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347 Potential improvement of liver repopulation by transplanted mouse hepatocytes in propranolol pre-treated mice after partial hepatectomy
Category 4d: Molecular and Cellular Biology: Liver Regeneration and Experimental Oncology used to investigate mRNA and protein level. The expression of multidrug resistance gene (MDR1), multidrug resistance protein (MRP) and lung resistance protein (LRP) was increased in a time-dependent manner. We found simultaneously that hypoxia inducible factor-1-alpha (HIF-1-α) was also increased by glucose starvation. And the alteration of HIF-1-α was almost consistent with that of MDR1, MRP and LRP. The transient transfection of the hepG2 cells with a HIF-1-α plasmid confirmed that HIF-1-α promoted the expression of correlative multidrug resistance genes. All these results indicated that glucose metabolism is very important in chemoresistance of HCC. Glucose deprivation induces HIF-1-α-dependent multidrug resistance of HCC. Inhibition of expression of HIF-1-α can become a novel approach to circumventing multidrug resistance of HCC.
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ated gene transfer in vitro. Two days after PH, up to 5x105 hepatocytes were transplanted into the spleen of propPH or PH treated mice. Clusters of fluorescent hepatocytes were detected microscopically in the livers of propPH mice 19 days after cell transplantation. Conclusions: 1. propPH treatment temporarily reduced the regenerative capacity of the liver. 2. dsRED labelled donor hepatocytes allow for a rapid screening for transplanted cells. 3. By temporarily blocking cell cycle progression in the host hepatocytes, propranolol pre-treatment of recipient mice might improve liver repopulation by transplanted hepatocytes.
348 TELOMERE SHORTENING HAS A DIVERSE EFFECT ON TUMOR SUPRESSION, LIVER REGENERATION AND SURVIVAL IN MTERC DEFICIENT MICE
346 TUPAIA HEPATOCYTES BUT NOT NONPARENCHYMAL LIVER CELLS CAN EXTENSIVELY REPOPULATE THE LIVERS OF UPA/RAG-2 MICE
M.R. Burda 1 , K. Wursthorn 1,2 , M. Dandri 1,2 , E. Toeroek 3 , J.M. Pollok 3 , X. Rogiers 3 , H. Greten 1 , J. Koeck 4 , F. von Weizsaecker 4 , H.E. Blum 4 , J. Petersen 1,2 . 1 Department of Medicine, University Hospital Hamburg, Eppendorf; 2 Heinrich Pette Institute For Experimental Virology, University of Hamburg, Hamburg; 3 Department of Hepatobiliary Surgery and Transplantation, University Hospital Hamburg, Eppendorf; 4 Department of Medicine, University of Freiburg, Freiburg, Germany The tree shrew species Tupaia belangeri has been used for the study of HBV infection both in vitro and in vivo, taking advantage of the adaptability of these animals to the laboratory environment and their close evolutionary relationship to primates. We developed a novel immunodeficient chimeric mouse model (uPA/RAG-2), in which hepatocyte-targeted expression of a hepatotoxic urokinase-type plasminogen activator (uPA) transgene depletes host hepatocytes progressively. The uPA/RAG-2 mice lack mature T and B lymphocytes due to a deletion in the recombination activation gene 2 (RAG-2). We have shown that transplantation of Tupaia hepatocytes in uPA/RAG-2 mice allowed extensive proliferation of both freshly isolated and cryopreserved xenografted cells in the mouse liver (AASLD 2002). It is speculated that nonparenchymal liver cells contain liver stem cells or progenitor cells. Here we show that purified nonparenchymal cells from Tupaia livers were not able to contribute to the repopulation of mouse livers after transplantation. Nonparenchymal cells were not able to differentiate into hepatocyte like cells as demonstrated by the absence of hepatocyte specific proteins.
S.U. Wiemann 1 , K. Kamino 2 , M.P. Manns 1 , K.L. Rudolph 1 . 1 Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Hannover, Germany; 2 Institut Fur Zell- Und Molekularpathologie, Medical School Hannover, Hannover, Germany Telomere shortening limits tumor progression but also restrains the regenerative capacity of cells. The use of telomerase activators for treatment of chronic liver diseases will depend on its effects on regeneration and cancer. The impact of telomere shortening on heptocellular cancer (HCC) and liver regeneration was tested in HBV-transgenic mice, a model of chronic liver damage, crossed with telomerase deficient (mTERC-/-) mice. We analysed mTERC+/+ wildtype mice and late generations G3mTERC-/- mice, both either HBV-positive or negative. The animals were sacrified at 5, 10 and 15 month resp. and livers were histologically characterised. HBV-negative mice showed no pathological or survival phenotype irrespective of the telomere status. In contrast, HBV-positive mice developed microscopic and macroscopic liver tumors, and showed signs of chronic liver damage and hepatitis. G3mTERC-/- mice had a significant supression of foci compared to mTERC+/+ mice and a complete suppression of HCC, which developed in 45% of mTERC+/+ mice. Despite suppressed tumor progression the survival of HBV-positive G3mTERC-/- mice was significant reduced. In correlation with reduced survival HBV-positive G3mTERC-/mice showed higher rates of apoptotic cells compared to mTERC+/+ mice. Especially the ratio of cell apoptosis/regeneration was disturbed in HBVpositive G3mTERC-/- mice compared to mTERC+/+ mice. Together our study shows telomere shortening in the setting of chronic liver damage supresses HCC formation but at the same time reduces survival by disturbing the balance between liver regeneration and liver cell apoptosis. These data support the potential use of telomerase activators for end-stage chronic liver disease limited by cirrhosis progression.
347 POTENTIAL IMPROVEMENT OF LIVER REPOPULATION BY TRANSPLANTED MOUSE HEPATOCYTES IN PROPRANOLOL PRE-TREATED MICE AFTER PARTIAL HEPATECTOMY
349 MULTIDRUG RESISTANCE INDUCED BY HYPOXIA IN HEPATOCELLULAR CARCINOMA
J. Walldorf, R. Haftendorn, H. Aurich, W.E. Fleig, B. Christ. Department For Internal Medicine I, Section Molecular Hepatology, University of Halle-Wittenberg, Halle-Wittenberg, Germany
H. Zhu, S.F. Luo, J. Guan, W.G. Zhang, B.X. Zhang, X.P. Chen. Hepatic Surgery Center, Tongji Hospital, Wuhan City, China
Introduction: Hepatocyte transplantation is an alternative to liver transplantation for the treatment of liver disease. In normal animals, liver repopulation after partial hepatectomy (PH) by transplanted hepatocytes is discontenting, because the transplanted cells do not have a growth advantage over the host cells. Protocols to block recipient hepatocyte proliferation are not applicable clinically because of side effects of the applied drugs. Methods and Results: To temporarily block the proliferation of the host hepatocytes, female C57/Bl6 mice were treated with propranolol p.o. (15 µg/kg BW per day) for five days prior to PH. As compared to PH alone, liver regeneration after PH and propranolol pre-treatment (propPH) was impaired as demonstrated by higher serum bilirubin levels, lower liver/body weight-ratio and lower expression of cell cycle proteins (Western blotting). For transplantation, hepatocytes were isolated from male syngeneic donors and labelled by a red fluorescent protein (dsRED) via lipid medi-
The rapid tumor growth results in increasing oxygen consume and relatively decreasing blood supply, making for hypoxia of local microenvironment in hepatocellular carcinoma (HCC). Hypoxia activates a number of hypoxia responsive elements. Here we demonstrate the expression of several correlative multidrug resistance genes induced by hypoxia in HCC. HepG2 cells were exposed to hypoxia (pO2 2%, N2 90%, CO2 5%). We analyzed the revealed multidrug resistance gene (MDR1), multidrug resistance protein (MRP), lung resistance protein (LRP) at the mRNA and protein level in a continuous series of phases (total 72 hours) respectively using real-time fluorescent quantitative PCR and Western-blot. Simultaneously hypoxia inducible factor-1-alpha (HIF-1-alpha) was also investigated. MDR1 and MRP RNA level were stepped up in hypoxia 6, 12, 24, 36, 48 hours and the changes of their protein level were also coincident. Furthermore, they were synchronous with the changes of HIF1-alpha. But the expression of LRP RNA and protein was significantly
105
ated gene transfer in vitro. Two days after PH, up to 5x105 hepatocytes were transplanted into the spleen of propPH or PH treated mice. Clusters of fluorescent hepatocytes were detected microscopically in the livers of propPH mice 19 days after cell transplantation. Conclusions: 1. propPH treatment temporarily reduced the regenerative capacity of the liver. 2. dsRED labelled donor hepatocytes allow for a rapid screening for transplanted cells. 3. By temporarily blocking cell cycle progression in the host hepatocytes, propranolol pre-treatment of recipient mice might improve liver repopulation by transplanted hepatocytes.
348 TELOMERE SHORTENING HAS A DIVERSE EFFECT ON TUMOR SUPRESSION, LIVER REGENERATION AND SURVIVAL IN MTERC DEFICIENT MICE
346 TUPAIA HEPATOCYTES BUT NOT NONPARENCHYMAL LIVER CELLS CAN EXTENSIVELY REPOPULATE THE LIVERS OF UPA/RAG-2 MICE
M.R. Burda 1 , K. Wursthorn 1,2 , M. Dandri 1,2 , E. Toeroek 3 , J.M. Pollok 3 , X. Rogiers 3 , H. Greten 1 , J. Koeck 4 , F. von Weizsaecker 4 , H.E. Blum 4 , J. Petersen 1,2 . 1 Department of Medicine, University Hospital Hamburg, Eppendorf; 2 Heinrich Pette Institute For Experimental Virology, University of Hamburg, Hamburg; 3 Department of Hepatobiliary Surgery and Transplantation, University Hospital Hamburg, Eppendorf; 4 Department of Medicine, University of Freiburg, Freiburg, Germany The tree shrew species Tupaia belangeri has been used for the study of HBV infection both in vitro and in vivo, taking advantage of the adaptability of these animals to the laboratory environment and their close evolutionary relationship to primates. We developed a novel immunodeficient chimeric mouse model (uPA/RAG-2), in which hepatocyte-targeted expression of a hepatotoxic urokinase-type plasminogen activator (uPA) transgene depletes host hepatocytes progressively. The uPA/RAG-2 mice lack mature T and B lymphocytes due to a deletion in the recombination activation gene 2 (RAG-2). We have shown that transplantation of Tupaia hepatocytes in uPA/RAG-2 mice allowed extensive proliferation of both freshly isolated and cryopreserved xenografted cells in the mouse liver (AASLD 2002). It is speculated that nonparenchymal liver cells contain liver stem cells or progenitor cells. Here we show that purified nonparenchymal cells from Tupaia livers were not able to contribute to the repopulation of mouse livers after transplantation. Nonparenchymal cells were not able to differentiate into hepatocyte like cells as demonstrated by the absence of hepatocyte specific proteins.
S.U. Wiemann 1 , K. Kamino 2 , M.P. Manns 1 , K.L. Rudolph 1 . 1 Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Hannover, Germany; 2 Institut Fur Zell- Und Molekularpathologie, Medical School Hannover, Hannover, Germany Telomere shortening limits tumor progression but also restrains the regenerative capacity of cells. The use of telomerase activators for treatment of chronic liver diseases will depend on its effects on regeneration and cancer. The impact of telomere shortening on heptocellular cancer (HCC) and liver regeneration was tested in HBV-transgenic mice, a model of chronic liver damage, crossed with telomerase deficient (mTERC-/-) mice. We analysed mTERC+/+ wildtype mice and late generations G3mTERC-/- mice, both either HBV-positive or negative. The animals were sacrified at 5, 10 and 15 month resp. and livers were histologically characterised. HBV-negative mice showed no pathological or survival phenotype irrespective of the telomere status. In contrast, HBV-positive mice developed microscopic and macroscopic liver tumors, and showed signs of chronic liver damage and hepatitis. G3mTERC-/- mice had a significant supression of foci compared to mTERC+/+ mice and a complete suppression of HCC, which developed in 45% of mTERC+/+ mice. Despite suppressed tumor progression the survival of HBV-positive G3mTERC-/- mice was significant reduced. In correlation with reduced survival HBV-positive G3mTERC-/mice showed higher rates of apoptotic cells compared to mTERC+/+ mice. Especially the ratio of cell apoptosis/regeneration was disturbed in HBVpositive G3mTERC-/- mice compared to mTERC+/+ mice. Together our study shows telomere shortening in the setting of chronic liver damage supresses HCC formation but at the same time reduces survival by disturbing the balance between liver regeneration and liver cell apoptosis. These data support the potential use of telomerase activators for end-stage chronic liver disease limited by cirrhosis progression.
347 POTENTIAL IMPROVEMENT OF LIVER REPOPULATION BY TRANSPLANTED MOUSE HEPATOCYTES IN PROPRANOLOL PRE-TREATED MICE AFTER PARTIAL HEPATECTOMY
349 MULTIDRUG RESISTANCE INDUCED BY HYPOXIA IN HEPATOCELLULAR CARCINOMA
J. Walldorf, R. Haftendorn, H. Aurich, W.E. Fleig, B. Christ. Department For Internal Medicine I, Section Molecular Hepatology, University of Halle-Wittenberg, Halle-Wittenberg, Germany
H. Zhu, S.F. Luo, J. Guan, W.G. Zhang, B.X. Zhang, X.P. Chen. Hepatic Surgery Center, Tongji Hospital, Wuhan City, China
Introduction: Hepatocyte transplantation is an alternative to liver transplantation for the treatment of liver disease. In normal animals, liver repopulation after partial hepatectomy (PH) by transplanted hepatocytes is discontenting, because the transplanted cells do not have a growth advantage over the host cells. Protocols to block recipient hepatocyte proliferation are not applicable clinically because of side effects of the applied drugs. Methods and Results: To temporarily block the proliferation of the host hepatocytes, female C57/Bl6 mice were treated with propranolol p.o. (15 µg/kg BW per day) for five days prior to PH. As compared to PH alone, liver regeneration after PH and propranolol pre-treatment (propPH) was impaired as demonstrated by higher serum bilirubin levels, lower liver/body weight-ratio and lower expression of cell cycle proteins (Western blotting). For transplantation, hepatocytes were isolated from male syngeneic donors and labelled by a red fluorescent protein (dsRED) via lipid medi-
The rapid tumor growth results in increasing oxygen consume and relatively decreasing blood supply, making for hypoxia of local microenvironment in hepatocellular carcinoma (HCC). Hypoxia activates a number of hypoxia responsive elements. Here we demonstrate the expression of several correlative multidrug resistance genes induced by hypoxia in HCC. HepG2 cells were exposed to hypoxia (pO2 2%, N2 90%, CO2 5%). We analyzed the revealed multidrug resistance gene (MDR1), multidrug resistance protein (MRP), lung resistance protein (LRP) at the mRNA and protein level in a continuous series of phases (total 72 hours) respectively using real-time fluorescent quantitative PCR and Western-blot. Simultaneously hypoxia inducible factor-1-alpha (HIF-1-alpha) was also investigated. MDR1 and MRP RNA level were stepped up in hypoxia 6, 12, 24, 36, 48 hours and the changes of their protein level were also coincident. Furthermore, they were synchronous with the changes of HIF1-alpha. But the expression of LRP RNA and protein was significantly