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Increassed susceptibility of rat cholangiocytes to tumor necrosis factor α (TNFα) cytotoxicity following bile duct ligation
1859 Brain-Specific Angiogenesis Inhibitor 1 (BAIl) Inhibits the Angingenesis of Hepatocellular Carcinoma (HCC). Fumihiko Komine, HikarigaokaHosp Nihon Univ Sch of Medicine, Tokyo Japan; Kenzaburo Tani, Xixiong Kang, Yusuke Nakamura, Institute of Medical Science, Univ of Tokyo, Tokyo Japan; Masaki Shimojima, HikarigaokaHosp Nihon Univ Sch of Medicine, Tokyo Japan; Tamiko Saito, Hiroshi Aoki, Toshihiro Shimizu, Yasuyuki Arakawa, Nihon Univ Scit of Medicine, Tokyo Japan; ShigetakaAsano, Institute of Medical Science, Univ of Tokyo, Tokyo Japan; Yukimoto Ishii, Nihon Univ Sch of Medicine, Tokyo Japan BACKGROUND/OBJECTIVESHCC develops mostly from chronic viral diseases, and it is characterizedby its multicentric carcinogenesis.During repeatedtreatments, most of patients die as a result of hepatic insufficiency. In addition, no treatment is available for advanced HCCs at present. The possibility of liver transplantation is limited becauseof a deficiency of donors. We are developinga new treatment by meansof an angiogenesisinhibitor to suppress progression of cancers and bring them into a dormant state while maintaining the functional reserve of the liver intact. METHODSWe examined, using the Northern blotting method, the presence or absence of expression of brain-sgecific angioganesis inhibitor 1 (BAIl) in HCC cell lines (HUH7, HepG2, PLC/PRF/5, HLF). Additionally, we examinedthe expressionof BAIl in cancerous and non-cancerous regions (but with cirrhosis) in specimens excised from 8 cases with HCC. Then, we examined, using the PCR method, the homology between the nucleotide sequenceof BAIl mRNA in the brain and that in HCC. In addition, we constructed a recombinantadenoviral vector, which contained the BAIl cDNA (AdBAI1), and tran~ucted this gene into the HCCcells ex vivo. Human HCCcells infected with AdBAIls were transplanted into SCID mice with skin-chambers in order to observe the angiogenesisin vivo. RESULTS In normal tissues, BAIl has been reported to be expressedonly in the brain. In the present study, its expression was found in all of the HCC cell lines. In 8 patients, BAIl mRNA was expressed in 6 cancerous regions and 4 non-cancerous regions. But the expression in noncancerous regions were weaker than cancerous regions in the same patient. Interestingly, the nucleotidesequencesof BAIl mRNAin the brain and in HCCwere found to be homologous. The angiogenesiswas found to be suppressedmore markedlyin HCCcells infectedwith AdBAI1 than in non-infected HCC cells. CONCLUSIONSThe angiogenesisof HCC was suppressed by transduction of the BAIl gene into cancer cells. We are planning to make muitifocal HCC mice models by splenic transplantation of humanhepatomacells to NOD/SCIDmice followed by intravenousadministration of BAI-1 edenovirusa few weeks later. The experimentalmodel would clarify whether the BAIl gene therapy HCC is helpful to suppress in vivo as a dormancy therapy.
1860 The Status Of The p53 Geee in Human Hepatocellular Carcinoma Cells influences The Responses Of Cell Cycle-Associated Gene (Cyclin A, CDC2, RB, p21) By Geee Transfer Of p53. Daiju Nakayama,Yasushi Magami, Masaya Furukawa, Fuminori Modyasu, Tokyo Medical Univ, Tokyo Japan; Toshio Nikaido, Sch of Medicine, Shishu Univ, Matsumoto Japan; Toshiyuki Sakai, Kyoto Prefectural Univ of Medicine, Kyoto Japan Backgroundand purpose: Hepatocellularcarcinomasare among the most common malignancies in the world, and prognosis is very poor. It has been shown that 60% of hepatncellular carcinomas have mutant type p53, and recently it's treatment has been attempted by using p53 gene. However, the relationship and interaction betweenthe expression of various cell cycle-associatedgenes and the introduction of p53 gene in the tumorigenesis and growth of hepatocellularcarcinomas remains poorly understood.To clarify the difference of the effect ot the p53 gene on hepatocellularcarcinomas with different p53 gene status, we examined the transcriptional activities of cell cycle-associatedgenes(cyclinA, RB, cdc2, p21) by transtection with p53 gene. Method: The hepatocellularcarcinoma cell lines examinedto determine cyclinA, RB, cdc2 and p21 promoter activities were HepG2, HLE, HLF, Huh6, Huh7 and PLC. Mutation of p53 has been reported in HLE, HLF, Huh6, Huh7 and PLC, and wild type p53 in HepG2. A plasmid expressingwild or mutant type p53 was cotransfected into these cell lines with cyclinA, RB, cdc2, p21 fused to luciferasecDNAusing lipofectin, then 24 h after completion of transfection, cyclinA, RB, cdc2, p21 promoter activity levels were determined by luciferase assay. Results: CyclinA, RB and cdc2 promoter activities were remarkablysupressed by wild type p53 in all cell lines. On the other hand, p21 promoter activity showed various responses. Wild type p53 upregulated p21 promoter activity in HLF, Huh6, Huh7 and PLC, however it was downregulatedin HepG2and HLE. Mutant type p53 upregulatedcyclinA promoter activity in HepG2,HLE, HLF, Huh6, and RB promoter activity in Huh6, Huh7, and cdc2 promoter activity in all cell lines. Conclusion: CyclinA, RB and cdc2 promoter activities were downregulated in all cell lines due to introduction of wild type p53. The p21 promoter activity was downregulated by introduction with wild type p53 in cell line with normal for p53 status, and upregulated in cell lines with mutation of p53, except for HLE.
1861 An NO-derivative of Acataminophen, NCX-701, Spores the Liver By Inhibiting Fasdependent Apoptosis Stefano Fiorucci, ElisabettaAntonelli, Andrea Mencarelli, Barbara Palazzetti,Univ of Perugia, Perugia Italy; Piero Del Soldato, Sophia Antipolis, Nice France; Antonio Morelli, Univ of Perugia, Perugia Italy Background.Acetaminophen(APAP) is a widely used analgesic and antipyretic that can lead to severe liver damagewhen taken at excessivedoses. APAP overdose is a leading cause of liver transplantation in Western countries. NCX-701 is an NO, nitric oxide, derivative of APAP with enhanced anti-inflammatory and analgesic activity. Aim. To investigate whether NCX701 sparethe liver and define mechanism involved in protection. Methods. BALB/c mice were injected with increasing doses, 100, 300 and 500 mg/kg, of APAP or NCX-701. Both drug were also administered at the same doses in animals with hepatitis caused by concanavaltin A (Con A). Results. APAP, but not NCX-701 caused a dose-dependentliver toxicity. At the dose of 500 mg/kg it caused an _< fold increase of AST plasma levels and liver necrosis.
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NCX-701, at any dose, did not increase AST release.APAP, but not NCX-701, caused IL-1,8, IFN-/and TNFa release and upregutatedliver Fas/FasL mRNA expression. In animals treated with Con A, APAPworsenedthe severityof liver injury. In contrast, NCX-701 inhibited cytokine releaseand liver Fas/FasLmRNAexpressionand reverteddamageinducedby Con A. In isolated mouse hepatocytes,APAP, but not NCX-701, increasedthe translocation of cytoplasmic Fas to the cell surface, resulting in an Fas expression as measured at flow cytometry analysis and increased sensitivity to the Fas agonistic monoclonal antibody Jo2. Incubating mouse hepatocyteswith NCX-701 resulted in a concentration and time-dependentformation of NO. Conclusions. NCX-701, an NO-derivativeof APAP,spares the liver in intact and compromised animals by inhibiting pro-inflammatory cytokine release and Fas-dependentapoptosis and inflammation by acting at several checkpoints in the Fas pathway.
TraMfonMng Growth Factor Beta-induced Apoptosis in Primary Murine Hepatocytes
is Cmqsuee-8Dependent. Charles M. Samson, Patty A. Lange, Mark A. Bird, Melissa A. Hayden,David A. Brenner, Kevtn E. Bahms, Univ of North Carolina at Chapel Hill, Chapel Hill, NC Background:TGF,8regulateshepatocytedifferentiation, growth, fibrosis, and apoptosis.TGF/~induced apoptosis appearsto be caspasedependent.The precise caspasasinduced by TGF/~, however, have not yet been well characterized.Aims: To determinethe necessityof easpase3 and caspase-8in TGF/]-induced apoptosis. We hypothesizedthat TGF/3-inducedapoptosis would be cospase-3 dependent, but caspase-8 independent. Methods: Hepatocytes were isolated from BALB/c mice by collagenaseperfusion. After plating, cells were serum starved and hepatocyteswere pretreated either with 1 p.M DEVD-CHO(caspase-3 inhibitor), 1 /~M Z-IETD-FMK (caspase-8 inhibitor) or 1 p.M DMSO (control). Two hours later, 5 ng/ml of recombinant human TGF,8 cytokine was added. Isolated DNA was separated on a 1.5 % agarose gel to detect DNA laddering. Other markers used to detect hepatocyte apoptosis includedprOpldium iodine (PI) staining for morphologic assessmentand PARPcleavagefor caspaseinvolvement. Protein from whole cell extracts were separatedon a 10% SDS gel and western blot analyses ware performed for cytochrome c release.Caspase-3and -8 specific activities ware measured by detecting cleavageof the fluorometric substrates DEVD-AFCand LETDoAFC,respectively. Results: Apoptosis in primary murine hepatocytes was confirmed within 48 h of TGF/]treatment by biochemicalassessmentincluding DNA ladderingand PARP cleavage, and morphologically by PI staining. Cytochrome c release was detected within 24 h of treatment. Both caspases-3 and -8 were activated at 48 h, with larger increases in activities by 72 h. Hepatocytaapoptosis was blockedwith the pretreatmentof either caspase3 or-8 inhibitor. Pretreatmentwith caspase-8inhibitor not only blockedTGF~induced caspase8 aclhrahon, but it also blocked caspase-3 activation. Conversely, inhibition of caspase-3 decreasedbut did not abolish caspase-8activity. Summary: TGF/Yinduces murine hepatocyte apoptosis with cytochrome c releaseand activation of both caspases-3and -8. Inhibition of either caspase-3 or caspase-8 blocks hepatocyteapoptosis. Caspase-3activity is caspase-8 dependent, as pre~eatment with the caspasa-8 inhibitor blocks TGF,8-induced caspase-3 activation.C a s ~ activity, however,is not caspase-3dependent.Conclusion: In conclusion, caspaea-8inhibition blocks both TGF/Y-inducedapoptosisand caspase-3activation.Therefore, TGF/Y-indueadapoptosis in murine hepatocytesis dependenton caspase-8activation.
InuSased Susceptibility of Rat Choluegiocytesto Tumor Necrosis Factora (TNFa) Cl~Modcity Following Bile Ouct LiBation. Giardraoco A/pini, Laura Tadiock, ShannonGlaser, Gene Lesage, Tushar Patel, Scott and White Clinic; Texas A&M Univ Sys Hlth Sci Ctr, Temple, TX Cholangiocyteinjury during cholestasishas beenassociatedwith activation of death receptors such as TNFa and Fas.Although these receptorsinduce cell deaththrough similar intracellular pathways,their individual contributions to cholangiocyteinjury during biliary tract obstruction remain unknown. Our AIMS were to assess the relative roles of the death receptors TNF~ and Fas in mediating cholangiocyteinjury following experimentalbile duct obstruction, and to identify cellular mechanismsdeterminingsusceptibilityto deathreceptor mediatedcytotoxicity. METHODS:Cholangiocyteswere isolatedfrom normal or 1-week bile duct ligated (BDL) rats. TNF-R1 receptorexpressionwas determinedby Western blot analysis. Cytotoxicityin response to TNF~ or egonistic antibodiesto the Fas receptor (anti-Fas) was assessedusing a tetrazolium salt bioreduction assay for viable cells. Actinomycin O was used as an inhibitor of RNA synthesis. Expressionof Fas and FasL mRNA, as well as mRNA expressionof the apoptosisregulatorygenes, Baxand Bcl-2, were detectedby ribonucleaseprotection assays,and quantitated relativeto GAPDHexpression.RESULTS:Expressionof the TNF-R1 receptorwas markedly increased in cholangiocytesfrom BDL rats as comparedto normal. However,cholangiocytes from both normal and bile duct ligated rats were highly resistant to TNFa (0-100 og/ml) as well as to Fas receptor stimulation with anti-Fas (0-10 pg/ml). Co-incubation with 1 /.;,M Actinomycin D sensitized cholangiocytesfrom BDL rats, but not normal cholangiocytes,to TNFatoxicity. The ratio of Bcl-2 to Bax geneexpressionwas 30% lower in BDL cholangiocytes compared to normal cholangiocytes.However,the sensitivity of BDL cholangiocytesto antiFascytotoxicity was not altered by Actinomycin D. Fas mRNAexpressionin BDL cholangiocytes was decreased to approx. 65% of normals, but FasL mRNA expression was not altered. SUMMARYand CONCLUSIONS:Cholangiocytesare susceptibleto TNFa but not Fas cytotoxicity by inhibition of RNA synthesis following BDL. Although the relative expression of the proapoptotic Bax gene is increasedfollowing BDL, Fas mRNA expression is decreasedand the sensitivity of BDL cholangiocytesto Fasstimulation is not altered.Theseobservationssuggest that TNFa but not Fasmediatescholangiocyteinjury and that down-regulationof Fasexpression may represent a protective mechanism during biliary tract obstruction.
1860 The Status Of The p53 Geee in Human Hepatocellular Carcinoma Cells influences The Responses Of Cell Cycle-Associated Gene (Cyclin A, CDC2, RB, p21) By Geee Transfer Of p53. Daiju Nakayama,Yasushi Magami, Masaya Furukawa, Fuminori Modyasu, Tokyo Medical Univ, Tokyo Japan; Toshio Nikaido, Sch of Medicine, Shishu Univ, Matsumoto Japan; Toshiyuki Sakai, Kyoto Prefectural Univ of Medicine, Kyoto Japan Backgroundand purpose: Hepatocellularcarcinomasare among the most common malignancies in the world, and prognosis is very poor. It has been shown that 60% of hepatncellular carcinomas have mutant type p53, and recently it's treatment has been attempted by using p53 gene. However, the relationship and interaction betweenthe expression of various cell cycle-associatedgenes and the introduction of p53 gene in the tumorigenesis and growth of hepatocellularcarcinomas remains poorly understood.To clarify the difference of the effect ot the p53 gene on hepatocellularcarcinomas with different p53 gene status, we examined the transcriptional activities of cell cycle-associatedgenes(cyclinA, RB, cdc2, p21) by transtection with p53 gene. Method: The hepatocellularcarcinoma cell lines examinedto determine cyclinA, RB, cdc2 and p21 promoter activities were HepG2, HLE, HLF, Huh6, Huh7 and PLC. Mutation of p53 has been reported in HLE, HLF, Huh6, Huh7 and PLC, and wild type p53 in HepG2. A plasmid expressingwild or mutant type p53 was cotransfected into these cell lines with cyclinA, RB, cdc2, p21 fused to luciferasecDNAusing lipofectin, then 24 h after completion of transfection, cyclinA, RB, cdc2, p21 promoter activity levels were determined by luciferase assay. Results: CyclinA, RB and cdc2 promoter activities were remarkablysupressed by wild type p53 in all cell lines. On the other hand, p21 promoter activity showed various responses. Wild type p53 upregulated p21 promoter activity in HLF, Huh6, Huh7 and PLC, however it was downregulatedin HepG2and HLE. Mutant type p53 upregulatedcyclinA promoter activity in HepG2,HLE, HLF, Huh6, and RB promoter activity in Huh6, Huh7, and cdc2 promoter activity in all cell lines. Conclusion: CyclinA, RB and cdc2 promoter activities were downregulated in all cell lines due to introduction of wild type p53. The p21 promoter activity was downregulated by introduction with wild type p53 in cell line with normal for p53 status, and upregulated in cell lines with mutation of p53, except for HLE.
1861 An NO-derivative of Acataminophen, NCX-701, Spores the Liver By Inhibiting Fasdependent Apoptosis Stefano Fiorucci, ElisabettaAntonelli, Andrea Mencarelli, Barbara Palazzetti,Univ of Perugia, Perugia Italy; Piero Del Soldato, Sophia Antipolis, Nice France; Antonio Morelli, Univ of Perugia, Perugia Italy Background.Acetaminophen(APAP) is a widely used analgesic and antipyretic that can lead to severe liver damagewhen taken at excessivedoses. APAP overdose is a leading cause of liver transplantation in Western countries. NCX-701 is an NO, nitric oxide, derivative of APAP with enhanced anti-inflammatory and analgesic activity. Aim. To investigate whether NCX701 sparethe liver and define mechanism involved in protection. Methods. BALB/c mice were injected with increasing doses, 100, 300 and 500 mg/kg, of APAP or NCX-701. Both drug were also administered at the same doses in animals with hepatitis caused by concanavaltin A (Con A). Results. APAP, but not NCX-701 caused a dose-dependentliver toxicity. At the dose of 500 mg/kg it caused an _< fold increase of AST plasma levels and liver necrosis.
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NCX-701, at any dose, did not increase AST release.APAP, but not NCX-701, caused IL-1,8, IFN-/and TNFa release and upregutatedliver Fas/FasL mRNA expression. In animals treated with Con A, APAPworsenedthe severityof liver injury. In contrast, NCX-701 inhibited cytokine releaseand liver Fas/FasLmRNAexpressionand reverteddamageinducedby Con A. In isolated mouse hepatocytes,APAP, but not NCX-701, increasedthe translocation of cytoplasmic Fas to the cell surface, resulting in an Fas expression as measured at flow cytometry analysis and increased sensitivity to the Fas agonistic monoclonal antibody Jo2. Incubating mouse hepatocyteswith NCX-701 resulted in a concentration and time-dependentformation of NO. Conclusions. NCX-701, an NO-derivativeof APAP,spares the liver in intact and compromised animals by inhibiting pro-inflammatory cytokine release and Fas-dependentapoptosis and inflammation by acting at several checkpoints in the Fas pathway.
TraMfonMng Growth Factor Beta-induced Apoptosis in Primary Murine Hepatocytes
is Cmqsuee-8Dependent. Charles M. Samson, Patty A. Lange, Mark A. Bird, Melissa A. Hayden,David A. Brenner, Kevtn E. Bahms, Univ of North Carolina at Chapel Hill, Chapel Hill, NC Background:TGF,8regulateshepatocytedifferentiation, growth, fibrosis, and apoptosis.TGF/~induced apoptosis appearsto be caspasedependent.The precise caspasasinduced by TGF/~, however, have not yet been well characterized.Aims: To determinethe necessityof easpase3 and caspase-8in TGF/]-induced apoptosis. We hypothesizedthat TGF/3-inducedapoptosis would be cospase-3 dependent, but caspase-8 independent. Methods: Hepatocytes were isolated from BALB/c mice by collagenaseperfusion. After plating, cells were serum starved and hepatocyteswere pretreated either with 1 p.M DEVD-CHO(caspase-3 inhibitor), 1 /~M Z-IETD-FMK (caspase-8 inhibitor) or 1 p.M DMSO (control). Two hours later, 5 ng/ml of recombinant human TGF,8 cytokine was added. Isolated DNA was separated on a 1.5 % agarose gel to detect DNA laddering. Other markers used to detect hepatocyte apoptosis includedprOpldium iodine (PI) staining for morphologic assessmentand PARPcleavagefor caspaseinvolvement. Protein from whole cell extracts were separatedon a 10% SDS gel and western blot analyses ware performed for cytochrome c release.Caspase-3and -8 specific activities ware measured by detecting cleavageof the fluorometric substrates DEVD-AFCand LETDoAFC,respectively. Results: Apoptosis in primary murine hepatocytes was confirmed within 48 h of TGF/]treatment by biochemicalassessmentincluding DNA ladderingand PARP cleavage, and morphologically by PI staining. Cytochrome c release was detected within 24 h of treatment. Both caspases-3 and -8 were activated at 48 h, with larger increases in activities by 72 h. Hepatocytaapoptosis was blockedwith the pretreatmentof either caspase3 or-8 inhibitor. Pretreatmentwith caspase-8inhibitor not only blockedTGF~induced caspase8 aclhrahon, but it also blocked caspase-3 activation. Conversely, inhibition of caspase-3 decreasedbut did not abolish caspase-8activity. Summary: TGF/Yinduces murine hepatocyte apoptosis with cytochrome c releaseand activation of both caspases-3and -8. Inhibition of either caspase-3 or caspase-8 blocks hepatocyteapoptosis. Caspase-3activity is caspase-8 dependent, as pre~eatment with the caspasa-8 inhibitor blocks TGF,8-induced caspase-3 activation.C a s ~ activity, however,is not caspase-3dependent.Conclusion: In conclusion, caspaea-8inhibition blocks both TGF/Y-inducedapoptosisand caspase-3activation.Therefore, TGF/Y-indueadapoptosis in murine hepatocytesis dependenton caspase-8activation.
InuSased Susceptibility of Rat Choluegiocytesto Tumor Necrosis Factora (TNFa) Cl~Modcity Following Bile Ouct LiBation. Giardraoco A/pini, Laura Tadiock, ShannonGlaser, Gene Lesage, Tushar Patel, Scott and White Clinic; Texas A&M Univ Sys Hlth Sci Ctr, Temple, TX Cholangiocyteinjury during cholestasishas beenassociatedwith activation of death receptors such as TNFa and Fas.Although these receptorsinduce cell deaththrough similar intracellular pathways,their individual contributions to cholangiocyteinjury during biliary tract obstruction remain unknown. Our AIMS were to assess the relative roles of the death receptors TNF~ and Fas in mediating cholangiocyteinjury following experimentalbile duct obstruction, and to identify cellular mechanismsdeterminingsusceptibilityto deathreceptor mediatedcytotoxicity. METHODS:Cholangiocyteswere isolatedfrom normal or 1-week bile duct ligated (BDL) rats. TNF-R1 receptorexpressionwas determinedby Western blot analysis. Cytotoxicityin response to TNF~ or egonistic antibodiesto the Fas receptor (anti-Fas) was assessedusing a tetrazolium salt bioreduction assay for viable cells. Actinomycin O was used as an inhibitor of RNA synthesis. Expressionof Fas and FasL mRNA, as well as mRNA expressionof the apoptosisregulatorygenes, Baxand Bcl-2, were detectedby ribonucleaseprotection assays,and quantitated relativeto GAPDHexpression.RESULTS:Expressionof the TNF-R1 receptorwas markedly increased in cholangiocytesfrom BDL rats as comparedto normal. However,cholangiocytes from both normal and bile duct ligated rats were highly resistant to TNFa (0-100 og/ml) as well as to Fas receptor stimulation with anti-Fas (0-10 pg/ml). Co-incubation with 1 /.;,M Actinomycin D sensitized cholangiocytesfrom BDL rats, but not normal cholangiocytes,to TNFatoxicity. The ratio of Bcl-2 to Bax geneexpressionwas 30% lower in BDL cholangiocytes compared to normal cholangiocytes.However,the sensitivity of BDL cholangiocytesto antiFascytotoxicity was not altered by Actinomycin D. Fas mRNAexpressionin BDL cholangiocytes was decreased to approx. 65% of normals, but FasL mRNA expression was not altered. SUMMARYand CONCLUSIONS:Cholangiocytesare susceptibleto TNFa but not Fas cytotoxicity by inhibition of RNA synthesis following BDL. Although the relative expression of the proapoptotic Bax gene is increasedfollowing BDL, Fas mRNA expression is decreasedand the sensitivity of BDL cholangiocytesto Fasstimulation is not altered.Theseobservationssuggest that TNFa but not Fasmediatescholangiocyteinjury and that down-regulationof Fasexpression may represent a protective mechanism during biliary tract obstruction.