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M1670 Inducible Expression of Hnf4alpha in Huh 7.5 Cell Line Has a Strong Antiproliferative Effect
M1669
gallbladder cancer who underwent resection at the Samsung Medical Center(Seoul, Korea) between march 1999 and may 2008 were included. Gallbladder adenoma(25 cases) and normal gallbladder(10 cases) specimens were also evaluated. We performed tissue microarray construction and immunohistochemistry. The staining results were estimated semi-quantitatively by one pathologist, based on the intensity and the percentage of positive cells:0,Absent;1,weak and focal(<10%);2,partial(10-50%);3,strong and diffuse(>50%). Maspin labeling was reported as negative(score 0 or 1) or positive(score 2 or 3). Results: The positive rate of maspin expression was 59.4%(60/101) in gallbladder cancer, whereas no maspin expression was observed in adenoma and normal gallbladder specimens. No significant difference in positive rate of maspin expression between early and advanced cancer was observed (positive rate, 49 versus 60 %; p=0.731). Conclusions: This results may suggest that maspin have influence on the early step of gallbladder carcinogenesis. Maspin expression in gallbladder cancer, adenoma and normal gallbladder specimens
AASLD Abstracts
Inhibition of Acid Sphingomyelinase Causes Hepatic Glucose Intolerance and Insulin Resistance Yosuke Osawa, Hiroyasu Ito, Atsushi Suetsugu, Masahito Nagaki, Hisataka Moriwaki, Mitsuru Seishima Background and Aims: Acid sphingomyelinase (ASM) regulates sphingolipids homeostasis including ceramides and sphingosine-1-phosphate. Although the sphingolipids are involved in AKT activation, little information is available for the role of ASM in glucose and lipid metabolism. To explore this issue, we investigated glucose and lipid metabolisms in ASMinhibited hepatocytes. Methods: Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed on ASM+/+ and ASM-/- mice. Primary cultured hepatocytes were isolated and the effects of ASM inhibitor imipramine or ASM deficiency were examined for AKT activation, glucose up-take, glycogen deposition, and lipid accumulation. Results: Food intake decreased most of sphingomyelin species in mice liver. IPGTT and ITT showed glucose intolerance accompanied with hepatic insulin resistance in ASM-/- mice. In primary cultured hepatocytes, insulin treatment alone was unable to increase glucose uptake and glycogen because the cells had high basal nuclear factor (NF)-kappaB activity due to the stimulation by the culturing on collagen-coated plastic dishes. When NF-kappaB was inhibited by either pretreatment of the IkappaB kinase inhibitor PS-1145 or introduction of an adenoviral IkappaB super repressor (Ad5IkappaB), insulin treatment increased glucose uptake and glycogen levels in the hepatocytes. Thus, Ad5IkappaB-infected hepatocytes were used in case of insulin treatment. Pretreatment with imipramine inhibited glucose uptake by insulin, and also inhibited glycogen deposition by insulin or high dose of glucose in rat hepatocytes as did the knockout of ASM in mouse hepatocytes. Moreover, either insulin or high dose of glucose treatment increased lipid droplets in primary hepatocytes, and imipramine or ASM deficiency suppressed this increase. Insulin induced phosphorylation of AKT in primary hepatocytes, and overexpression of dominant negative-AKT by adenovirus abrogated the phosphorylation of endogenous AKT by insulin. DN-AKT inhibited glucose uptake, glycogen deposition, and lipid accumulation by insulin or high dose of glucose suggesting that AKT has central roles in hepatic glucose and lipid metabolisms. However, pretreatment of imipramine or knockout of ASM did not affect AKT phosphorylation induced by insulin. Conclusion: ASM deficiency causes glucose intolerance and hepatic insulin resistance without affecting insulin-induced AKT activation in hepatocytes.
Clinical charicteristics and maspin expression pattern of the early and advanced cancers
NS:not significant M1672
M1670 An Analysis of CD133 Positive Bone Marrow in Oval Cell Induced Model and a Role of CD133 Bone Marrow in Mice With Liver Fibrosis Atsushi Suetsugu, Masahito Nagaki, Yosuke Osawa, Robert M. Hoffman, Hisataka Moriwaki
Inducible Expression of Hnf4alpha in Huh 7.5 Cell Line Has a Strong Antiproliferative Effect Razvan Iacob, Urda Ruedrich, Michael Rothe, Michael Bock, Benjamin Maasoumy, Ina Rittelmeyer, Marcus Iken, Julia Bitzegeio, Yuan Qinggong, Irinel Popescu, Michael P. Manns, Michael Ott HNF4alpha is one of the key transcription factors coordinating cell proliferation, differentiation, and the maintenance of a hepatocyte phenotype. Transcriptional repression of HNF4alpha has been associated with progression and dedifferentiation of hepatic tumors. The aim of our study was to generate an In Vitro model for the study of HNF4alpha antiproliferative effects using a lentiviral inducible expression system. Methods. A lentiviral Tet-ON coexpression system for HNF4alpha and eGFP under a bi-directional Tet-promoter has been generated. HUH 7.5 cells have been transduced with this lentivirus and single cell clones have been created. After establishing a stable transgenic cell line, cells membranes were labeled with PKH26 and cultured in the presence of doxycycline, in order to induce HNF4alpha expression. The cells cultured without doxycycline and non transduced HUH 7,5 cells were used as controls. The PKH26 intensity has been measured by FACS at day 1, 3 and 5 and the mean doubling time for each cell population has been calculated. Gene expression for genes with antiproliferative effects, known to be upregulated by HNF4alpha in non-hepatic cancer cell lines, as well as for liver specific genes have been investigated by quantitative RTPCR at day 4 and 7, in the presence or absence of doxycycline. Results. A strong antiproliferative effect has been noted in HUH7.5 cells after HNF4alpha induction, with a 10 fold mean doubling time increase. A significant increase in cell size has also been observed. The quantitative RT-PCR analysis indicated a significant upregulation of HNF4 alpha and also of the following genes with a documented antiproliferative effect: p21, desmocollin2, ankyrin3, ADAMTS1, Nell2, Sepp1. A decrease in AFP expression has been noted. Conclusion. HNF4 alpha overexpression in HUH 7,5 human hepatocarcinoma cell line leads to a marked reduction of cell proliferation. HNF4alpha upregulates a variety of genes with antiproliferative effect involved in cell cycle, cell adhesion, cytoscheletal anchoring, metabolism and which are not all liver specific. The new HUH 7,5 transgenic cell line could be used as an In Vitro model for further studies of the antiproliferative effects of HNF4 alpha, leading to a better understanding of hepatocyte growth control. It could also serve as a model for lentiviral insertional mutagenesis studies or In Vivo and In Vitro HCV infectivity and replication studies.
Background: Bone marrow (BM) cells have been reported to behave as tissue-specific stem cells in some organs. However, it is still unknown in the liver whether BM cells are a source for liver stem cells or oval cells and these progenitor cells differentiate to mature liver cells. To examine this issue, we examined the characteristics of oval cells induced by 3,5diethoxycarbonyl-1,4-dihydrocollidine (DDC) using GFP-chimeric mice which contain GFP positive BM cells, and examined whether stem cells from BM cells can be a candidate for cell therapy against liver injury. Methods: Total BM cells of GFP mice were injected (1×107) to irradiated mice (11 Gy). After 6 weeks, the mice were fed with DDC containing chow for 4 or 8 weeks to induce oval cells. Liver cells were isolated by collagenase, and the BMderived GFP positive cells were analyzed using a flow cytometer. In addition, the expressions of albumin, AFP, and CK19 were examined using immunocytochemistry to determine the potential for the differentiation of the BM-driven oval cells. Next, stem cells in BM cells from GFP mice were separated by flow cytometory according to presence or absence of CD133. The separated cells were injected into carbon tetrachloride (CCL4) treated mice and the liver injury and fibrosis were examined by serum transaminase level, hydroxyproline content, HE staining, and Sirius red staining. Results: BM-derived GFP positive cells were mainly observed in periportal lesions in the GFP-chimeric mice 4 weeks after DDC treatment. At 8 weeks, these cells had spread toward the central vein regions. GFP positive cells were detected in 28.9% among the isolated liver cells at 8 weeks. A part of the BM-derived GFP positive cells expressed albumin and CK19 suggesting the cells differentiated and had characteristics of hepatocytes and cholangiocytes. Especially, CD133 positive cells, which were contained 26.9% in the BM derived GFP positive cells, expressed higher mRNA expression of albumin compared with CD133 negative cells. The injection of CD133 positive BM cells significantly improved liver injury and fibrosis in the CCL4 treated mice. In contrast, the transplantion of the CD133 negative cell promoted liver damage and fibrosis. Conclusions: A part of oval cells in the liver are mobilized from BM, and the BM derived stem cells have characteristics of hepatocyte and cholangiocyte progenitor/stem cells. Among the BM derived cells, CD133 positive cells were enriched for the hepatocyte/cholangiocyte marker. CD133 positive BM cells may be useful as a source for cell therapy against liver damages.
M1671
M1673
Aberrant Maspin Expression in Gallbladder Cancer Jeong Kim, Kee Taek Jang, Sun Youn Bae, Kwang Hyuck Lee, Jong Kyun Lee, Jong Chul Rhee, Kyu Taek Lee
Palmitate Induced Interleukin-8 Production From Hepatic Stellate Cells is Toll-Like Receptor 4 Dependent Ming Song, Theresa S. Chen, Shirish Barve, Craig J. McClain
Background/Aims: Maspin(mammary serine protease inhibitor) is expressed in normal human mammary epithelial cells and down-regulated during cancer progression. But, aberrant maspin expression was noted in pancreatic cancers and ovarian cancers. The tissue-specific expression of maspin is epigenetically controlled, and aberrant maspin expression in biliary tract carcinomas is closely associated with demethylation at the promoter region. Recently, We had identified several genes that may be tumor markers for gallbladder cancer using DNA microarray method. Among them maspin seemed to be the most interesting gene. To our knowledge, there is no published data regarding maspin expression in gallbladder cancer. The aim of this study were to evaluate the maspin expression in gallbladder cancer group compared with adenoma and normal GB groups and to compare the expression pattern of early and advanced gallbladder cancers. Methods: A total of 101 patients with primary
Background/Aims: Emerging evidence suggests that elevated circulating free fatty acids (FFAs) play an etiologic role in non-alcoholic steatohepatitis (NASH). Several studies have shown that saturated FFAs exert their pro-inflammatory effects in certain cell types (including adipocytes and macrophages), through toll-like receptor 4 (TLR4) signaling pathway, which contributes to obesity-associated inflammation and insulin resistance. However, the direct effect of FFAs on hepatic stellate cells (HSCs), the main fibrogenic cell type, is unknown. IL-8, a CXC chemokine which plays a major role in neutrophil infiltration/activation, is significantly elevated in the serum of NASH patients. The aim of this study was to investigate whether palmitate could induce IL-8 production in hepatic stellate cell and whether it is TLR4 dependent. Methods: LX2 cells (human hepatic stellate cell line) were exposed to various doses of palmitate for 48 hours. Culture supernatants or cell lysates were collected.
AASLD Abstracts
S-826
gallbladder cancer who underwent resection at the Samsung Medical Center(Seoul, Korea) between march 1999 and may 2008 were included. Gallbladder adenoma(25 cases) and normal gallbladder(10 cases) specimens were also evaluated. We performed tissue microarray construction and immunohistochemistry. The staining results were estimated semi-quantitatively by one pathologist, based on the intensity and the percentage of positive cells:0,Absent;1,weak and focal(<10%);2,partial(10-50%);3,strong and diffuse(>50%). Maspin labeling was reported as negative(score 0 or 1) or positive(score 2 or 3). Results: The positive rate of maspin expression was 59.4%(60/101) in gallbladder cancer, whereas no maspin expression was observed in adenoma and normal gallbladder specimens. No significant difference in positive rate of maspin expression between early and advanced cancer was observed (positive rate, 49 versus 60 %; p=0.731). Conclusions: This results may suggest that maspin have influence on the early step of gallbladder carcinogenesis. Maspin expression in gallbladder cancer, adenoma and normal gallbladder specimens
AASLD Abstracts
Inhibition of Acid Sphingomyelinase Causes Hepatic Glucose Intolerance and Insulin Resistance Yosuke Osawa, Hiroyasu Ito, Atsushi Suetsugu, Masahito Nagaki, Hisataka Moriwaki, Mitsuru Seishima Background and Aims: Acid sphingomyelinase (ASM) regulates sphingolipids homeostasis including ceramides and sphingosine-1-phosphate. Although the sphingolipids are involved in AKT activation, little information is available for the role of ASM in glucose and lipid metabolism. To explore this issue, we investigated glucose and lipid metabolisms in ASMinhibited hepatocytes. Methods: Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed on ASM+/+ and ASM-/- mice. Primary cultured hepatocytes were isolated and the effects of ASM inhibitor imipramine or ASM deficiency were examined for AKT activation, glucose up-take, glycogen deposition, and lipid accumulation. Results: Food intake decreased most of sphingomyelin species in mice liver. IPGTT and ITT showed glucose intolerance accompanied with hepatic insulin resistance in ASM-/- mice. In primary cultured hepatocytes, insulin treatment alone was unable to increase glucose uptake and glycogen because the cells had high basal nuclear factor (NF)-kappaB activity due to the stimulation by the culturing on collagen-coated plastic dishes. When NF-kappaB was inhibited by either pretreatment of the IkappaB kinase inhibitor PS-1145 or introduction of an adenoviral IkappaB super repressor (Ad5IkappaB), insulin treatment increased glucose uptake and glycogen levels in the hepatocytes. Thus, Ad5IkappaB-infected hepatocytes were used in case of insulin treatment. Pretreatment with imipramine inhibited glucose uptake by insulin, and also inhibited glycogen deposition by insulin or high dose of glucose in rat hepatocytes as did the knockout of ASM in mouse hepatocytes. Moreover, either insulin or high dose of glucose treatment increased lipid droplets in primary hepatocytes, and imipramine or ASM deficiency suppressed this increase. Insulin induced phosphorylation of AKT in primary hepatocytes, and overexpression of dominant negative-AKT by adenovirus abrogated the phosphorylation of endogenous AKT by insulin. DN-AKT inhibited glucose uptake, glycogen deposition, and lipid accumulation by insulin or high dose of glucose suggesting that AKT has central roles in hepatic glucose and lipid metabolisms. However, pretreatment of imipramine or knockout of ASM did not affect AKT phosphorylation induced by insulin. Conclusion: ASM deficiency causes glucose intolerance and hepatic insulin resistance without affecting insulin-induced AKT activation in hepatocytes.
Clinical charicteristics and maspin expression pattern of the early and advanced cancers
NS:not significant M1672
M1670 An Analysis of CD133 Positive Bone Marrow in Oval Cell Induced Model and a Role of CD133 Bone Marrow in Mice With Liver Fibrosis Atsushi Suetsugu, Masahito Nagaki, Yosuke Osawa, Robert M. Hoffman, Hisataka Moriwaki
Inducible Expression of Hnf4alpha in Huh 7.5 Cell Line Has a Strong Antiproliferative Effect Razvan Iacob, Urda Ruedrich, Michael Rothe, Michael Bock, Benjamin Maasoumy, Ina Rittelmeyer, Marcus Iken, Julia Bitzegeio, Yuan Qinggong, Irinel Popescu, Michael P. Manns, Michael Ott HNF4alpha is one of the key transcription factors coordinating cell proliferation, differentiation, and the maintenance of a hepatocyte phenotype. Transcriptional repression of HNF4alpha has been associated with progression and dedifferentiation of hepatic tumors. The aim of our study was to generate an In Vitro model for the study of HNF4alpha antiproliferative effects using a lentiviral inducible expression system. Methods. A lentiviral Tet-ON coexpression system for HNF4alpha and eGFP under a bi-directional Tet-promoter has been generated. HUH 7.5 cells have been transduced with this lentivirus and single cell clones have been created. After establishing a stable transgenic cell line, cells membranes were labeled with PKH26 and cultured in the presence of doxycycline, in order to induce HNF4alpha expression. The cells cultured without doxycycline and non transduced HUH 7,5 cells were used as controls. The PKH26 intensity has been measured by FACS at day 1, 3 and 5 and the mean doubling time for each cell population has been calculated. Gene expression for genes with antiproliferative effects, known to be upregulated by HNF4alpha in non-hepatic cancer cell lines, as well as for liver specific genes have been investigated by quantitative RTPCR at day 4 and 7, in the presence or absence of doxycycline. Results. A strong antiproliferative effect has been noted in HUH7.5 cells after HNF4alpha induction, with a 10 fold mean doubling time increase. A significant increase in cell size has also been observed. The quantitative RT-PCR analysis indicated a significant upregulation of HNF4 alpha and also of the following genes with a documented antiproliferative effect: p21, desmocollin2, ankyrin3, ADAMTS1, Nell2, Sepp1. A decrease in AFP expression has been noted. Conclusion. HNF4 alpha overexpression in HUH 7,5 human hepatocarcinoma cell line leads to a marked reduction of cell proliferation. HNF4alpha upregulates a variety of genes with antiproliferative effect involved in cell cycle, cell adhesion, cytoscheletal anchoring, metabolism and which are not all liver specific. The new HUH 7,5 transgenic cell line could be used as an In Vitro model for further studies of the antiproliferative effects of HNF4 alpha, leading to a better understanding of hepatocyte growth control. It could also serve as a model for lentiviral insertional mutagenesis studies or In Vivo and In Vitro HCV infectivity and replication studies.
Background: Bone marrow (BM) cells have been reported to behave as tissue-specific stem cells in some organs. However, it is still unknown in the liver whether BM cells are a source for liver stem cells or oval cells and these progenitor cells differentiate to mature liver cells. To examine this issue, we examined the characteristics of oval cells induced by 3,5diethoxycarbonyl-1,4-dihydrocollidine (DDC) using GFP-chimeric mice which contain GFP positive BM cells, and examined whether stem cells from BM cells can be a candidate for cell therapy against liver injury. Methods: Total BM cells of GFP mice were injected (1×107) to irradiated mice (11 Gy). After 6 weeks, the mice were fed with DDC containing chow for 4 or 8 weeks to induce oval cells. Liver cells were isolated by collagenase, and the BMderived GFP positive cells were analyzed using a flow cytometer. In addition, the expressions of albumin, AFP, and CK19 were examined using immunocytochemistry to determine the potential for the differentiation of the BM-driven oval cells. Next, stem cells in BM cells from GFP mice were separated by flow cytometory according to presence or absence of CD133. The separated cells were injected into carbon tetrachloride (CCL4) treated mice and the liver injury and fibrosis were examined by serum transaminase level, hydroxyproline content, HE staining, and Sirius red staining. Results: BM-derived GFP positive cells were mainly observed in periportal lesions in the GFP-chimeric mice 4 weeks after DDC treatment. At 8 weeks, these cells had spread toward the central vein regions. GFP positive cells were detected in 28.9% among the isolated liver cells at 8 weeks. A part of the BM-derived GFP positive cells expressed albumin and CK19 suggesting the cells differentiated and had characteristics of hepatocytes and cholangiocytes. Especially, CD133 positive cells, which were contained 26.9% in the BM derived GFP positive cells, expressed higher mRNA expression of albumin compared with CD133 negative cells. The injection of CD133 positive BM cells significantly improved liver injury and fibrosis in the CCL4 treated mice. In contrast, the transplantion of the CD133 negative cell promoted liver damage and fibrosis. Conclusions: A part of oval cells in the liver are mobilized from BM, and the BM derived stem cells have characteristics of hepatocyte and cholangiocyte progenitor/stem cells. Among the BM derived cells, CD133 positive cells were enriched for the hepatocyte/cholangiocyte marker. CD133 positive BM cells may be useful as a source for cell therapy against liver damages.
M1671
M1673
Aberrant Maspin Expression in Gallbladder Cancer Jeong Kim, Kee Taek Jang, Sun Youn Bae, Kwang Hyuck Lee, Jong Kyun Lee, Jong Chul Rhee, Kyu Taek Lee
Palmitate Induced Interleukin-8 Production From Hepatic Stellate Cells is Toll-Like Receptor 4 Dependent Ming Song, Theresa S. Chen, Shirish Barve, Craig J. McClain
Background/Aims: Maspin(mammary serine protease inhibitor) is expressed in normal human mammary epithelial cells and down-regulated during cancer progression. But, aberrant maspin expression was noted in pancreatic cancers and ovarian cancers. The tissue-specific expression of maspin is epigenetically controlled, and aberrant maspin expression in biliary tract carcinomas is closely associated with demethylation at the promoter region. Recently, We had identified several genes that may be tumor markers for gallbladder cancer using DNA microarray method. Among them maspin seemed to be the most interesting gene. To our knowledge, there is no published data regarding maspin expression in gallbladder cancer. The aim of this study were to evaluate the maspin expression in gallbladder cancer group compared with adenoma and normal GB groups and to compare the expression pattern of early and advanced gallbladder cancers. Methods: A total of 101 patients with primary
Background/Aims: Emerging evidence suggests that elevated circulating free fatty acids (FFAs) play an etiologic role in non-alcoholic steatohepatitis (NASH). Several studies have shown that saturated FFAs exert their pro-inflammatory effects in certain cell types (including adipocytes and macrophages), through toll-like receptor 4 (TLR4) signaling pathway, which contributes to obesity-associated inflammation and insulin resistance. However, the direct effect of FFAs on hepatic stellate cells (HSCs), the main fibrogenic cell type, is unknown. IL-8, a CXC chemokine which plays a major role in neutrophil infiltration/activation, is significantly elevated in the serum of NASH patients. The aim of this study was to investigate whether palmitate could induce IL-8 production in hepatic stellate cell and whether it is TLR4 dependent. Methods: LX2 cells (human hepatic stellate cell line) were exposed to various doses of palmitate for 48 hours. Culture supernatants or cell lysates were collected.
AASLD Abstracts
S-826