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Transcriptional factors induced by hepatocyte growth factor and analysis of transcriptional regulatory sequences of rat cyclin D1 gene
Poster Sessions
104
of liver function in 4 and gastroduodenitis in 9 however, there was no treatment-related death. In clonclusion, external radiotherapy using 3-DCRP achieved a substantial tumor response and long term survival with acceptable toxicity.
I 1166
STAGING OF HEPATOCELLULAR CARCINOMA USING THE CLIP PROTOCOL
M. Sherman, I. Levy, P.D. Greig, S. Gallinger, B. Langer, M. Asch, J. Chan. Medicine, University Health Network, CaMda Background: A new staging system for hepatocellular carcinoma (HCC) has recently been reported from Italy (CLIP classification). It combines the Child-Pugh staging with tumor criteria: tumor morphology, portal invasion and alpha-fetoprotein. Aims: To validate the use of the CLIP staging in a cohort of HCC patients and compare it with the Okuda staging. Patients and Methods: A retrospective analysis of patients with HCC diagnosed in the Toronto General Hospital between 10/1994-120998. Results: Three hundred and thirteen patients were identified, 19 patient with insufficient data and 37 transplant patients were excluded. Two hundred and fifty seven patients in whom complete data for clinical staging was available were included in the study. The median survival of the cohort was 22.8 months. The CLIP stage 0 group (23.1% of the cohort) and the Okuda stage 1 group (50.7% of the cohort) had a 5 year survival rate of 67% and 35% respectively (p < 0.02). The CLIP stage 0 criteria more accurately defined the patients with a good prognosis. The Okuda classification also failed to identify two thirds of the 37 patients with a poor prognosis, who were identified by the CLIP criteria. Patients in CLIP score &#!61619;4 shared very poor prognosis (median survival of l-3 months), Further classification above stage 4 was unnecessary. Summary: The CLIP classification for HCC is easy to implement and more accurate than the Okuda classification. Our cohort was different from the CLIP cohort (more hepatitis B) but the results were still consistent.
I 1194
TRANSCRIPTIONAL FACTORS INDUCED BY HEPATOCYTE GROWTH FACTOR AND ANALYSIS OF TRANSCRIPTIONAL REGULATORY SEQUENCES OF RAT CYCLIN Dl GENE
Conclusion: These results suggest the possibility that CDlE0.7 is bound by a novel transcription factor induced by growth factors in hepatocytes, and indicated that CREB bind to the CRE in the cyclin Dl promoter.
I 1213
J. Wang, F. Oberti, F. Moal, V. Croquet, E. Vuillemin, J. Roux, 0. Douay, Y. Gallois, P. Cales. University of Angers, HIFIH Laboratory, France
Introduction: Some reports suggested that amiloride, one specific Na+/H+ exchanger inhibitor, could prevent the development of cardiac and renal fibrosis. The preventive effect of early and chronic administration of amiloride on the development of portal hypertension and liver fibrosis has not been studied. Aim: To assess the hemodynamic and antitibrotic effects of chronic administration of amiloride in rats with bile duct ligation. Methods: 30 male SD rats were divided in 2 groups: placebo group (saline 1 ml/day); amiloride group (amiloride, 1 mg/kg/day). Saline or amiloride were administrated by daily gavage after bile duct ligation for 4 weeks. At day 28, hemodynamic measurements included mean arterial pressure (MAP), heart rate (HR), portal pressure (PP), cardiac index (CI), splenorenal shunt blood flow (SRS BF), systemic vascular resistance (SVR). Assesment of liver fibrosis was performed by image analysis, liver hydroxyproline content and plasma hyaluronate level. Results: amiloride did not significantly change systemic hemodynamics (MAP, HR, SVR). However, amiloride significantly decreased SRS BF compared to placebo group (0.8 f 0.8 vs 3.2 f 3.1 ml/mitt, p < 0.05), without significantly change in PP (15 f 2 vs 16 f 2 mmHg, NS). Liver fibrosis was decreased in amiloride-treated rats with a significant decrease in the area of liver fibrosis (6.7 f 2.6 vs 10.1 f 4.1%, p < 0.001) and hepatic hydroxyproline content (0.17 f 0.17 vs 0.29 f 0.12 nglpg prot, p < 0.05). Conclusions: this study suggests that the early and chronic administration of amiloride prevents the development of collateral circulation and of liver fibrosis in bile duct-ligated rats.
A. Moriuchi, A. Ido, M. Onaga, K. Nagata, T. Hori, S. Hirono, K. Hayashi, H. Tsuboucbi. Department Internal Medicine II, Miyazaki Medical College, Miyazaki, Japan
Background: Hepatocyte growth factor (HGF) plays an important role in liver regeneration, and enhances the expression of cyclin Dl mRNA mainly through the activation of Ras/MAPK pathway in hepatocytes. The aim of this study is to clarify the molecular mechanisms goveming the transcriptional regulation of cyclin Dl gene by growth factor signaling in hepatoma cells and primary cultured hepatocyes. Methods: A genomic clone of rat cyclin Dl genes was isolated, and the transcriptional activity of the 5’-flanking region was measured by reporter gene assays. To clarify nuclear factors regulating the rat cyclin Dl promoter, electrophoretic gel mobility shift assays (EMSAs) were performed. Results: The clone of cyclin Dl gene contained the 5.0 kb of 5’-flanking region and exon 1,2 and 3. The transcriptional activity was found in the I 0.7 kb upstream region of the initiation site (CDlE0.7) and the 64 bp promoter. EMSAs showed a specific interaction of a nuclear protein with the CDlE0.7, and this interaction was increased when using proteins extracted from HGF-treated hepatocytes. Although CDlE0.7 revealed high homology with binding sites for HNF-3beta, NF-AT and Ets family, CDlE0.7 did not interact with these nuclear factors. On the other hand, the CREB-binding CAMP responsive element (CRE) was found in the 64 bp promoter.
AMILORIDE PREVENTS THE DEVELOPMENT OF SPLANCHNIC COLLATERAL CIRCULATION AND LIVER FIBROSIS IN RATS WITH SECONDARY BILIARY CIRRHOSIS
I
1219
DETECTION OF CD34-POSlTlVE BUT VON WILLEBRAND FACTOR-NEGATIVE SINUSOIDAL ENDOTHELIAL CELLS IN HCV-ASSOCIATED CHRONIC LIVER DISEASE
S. Ohmori, K. Shit&i, T. Sakai, K. Fujikawa, H. Wagayama, K. Takase, T. Nakano. First department of internal medicine, Mie university school of medicine, Japan
Recently it has been clarified that CD3Cpositive, vWF-negative endothelial progenitor cells play a significant role in angiogenesis. We investigated the relationship between angiogenesis and hepatocarcinogenesis by the detection of CD3Cpositive, vWF-negative endothelial progenitor cells and by the measurement of vascular endothelial growth factor (VEGF) in the human liver tissues with HCV-associated chronic liver diseases. Methods: 34 liver specimens which were obtained from patients with HCV-associated liver cirrhosis (LC) (16 patients progressed to hepatocellular carcinoma (HCC) within 5 years 〔LC/HCC〕, and 18 did not〔LC〕) were embedded in paraffin, and 4 pm histological sections were prepared. They were immunostained with anti-CD34 monoclonal antibody, anti-vWF monoclonal antibody, and anti-VEGF monoclonal antibody. The standard avidin-biodin-peroxidase complex technique was applied for immunostaining. VEGF score was classified 4 levels by the strength of immunostaining. CD3Cpositive cell
104
of liver function in 4 and gastroduodenitis in 9 however, there was no treatment-related death. In clonclusion, external radiotherapy using 3-DCRP achieved a substantial tumor response and long term survival with acceptable toxicity.
I 1166
STAGING OF HEPATOCELLULAR CARCINOMA USING THE CLIP PROTOCOL
M. Sherman, I. Levy, P.D. Greig, S. Gallinger, B. Langer, M. Asch, J. Chan. Medicine, University Health Network, CaMda Background: A new staging system for hepatocellular carcinoma (HCC) has recently been reported from Italy (CLIP classification). It combines the Child-Pugh staging with tumor criteria: tumor morphology, portal invasion and alpha-fetoprotein. Aims: To validate the use of the CLIP staging in a cohort of HCC patients and compare it with the Okuda staging. Patients and Methods: A retrospective analysis of patients with HCC diagnosed in the Toronto General Hospital between 10/1994-120998. Results: Three hundred and thirteen patients were identified, 19 patient with insufficient data and 37 transplant patients were excluded. Two hundred and fifty seven patients in whom complete data for clinical staging was available were included in the study. The median survival of the cohort was 22.8 months. The CLIP stage 0 group (23.1% of the cohort) and the Okuda stage 1 group (50.7% of the cohort) had a 5 year survival rate of 67% and 35% respectively (p < 0.02). The CLIP stage 0 criteria more accurately defined the patients with a good prognosis. The Okuda classification also failed to identify two thirds of the 37 patients with a poor prognosis, who were identified by the CLIP criteria. Patients in CLIP score &#!61619;4 shared very poor prognosis (median survival of l-3 months), Further classification above stage 4 was unnecessary. Summary: The CLIP classification for HCC is easy to implement and more accurate than the Okuda classification. Our cohort was different from the CLIP cohort (more hepatitis B) but the results were still consistent.
I 1194
TRANSCRIPTIONAL FACTORS INDUCED BY HEPATOCYTE GROWTH FACTOR AND ANALYSIS OF TRANSCRIPTIONAL REGULATORY SEQUENCES OF RAT CYCLIN Dl GENE
Conclusion: These results suggest the possibility that CDlE0.7 is bound by a novel transcription factor induced by growth factors in hepatocytes, and indicated that CREB bind to the CRE in the cyclin Dl promoter.
I 1213
J. Wang, F. Oberti, F. Moal, V. Croquet, E. Vuillemin, J. Roux, 0. Douay, Y. Gallois, P. Cales. University of Angers, HIFIH Laboratory, France
Introduction: Some reports suggested that amiloride, one specific Na+/H+ exchanger inhibitor, could prevent the development of cardiac and renal fibrosis. The preventive effect of early and chronic administration of amiloride on the development of portal hypertension and liver fibrosis has not been studied. Aim: To assess the hemodynamic and antitibrotic effects of chronic administration of amiloride in rats with bile duct ligation. Methods: 30 male SD rats were divided in 2 groups: placebo group (saline 1 ml/day); amiloride group (amiloride, 1 mg/kg/day). Saline or amiloride were administrated by daily gavage after bile duct ligation for 4 weeks. At day 28, hemodynamic measurements included mean arterial pressure (MAP), heart rate (HR), portal pressure (PP), cardiac index (CI), splenorenal shunt blood flow (SRS BF), systemic vascular resistance (SVR). Assesment of liver fibrosis was performed by image analysis, liver hydroxyproline content and plasma hyaluronate level. Results: amiloride did not significantly change systemic hemodynamics (MAP, HR, SVR). However, amiloride significantly decreased SRS BF compared to placebo group (0.8 f 0.8 vs 3.2 f 3.1 ml/mitt, p < 0.05), without significantly change in PP (15 f 2 vs 16 f 2 mmHg, NS). Liver fibrosis was decreased in amiloride-treated rats with a significant decrease in the area of liver fibrosis (6.7 f 2.6 vs 10.1 f 4.1%, p < 0.001) and hepatic hydroxyproline content (0.17 f 0.17 vs 0.29 f 0.12 nglpg prot, p < 0.05). Conclusions: this study suggests that the early and chronic administration of amiloride prevents the development of collateral circulation and of liver fibrosis in bile duct-ligated rats.
A. Moriuchi, A. Ido, M. Onaga, K. Nagata, T. Hori, S. Hirono, K. Hayashi, H. Tsuboucbi. Department Internal Medicine II, Miyazaki Medical College, Miyazaki, Japan
Background: Hepatocyte growth factor (HGF) plays an important role in liver regeneration, and enhances the expression of cyclin Dl mRNA mainly through the activation of Ras/MAPK pathway in hepatocytes. The aim of this study is to clarify the molecular mechanisms goveming the transcriptional regulation of cyclin Dl gene by growth factor signaling in hepatoma cells and primary cultured hepatocyes. Methods: A genomic clone of rat cyclin Dl genes was isolated, and the transcriptional activity of the 5’-flanking region was measured by reporter gene assays. To clarify nuclear factors regulating the rat cyclin Dl promoter, electrophoretic gel mobility shift assays (EMSAs) were performed. Results: The clone of cyclin Dl gene contained the 5.0 kb of 5’-flanking region and exon 1,2 and 3. The transcriptional activity was found in the I 0.7 kb upstream region of the initiation site (CDlE0.7) and the 64 bp promoter. EMSAs showed a specific interaction of a nuclear protein with the CDlE0.7, and this interaction was increased when using proteins extracted from HGF-treated hepatocytes. Although CDlE0.7 revealed high homology with binding sites for HNF-3beta, NF-AT and Ets family, CDlE0.7 did not interact with these nuclear factors. On the other hand, the CREB-binding CAMP responsive element (CRE) was found in the 64 bp promoter.
AMILORIDE PREVENTS THE DEVELOPMENT OF SPLANCHNIC COLLATERAL CIRCULATION AND LIVER FIBROSIS IN RATS WITH SECONDARY BILIARY CIRRHOSIS
I
1219
DETECTION OF CD34-POSlTlVE BUT VON WILLEBRAND FACTOR-NEGATIVE SINUSOIDAL ENDOTHELIAL CELLS IN HCV-ASSOCIATED CHRONIC LIVER DISEASE
S. Ohmori, K. Shit&i, T. Sakai, K. Fujikawa, H. Wagayama, K. Takase, T. Nakano. First department of internal medicine, Mie university school of medicine, Japan
Recently it has been clarified that CD3Cpositive, vWF-negative endothelial progenitor cells play a significant role in angiogenesis. We investigated the relationship between angiogenesis and hepatocarcinogenesis by the detection of CD3Cpositive, vWF-negative endothelial progenitor cells and by the measurement of vascular endothelial growth factor (VEGF) in the human liver tissues with HCV-associated chronic liver diseases. Methods: 34 liver specimens which were obtained from patients with HCV-associated liver cirrhosis (LC) (16 patients progressed to hepatocellular carcinoma (HCC) within 5 years 〔LC/HCC〕, and 18 did not〔LC〕) were embedded in paraffin, and 4 pm histological sections were prepared. They were immunostained with anti-CD34 monoclonal antibody, anti-vWF monoclonal antibody, and anti-VEGF monoclonal antibody. The standard avidin-biodin-peroxidase complex technique was applied for immunostaining. VEGF score was classified 4 levels by the strength of immunostaining. CD3Cpositive cell