Hepatocellular Carcinoma Cell Line

Invasiveness of Hepatocellular Carcinoma Cell Lines:Contribution of Hepatocyte Growth Factor,c-met,and Transcription Factor Ets-1

Yan Jiang,*Wangxiang Xu,*Jun Lu,?Fuchu He,*and Xiaoming Yang*,1

*Beijing Institute of Radiation Medicine,Beijing,100850,People’s Republic of China;and ?Zhejiang University,Hangzhou,710006,People’s Republic of China

Received August 8,2001

To understand the mechanism of invasion and me-tastasis of hepatocellular carcinoma (HCC),the ex-pression of c-met and Ets-1,and the effect of HGF on these cell’s motility and invasion ability were exam-ined in four hepatoma cell lines.The analysis re-vealed that the overexpression of c-met and Ets-1is closely connected with the motility and invasion ability of the HCC cell lines.Invasion activity of HepG2and HLE cells were enhanced by the addition of HGF to medium.HGF regulated c-met transcrip-tion in HepG2and Bel-7402cells,HGF also induced Ets-1transcription in Bel-7402cell.Bel-7402cells stably transduced with the human Ets-1gene showed signi?cantly increased invasion potentials compared to parental and mock-transfected cells.The expression level of c-met,MMP1,MMP9,and u-PA in Bel-7402cells transfected with Ets-1were markedly increased,and as a consequence of c-met expression increase.Bel-7402cells transfected with Ets-1were more responsive to exogenous HGF stim-ulation in invasiveness and motility ability.In addi-tion,conditioned by antisense Ets-1oligonucleotide-treat-Bel-7402cells transfected with Ets-1gene and HLE hepatoma cells showed markedly reduced inva-sion activity,and down-regulated the transcription of Ets-1,c-met,u-PA,MMP-1,and MMP-9.These re-sults strongly suggest that Ets-1has a crucial role in the invasive property in hepatoma cell lines,and there may exist a loop to enhance the invasive abil-ity of hepatoma cell lines.?2001Academic Press

Hepatocellular carcinoma (HCC)is one of the most frequent primary tumors in the world.Invasion is a characteristic feature of HCC,it frequently shows early invasion into blood vessels as well as intrahepatic metastasis and later shows extrahepatic metastasis.Although many factors have been reported to be in-volved in this process,the molecular mechanisms of HCC progression are unclear.Hepatocyte growth fac-tor (HGF,also known as scatter factor,SF),a mesenchymally-derived heparin-binding polypeptide,was ?rst identi?ed as a potent mitogen for adult rat hepatocytes in primary culture (1).It is a pleiotropic factor,including induction of mitogenesis,motogen-esis,morphogenesis,and metastogenesis on a variety of epithelial cells (2).These pleiotropic effects of HGF/SF could contribute to tumor invasion and metas-tasis (3,4).The receptor for HGF is a receptor type tyrosine kinase encoded by the c-met proto-oncogene which was originally identi?ed as a transforming gene formed by a DNA rearrangement in carcinogen-treated cells (5).c-met is frequently found to be overexpressed in a variety of tumor tissues including HCC (6,7),proving further evidence for their involvement in tu-mor formation/metastasis.

The Ets gene superfamily encodes a class of evolu-tionarily well-conserved transacting phosphoproteins including more than 30members.Ets-1is a member of Ets family and was originally characterized as the v-ets retroviral gene,one of the two oncogenes (v-myb and v-ets)in the avian leukemia retrovirus E2b (8).Ets-1plays a role in the regulation of physiological processes such as cell proliferation and differentiation (9),and Ets-1has also been involved in cell transfor-mation and notably in invasion and metastasis (10,11).It regulates the expression of urokinase-type plas-minogen activator and MMP-1and the migration of vascular endothelial cells (12).Another feature shared by c-met and Ets-1is angiogenesis,which requires proliferation,matrix degradation,and organization of

Abbreviations used:HCC,hepatocellular carcinoma;HGF,hepa-tocyte growth factor;FGF,?broblast growth factor;EGF,epidermal growth factor;MMP,matrix metalloprotease;u-PA,urokinase-type plasminogen activator;AP-1,activator protein 1;MDCK,Madin-Darby canine kidney;G3PDH,glyceraldehydes-3-phosphate dehy-drogenase.1

To whom correspondence should be addressed at Beijing Insti-tute of Radiation Medicine,27Taiping Road,Beijing,100850.P.R.China.Fax:(86)-10-68214653.E-mail:xmyang2@https://www.360docs.net/doc/7e6472896.html,.

Biochemical and Biophysical Research Communications 286,1123–1130(2001)doi:10.1006/bbrc.2001.5521,available online at https://www.360docs.net/doc/7e6472896.html, on

polarized tubular structures.It has been reported that HGF enhanced MT1-MMP synthesis and induced MMP-2activation in two human EC lines(13).Fur-thermore,HGF accelerated EC invasion into matrix. The concomitant involvement of member of the Ets-1 and HGF/c-met in physiological as well as pathological events characterized by cell growth and extracellular matrix invasion suggest a functionary link between the two genes.Interestingly,it has been shown that Ets-1 enhances the activity of the300bp isolated promoter region of c-met in vitro and the activity of the natural promoter in stably transfected cells(14).The region contains four putative binding sites for member of the Ets transcription factor family,and increased expres-sion of the c-met was observed in cells stably trans-fected with Ets-1.More interesting,c-met activation induced transcription of Ets-1mRNA,showing that Ets proteins act both upstream and downstream to c-met expression,generating a positive feed-back loop. The link of c-met or Ets-1expression with human hepatocellular carcinoma has been observed(15–19). However,the correlation between of HGF/c-met signal-ing and Ets-1in liver carcinogenesis is somewhat con-troversial and not clear.We investigated the expres-sion of Ets-1,c-met,and the roles in enhancing the invasion activity in hepatoma cell lines.Our results strongly suggest that Ets-1has a crucial role in the invasive property in hepatoma cell lines,and there may exist a loop to enhance the invasive ability of hepatoma cell lines.

MATERIALS AND METHODS

Cell lines and culture conditions.Human hepatoma cell lines HepG2and HLE were purchased from the American Type Culture Collection(Rockville,MD).Bel-7402(epithelial hepatoma cell lines, derived from male,75years)and SMMC-7721(epithelial hepatoma cell lines,derived from male,50years)were purchased from the Cell Institute of Chinese Academy of Science.All cell lines were main-tained in Dulbecco’s modi?ed Eagles’minimum essential medium (DMEM)supplemented with10%heat-inactivated FBS(Life Tech-nologies,Inc.,Gaithersburg,MD)and antibiotic agents(100U/ml penicillin G and80?g/ml streptomycin)in a humidi?ed atmosphere of5%CO2and95%air.For the experiments,subcon?uent cells were seeded in T25?asks at a density of0.6?106cell/ml and maintained for24h in culture medium containing10%FBS.The cells were then incubated for24h in the starvation medium(0.1%FBS)that was thereafter replaced with5ml of fresh medium without serum and containing HGF(Sigma).

RT-PCR.Forward and reverse primers for human Ets-1,c-met, u-PA,MMP-1,MMP-9,and G3PDH genes were designed and applied to amplify transcripts.Brie?y,total RNAs were extracted with Trizol (GibcoBrl,Life Technologies).Reverse-transcribed and ampli?ed for 30cycles,using RT and PCR kit(Promega Corporation,Madison, WI),according to the manufacturer’s instructions.The expression of G3PDH mRNA was detected as an internal control.Six pairs of oligonucleotide primers for human Ets-1,c-met,u-PA,MMP-1, MMP-9,and G3PDH genes were used:Ets-1sense:5?-ATG AAG GCG GCC GTC GAT CTC AAG-3?;antisense:5?-TCA CTC GTC GGC ATC TGG CTT GAC-3?;c-met sense:5?-GAA AGA TAA ACC TCT CAT AAT GAA GG-3?;antisense:5?-CAC CAC ACA GTC AGG ACA CTG-3?;u-PA sense:5?-AGA ATT CTA CCG ACT ATC TC-3?;anti-sense:5?-ATT CTC TTC CTT GGT GTG AC-3?;MMP-1sense:5?-ACA ACT GCC AAA TGG GCT TG-3?;antisense:5?-TCC TGC AGT TGA ACC AGC TA-3?;MMP-9sense:5?-GAC TCG GTC TTT GAG GAG CC-3?;antisense:5?-GAA CTC ACG CGC CAG TAG AA-3?; G3PDH sense:5?-ACC ACA GTC CAT GCC ATC AC-3?;antisense: 5?-TCC ACC ACC CTG TTG CTG TA-3?.The expected size of the ampli?ed DNA fragments for Ets-1,c-met,u-PA,MMP-1,MMP-9, and G3PDH were1400,500,300,400,350,and1000bp.The PCR products were separated on1%agarose gel with ethidium bromide staining and photographed under UV.The photographs of the gels were analysed using Bio-Print(M&S Instruments Trading Inc.,To-kyo,Japan).The values of these DNA fragments were calculated as relative intensity against G3PDH mRNA.

Western blot analysis.Protein was extracted from different cells and the protein concentration was determined by Coomassie bril-liant blue G-250staining.Samples containing equivalent amounts (50?g)were separated on a8%(detecting c-met)or10%(detecting Ets-1)SDS–polycocrylamide gel under reducing conditions and transferred to a Hybond-N nitrocellulose membrane(Amersham, Arlington Heights,IL).The membrane was incubated expectively with rabbit polyclonal anti-human c-met antibody and sheep poly-clonal anti-human Ets-1antibody(Santa Cruz Biotech,Santa Cruz, CA),and developed with an ECL Western blotting detection system (Santa Cruz)using horseradish peroxidase-conjugated second anti-body(Santa Cruz).

Scattering and invasiveness assay.For the stimulatory effect on cell scatter,cells were seeded at a density of1?103cell/well for cells on96-well plates(Nun).After the cells had become attached to the plate,medium with20ng/ml HGF or without were replaced,and the cultures were maintained for24h.The cells then were stained with 0.2%crystal violet in20%ethanol,photographed.Two?elds per well were selected,each sample was repeated with three wells,and all experiments were independently performed three times.

For the invasiveness assay,cells(1?105cells in1ml serum-free medium DMEM?0.1%BSA)with or without Ets-1antisense oligo-nucleotide(8?M)were placed in Boyden chambers with8?m ma-trigel coated?lters(24-well format)purchased from Becton Dickin-son Labware(Franklin Lakes,New Jersey).Brie?y,the lower compartment contained400?l DMEM with or without20ng/ml HGF.After incubated at37°C in a5%CO2/95%water saturated atmosphere for10h,cells attached to the upper side of the?lters were then mechanically removed by wiping with a cotton swab,while cells that had migrated to the lower side were?xed with ethanol, stained with crystal violet,and counted.Six?elds per?lter were selected for cell counts;each sample was repeated with three wells, and all experiments were independently performed three times. Northern blot.Total RNA(20?g)was fractionated in1%agarose/ 2.2M formaldehyde gel transferred to Genescreen plus membrane and hybridized with32P-labeled cDNA probes for the C-met and Ets-1gene.The DNA probes used for hybridization were as follows. The plasmid containing the cDNA for human c-met(20)was used as template,a500bp fragment was ampli?ed by PCR as previous described and used as probe.1.4kb Ets-1cDNA was obtained by PCR from the human fetal thymus cDNA and sequenced. Construction of Ets-1expression vector and transfection.The hu-man full-length cDNA coding human Ets-1was obtained through PCR from the human fetal thymus cDNA(Clontech).The1.4kb PCR product was cloned into pGEM-T Easy vector(Promega),and the sequence was con?rmed to be identical to full-length Ets-1cDNA. The Not1fragment containing full-length Ets-1cDNA was inserted into the Not1site of pcDNA3.1(?)plasmid.The plasmids in correct orientation and reverse orientation were isolated and used for trans-fection.Bel-7402cells were subsequently harvested and reseeded at a density of1?106cells/100-mm plate.The cells were transfected the next day with different plasmids(2?g)using lipofectamine reagent(Gibco BRL,Gaithersburg,MD)and selected in medium

containing 400?g/ml G418for 14days.G418-resistant clones were isolated and grown in a medium containing G418to maintain the phenotype.

Treatment with antisense oligonucleotides.Antisense or sense phosphorothioate oligonucleotide corresponding to Ets-1gene and in-corporating an initiation codon was purchased from BioAsia Tech.(Shanghai,China).The sequence were 5?-ATGAAGGCGGCCG-TCGA-3?(sense)and 5?-AGATCGACGGCCGCCTTCAT-3?(antisense).A detailed analysis of the properties of this antisense have been pre-sented by Clausen (21).The oligonucleotides were puri?ed by high-pressure liquid chromatography.Cells were grown in DMEM under similar condition as mentioned above.Cells at 70%con?uence were washed brie?y in PBS and incubated in serum free medium for 36h.Thereafter,cells were treated with 10?M of either the sense or anti-sense Ets-1oligonucleotides.After 24h of incubation with Ets-1oligo-nucleotides,serum was added to the dishes to make a ?nal concentra-tion of 10%.Total RNA was isolated after 8h of exposure to the serum.Each sample was repeated with three dishes,and all experiments were independently performed two times.

RESULTS

Correlation between Expression of Ets-1and c-met in Hepatoma Cell Lines To examine the relationship between Ets-1and c-met expression pro?les in hepatoma cell lines,the expression of c-met and Ets-1were determined by semiquantitative RT-PCR and Western blotting.As Figs.1A and 1B show,the cell lines show interesting and revealing differences in the levels of Ets-1and c-met,HepG2and HLE hepatoma cell lines expressed c-met and Ets-1in high levels.However,expression of c-met and Ets-1was undetected in Bel-7402and SMMC-7721cells (Fig.1A).Similarly,we only ob-

served Ets-1and c-met protein in HepG2and HLE cell lines (Fig.1B).

Scattering and Invasive Activity of Hepatoma Cells and Effect by HGF As an initial step in elucidating the roles of expres-sion of Ets-1and c-met in hepatoma cells,we ?rstly examined whether HGF would stimulate the motility of these hepatoma cells.For MDCK cells,which are used as responder cells for the puri?cation of scatter factor,HGF caused a dissociation of the colonies and there was a remarkable scattering of the cells.Similar to the effect on MDCK cells,HGF scattered cell colo-nies of HepG2and HLE,which highly expressed c-met,but only a weak effect on Bel-7402and SMMC-7721(Fig.2A).

Next,we measured the invasive properties of hepa-toma cells by using a hybrid matrix composed of a mixture of basement membrane-like matrix,the re-sults show that invasiveness activity of HepG2and HLE,which highly express c-met and Ets-1,is stronger than that of Bel-7402and SMMC-7721(Fig.2B).The addition of HGF/SF markedly stimulated the HLE and HepG2cell lines to invade,the factor increased the invasive ?vefold,which only a weak induction was observed in both SMMC-7721and Bel-7402cells after adding HGF (20ng/ml)to the culture medium (Fig.2B).

Effect of HGF on Levels of c-met and Ets-1in Hepatoma Cell Lines

To assess whether HGF induces its receptor expres-sion,c-met mRNA levels were evaluated by Northern blot at various time after stimulation with HGF in serum-starved HepG2and Bel-7402(Fig.3A).The c-met mRNA was very low in resting cells and in-creased triple after HGF treatment 1h in HepG2cells,reached a maximum at 4h (sixfold),and declined at 8h.Similarly,c-met mRNA was induced in 7402cells.We subsequently tested the ability of HGF to induce Ets-1expression in serum-starved Bel-7402cells by RT-PCR and Western blotting.As Fig.3B shows,the amount of Ets-1mRNA in unstimulated cells was un-detected and HGF could induce Ets-1mRNA expres-sion at 2h after treatment (upper panel).The protein of Ets-1was also determined to expression after treat-ment with HGF (20ng/ml)at 2h (lower panel).Effect of Overexpression of Ets-1in Bel-7402on Invasion Activity

Using the lipofectamine agent,Bel-7402cells were stably transfected with pcDNA3.1or pcDNA3.1-Ets-1in correct orientation and reverse orientation.Expres-sion of Ets-1protein was analyzed by Western blotting using antibody against Ets-1protein.As shown in

Fig.

FIG.1.c-met,Ets-1expression,and invasive activity in human hepatoma cell lines.(A)Total RNA were extracted from hepatoma cells.1.0?g total RNA was used as template in RT-PCR as described under Materials and Methods.G3PDH was used as an internal control.(B)The expression levels of c-met and Ets-1were determined by Western blot as described under Materials and Methods.

4A,a protein band with an apparent molecular weight of 51kDa only was detected in the Ets-1transfected Bel-7402cells.To determine if Bel-7402cells stably expressed Ets-1displayed invasive activity in vitro,we assayed the invasive activity in the Boyden chamber with Matrigel-coated ?lter.Cells that passed through the ?lter pores and migrated to lower surface of the ?lters were counted as shown in Fig.4B.The invasive activity of nontransfected Bel-7402cells,Bel-7402pcDNA3.1cells (transfected pcDNA3.1),Bel-7402Ets(?)(transfected pcDNAEts-1in reverse orientation)was low,by stable expression Ets-1in Bel-7402cells (Bel-7402Ets(?)),the invasive activity was increased by 3.9-fold in the absence of exogenous HGF/SF in the lower chamber (Fig.4B).We also observed the antisense,but not sense,Ets-1oligonucleotide signi?cantly inhibited

invasiveness activity of Bel-7402Ets(?)toward the level of nontransfected Bel-7402and 7402pcDNA3.1(P ?0.01,Fig.4B).

Expression of u-PA,MMP-1,and MMP-9in Ets-1Stably Expressed 7402Cells To clarify the mechanism of the Ets-1expression enhanced cell invasion,we examined the expression of some Ets-1’s target genes in Bel-7402Ets(?)by RT-PCR.As Fig.5shows,mock-transfected and pcDNA Ets-1in reverse orientation transfected 7402cells showed only weak expression of u-PA,MMP-1,and MMP-9mRNAs.However,Bel-7402cells transfected with Ets-1showed a signi?cant increase in expression of u-PA,MMP-1,and MMP-9

mRNAs.

FIG.2.Enhancement of cell motility by HGF in MDCK,HepG2,Bel-7402,and SMMC-7721cells.(A)Cells were plated in 96-well plates at a density of 1?103cells/well and cultured for 24h in the presence (lower lane)or absence of 20ng/ml HGF (upper lane).The cells were ?xed,stained with 0.2%crystal violet,and photographed.(B)Invasive activity was determined by invasiveness assay as described under Materials and Methods.Two ?elds per well were selected,each sample being repeated with three wells,and all experiments were independently performed in triplicate.Shown is mean ?SD.

Effect of HGF on Motility and Invasive Activity in 7402Cell Transfected with Ets-1To examine the response of transduced 7402cell to stimulation by HGF,the cells were treated with HGF (20ng/ml),and resulted in causing a remarkable scat-tering of the cells (date not shown)and increase of cells that migrated through the Matrigel coated ?lter,but no evident effect on nontransfected 7402cells,mock-transfected,and 7402Ets-1(?)cells (Fig.4B).If the en-hanced response of Ets-1transduced 7402cell to stim-ulation by HGF is caused by increasing expression of c-met,the c-met expression was detected by semiquan-titative RT-PCR and Western blotting.As Fig.5and Fig.4A show,the c-met mRNA and protein was very lowly expressed in nontransfected,mock-transfected 7402cells,and 7402ets(?)cells,however,signi?cantly increased expression in transduced 7402cells.Effect of Antisense Ets-1Oligonucleotide on Gene Expression in HLE Hepatoma Cells Our ?nding above suggests that Ets-1transcription factor mediates u-PA,c-met,MMP-1,and MMP-9ex-pression in the hepatoma cells.To verify this view,the human hepatoma cell line HLE cells,which highly express Ets-1,c-met,MMP-1,MMP-9,and

u-PA

FIG.3.HGF-induced Ets-1and c-met expression in human hep-atoma cell lines.(A)Time courses of the mRNA of c-met in HepG2(left)and 7402(right)cells treated with 20ng/ml HGF.24-h starved cells (0h)were treated with HGF for a various time.Total RNA was puri?ed and analysed by Northern blotting (the lower lane).The experiment was performed twice with similar results.The bottom panel is a photograph of 28S rRNA band.(B)Time courses of Ets-1in Bel-7402cells treated with HGF by RT-PCR (upper panel)and Western blotting (lower panel).All experiments were performed twice with similar

results.

FIG.4.Expression of Ets-1in 7402hepatoma cells.(A)Ets-1and c-met expression in Bel-7402,7402Ets(?),7402Ets(?),and 7402pcDNA3.1.The levels of Ets-1and c-met protein were determined by Western blot analysis as described under Materials and Methods.(B)Antisense or sense Ets-1oligonucleotide treated for 24h Bel-7402,7402Ets(?),7402Ets(?),and 7402pcDNA3.1with or without 20ng/ml HGF were added to the upper compartment of chambers.The migration of cells to the lower surface of the ?lter was assayed as described under Materials and Methods.Each sample being repeated with three wells,the experiments were performed twice and shown are mean ?SD.

mRNA were treated with a phosphorothioate-modi?ed antisense or sense Ets-1oligonucleotide,and Ets-1,c-met,MMP-1,MMP-9,and u-PA mRNAs were ana-lyzed by semiquantive RT-PCR.As shown in Fig.6,Ets-1mRNA was down-regulated in HLE cells treated with antisense oligonucleotides but was not down-regulated in HLE cells treated with sense oligonucleo-tides.c-met,MMP-1,MMP-9,and u-PA mRNAs also were down-regulated,although sense oligonucleotide did not show any effect on their mRNA levels,G3PDH did not show any change in HLE cells treated with sense or antisense oligonucleotide.

Effect of Antisense Ets-1Oligonucleotide on Invasive Activity in Hepatoma Cells Lastly,we examined the potential of antisense Ets-1oligonucleotides to modulate hepatoma cell invasion in

the absence or presence HGF (20ng/ml).The addition of antisense Ets-1oligonucleotide to cultures of HepG2and HLE cells caused evident inhibition in invasive activity,but had no evident effect on Bel-7402cells (Fig.7).The inhibition in invasive activity of HLE displayed dose-dependent manner (data not shown).The addition of sense Ets-1oligonucleotide had no sig-ni?cant effect on invasive activity in these cells (P ?0.01).DISCUSSION

In this paper,we have reported the association be-tween Ets-1transcription factor and HGF/c-met in the regulation of hepatoma cell lines invasiveness.We found differential expression of c-met in four human hepatoma cell lines that were maintained in usual serum-supplemented medium,and HGF only en-hanced the invasive activity in HLE and HepG2hep-atoma cells that contain overexpression of a high num-ber of receptor for HGF,and only very weak effect on the 7402and 7721hepatoma cells.This shows that the c-met expression has a close correlation with cell’s in-vasive phenotypes in presence of HGF.In spite of its overexpression,c-met is not ampli?ed in the majority of primary tumors overexpressing the receptor (22,23),and the previous reports have shown that c-met is an inducible gene,which could be induced expression by many growth factors including HGF itself (24).In the present work,we demonstrate that the HGF was ca-pable of inducing its receptor in human hepatoma cells HepG2since a precocious and transient accumulation of c-met mRNA.This ?nding is consistent with other results showing that c-met behaves as a growth re-sponse gene (25).

Ets-1genes have been involved in cell transforma-tion and notably in invasion and metastasis.Our re-sults show that invasiveness activity of HepG2and HLE,which highly express Ets-1,is stronger than

that

FIG.5.Expression of uPA,MMP-1,MMP-9,and c-met in Ets-1transfected 7402cells.uPA,MMP-1,MMP-9,c-met,and G3PDH were detected by semiquantitative RT-PCR in 7402,7402Ets(?),7402Ets(?),and 7402pcDNA3.1cells.G3PDH was used as an internal control.The experiment was performed three times with similar

results.

FIG.6.Effect of antisense Ets-1oligonucleotide in hepatoma cells.Effect of antisense and sense Ets-1oligonucleotide on gene expression in HLE hepatoma cells.HLE hepatoma cell was treated with 10?M antisense or sense Ets-1oligonucleotide for 36h,then the total RNA was puri?ed and the mRNA levels of Ets-1,c-met,MMP-1,MMP-9,and u-PA were analyzed by semiquantive RT-PCR.G3PDH was used as an internal

control.

FIG.7.Effect of antisense Ets-1oligonucleotide on invasive ac-tivity in hepatoma cells.The effect of antisense or sense Ets-1oligo-nucleotide on invasive activity in hepatoma cells.Invasive activity was determined by invasiveness assay as described under Materials and Methods.Shown are mean ?SD.All experiments were inde-pendently performed three times.

of Bel-7402and SMMC-7721.The addition of antisense Ets-1oligonucleotide to cultures of HepG2and HLE cells caused evident inhibition in invasive activity,but had no evident effect on Bel-7402cells.These sug-gested that Ets-1may play a role in invasive properties of hepatoma cells.Previous studies have also shown that the spatial distribution of Ets-1transcripts was frequently observed in various invasive tumors,and suggested that soluble factors produced by neighboring stromal of tumor cells may induce Ets-1expression (26),and this view is supported by the report that the Ets-1gene is a new early-response gene regulated by cytokines and growth factors in human?broblasts(27). We were tempted by this initial observation to think that HGF may regulate Ets-1expression in hepatoma cells.Our results showed that HGF was a potent Ets-1 stimulator,inducing rapid(within2h)increases of Ets-1mRNA and protein expression,it seems that expression of Ets-1could be regulated in hepatoma cell by paracrine pathway.Stably transfected human Ets-1 cDNA into Bel-7402cell lines,in which the expression of c-met and Ets-1was undetected in the parent cell line,results in induction of invasive activities,and the invasion is inhibited when the cell lines are treated with antisense Ets-1oligonucleotide.Thus,the Ets-1 transcription factor participates in hepatoma cell inva-sion.It was well known that degradation of extracel-lular matrix is responsible for invasion of surrounding tissues(28–30).It were reported that proteases such as u-PA,MMP-1,MMP-3,and MMP-9contain Ets-binding motifs in their cis-acting elements and the mutation of Ets-1-binding motif leads to the severe impairment of expression of these proteases(31–36). Therefore,overexpression of Ets-1may contribute to the invasion and metastasis of HCC through stimulat-ing transcription of a set of genes overlapping with those downstream Ets-1,and this view is supported by our results that transfected Ets-1cDNA into hepatoma cell line7402enhance the expression of u-PA,MMP1, and MMP-9.Furthermore,inhibition of Ets-1expres-sion using a speci?c antisense Ets-1oligonucleotide in HLE human hepatoma cells,which express high level of Ets-1,showed a dramatic decrease in u-PA,MMP-1, MMP-9,c-met transcription,and cell’s invasive activ-ity,although sense oligonucleotide had on effect.It has also been reported that Ets-1enhances the activity of the300-bp isolated promoter region of c-met in vitro and stably transfected a human Ets-1cDNA into the murine cell line MLP could regulate c-met transcrip-tion in vivo(14).Similarly,we found that,as a conse-quence of Ets-1overexpression,transfected Ets-1into human hepatoma cell line7402enhanced expression of the c-met mRNA and protein.The population of cells transfected by Ets-1showed an increased response to HGF,this is observed in invasion and motility assay. These data strongly suggest that Ets-1seems to play a crucial role in the invasive ability in hepatoma cell lines.

To summarize,the data presented here show that Ets-1seem to play important roles in regulation of invasive ability of human hepatoma cells,and there may exist a positive loop to enhance the invasiveness: HGF induces Ets-1transcription,and Ets-1from one side up-regulates many genes involved in cancer inva-sion including u-PA,MMP-1,and MMP-9,from the other side,Ets-1stimulates c-met transcription,en-hances the response to stimulation by HGF,in turn, activates Ets-1transcription and consequently cause the increase of invasiveness together. ACKNOWLEDGMENTS

This project was supported partially by Chinese State Key Projects for Basic Research(G1998051122)and Chinese National Distin-guished Young Scholar Award(30025018).We thank Dr.Toshikazu Nakamura(Department of Oncology,Osaka University Medical School,Osaka,Japan)for the gift of the plasmid containing human c-met cDNA.

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