized HIF-1� isranslocated to the nucleus, heterodimerized with HIF-1� andctivates an impressive array of target genes through bindingo the hypoxia-responsive element (HRE) in the cis-actingequences so as to overcome hypoxic stress.

Since first identified as a nuclear factor induced underypoxia, HIF-1 has been attracting a tremendous amount ofnsight because of its involvement in fundamental biologicalrocesses, including but not limited to tumor metabolism,ngiogenesis, metastasis, inflammation, and its potentialole as a therapeutic target [22]. Although the roles ofypoxia in solid tumors have been widely studied, few stud-es are reported regarding the possible effects of hypoxia oneukemic cells. At the same time, the in vitro and in vivobservations mentioned above drive us to extend research ofypoxia to leukemia, which, surprisingly, uncover the two-dged sword effect of hypoxia/HIF-1�. Herein, this reviewill focus on the recent research of the differentiation induc-

ion effects of hypoxia/HIF-1� and the related pathways ineukemic cells.

. Hypoxia induces AML cell differentiation: in vitrond in vivo evidence

Hypoxia-mediated modulation of hematopoietic progen-tor behavior by oxygen tension has been reported [23,24].nexpectedly, on the way to unravel the possible effect of

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J. Zhang, G.-Q. Chen / Pat

s2O3 on APL cells under hypoxia, we found that mildypoxia (2–3% O2) and non-toxic concentrations of hypoxia-imetic agents such as cobalt chloride (CoCl2, 12.5–50 �M)

nd desferrioxamine (DFO, 5–20 �M) trigger differentia-ion in APL cell lines NB4, as assessed by morphologicalriteria and differentiation-associated antigens [25,26]. How-ver, unlike ATRA/As2O3 which could cleave/degrade thepecific fusion proteins PML-RAR� resulting in differentia-ion of APL cells, treatment with CoCl2 does not modulateML-RAR�, suggesting that the differentiation inductionffect of hypoxia and hypoxia-mimetic agents might existn other leukemic cells. Indeed, the experiments indicatedhat hypoxia and hypoxia-mimetic agents could also differ-ntiate other subtypes of AML cells such as U937 in a similaranner. Of importance, CoCl2 induces primary leukemic

ells from some AML patients to undergo differentiation,hich greatly supports the results observed in the cell lines.urthermore, non-toxic concentrations of CoCl2 and DFOach potentiate the growth-arresting effect and differentia-ion induction of low-dose As2O3 in APL cell line NB4 cells27]. In parallel, the differentiation induction by these treat-ents is accompanied by the increase of HIF-1� protein and

ts DNA-binding activity, which suggests that HIF-1 may playn active role in the differentiation of AML cells. Of note,igher concentrations of CoCl2 and DFO trigger leukemicells to undergo apoptosis through mitochondrial-dependentnd HIF-1�-independent mechanisms [28].

Based on these in vitro findings, we investigated the inivo effects of hypoxia on AML mice [29]. For this set ofxperiments, leukemic blasts from PML-RAR� transgenicice were injected into the tail vein of 7–10-week-old syn-

enic FVB-NICO (FVB/N) mice after sublethal irradiationotaling 4.5 Gy and the leukemic mice were then housed in

hypoxic chamber equivalent to an altitude of 6000 m for8 h each consecutive day. Results showed that the survivalime of leukemic mice is significantly prolonged with thencubation of intermittent hypoxia while normal mice keeplive under intermittent hypoxia for 60 days without any evi-ence for hypoxic damage. More importantly, intermittentypoxia reduces leukemic cell infiltration by inhibiting theroliferation without apoptosis induction and inducing dif-erentiation of leukemic cells in the peripheral blood, bonearrow and peripheral tissues such as spleen and liver, with

harp decrease in monomorphic, immature, promyelocyte-ike cells and emergence of a percentage of mainly ring-likeerminally differentiated cells.

Moreover, there are several other reports to substanti-te our observations. For example, differentiation of normaluman CD34+ progenitors cells along all the erythrocytic,egakaryocytic and granulocytic pathways is accelerated

n low versus high oxygen tension conditions and murine2Dcl3 myeloid cells also show accelerated granulocytic dif-

erentiation in low oxygen tension in response to granulocyteolony-stimulating factor, which is inhibited by the expres-ion of TEL-ARNT fusion protein [30]. Under low oxygenension, bone marrow stromal cells (MSCs) exhibit growth-

bma

iology 16 (2009) 297–303 299

rrest and differentiation behavior changes. When culturedn adipogenic medium, there is a 5–6-fold increase in theumber of lipid droplets under hypoxic conditions comparedith that in normoxic culture [31]. Taken together, these find-

ngs underscore the differentiation induction of hypoxia orypoxia-mimic agents in AML cells, which is possibly of sig-ificance to explore clinical potentials and novel target-basedrugs for differentiation therapy of leukemia.

. The role of HIF-1� in AML cell differentation

There is a consistence between the differentiation induc-ion induced by hypoxia/hypoxia-mimic agents and theccumulation of HIF-1� protein under these treatments,hich is the master transcriptional factor in cellular response

o hypoxia [25,26]. In contrast, while nitric oxide (NO)onor 3-morpholinosydnonimine(SIN-1) abrogates CoCl2-rigerred HIF-1� accumulation and the HRE-binding activity,t significantly antagonizes CoCl2-induced differentiation ofML cells. Meanwhile, metavanadate antagonizes DFO-

nduced growth-arrest and differentiation with the inhibitionf HIF-1� protein stabilization in leukemic cells [25,32]. Inddition, Zhong et al. reported that nuclear expression of HIF-� protein is heterogeneous in human malignant cells underormoxic conditions [33]. All of these suggest a potentialole of HIF-1� protein in leukemic cell differentiation.

To confirm the role of HIF-1� in AML cell differen-iation, HIF-1�-expressing vector and empty vector wereransfected into U937 cells, the results indicated that withecreased proliferative ability, some maturating cells can bebserved under microscope and CD11b+ cells% is signifi-antly higher in HIF-1�-transfected cells than those in emptyector-transfected cells [26]. To provide more direct evi-ence, we generate myeloid leukemic U937T transformants,n which HIF-1� is tightly induced by tetracycline withdrawal34]. The results showed 3–4 days after the induction of HIF-�, the proliferation is substantially slowed down and the1-S phase transition is blocked. More importantly, HIF-1�

nduction directly triggers myeloid leukemic cells to undergoifferentiation, as determined by differentiation-related mor-hological changes, increased CD11/NBT reduction, andxpressions of differentiation signatures such as neutrophilytosolic factor-1 (NCF1), interleukin-1 receptor antagonistIL1RN) and secreted phosphoprotein 1 (SPP1). Further-ore, two pairs of shRNAs specifically against HIF-1�RNA were respectively transfected into the parental U937

ells with negative shRNA as a control [34]. Under theffective interference of HIF-1� protein, CoCl2 and 2% O2-nduced differentiation of U937 cells is remarkably inhibited,ndicating the direct contribution of HIF-1� to hypoxia andoCl2-induced differentiation of myeloid leukemic cells.

On the other hand, it is reported that HIF-1� protein coulde accumulated in differentiating U937 macrophages in nor-oxia [35]. Hence, we next investigated whether or not it is

ssociated with the differentiation effect triggered by other

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00 J. Zhang, G.-Q. Chen / Pat

ifferentiation induction agents, choosing ATRA as a model36]. To our surprise, the results showed that ATRA rapidlyccumulates endogenous and inducible expressed or CoCl2-tabilized HIF-1� protein of leukemic cells under normoxicxygen tension. More importantly, suppression of HIF-1�xpression by specific shRNAs partially but significantlyepresses while conditional HIF-1� induction and HIF-1�-tabilizing CoCl2 treatment greatly enhances ATRA-inducedeukemic differentiation. These observations indicate that as arotein rapidly responsive to ATRA, HIF-1� does exert a rolen the differentiation induction of ATRA on AML cells whichrovided a new signal pathway with clinical potential forTRA-induced leukemic differentiation and further supports

he role of HIF-1� protein in leukemic cell differentiation. Inonsistence, Kim et al. also found that tiron, a widely usednti-oxidant and non-toxic chelator to alleviate an acute metalverload, causes HL-60 cells to induce differentiation-relatedlterations such as the increase in CD11b and CD14 expres-ion or chromatin condensation through increasing HIF-1�xpression [37].

. Interaction of HIF-1� with C/EBP� and Runx1

As we confirm the role of HIF-1� in leukemic cell dif-erentiation, in the next step we dedicate to unravel thenderlying mechanism. It is well known that HIF-1� proteinxerts its role in a series of biological processes through theranscriptional activation of its target genes, which requireshe heterodimerization with HIF-1�. Hence, we first deter-

ine whether HIF-1�-mediated differentiation involves theranscriptional activity of HIF-1� protein. To this end, twoffective pairs of shRNAs specially against HIF-1� mRNAere transfected into the parental U937 cell line. While the

uppression of HIF-1� protein by shRNAs damages the tran-criptional activity of HIF-1, which is evidenced by the inhi-ition of HIF-1 target genes, 50 �M CoCl2 or 2% O2 treat-ent for 6 days still induces differentiation of parental U937

ells to the same degree as that in the negative control trans-ection. Similarly, the silencing of HIF-1� protein does notmpinge on HIF-1� induction-triggered differentiation afteretracycline withdrawal. Thus, HIF-1� is not necessary forypoxia/HIF-1�-mediated myeloid cell differentiation [34].

On the other hand, our results showed that comparedith other leukemic cells, the differentiation induction effectf hypoxia/CoCl2/DFO is less obvious in the Kasummi-cell, which expresses a high level of the chromosomal

ranslocation t(8;21)-generated leukemogenic AML1-ETOusion protein [26]. As the fusion genes are reportedo exhibit the inhibitory effect on the differentiation ofML cells induced by a wide spectrum of differentiation-

nducing agents [38], we extend our study to investigate

he potential inhibitory effect of AML1-ETO expression onypoxia/CoCl2/DFO-induced leukemic differentiation. Forhis purpose, U937-A/E 9/14/18 cells were incubated withonasterone A, which induced AML1-ETO expression in

ae1o

iology 16 (2009) 297–303

he cells in a time-dependent manner [39–44]. While DFOt 5 �M and 10 �M induces U937-A/E 9/14/18 cells to dif-erentiate in the absence of ponasterone A, the additionf ponasterone A with inducible expression of AML1-TO significantly inhibits DFO-induced differentiation in

he cells, which indicates that AML1-ETO expression blocksFO-induced leukemic differentiation. Unexpectedly, the

reatment with ponasterone A increases HIF-1� mRNA time-ependently in U937-A/E 9/14/18 cells, but it fails to stabilizeIF-1� protein in the alone treatment alone while elevatingFO-accumulated HIF-1� protein [26].Combining the observations that the suppression of

IF-1� does not influence hypoxia/HIF-1�-mediated differ-ntiation and the contradictory discovery that AML1-ETOxpression blocks DFO-induced differentiation but potenti-tes HIF-1� protein expression in combination with DFO,e hypothesize that HIF-1� induces leukemic differentia-

ion by regulating some other differentiation-related factorsndependent of its transcriptional activity and inhibitedy the AML1-ETO fusion protein. As it is reported thatML1-ETO expression results in the inhibition of C/EBP�

xpression [45], in the next step we investigated the poten-ial role of C/EBP� in HIF-1�-induced differentiation. Tohis end, we examined whether HIF-1� interacts with andffects the transcriptional activity of C/EBP� [26,46]. Co-mmunoprecipitation indicated that not only ectopicallyut also endogenously expressed HIF-1� protein interactsith C/EBP�. GST pull-down assay proposed that therotein–protein interaction is direct and demonstrated that/EBP� competes with HIF-1� for direct binding to HIF-1�rotein. The functional analysis such as EMSA, chromatinmmunoprecipitation, luciferase assay and the expression ofIF-1-targeted genes showed that HIF-1� protein enhances

ranscriptional activity of C/EBP� while C/EBP� signifi-antly inhibits the DNA-binding ability and transcriptionalctivity of HIF-1�. The inhibition of HIF-1 function leadso the reduced expression of VEGF, inhibiting angiogenesiso form a hypoxic microenvironment and thus for leukemicell differentiation, which provides new evidence of the anti-umor potential of C/EBP�. Of note, Kim et al. found thatiron also increases HIF-1� protein and thus the activity of/EBP� [37].

Considering that C/EBP�, as one of the most importantranscriptional factors in hematopoiesis, is always absent in

ost solid tumors, we extend our study to other importantranscriptional factors particularly expressed in hematopoi-tic system, such as PU.1 and Runt-related protein 1 (Runx1,lso known as acute myeloid leukemia-1, AML1) whichre also reported to play a role in angiogenesis [47].he results showed that ectopically expressing Runx1 pro-

ein directly interacts with HIF-1� protein to a degree,s determined by LacO array, Co-IP and GST pull-down

ssay, and such an interaction can also be detected inndogenously expressed Runx1 and CoCl2-stabilized HIF-� protein. Runx1 inhibits transcription-dependent functionf HIF-1 as over-expression of Runx1 suppresses DNA-

J. Zhang, G.-Q. Chen / Pathophysiology 16 (2009) 297–303 301

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ig. 1. HIF-1� expression enhances the transcriptional activities of PU.1,ottom, together with pM-CSFR-Luc and pSV40-Renilla. Twenty-four hoelative M-CSFR-Luc activity was normalized by pSV40-Renilla, and foldhan 0.01 between the lanes indicated.

inding and transcriptional activity of HIF-1 protein withecreased expression of HIF-1-targeted genes, while abro-ation of Runx1 expression by specific shRNAs significantlyncreases transcriptional activity of HIF-1 protein; vice versa,IF-1� enhances DNA-binding ability and transcriptional

ctivity of Runx1 protein [48].It has been known that M-CSFR is also synergistically

egulated by C/EBP�, Runx1 and PU.1, which are cru-ial for normal myeloid hematopoiesis [49]. Hence, in theext phase of analysis, we attempted to understand whether

IF-1� impinges on transcriptional activity of PU.1 and

ynergy of these three transcriptional factors. As for this,U.1, C/EBP�, Runx1 and HIF-1�, alone or their combina-

ion, together with pM-CSFR-Luc plasmids, which contains

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ig. 2. A recapitulation to describe the mechanism of hypoxia-induced leukemicifferentiation with the accumulation of HIF-1� protein, which physically interactsownstream effectors of HIF-1� protein to finally induce leukemic differentiation. Arotein contributes to ATRA-induced myeloid leukemic differentiation. AML1rotein AML1-ETO suppresses the expression of C/EBP� as well as its transcriifferentiation is partially but significantly inhibited.

and Runx1. 293 cells were transfected with plasmids as indicated in ther, western blots were performed to confirm the effective transfection. Thereases against lane 1 were calculated. Symbol*represents p values of less

inding sites for PU.1, Runx1 and C/EBP�, were trans-ected into 293 cells effectively. The results showed thatlone transfection of HIF-1�, Runx1, C/EBP� and PU.1lanes 2, 3, 5 and 7, respectively, Fig. 1) increased tran-criptional activity of M-CSFR promoter. Also, combinationf HIF-1�/Runx1 (lane 4, Fig. 1), HIF-1�/C/EBP� (lane, Fig. 1), Runx1/C/EBP� (lane 9, Fig. 1), C/EBP�/PU.1lane 11, Fig. 1), Runx1/PU.1 (lane 13, Fig. 1), andunx1/C/EBP�/PU.1 (lane 15, Fig. 1) also synergistically

ncreased M-CSFR-Luc activity. More intriguingly, such a

ynergy could also be seen between HIF-1� and PU.1 (lane, Fig. 1). To our excitement, HIF-1� also remarkably potenti-ted the transcriptional synergy of Runx1/C/EBP� (lane 10,ig. 1), C/EBP�/PU.1 (lane 12, Fig. 1), Runx1/PU.1 (lane

differentiation. Hypoxia and hypoxia-mimic agents triggers leukemicwith and enhances the transcriptional activities of C/EBP� and Runx1, thelso, HIF-1� increased the transcriptional activity of PU.1. Of note, HIF-1�

-ETO increases the mRNA level of HIF-1� but fails to stabilize HIF-1�

ptional activity. Accordingly, in the presence of AML1-ETO, the leukemic

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4, Fig. 1), and Runx1/C/EBP�/PU.1 (lane 16, Fig. 1) on M-SFR-luc activity. These findings could also be confirmed inos-7 cells.

Finally, we investigated the potential contribution of/EBP�, Runx1 and PU.1 on hypoxia/HIF-1�-inducedifferentiation [34,36]. Thus, shRNAs specifically against/EBP�, Runx1 or PU.1 were transfected into leukemic937Tclone cells, together with a negative vector transfection

s a control (NC). These shRNAs effectively reduce expres-ions of their cognate targeted genes with no influence ofIF-1� protein induction after tetracycline withdrawal. More

ntriguingly, the reduced C/EBP�, Runx1 or PU.1 expressionignificantly inhibits HIF-1� induction-triggered differentia-ion in the U937Tclone cell line in the absence of tetracycline,s determined by CD11 expression. Such inhibition can alsoe observed in CoCl2-induced differentiation in the cell linen the presence of tetracycline. By the way, differentiationynergy of ATRA and HIF-1� induction can also be signif-cantly inhibited by the suppression of expressions of theseematopoietic transcriptional factors.

. Conclusion

Taken together, our results put forward with the notionhat hypoxia/hypoxia-mimic agents/HIF-1� can trigger dif-erentiation in myloid leukemic cells, in which, HIF-1�lays a critical role independent of its transcriptional activityhile C/EBP� and Runx1 act as the effectors downstream toIF-1� protein through direct interactions to enhance their

ranscriptional activities. Also, HIF-1� contributes to ATRA-nduced leukemic differentiation (Fig. 2). These discoveriesould shed new insights for understanding mechanismsnderlying leukemogenesis and designing new therapeuticsor differentiation induction of AML.

Albeit, some directions still deserve further investigation.ake the clinical potential of hypoxia/hypoxia-mimic agentsor example, it may be of great interest to set the leukemicatients on Qinghai-Tibet Plateau which has an altitude ofhousands of miles with lower oxygen tension and to observehether their survival time is longer than those under the

ame treatment in the places with lower altitude. In addition,ther chelators which might act as hypoxia-mimic agents totabilize HIF-1� protein deserve further exploration. Futher-ore, as we figure out C/EBP� and Runx1 as the effectors

ownstream to HIF-1� protein, some other factors might alsoink HIF-1� protein to leukemic differentiation due to the net-ork of the hematopoietic regulation. Actually, we exert the

omparative proteomic analysis of hypoxia/CoCl2-treatednd untreated human leukemic U937 cells by 2-DE coupledith MALDI–TOF/TOF MS/MS [50] and have identified

ome proteins regulated by hypoxia and CoCl2 treatment,or example, N-myc downstream regulated gene 1 (NDRG1),putative differentiation-related gene. These would provideew clues for uncovering mechanisms by which leukemicells respond to hypoxia.

[

iology 16 (2009) 297–303

cknowledgements

Due to word limitation, the paper mainly lists literatures inhe authors’ group. We apologize for losing many referencesrom other groups which have made excellent contributions tohe related work. We also appreciate all colleagues in Shang-ai Jiao-Tong University School of Medicine and Institutef Health Sciences, for their contributions to this work. Thisork was supported in part by National Key Program (973)

or Basic Research of China, National Natural Science Foun-ation of China, and Grants from Science and Technologyommittee of Shanghai.

eferences

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