Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

Transcription

1 P.O.Box 2345, Beijing ,Chin World J Gstroenterol 2002;8(2): Fx: World Journl of Gstroenterology E-mil: wcjd@public.bt.net.cn Copyright 2002 by The WJG Press ISSN ESOPHAGEAL CANCER Trnscription fctor EGR-1 inhibits growth of heptocellulr crcinom nd esophgel crcinom cell lines Mio-Wng Ho, Ying-Rui Ling, Yn-Fng Liu, Li Liu, Ming-Yo Wu, Hun-Xing Yng Mio-Wng Ho, Li Liu, Deprtment of Internl Medicine, Tngdu Hospitl, Xi n , Shnxi Province, Chin Ying-Rui Ling, Ming-Yo Wu, Hun-Xing Yng, Deprtment of Pthology, Medicl College of Shntou University, Shntou , Gungdong Province, Chin Yn-Fng Liu, Deprtment of Pthology, Fourth Militry Medicl University, Xi n , Shnxi Province, Chin Supported by the Ntionl Nturl Scientific Foundtion of Chin, No Correspondence to: Dr. Mio-Wng Ho, Deprtment of Internl Medicine, Tngdu Hospitl, Fourth Militry Medicl University, Xi n , Shnxi Provioce, Chin. homwg@fmmu.edu.cn Telephone: Received Accepted Abstrct AIM: The trnscription fctor EGR-1 (erly growth response gene-1) plys n importnt role in cell growth, differentition nd development. It hs identified tht EGR-1 hs significnt trnsformtion suppression ctivity in some neoplsms, such s fibrosrcom, brest crcinom. This experiment ws designed to investigte the role of egr-1 in the cncerous process of heptocellulr crcinom (HCC) nd esophgel crcinom (EC), nd then to pprise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the trnscription nd expression of egr-1 in HCC nd EC, prcncerous tissues nd their norml counterprt prts were detected by in situ hybridiztion nd immunohistochemistry, with norml humn brest nd mouse brin tissues s positive controls. egr-1 gene ws then trnsfected into HCC (HHCC, SMMC7721) nd EC (EC109) cell lines in which no egr-1 trnscription nd expression were present. The cell growth speed, FCM cell cycle, plte clone formtion nd tumorigenicity in nude mice were observed nd the controls were the cell lines trnsfected with vector only. RESULTS: Little or no egr-1 trnscription nd expression were detected in HCC, EC nd norml liver tissues. The expression of egr-1 were found higher in heptocellulr prcncerous tissue (trnscription level P=0.000; expression level P=0.143, probbly becuse fewer in number of cses) nd dysplstic tissue of esophgel cncer (trnscription level P=0.000; expression level P=0.001). The growth rte of egr-1-trnsfected HHCC (HCC cell line) cells nd EC109 (EC cell line) cells ws much slower thn tht of the controls. The proportion of S phse cell, clone formtion nd tumorigenicity were significntly lower thn these of the controls (decresed 45.5% in HHCC cells nd 34.1% in EC109 cells; 46.6% nd 41.8%; 80.4% nd 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-trnsfected cells nd the controls with regrd to the bove items. CONCLUSION: The decresed expression of egr-1 might ply role in the dysregultion of norml growth in the cncerous process of HCC nd EC. egr-1 gene of trnsfected HHCC nd EC109 cells showed obvious suppression of the cell growth nd mlignnt phenotypes, but no suppression in SMMC7721 (HCC cell line) cells. Ho MW, Ling YR, Liu YF, Liu L, Wu MY, Yng HX. Trnscription fctor EGR-1 inhibits growth of heptocellulr crcinom nd esophgel crcinom cells lines.world J Gstroenterol 2002;8(2): INTRODUCTION The tumor suppressorgene therpy reserch hs become incresingly interested. Tumor suppressor genes hve two ctegories: Type 1, they undergo muttion or deletion, such s p53, Rb, WT1 nd BRCA1 etc. Type 2, they express little or nil nd with rre muttion or deletion, such s cx34, mspin nd integrin α6 etc [1]. In designing the tumor gene therpy, the type IItumor suppressor genes re evidently more dvntgeous. We only need to induce or to promote more expression of the genes to get rid of mny technicl difficulties such s replcing the deleted gene or knocking out the mutted gene. The erly growth response gene, egr-1 (Zfp-6 in stndrdized genetic Nomenclture for mice [2], lso known s NGFI-A, Krox-24, Zif-268, or TIS-8) is one of the importnt members of the immedite erly gene fmily. It encodes protein EGR-1 with 3 djcent zincfinger motifs, nd structures tht re present in mny DNA-binding trnscription fctors. Its specil zinc-finger structure cn bind with GC-rich DNA regultory elements nd cn control tht prticulr gene to trnscribe [3]. The expression of egr-1 is closely relted to cell growth [4] nd differentition [5]. Some reserching results hve reveled tht EGR-1 cn reverse the mlignnt phenotype of HT fibrosrcom nd ZR-75-1 mmmry crcinom cell lines, nd cn lso EGR-1 inhibit the tumorigenicity nd trnsforming growth of these cells [6-8]. egr-1 my ct s type II tumor suppressor gene. Both heptocellulr crcinom (HCC) nd esophgel crcinom (EC) re common mlignnt tumors in Chin. Mny reserch works on HCC nd EC [9-24], especilly on the effects of the tumor suppressor genes such s p53, p16 nd p21 etc [25-38] hd been reported, but the reports bout egr-1 for these two cncers re still lcking. In this pper, the trnscription nd expression of egr-1 in HCC,EC tissues nd their norml counterprts were detected by in situ hybridiztion nd immunohistochemistry in order to investigte the role of egr-1 in the cncerous process of HCC nd EC. The constructed egr-1 eukryoexpressing vector ws trnsfected into HCC nd EC lines, where there ws bsence of expression of egr-1, with view to observe how the high level of exogenous expression of EGR-1 in the cncer cell influenced their growth, clone formtion nd tumorigenicity. The inhibitory ction of egr-1 on HCC nd EC is to be probed so s to provide n experimentl evidence for the study of gene therpy of the two cncers. MATERIALS AND METHODS Mterils The plsmids, pac-h-egr-1 (frgment of single nd double enzyme digestion by BmH I nd/or SlI ws determined by 10g L -1 grose gel electrophoresis. The size of the frgments ws identicl to tht of

2 204 ISSN CN / R World J Gstroenterol April 15, 2002 Volume 8 Number 2 plsmid mp), contining the whole cdna frgment of humn wildtype egr-1 gene nd pac-φ eukryo-expressing vectors, were kindly grnted by Dr. JG Monore, Pennsylvni University School of Medicine [39]. Humn heptocellulr, esophgel crcinoms nd brest tissue specimens were collected from the Pthology Deprtment of Fourth Militry Medicl University nd Medicl College of Shntou University. All ptients providing the specimens were not treted by rdiotherpy or chemotherpy before opertion. The specimens were fixed with 40g L -1 formldehyde solution (forin situhybridiztion) nd Crnoy solution (for immuno-histochemistry). Mouse brin tissues were from Blb/c mice. The cell lines HHCC nd SMMC7721 of HCC were purchsed from Shnghi Cytology Institute, Chin. The EC cell line EC109 ws from Cytology Institute of Chinese Medicl Acdemy. All the cell lines were cultured in RPMI 1640 medium supplemented with 100mL L -1 new born bovine serum nd grown in the circumstnce of 37, 50mL L -1 CO 2 nd sturted humidity. Cell numbers were determined by Coulter counting method. The Lipofectmine TM, G418 nd TRIzol TM totl RNA extrction kit were purchsed from Gibco/BRL Co. The Wizrd TM plus Minipreps DNA purifiction kit ws the product of Promeg Co. The polyclonl rbbit nti-humn EGR-1 (C # -19) ntibody ws from Snt Cruz Co.in situhybridiztion (POD) detection kit nd SABC immunohistochemistry kit were from Boster Co, Wuhn,Chin. The Advntge TM PCR purifiction kit ws from Clontech Co, nd γ- 32 P- datp ws provided by Yhui Biomedicl Engineering Co, Beijing. Methods Probe preprtion nd lbeling The plsmid pac-h-egr-1 ws digested by both Sl I nd BmH I, nd the products were determined by grose gel electrophoresis; the 400 bp DNA frgments were recovered by promeg DNA purifiction kit. The recovered DNA ws dissolved in 20ul sterile three-distilled wter nd ultrviolet spectrophotometry ws tken for quntittion nlysis. γ- 32 P-dATP 5 end Lbel method ws used for probe lbeling. After lcohol precipittion, the probe ws dissolved in TE; the specific rdition ctivity ws determined by TAC method, nd stored t -20. In situ hybridiztion nd Immunohistochemistry In situ hybridiztion ws performed ccording to the kit instructions on the formldehyde-fixed nd prffin-embedded sections. Humn norml brest nd mouse brin tissue sections were used s positive controls. According to the SABC stin instructions, immunohistochemistry ws done on the Crnoy-fixed or frozen section. Positive control ws frozen section of humn norml mmmry denosis, nd negtive control ws primry ntibody-blnk. Gene trnsfection nd identifiction fter trnsfection Trnsfection ws performed ccording to the legend of Lipofectmine TM kit, nd 48h lter selective culture ws dded with 600mg L -1 G418.2 weeks lter, the cells nd clones ppered ll (the cells of blnk control died). The concentrtion of G418 ws chnged to 400mg L -1 G418 for mintennce. The clones were digested in situ, nd trnsferred to 6-well plte for mplifiction, which were used for detection of every item. mrna dot hybridiztion: cells were collected nd totl RNA ws extrcted ccording to TRIzol TM totl RNA extrction kit. The extrcted RNA ws dissolved in the RNse-free wter. A bsorbnce vlue ws determined, RNA quntity ws clculted nd purified ws identified; stored t -80 ; procedures of RNA dot blot ws ccording to the directions. Western blot hybridiztion nd immunohistochemistry: Western blot hybridiztion ws performed ccording to the directions. The cell crwling slide in logrithm stge ws collected, rinsed with PBS 2, fixed in 700mL L -1 lcohol nd stined by SABC kit. The positive control ws frozen section of mmmry denosis, nd the negtive control ws primry ntibody-blnk. Assy of cell phenotype FCM cell cycle exmintion: The trnsfected cells were digested by 2.5g L -1 trypsin to single cell suspension when they were 80% confluent, counted, cells were fixed in 700mL L -1 lcohol nd FCM ws performed. Cell growth curve: cells of trnsfected nd blnk vector groups were digested by trypsin nd counted in cells. Ech group ws inoculted in 24-well plte, nd 1ml RPMI 1640 medium contining 100mL L -1 FCS ws dded. In the culture period, cells of 3 wells were digested nd counted every dy, nd the men vlues were lso clculted. Of the reminder, the medium ws chnged every 3-4d nd counted every 6-7d ccording to the cell growth conditions, growth curves were then mde. Exmintion of plte cloning: HCC nd EC lines, which hd been trnsfected with egr-1 gene, G418 selected nd mplified were digested to single cell suspension. Cells counted were inoculted into 6-well tissue culture pltes; ech well contined 100 cells. The control cells were trnsfected with blnk vector. Chnging the medium 1 2 times per wk; by bout 2 wk pltes were tken out to observe the clone formtion; fixed in methyl lcohol, Giems stined, clones over 50 cells were counted, nd the men vlue ws dopted nd clone formtion rtes were mde. Nude mice tumorigenicity: After trypsin digestion, the cells of the experimentl nd control groups were inoculted into nude mice subcutneously with cell (bout mL) for ech mouse. About 3wk lter the mice were killed ccording to the tumor growth, nd then pictures were tken. The tumor mss ws seprted crefully nd weighed, nd the dily growing mss ws clculted then. Tumor tissues were fixed, prffin embedded, HE stined nd observed microscopiclly. Sttisticl nlysis Results were expressed s men±se. To compre the men vlues, Stt sttisticl softwre ws used. P<0.05 ws considered significnt. RESULTS Egr-1 mrna nd protein expression in HCC nd EC In situ hybridiztion nd SABC immunohistochemistry stining for egr-1 mrna nd protein in humn HCC, EC tissues nd their norml counterprts were performed. Little or no egr-1 trnscription ws detected in HCC nd norml liver tissues. Higher trnscription of egr-1 ws detected in heptocellulr prcncerous tissues such s lrge cell hyperplsi (LCD) nd trophic-like liver plte (reluctnt LCD). egr-1 mrna signl ws present in the cytoplsm. EGR-1 expression in HCC, norml liver nd heptocellullr prcncerous tissues ws consistent with the trnscription (When EGR-1expression in HCC compred with the expression in the prcncerous tissue, P= Tble 1, Figure 1). EGR-1 protein signl ws in the nucleus. Little or no egr-1 trnscription ws detected in the ggressive EC nd norml esophgel epithelil tissues (not including bsl lyer cells). Higher trnscription of egr-1 ws detected in dysplstic esophgel epithelium. EGR-1 expression in ggressive EC, norml esophgel epithelium nd dysplstic tissues ws lso consistent with the trnscription (Tble 2, Figure 2).

3 Ho MW, et l. EGR-1 inhibits liver & esophgel crcinomtous growth 205 Tble 1 egr-1 trnscription nd expression in HCC tissue nd its norml counterprts Tissue n mrna (%) n Protein (%) Norml liver 8 0(0.0) 2 0(0.0) Heptic prcncerous tissue 22 21(95.5) 5 4(80.0) Liver crcinom 24 6(25.0) 3 0(0.0) b P=0.000, b P=0.143, vs heptic prcncerous tissue Mlignnt phenotype exmintion Curves of cell growth The growth rtes of the egr-1 trnsfected HHCC liver crcinom cells nd EC109 esophgel crcinom cells were obviously slower thn tht of the controls. But the difference ws not significnt in the SMMC7721 liver crcinom cell line (Figure 3). Tble 2 egr-1 trnscription nd expression in EC tissue nd its norml counterprts Tissue n mrna(%) Protein (%) Agressive crcinom 47 8(17.0) 6(12.8) b Dysplsi (62.1) 14 (48.3) Norml epithelium 28 9(32.1) 2(7.1) P=0.000, b P=0.001, vs dysplsi Figure 3 Cell growth curve of liver nd esophgel crcinom cells trnsfected egr-1 nd vector FCM cell cycle ssy In egr-1 trnsfected HHCC, the number of frction of S phse ccounted for 0.15, lower thn tht of the controls (0.28, P=0.038); i. e., the proportion of S phse ws 0.46 lower thn tht of the control (Tble 3). In EC109, the number of frction of S phse ccounted for 0.18 (control 0.27, P=0.048), nd ws 0.34 lower thn tht of the control. In SMMC7721 (HCC), there ws no mrked difference between the experimentl nd the control groups (0.21 to 0.22, P=1.000). As for the G 1 nd G 2 /M phses, there were neither significnt differences between experiment groups nd control groups. Tble 3 Cell cycle of HCC nd EC cells expressing EGR-1 with FCM (number of frction) Cell clone G 1 G 2/M S Figure 1 Positive signl of egr-1 mrna in the cytoplsm prcncerous trophic-like liver plte tissue of HCC. SP 400 Figure 2 Positive signl of EGR-1 mrna in the cytoplsm of modertely dysplstic esophgel epithelium. SP 400 Egr-1 mrna nd protein detection in trnsfected tumor cells mrna level (mrna dot blot): The totl RNA of egr-1 nd vector trnsfected HCC nd EC cells were dot hybridized by 32 P lbeled egr- 1 probe. The result reveled tht egr-1 group hd stronger signls thn tht of the control. Western blot nd immunohistochemistry: The proteins of egr-1 nd vector trnsfected HCC nd EC cells were extrcted nd quntittively nlyzed. Then both groups with identicl quntittive proteins were determined by SDS-PAGE; electricl trnsfer onto the nitro-cellulose membrne ws mde, nd Western blot hybridiztion ws performed. The results reveled tht in egr-1 trnsfected groups of both HCC nd EC cells, there were strongly stined bnds just t the moleculr mss of 59ku, which ws identicl to tht of EGR-1, wheres in ll the controls there were no such bnds present. This suggested tht the tumor cells trnsfected by egr-1 could strongly express EGR-1 protein. The results of immunohistochemistry by primry ntibody EGR-1 nd SABC method showed 50-70% positive stining of egr-1 trnsfected cells were, while in the controls the results were ll negtive; which ws consistent with tht of the Western blot. HHCC/EGR HHCC/ϕ SMMC7721/EGR b SMMC7721/ϕ EC109/EGR c EC109/ϕ P=0.038, b P=1.000, c P=0.048, vs their controls Plte cloning formtion In HHCC cell line, the clone forming rte of trnsfected cells ws 49.0±10.6%, 42.7% lower thn tht of control 91.7±6.1%. In EC109 line, the clone forming rte of trnsfected cells ws 45.0±5.3%, 32.3% lower thn tht of control 77.3±3.1%. In SMMC7721, There were no sttisticl difference between the experimentl group 26.0±2.6% nd the control group 23.3±2.1% (Figure 4). Figure 4 Clone formtion rtes of exogenous high EGR-1 expression HCC nd EC

4 206 ISSN CN / R World J Gstroenterol April 15, 2002 Volume 8 Number 2 Nude mice tumorigenicity All the inoculted nude mice hd tumor mss growth except in one cse (one of the three SMMC7721/ϕ inoculted mice). In HHCC cell line, the tumor growth rte of trnsfected group ws 80.7%, lower thn tht of the control (P=0.001). In EC109 cell, the growth rte of trnsfected group ws 72.6%, lso lower thn tht of the control (P=0.001). In SMMC7721, there ws no growth rte difference between the experimentl nd the control group(p=0.681)(tble 4). Tble 4 Nude mice tumorigenicity of gene trnsfected groups in HCC nd EC cells Cell clone n Tumorigenicity rte Tumor growth rte (x±s, mg d -1 ) HHCC/EGR-1 3 3/3 13.9±5.2 HHCC/ϕ 3 3/3 70.9±7.8 SMMC7721/EGR-1 3 3/3 18.7±4.3 b SMMC7721/ϕ 3 2/3 15.2±13.0 EC109/EGR-1 4 4/4 12.9±2.1 c EC109/ϕ 4 4/4 47.1±11.3 P=0.001, b P=0.681, P=0.001, vs their controls DISCUSSION Althrough egr-1 is member of immedite erly genes, its expression is lso elevted during the process of growth [4] nd differentition [5]. Observtions suggest tht EGR-1 hve pleiotropic roles such s growth, development, differentition etc. This is Supported by the finding tht the GC-rich DNA-binding element for egr-1 (GCE) is present in lrge number of gene regulting regions, including growth fctors, signl trnsduction genes, other trnscription fctors nd oncogenes. In this report, we showed tht egr-1 trnscription nd expression decresed obviously in HCC nd EC tissues compred with their prcncerous tissues. Another (lrge) study lso indicted tht humn smll cell lung tumors express little or no egr-1 mrna compred with djcent norml tissues [40], further supporting our findings. Except tht the egr-1 gene ws deleted only in ll 5q - syndrome cses [41], egr-1 expression ws profoundly decresed in number of other humn tumor cell lines such fibroblstom, glioblstom, osteosrcom nd lung cncer [6].Therefore it seems likely tht the inctivtion of egr-1 expression is generlized phenomenon in the developmentl process of number of tumors. Egr-1 is ubiquitously expressed t low levels but ccumultes to reltively high levels in only few dult orgns s brin, hert, lung, kidney [42] nd brest [1]. We showed here tht norml liver nd esophgel epithelil tissues expressed undetectble level of egr-1, but the egr-1 gene ws ctivted in the tissues of heptic prcncerous tissue such s LCD nd trophic liver plte (reluctnt LCD) nd dysplstic tissue of esophgel crcinom. During regenertion of norml liver tissue, incresed egr-1 expression is induced [43]. In mouse fibroblst NIH3T3 cells, egr-1 expression increses two-fold 10min fter UV irrdition, which rises to mximum (eightfold induction) fter 2h [6]. Humn HT1080 fibrosrcom subclone, cells H4, express little or no egr-1. Phorbol ester tretment only cn elicit smll increse in egr-1 expression in H4, in contrst to the normlly rpid, high trnsient expression of egr-1 observed fter the ddition of wide rnge of stimulting gents to norml or immortlized cell lines [7]. Therefore, the mening of little or no egr-1 expression in liver nd esophgel crcinoms is different to their counterprts. egr-1 gene in the heptic prcncerous tissue such s LCD nd dysplsi of esophgel crcinom cn be ctivted when they re stimulted, but the stimultions is invlidted in crcinoms. The decresed expression of egr-1 my ply role in the dysregultion of norml growth in the cncerous process of HCC nd EC. To confirm the development of liver nd esophgel crcinoms involves the inctivtion of egr-1. It would deserve further investigtion of the effects of exogenous EGR-1 on the growth nd mlignnt phenotypes of the cncer. Like tumor suppressor gene WT1, EGR-1 cn lso bind with GCE elements [44], this suggests tht EGR-1 might hve inhibitory ctivity on cncer growth. In the experimentl report [8], EGR-1 could reverse the mlignnt phenotype of v-sis trnsformed NIH3T3 cells. The results of this pper lso showd tht stbly expressed EGR-1 could inhibit the cncer s growth rte, reduce the number of frction of S phse, decrese the plte clone formtion rte nd reduce the tumorigencity of HHCC nd EC109 cell lines. In recent yers, studies hve reveled tht in humn tumors, EGR-1 expression suppressing tumor cell growth is common phenomenon. In humn HT-1080 fibrosrcom cells, the growth rtes in 6 groups of expressing different EGR-1 level re inhibited proportiontely nd re dose-dependent. The totl inhibition rte of these cells tumorigencity is over 50%. The growth of humn ZR-75-1 brest crcinom, U251 glioblstom nd SAOS-2 osteosrcom cells hs lso been demonstrted to be suppressed by EGR-1 in different degree [6]. However, in this experiment EGR-1 did not suppress the mlignnt phenotypes of SMMC7721, HCC cell line, this might be due to different trnsformtion mechnism in this cell line, which needs investigtion further. The mechnism of EGR-1 inhibition on cncer growth is complicted, which intercts with TGF-β1, PDGF nd WT1. TGFβ1 specificlly inhibits PDGFβ-dependent cells growth [4], while EGR- 1 inhibits the mlignnt phenotype nd growth of the PDGFβ/v-sis tht were trnsformed from NIH3T3 cells. This suggests tht EGR-1 is closely relted with TGF-β1 nd PDGF. Menwhile, EGR-1 cn lso ctivte the trnscription of TGF-β1 in dose-dependent mnner, this ctivtion might be one of the potentil mechnisms of EGR-1 of inhibiting tumor growth [45]. Interestingly, WT1 cn bind with the GC element of TGF-β1 promoter, nd then inhibit the ctivity of tht promoter (but this rection is weker thn tht of inhibition on PDGFβ). One recent study hs shown tht the inhibitory rection of EGR-1 on cncer growth is lso by inducing the expression of TGF-β1, fibronectin, p21 nd focl dhesion kinse (FAK) in humn fibrosrcom cell line [46]. EGR-1 my lso regulte cell interction with the extrcellulr mtrix by synergistic induction of TGF-bet1, FN, nd PAI-1 in humn HT-1080 glioblstom cells [47] nd directly trnsctivtes the fibronectin gene nd enhnces ttchment of humn glioblstom cell line U251 [48]. Other reports show tht the suppression by egr-1 lso involves down-regultion of bcl-2 [49] nd stimultes poptosis by trnsctivtion of the p53 gene [50]. Recent studies [6, 8, 50] s well s this experiment suggest tht EGR- 1 cn ct s tumor suppressor fctor. EGR-1 hs been identified to be little or no expression in severl humn mlignnt tumors such s fibroblstom, glioblstom, osteosrcom, lung cncer, brest crcinom (including cncer cells nd tumor tissues) [1,6], s well s heptocellulr crcinom nd esophgel crcinom in this experiment. Another (lrger) study lso indictes tht humn smll cell lung tumors express little or no egr-1 mrna compred with djcent norml tissues [40]. Furthermore, 5q-syndrome is the exclusive tumor with egr-1 gene deletion [41]. Hence we suspect tht egr-1 might be belonged to typeii tumor suppressor genes s mentioned bove. The reintroduction of egr-1 gene products by drugs might be promising pproch to normlize growth regultion, which cn void the difficult problem of replcing defective gene DNA. AKNOWLEDGEMENT Thnks to Prof. Bo-Rong Pn in the Oncology Center of Xijing Hospitl for his help in the preprtion of this rticle. REFERENCES 1 Hung RP, Fn Y, de-belle I, Niemeyer C, Gottrdis MM, Mercol D, Admson ED. Decresed egr-1 expression in humn, mouse nd rt mmmry cells nd tissues correltes with tumor formtion. Int J Cncer 1997;72: Drlnd T, Smuels M, Edwrds SA, Sukhtme VP, Admson ED. Regultion of Egr-1 (Zfp-6) nd c-fos expression in differentiting embryonl crcinom cells. Oncogene 1991;6: Gshler AL, Swminthn S, Sukhtme VP. A novel repression module, n extensive ctivtion domin, nd biprtite nucler locliztion signl defined in the immedite-erly trnscription fctor egr-1. Mol Cell Biol

5 Ho MW, et l. EGR-1 inhibits liver & esophgel crcinomtous growth ;13: Liu C, Clogero A, Rgon G, Admson E, Mercol D. EGR-1, the reluctnt suppression fctor: EGR-1 is known to function in the regultion of growth, differentition, nd lso hs significnt tumor suppressor ctivity nd mechnism involving the induction of TGF-bet1 is postulted to ccount for this suppressor ctivity. Crit Rev Oncog 1996;7: Nguyen HQ, Hoffmn Liebermnn B, Liebermnn DA. The zinc finger trnscription fctor egr-1 is essentil for nd restricts differentition long the mcrophge linege. Cell 1993;7: Hung RP, Liu C, Fn Y, Mercol D, Admson ED. egr-1 negtively regultes humn tumor cell growth vi the DNA-binding domin. Cncer Res 1995;55: Liu C, Admson E, Mercol D. Trnscription fctor EGR-1 suppresses the growth nd trnsformtion of humn HT-1080 fibrosrcom cells by induction of trnsforming growth fctor bet1. Proc Ntl Acd Sci USA 1996;93: Hung RP, Drlnd T, Okmur D, Mercol D, Admson ED. Suppression of v-sis-dependent trnsformtion by the trnscription fctor, egr-1. Oncogene 1994;9: Tng ZY. Schmid. Advnces in clinicl reserch of heptocellulr crcinom in Chin. World J Gstroenterol 1998;4(Suppl 2): Wu XY, Zhng XF, Yin FS, Lu HS, Gun GX. Clinicl study on surgicl tretment of esophgel crcinom in ptients fter subtotl gstrectomy. World J Gstroenterol 1998;4(Suppl 2): Bi YM, Wng LD, Seril DN, Lio J, Yng GY, Yng CS. Expression of hmsh2 in humn esophgel cncer from ptients in high incidence re in Henn, Chin. World J Gstroenterol 1998; 4(Suppl 2): Qi YJ, Wng LD, Nie Y, Ci C, Yng GY, Xing EP, Yng CS. Altertion of p19 mrna expression in esophgel cncer tissue from ptients t high incidence re in northern Chin. World J Gstroenterol 1998; 4(Suppl 2): Qin QJ, Xue HB, Qu ZQ, Fng SG, Co HF, Wu MC.in situdetection of tumor infiltrting lymphocytes expressing perforin nd fs lignd genes in humn HCC. World J Gstroenterol 1999; 5: Go SS,Zhou Q,Li YX,Bi YM,Zheng ZY,Zou JX,Liu G,Fn ZM,Qi YJ, Zho X,Wng LD.Comprtive studies on epithelil lesions t gstric crdi nd pyloric ntrum in subjects from high incidence re for esophgel cncer in Henn,Chin. World J Gstroenterol 1998;4: Ling YR, Zheng SY, Shen YQ, Wu XY, Hung ZZ. Reltionship between expression of poptosis relted ntigens in heptocellulr crcinom nd in situ end lbeling. World J Gstroenterol 1998; 4: Xio L, Zhou HY, Luo ZC, Liu J. Telomeric ssocitions of chromosomes in ptients with esophgel squmous cell crcinoms. World J Gstroenterol 1998; 4: Hung B, Wu ZB, Run YB. Expression of nm23 gene in heptocellulr crcinom tissue nd its reltion with metstsis. World J Gstroenterol 1998; 4: Sun HC, Li XM, Xue Q, Chen J, Go DM, Tng ZY. Study of ngiogenesis induced by metsttic nd non metsttic liver cncer by cornel micropocket model in nude mice. World J Gstroenterol 1999; 5: Luo YQ, Wu MC, Cong WM. Gene expression of heptocyte growth fctor nd its receptor in HCC nd nontumorous liver tissues. World J Gstroenterol 1999; 5: Liu CG, Zhu MC, Chen ZN. Preprtion nd purifiction of F(b ) 2 frgment from nti heptom mouse IgG1 mab. World J Gstroenterol 1999; 5: M XD, Sui YF, Wng WL. Expression of gp junction genes connexin 32, connexin 43 nd their proteins in heptocellulr crcinom nd norml liver tissues. World J Gstroenterol 2000; 6: Tng YC, Li Y, Qin GX. Reduction of tumorigenicity of SMMC-7721 heptom cells by vsculr endothelil growth fctor ntisense gene therpy. World J Gstroenterol 2001; 7: Feng DY,Zheng H,Tn Y,Cheng RX.Effects of phosphoryltion of MARK nd Stt3 nd expression of c-fos nd c-jun proteins on heptocrcinogenesis nd their clinicl significnce. World J Gstroenterol 2001; 7: Fn ZR, Yng DH, Cui J, Qin HR, Hung CC. Expression of insulinlike growth fctor II receptor in heptocellulr crcinogenesis. World J Gstroenterol 2001; 7: Yng SM, Zhou H, Chen RC, Wng YF, Chen F, Zhng CG, Zhen Y, Yn JH, Su JH. Sequencing of P53 muttion in estblished humn heptocellulr crcinom cell line of HHC4 nd HHC15 in nude mice. World J Gstroenterol 1998; 4: Luo F, Kn B, Lei S, Yn LN, Mo YQ, Zou LQ, Yng YX, Wei YQ. Study on P53 protein nd C erbb2 protein expression in primry heptic cncer nd colorectl cncer by flow cytometry. World J Gstroenterol 1998; 4(Suppl 2): Zhou Q, Wng LD, Go SS, Li YX, Zho X, Wng LX. P53 immunostining positive cells correlted positively with S phse cells s mesured by BrdU in the esophgel precncerous lesions from the subjects t high incidence re for esophgel cncer in northern Chin. World J Gstroenterol 1998; 4 (Suppl 2): Feng DY, Chen RX, Peng Y, Zheng H, Yn YH. Effect of HCV NS3 protein on P53 protein expression in heptocrcinogenesis. World J Gstroenterol 1999; 5: Csson AG. Moleculr biology of Brrett's esophgus nd esophgel cncer: role of P53. World J Gstroenterol 1998;4: Wng LD, Zhou Q, Wei JP, Yng WC, Zho X, Wng LX, Zou JX, Go SS, Li YX, Yng CS. Apoptosis nd its reltionship with cell prolifertion, P53, Wf1p21, bcl-2 nd c-myc in esophgel crcinogenesis studied with high risk popultion in northern Chin. World J Gstroenterol 1998;4: Yu GQ, Zhou Q, Ding I, Go SS, Zheng ZY, Zou JX, Li YX, Wng LD. Chnges of P53 protein blood level in esophgel cncer ptients nd norml subjects from high incidence re in Henn, Chin. World J Gstroenterol 1998; 4: Deng ZL, M Y. Afltoxin sufferer nd P53 gene muttion in heptocellulr crcinom. World J Gstroenterol 1998; 4: Qio GB, Hn CL, Jing RC, Sun CS, Wng Y, Wng YJ. Overexpression of P53 nd its risk fctors in esophgel cncer in urbn res of Xi n. World J Gstroenterol 1998; 4: Luo D, Liu QF, Gove C, Nomov NV, Su JJ, Willims R. Anlysis of N rs gene muttion nd P53 gene expression in humn heptocellulr crcinoms. World J Gstroenterol 1998; 4: Peng XM, Peng WW, Yo JL. Codon 249 muttions of P53 gene in development of heptocellulr crcinom. World J Gstroenterol 1998; 4: Jio LH,Wng LD,Xing EP,Yng G,Yu Y,Yng CS.Frequent inctivtion of P16 nd P15 expression in humn esophgel squmous cell crcinom detected by RT-PCR. World J Gstroenterol 1998;4(Suppl 2): Liu LH, Xio WH, Liu WW. Effects of 5-Az-2'-deoxycytidine on the P16 tumor suppressor gene in heptocellulr crcinom cell line HepG2. World J Gstroenterol 2001; 7: Qin LL, Su JJ, Li Y, Yng C, Bn KC, Yin RQ. Expression of IGFII, p53, P21 nd HBxAg in precncerous events of heptocrcinogenesis induced by AFB1 nd/or HBV in tree shrews. World J Gstroenterol 2000; 6: Mltzmn JS, Crmn JA, Monroe JG. Role of EGR1 in regultion of stimulus-dependent CD44 trnscription in B lymphocytes. Mol Cell Biol 1996; 16: Levin WJ, Press MF, Gynor RB, Sukhtme VP, Boone TC, Reissmnn PT, Figlin RA, Holmes EC, Souz LM, Slmon DJ. Expression ptterns of immedite erly trnscription fctors in humn non-smll cell lung cncer. The Lung Cncer Study Group. Oncogene 1995; 11: Le Beu MM, Espinos R, Neumn WL, Stock W, Roulston D, Lrson RA, Keinnen M, Westbrook CA. Cytogenetic nd moleculr delinetion of the smllest commonly deleted region of chromosome 5 in mlignnt myeloid diseses. Proc Ntl Acd Sci USA 1993; 90: Herms J, Zurmohle U, Schlingensiepen R, Brysch W, Schlingensiepen KH. Developmentl expression of the trnscription fctor zif268 in rt brin. Neurosci Lett 1994; 165: Columbno A, Shinozuk H. Liver regenertion versus direct hyperplsi. FASEB J 1996; 10: Ruscher FJ 3d, Morris JF,Tourny OE, Cook DM, Currn T. Binding of the Wilms tumor locus zinc finger protein to the EGR-1 consensus sequence. Science 1990; 250: Dey BR,Sukhtme VP,Roberts AB,Sporn MB,Ruscher FJ 3rd,Kim SJ. Repression of the trnsforming growth fctor-bet 1 gene by the Wilms' tumor suppressor WT1 gene product. Mol Endocrinol 1994;8: Belle I, Hung RP, Fn Y, Liu C, Mercol D, Admson ED. P53 nd egr-1 dditively suppress trnsformed growth in HT1080 cells but egr- 1 countercts P53-dependent poptosis. Oncogene 1999; 18: Liu C, Yo J, de Belle I, Hung RP, Admson E, Mercol D. The trnscription fctor EGR-1 suppresses trnsformtion of humn fibrosrcom HT1080 cells by coordinted induction of trnsforming growth fctor-bet1, fibronectin, nd plsminogen ctivtor inhibitor-1. J Biol Chem 1999; 274: Liu C, Yo J, Mercol D, Admson E. The trnscription fctor EGR-1 directly trnsctivtes the fibronectin gene nd enhnces ttchment of humn glioblstom cell line U251. J Biol Chem 2000; 275: Hung RP,Fn Y,Peng A,Zeng ZL,Reed JC,Admson ED,Boynton AL. Suppression of humn fibrosrcom cell growth by trnscription fctor, egr-1,involves down-regultion of Bcl-2. Int J Cncer 1998;77: Liu C, Rngnekr VM, Admson E, Mercol D. Suppression of growth nd trnsformtion nd induction of poptosis by EGR-1. Cncer Gene Ther 1998; 5: 3-28 Edited by Wu XN