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Long-term survival post early liver transplantation in organic acidaemias
HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001
AASLD ABSTRACTS
503A
1323
1324
FREQUENT IMPAIRMENT OF THE SPINDLE ASSEMBLY CHECKPOINT IN HEPATOCELLULAR CARCINOMA. Ayuko Saeki, Shinji Tamura, Nobuyuki tto, Shinichi Kiso, Grad Sch of Medicine, Osaka Univ, Osaka Japan; Yasuo Matsuda, Iwao Yabuuchi, Otemae Hosp, Osaka Japan; Sumio Kawata, Yamagata Univ, Yamagata Japan; Yuji Matsuzawa, Grad Sch of Medicine, Osaka Univ, Osaka Japan
A NOVEL DIFFERENTIALLY EXPRESSED GENE PROMOTES APOPTOSIS OF HEPATOCELLULAR CARCINOMA CELLS. Jin-Ping Lai, Rajeswari Avula, Damian P Montoya, Ileana Aderca, Chao Yu, Ahmad H Bani-Hani, David I Smith, Viji Shridhar, Lewis R Roberts, Mayo Clinic, Rochester, MN
Background & Aims: Genetic instability is one of the characteristic features of various types of cancers and is thought to be important in terms of carcinogenesis and tumor growth. Chromosomal instability (CI), which is characterized by aneuploidy, is one form of genetic instability, and recent work has suggested that CI would be induced by a spindle assembly checkpoint defect. The aim of this study was to determine the frequency of a defect of the spindle assembly checkpoint in hepatoceUular carcinoma (HCC) and whether the alterations of hBUB1 gene, one of components of the spindle assembly checkpoint, is associated with CI in HCC. Methods: Eight HCC cell lines, Hep3B, HepG2, HLE, HT17, HUH-7, Li-7, Mahlavu, and PLC/PRF/5, and 50 samples of histologically confirmed HCC from 50 patients (43 males and 7 female, ages 43-83 years) were used in this study. (1) Aneuploidy was analyzed using DNA flow cytometry. HCC cefllines and lymphocytes, used as an internal standard, were stained with propidium iodide and used to flow cytometry. (2) The function of spindle assembly checkpoint was examined using morphological analysis with microtubule-disrupting drugs (nocodazole or colcemid). Cells were plated in 6cm dishes, After 48 hours, nocodazole or colcemid was added to the media, respectively. Cells were harvested at 6-h time intervals thereafter, and then fixed with glutalaldehyde, stained with Hoechst 33258 and analyzed with fluorescence microscopy. (3) To explore the molecular basis, we isolated RNA from 8 HCC cell lines and 50 HCC specimens and examined the expression and alterations of hBUB1 gene using Northern hybridization and direct sequencing. Results: (1) All HCC cell lines that were examined in this study exhibited aneuploidy. The DNA index that was calculated relative to an internal standard, of Hep3B, HepG2, HLE, HT17, HUH-7, Li-7, Mahlavu, and PLC/PRF/5 were 1.69, 2.34, 1.50, 1.82, 1.55, 1.55, 1.46, and 1.57, respectively. (2) By treatment with nocodazole or colcemid, three cell lines (HLE, Mahlavu, and PLC/PRF/5) exhibited an accumulation of ceils that were arrested at the M phase. However, other ceil lines (Hep3B, HepG2, HT17, HaH-7, and Li-7) showed no accumulation at any time point, including that impaired spindle assembly checkpoint was presentedtn 62.5% of the HCC cell lines. (3) TranscriptionaI expressions of hBUB1 genes were appeared in all cell lines and no significant difference in the amount of transcript was detected between the normal and abnormal checkpoint ceils. No mutations were found in the hBUB1 gene by direct sequencing analysis in either the HCC cell lines or HCC specimens, although we were able to identify the new sequence variants. One variant substitutes serine (TCC) for phenylalanine (TTC) at codon 375, and the other substitutes lysin (AAG) for arginine (AGG) at codon 566. Conclusion: The spindle assembly checkpoint defect was occurred with a high frequency in HCC with CI and might be critical for CI in HCC. However, our results suggested that other mechanisms might contribute to CI in HCC. The mutations of the hBUB1 gene were very rare and did not seem to be involved in tumor development in HCC.
Background: Most human hepatocellular carcinomas (HCC) are detected at an advanced, untreatable stage. Improved understanding of the pathogenesis of HCC will allow the exploration of novel therapeutic interventions for HCC. Aberrations in expression or function of genes regulating the cell cycle, cell death, angiogenesis, and tumor invasion mediate tumor initiation and progression. The aim of this study was to examine the role of a novel differentially expressed protein in the pathogenesis of HCC. Dr. Viii Shridhar provided the construct of the novel cDNA used in this study. Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to investigate expression of the novel gene in 11 HCC cell lines (Hep3B, HepG2, HUH7, PLC5, SKHepl, SNU182, SNU387, SNU398, SNU423, SNU449, and SNU475) as well as in normal liver tissue. High levels of expression were found in SNUI82 and SNU475 cefls. SNU449 cells, which did not express the mRNA, were transfected with either a vector containing the open reading frame (designated pcORF) or pcDNA3.1 vector plasmid DNA by lipofection. To investigate the role of the gene in hepatocellular apoptosis, cell lines either expressing high levels of the mRNA or showing no expression of the mRNA were treated with Staurosporine, a potent protein kinase C inhibitor that induces apoptosis through the mitochondrial pathway. Forty-eight hours after transfection, SNU449-pcORF, SNU449-pcDNA3.1, or untransfected SNU449, SNU 182 and SNU475 cells were treated with Staurosporine (1 uM) for 5 hours. Apoptosis of HCC cells was assessed by both fluorescence microscopy after DAPI staining and agarose get electrophoresis of genomic DNA to assess DNA fragmentation. Results: Expression of the novel mRNA in normal liver tissue was low. Two of the 11 HCC cell lines, SNU182 and SNU475, expressed high levels of the mRNA, whereas the other 9 celllines were negative. Five hours after treatment with Staurosporine, the percent apoptosis of SNU449, SNU449-pcDNA3.1, SNU449-pcORF, SNU182 and SNU475 were 4.34---0.68%, 8.65+1.31%, 42.06-+2.35%, 59.75-+1.17%, 54.01---3.37%, respectively. High-level expression of the differentially expressed gene was significantly associated with promotion of apoptosis in SNU 182, SNU475 and SNU449-pcORF cells (p= 0.0010.0002). A DNA ladder characteristic of apoptosis was found in genomic DNA isolated from 5NU182, SNU475 and SNU449-pcORF cells treated with Staurosporine for 5 hours; no DNA fragmentation was found in genomic DNA from Staurosporine,treated SNU449 and SNU449-pcDNA3.1 cells, or in untreated controls. Conclusion: The novel differentially expressed gene promotes apoptosis of HCC cells. This property may be useful for the development of novel therapeutic approaches for HCC.
1325
1326
PRECOCITY OF LIVER DISEASE IN CYSTIC FIBROSIS (CF). Cecilia A Escanhoela, University of Campinas - UNICAMP, Campinas, SP Brazil; Luciana Meirelles, Konradin Metze, Gabriel Hessel, University of Campinas - UNICAMP, Campinas Brazil
LONG-TERM SURVIVAL POST EARLY LIVER TRANSPLANTATION IN ORGANIC ACIDAEMIAS. Paul Gissen, The Liver Unit, The Birmingham Childrens Hospital, Birmingham Uk; A Chakrapani, J E Wraith, J H Walter, Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester Uk; J V Leonard, Institute of Child Health and Great Ormond Street Hospital for Children, London Uk; J Buckels, D Mayer, J De Ville de Goyet, S V Beath, D A Kelly, P J McKiernan, The Liver Unit, The Birmingham Childrens Hospital, Birmingham Uk
Introduction: CF is an autosomal recessive inherited disorder of ion transport inducing clinical disease in a variety of organs including lungs, exocrine pancreas, liver and gut. It is the most common potentially lethal genetic disorder of the Caucasian population. As the survival of pacients with CF has increased worldwide, hepatic lesions are more evident. Focal biliary cirrhosis is considered the pathognomic hepatic alteration and represents the end-resuh of bile duct plugging with iuspissated secretions. Objective: The aim of this study was to evaluate the natural history of CF-associated liver disease based on quantitative histologic features. Methodology: This study was based on the material obtained by percutaneous liverbiopsy (10 patients) or autopsy (4 patients), in all patients, CF had been diagnosed based on clinical, laboratory and/or PCR findings. Routine histologic slides of formalin-fixed and paraffin-embedded material were analysed quantitatively. We defined as the Portal Biliary Reaction Coefficient (PBRC), the porcentage of portal spaces with bile duct proliferation. In a similar way, the porcentage of portal spaces with acute cholangitis and eosinophilic material in dilated ductules. Furthermore, we determined the ratio between the number of fibrous septs/number of portal spaces, in order to indicate the degree of architectural disturbances. Results: Portal Biliary Reaction (PBR) was observed in 8 patients with early onset in some children (figure). There was a direct correlation between PE;RC and the age (r=0,44; p=0,05). As well as with frequency of eosinophilic material (r~0,46; p=0,035) or acute cholangitis (r=0,46; p=0,036). There was also direct correlation between PBR and septal proliferation (r=0,57; p=0,01). This result was independent of the variable age in a partial correlation (r=0,61). Conclusions: The precocity of CF portal space alterations in some patients supports the necessity of routine liver biopsies, since PCR examination showed only one type of genetic alteration. There are other unknown factor contributing to the development of liver disease. % 100
00 75
so
0
0 0
2~ 0 ~00
6
•
s'o
18o " t~o Age (months)
Introduction: Conventional management of early-onset methylmatonic acidaemia (MMA) and propionic acidaemia (PA) is associated with a poor outcome and thus early liver transplantation (OLT) has been proposed as a potentially "curative" treatment in order to prevent neurological damage. Objective: To review the outcome of early OLT in the treatment of PA and MMA. Patients and Methods: Retrospective case-note review of all patients who have undergone OLT for PA and MMA in a single centre. 8 patients with early-onset organic acidaemias (2 MMA, 2 F, 6 PA, 3 F) underwent OLT between 1991 and 1999. The median age at OLT was 15 months (range 7-29 months). Results: There was a higher than average incidence of serious perioperative complications, relating to metabolic instability. 3 patients survived more than i year (1 MMA, 2 PA) and are now 4, 6 and 8 years post OLT (mean survival 5.7 years) with mean age of 7.5 yrs. All have improved metabolic control. 2 patients with PA have had no metabolic decompensation on a normal diet, are making developmental progress and attend mainstream schools. The surviving patient with MMA initially made developmental progress. She subsequently developed portal vein thrombosis, requiring porto-systemic shunting. This surgery was followed by persistent hyperammonaemia. One year following this, she developed basal ganglia necrosis during an episode of pneumonia, with resultant handicap. Conclusions: Patients with organic acidaemias are a high-risk group for OLT. The potential benefits of improved quality of life, reduction in metabolic decompensation and improved protein tolerance must be weighed against high perioperative mortality. The small number of survivors makes it difficult to assess the neurological benefits of early OLT in this group of patients, and aggressive medical management in infancy should be the priority, with recourse to OLT being an option in an appropriately selected subgroup.
AASLD ABSTRACTS
503A
1323
1324
FREQUENT IMPAIRMENT OF THE SPINDLE ASSEMBLY CHECKPOINT IN HEPATOCELLULAR CARCINOMA. Ayuko Saeki, Shinji Tamura, Nobuyuki tto, Shinichi Kiso, Grad Sch of Medicine, Osaka Univ, Osaka Japan; Yasuo Matsuda, Iwao Yabuuchi, Otemae Hosp, Osaka Japan; Sumio Kawata, Yamagata Univ, Yamagata Japan; Yuji Matsuzawa, Grad Sch of Medicine, Osaka Univ, Osaka Japan
A NOVEL DIFFERENTIALLY EXPRESSED GENE PROMOTES APOPTOSIS OF HEPATOCELLULAR CARCINOMA CELLS. Jin-Ping Lai, Rajeswari Avula, Damian P Montoya, Ileana Aderca, Chao Yu, Ahmad H Bani-Hani, David I Smith, Viji Shridhar, Lewis R Roberts, Mayo Clinic, Rochester, MN
Background & Aims: Genetic instability is one of the characteristic features of various types of cancers and is thought to be important in terms of carcinogenesis and tumor growth. Chromosomal instability (CI), which is characterized by aneuploidy, is one form of genetic instability, and recent work has suggested that CI would be induced by a spindle assembly checkpoint defect. The aim of this study was to determine the frequency of a defect of the spindle assembly checkpoint in hepatoceUular carcinoma (HCC) and whether the alterations of hBUB1 gene, one of components of the spindle assembly checkpoint, is associated with CI in HCC. Methods: Eight HCC cell lines, Hep3B, HepG2, HLE, HT17, HUH-7, Li-7, Mahlavu, and PLC/PRF/5, and 50 samples of histologically confirmed HCC from 50 patients (43 males and 7 female, ages 43-83 years) were used in this study. (1) Aneuploidy was analyzed using DNA flow cytometry. HCC cefllines and lymphocytes, used as an internal standard, were stained with propidium iodide and used to flow cytometry. (2) The function of spindle assembly checkpoint was examined using morphological analysis with microtubule-disrupting drugs (nocodazole or colcemid). Cells were plated in 6cm dishes, After 48 hours, nocodazole or colcemid was added to the media, respectively. Cells were harvested at 6-h time intervals thereafter, and then fixed with glutalaldehyde, stained with Hoechst 33258 and analyzed with fluorescence microscopy. (3) To explore the molecular basis, we isolated RNA from 8 HCC cell lines and 50 HCC specimens and examined the expression and alterations of hBUB1 gene using Northern hybridization and direct sequencing. Results: (1) All HCC cell lines that were examined in this study exhibited aneuploidy. The DNA index that was calculated relative to an internal standard, of Hep3B, HepG2, HLE, HT17, HUH-7, Li-7, Mahlavu, and PLC/PRF/5 were 1.69, 2.34, 1.50, 1.82, 1.55, 1.55, 1.46, and 1.57, respectively. (2) By treatment with nocodazole or colcemid, three cell lines (HLE, Mahlavu, and PLC/PRF/5) exhibited an accumulation of ceils that were arrested at the M phase. However, other ceil lines (Hep3B, HepG2, HT17, HaH-7, and Li-7) showed no accumulation at any time point, including that impaired spindle assembly checkpoint was presentedtn 62.5% of the HCC cell lines. (3) TranscriptionaI expressions of hBUB1 genes were appeared in all cell lines and no significant difference in the amount of transcript was detected between the normal and abnormal checkpoint ceils. No mutations were found in the hBUB1 gene by direct sequencing analysis in either the HCC cell lines or HCC specimens, although we were able to identify the new sequence variants. One variant substitutes serine (TCC) for phenylalanine (TTC) at codon 375, and the other substitutes lysin (AAG) for arginine (AGG) at codon 566. Conclusion: The spindle assembly checkpoint defect was occurred with a high frequency in HCC with CI and might be critical for CI in HCC. However, our results suggested that other mechanisms might contribute to CI in HCC. The mutations of the hBUB1 gene were very rare and did not seem to be involved in tumor development in HCC.
Background: Most human hepatocellular carcinomas (HCC) are detected at an advanced, untreatable stage. Improved understanding of the pathogenesis of HCC will allow the exploration of novel therapeutic interventions for HCC. Aberrations in expression or function of genes regulating the cell cycle, cell death, angiogenesis, and tumor invasion mediate tumor initiation and progression. The aim of this study was to examine the role of a novel differentially expressed protein in the pathogenesis of HCC. Dr. Viii Shridhar provided the construct of the novel cDNA used in this study. Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to investigate expression of the novel gene in 11 HCC cell lines (Hep3B, HepG2, HUH7, PLC5, SKHepl, SNU182, SNU387, SNU398, SNU423, SNU449, and SNU475) as well as in normal liver tissue. High levels of expression were found in SNUI82 and SNU475 cefls. SNU449 cells, which did not express the mRNA, were transfected with either a vector containing the open reading frame (designated pcORF) or pcDNA3.1 vector plasmid DNA by lipofection. To investigate the role of the gene in hepatocellular apoptosis, cell lines either expressing high levels of the mRNA or showing no expression of the mRNA were treated with Staurosporine, a potent protein kinase C inhibitor that induces apoptosis through the mitochondrial pathway. Forty-eight hours after transfection, SNU449-pcORF, SNU449-pcDNA3.1, or untransfected SNU449, SNU 182 and SNU475 cells were treated with Staurosporine (1 uM) for 5 hours. Apoptosis of HCC cells was assessed by both fluorescence microscopy after DAPI staining and agarose get electrophoresis of genomic DNA to assess DNA fragmentation. Results: Expression of the novel mRNA in normal liver tissue was low. Two of the 11 HCC cell lines, SNU182 and SNU475, expressed high levels of the mRNA, whereas the other 9 celllines were negative. Five hours after treatment with Staurosporine, the percent apoptosis of SNU449, SNU449-pcDNA3.1, SNU449-pcORF, SNU182 and SNU475 were 4.34---0.68%, 8.65+1.31%, 42.06-+2.35%, 59.75-+1.17%, 54.01---3.37%, respectively. High-level expression of the differentially expressed gene was significantly associated with promotion of apoptosis in SNU 182, SNU475 and SNU449-pcORF cells (p= 0.0010.0002). A DNA ladder characteristic of apoptosis was found in genomic DNA isolated from 5NU182, SNU475 and SNU449-pcORF cells treated with Staurosporine for 5 hours; no DNA fragmentation was found in genomic DNA from Staurosporine,treated SNU449 and SNU449-pcDNA3.1 cells, or in untreated controls. Conclusion: The novel differentially expressed gene promotes apoptosis of HCC cells. This property may be useful for the development of novel therapeutic approaches for HCC.
1325
1326
PRECOCITY OF LIVER DISEASE IN CYSTIC FIBROSIS (CF). Cecilia A Escanhoela, University of Campinas - UNICAMP, Campinas, SP Brazil; Luciana Meirelles, Konradin Metze, Gabriel Hessel, University of Campinas - UNICAMP, Campinas Brazil
LONG-TERM SURVIVAL POST EARLY LIVER TRANSPLANTATION IN ORGANIC ACIDAEMIAS. Paul Gissen, The Liver Unit, The Birmingham Childrens Hospital, Birmingham Uk; A Chakrapani, J E Wraith, J H Walter, Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester Uk; J V Leonard, Institute of Child Health and Great Ormond Street Hospital for Children, London Uk; J Buckels, D Mayer, J De Ville de Goyet, S V Beath, D A Kelly, P J McKiernan, The Liver Unit, The Birmingham Childrens Hospital, Birmingham Uk
Introduction: CF is an autosomal recessive inherited disorder of ion transport inducing clinical disease in a variety of organs including lungs, exocrine pancreas, liver and gut. It is the most common potentially lethal genetic disorder of the Caucasian population. As the survival of pacients with CF has increased worldwide, hepatic lesions are more evident. Focal biliary cirrhosis is considered the pathognomic hepatic alteration and represents the end-resuh of bile duct plugging with iuspissated secretions. Objective: The aim of this study was to evaluate the natural history of CF-associated liver disease based on quantitative histologic features. Methodology: This study was based on the material obtained by percutaneous liverbiopsy (10 patients) or autopsy (4 patients), in all patients, CF had been diagnosed based on clinical, laboratory and/or PCR findings. Routine histologic slides of formalin-fixed and paraffin-embedded material were analysed quantitatively. We defined as the Portal Biliary Reaction Coefficient (PBRC), the porcentage of portal spaces with bile duct proliferation. In a similar way, the porcentage of portal spaces with acute cholangitis and eosinophilic material in dilated ductules. Furthermore, we determined the ratio between the number of fibrous septs/number of portal spaces, in order to indicate the degree of architectural disturbances. Results: Portal Biliary Reaction (PBR) was observed in 8 patients with early onset in some children (figure). There was a direct correlation between PE;RC and the age (r=0,44; p=0,05). As well as with frequency of eosinophilic material (r~0,46; p=0,035) or acute cholangitis (r=0,46; p=0,036). There was also direct correlation between PBR and septal proliferation (r=0,57; p=0,01). This result was independent of the variable age in a partial correlation (r=0,61). Conclusions: The precocity of CF portal space alterations in some patients supports the necessity of routine liver biopsies, since PCR examination showed only one type of genetic alteration. There are other unknown factor contributing to the development of liver disease. % 100
00 75
so
0
0 0
2~ 0 ~00
6
•
s'o
18o " t~o Age (months)
Introduction: Conventional management of early-onset methylmatonic acidaemia (MMA) and propionic acidaemia (PA) is associated with a poor outcome and thus early liver transplantation (OLT) has been proposed as a potentially "curative" treatment in order to prevent neurological damage. Objective: To review the outcome of early OLT in the treatment of PA and MMA. Patients and Methods: Retrospective case-note review of all patients who have undergone OLT for PA and MMA in a single centre. 8 patients with early-onset organic acidaemias (2 MMA, 2 F, 6 PA, 3 F) underwent OLT between 1991 and 1999. The median age at OLT was 15 months (range 7-29 months). Results: There was a higher than average incidence of serious perioperative complications, relating to metabolic instability. 3 patients survived more than i year (1 MMA, 2 PA) and are now 4, 6 and 8 years post OLT (mean survival 5.7 years) with mean age of 7.5 yrs. All have improved metabolic control. 2 patients with PA have had no metabolic decompensation on a normal diet, are making developmental progress and attend mainstream schools. The surviving patient with MMA initially made developmental progress. She subsequently developed portal vein thrombosis, requiring porto-systemic shunting. This surgery was followed by persistent hyperammonaemia. One year following this, she developed basal ganglia necrosis during an episode of pneumonia, with resultant handicap. Conclusions: Patients with organic acidaemias are a high-risk group for OLT. The potential benefits of improved quality of life, reduction in metabolic decompensation and improved protein tolerance must be weighed against high perioperative mortality. The small number of survivors makes it difficult to assess the neurological benefits of early OLT in this group of patients, and aggressive medical management in infancy should be the priority, with recourse to OLT being an option in an appropriately selected subgroup.