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53 Regulation of interleukin 12 expression in the liver for the treatment of hepatic tumors
Parallel Session 6: Liver Cancer and Regeneration 51 NF-KB ACTIVATION VIA TYROSINE PHOSHORYLATION OF I-KB-ALPHA RESULTS IN THE SELECTIVE MODULATION OF THE NF-KB TRANSCRIPTOME AND ACTIVATION OF PROGENITOR CELLS MARKERS
F. Moretti 1 , E. Palescandolo 1 , C. Serra 1,2 , S. Vossio 1 , A. Costanzo 1,3 , M. Levrero 1,4 . 1 Fond. A. Cesalpino - CRS.IRE, Rome, Italy; 2 Telethon Dulbecco Institute, Rome, Italy; 3 Dept. of Internal Medicine and Dermatology, University of Tor Vergata, Rome, Italy; 4 Dept. of Internal Medicine - Università Di Cagliari, Cagliari, Italy In addition to its well established role in activating genes involved in immunological responses, NFkB has been also implicated in promoting cell growth, cell survival and, more recently, stem cell-like phenotype. In several hepatocarcinogenetic conditions (HBV and HCV infections, ALD, NAFLD) activation of both NFkB pathways and of hepatic progenitor cells (HPCs) is commonly observed. Indeed, Displastic lesions and HCCs may express progenitor cell markers as a result both of hepatocytes dedifferentiation and of HPC maturation arrest. In response to most stimuli activation of NFkB occurs via IkB kinase (IKK) complex-mediated Ser32,36 phosphorylation of the cytoplasmic inhibitor IkBa followed by ubiquitinproteasome dependent degradation. Alternatively, NFkB activation may follow dissociation of IkBa-NFkB complexes after Tyr42 phosphorylation of IkBa. We show here that bpV, a pervanadate derivative that acts as a tyrosine phosphatase inhibitor, induces Tyr42 phosphorylation of IkBa and sustained NFkB transcriptional activation in human HCC cell lines (Huh6, Hep3B, HepG2) and in well differentiated immortalized Met Murine Hepatocytes D3 (MMH-D3) cells. BpV-induced Tyr42 phosphorylation is independent from IKK-mediated Ser32,36 phopshorylation of IkBa and results in sustained nuclear traslocation of p65 and p50 NFkB species. Chromatin immunoprecipitation experiments in Huh6 cells show that bpV induces p65 recruitment onto the promoter of DNp73, a member of the p53/p73 family with pro-proliferative and anti-apoptotic activity in hepatocytes. Finally, exposure of both HCC and MMH-D3 cells to bpV results in cell cycle changes, striking morphological alterations and expression of the progenitor cells markers Sca1, cKit and CD45, suggesting a selective modulation of the NFkB transcriptome.
52 PARTIAL HEPATECTOMY INDUCES MOBILIZATION OF A
19
positive staining with monoclonal antibodies specific for cytokeratin (CK) 8, CK18, alpha-fetoprotein and human albumin. In conclusion, PH induces the mobilization of a distinct population of hematopoietic progenitor cells that can differentiate into the hepatic lineage in vitro. Thus, mobilized AC133+CD45+CD14+ progenitor cells may play a role in liver regeneration after PH in humans.
53 REGULATION OF INTERLEUKIN 12 EXPRESSION IN THE LIVER FOR THE TREATMENT OF HEPATIC TUMORS
M. Zabala 1 , L. Wang 3 , R. Hernandez-Alcoceba 1 , W. Hillen 2 , C. Qian 1 , J. Prieto 1 , M.G. Kramer 1 . 1 University of Navarra-FIMA. Clinica Universitaria/School of Medicine. Division of Gene Therapy and Hepatology. Pamplona, Navarra, Spain; 2 Instität Fur Mikrobiologie, Biochemie Und Genetik, Friedrich-Alexander Universität, Erlangen, Germany; 3 Jilin University, Department of Pathology, Changchun, China IL-12 is a potent cytokine with marked antitumoral activity but potential toxicity. Therefore, when using long-term expression vectors encoding IL12 as a therapy for hepatic tumors, tight regulation of IL-12 expression within the liver is required. The Tet-on system is composed of a chimeric protein (rtTA2S-M2) that induces transcription from a minimal promoter in the presence of doxycycline (Dox). This system, however, is limited by its high basal activity. This work was aimed to optimize the Tet-on system for tight and liver-specific IL-12 regulation and to use it for the treatment of liver cancer. In hepatocytic cell lines we found that basal activity of the Tet-on system was markedly reduced by substituting the CMVm region of the inducible promoter by a sequence of the mouse albumin promoter. The vector that allowed optimal gene regulation in vitro was selected to control human (hIL-12) or mouse (mIL-12) interleukin 12 expression and was transferred to mice using the hydrodynamics-based procedure. In vivo administration of Dox enhanced expression of hIL-12 in a dose-dependent manner, while undetectable levels were observed in the non-induced status. Gene activation could be re-induced several times after plasmid administration and sustained hIL-12 expression was achieved with daily infusion of Dox. All mice with metastatic colon cancer in the liver treated with the vector encoding mIL-12 rejected the tumor when given Dox for 10 days. In summary our data describe an improved Tet-on system which may prove useful as a therapy of liver tumors.
DISTINCT POPULATION OF PROGENITOR CELLS IN HEALTHY LIVER DONORS
U.M. Gehling 1 , M. Willems 2 , K. Schlagner 1 , C. Faltz 1 , D.K. Hossfeld 1 , X. Rogiers 2 . 1 Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 2 Department of Hepatobiliary Surgery and Transplantation, University Hospital Hamburg-Eppendorf, Hamburg, Germany Recent transplantation studies in animal models and humans suggest that hematopoietic stem cells, residing in the bone marrow, can contribute to liver regeneration after injury. However, regeneration after hepatectomy is not believed to involve such cells. Here, we report that liver resection induces mobilization of hematopoietic precursor cells into the circulation. Peripheral blood samples were obtained from 11 healthy donors one day before and at 12 hours after classic partial hepatectomy (PH). As revealed by flow cytometry, a significant increase in the percentage of cells expressing the stem cell marker AC133 could be observed in the postoperative samples. Two-color flow cytometry showed that all AC133positive (+) cells coexpressed the common leucocyte antigen CD45. Unexpectedly, virtually all AC133+ cells also stained positive for the monocyte marker CD14, whereas only a small subset coexpressed the stem cell marker CD34. Using an immunomagnetic device, AC133+ cells were isolated and placed into culture. In semisolid medium containing a combination of hematopoietic growth factors, enriched AC133+ cells gave rise to monocytic/macrophage colonies. On transfer to liquid cultures supplemented with cytokines that support hepatic differentiation, AC133+ cells generated an adherent cell population that immunocytochemically showed
54 LOSS OF MATERNAL-SPECIFIC METHYLATION AT THE L’INSULINE GROWTH FACTOR 2 (IGF2) LOCUS IS A PREDICTIVE FACTOR FOR THE OCCURRENCE OF HEPATOCELLULAR CARCINOMA (HCC) IN HEPATITIS C VIRUS (HCV) CIRRHOSIS
N. Ganne-Carrié 1 , J. Paries 2 , A. Carrié 3 , P. Couvert 3 , A. Kerjean 3 , J.C. Trinchet 1 , M. Beaugrand 1 . 1 Liver Unit, Hospital Jean Verdier, AP-HP, Bondy 93140 and UPRES EA3409, University Paris 13, Paris, France; 2 Public Health Department; Hospital Jean Verdier, AP-HP, Bondy, France; 3 GDPM, Institut Cochin, 24 Rue Du Faubourg Saint-Jacques, Paris, France IGF2 is controlled by an inherited epigenetic process that leads to monoallelic expression. Loss of maternal-specific methylation (LMSM) at the IGF2 locus was observed in HCC suggesting the role of IGF2 in hepatocarcinogenesis. The aim of this study was to assess the predictive value of LMSM at the IGF2 locus for HCC occurrence in patients with HCV cirrhosis. Patients were selected according the following criteria: a) biopsy-proven HCV cirrhosis; b) absence of viral co-infection; c) regular US screening and follow-up untill death; d) absence of detectable HCC at enrollment. Methods: After DNA extraction from frozen liver biopsy, unbiased PCR amplification and DHPLC analysis were used for methylation analysis at IGF2 locus. The predictive value for HCC was assessed by Kaplan-Meier method and Cox model.
F. Moretti 1 , E. Palescandolo 1 , C. Serra 1,2 , S. Vossio 1 , A. Costanzo 1,3 , M. Levrero 1,4 . 1 Fond. A. Cesalpino - CRS.IRE, Rome, Italy; 2 Telethon Dulbecco Institute, Rome, Italy; 3 Dept. of Internal Medicine and Dermatology, University of Tor Vergata, Rome, Italy; 4 Dept. of Internal Medicine - Università Di Cagliari, Cagliari, Italy In addition to its well established role in activating genes involved in immunological responses, NFkB has been also implicated in promoting cell growth, cell survival and, more recently, stem cell-like phenotype. In several hepatocarcinogenetic conditions (HBV and HCV infections, ALD, NAFLD) activation of both NFkB pathways and of hepatic progenitor cells (HPCs) is commonly observed. Indeed, Displastic lesions and HCCs may express progenitor cell markers as a result both of hepatocytes dedifferentiation and of HPC maturation arrest. In response to most stimuli activation of NFkB occurs via IkB kinase (IKK) complex-mediated Ser32,36 phosphorylation of the cytoplasmic inhibitor IkBa followed by ubiquitinproteasome dependent degradation. Alternatively, NFkB activation may follow dissociation of IkBa-NFkB complexes after Tyr42 phosphorylation of IkBa. We show here that bpV, a pervanadate derivative that acts as a tyrosine phosphatase inhibitor, induces Tyr42 phosphorylation of IkBa and sustained NFkB transcriptional activation in human HCC cell lines (Huh6, Hep3B, HepG2) and in well differentiated immortalized Met Murine Hepatocytes D3 (MMH-D3) cells. BpV-induced Tyr42 phosphorylation is independent from IKK-mediated Ser32,36 phopshorylation of IkBa and results in sustained nuclear traslocation of p65 and p50 NFkB species. Chromatin immunoprecipitation experiments in Huh6 cells show that bpV induces p65 recruitment onto the promoter of DNp73, a member of the p53/p73 family with pro-proliferative and anti-apoptotic activity in hepatocytes. Finally, exposure of both HCC and MMH-D3 cells to bpV results in cell cycle changes, striking morphological alterations and expression of the progenitor cells markers Sca1, cKit and CD45, suggesting a selective modulation of the NFkB transcriptome.
52 PARTIAL HEPATECTOMY INDUCES MOBILIZATION OF A
19
positive staining with monoclonal antibodies specific for cytokeratin (CK) 8, CK18, alpha-fetoprotein and human albumin. In conclusion, PH induces the mobilization of a distinct population of hematopoietic progenitor cells that can differentiate into the hepatic lineage in vitro. Thus, mobilized AC133+CD45+CD14+ progenitor cells may play a role in liver regeneration after PH in humans.
53 REGULATION OF INTERLEUKIN 12 EXPRESSION IN THE LIVER FOR THE TREATMENT OF HEPATIC TUMORS
M. Zabala 1 , L. Wang 3 , R. Hernandez-Alcoceba 1 , W. Hillen 2 , C. Qian 1 , J. Prieto 1 , M.G. Kramer 1 . 1 University of Navarra-FIMA. Clinica Universitaria/School of Medicine. Division of Gene Therapy and Hepatology. Pamplona, Navarra, Spain; 2 Instität Fur Mikrobiologie, Biochemie Und Genetik, Friedrich-Alexander Universität, Erlangen, Germany; 3 Jilin University, Department of Pathology, Changchun, China IL-12 is a potent cytokine with marked antitumoral activity but potential toxicity. Therefore, when using long-term expression vectors encoding IL12 as a therapy for hepatic tumors, tight regulation of IL-12 expression within the liver is required. The Tet-on system is composed of a chimeric protein (rtTA2S-M2) that induces transcription from a minimal promoter in the presence of doxycycline (Dox). This system, however, is limited by its high basal activity. This work was aimed to optimize the Tet-on system for tight and liver-specific IL-12 regulation and to use it for the treatment of liver cancer. In hepatocytic cell lines we found that basal activity of the Tet-on system was markedly reduced by substituting the CMVm region of the inducible promoter by a sequence of the mouse albumin promoter. The vector that allowed optimal gene regulation in vitro was selected to control human (hIL-12) or mouse (mIL-12) interleukin 12 expression and was transferred to mice using the hydrodynamics-based procedure. In vivo administration of Dox enhanced expression of hIL-12 in a dose-dependent manner, while undetectable levels were observed in the non-induced status. Gene activation could be re-induced several times after plasmid administration and sustained hIL-12 expression was achieved with daily infusion of Dox. All mice with metastatic colon cancer in the liver treated with the vector encoding mIL-12 rejected the tumor when given Dox for 10 days. In summary our data describe an improved Tet-on system which may prove useful as a therapy of liver tumors.
DISTINCT POPULATION OF PROGENITOR CELLS IN HEALTHY LIVER DONORS
U.M. Gehling 1 , M. Willems 2 , K. Schlagner 1 , C. Faltz 1 , D.K. Hossfeld 1 , X. Rogiers 2 . 1 Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 2 Department of Hepatobiliary Surgery and Transplantation, University Hospital Hamburg-Eppendorf, Hamburg, Germany Recent transplantation studies in animal models and humans suggest that hematopoietic stem cells, residing in the bone marrow, can contribute to liver regeneration after injury. However, regeneration after hepatectomy is not believed to involve such cells. Here, we report that liver resection induces mobilization of hematopoietic precursor cells into the circulation. Peripheral blood samples were obtained from 11 healthy donors one day before and at 12 hours after classic partial hepatectomy (PH). As revealed by flow cytometry, a significant increase in the percentage of cells expressing the stem cell marker AC133 could be observed in the postoperative samples. Two-color flow cytometry showed that all AC133positive (+) cells coexpressed the common leucocyte antigen CD45. Unexpectedly, virtually all AC133+ cells also stained positive for the monocyte marker CD14, whereas only a small subset coexpressed the stem cell marker CD34. Using an immunomagnetic device, AC133+ cells were isolated and placed into culture. In semisolid medium containing a combination of hematopoietic growth factors, enriched AC133+ cells gave rise to monocytic/macrophage colonies. On transfer to liquid cultures supplemented with cytokines that support hepatic differentiation, AC133+ cells generated an adherent cell population that immunocytochemically showed
54 LOSS OF MATERNAL-SPECIFIC METHYLATION AT THE L’INSULINE GROWTH FACTOR 2 (IGF2) LOCUS IS A PREDICTIVE FACTOR FOR THE OCCURRENCE OF HEPATOCELLULAR CARCINOMA (HCC) IN HEPATITIS C VIRUS (HCV) CIRRHOSIS
N. Ganne-Carrié 1 , J. Paries 2 , A. Carrié 3 , P. Couvert 3 , A. Kerjean 3 , J.C. Trinchet 1 , M. Beaugrand 1 . 1 Liver Unit, Hospital Jean Verdier, AP-HP, Bondy 93140 and UPRES EA3409, University Paris 13, Paris, France; 2 Public Health Department; Hospital Jean Verdier, AP-HP, Bondy, France; 3 GDPM, Institut Cochin, 24 Rue Du Faubourg Saint-Jacques, Paris, France IGF2 is controlled by an inherited epigenetic process that leads to monoallelic expression. Loss of maternal-specific methylation (LMSM) at the IGF2 locus was observed in HCC suggesting the role of IGF2 in hepatocarcinogenesis. The aim of this study was to assess the predictive value of LMSM at the IGF2 locus for HCC occurrence in patients with HCV cirrhosis. Patients were selected according the following criteria: a) biopsy-proven HCV cirrhosis; b) absence of viral co-infection; c) regular US screening and follow-up untill death; d) absence of detectable HCC at enrollment. Methods: After DNA extraction from frozen liver biopsy, unbiased PCR amplification and DHPLC analysis were used for methylation analysis at IGF2 locus. The predictive value for HCC was assessed by Kaplan-Meier method and Cox model.