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Differential display and cDNA expression array analyses of cancer cell differentiation in hepatocellular carcinoma
others are antagonists provides further support for the hypothesis that, in tissues exposed to sufficient bile concentrations,these agents may serve a cellular signaling function.
1839 Replication-induced Expression of Glucegon Receptor-mRHA in Rat Liver Lars Mueller, Dieter C. Broefing, Jannine Meyer, Yogesh Vashist, Xavier Rngiers, Univ Hosp Hamburg-Eppendorf,Hamburg Germany Background: Glucagon-dependentsignaling with intrecellular, G-protein-mediatedrelease of cAMP might display promltotic properties in the liver. However,the role of glucagoo receptor (GR)-gene expression during experimental liver regeneration has not been assessed so far. In this study, we examined the differential expression level of hepatic GR-mRNA, using two different models of liver regeneration, the partial (70%)-hepatectomy model and the model of 7O%-portal-branch-lig~on. The latter technique yields both growing and regressive liver tissue, since early prereplicativechanges after partial hepateetomydid net always prove to be restricted to regenerationper se. Experimentaldesign: A total of 133 male W'~tar-rats of 200-250 grams were subjected to eltber sham-operation (SO), partial hepatectomy (Hx) or portal branch libation (PBL) and sacriflod after 6 hours (h), 12 h, 24 h, 48 h, 96 b, 192 h and 2 weeks. After determining segmental liver weights, liver tissue was snap frozen in liquid nitrogen and stored, and total RNA preparation was executed. Mescenger-RNAexpression analysiswas basedon RT-PCRand Northem-hybridisation-experiments.eDNA-probes,specific to Histone-protein2b (H2b) as a replicationmarker, as well as OR, were Digo)dganin-laholodand detected by chemoluminescenceand followed by scanning densltomatryafter normalizationto 28S-RNA. Results: All operative procedures were well tolerated, and perioperativemortality was below 5%. Maximal replicative response, as assessedby maximal peaks in R 2 b - ~ sign, was shifted from 24 h after Hx to 48 b after PBL in the non-ligeted lobes. Donsitomatric analysis revealed a maximal increase (6,1fold) after 24h in the Hx-group and a 5,4fold peak in the non-ligated lobes 48h after PBL. In the ligated/obas after PBL, no enhancedexpression of H2b-mRNAwas detected at any time point io comparison to control liver tissue or shamoperatedanimals. A coincidental induction of GR-mRNAwas detectedafter 24h and 4811after both Hx and PBL in the non-lipated lobe (1,6fold and 1,6fold vs. SO, respectively),whereas in the ligated lobe, the OR-expressiondeclined (100% vs SO after 24h). Conclusion: The transcriptional activation of Glucagon-receptor-expressionbatweeo24h and 48h after Hx and PBL in the non-ligated lobes correlates with the onset of replication. Glucagon-depeodant intracellular signalling might be modulated by enhanced receptor expression in this stage of the regenerativeresponse.
expression array. Methods: Total RNA was extracted from a human HCC cell line, HCC-T,at various time points during 8 hours after stimulation with butyrate.To find out unknown genes responsible for differentiation, we performed a differential display assay. Genes drastically changed their expression levels were picked up and homology search was performed. To investigate the general aspect of changes of gene expression during the first 2 hours of differentiation, the cDNAexpressionarray (Human Cancer1.2k, Clontech,CA) was performed. Results: Several significant genes were found out by differential display analyses. We found that mitochooddal DNA, laminin, keratin 18 and severalunidatifled genes were overexprassed during 8 hours. On the other hand,down-regulation of the geoe expressionin signal sequence receptor (SSR) 2 and T-cell cyclophilin was found out. Wesfam blot analysis sbowad that the laminin production increased6 hours after stimulation with butyrate. The results of cDNA expression array revealedup-regulation of McI-1/EAT, STAT2, STAT3, IRF-1, p21w~l, RhoC, RhoE,MEGFand APC,while down-regulationof CDK2and GI/S specific cyclin E. Theseresults were compatiblewith those in our previous studies. Conclusion:The results that up-regulation of p21=~', Bd-2 and McI-1/EAI was observed in both Western blotting and cDNAexpression array analysis suggestedthat these genes are important for the cellular differentiation of HCC cells. The cytoskel~ change indicated by up-regulation of laminin and keratin 18 may be another important factor in the differentiationof cancercells. This study suggestsseveraltarget genesfor the future genetherapypreventingdevelopmentof cancerfrom pre-malignanttissues.
1842 Inhlblthq Effeofs of an Herbal Madisine "Sho-Saiko-To" on POGF-BBInduced Tyrosime Phosphoqlofion and Cell I~oliferaUon in Human Hepatic Stellate Cells Ryuichiro Saketa,Takato Ueno, Toru Nakemura, Osamu Hashimoto, Rina Kimura, Maeaharu Sahamoto,Takuji Torimura, Michio Sata, Kurume Univ Sch of Medicine, Kurume Japan Backgroundand Aim: Sbo-eaiko-to (TJ-9), one of the oriental herbal medicines is commonly administrated to patients with chronic liver diseases. The previous study demonstrated that sho--saiko-toinhibits collagen I mRNAexpressionand cell proliferation of hepaticstellatecells (HSCs). Proliferation of HSCsis a critical process for the developmentof liver fibrosis. PDGFBB is the most potent mitogen for HSCs. Although PDGF-BBstimulates cell proliferation by ectWating key effectors, including tyrosine autophosphorylationof PDGF-raceptorand activatioo of intreceltular signaling pathways (e.g. MEK and Akt), the effects of sho-saiko-to in these pathways are little understood. This study is designedto investigate how sho-saiko-to inhibits the PDGF-BB-ioducodactivation of intracellular signaling pathways in human HSCs. Materials and Methods: A cultured human hepatic stellate cell line LI90 was used for this study. LIgO cells ware serum-starved for 48 hr and exposedto PDGF (lOng/ml). Sho-saikoto powder (TJ-g; Tsumura & Co., Tokyo) was dissolved in basal medium and prepared at 500,1000 and 2000 #Q/ml concentrations.Proliferationwas determinedby BrdU incorporation 24 hr after POGFtreatment and cell cycle analysis was performed by flow cytometory (FACS; Becton Dickinson). MEK and Akt pbosphorylation was detected by Western immunoblotting analysis using anti phospbo-MEKand anti pbospho-AId antibody (Cell Signaling Tech. Inc., IDA). Tyrosine phospborylabon was detected by coofocal microscopy and Western immunol)lothng analysis using anti phospho-tyrosine antibody (Upstate Biotechnology Inc., NY), Results: PDGF-OBtraabneat inducedMEKand Akt phospborylationand increasedcell proliferaof LIgO cells. Sbo-saiko-to inhibited the increases in MEK and Akt phospborylation and cell proliferation induced by PDGF-BB. Confocal images of immunostaining and Western immunobloffing analysis showed that PDGF-BB-inducedtyrosine phosphorylationwas inhibited by sho-saiko-to.Conclusion:Sho-saiko-toinhibited PDGF-BB-inducedtyrosine phosphorylation. It also inhibited MEK and Akt phosphorylation that led to mitogenic effect of HSCs. These results suggest that sho-caiko-to may act on PDGF-receptoror its downstream and inhibits the proliferation of HSCs. The relation of sho-saiko-to to the promitogenic signaling pathways in HSCs may be important to know the exact functional mechanisms of sho-saikoto in HSCs.
1840 Differential UGT GeM Regulation and the Development of Hifllal Ofllg Glucuronidofion in Young Children and Mulls Christian P. Strassburg, Ahlke Strassburg, Susanne Kneip, Ayse Buret, Robert H. Take/, Burkbard Rodeck, Michael P. Manns, Hannover Medical Sch, HannoverGermany Background:The liver representsone of the major sites of human drug glucuronidation. UDPo glucurcnosyltransterasestarget manytherapeuticdragsto catalyzethe formation of hydrophilic and mostly inactive glucuronides. Hepatic glucuronidation undergoes significant changes during fetal and neonatal developmentrequiring age-adapteddrug therapy. The regulation of individual human UGT genes during hepatic development has not been defined. Methods: The expression of 13 UGTgeoes and specific glucuronidation activities were analyzedusing quantitative RT-PCR, isoform specific Western blot and specific catalytic UGT activity assays in 16 pediatric liver samplesfrom children aged7 and 24 months undergoingliver transptantation for extrahepaticbiliary atresla, 2 fetal samples, and 12 samplesfrom adult liver aged 25 to 75 years undergoinghepatic resection or liver transplantationfor hepatocallularcarcinoma. All samples were frozen during surgery and were free of tumor or necrosis by histology. Results: UGT transcripts were undetectableby RT-PCRin fetal liver at 20 weeks of gestation. However, after 6 months, UGT1A1, UGT1A3, UGT1A4,UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2BIO, and UGT2815traoscripts were expressedwithout interfndividualvariation in all 28 hepatic samples.Expressionwas confirmed by Western blot analysis using availableantibody for UGT1A1, UGT1A6 and UGT2B7, The analysis of UGT1A9 and UG'I'21Ngene expression identified significant differential down-regulation exclusively in child liver. UGT1A9is capable of significant phenolic drug glucuronidation and therefore clinical relevancewas tested by specific UGTactivity analysis.HepaticUGTactivity with ibuprofen, amitriptylin, 4-tert-betylpbenol, estrone and buprenorphin was 24-, 16-, 40-, 1B-, and 12-fold lower at 13-24 months than in adult liver. Conclusions: These findings identify an early phase of qualitative and a later phase of quantitative UGT regulation in human hepatic development. The differential regulation of UGT1A9and UGT2B4expression extends beyond 2 years and may be capable of influencing the overall hepatic glucuronidaflon of common therapeutic drugs io children. The demonstrated development of hepatic UGT activities is significant for pediatric drug therapy and the prevention of adverse drug effects.
Fibronectin Receptor ( a ~ ) Changes the Iofracellular pH during Activation of Slofhm Cells Michael T. Milllano, Bruce A. Luxon, Saint Louis Univ, St. Louis, MD Background: Hepatic stellate cells (HSC) play an integral role in hepatic fibrosis. Activated HSC synthesize extracellular matrix (ECM) components, including fibronectin and type I collagen. HSC activation causes increased as/~ expression (fibronectin receptor). Binding of the ~ integrin to the ECM increasescollagensynthesisand HSCproliferation. HSCactivation also increases intracellular pH (pH,) by increasing Na+/H+ pump activity. It is unclear how stimulating the c,5#~receptor Jnitiatesthese changes. Aim: To determine changes in pH, after ~,& stimulation dunng HSC activation. Methods: HSC were isolated from male Sprague Dawiny rats and plated on plastic coated with d-lysine, collagen type I or ROD (100/~g/ml). Only ROD interacts with the o~/~ receptor. Primary HSC were studied 2 hours after isolation. Plated HSC were incubated in Ham's DMEM for 7 days. pH, was determined in the presence and absence of bicarbonate using the fluorescent indicator BCEMF. Immunohistochemistry was done using antibodies against the as and ~ subunits. Activation of HSC was assessed by ~-smooth muscle aefin (.-SMA) expression. Results: HSC becameactivatedwhen plated on plastic, d-lysine or type I collagenas judged by increased.-SMA expression.~ receptor expression was also increased. HSC plated on ROD becameactivated bet had decreased.SMA and ~ receptor expression.Activation increasedpH, and Na+/H+ pump activity. HSC binding to ROD decreasedo ~ receptor expression did not change pHi. Binding to collagen type i returned intracellular pH to quiescent levels without stimulating the o~/~ receptor, Conclusions: Activation of HSC causes an increase in pH, and Na÷/H÷ pump activity. Binding of HSC to ROD decreasesintegrin expression consistent with a negative feedback loop but does not alter pH, and Na*/H ÷ pump activity. Type I collagen increases expression of the fibronectin receptor without directly binding to it and rstums pH, and Na*/H + pump activity to levels found in quiescent HSC.
1841 Differential Display and cDNA Expression Anay Analyses of Canun cell Differentiation in Hepatocellular Carcinoma Kanji Wakabayashi,Hidctsugu Saito, MaeayukiAdachi, Tamako Takegi, YoshimaeaSanto, Nobohiro Nakemoto, Satoshi Kurita, Mitsuyasu Nakamura, Hirotoshi Ebinuma, Hiromasa Ishii, Keio Univ Sch of Medicine, Tokyo Japan Background: Sodium butyrats is a short-chain fatty acid produced by fermentation in the body. It induces differenti~on of several kinds of cancer by inhibiting histone deacetylase activity. We have reported that betyrate differentiated hapatocedularcarcinoma (HCC) ceils into their normal counterparts,Alteration in the expressionof severalgenes has beenidentified in our previous studies. However,the gene expression responsiblefor the differentiation of cancer cell is not understood at all. The aim of this study was to find out the responsible gene for the differentiation of HCC cells. To that effect, we analyzedgane expressions in the early stage of differentiation induced by butyrate using a differential display assay and a cDNA
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1839 Replication-induced Expression of Glucegon Receptor-mRHA in Rat Liver Lars Mueller, Dieter C. Broefing, Jannine Meyer, Yogesh Vashist, Xavier Rngiers, Univ Hosp Hamburg-Eppendorf,Hamburg Germany Background: Glucagon-dependentsignaling with intrecellular, G-protein-mediatedrelease of cAMP might display promltotic properties in the liver. However,the role of glucagoo receptor (GR)-gene expression during experimental liver regeneration has not been assessed so far. In this study, we examined the differential expression level of hepatic GR-mRNA, using two different models of liver regeneration, the partial (70%)-hepatectomy model and the model of 7O%-portal-branch-lig~on. The latter technique yields both growing and regressive liver tissue, since early prereplicativechanges after partial hepateetomydid net always prove to be restricted to regenerationper se. Experimentaldesign: A total of 133 male W'~tar-rats of 200-250 grams were subjected to eltber sham-operation (SO), partial hepatectomy (Hx) or portal branch libation (PBL) and sacriflod after 6 hours (h), 12 h, 24 h, 48 h, 96 b, 192 h and 2 weeks. After determining segmental liver weights, liver tissue was snap frozen in liquid nitrogen and stored, and total RNA preparation was executed. Mescenger-RNAexpression analysiswas basedon RT-PCRand Northem-hybridisation-experiments.eDNA-probes,specific to Histone-protein2b (H2b) as a replicationmarker, as well as OR, were Digo)dganin-laholodand detected by chemoluminescenceand followed by scanning densltomatryafter normalizationto 28S-RNA. Results: All operative procedures were well tolerated, and perioperativemortality was below 5%. Maximal replicative response, as assessedby maximal peaks in R 2 b - ~ sign, was shifted from 24 h after Hx to 48 b after PBL in the non-ligeted lobes. Donsitomatric analysis revealed a maximal increase (6,1fold) after 24h in the Hx-group and a 5,4fold peak in the non-ligated lobes 48h after PBL. In the ligated/obas after PBL, no enhancedexpression of H2b-mRNAwas detected at any time point io comparison to control liver tissue or shamoperatedanimals. A coincidental induction of GR-mRNAwas detectedafter 24h and 4811after both Hx and PBL in the non-lipated lobe (1,6fold and 1,6fold vs. SO, respectively),whereas in the ligated lobe, the OR-expressiondeclined (100% vs SO after 24h). Conclusion: The transcriptional activation of Glucagon-receptor-expressionbatweeo24h and 48h after Hx and PBL in the non-ligated lobes correlates with the onset of replication. Glucagon-depeodant intracellular signalling might be modulated by enhanced receptor expression in this stage of the regenerativeresponse.
expression array. Methods: Total RNA was extracted from a human HCC cell line, HCC-T,at various time points during 8 hours after stimulation with butyrate.To find out unknown genes responsible for differentiation, we performed a differential display assay. Genes drastically changed their expression levels were picked up and homology search was performed. To investigate the general aspect of changes of gene expression during the first 2 hours of differentiation, the cDNAexpressionarray (Human Cancer1.2k, Clontech,CA) was performed. Results: Several significant genes were found out by differential display analyses. We found that mitochooddal DNA, laminin, keratin 18 and severalunidatifled genes were overexprassed during 8 hours. On the other hand,down-regulation of the geoe expressionin signal sequence receptor (SSR) 2 and T-cell cyclophilin was found out. Wesfam blot analysis sbowad that the laminin production increased6 hours after stimulation with butyrate. The results of cDNA expression array revealedup-regulation of McI-1/EAT, STAT2, STAT3, IRF-1, p21w~l, RhoC, RhoE,MEGFand APC,while down-regulationof CDK2and GI/S specific cyclin E. Theseresults were compatiblewith those in our previous studies. Conclusion:The results that up-regulation of p21=~', Bd-2 and McI-1/EAI was observed in both Western blotting and cDNAexpression array analysis suggestedthat these genes are important for the cellular differentiation of HCC cells. The cytoskel~ change indicated by up-regulation of laminin and keratin 18 may be another important factor in the differentiationof cancercells. This study suggestsseveraltarget genesfor the future genetherapypreventingdevelopmentof cancerfrom pre-malignanttissues.
1842 Inhlblthq Effeofs of an Herbal Madisine "Sho-Saiko-To" on POGF-BBInduced Tyrosime Phosphoqlofion and Cell I~oliferaUon in Human Hepatic Stellate Cells Ryuichiro Saketa,Takato Ueno, Toru Nakemura, Osamu Hashimoto, Rina Kimura, Maeaharu Sahamoto,Takuji Torimura, Michio Sata, Kurume Univ Sch of Medicine, Kurume Japan Backgroundand Aim: Sbo-eaiko-to (TJ-9), one of the oriental herbal medicines is commonly administrated to patients with chronic liver diseases. The previous study demonstrated that sho--saiko-toinhibits collagen I mRNAexpressionand cell proliferation of hepaticstellatecells (HSCs). Proliferation of HSCsis a critical process for the developmentof liver fibrosis. PDGFBB is the most potent mitogen for HSCs. Although PDGF-BBstimulates cell proliferation by ectWating key effectors, including tyrosine autophosphorylationof PDGF-raceptorand activatioo of intreceltular signaling pathways (e.g. MEK and Akt), the effects of sho-saiko-to in these pathways are little understood. This study is designedto investigate how sho-saiko-to inhibits the PDGF-BB-ioducodactivation of intracellular signaling pathways in human HSCs. Materials and Methods: A cultured human hepatic stellate cell line LI90 was used for this study. LIgO cells ware serum-starved for 48 hr and exposedto PDGF (lOng/ml). Sho-saikoto powder (TJ-g; Tsumura & Co., Tokyo) was dissolved in basal medium and prepared at 500,1000 and 2000 #Q/ml concentrations.Proliferationwas determinedby BrdU incorporation 24 hr after POGFtreatment and cell cycle analysis was performed by flow cytometory (FACS; Becton Dickinson). MEK and Akt pbosphorylation was detected by Western immunoblotting analysis using anti phospbo-MEKand anti pbospho-AId antibody (Cell Signaling Tech. Inc., IDA). Tyrosine phospborylabon was detected by coofocal microscopy and Western immunol)lothng analysis using anti phospho-tyrosine antibody (Upstate Biotechnology Inc., NY), Results: PDGF-OBtraabneat inducedMEKand Akt phospborylationand increasedcell proliferaof LIgO cells. Sbo-saiko-to inhibited the increases in MEK and Akt phospborylation and cell proliferation induced by PDGF-BB. Confocal images of immunostaining and Western immunobloffing analysis showed that PDGF-BB-inducedtyrosine phosphorylationwas inhibited by sho-saiko-to.Conclusion:Sho-saiko-toinhibited PDGF-BB-inducedtyrosine phosphorylation. It also inhibited MEK and Akt phosphorylation that led to mitogenic effect of HSCs. These results suggest that sho-caiko-to may act on PDGF-receptoror its downstream and inhibits the proliferation of HSCs. The relation of sho-saiko-to to the promitogenic signaling pathways in HSCs may be important to know the exact functional mechanisms of sho-saikoto in HSCs.
1840 Differential UGT GeM Regulation and the Development of Hifllal Ofllg Glucuronidofion in Young Children and Mulls Christian P. Strassburg, Ahlke Strassburg, Susanne Kneip, Ayse Buret, Robert H. Take/, Burkbard Rodeck, Michael P. Manns, Hannover Medical Sch, HannoverGermany Background:The liver representsone of the major sites of human drug glucuronidation. UDPo glucurcnosyltransterasestarget manytherapeuticdragsto catalyzethe formation of hydrophilic and mostly inactive glucuronides. Hepatic glucuronidation undergoes significant changes during fetal and neonatal developmentrequiring age-adapteddrug therapy. The regulation of individual human UGT genes during hepatic development has not been defined. Methods: The expression of 13 UGTgeoes and specific glucuronidation activities were analyzedusing quantitative RT-PCR, isoform specific Western blot and specific catalytic UGT activity assays in 16 pediatric liver samplesfrom children aged7 and 24 months undergoingliver transptantation for extrahepaticbiliary atresla, 2 fetal samples, and 12 samplesfrom adult liver aged 25 to 75 years undergoinghepatic resection or liver transplantationfor hepatocallularcarcinoma. All samples were frozen during surgery and were free of tumor or necrosis by histology. Results: UGT transcripts were undetectableby RT-PCRin fetal liver at 20 weeks of gestation. However, after 6 months, UGT1A1, UGT1A3, UGT1A4,UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2BIO, and UGT2815traoscripts were expressedwithout interfndividualvariation in all 28 hepatic samples.Expressionwas confirmed by Western blot analysis using availableantibody for UGT1A1, UGT1A6 and UGT2B7, The analysis of UGT1A9 and UG'I'21Ngene expression identified significant differential down-regulation exclusively in child liver. UGT1A9is capable of significant phenolic drug glucuronidation and therefore clinical relevancewas tested by specific UGTactivity analysis.HepaticUGTactivity with ibuprofen, amitriptylin, 4-tert-betylpbenol, estrone and buprenorphin was 24-, 16-, 40-, 1B-, and 12-fold lower at 13-24 months than in adult liver. Conclusions: These findings identify an early phase of qualitative and a later phase of quantitative UGT regulation in human hepatic development. The differential regulation of UGT1A9and UGT2B4expression extends beyond 2 years and may be capable of influencing the overall hepatic glucuronidaflon of common therapeutic drugs io children. The demonstrated development of hepatic UGT activities is significant for pediatric drug therapy and the prevention of adverse drug effects.
Fibronectin Receptor ( a ~ ) Changes the Iofracellular pH during Activation of Slofhm Cells Michael T. Milllano, Bruce A. Luxon, Saint Louis Univ, St. Louis, MD Background: Hepatic stellate cells (HSC) play an integral role in hepatic fibrosis. Activated HSC synthesize extracellular matrix (ECM) components, including fibronectin and type I collagen. HSC activation causes increased as/~ expression (fibronectin receptor). Binding of the ~ integrin to the ECM increasescollagensynthesisand HSCproliferation. HSCactivation also increases intracellular pH (pH,) by increasing Na+/H+ pump activity. It is unclear how stimulating the c,5#~receptor Jnitiatesthese changes. Aim: To determine changes in pH, after ~,& stimulation dunng HSC activation. Methods: HSC were isolated from male Sprague Dawiny rats and plated on plastic coated with d-lysine, collagen type I or ROD (100/~g/ml). Only ROD interacts with the o~/~ receptor. Primary HSC were studied 2 hours after isolation. Plated HSC were incubated in Ham's DMEM for 7 days. pH, was determined in the presence and absence of bicarbonate using the fluorescent indicator BCEMF. Immunohistochemistry was done using antibodies against the as and ~ subunits. Activation of HSC was assessed by ~-smooth muscle aefin (.-SMA) expression. Results: HSC becameactivatedwhen plated on plastic, d-lysine or type I collagenas judged by increased.-SMA expression.~ receptor expression was also increased. HSC plated on ROD becameactivated bet had decreased.SMA and ~ receptor expression.Activation increasedpH, and Na+/H+ pump activity. HSC binding to ROD decreasedo ~ receptor expression did not change pHi. Binding to collagen type i returned intracellular pH to quiescent levels without stimulating the o~/~ receptor, Conclusions: Activation of HSC causes an increase in pH, and Na÷/H÷ pump activity. Binding of HSC to ROD decreasesintegrin expression consistent with a negative feedback loop but does not alter pH, and Na*/H ÷ pump activity. Type I collagen increases expression of the fibronectin receptor without directly binding to it and rstums pH, and Na*/H + pump activity to levels found in quiescent HSC.
1841 Differential Display and cDNA Expression Anay Analyses of Canun cell Differentiation in Hepatocellular Carcinoma Kanji Wakabayashi,Hidctsugu Saito, MaeayukiAdachi, Tamako Takegi, YoshimaeaSanto, Nobohiro Nakemoto, Satoshi Kurita, Mitsuyasu Nakamura, Hirotoshi Ebinuma, Hiromasa Ishii, Keio Univ Sch of Medicine, Tokyo Japan Background: Sodium butyrats is a short-chain fatty acid produced by fermentation in the body. It induces differenti~on of several kinds of cancer by inhibiting histone deacetylase activity. We have reported that betyrate differentiated hapatocedularcarcinoma (HCC) ceils into their normal counterparts,Alteration in the expressionof severalgenes has beenidentified in our previous studies. However,the gene expression responsiblefor the differentiation of cancer cell is not understood at all. The aim of this study was to find out the responsible gene for the differentiation of HCC cells. To that effect, we analyzedgane expressions in the early stage of differentiation induced by butyrate using a differential display assay and a cDNA
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