Heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor expression by...

transcript

Heparin-Binding Epidermal Growth Factor–Like Growth Factor/DiphtheriaToxin Receptor Expression by Acute Myeloid Leukemia Cells

By Fabrizio Vinante, Antonella Rigo, Emanuele Papini, Marco A. Cassatella, and Giovanni Pizzolo

Heparin-binding epidermal growth factor–like growth factor

(HB-EGF) is an EGF family member expressed by numerous

cell types that binds to EGF receptor 1 (HER-1) or 4 (HER-4)

inducing mitogenic and/or chemotactic activities. Membrane-

bound HB-EGF retains growth activity and adhesion capabili-

ties and the unique property of being the receptor for

diphtheria toxin (DT). The interest in studying HB-EGF in

acute leukemia stems from these mitogenic, chemotactic,

and receptor functions. We analyzed the expression of

HB-EGF in L428, Raji, Jurkat, Karpas 299, L540, 2C8, HL-60,

U937, THP-1, ML-3, and K562 cell lines and in primary blasts

from 12 acute myeloid leukemia (AML) cases, by reverse-

transcriptase polymerase chain reaction (RT-PCR) and North-

ern blot and by the evaluation of sensitivity to DT. The

release of functional HB-EGF was assessed by evaluation of

its proliferative effects on the HB-EGF–sensitive Balb/c 3T3

cell line. HB-EGF was expressed by all myeloid and T, but not

B (L428, Raji), lymphoid cell lines tested, as well as by the

majority (8 of 12) of ex vivo AML blasts. Cell lines (except for

the K562 cell line) and AML blasts expressing HB-EGF mRNA

underwent apoptotic death following exposure to DT, thus

demonstrating the presence of the HB-EGF molecule on their

membrane. Leukemic cells also released a fully functional

HB-EGF molecule that was mitogenic for the Balb/c 3T3 cell

line. Factors relevant to the biology of leukemic growth,

such as tumor necrosis factor-a (TNF-a), 1a,25-(OH)2D3, and

especially all-trans retinoic acid (ATRA), upregulated HB-EGF

mRNA in HL-60 or ML-3 cells. Granulocyte-macrophage

colony-stimulating factor (GM-CSF) induced HB-EGF mRNA

and acquisition of sensitivity to DT in one previously HB-EGF–

negative leukemia case. Moreover, the U937 and Karpas 299

cell lines expressed HER-4 mRNA. This work shows that

HB-EGF is a growth factor produced by primary leukemic

cells and regulated by ATRA, 1a,25-(OH)2D3, and GM-CSF.

r 1999 by The American Society of Hematology.

HEPARIN-BINDING epidermal growth factor–like growthfactor (HB-EGF) is a heavily glycosylated EGF family

member of approximately 22 kD capable of binding to heparin.It was originally identified in human monocytes and U937monocytic cell line–conditioned medium.1-3 Subsequently, HB-EGF expression was found in a wide range of cell types,including monocytes,1 CD41 lymphocytes,4 eosinophils,5 smoothmuscle cells (SMC),6 and endothelial7 and normal or neoplasticepithelial cells.1,8 HB-EGF can be released from the cellmembrane through proteolytic mechanisms,9 but multiple splicedmRNAs are likely to be produced and the cDNA correspondingto a short HB-EGF form lacking intramembrane and intracyto-plasmic domains has been cloned.10 HB-EGF binds to EGFreceptor 1 (HER-1) and 4 (HER-4)1,2,11 eliciting different bio-logic responses.11 It is a potent mitogenic and a chemotacticfactor for fibroblasts4 and SMC,12 mitogenic factor for keratino-cytes,8 and chemotactic factor for endothelial cells13 andastrocytes.14 Moreover, HB-EGF has been shown to participatein autocrine-paracrine loops, which are active in a number ofepithelial neoplasia,15 and to be involved in stromal prolifera-tion following decidualization.16

Membrane-bound HB-EGF retains growth activity and adhe-sion capabilities. Macrophages infiltrating atheromatous plaquesactively induce SMC hyperplasia through HB-EGF.17 Ratblastocyst implantation has been reported to be associated withHB-EGF expression and adhesion activity.18 Finally, membrane-bound HB-EGF has the unique property of acting as thereceptor for the diphtheria toxin (DT),19 a protein translationinhibitor capable of triggering apoptotic death.20 CD9 coexpres-sion enhances the mitogenic activity of membrane-boundHB-EGF,17 as well as the sensitivity to DT.21

Attention has been devoted to the role of HB-EGF inreproductive biology,16,18 wound healing,22 atheromatous phe-nomena,17,23angiogenesis,13 and epithelial neoplastic prolifera-tive events.15 The production of HB-EGF by monocytes, CD41

lymphocytes, and eosinophils suggests that it may also beproduced by other normal and neoplastic hematologic celltypes. In the present study, we analyzed the expression of

HB-EGF in a panel of human hematologic cell lines derivedfrom different lineages, and in primary blast cells from patientswith acute myeloid leukemia (AML). The interest in studyingHB-EGF in AML stems from its mitogenic, chemotactic, andreceptor functions.

We found that HB-EGF was expressed by the human myeloidand T, but not B, lymphoid cell lines tested, as well as by ex vivoblast cells in a number of AML cases. Thus, HB-EGF is anadditional growth factor produced by primary leukemic cells.

MATERIALS AND METHODS

Cell lines. The following cell lines available in our laboratory werestudied: B-cell lines—L428 (Hodgkin-derived)24 and Raji (non-Hodgkin’s lymphoma)24; T-cell lines—Jurkat (acute T-cell leukemia),25

Karpas 299 (anaplastic large-cell lymphoma),24 and L540 (Hodgkin-derived)24; natural killer (NK)-cell line—2C826; myeloid cell lines—HL-60 (AML)24, U937 (monocytic leukemia),24 THP-1 (acute mono-cytic leukemia),27 ML-3 (acute myelomonocytic leukemia),28 and K562(blastic phase of chronic myeloid leukemia).24

Patients and isolation of leukemic cells.The main clinical charac-teristics of the patients whose leukemic cells were investigated are listedin Table 1. All cases were untreated AML patients at diagnosis with ahigh percentage ($90%) of blasts and minimal residual contaminationby normal cells. The diagnosis of AML and its French-American-

From the Departments of Hematology and General Pathology,University of Verona, Verona; and CNR Center for Biomembranes,University of Padua, Padua, Italy.

Submitted August 10, 1998; accepted October 23, 1998.Supported by grants from Associazione Italiana per la Ricerca sul

Cancro (AIRC, Milano), Progetto Sanita 96/97, Fondazione Cariverona(Verona), and Italy-USA Program on ‘‘Therapy of Tumors’’ (’96-’98).

Address reprint requests to Fabrizio Vinante, MD, Cattedra diEmatologia, Ospedale Policlinico, 37134 Verona, Italy.

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked‘‘adver-tisement’’ in accordance with 18 U.S.C. section 1734 solely to indicatethis fact.

r 1999 by The American Society of Hematology.0006-4971/99/9305-0013$3.00/0

Blood, Vol 93, No 5 (March 1), 1999: pp 1715-1723 1715

For personal use only.on March 17, 2016. by guest www.bloodjournal.orgFrom

British (FAB) subtypes was based on clinical findings and on estab-lished morphologic, cytochemical, and cytofluorimetric parameters ofperipheral blood and/or bone marrow cells. Viable leukemic cells fromfreshly heparinized peripheral blood or bone marrow were separated bycentrifugation on Lymphoprep (Nycomed Pharma AS, Oslo, Norway),washed twice with phosphate-buffered saline (PBS), and either immedi-ately used or stored frozen in liquid nitrogen. In all cases, frozen cellsamples contained greater than 95% blasts.

Cell storage. The cell lines and aliquots of ex vivo blasts werestored frozen in liquid nitrogen in 70% RPMI 1640 (Gibco-BRL LifeTechnologies, Paisley, UK), 20% dimethyl sulfoxide (DMSO), and 10%heat-inactivated fetal calf serum (FCS; Gibco-BRL). Frozen cells werethawed in 20% FCS/80% RPMI 1640, immediately centrifuged, andwashed once with culture medium. Cell viability after thawing wasalways greater than 90%, as assessed by Trypan-blue staining. Freezingprocedures did not modify the expression of HB-EGF.

DT sensitivity assay. Highly purified DT at 10211, 10210, 1029, and1028 mol/L concentration was added to 13 106 cells/mL (cell lines orex vivo blasts) in a 96-well plate (Falcon, Lincoln Park, NJ) andincubated for 24 or 48 hours at 37°C in 5% CO2 in RPMI 1640supplemented with 10% heat-inactivated FCS and penicillin (100IU/mL)/streptomycin (100 µg/mL). The sensitivity to DT was evaluatedas cytotoxic activity assessed by the modified 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Ten microliters perwell of 5 mg/mL MTT solution was added and the plates were incubatedat 37°C for 4 hours. After adding 100 µL/well of 0.04N HCl inisopropanol and thoroughly mixing, the plates were read (wavelengths:test, 570; reference, 630 nm) on an AutoReader III (Ortho DiagnosticSystems, Raritan, NJ).29,30Resting cells were considered to be sensitiveto DT when more than 50% of them were killed with a typicaldose-response curve in the 10211 to 1028 mol/L DT concentration rangeduring a 48-hour period. When the acquisition of sensitivity to DT wasevaluated in stimulatory experiments on previously insensitive cells, theappearance of statistically significant cytotoxicity after stimulation wasconsidered.

Assessment of apoptosis: DNA laddering and morphologic changes.After 48 hours, 1028 mol/L DT-incubated cells (13 106 cells/mL) andtheir controls were harvested and subdivided into three aliquots toevaluate apoptotic death. (a) One milliliter of cells was added with 200µL of lysing buffer (2 mol/L NaCl, 50 mmol/L Tris/HCl, 10 mmol/LEDTA, pH 8), 50 µL of 20% sodium dodecyl sulfate (SDS), and 10 µLof proteinase K, and incubated for 45 minutes at 60°C and overnight at

37°C. Following the addition of 400 µL of 5 mol/L NaCl. the cells werecentrifuged for 30 minutes at 3,500 rpm. Supernatant (SN) wastransferred to a new tube, DNA precipitated for 3 hours with 5 mLethanol 96% at280°C, and centrifuged at 4,000 rpm for 30 minutes at4°C. The dried pellet was resuspended in Tris-EDTA (TE)-RNAse (1µg/µL) and incubated for 1 hour at 37°C. DNA was loaded onto 1%agarose gel and stained with ethidium bromide. (b) Cell shrinkage andacridine orange staining by phase contrast and fluorescence microscopywere evaluated. (c) Cells were analyzed with a FACScan cytometer(Becton Dickinson, San Jose, CA). A gate was depicted including theliving population in the forward and side light scatter cytogram afteracquiring 13 105 untreated cells, and the percentage of DT-treated cellsfalling outside the defined gate due to forward or side light scatterchanges was calculated.

Transwell cultures and HB-EGF activity detection.Transwell cul-tures of the Balb/c 3T3 cell line, proliferation of which is induced byHB-EGF,2 and of leukemic cells were performed to assess whetherHB-EGF released by the ML-3 cell line and ex vivo AML blastsretained its mitogenic capability on Balb/c 3T3 cells. Confluent Balb/c3T3 cells were trypsinized and resuspended at 53 104 cells/mL.Aliquots of 200 µL were plated in Dulbecco’s modified Eagle’s medium(DMEM)/10% FCS in 24-well plates (Falcon). The cells were incubatedfor 5 days after reaching confluence to deplete the media of growthfactors. Transwells (12-mm diameter, 0.4-µm pore size; Costar, Cam-bridge, MA) were introduced into the wells of the 24-well platecontaining Balb/c 3T3 cells and ML-3 or myeloid blast cells (53 105

cells/mL) were cocultured with or without 40 ng/mL phorbol myristateacetate (PMA) in order to favor HB-EGF release from the cell mem-brane.31,32A 100-µg/mL quantity of goat antihuman HB-EGF neutralizingantibody (R&D Systems, Minneapolis, MN) was used to block HB-EGFactivity. Such activity was evaluated after 72 hours by measuring the numberof Balb/c 3T3 cells using the modified MTT assay,29,30after constructing astandard curve based on the absorbance/cell number ratio. The results wereexpressed as percentage increase in cell number as opposed to control.

Stimulation of cells in culture. The cell lines and ex vivo blastswere cultured in RPMI medium supplemented with 10% FCS at aconcentration of 13 106 cells/mL. When indicated, cells were treatedwith PMA (40 ng/mL), all-trans retinoic acid (ATRA, 1025 mol/L),tumor necrosis factor-a (TNF-a, 100 U/mL), interferon-gamma (IFN-g,1,000 U/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL), 1a,25-(OH)2D3 (vitamin D3, 10 21 mol/L), DMSO(1.6%), or a combination of two of these factors. After culture for the

Table 1. Patient Characteristics

PatientNo.

FABType

Sex/Age(yr) PB/BM

% ofBlasts*

Responseto Therapy

HB-EGFmRNA†

DTSensitivity‡ CD9§ HER-1§

1 M0 M/61 PB 95 No 1 1 2 2

2 M0 F/56 PB 90 CR 1 1 nd 2

3 M1 F/67 PB 95 CR 1 1 2 2

4 M1 M/27 PB 100 CR 2 2 2 nd5 M1 M/24 PB 93 No 1 1 2 2

6 M2 F/69 PB 100 CR 2 2 nd nd7 M2 F/62 PB 90 CR 1 2 2 nd8 M3 F/43 BM 95 CR 1 1 nd 2

9 M4 M/70 PB 95 No 1 1 2 nd10 M4 M/66 PB 95 CR 2 2 1 nd11 M5 M/75 PB 90 No 1 1 1 2

12 M5 M/43 PB 99 CR 2 2 nd 2

Abbreviations: M, male; F, female; PB, peripheral blood; BM, bone marrow; CR, complete remission; nd, not done.*In vivo.†By Northern blot.‡Blasts were considered to be sensitive (1) to DT when more than 50% of them were killed with a typical dose-response curve in the 10211 to 1028

mol/L DT concentration range during a 48-hour period, as evaluated by the MTT assay.§By flow cytometry.

1716 VINANTE ET AL

indicated times, the cells were harvested and RNA was extracted, aspreviously reported,33 and analyzed for HB-EGF gene expression byreverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot.

RT-PCR analysis of HB-EGF and HER-4; plasmid insertion ofHB-EGF cDNA. Total cellular RNAs were isolated and 4 µg of RNAwas reverse-transcribed (universal primers, 1.25 U of AMV RT[Gibco-BRL Life Technologies]) as previously described.33,34 cDNAwas PCR-amplified using the following primers (Genenco, m-medical,Florence, Italy). (1) HB-EGF sense 58-TGGTGCTGAAGCTCTTTC-TGG-38and antisense 58-GTGGGAA TTAGTCATGCCCAA-38; theseprimers were designed to span exons 1 to 5 of the gene giving afragment of 605 bp (complete form of HB-EGF cDNA)35 or a fragmentof 605 1 94 bp (short form of HB-EGF cDNA).10 (2) HB-EGFantisense 58-TCAAGTAACATCTTTCTGCCCAGC-83 specific for asequence on the 94-bp insert present in the short HB-EGF10 expected togive a 407-bp fragment when associated with the above-specified senseprimer. (3) HER-4 sense 58-AGATGGAGGTTTTGCTGCTGAA CA-38and antisense 58-TTACACCACAGTATTCCGGTGTCT-38(726-bp frag-ment)36; (4) vimentin sense 58-GCTCAGATTCAGGAACAGCAT-38,and antisense 58-TAAGGGCATCCACTTCACAGG-38 (266-bp frag-ment). The cDNA was denatured for 5 minutes at 94°C before 35runs in a thermal cycler (GeneAmp PCR System 2400; PerkinElmer, Norwalk, CT) using 1.25 U of Taq polymerase (PerkinElmer, Branchburg, NJ) in 50 µL (94°C 40 seconds, 57°C 40 seconds,72°C 50 seconds) followed by 5 minutes at 72°C. PCR products wereseparated by electrophoresis on 1.5% agarose gel. The HB-EGFRT-PCR product was analyzed for theSmaI (Gibco-BRL) restrictionsite (which gave the expected HB-EGF fragments of 388 and 217 bp),and was sequenced (Sequenase 2.0 sequencing kit; USB, Cleveland,OH) as a plasmid insert (TA cloning kit; Invitrogen, San Diego, CA)from which the HB-EGF probe was generated for Northern blotanalysis.

Northern blot analysis. Total RNA preparation and Northern blotanalysis (10 µg of RNA per lane) were performed as previouslydescribed.33 The RNA blots were hybridized with the32P-labeled cDNAprobe to HB-EGF obtained as specified above and with a32P-labeledplasmid containing a cDNA probe to G6PDH or beta-actin.

Immunostaining. Surface expression of HER-1 and CD9 wasassessed by incubation of 13 106 cells with 10 µL fluoresceinisothiocyanate (FITC)-conjugated anti–HER-1 monoclonal antibody(mAb) (Medac, Hamburg, Germany) and 10 µL phycoerythrin (PE)-conjugated anti-CD9 mAb (SBA, Birmingham, AL) for 30 minutes at4°C. Cells were washed twice in PBS. Irrelevant FITC- or PE-conjugated IgG2b mAbs (Immunotech, Westbrook, MA) were used as acontrol. The analysis was performed with a FACScan cytometer(Becton Dickinson).

Statistics. Student’st-test, the Mann-WhitneyU test, and Kruskall-Wallis analysis of variance (ANOVA) by ranks were used.When needed, a logarithmic transformation was performed. Differenceswere considered statistically significant when theP value was lessthan .05.

RESULTS

HB-EGF mRNA in cell lines and blasts.We examined thepresence of mRNA for HB-EGF in a panel of cell lines and in exvivo AML blasts. The main findings are listed in Tables 1 and 2for patients and cell lines, respectively. The results of mRNAanalyses in AML blasts and cell lines are detailed in Figs 1 and2. HB-EGF mRNA expression by cell lines was studied usingRT-PCR. As shown in Fig 1, B-derived cell lines (L428, Raji)were negative, whereas the remaining cell lines (Jurkat, Karpas299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562)shared a band of 605 bp corresponding to cDNA encoding thecomplete form of HB-EGF. At the resolution level adopted, only

one clear-cut 605-bp band for HB-EGF was amplified, corre-sponding to the complete HB-EGF molecule. In no cell lineswere we able to reamplify an HB-EGF cDNA corresponding tothe short form of the molecule.10 Restriction and base sequenceanalysis of the PCR product confirmed that it was amplifiedfrom HB-EGF mRNA. The 605-bp cDNA obtained from PCRwas used as a probe for HB-EGF in the Northern blot analysisof the leukemic cells from patients. HB-EGF transcript, evalu-ated by Northern blot, was present in 8 of 12 cases with adistribution apparently independent of FAB subtype (Table 1and Fig 2).

Membrane-bound HB-EGF assessed by sensitivity to DT.To evaluate whether the HB-EGF molecule was expressed oncells positive for HB-EGF mRNA, we tested their sensitivity toDT. We found that all T-cell lines and patient blasts positive forHB-EGF mRNA were sensitive to DT-induced cytolysis, asevaluated at 24 and 48 hours by a dose-response curvecomprising 10211 to 1028 mol/L concentrations of DT. Bycontrast, B-cell lines and four patients negative for HB-EGFmRNA expression were insensitive to DT (Tables 1 and 2, Figs1 and 2). The sole exception was the myeloid cell line K562,which, though positive for HB-EGF mRNA, was fairly resistantto DT (Fig 1). DT-related apoptosis was evaluated by differenttechniques. Microscopy and flow cytometry analysis showedmorphologic changes usually associated with apoptotic death inthe majority of DT-sensitive cases. In the same cases, DNAladdering was documented. No evidence of apoptosis in DT-insensitive cases was observed.

Expression of HER-1, HER-4, and CD9.Since HB-EGFbinds to HER-1 or HER-4, we evaluated whether these recep-tors were expressed by cell lines and ex vivo AML cells. Wefailed to detect HER-1 expression by such cells (Tables 1 and2). HER-4 mRNA was detectable in U937 and Karpas 299 celllines in basal conditions, whereas a very low expression wasfound in ML-3 and HL-60 (Table 2). CD9, a coreceptor ofmembrane-bound HB-EGF,3,17,21was present on a minority ofcell lines (Table 2) and ex vivo AML cells (Table 1).

Regulation of HB-EGF expression in leukemic cells.Weanalyzed whether the spontaneous expression of HB-EGF

Table 2. HB-EGF, HER-1, HER-4, and CD9 Expression in Cell Lines in

Basal Conditions

PatientNo.

CellLines*

HB-EGFmRNA†

DTSensitivity‡ HER-1§

HER-4mRNA† CD9§

1 U937 1 1 2 1 2

2 THP-1 1 1 2 2 1

3 ML-3 1 1 2 6 2

4 HL-60 1 1 2 6 2

5 K562 1 2 2 2 1

6 Jurkat 1 1 2 2 1

7 Karpas 299 1 1 2 1 2

8 L540 1 1 2 2 2

9 L428 2 2 2 2 2

10 Raji 2 2 2 2 2

11 2C8 2 2 2 2 nd

Abbreviation: nd, not done.*See Materials and Methods for lineage assignment.†By RT-PCR.‡DT sensitivity was evaluated by the MTT assay (see Table 1).§By flow cytometry.

HB-EGF IN AML 1717

mRNA in HL-60 and ML-3 cell lines could be modified byexogenous agents. Cell lines were stimulated with a panel ofmolecules, including DMSO, PMA, ATRA, IFN-g, 1a,25-(OH)2D3, and TNF-a, known to induce biologic effects onHL-60 or ML-3 cells, such as proliferation or differentiation.PMA, DMSO, and, more interestingly, TNF-a, 1a,25-(OH)2D3,and especially ATRA and costimulation with TNF-a and ATRAinduced an increase in transcripts for HB-EGF (Figs 3 and 4).However, TNF-aantagonized the effects of ATRA in ML-3cells (Fig 4). When we used GM-CSF to stimulate AML blastsfrom a patient shown to be negative for HB-EGF mRNA andinsensitive to DT (patient no. 6 in Table 1), we induced bothexpression of HB-EGF mRNA and sensitivity to DT (GM-CSF–treatedv untreated blasts,P 5 .002) (Fig 5).

HB-EGF proliferation assay. We examined whether HB-EGF expressed by cell lines and leukemic cells was released asa functional molecule mitogenic for Balb/c 3T3 cells incoculture tests. As shown in Fig 6, the stimulation of HB-EGF–positive ML-3 cells with PMA, which increases HB-EGFexpression and release from the cell membrane,31,32 induced a1.67-fold increase in Balb/c 3T3 cell number as compared withcontrols (P, .01). In addition, antihuman HB-EGF antibodyinhibited this proliferative effect, as expected.8,37 Ex vivo AMLblasts presented a different pattern, characterized by a higherHB-EGF proliferative effect on Balb/c 3T3 in basal conditions

than ML-3 cells, whereas PMA had no effect on HB-EGFactivity (Fig 6).

DISCUSSION

In this study, we found that all human hematologic cell linesinvestigated, except for the B-derived ones, and blasts from asubstantial proportion of AML cases produce, bear on theirmembrane, and release a fully functional HB-EGF molecule.This evidence is based on the following : (1) the demonstrationof HB-EGF mRNA expression, (2) the cytolytic effect of DTexposure exerted solely on HB-EGF mRNA-expressing cells,and (3) the proliferative effect of HB-EGF released by ML-3cells or by AML blasts on the Balb/c 3T3 cell line. We alsofound that factors relevant to the biology of leukemic growthmodified the expression of HB-EGF mRNA. TNF-a, 1a,25-(OH)2D3, and especially ATRA increased the expression ofHB-EGF mRNA in HL-60 and ML-3 cells. GM-CSF inducedHB-EGF mRNA and acquisition of sensitivity to DT in onepreviously HB-EGF–negative AML case. At variance withanother report,10 we failed to demonstrate the expression of thespliced short HB-EGF mRNA in the Raji and Daudi (the latterwas studied only for this purpose) cell lines, at least at theagarose gel electrophoresis level. Finally, the U937 and Karpas299 cell lines expressed HER-4 mRNA.

HB-EGF has been shown to play a role in the context of

Fig 1. (A) RT-PCR analysis of HB-EGF mRNA ex-

pression in a number of leukemic, Hodgkin-, and

NK-derived cell lines. HB-EGF: 605 bp; vimentin: 266

bp; marker: 100-bp DNA ladder. (B) 48-hour DT dose-

sensitivity curve as evaluated by the MTT method.

Only HB-EGF mRNA-positive cell lines were sensitive

to DT (ie, G50% death in the range of tested concen-

trations), indicating membrane expression of the

HB-EGF molecule.

1718 VINANTE ET AL

proliferative and chemotactic phenomena related to the inflam-matory response.3 More recently, a number of reports havedemonstrated that HB-EGF is involved in epithelial neoplasticproliferation15 and may stimulate angiogenesis through theinduction of vascular-endothelial growth factor (VEGF) invascular SMC.13 In such contexts, HB-EGF plays a relevantpart as a cytokine whose activity is mediated through paracrine,cell-to-cell, or autocrine interactions.

The role, if any, played by HB-EGF in leukemic expansionsis less intuitive. The lack of HER-1 on AML cells suggests that

this cytokine is not directly involved in leukemic proliferation.However, it has been shown that THP-1 cell line can be inducedto express HER-4 upon adequate stimulation36 and we foundHER-4 mRNA at least in resting U937 and Karpas 299 celllines. Leukemia-derived HB-EGF may be active on bystandercells via paracrine or juxtacrine mechanisms either directly orthrough induction of secondary factors, including VEGF, whichis expressed by AML cells38 and, though in a different context,has been shown to be induced by HB-EGF.13 Transwellcostimulatory experiments demonstrated that HB-EGF was

Fig 2. (A) Northern blot analy-

sis of HB-EGF mRNA expression

in different subtypes of ex vivo

AML blasts. Human umbilical

vein endothelial cells (HUVEC)

were used as positive control.

(B) 48-hour DT dose-sensitivity

curve for 8 representative cases

(2, 3, 4, 6, 7, 9, 10, 12 in A), as

evaluated by the MTT method.

Only HB-EGF mRNA-positive leu-

kemic cells (cases 2, 3, 7, 9) were

sensitive to DT, indicating mem-

brane expression of the HB-EGF

molecule.

Fig 3. HB-EGF mRNA expres-

sion by HL-60 cell line in basal

conditions and after different

stimuli. (A) After 24 hours, PMA,

ATRA, and ATRA 1 TNF-a were

the strongest inducers of HB-

EGF mRNA. (B) After 96 hours,

PMA-induced band was more

evident than at 24 hours.

HB-EGF IN AML 1719

released by both the ML-3 cell line and ex vivo AML blastsinducing the Balb/c 3T3 cell line to proliferate. Ex vivo blastsreleased more HB-EGF as compared with ML-3 cells in basalconditions, but PMA was less effective on AML blasts, possibly

due to a direct cytotoxic effect (Fig 6). Interestingly, factorsinvolved in differentiation or proliferation of acute leukemia,such as ATRA, 1a,25-(OH)2D3, TNF-a, and GM-CSF, couldmodify the expression of HB-EGF mRNA in leukemic cell

Fig 4. HB-EGF mRNA expres-

sion by ML-3 cell line in basal

conditions and after different

stimuli. (A) After 24 hours, HB-

EGF mRNA was strongly induced

by ATRA, 1a,25-(OH)2D3 (vitamin

D3), DMSO, and PMA. (B) After

96 hours, ATRA- and 1a,25-

(OH)2D3-induced bands were still

present.

Fig 5. HB-EGF induction by GM-CSF treatment in AML blasts. Resting blasts were negative for HB-EGF mRNA and insensitive to DT, but after

GM-CSF they acquired HB-EGF transcripts and sensitivity to DT-induced cytolysis (P 5 .002). (A) Dose-response curves testing the different

sensitivity to DT in ex vivo myeloid blasts from case 6 in Table 1 before and after treatment with GM-CSF. Results were expressed as percentage

of controls and represented the mean 6 SD of 5 experiments. (B) The percentage of cell viability at the 1028 mol/L DT concentration in two

representative experiments is associated with the corresponding pattern of RT-PCR analysis, showing the induction of the transcripts for HB-EGF

after exposure to GM-CSF (HB-EGF: 605 bp; vimentin: 266 bp).

1720 VINANTE ET AL

lines. In HB-EGF promoter, putative binding sites for NFkBand AP1 sites have been identified.3,35 In addition, it has beenshown that HB-EGF could be induced through Ras pathwayactivation.3,39,40Actually, TNF-a has been reported to mobilizeNFkB41; the receptors for vitamins A and D (including 1a,25-(OH)2D3) recognize common response elements containing theAP1 site42; by binding to the beta subunit of its receptor,GM-CSF activates Ras and Raf-1 and the MAP kinase path-way.43Thus, it is likely that TNF-a, ATRA, 1a,25-(OH)2D3, andGM-CSF had the HB-EGF gene as a downstream target.Whereas HB-EGF induction by TNF-a has been reported byothers,7,35 to the best of our knowledge the role of ATRA,1a,25-(OH)2D3, and GM-CSF has yet to be characterized.

These findings may lead to a better understanding of anumber of biologic aspects of leukemic blasts, including theiroutgrowth capability in the context of skin or mucosal tissues orthe induction of fibrosis, which characterize some FAB sub-types33,34 and are believed to play a part in limiting theeffectiveness of the therapy. However, it is not clear how and towhat extent AML cells may be influenced by HB-EGF or byfactors similar to, or induced by, HB-EGF.

The presence of HB-EGF protein on the membrane ofleukemic cells was also assessed by evaluation of the degree ofcytolysis induced by DT.20 In general, DT cytolysis wasassociated with DNA laddering. We found a clear-cut correla-tion between the expression of HB-EGF mRNA and thesusceptibility to death from DT in both cell lines and ex vivoAML cells. Worthy of note is the fact that the K562 cell line wasthe only case in which HB-EGF mRNA encoding for thecomplete molecule was expressed, but DT did not inducecytotoxicity. Chang et al have reported that actually DT isinternalized by K562 cells in which DT severely reduces protein

synthesis.20 The lack of a cytolytic effect in this cell line mightbe related to the expression of antiapoptotic factors, such as thechimeric protein BCR/ABL being translated as a consequenceof t(9;22), which is known to confer resistance to a variety ofapoptotic signals.44,45Hence, our data suggest, albeit indirectly,that DT internalization after binding to HB-EGF and inhibitionof protein translation do not imply straightforward apoptoticdeath in leukemic cell lines.20,46 DT receptor density, corecep-tors, efficiency of internalization, and, in general, the status ofpathways allowing transduction of apoptotic signals (ie, overex-pression of antiapoptotic factors) should be considered in agiven cell type. Yet, on the whole, apoptotic mechanismsactivated in response to DT were extremely efficient in theHB-EGF–positive leukemic cells tested.

In summary, HB-EGF was expressed in the majority of myeloidblasts. It was inducible in HB-EGF–negative leukemic cells, at leastby TNF-a, GM-CSF,ATRA, and 1a,25-(OH)2D3. It was released byleukemic blasts in a fully functional form mitogenic for the Balb/c3T3 cell line. The expression of at least HER-4 by a number ofleukemic cell lines adds significance to these findings. Finally,membrane-bound HB-EGF was found to be a functional DTreceptor efficiently mediating DT-induced cytotoxicity in ex vivoAML blasts. Since DT per se is not practical for therapeuticpurposes, due to the wide range of cell types expressing HB-EGF,DT fusion proteins binding to GM-CSF and interleukin-3 (IL-3)receptors have been generated and reported to be effective and moreselective in targeting leukemic cells.46-50

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Fig 6. Mitogenic effect on

Balb/c 3T3 cells of the HB-EGF

molecule released by the ML-3

cell line and by ex vivo myeloid

blasts. ML-3 and AML blasts were

stimulated with PMA to induce

both new transcripts and release

of HB-EGF (see text). Results

were expressed as percentage

increase in Balb/c 3T3 cell num-

ber versus untreated controls 6

SD (P F .01). Each test was repli-

cated five times.

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HB-EGF IN AML 1723

1999 93: 1715-1723

Fabrizio Vinante, Antonella Rigo, Emanuele Papini, Marco A. Cassatella and Giovanni Pizzolo Toxin Receptor Expression by Acute Myeloid Leukemia Cells

Like Growth Factor/Diphtheria−Heparin-Binding Epidermal Growth Factor

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