RESUMO
Perturbations to the Wnt signaling pathway have been implicated in a large proportion of human hepatocellular carcinomas (HCCs). Activating beta-catenin mutations and loss of function mutations in Axin1 are thought to be functionally equivalent. We examined the Wnt pathway in HCC by comparing the expression of beta-catenin target genes and the level of beta-catenin-dependent transcriptional activation, in 45 HCC tumors and four cell lines. Among these samples, beta-catenin and AXIN1 were mutated in 20 and seven cases, respectively. We found a significant correlation between activated beta-catenin mutations and overexpression of mRNA for the target genes glutamine synthetase (GS), G-protein-coupled receptor (GPR)49 and glutamate transporter (GLT)-1 (P=0.0001), but not for the genes ornithine aminotransferase, LECT2, c-myc and cyclin D1. We also showed that GS is a good immunohistochemical marker of beta-catenin activation in HCC. However, we observed no induction of GS, GPR49 or GLT-1 in the five inactivated Axin1 tumors. Beta-catenin-dependent transcriptional activation in two Axin1-mutated HCC cell lines was much weaker than in beta-catenin-mutated cell lines. Our results strongly suggest that in HCC, contrary to expectation, the loss of function of Axin1 is not equivalent to the gain of function of beta-catenin. Our results also suggest that the tumor suppressor function of Axin1 in HCC may be related to another, non-Wnt pathway.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mutação/genética , Proteínas Repressoras/genética , beta Catenina/genética , Proteína Axina , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Células Tumorais Cultivadas , beta Catenina/metabolismoRESUMO
The Wnt/beta-catenin signaling pathway is activated in many human hepatocellular carcinomas (HCC). We tried to identify the genes involved in carcinogenesis and progression of HCC with beta-catenin mutations. We used PCR-based subtractive hybridization to compare gene expression between malignant and benign components of a human HCC occurring in pre-existing adenoma activated for beta-catenin. Two of the genes identified belong to the Regenerating gene (REG) family. They encode the Regenerating islet-derived 3 alpha (REG3A/HIP/PAP/REG-III) and 1 alpha (REG1A) proteins, both involved in liver and pancreatic regeneration and proliferation. Using siRNA directed against beta-catenin, we demonstrated that REG3A is a target of beta-catenin signaling in Huh7 hepatoma cells. The upregulation of REG3A and REG1A expression is significantly correlated to the beta-catenin status in 42 HCC and 28 hepatoblastomas characterized for their beta-catenin status. Thus, we report strong evidence that both genes are downstream targets of the Wnt pathway during liver tumorigenesis.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Lectinas Tipo C/genética , Litostatina/genética , Neoplasias Hepáticas/genética , Mutação , beta Catenina/genética , Adenoma/genética , Adulto , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Hepatoblastoma/genética , Humanos , Masculino , Proteínas Associadas a Pancreatite , Transdução de SinaisRESUMO
It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.
Assuntos
Regulação Viral da Expressão Gênica/efeitos da radiação , Repetição Terminal Longa de HIV/efeitos da radiação , Animais , Humanos , Óperon Lac , Camundongos , Camundongos Transgênicos , Pele/microbiologia , Raios Ultravioleta , beta-Galactosidase/análiseRESUMO
The role of EGF in the evolution of renal lesions after injury is still controversial. To determine whether the EGF expression is beneficial or detrimental, we generated transgenic mice expressing a COOH-terminal-truncated EGF-R under the control of the kidney-specific type 1 gamma-glutamyl transpeptidase promoter. As expected, the transgene was expressed exclusively at the basolateral membrane of proximal tubular cells. Under basal conditions, transgenic mice showed normal renal morphology and function. Infusion of EGF to transgenic animals revealed that the mutant receptor behaved in a dominant-negative manner and prevented EGF-signaled EGF-R autophosphorylation. We next evaluated the impact of transgene expression on the development of renal lesions in two models of renal injury. After 75% reduction of renal mass, tubular dilations were less severe in transgenic mice than in wild-type animals. After prolonged renal ischemia, tubular atrophy and interstitial fibrosis were reduced in transgenic mice as compared with wild-type mice. The beneficial effect of the transgene included a reduction of tubular cell proliferation, interstitial collagen accumulation, and mononuclear cell infiltration. In conclusion, functional inactivation of the EGF-R in renal proximal tubular cells reduced tubulo-interstitial lesions after renal injury. These data suggest that blocking the EGF pathway may be a therapeutic strategy to reduce the progression of chronic renal failure.
Assuntos
Receptores ErbB/genética , Isquemia/complicações , Nefropatias/fisiopatologia , Túbulos Renais/patologia , Nefrectomia/efeitos adversos , Animais , Divisão Celular , Colágeno/biossíntese , Heterozigoto , Homozigoto , Testes de Função Renal , Camundongos , Camundongos Transgênicos , Fosforilação , Insuficiência Renal/terapia , Transdução de SinaisRESUMO
Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Leucemia Eritroblástica Aguda/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Éxons , Vírus da Leucemia Murina de Friend/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/patologia , Homozigoto , Leucemia Eritroblástica Aguda/patologia , Leucemia Eritroblástica Aguda/fisiopatologia , Fígado/citologia , Fígado/patologia , Camundongos , Camundongos Transgênicos , Mutagênese Insercional , Especificidade de Órgãos , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/genética , Baço/citologia , Baço/patologia , Transcrição Gênica , TransfecçãoRESUMO
The liver-type pyruvate kinase (L-PK) gene is controlled positively by insulin and carbohydrates, negatively by glucagon and fasting. Diet-inducible models of carcinogenesis were obtained using the L-PK gene promoter and regulatory sequences to control the expression of c-myc and SV40 T oncogenes in transgenic mice. L-PK/c-myc and L-PK/Tag animals fed a carbohydrate-rich diet developed hepatocarcinomas. In addition, L-PK/Tag animals developed diet-dependent, aggressive endocrine pancreatic tumors, preceded by islet hyperplasia involving the different analysed cell populations (alpha, beta and delta). Expression of the L-PK gene was demonstrated in pancreatic tumors, in rat isolated islets and in rat insulinoma-derived cells (RIN line), revealing a new tissue specificity of the L-PK gene. Our results suggest that this gene may be expressed in islet progenitor cells from which the different mature endocrine cells derive.
Assuntos
Carboidratos da Dieta/efeitos adversos , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Pancreáticas/etiologia , Piruvato Quinase/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Sequência de Bases , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , Regiões Promotoras GenéticasRESUMO
To analyse the effect of p53 on liver tumor development, we generated transgenic mice overexpressing wild-type p53 in the liver and crossed them with transgenic mice in which the expression of the SV40 large T antigen (TAg) induces hepatic tumors. Remarkably, whereas preneoplastic TAg liver exhibited anisocaryosis and anisocytosis, TAg/p53 liver never presented any dysplastic cells. Moreover, whereas expression of p53 did not affect hepatic development, its constitutive expression in tumorigenic livers resulted in a significantly enhanced apoptosis once nodules had appeared. In contrast, p53 overexpression did not modify the elevated proliferation of TAg-transformed hepatocytes and had no effect on hepatocarcinoma progression. In vitro analysis of primary hepatocytes exposed to various genotoxic agents showed that p53 failed to sensitize normal or TAg-transformed hepatocytes to apoptosis, except when high doses of doxorubicin, UV-B and UV-C radiation were used. Our results confirmed that the hepatocyte cell type is very resistant to genotoxic agents and showed that constitutive expression of p53 failed to improve their responsiveness. In addition, our results showed that suppression of dysplastic cells, probably by restoring normal cytokinesis and karyokinesis, and enhancement of apoptosis by means of p53 overexpression were insufficient to counteract or delay the TAg-induced liver tumoral progression. Oncogene (2000) 19, 3498 - 3507
Assuntos
Dano ao DNA/genética , Doxorrubicina/toxicidade , Raios gama/efeitos adversos , Regulação da Expressão Gênica/genética , Genes p53 , Neoplasias Hepáticas Experimentais/genética , Metotrexato/toxicidade , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Supressora de Tumor p53/fisiologia , Raios Ultravioleta/efeitos adversos , Animais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Sítios de Ligação , Peso Corporal , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/efeitos da radiação , Cruzamentos Genéticos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genótipo , Hiperplasia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/efeitos da radiação , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Tamanho do Órgão , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Vírus 40 dos Símios/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína X Associada a bcl-2RESUMO
Transgenesis allows the in vivo determination of the effects of oncogene expression in normal tissues. In an attempt to understand the mechanism underlying liver transformation, we have previously created transgenic mice carrying the SV40 early gene sequences, which developed hepatocarcinoma in a reproducible way. In the present study, we show that constant expression of the transgene was directly correlated to an abnormally increased hepatocyte proliferation, even at the adult stage. We further demonstrate in this model that the preneoplastic stage of hepatocarcinoma is characterized by marked ploidy alterations as early as 1 month, including the emergence of aneuploid and hyperpolyploid cells, and the persistence of an important diploid cell population. We show that this elevated proliferation is early and transiently counterbalanced by a mechanism of apoptosis, which maintains liver homeostasis. The disappearance of this programmed cell death response effective during preneoplasia might signal the commitment of the liver to neoplasia.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Apoptose , Neoplasias Hepáticas Experimentais/patologia , Fígado/patologia , Lesões Pré-Cancerosas/patologia , Vírus 40 dos Símios/imunologia , Animais , Genes do Retinoblastoma/fisiologia , Genes p53/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos TransgênicosRESUMO
The transforming activity of SV40 large T-antigen (Tag) depends on its binding to cellular proteins involved in the control of the cell cycle (p53, pRb, p300..) and on the J-domain region in the amino-terminus. We established transgenic lines expressing wild-type or Tag mutant proteins lacking one of the three transforming domains, to determine the respective contributions of these domains to hepatic tumour formation. Tag mutants with no pRb-binding domain or N-terminal fragment did not cause neoplastic liver abnormalities. The d11137 Tag mutant protein, which inhibits pRb function without affecting p53, induced hepatic tumours. These tumours grew significantly faster than those induced by wild-type Tag. Our results demonstrate different requirements for each of the inactivating functions of SV40 Tag in hepatocyte transformation and show that the loss of p53 function has only a moderate effect on hepatic tumour formation.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Neoplasias Hepáticas Experimentais/genética , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Antitrombina III/genética , Sítios de Ligação , Carcinoma Hepatocelular/genética , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
1 and 10 mmol/l isovalerate strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mmol/l alanine and 3 mmol/l ornithine. Isovalerate also markedly decreased N-acetylglutamate levels, and the decrease correlated with the inhibition of urea synthesis by isovalerate. This compound also lowered cellular levels of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1). Isovalerate did not significantly affect the cellular levels of ATP and had no direct effect on N-acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by isovalerate is due to decrease in N-acetylglutamate levels.
Assuntos
Acetilcoenzima A/metabolismo , Glutamatos/metabolismo , Fígado/metabolismo , Ácidos Pentanoicos/farmacologia , Ureia/biossíntese , Valeratos/farmacologia , Alanina/metabolismo , Animais , Hemiterpenos , Técnicas In Vitro , Masculino , Ornitina/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Apoptosis is crucial for the normal development of multicellular organisms and is also important for clearing injured cells, such as virus-infected cells or cancer cells. Defective regulation of apoptosis may contribute to viral pathogenesis and aetiology of cancer. Apoptosis of injured cells is principally triggered by the immune system through cytokines such as Fas-ligand and TNF-alpha. Thus, one of the functions of a viral oncogene, such as SV40T-antigen, may be to inhibit cytokine-mediated apoptosis. We previously demonstrated that Fas-mediated apoptosis of hepatocytes is blocked by the wild-type SV40T-antigen during hepatocarcinogenesis. We determined whether this inhibition was directly related to the T-antigen or whether it is a secondary event of cell transformation, by generating transgenic mice expressing a non-transforming T-antigen mutant able to bind endogenous p53 in the liver. This T-antigen mutant cannot induce hepatocarcinoma, unlike the wild-type T-antigen. However, like the wild-type T-antigen, the mutant was a potent inhibitor of apoptosis induced by the Fas-receptor, but not by the TNF-receptor. Therefore, SV40T-antigen has a new property; the inhibition of Fas-mediated apoptosis, which could facilitate the emergence of transformed hepatocytes, but is not sufficient to induce it.
RESUMO
A low citrullinogenesis (less than 60 per cent of the adult value) was observed throughout the suckling period when mitochondria isolated from newborn rat liver were incubated in vitro with L-glutamate or succinate as oxidizable substrates. The adult value was reached after weaning. From birth to weaning, intact mitochondria synthesized more citrulline when supplemented with L-glutamate than with succinate. The low citrullinogenesis could not be explained by low carbamoylphosphate synthetase-I and ornithine transcarbamoylase activities that reached adult values at birth. The decreased citrullinogenesis seen for the first three days of life seemed to be related to the low intramitochondrial concentration of N-acetylglutamate, an activator of the carbamoylphosphate synthetase-I. The concentration of this activator did not differ from that reported for adult rat liver mitochondria after the fourth day of life. The discrepancy between the normal value of N-acetylglutamate concentration and the low activity of the N-acetylglutamate synthetase (15 to 30 per cent of the adult activity) is discussed on the basis of acetyl-CoA or L-glutamate availability in mitochondria isolated from newborn or young rats.
Assuntos
Acetiltransferases/análise , Citrulina/biossíntese , Glutamatos/análise , Fígado/crescimento & desenvolvimento , Mitocôndrias Hepáticas/metabolismo , Aminoácido N-Acetiltransferase , Animais , Feminino , Masculino , Mitocôndrias Hepáticas/análise , Gravidez , Propionatos/farmacologia , Ratos , Ratos EndogâmicosRESUMO
A mouse line transgenic for nerve growth factor (NGF) was developed using the mouse prepro-NGF cDNA inserted within a plasmid containing the proximal region (-10 to -550 bp) of the c-fos promoter and the transcription termination and polyadenylation signals of the rabbit beta-globin gene. No significant modification of gross behavior or central nervous system anatomy was detected in adult animals as assessed by immunohistochemistry and in situ hybridization for NGF and choline acetyltransferase. The expression of the transgene and the possible regulation of its expression by agents acting on the promoter were investigated in vitro. Despite the presence of an additional pool of NGF mRNA specific to the transgene, basal levels of NGF in the supernatant of transgenic astrocytes were similar to normal ones. On the other hand, transgenic neurons spontaneously synthesized and released levels of NGF two to three times higher than normal neurons, while mRNA levels were barely detectable by conventional Northern blotting. The tissue-specificity of NGF expression was respected, with higher levels in hippocampal than neocortical neurons. Increases of NGF mRNA by agents acting on the promoter could be observed in normal and transgenic astrocytes only after inhibition of the protein synthesis by cycloheximide, suggesting a similar rapid turnover of normal and transgenic transcripts. Cyclic AMP agonists specifically increased the secretion of NGF protein by transgenic astrocytes and neurons, while activators of the protein kinase C had a similar effect on transgenic and normal cells. Differences between amounts of NGF secreted by neurons and astrocytes with regards to their respective content in mRNA suggest that transgenic transcripts are subject to normal cell- and tissue-specific post-transcriptional regulations. Agents acting on the c-fos promoter through the protein kinase C or cyclic AMP routes differentially increased the secretion of NGF by transgenic astrocytes or neurons, supporting this hypothesis.
Assuntos
Astrócitos/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes fos , Fatores de Crescimento Neural/genética , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Clonagem Molecular , Desenvolvimento Embrionário e Fetal/genética , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Transcrição Gênica/efeitos dos fármacosRESUMO
It has previously been shown that the hepatitis B virus (HBV) X gene product, HBx, transactivates homologous and heterologous transcriptional regulatory sequences of viruses, including the human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR), and various cellular genes in vitro. To evaluate the transactivating function of HBx in vivo, we generated transgenic mice carrying the X open reading frame under the control of the human antithrombin III (ATIII) gene regulatory sequences. These mice express the 16 Kd HBx protein in the liver, as demonstrated by immunoprecipitation studies. Crossbreeding of HBx mice with transgenics carrying either the chloramphenicol acetyl transferase (CAT) bacterial or the lacZ reporter gene driven by the HIV1-LTR allowed us to demonstrate, for the first time, the in vivo transactivating function of HBx protein.
Assuntos
HIV-1/genética , Vírus da Hepatite B/genética , Sequências Repetitivas de Ácido Nucleico/genética , Transativadores/genética , Ativação Transcricional , Animais , Antitrombina III/genética , Cloranfenicol O-Acetiltransferase/genética , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Fases de Leitura Aberta , Proteínas Virais Reguladoras e Acessórias , beta-Galactosidase/genéticaRESUMO
It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Down's syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammon's horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Down's syndrome patients.
Assuntos
Encéfalo/enzimologia , Síndrome de Down/genética , Neurônios/enzimologia , Superóxido Dismutase/genética , Envelhecimento , Animais , Northern Blotting , Southern Blotting , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , DNA/genética , Modelos Animais de Doenças , Síndrome de Down/enzimologia , Humanos , Imuno-Histoquímica , Peroxidação de Lipídeos , Fígado/crescimento & desenvolvimento , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Especificidade de Órgãos , RNA/genética , RNA/isolamento & purificação , Valores de ReferênciaRESUMO
The synthesis of hippurate from benzoate as compared to ureagenesis has been investigated in isolated rat hepatocytes. Half-maximal synthesis of hippurate was observed at 0.3 mmol/l benzoate. In the presence of 1 mmol/l benzoate, hippurate synthesis proceeded linearly with time at a rate of 40 +/- 10 mumol X h-1 X g-1 dry weight. This provided less than 10% of nitrogen epuration supported by concomitant urea synthesis (350 +/- 82 mumol X h-1 X g-1 dry weight). The incorporation of benzoate to hippurate was markedly limited by the availability of glycine. Half-maximal hippurate synthesis was observed at 2 mmol/l glycine. In the absence of glycine, piridoxilate, a glyoxylate derivative, markedly potentiated hippurate synthesis. Half-maximal stimulation was observed at 10 mmol/l piridoxilate. In the presence of 10 mmol/l piridoxilate, hippurate synthesis (420 +/- 35 mumol X h-1 X g-1 dry weight) provided more than 50% of nitrogen epuration supported by urea synthesis. It is concluded that supplementation with nitrogen-free analogues of glycine such as piridoxilate are required to potentiate hippurate synthesis in an attempt to replace ureagenesis as an alternative pathway of waste nitrogen excretion in inborn errors of urea synthesis.
Assuntos
Benzoatos/metabolismo , Glioxilatos/farmacologia , Hipuratos/biossíntese , Fígado/metabolismo , Piridoxina/análogos & derivados , Ureia/biossíntese , Alanina/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Glicina/farmacologia , Hipuratos/urina , Masculino , Ornitina/metabolismo , Piridoxina/farmacologia , Ratos , Ratos Endogâmicos , Estimulação QuímicaRESUMO
The Wnt/beta-catenin signalling pathway is activated in many human hepatocellular carcinomas (HCC). Identification of beta-catenin mutation relies mostly on sequence analysis and/or immunohistochemistry. beta-catenin mutation may also be detected by analysing the expression of its target genes. The GLUL gene encoding glutamine synthetase (GS), for example, appears to be a pertinent marker. The aim of this study was to correlate GS immunostaining and beta-catenin mutations with clinicopathological features in HCC. We found that GS immunostaining had a sensitivity of 90% for the detection of beta-catenin mutations, with 98% specificity, whereas beta-catenin immunostaining had a sensitivity of 63% with 98% specificity. We used the sensitive GS marker to characterize 190 HCC cases. Sixty-eight (36%) cases displayed Wnt/beta-catenin activation. In addition to their well-differentiated pattern, these tumours exhibited significant features such as a homogeneous microtrabeculo-acinar pattern, low-grade cellular atypia, and cholestasis. As these tumours exhibited cholestasis, we hypothesized that beta-catenin acts on specific bile synthesis and/or transport pathways. In conclusion, we propose that GS immunostaining and a cholestatic pattern are relevant criteria for the identification of HCC with beta-catenin mutations.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/patologia , Colestase/patologia , Glutamato-Amônia Ligase/análise , Neoplasias Hepáticas/patologia , beta Catenina/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Distribuição de Qui-Quadrado , Colestase/genética , Colestase/metabolismo , Análise Mutacional de DNA , Expressão Gênica , Glutamato-Amônia Ligase/genética , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Transdução de Sinais , Proteína Wnt1/metabolismo , beta Catenina/análise , beta Catenina/metabolismoRESUMO
Pent-4-enoic acid inhibited ureagenesis approximatively 90% in rat hepatocytes incubated with pyruvate, ammonia and ornithine. Inhibition of ureagenesis was much less with alanine as substrate (approximatively 10%). The addition of ammonia led to a drastic dose-dependent inhibition of ureagenesis by pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of N-acetylglutamate were also drastically modified by the addition of ammonia as was the accumulation of citrulline. These data suggest that ammonia may seriously interfere with the metabolism of pent-4-enoic acid and leads to a dramatic potentiation of its toxicity.