Báo cáo Y học: N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma

Eur. J. Biochem. 269, 3732–3741 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03066.x N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma Elissavet Hatzi1, Carol Murphy1,2, Andreas Zoephel3, Horst Ahorn,3 Ulrike Tontsch3, Ana-Maria Bamberger4, Keiko Yamauchi-Takihara5, Lothar Schweigerer6 and Theodore Fotsis1 1Laboratory of Biological Chemistry, Medical School, University of Ioannina, Greece; 2Biomedical Research Institute, Ioannina, Greece; 3Boehringer Ingelheim Austria GmbH, Vienna, Austria; 4Institute of Pathology, Department of Gynecophathology, University Hospital Hamburg Eppendorf, Hamburg, Germany; 5Department of Molecular Medicine, Osaka University Graduate School of Medicine, Suita, Japan; 6Abt. Hamatologie, Onkologie und Endokrinologie, Universitats-Kinderklinik Essen, Germany Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the pro-gression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regu-late angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regu-lation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demon-strated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a The N-myc proto-oncogene encodes a 64-kDa nucleopro-tein (N-Myc) which associates with a 21- to 22-kDa Max proteintoformN-Myc/Maxheterodimers[1].Thesedimers can bind to the E-box consensus sequence (CACGTG) in the promoter regions of target genes [2], including alpha prothymosin and ornithine decarboxylase, eventually inducing their up-regulation [3]. Thephysiologicalfunctions of N-Myc have remained elusive although there is evidence for developmentally important activities [4–6]. In the neural crest, enhanced N-Myc expression may facilitate prolifera-tion of immature neuronal precursor cells at the expense of differentiation [7,8]. N-myc is implicated in the pathogenesis of neural crest-derived tumors including neuroblastoma [9], the most frequent solid malignancy of infants. Amplification of N-myc oncogene is a frequent event in advanced stages (III and IV) of human neuroblastoma [10] and correlates with poor prognosis [11]. In fact, N-myc amplification is nearly exclusively observed in neuroblastoma. N-myc amplifica-tion results in high N-Myc protein levels that could perturb Correspondence to T. Fotsis, Laboratory of Biological Chemistry, Medical School, University of Ioannina, 45110 Ioannina, Greece. Tel.: + 30 65197560, Fax: + 30 65197868, E-mail: thfotsis@cc.uoi.gr Abbreviations:LIF,leukemiainhibitoryfactor;bFGF,basicfibroblast growth factor; VEGF, vascular endothelial growth factor; TIMP, tissue inhibitor of matrix metalloproteinases. (Received 20 February 2002, revised 30 May 2002, accepted 21 June 2002) contiguous N-terminal N-Myc domain. STAT3, a down-stream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensi-tive to the antiproliferative action of LIF. LIF did not influenceneuroblastomacellproliferationsuggestingthat,at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene ampli-fication enhances the malignant phenotype in neuroblas-toma. Keywords: N-myc; LIF; STAT3; endothelial cell; neuro-blastoma. thefinelytunedinterplayofN-MycandMaxandeventually induce abnormal expression patterns of target genes [1]. However, few N-Myc target genes have been identified so far [1]. A number of data indicate that some of the target genes might modulate the cardiovascular system. Indeed, whereas N-myc knockout mice die in utero [12,13], com-pound heterozygotes suffer from serious heart defects [6]. Moreover, N-myc may also regulate the growth of tumor vessels as neuroblastoma with N-myc amplification exhibit enhanced neovascularization [14] suggesting that N-Myc oncogene could stimulate tumor angiogenesis and thereby enhance neuroblastoma progression. Indeed, stable trans-fection and 100-fold overexpression of N-Myc in a neuro-blastoma cell line (SH-EP) resulted in an enhanced malignant phenotype of the transfectants (WAC2) and the ability to form well vascularized tumors in nude mice [15]. As tumor angiogenesis derives from an imbalance between angiogenic factors and inhibitors [16,17], it is conceivable that the genetic changes of cancer could initiate angiogenesisbydisturbingthisbalanceinthetumorvicinity. Indeed, normal p53 regulates the expression of the angio-genesis inhibitors thrombospondin [18] and glioma-derived angiogenesis inhibitory factor [19]. Also, activation of oncogenes, such as ras, has been shown to cause both up-regulation of the expression of angiogenesis stimulators, such as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), and down-regulation of angiogenesis inhibitors such as tissue inhibitor of matrix metalloproteinases (TIMP) and thrombospondin [20]. Towards the aim of identifying N-myc-regulated mole-cules modulating neuroblastoma tumor angiogenesis, we Ó FEBS 2002 N-myc oncogene down-regulates LIF (Eur. J. Biochem. 269) 3733 have previously screened conditioned media from SH-EP control transfectants (SH-EP007) and WAC2 cells for the presence of inhibitors or stimulators of endothelial cell proliferation [21]. We were able to demonstrate that three endothelialcellproliferationinhibitorspresentinSH-EP007 supernatants (SI.1, SI.2, and SI.3) were completely down-regulated in WAC2 cells [21]. In a further study, we have identified SI.3 as being activin A and documented antian-giogenic properties for this TGF-family member [22]. The present study deals with the structural and functional characterization of SI.1. MATERIALS AND METHODS Cell culture and cell proliferation assays Dishes, media and recombinant growth factors have been describedpreviously[21,23].Cellswereculturedasdescribed [15,23,24]. Collection of conditioned medium from SH-EP007 cells was carried out as mentioned before [21]. Cell proliferation assays using BBCE cells were previously described[21,23].Briefly,cellswereseeded(day 0)in12-well tissue culture plates at a density of 1250 cellsÆcm)2 (5000 cells per well) and the following day (day 1), wells received 10 lL of the fractions to be tested and 2.5 ngÆmL)1 bFGF. This treatment was repeated after two days (day 3). On day 5 or 6, cells in duplicate wells were trypsinized and counted using a Coulter particle counter. SHEP-007 and WAC2 cells were seeded at a density of 2500 cellsÆcm)2 (10 000 cells per well). For growth curves of WAC2 stable transfectants, cells were seeded in 12-well tissue culture plates at a density of 2500 cellsÆcm)2 (10 000 cells per well) and counted daily using a Coulter particle counter. Goat anti-human polyclonal LIF neutralizing antibody was obtained from R & D systems. Purification of SI.1 Concentration of conditioned medium (47 L), acidification and SP-Sepharose Fast Flow chromatography were carried out as described previously [21]. Fractions containing the SI.1 were subjected to concavalin A affinity chromatogra-phy. A concavalin A–Sepharose column was used (11-mL bed volume, Pharmacia) equilibrated in 50 mM Tris/HCl, pH 7.2 containing 500 mM NaCl, 0.1% Chaps, 1 mM CaCl2 and 1 mM MnCl2. Samples were applied in equil-ibrationbuffer,andelutionwascarriedoutusing:(a)10bed volumes of equilibration buffer; (b) a linear gradient consisting of 5 bed volumes of equilibration buffer and 5 bed volumes of equilibration buffer containing 500 mM a-methyl mannopyranoside; and (c) 5 bed volumes of equilibration buffer containing 500 mM a-methyl manno-pyranoside.Aflowrateof12 cmÆh)1 wasusedandfractions of 2.5 mL were collected. The active fractions were desalted, lyophilized and subjected to a preparative SDS/PAGE electrophoresis in tubes (150 · 5 mm) containing 80 · 5 mm of resolving (12%)and30 · 5 mmofstacking(4%)polyacrylamidegel. The tubes and buffers were according to Laemmli [21a] and electrophoresis was carried out with buffer recirculation at 2 mA per tube (1 W maximun power). Following electro-phoresis the tubes were washed for 30 min in 1% Chaps in NaCl/Pi followed by a further 30 min wash in NaCl/Pi. Afterexchangeofdetergentfromthedenaturing SDStothe nondenaturing Chaps, the polyacrylamide tubes were cut in 2.5 mm slices and a small piece of each slice was allowed to diffuse, at 4 °C overnight, in 700 lL of endothelial cell medium. The diffusates and/or their dilutions were tested for inhibitory activity on endothelial cell proliferation. The slices corresponding to the active fractions were then lyophilizedandtheboundproteinswereallowedtodifuseto tissue culture tested water. Following concentration, they dissolved in 0.25% trifluoroacetic acid/distilled H2O and loaded onto a Bakerbond Wide-Pore C18 RP-HPLC) column (4.6 · 250 mm; Malinckrodt Baker, Philipsburg, NJ, USA) equilibrated with 0.1% trifluoroacetic acid. Bound material was eluted with a linear gradient from 0 to 30% acetonitrile in 15 min and 30–60% acetonitrile in 45 min at a flow rate of 1 mLÆmin)1. Fractions of 1 mL were collected, lyophilized and dissolved in tissue culture water for activity evaluation on the proliferation of endothelial cells and amino-acid sequencing. Mass fingerprint analysis and microsequencing The active fractions from the HPLC step were electropho-resed on a 12.5% SDS-polyacrylamide gel under nonreduc-ing conditions and stained with Coomassie blue R250. Protein bands were excised from the stained gel, destained, reducedandalkylatedwithiodoacetamide,anddigestedwith trypsin. The resulting peptides were subjected to mass spectroscopy analysis using a MALDI-TOF instrument (VoyagerDE-STR,PerSeptiveBiosystems).Peptidesamples werepreparedusingdihydroxybenzoicacidasmatrix.From the calibrated MS peptide mass map a peak table list of the peptide-mass fingerprint were designed omitting signals observed in the chemical background spectrum. The peak table list served as input data and the MS-FIT software program (K. Clauser & P. Baker, available from http:// prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) was used for searching the SWISS Prot and NCBI protein databases for sequence similarities and thus suggesting the identity of the protein.Parametersweresetformasstoleranceat50 p.p.m., minimum number of peptides required at 4, molecular mass ofproteinsfrom1000to10 000 Da,proteinpIfrom3to10, cysteines modified as amidomthylated. MS-FIT search results were checked regarding the MOWSE score, molcular mass (Da),pI,speciesand%massesmatched.Edmansequencing of the RP-HPLC separated tryptic peptides was performed using an 494 cLC ABI-PerkinElmer apparatus. Transfections and LIF promoter reporter assays Full-lengthN-myc(pcDNA3-N-myc)wasgeneratedbyPCR amplification using pN-myc as template (a gift from M. Schwab, German Cancer Research Center, Heidelberg, Germany) [24]. All deletion mutants of N-myc were gener-ated from the above construct by ligation of PCR products. All constructs were sequenced. Transient transfections were performed using lipofectamine-plus transfection reagent (Invitrogen) according to the manufacturer’s instructions. A reporter luciferase construct containing the 666 bp human LIF promoter fragment (phLIF-Luc) was used [25]. SH-EP007 cells were plated at a density of 5 · 105 cells per well in a six-well plate were transfected with 0.4 lL of the phLIF-Lucconstruct,aloneorincombinationwith0.4 lgof 3734 E. Hatzi et al. (Eur. J. Biochem. 269) wild-type or mutated N-myc expression vectors for 28 h. In each transfection a constant total DNA concentration was used.Cellswerecollectedandanalyzedforluciferaseactivity using a kit according to the manufacturer (Promega, Madison, WI, USA) and a luminometer (EG & G Junior). As internal control of transfection efficiency, 0.2 lg of b-galactosidaseexpressionvector(CMV-b-gal)wascotrans-fected and the enzymatic activity was measured. The expression and nuclear localization of all constructs was determined by Western blot using a mouse antihuman N-mycantibody(0.3 lgÆmL)1,CymbusBiotechnologyLtd). The full-length cDNA encoding human LIF, kindly donated by Y. Jacques (INSERM U211, Nantes, France), was subcloned into the XhoI site of a CMV promoter construct. WAC2 cells (70% confluent) were transfected with effectene (Qiagen) using 1 lg of the LIF construct and 0.1 lg of a construct containing puromycin resistance gene (Clontech). Clones were selected in medium containing 0.7 lgÆmL)1 puromycin and expression of LIF was evalu-ated by Western blot analysis of supernatants using a goat anti-LIF Ig (R & D). Several independently derived clones were obtained for each construct. Control transfectants were generated using the empty CMV promoter construct. Phosphorylation of STAT3 protein in BBCE cells BBCE cells were seeded in 12-well plates (400 000 cells per well), cultured for 2 days in DMEM containing 0.5% (v/v) newborn calf serum and STAT3 and phospho-STAT3 (Tyr705) were detected as described in a kit purchased from New England Biolabs. Preparation of adenoviruses expressing STAT3 and [3H]thymidine incorporation assays Recombinantadenoviruses harbouring wild-type(AD/WT) and dominant negative (Y705F) (AD/DN) Stat3 cDNAs as well as a control adenovirus carrying only the vector (AD) were prepared as described previously [26]. Adenoviruses were amplified in 293 cells. The efficiency of infection and the expression of the proteins were monitored by immuno-fluorescence(Zeiss,AxiovertS100microscope)andWestern blot analysis. BBCE cells grown to subconfluency were infected with recombinant adenoviruses at a multiplicity of infection of 5 : 1 for 2 h in full medium. The medium was renewed for another 6 h allowing the constructs to be expressed and then various concentrations of recombinant LIF (R & D Systems) were added for an additional day. Then, 1 lCiÆmL)1 of [3H]methyl-thymidine (ICN) was added to each of the wells for the last 4 h of the incubation. Culture medium was removed and the cells were fixed with ice-cold 10% trichloroacetic acid for 20 min at 4 °C, washed three times with water and solubilized in 0.1 M NaOH overnight at 4 °C. The radioactivity was counted in a beta liquid scintillation counter (LKB). RESULTS SI.1 is identified as LIF The flow-through of the initial cation exchange chroma-tography step containing SI.1 activity [21] was sequentially Ó FEBS 2002 subjected to concanvalin A–Sepharose affinity chromatog-raphy (Fig. 1A) and preparative SDS/PAGE tube electro-phoresis (Fig. 1B). Following exchange to a nondenaturing detergent,theactivity wasevaluatedandtheactivefractions were subjected to a final C18 RP-HPLC chromatography (Fig. 1C). The main SDS/PAGE band exhibited a large size distribution (40–60 kDa) as a result of heterogeneity and glycosylation (Fig. 1D). Sequence analysis of five different segments of the main band revealed that one of the candidate proteins was LIF, a cytokine that has been previously shown to exhibit inhibitory activity on endothe-lial cells (Table 1). The rest of the sequenced peptides belonged to proteins that appeared irrelevant concerning inhibition of endothelial cell proliferation (Table 1). Con-firmation of SI.1 identity as LIF was further obtained by excising the corresponding SDS/PAGE gel segment, to that containing the sequenced LIF peptides, from another lane of the final SDS/PAGE gel. Following renaturation, this gel segment showed both inhibitory activity on endothelial cells (Fig. 2A) and the presence of LIF by Western blot analysis with a specific LIF antibody (Fig. 2B). More importantly, neutralizing LIF antibodies abolished the antiproliferative SI.1 activity on endothelial cells (Fig. 2C). LIF expression is down-regulated by N-Myc Detection and isolation of LIF in SH-EP007 cells (vs. WAC2) suggested that N-Myc down-regulated LIF. To substantiate N-Myc-induced down-regulation of LIF, we used promoter–reporter assays. Upon transfection of SH-EP007 cells with a vector containing wild-type N-myc cDNA (Fig. 3A), LIF promoter activity was suppressed substantially (Fig. 3B). In order to identify the domain(s) responsible for this inhibition, several deletions within the N-myc cDNA were generated and tested (Fig. 3A). All mutant N-Myc proteins were equally well expressed (data not shown). Transfection of N-myc mutants lacking the DNA-binding [d(381–395)] or the helix/loop/helix leucine zipper [d(350–464)] regions inhibited LIF promoter-luc activity, in a manner similar to the wild type N-myc. In contrast, the N-terminal part of N-myc seemed to be important for the inhibition of the LIF promoter transcrip-tion as the mutants d(1–134) and d(1–300) had completely lost this ability. As deletion of either the MbI [d(20–90)] or the MbII [d(96–140)] domains, together with flanking sequences, did not result in derepression of the inhibitory activity (Fig. 3B), the results indicate that a contiguous N-terminal N-Myc domain is essential for suppression of the LIF promoter transcription. These data indicate an N-myc-specific role in inhibiting LIF promoter activity. LIF inhibits the proliferation of endothelial cells but not that of WAC2 neuroblastoma cells We had identified LIF due to its ability to inhibit an important step of angiogenesis, i.e. vascular endothelial cell proliferation. In agreement with this finding, in addition to bovinebraincapillaryendothelialcells,recombinanthuman LIF (rhLIF) strongly inhibited the bFGF-stimulated pro-liferation of aorta (BAE) and human dermal microvascular endothelial cells (Fig. 4A, and data not shown). However, endothelial cells from other tissue origin, such as those from adrenal cortex (ACE), were weakly inhibited (Fig. 4A). At Ó FEBS 2002 N-myc oncogene down-regulates LIF (Eur. J. Biochem. 269) 3735 Fig. 1.PurificationandisolationofSI.1.(A)Theflowthroughfractionsofthepreviouscationexchangechromatographystep,containingSI.1,were ultrafiltrated, lyophilized, dissolved in equilibration buffer and applied to a concanvalin A–Sepharose column. Bound material was eluted with linear gradients of a-methyl-mannopyranoside as indicated (–). Aliquots of the fractions were tested for protein content (s) and activity on endothelial (BBCE) cell proliferation (d). Ten microliters of the fractions were added every other day with 2.5 ngÆmL)1 bFGF. The results were expressed as percentage of control (cells receiving buffer only). (B) The active fractions from concanvalin A–Sepharose chromatography were concentrated and diafiltrated using ultrafiltration and were subjected to SDS/PAGE in tubes as described under Material and methods. Following electrophoresis,thetubeswerewashedwithNaCl/Pi/1%Chapsfollowedbyafurther30-minwashinNaCl/Pi.The12%polyacrylamidetubeswere cut into 2.5-mm slices and a small piece of each slice was allowed to diffuse, overnight at 4 °C with gentle agitation, in 700 lL of endothelial cell medium.Thediffusatesweretestedforinhibitoryactivityonendothelialcellproliferation(d).(C)Theproteinsoftheactivegelpieceswereelutedin dH2O and lyophilized for application onto a C18 HPLC column. The application was performed in 0.1% TFA/dH20 and elution was carried out withalineargradientfrom0to30%acetonitrilein10 minand30–60%acetonitrilein45 min.Aflowrateof1 mLÆmin)1 wasusedandfractionsof 1 mL were collected, lyophilized and resuspended into 100 lL culture-tested water for activity evaluation. Ten microliters of 100-fold dilutions of the fractions were used for BBCE proliferation test (d). (D) The fractions corresponding to the active peak of the HPLC column in Fig. 1C were pooled, lyophilized, and separated on a 12.5% SDS-polyacrylamide gel under reducing conditions. The bands were excised from Coomassie blue stained gel and subjected to in-gel digestion followed by mass fingerprint analysis as described in material and methods. Left lane, pooled HPLC fractions; right lane, molecular markers in kDa. Table 1. ProteinsidentifiedbymassanalysisandEdmansequencingoftrypticpeptides.Thepositionofpeptideaminoacidinproteinwasdetermined by Edman sequencing. Band No. number 1, minor 2, major 3, major 4, major 5, minor Protein position Metabotropic glutamate receptor 2 Signal recognation particle Alpha antitrypsin Carboxypeptidase H Prec Carboxypeptidase H Prec Alpha antitrypsin Leukemia inhibitory factor Keratin I Keratin II SWISS-PROT accession Q14416 P13624 P34955 P16870 P16870 P34955 P15018 P13645 P04264 Amino-acid 20–34 29–43 323–337 87–107 148–157 205–213 23–37, 61–80, 193–202, 202–107 3736 E. Hatzi et al. (Eur. J. Biochem. 269) Fig. 2.IdentificationofSI.1asLIF. Asmallfractionofthesampleused for the sequencing of SI.1 was subjected to the same SDS/PAGE electrophoresis under nonreducing conditions and treated as in Fig. 1B. The gel pieces were analyzed for cell growth inhibition and correlation with LIF immunoreactivity. (A) Part of the eluates of the gel pieces (presented in this figure the gel pieces 7, 13, 17) was exami-nated for their ability to inhibit bFGF-stimulated proliferation of BBCEcells.(B)AliquotsofthesameeluateswereanalyzedbyWestern blot using a goat anti-LIF Ig (1 lgÆmL)1). The bound antibodies were detected with horseradish peroxidase conjugated anti-goat IgG fol-lowed by ECL detection system. (C) Neutralization of SI.1 activity. BBCEcellsweretreatedeitherwithrhLIF(2.5 ngÆmL)1)oraliquotsof the gel piece 13 alone or in combination with antibodies against LIF (anti-LIF, 1 lgÆmL)1) as described in material and methods. The results were expressed as percentofcontrol (cellsreceived only bFGF). theconcentrationsused,rhLIFwasnotcytotoxic,asseenby microscopic evaluation and by the fact that cell densities never fell below those present at seeding. rhLIF also inhibited basal proliferation of BBCE cells (data not shown and Fig. 5). The inhibitory effect of LIF on endothelial cell proliferation did not, however, exclude a direct autocrine inhibitory effectof LIF on neuroblastoma cell proliferation. Towards this end, rhLIF had no effect on the proliferation of SH-EP007 and WAC2 neuroblastoma cells, even at very high concentrations (Fig. 4B). The same results were obtained when we transfected WAC2 neuroblastoma cells with a vector containing the human LIF cDNA under the control of a CMV promoter (not down-regulated by N-Myc) compared to cells transfected with the empty vector. The proliferation potential of the LIF-expressing clones was either similar (c6) or higher (c11, c13) than the control clones (cve-1 and cve-2) (Fig. 4C). Indeed, in the case of c11 and c13 the proliferation rate was similar to that of the parental WAC2 cells and clearly distinct from that of the low N-myc expressing SH-EP007 cells (Fig. 4C). Thus, forced overexpression of LIF in WAC2 cells did not inhibit their in vitro proliferation. LIF inhibits endothelial cell proliferation via the STAT3 pathway LIF can mediate its effects by binding to specific cell surface receptors with subsequent phosphorylation of the transcrip-tion factor STAT3 at Tyr705. When rhLIF was added to BBCE cells, it was, in fact, able to phosphorylate STAT3 in a time-dependent manner (Fig. 5A). Phosphorylation of STAT3 Tyr705 was observed at 2.5 min post induction, reached a maximum by 20 min and declined slowly over Ó FEBS 2002 80 min. Simultaneous administration of angiogenic factors such as bFGF did not alter the phosphorylation pattern of STAT3 (data not shown). We wished to determine whether the STAT3 pathway was necessary and crucial for the LIF-induced inhibition of vascular endothelial cell proliferation. To that aim, we infected BBCE cells with recombinant adenoviruses con-taining various stat3 forms and investigated the ability of rhLIF to inhibitbFGF-induced proliferation ofthe infected cells. rhLIF was able to inhibit proliferation of BBCE cells infected with the empty vector controls (AD) or with the vectorcontainingthewild-typestat3(AD/WT)(Fig. 5B).In contrast,rhLIFwasunabletoinhibitproliferationofBBCE cellsinfectedwiththedominant-negativestat3mutant(AD/ DN) (Fig. 5B). Thus, the STAT3 pathway is important for mediating the inhibitory signals of LIF regarding endothe-lial cell proliferation. DISCUSSION We have previously shown that neuroblastoma cell super-natants contained three endothelial cell proliferation inhib-itors (SI.1, SI.2 and SI.3), the expression of which were dramatically down-regulated upon N-Myc overexpression [21].Inthepresentstudy,SI.1activitywasisolatedfrom47L of SH-EP007 supernatants by a series of chromatographic steps using inhibition of endothelial cell proliferation as a marker for biological activity. The main SDS/PAGE band exhibited a large size distribution (40–60 kDa) as a result of heterogeneity and glycosylation. Sequence analysis of tryptic peptides combined with detection with specific antibodies identified SI.1 as LIF. A conclusion further supported by inhibition of the SI.1 activity by specific anti-LIF neutralizing antibodies. Identification of SI.1 as LIF strongly implied that N-Myc overexpression down-regulat-ed LIF expression in neuroblastoma. Indeed, reporter assay experiments revealed, for the first time, that N-Myc overexpression dramatically down-regulated LIF promoter transcription.LIFhasneverbeenreportedtobelongtoaset of genes shown to be regulated by the myc gene family members using various assays [1,27], including crosslinking [28], and cDNA microarrays [29]. Myc/Max interactions play an important role in Myc-induced transcriptional regulation. Indeed, the C-terminus of N-Myc contains the HLH and Zip domains, which mediate heterodimerization with Max [2,30,31], and the BR domain, which is required for binding of the N-Myc/ Max heterodimers to consensus sites known as E boxes [30,32,33]. Our data suggest that down-regulation of LIF is independent of N-Myc/Max interaction and DNA binding as neither the HLH-Zip nor the BR deletion released the transcriptional repression. This excludes an indirect repres-sion of LIF gene via some of the known N-Myc/Max regulated genes [1]. Our data, however, reserve an important role for the N-terminal domain of N-Myc in down-regulation of LIF. Indeed, deletion of amino acids 1–134 abolished almost all of the N-Myc repressing activity. The N-terminal domain contains Myc boxI (MbI) and Myc boxII (MbII), two highly conserved domains of the myc family that are considered important sites for protein interactions. In most transcriptional assays, MbII domain is associated with repression rather than transactivation [34,35]. Indeed, MbII ... - tailieumienphi.vn