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Expression and processing of gastrin in patients with hepatocellular carcinoma, fibrolamellar carcinoma and cholangiocarcinoma
AASLD A1219
April 1998 Results Seven of the 24 subjects (29%, 95% C1 11-47%) showed worsening of alanine transaminase and aspartate transaminase of similar proportion. There was no change in bilirubin, gamma GT, creatine kinase or alkaline phosphatase. This group did not differ in other clinical or laboratory parameters from the others who tended to show improving transaminases. Plasma TIMP-1 levels did not change following alcohol withdrawal, but were markedly increased in alcoholics versus control subjects (median 1158 vs 187 ng/ml, p<0.001). Conclusions A proportion of alcoholics show spontaneous mild worsening of hepatic transaminases following alcohol withdrawal. TIMP1 levels do not increase following alcohol withdrawal, but are markedly elevated in alcoholics who have clinically mild liver disease compared to controls. This elevation suggests that inhibition of collagen degradation may be present even at an early stage of alcoholic liver disease.
ciclosporin levels within normal range after treatment. Four patients, who had had headaches since liver transplantation, were asymptomatic after three months of UDC therapy. One of these was hypertensive and had normalization of blood pressure after three months of treayment. Conclusion: UDC therapy improves liver and renal functions in liver transplant patients treated with Neoral despite unchanged cyclosporin levels.
• L0083 EXPRESSION AND PROCESSING OF GASTRIN IN PATIENTS WITH HEPATOCELLULAR CARCINOMA, FIBROLAMELLAR CARCINOMA AND CHOLANGIOCARCINOMA. M.E. Caplin 1, K. Savage 1, K. Khan l, J. Rode 2, B. Brett 1, A. Varro 3, D. Michaeli 4, R.E. Pounder 1, A.P. Dhillnn t. lRoyal Free Hospital School of Medicine, London NW3 2QG. 2Flinders University, Darwin, Australia. 3University of Liverpool, Liverpool L69 3BX. 4Aphton Corporation, Woodland, CA. BACKGROUND: Gastrin and its precursors have been shown to be trophic to some gastrointestinal tumours. Cell culture studies and transgenic mouse models suggest there may be a role for gastrin in the proliferation of some liver tumours. AIM: To assess the expression of gastrin receptor and post-translational forms of gastrin in human hepatocellular carcinoma (HCC), fibrolamellar carcinoma (FLC) cholangiocarcinoma (CC) and normal liver sections. METHODS: Immunocytochemistry was performed on paraffin sections from 23 consecutive patients with HCC, 10 with FLC, 5 with CC and 10 normal liver biopsies. Antibodies directed against CCK-B/gastrin receptor (CCK-BR), progastrin (ProG), glycine-extended gastrin (G-gly) and amidated gastrin (G) were used. Standard avidin-biotin method was used and specificity was demonstrated by pre-absorbance of antibodies with epitope. RESULTS:
Background. Substantial amounts of lipids, mainly represented by phosphatidylcholine (PC) and cholesterol (CH), are daily secreted in bile. However, their precursor sources and secretory mechanisms are still under scrutiny. Particularly, it is not clear whether, besides preformed sources, the biliary PC and CH secretion may also connect with their hepatic synthesis. Previous studies in rats fed with pravastatin had shown a large increase in biliary CH and PC secretion occurring in association with an induced hepatic CH synthesis. Aim. In the present study using the same rat model, the main pathway for PC synthesis was examined in relation to biliary lipid secretion. Methods. Male Sprague-Dawley rats were fed for 3 weeks with 0.075% pravastatin mixed with ground chow and compared to matched animals receiving ground chow only. At the end of treatment bile was obtained by bile fistula for the determination of CH, PC, and total bile acids. Subcellular liver fractions were prepared for the assay of the PC synthetic enzymes cytidylyltransferase (CT) and choline kinase, and of HMG CoA reductase (HMGR). CT protein mass of microsomal and cytosolic fractions was determined by Western blot using a polyclonal CT specific antibody. Liver and bile phospholipid content and classes were assayed after extraction and TLC. Results. Biliary PC and CH secretion at the outset of bile fistula were 283% and 400% of controls. There was an increased CT activity: 2.37 -+ 0.374 vs 1.05 ± 0.314 nmol/mirdmg protein +- SD in microsomes (226% of controls, p<0.001), and 3.14 +_0.217 vs 2.14 +- 0.228 (146%, p<0.01) in the cytosol. Choline kinase activity also increased significantly by 75% indicating an upregulation of multiple steps in the main PC synthetic pathway. CT protein mass increased by 86.8% in microsomes (1945 -+484 vs 1041 -+ 185 arbitrary units, p<0.01) and 46% in cytosol (1617 +_204 vs 1145+_369, p<0.04), possibly due to an altered turnover. No changes were found in hepatic phospholipid content and classes, suggesting that the increased PC synthetic inflow was mainly balanced by export into bile. HMGR increased over 8-fold vs controls. Hepatic free CH was unvaried. Conclusions. The study establishes a coordinate relationship in the increases of PC and CH synthesis and biliary secretion under pravastatin treatment and argues for the involvement of molecular mechanisms regulating these processes.
Expression of CCK-BR and rest-translationalforms of asttin: CCK-BR P r o G G-gly G HCC 21/23 14/23 11/23 0/23 FLC 9/10 8/10 7/10 1/10 CC 5/5 4/5 2/5 2/5 NORMAL 5/10 0/10 0/10 0/10 CONCLUSION: There is expression of the CCK-B/gastrin receptor in most liver tumours and processing of precursor gastrin to ProG and G-gly in a significant proportion of liver tumours which may contribute to tumour proliferation through an autocrine mechanism. Dr M. Caplin and Dr B. Brett have research funding from Aphton Co. CA L0084 URSODEOXYCHOLIC ACID IMPROVES LIVER AND RENAL BIOLOGICAL PARAMETERS IN LIVER TRANSPLANT PATIENTS TREATED WITH NEORAL (CYCLOSPORIN A). FX Car01i-Bosc (1-2), JC Montet (2), EP Peten (1), N Demuth(1), JF Demarquay (1), JP Delmont (1). 1.Service d'H~patologie, H6pital Archet II, Nice, France. 2. INSERM d'H~patologie, Marseille, France. Ursodeoxycholic acid (UDC) is a biliary acid with hepatoprotective properties which have been recently demonstrated for cyclosporin A induced cholostasis in long term treated rats. Aims of the study: to investigate the effect of UDC on liver and renal functions in liver transplant patients treated with Neoral. Patients and Methods: 14 liver transplant cases (9 males), mean age 47,6 years (range: 24-59) were treated for 3 months with the association of UDC (12mg/Kg/d) and Neoral (3,4 mg/Kg/d). Indications for liver transplantation were: alcoholic cirrhosis (n=7), post viral cirrhosis (n=2), secondary biliary cirrhosis (n=l), Wilson's disease (n=l), Budd-Chiari (n=l), myofibromatosis (n=l), fulminant toxic hepatitis (n=l). The mean delay after transplantation was 6,9 years (range 3-10). Clinical examination, blood liver and renal parameters, and residual serum levels of cyclosporin were performed at day 10 and 90. Results: Liver and kidney blood results are shown in the following table: Day 0 increased serum creatinine 6 ALT 3 AST 5 GGT 8 AP 7 (AP, alkalinephosphatase)
normal 0 2 3 6 1
Day 90 improved 4 1 2 1 6
unchanged 2
1
Biochemical indices for cholestasis as well as markers for parenchymal damage were improved in all patients but one. All patients had residual
L0085 COORDINATE INCREASES IN HEPATIC PHOSPHATIDYLCHOLINE AND CHOLESTEROL SYNTHESIS AND BILIARY OUTPUT IN PRAVASTATIN-TREATED RATS. Massimino Can'ella*, Douglas Feldman§, Susanna Cogoi*, and Paul A. Weinhold§. Chair of Gastroenterology, University of Udine Medical School, Udine, Italy*, and Department of Biochemistry, University of Michigan Medical School, Ann Arbor, Michigan, USA§.
• L0086 GLUTATHIONE DEPLETION IN HUMAN CHOLANGIOCYTES IS ASSOCIATED WITH INCREASED BCL-2 DEGRADATION AND ENHANCED APOPTOSIS. A. Celli, F.G. Que, G.J. Gores, N.F. LaRusso, Mayo Medical School, Rochester, MN. Cholangiocytes are the target of a group of liver diseases that include the inflammatory cholangiopathies (i.e., conditions such as PBC) that are characterized by periductal inflammation and cholangiocyte apoptosis. Since inflammation is associated with oxidative stress resulting in glutathione (GSH) depletion, we initiated studies to assess the cellular consequences of GSH depletion. We previously demonstrated that cholangiocytes depleted of GSH by exposure to buthionine sulfoximine (BSO) show decreased Bcl-2 protein expression and increased susceptibility to experimentally-induced apoptosis (Gastroenterology 112(4): A1238, 1997). Nevertheless, a direct canse-effect relationship between GSH depletion and decreased Bcl-2 protein expression was not demonstrated nor was the mechanism of Bcl-2 depletion defined. Our AIM here was to address these two issues. METHODS: Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using BSO; in some experiments, glutathione ethyl ester (GSHee) was added to maintain cellular GSH levels. Apoptosis was assessed morphologically and Bcl-2 protein and message expression measured by quantitative immunoblot and RNAse protection assay, respectively; Bcl-2 protein synthesis and degradation was determined by pulse-chase analysis. RESULTS: As with our previous studies, exposure of cholangiocytes to BSO reduced cellular GSH levels by 93% and Bcl-2 protein by 87%. Addition of GSHee in the presence of BSO reduced apoptosis in BSO treated cholangiocytes to control levels. Maintenance of GSH levels by GSHee supplementation also prevented the BSO-induced decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady state Bcl-2 message levels or Bcl-2 protein synthesis; however, Bcl-2 protein half-life decreased by 56% in BSO treated vs. untreated cells (10h vs. 23h). CONCLUSIONS: Our results using a human cholangiocyte cell line demonstrate for the first time that reduction in GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis and may be relevant to the pathways by which ductopenia occurs in the inflammatory cholangiopathies.
April 1998 Results Seven of the 24 subjects (29%, 95% C1 11-47%) showed worsening of alanine transaminase and aspartate transaminase of similar proportion. There was no change in bilirubin, gamma GT, creatine kinase or alkaline phosphatase. This group did not differ in other clinical or laboratory parameters from the others who tended to show improving transaminases. Plasma TIMP-1 levels did not change following alcohol withdrawal, but were markedly increased in alcoholics versus control subjects (median 1158 vs 187 ng/ml, p<0.001). Conclusions A proportion of alcoholics show spontaneous mild worsening of hepatic transaminases following alcohol withdrawal. TIMP1 levels do not increase following alcohol withdrawal, but are markedly elevated in alcoholics who have clinically mild liver disease compared to controls. This elevation suggests that inhibition of collagen degradation may be present even at an early stage of alcoholic liver disease.
ciclosporin levels within normal range after treatment. Four patients, who had had headaches since liver transplantation, were asymptomatic after three months of UDC therapy. One of these was hypertensive and had normalization of blood pressure after three months of treayment. Conclusion: UDC therapy improves liver and renal functions in liver transplant patients treated with Neoral despite unchanged cyclosporin levels.
• L0083 EXPRESSION AND PROCESSING OF GASTRIN IN PATIENTS WITH HEPATOCELLULAR CARCINOMA, FIBROLAMELLAR CARCINOMA AND CHOLANGIOCARCINOMA. M.E. Caplin 1, K. Savage 1, K. Khan l, J. Rode 2, B. Brett 1, A. Varro 3, D. Michaeli 4, R.E. Pounder 1, A.P. Dhillnn t. lRoyal Free Hospital School of Medicine, London NW3 2QG. 2Flinders University, Darwin, Australia. 3University of Liverpool, Liverpool L69 3BX. 4Aphton Corporation, Woodland, CA. BACKGROUND: Gastrin and its precursors have been shown to be trophic to some gastrointestinal tumours. Cell culture studies and transgenic mouse models suggest there may be a role for gastrin in the proliferation of some liver tumours. AIM: To assess the expression of gastrin receptor and post-translational forms of gastrin in human hepatocellular carcinoma (HCC), fibrolamellar carcinoma (FLC) cholangiocarcinoma (CC) and normal liver sections. METHODS: Immunocytochemistry was performed on paraffin sections from 23 consecutive patients with HCC, 10 with FLC, 5 with CC and 10 normal liver biopsies. Antibodies directed against CCK-B/gastrin receptor (CCK-BR), progastrin (ProG), glycine-extended gastrin (G-gly) and amidated gastrin (G) were used. Standard avidin-biotin method was used and specificity was demonstrated by pre-absorbance of antibodies with epitope. RESULTS:
Background. Substantial amounts of lipids, mainly represented by phosphatidylcholine (PC) and cholesterol (CH), are daily secreted in bile. However, their precursor sources and secretory mechanisms are still under scrutiny. Particularly, it is not clear whether, besides preformed sources, the biliary PC and CH secretion may also connect with their hepatic synthesis. Previous studies in rats fed with pravastatin had shown a large increase in biliary CH and PC secretion occurring in association with an induced hepatic CH synthesis. Aim. In the present study using the same rat model, the main pathway for PC synthesis was examined in relation to biliary lipid secretion. Methods. Male Sprague-Dawley rats were fed for 3 weeks with 0.075% pravastatin mixed with ground chow and compared to matched animals receiving ground chow only. At the end of treatment bile was obtained by bile fistula for the determination of CH, PC, and total bile acids. Subcellular liver fractions were prepared for the assay of the PC synthetic enzymes cytidylyltransferase (CT) and choline kinase, and of HMG CoA reductase (HMGR). CT protein mass of microsomal and cytosolic fractions was determined by Western blot using a polyclonal CT specific antibody. Liver and bile phospholipid content and classes were assayed after extraction and TLC. Results. Biliary PC and CH secretion at the outset of bile fistula were 283% and 400% of controls. There was an increased CT activity: 2.37 -+ 0.374 vs 1.05 ± 0.314 nmol/mirdmg protein +- SD in microsomes (226% of controls, p<0.001), and 3.14 +_0.217 vs 2.14 +- 0.228 (146%, p<0.01) in the cytosol. Choline kinase activity also increased significantly by 75% indicating an upregulation of multiple steps in the main PC synthetic pathway. CT protein mass increased by 86.8% in microsomes (1945 -+484 vs 1041 -+ 185 arbitrary units, p<0.01) and 46% in cytosol (1617 +_204 vs 1145+_369, p<0.04), possibly due to an altered turnover. No changes were found in hepatic phospholipid content and classes, suggesting that the increased PC synthetic inflow was mainly balanced by export into bile. HMGR increased over 8-fold vs controls. Hepatic free CH was unvaried. Conclusions. The study establishes a coordinate relationship in the increases of PC and CH synthesis and biliary secretion under pravastatin treatment and argues for the involvement of molecular mechanisms regulating these processes.
Expression of CCK-BR and rest-translationalforms of asttin: CCK-BR P r o G G-gly G HCC 21/23 14/23 11/23 0/23 FLC 9/10 8/10 7/10 1/10 CC 5/5 4/5 2/5 2/5 NORMAL 5/10 0/10 0/10 0/10 CONCLUSION: There is expression of the CCK-B/gastrin receptor in most liver tumours and processing of precursor gastrin to ProG and G-gly in a significant proportion of liver tumours which may contribute to tumour proliferation through an autocrine mechanism. Dr M. Caplin and Dr B. Brett have research funding from Aphton Co. CA L0084 URSODEOXYCHOLIC ACID IMPROVES LIVER AND RENAL BIOLOGICAL PARAMETERS IN LIVER TRANSPLANT PATIENTS TREATED WITH NEORAL (CYCLOSPORIN A). FX Car01i-Bosc (1-2), JC Montet (2), EP Peten (1), N Demuth(1), JF Demarquay (1), JP Delmont (1). 1.Service d'H~patologie, H6pital Archet II, Nice, France. 2. INSERM d'H~patologie, Marseille, France. Ursodeoxycholic acid (UDC) is a biliary acid with hepatoprotective properties which have been recently demonstrated for cyclosporin A induced cholostasis in long term treated rats. Aims of the study: to investigate the effect of UDC on liver and renal functions in liver transplant patients treated with Neoral. Patients and Methods: 14 liver transplant cases (9 males), mean age 47,6 years (range: 24-59) were treated for 3 months with the association of UDC (12mg/Kg/d) and Neoral (3,4 mg/Kg/d). Indications for liver transplantation were: alcoholic cirrhosis (n=7), post viral cirrhosis (n=2), secondary biliary cirrhosis (n=l), Wilson's disease (n=l), Budd-Chiari (n=l), myofibromatosis (n=l), fulminant toxic hepatitis (n=l). The mean delay after transplantation was 6,9 years (range 3-10). Clinical examination, blood liver and renal parameters, and residual serum levels of cyclosporin were performed at day 10 and 90. Results: Liver and kidney blood results are shown in the following table: Day 0 increased serum creatinine 6 ALT 3 AST 5 GGT 8 AP 7 (AP, alkalinephosphatase)
normal 0 2 3 6 1
Day 90 improved 4 1 2 1 6
unchanged 2
1
Biochemical indices for cholestasis as well as markers for parenchymal damage were improved in all patients but one. All patients had residual
L0085 COORDINATE INCREASES IN HEPATIC PHOSPHATIDYLCHOLINE AND CHOLESTEROL SYNTHESIS AND BILIARY OUTPUT IN PRAVASTATIN-TREATED RATS. Massimino Can'ella*, Douglas Feldman§, Susanna Cogoi*, and Paul A. Weinhold§. Chair of Gastroenterology, University of Udine Medical School, Udine, Italy*, and Department of Biochemistry, University of Michigan Medical School, Ann Arbor, Michigan, USA§.
• L0086 GLUTATHIONE DEPLETION IN HUMAN CHOLANGIOCYTES IS ASSOCIATED WITH INCREASED BCL-2 DEGRADATION AND ENHANCED APOPTOSIS. A. Celli, F.G. Que, G.J. Gores, N.F. LaRusso, Mayo Medical School, Rochester, MN. Cholangiocytes are the target of a group of liver diseases that include the inflammatory cholangiopathies (i.e., conditions such as PBC) that are characterized by periductal inflammation and cholangiocyte apoptosis. Since inflammation is associated with oxidative stress resulting in glutathione (GSH) depletion, we initiated studies to assess the cellular consequences of GSH depletion. We previously demonstrated that cholangiocytes depleted of GSH by exposure to buthionine sulfoximine (BSO) show decreased Bcl-2 protein expression and increased susceptibility to experimentally-induced apoptosis (Gastroenterology 112(4): A1238, 1997). Nevertheless, a direct canse-effect relationship between GSH depletion and decreased Bcl-2 protein expression was not demonstrated nor was the mechanism of Bcl-2 depletion defined. Our AIM here was to address these two issues. METHODS: Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using BSO; in some experiments, glutathione ethyl ester (GSHee) was added to maintain cellular GSH levels. Apoptosis was assessed morphologically and Bcl-2 protein and message expression measured by quantitative immunoblot and RNAse protection assay, respectively; Bcl-2 protein synthesis and degradation was determined by pulse-chase analysis. RESULTS: As with our previous studies, exposure of cholangiocytes to BSO reduced cellular GSH levels by 93% and Bcl-2 protein by 87%. Addition of GSHee in the presence of BSO reduced apoptosis in BSO treated cholangiocytes to control levels. Maintenance of GSH levels by GSHee supplementation also prevented the BSO-induced decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady state Bcl-2 message levels or Bcl-2 protein synthesis; however, Bcl-2 protein half-life decreased by 56% in BSO treated vs. untreated cells (10h vs. 23h). CONCLUSIONS: Our results using a human cholangiocyte cell line demonstrate for the first time that reduction in GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis and may be relevant to the pathways by which ductopenia occurs in the inflammatory cholangiopathies.