- Email: [email protected]
264 COMMON GENETIC VARIATION IN NATURAL KILLER CELL RECEPTOR PROTEIN G2D DOES NOT MODIFY SUSCEPTIBILITY TO SPORADIC CHOLANGIOCARCINOMA
POSTERS exposure. After withdrawal of sorafenib, these resistant cells show increased proliferation and metabolic activity. The HepG2 resistant cell lines have undergone an epithelial to mesenchymal transition (EMT) with loss of E-cadherin and high expression of vimentin, both on RNA- and protein level. The cells that displayed EMT became spindle shaped and were highly invasive. The resistant HepG2 cell line showed cross resistance for the mTOR inhibitor everolimus, but not for the PI3K-inhibitor LY294002. LY294002 also inhibited invasive potential at high dose. Conclusion: Long-term treatment with sorafenib can lead to the development of resistant cells with an aggressive phenotype, because the cells undergo EMT. Furthermore, we demonstrated that abrogation of treatment leads to rebound growth, suggesting the importance of aggressive management of side-effects. In addition, the resistant cell lines can be used to identify new therapies, for example PI3K-inhibitors, after progression with sorafenib. To understand the mechanism of resistance and find new therapeutic targets, we are currently performing microarray on the resistant cells. 262 SERPINB3 IS ASSOCIATED TO LONGER SURVIVAL IN MALE TRANSGENIC MICE G. Villano1 , M. Ruvoletto1 , F. Calabrese2 , S. Quarta1 , C. Turato3 , N. Tono3 , A. Biasiolo1 , C. Merkel1 , A. Gatta1 , P. Pontisso1 . 1 Clinical and Experimental Medicine, 2 Diagnostic Medical Sciences and Special Therapies, University of Padova, 3 Istituto Oncologico Veneto, I.O.V.-IRCCS, Padova, Italy E-mail: [email protected] Background: SERPINB3, a member of the ovalbumin family, is physiologically expressed in squamous epithelium and it is overexpressed in tumours of epithelial origin and in hepatocellular carcinoma. Different biological activities of SERPINb3 have been reported, including inhibition of apoptosis and, more recently, deregulation of adhesion processes and increased invasiveness potential supported by features of epithelial-mesenchymal transition. Aim: To assess the biological effects of the SERPINb3 in a transgenic mouse model, with a particular focus on survival and hepatic features. Methods: 123 mice transgenic for SERPINB3 and 148 wild type mice (strain C57BL/6J) have been followed up till death. Liver function parameters (ALT, AST, bilirubin and albumin) were measured in serum by commercial test. In a subgroup of mice (38 transgenic and 35 wild type) the liver was analyzed by histology and immunohistochemistry. In addition, SERPINB3 RNA expression in the liver was evaluated by real-time PCR (SYBR Green). Results: Transgenic mice showed a significantly longer survival than controls (mean±SD: 13.83±0.65 months vs 11.73±0.55 months, p = 0.0036). The difference was remarkably high in males (transgenic survival: 13.72±0.83 months vs controls: 11.19±0.71 months, p = 0.0085), while females survival was similar (transgenic: 13.96±1.03 months vs controls: 12.33±0.84 months, p = 0.1169). At hepatic level liver function tests revealed higher albumin concentration only in transgenic males (mean±SD, transgenic: 3.26±0.12 g/dL vs control males: 2.79±0.33 g/dL, p = 0.0312). Liver histology of transgenic mice and controls did not show any significant difference: steatosis accumulation and glycogen reduction more relevant at older age and these findings were confirmed by multivariate stepwise analysis. In addition, none of the animals developed neoplastic transformation foci. Conclusions: SERPINB3 induces cell protection and longer survival, especially in males, and it is not oncogenic by itself. The greater cell resistance to apoptotic cell death could be responsible for an increased risk of neoplastic transformation in chronic stress conditions. The interaction between SERPINB3 and estrogens activity remains to be determined. S108
263 NOVEL SPONTANEOUS HEPATOCELLULAR CARCINOMA (HCC) MODEL DEVELOPING IN FIBROTIC/CIRRHOTIC LIVER IN MICE A. Vogt1 , G. Decker1 , S. Conchon2 , E. Raskopf1 , T. Sauerbruch1 , M.A. Gonzalez-Carmona1 . 1 Department of Inner Medicine I, University of Bonn, Bonn, Germany; 2 Bioth´erapies H´epathiques, CHU Hotel Dieu, Nantes, France E-mail: [email protected] Background: The majority of HCC develop on basis of liver fibrosis or cirrhosis. In order to understand the influence of the the involving fibrotic/cirrhotic milieu on HCC and for the successful development of novel immunotherapies it is of upmost importance to establish a mouse tumor model that closely resembles the progression of human HCC. Therefore our aim was to establish a model of spontaneous developing multifocal HCC in liver with fibrosis/cirrhosis. Methods: N-nitrosodiethylamine (DEN) was injected intraperitoneally into 14 days old C3H-mice to induce spontaneous liver tumors. 50% of animals were additionally submitted to regular ethanol intake combined with an injection of Thioacetamid (TAA) to induce liver fibrosis. Survival time of animals with or without liver fibrosis/cirrhosis was monitored over 8 months. Thereafter animals were sacrificed, and tumor burden was analyzed. Portal fibrosis was verified by van Gieson staining. The expression of AFP in tumors was detected using western blot. Results: Eight months after DEN-treatment alone, 100% of mice developed multifocal HCC in a healthy liver. All mice additionally submitted to TAA/Ethanol developed multifocal HCC and liver fibrosis/cirrhosis (score F4). Tumor load was increased in the livers with fibrosis/cirrhosis. 30% of animals with liver fibrosis already died 6 months after DEN-treatment, whereas 100% of animals were alive in the group without fibrosis. Expression of murine AFP was detected in tumors of DEN-only- as well as in DEN-TAA-ethanol treated mice but not in healthy liver tissue. Amounts of AFP were significantly higher in the tumors from fibrotic livers compared to non-fibrotic livers. First analysis of the tumor milieu of HCC showed important differences in the expression pattern of chemokines/ cytokines between HCC developing in cirrhotic and non-cirrhotic environment. Conclusions: We were able to establish a novel spontaneous multifocal AFP-positive HCC model developing in a health liver but also in a fibrotic/cirrhotic liver. The presence of a liver fibrosis/cirrhosis seems to accelerate tumor progress. Thus, this model represents an opportunity to further analyze initiation and promotion of HCC as well as the development of new therapies. 264 COMMON GENETIC VARIATION IN NATURAL KILLER CELL RECEPTOR PROTEIN G2D DOES NOT MODIFY SUSCEPTIBILITY TO SPORADIC CHOLANGIOCARCINOMA C.A. Wadsworth1 , P.H. Dixon2 , A.A. Zabron1 , J.H. Wong1 , M.H. Chapman3 , S.C. McKay4 , D.R. Spalding4 , S.P. Pereira3 , H.S. Wasan5 , H.C. Thomas1 , S.D. Taylor-Robinson1 , J.C. Whittaker6 , C. Williamson2 , S.A. Khan1 . 1 Department of Hepatology & Gastroenterology, 2 Institute of Reproductive and Developmental Biology, Imperial College London, 3 Institute of Hepatology, University College London, 4 Department Surgery, 5 Department of Oncology, Imperial College London, 6 Statistical Genetics Unit, Royal School of Hygiene & Tropical Medicine, London, UK E-mail: [email protected] Background: Sporadic cholangiocarcinoma (CC) occurs in subjects with no known risk factor for the disease. Its pathogenesis is poorly understood but is likely to involve complex interaction of environmental and human factors, including genetic variation. Polymorphisms in natural killer cell receptor protein G2D (NKG2D) have been associated with numerous malignancies, including CC
Journal of Hepatology 2011 vol. 54 | S61–S208
POSTERS in subjects with primary sclerosing cholangitis (PSC). In a recent study, comparing 49 subjects with CC to 316 subjects with PSC and no CC, two single nucleotide polymorphisms (SNPs) in NKG2D were associated with an increased risk of CC: rs11053781 (OR 2.08, corrected p-value 0.011) and rs2617167 (OR 2.32, corrected p-value 0.0020). Aim: To test whether genetic variation in NKG2D modifies susceptibility to sporadic CC. Method: DNA was collected from 172 Caucasian subjects with sporadic CC and a comparable control cohort of 256. Haploview v4.2 was used to select SNPs capturing the majority of common genetic variation around the NKG2D gene (MAF >0.05, pairwise comparisons). This identified 7 SNPs to be genotyped, including rs11053781 and rs2617167. Genotyping was performed with a PCR based, robotic system. Hardy-Weinberg equilibrium testing, Cochran-Armitage trend testing and haplotype frequency comparisons were undertaken. Correction for multiple testing was performed using a permutation method. Results: All genotypes were in Hardy-Weinberg equilibrium. None of the individual SNPs were significantly associated with altered susceptibility to CC. Haplotype analysis revealed that no haplotypes significantly modified risk of CC. Conclusions: This is the first study to examine NKG2D polymorphisms in sporadic CC. The study was well powered. We found no relationship between variation in NKG2D and susceptibility to CC, in contrast to prior findings in PSC patients with CC. This finding may reflect an important difference between the pathogenesis of sporadic CC and that of PSC related CC. It might point to a false-positive result in the prior study of PSC related CC, but it was well executed and produced strong, positive results. This could be elucidated in an additional candidategene validation study. However, with increasing availability and affordability, a genome wide association study (GWAS) may prove a more efficient method for further exploring genetic factors in cholangiocarcinoma. 265 GENETIC VARIATION IN BILIARY TRANSPORTERS AS A SUSCEPTIBILITY FACTOR FOR CHOLANGIOCARCINOMA C.A. Wadsworth1 , P.H. Dixon2 , A.A. Zabron1 , J.H. Wong1 , M.H. Chapman3 , S.C. McKay4 , A.W. Sharif1 , D.R. Spalding4 , S.P. Pereira3 , H.S. Wasan5 , H.C. Thomas1 , S.D. Taylor-Robinson1 , J.C. Whittaker6 , C. Williamson2 , S.A. Khan1 . 1 Department of Hepatology & Gastroenterology, 2 Institute of Reproductive and Developmental Biology, Imperial College London, 3 Institute of Hepatology, University College London, 4 Department of Surgery, 5 Department of Oncology, Imperial College London, 6 Statistical Genetics Unit, Royal School of Hygiene & Tropical Medicine, London, UK E-mail: [email protected] Background: Cholangiocarcinoma (CC) is increasing in incidence and its pathogenesis remains poorly understood. Chronic inflammation of the bile duct and cholestasis are major risk factors but most cases in the West are idiopathic. Genetic polymorphisms in biliary transporter proteins modify bile flow and constituents and have been implicated in benign biliary disease. In the case of progressive familial cholestasis, such polymorphisms have been associated with childhood onset of CC. A recent case-control study of a single nucleotide polymorphism in ABCC2, rs3740066, reported an association with CC. Aim: To investigate the role of five candidate genes in the pathogenesis of CC: ABCB11; ABCB4; ABCC2; ATP8B1 and NR1H4. Methods: Germline DNA was collected from 172 Caucasian individuals with CC. A control cohort was formed by 256 healthy Caucasians. 73 SNPs were selected using the HapMap database in Haploview 4.1 (mean allele frequency >0.05, pair-wise comparisons
only) to capture the majority of common genetic variation around the five candidate loci. Genotyping was undertaken with a PCR based robotic system. Confirmation of Hardy-Weinberg equilibrium and Cochran-Armitage trend testing were performed. Haplotype frequencies were compared using Haplo Stats. Results: All 73 SNPs were in Hardy-Weinberg Equilibrium. Four SNPs in ABCB11 were initially associated with altered susceptibility to CC, one in ABCB4 and three in ATP8B1. The strongest association was in the V444A polymorphism (c.1331T>C, rs2287622, p < 0.007). These associations did not retain statistical significance after Bonferroni correction for multiple testing. None of the SNPs in ABCC2, including rs3740066, showed any association with CC. Haplotype analysis of the genotyped SNPs in ATP8B1 identified a significant differences in frequencies between cases and controls (corrected p-value 0.045). Discussion: This is the largest study to date of biliary transporter polymorphisms as susceptibility factors for CC. The previously reported association between SNP rs3740066 in ABCC2 and CC was not replicated. Haplotypes in ATP8B1 demonstrated a significant difference between CC and control groups. There was also a trend towards significant association of V444A with CC. Given the biological plausibility of polymorphisms in ABCB11 and ATP8B1 as risk modifiers for CC, further study in a validation cohort is required. 266 TAX1BP1 LIMITS TUMOR FORMATION IN EXPERIMENTAL LIVER CANCER O. Waidmann1,2 , A. Piiper1 , S. Zeuzem1 , I. Dikic2 . 1 Medizinische Klinik 1, 2 Institut f¨ ur Biochemie 2, Klinikum der J.W. Goethe-Universit¨ at, Frankfurt/Main, Germany E-mail: [email protected] Background and Aims: HCC (hepatocellular carcinoma) is an increasing burden in the western world. In most cases HCC develops in patients with chronic liver disease. The NFúB (nuclear factor kappa beta) signaling pathway is a central regulator of inflammatory and anti-apoptotic pathways. Inhibition of NFúB signaling can lead to slower tumor progression, but complete loss of central NFúB pathway members can also cause enhanced apoptosis of hepatocytes which results in enhanced cell turnover and cancer development. Tax1BP1 (Tax1 binding protein 1) is an ubiquitin binding regulator protein which inhibits NFúB signal transduction in cooperation with the proteins A20, ITCH and RNF11. Tax1BP1 knock-out mice show a strong activation of the NFúB signaling pathway especially in bone marrow derived cells. The aim of the study was to elucidate the role of Tax1BP1 in the DEN (diethylnitrosamine) induced liver damage and liver cancer model. Methods: For experimental liver damage 6–8 week old Tax1BP1+/+ or Tax1BP1−/− mice were injected with 100 mg/kg DEN or empty carrier and were sacrificed after 24 hours. Hepatic protein and mRNA were extracted and serum samples for measurement of liver enzymes were obtained. Western blotting was performed and the activation of several pathways was assessed with phosphospecific antibodies. Transcription of proinflammatory cytokines and antiapoptotic proteins were measured with quantitative PCR. For experimental liver cancer induction 15 days old mice were injected with 25 mg/kg DEN and were killed after eight months and the number of tumors was counted. Results: Tax1BP1−/− mice showed reduced elevation of serum ALT (alanine aminotransferase) after experimental liver damage (p < 0.05) but responded with enhanced NFúB and JNK activation and we observed a stronger transcription induction of the cMyc proto-oncogene mRNA (p < 0.05). Finally Tax1BP1−/− mice developed more tumors than their wildtype littermates (p < 0.05). Conclusions: Tax1BP1 inhibits activation of NFúB and JNK pathways and limits transcription induction of antiapoptotic proteins like
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S109
263 NOVEL SPONTANEOUS HEPATOCELLULAR CARCINOMA (HCC) MODEL DEVELOPING IN FIBROTIC/CIRRHOTIC LIVER IN MICE A. Vogt1 , G. Decker1 , S. Conchon2 , E. Raskopf1 , T. Sauerbruch1 , M.A. Gonzalez-Carmona1 . 1 Department of Inner Medicine I, University of Bonn, Bonn, Germany; 2 Bioth´erapies H´epathiques, CHU Hotel Dieu, Nantes, France E-mail: [email protected] Background: The majority of HCC develop on basis of liver fibrosis or cirrhosis. In order to understand the influence of the the involving fibrotic/cirrhotic milieu on HCC and for the successful development of novel immunotherapies it is of upmost importance to establish a mouse tumor model that closely resembles the progression of human HCC. Therefore our aim was to establish a model of spontaneous developing multifocal HCC in liver with fibrosis/cirrhosis. Methods: N-nitrosodiethylamine (DEN) was injected intraperitoneally into 14 days old C3H-mice to induce spontaneous liver tumors. 50% of animals were additionally submitted to regular ethanol intake combined with an injection of Thioacetamid (TAA) to induce liver fibrosis. Survival time of animals with or without liver fibrosis/cirrhosis was monitored over 8 months. Thereafter animals were sacrificed, and tumor burden was analyzed. Portal fibrosis was verified by van Gieson staining. The expression of AFP in tumors was detected using western blot. Results: Eight months after DEN-treatment alone, 100% of mice developed multifocal HCC in a healthy liver. All mice additionally submitted to TAA/Ethanol developed multifocal HCC and liver fibrosis/cirrhosis (score F4). Tumor load was increased in the livers with fibrosis/cirrhosis. 30% of animals with liver fibrosis already died 6 months after DEN-treatment, whereas 100% of animals were alive in the group without fibrosis. Expression of murine AFP was detected in tumors of DEN-only- as well as in DEN-TAA-ethanol treated mice but not in healthy liver tissue. Amounts of AFP were significantly higher in the tumors from fibrotic livers compared to non-fibrotic livers. First analysis of the tumor milieu of HCC showed important differences in the expression pattern of chemokines/ cytokines between HCC developing in cirrhotic and non-cirrhotic environment. Conclusions: We were able to establish a novel spontaneous multifocal AFP-positive HCC model developing in a health liver but also in a fibrotic/cirrhotic liver. The presence of a liver fibrosis/cirrhosis seems to accelerate tumor progress. Thus, this model represents an opportunity to further analyze initiation and promotion of HCC as well as the development of new therapies. 264 COMMON GENETIC VARIATION IN NATURAL KILLER CELL RECEPTOR PROTEIN G2D DOES NOT MODIFY SUSCEPTIBILITY TO SPORADIC CHOLANGIOCARCINOMA C.A. Wadsworth1 , P.H. Dixon2 , A.A. Zabron1 , J.H. Wong1 , M.H. Chapman3 , S.C. McKay4 , D.R. Spalding4 , S.P. Pereira3 , H.S. Wasan5 , H.C. Thomas1 , S.D. Taylor-Robinson1 , J.C. Whittaker6 , C. Williamson2 , S.A. Khan1 . 1 Department of Hepatology & Gastroenterology, 2 Institute of Reproductive and Developmental Biology, Imperial College London, 3 Institute of Hepatology, University College London, 4 Department Surgery, 5 Department of Oncology, Imperial College London, 6 Statistical Genetics Unit, Royal School of Hygiene & Tropical Medicine, London, UK E-mail: [email protected] Background: Sporadic cholangiocarcinoma (CC) occurs in subjects with no known risk factor for the disease. Its pathogenesis is poorly understood but is likely to involve complex interaction of environmental and human factors, including genetic variation. Polymorphisms in natural killer cell receptor protein G2D (NKG2D) have been associated with numerous malignancies, including CC
Journal of Hepatology 2011 vol. 54 | S61–S208
POSTERS in subjects with primary sclerosing cholangitis (PSC). In a recent study, comparing 49 subjects with CC to 316 subjects with PSC and no CC, two single nucleotide polymorphisms (SNPs) in NKG2D were associated with an increased risk of CC: rs11053781 (OR 2.08, corrected p-value 0.011) and rs2617167 (OR 2.32, corrected p-value 0.0020). Aim: To test whether genetic variation in NKG2D modifies susceptibility to sporadic CC. Method: DNA was collected from 172 Caucasian subjects with sporadic CC and a comparable control cohort of 256. Haploview v4.2 was used to select SNPs capturing the majority of common genetic variation around the NKG2D gene (MAF >0.05, pairwise comparisons). This identified 7 SNPs to be genotyped, including rs11053781 and rs2617167. Genotyping was performed with a PCR based, robotic system. Hardy-Weinberg equilibrium testing, Cochran-Armitage trend testing and haplotype frequency comparisons were undertaken. Correction for multiple testing was performed using a permutation method. Results: All genotypes were in Hardy-Weinberg equilibrium. None of the individual SNPs were significantly associated with altered susceptibility to CC. Haplotype analysis revealed that no haplotypes significantly modified risk of CC. Conclusions: This is the first study to examine NKG2D polymorphisms in sporadic CC. The study was well powered. We found no relationship between variation in NKG2D and susceptibility to CC, in contrast to prior findings in PSC patients with CC. This finding may reflect an important difference between the pathogenesis of sporadic CC and that of PSC related CC. It might point to a false-positive result in the prior study of PSC related CC, but it was well executed and produced strong, positive results. This could be elucidated in an additional candidategene validation study. However, with increasing availability and affordability, a genome wide association study (GWAS) may prove a more efficient method for further exploring genetic factors in cholangiocarcinoma. 265 GENETIC VARIATION IN BILIARY TRANSPORTERS AS A SUSCEPTIBILITY FACTOR FOR CHOLANGIOCARCINOMA C.A. Wadsworth1 , P.H. Dixon2 , A.A. Zabron1 , J.H. Wong1 , M.H. Chapman3 , S.C. McKay4 , A.W. Sharif1 , D.R. Spalding4 , S.P. Pereira3 , H.S. Wasan5 , H.C. Thomas1 , S.D. Taylor-Robinson1 , J.C. Whittaker6 , C. Williamson2 , S.A. Khan1 . 1 Department of Hepatology & Gastroenterology, 2 Institute of Reproductive and Developmental Biology, Imperial College London, 3 Institute of Hepatology, University College London, 4 Department of Surgery, 5 Department of Oncology, Imperial College London, 6 Statistical Genetics Unit, Royal School of Hygiene & Tropical Medicine, London, UK E-mail: [email protected] Background: Cholangiocarcinoma (CC) is increasing in incidence and its pathogenesis remains poorly understood. Chronic inflammation of the bile duct and cholestasis are major risk factors but most cases in the West are idiopathic. Genetic polymorphisms in biliary transporter proteins modify bile flow and constituents and have been implicated in benign biliary disease. In the case of progressive familial cholestasis, such polymorphisms have been associated with childhood onset of CC. A recent case-control study of a single nucleotide polymorphism in ABCC2, rs3740066, reported an association with CC. Aim: To investigate the role of five candidate genes in the pathogenesis of CC: ABCB11; ABCB4; ABCC2; ATP8B1 and NR1H4. Methods: Germline DNA was collected from 172 Caucasian individuals with CC. A control cohort was formed by 256 healthy Caucasians. 73 SNPs were selected using the HapMap database in Haploview 4.1 (mean allele frequency >0.05, pair-wise comparisons
only) to capture the majority of common genetic variation around the five candidate loci. Genotyping was undertaken with a PCR based robotic system. Confirmation of Hardy-Weinberg equilibrium and Cochran-Armitage trend testing were performed. Haplotype frequencies were compared using Haplo Stats. Results: All 73 SNPs were in Hardy-Weinberg Equilibrium. Four SNPs in ABCB11 were initially associated with altered susceptibility to CC, one in ABCB4 and three in ATP8B1. The strongest association was in the V444A polymorphism (c.1331T>C, rs2287622, p < 0.007). These associations did not retain statistical significance after Bonferroni correction for multiple testing. None of the SNPs in ABCC2, including rs3740066, showed any association with CC. Haplotype analysis of the genotyped SNPs in ATP8B1 identified a significant differences in frequencies between cases and controls (corrected p-value 0.045). Discussion: This is the largest study to date of biliary transporter polymorphisms as susceptibility factors for CC. The previously reported association between SNP rs3740066 in ABCC2 and CC was not replicated. Haplotypes in ATP8B1 demonstrated a significant difference between CC and control groups. There was also a trend towards significant association of V444A with CC. Given the biological plausibility of polymorphisms in ABCB11 and ATP8B1 as risk modifiers for CC, further study in a validation cohort is required. 266 TAX1BP1 LIMITS TUMOR FORMATION IN EXPERIMENTAL LIVER CANCER O. Waidmann1,2 , A. Piiper1 , S. Zeuzem1 , I. Dikic2 . 1 Medizinische Klinik 1, 2 Institut f¨ ur Biochemie 2, Klinikum der J.W. Goethe-Universit¨ at, Frankfurt/Main, Germany E-mail: [email protected] Background and Aims: HCC (hepatocellular carcinoma) is an increasing burden in the western world. In most cases HCC develops in patients with chronic liver disease. The NFúB (nuclear factor kappa beta) signaling pathway is a central regulator of inflammatory and anti-apoptotic pathways. Inhibition of NFúB signaling can lead to slower tumor progression, but complete loss of central NFúB pathway members can also cause enhanced apoptosis of hepatocytes which results in enhanced cell turnover and cancer development. Tax1BP1 (Tax1 binding protein 1) is an ubiquitin binding regulator protein which inhibits NFúB signal transduction in cooperation with the proteins A20, ITCH and RNF11. Tax1BP1 knock-out mice show a strong activation of the NFúB signaling pathway especially in bone marrow derived cells. The aim of the study was to elucidate the role of Tax1BP1 in the DEN (diethylnitrosamine) induced liver damage and liver cancer model. Methods: For experimental liver damage 6–8 week old Tax1BP1+/+ or Tax1BP1−/− mice were injected with 100 mg/kg DEN or empty carrier and were sacrificed after 24 hours. Hepatic protein and mRNA were extracted and serum samples for measurement of liver enzymes were obtained. Western blotting was performed and the activation of several pathways was assessed with phosphospecific antibodies. Transcription of proinflammatory cytokines and antiapoptotic proteins were measured with quantitative PCR. For experimental liver cancer induction 15 days old mice were injected with 25 mg/kg DEN and were killed after eight months and the number of tumors was counted. Results: Tax1BP1−/− mice showed reduced elevation of serum ALT (alanine aminotransferase) after experimental liver damage (p < 0.05) but responded with enhanced NFúB and JNK activation and we observed a stronger transcription induction of the cMyc proto-oncogene mRNA (p < 0.05). Finally Tax1BP1−/− mice developed more tumors than their wildtype littermates (p < 0.05). Conclusions: Tax1BP1 inhibits activation of NFúB and JNK pathways and limits transcription induction of antiapoptotic proteins like
Journal of Hepatology 2011 vol. 54 | S61–S208
S109