RESUMO
Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies.
Assuntos
Carcinogênese/patologia , Carcinoma Hepatocelular/patologia , DNA Topoisomerases Tipo I/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas/patologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Animais , Carcinógenos/toxicidade , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Núcleo Celular/metabolismo , Proliferação de Células , DNA Topoisomerases Tipo I/genética , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Conjuntos de Dados como Assunto , Metabolismo Energético , Feminino , Fibroblastos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , Células HCT116 , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Mitocôndrias/patologia , Prognóstico , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-κB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin ß1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFß1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFß1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Peptídeo Hidrolases/metabolismo , Complexo do Signalossomo COP9 , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Divisão Celular , Linhagem Celular Tumoral , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Peptídeo Hidrolases/genética , RNA Interferente Pequeno/genéticaRESUMO
The two dominant models of carcinogenesis postulate stochastic (clonal evolution) or hierarchic organization of tumor (cancer stem cell model). According to the latter, at the germinal center of tumor evolution is a cancer stem cell (CSC) which, similar to normal adult stem cells, possesses the capacity of self-renewal and a differentiation potential. Over the past few years, compelling evidence has emerged in support of the hierarchic cancer model for many solid tumors including hepatocellular cancers. The CSCs are posited to be responsible not only for tumor initiation but also for the generation of distant metastasis and relapse after therapy. These characteristics are particularly relevant for a multi-resistant tumor entity like human hepatocellular carcinoma and may herald a paradigm shift in the management of this deadly disease. Identification and detailed characterization of liver CSCs is therefore imperative for improving prevention approaches, enhancing early detection, and extending the limited treatment options. Despite the current progress in understanding the contribution of CSCs to the generation of heterogeneity of tumors, the molecular complexity and exact regulation of CSCs is poorly understood. This review focuses on the genetic and epigenetic mechanisms that regulate and define the unique CSC properties with an emphasis on key regulatory pathways of liver CSCs and their clinical significance.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Epigênese Genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Antígeno AC133 , Aldeído Desidrogenase/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Moléculas de Adesão Celular/metabolismo , Divisão Celular , Separação Celular/métodos , Molécula de Adesão da Célula Epitelial , Genes myc , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , Modelos Biológicos , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas Nucleares/genética , Peptídeos/metabolismo , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/genética , RNA Neoplásico/genética , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Antígenos Thy-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Growing evidence indicates that microRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. In this study, we evaluated the clinical and functional relevance of microRNA-122 (miR-122) expression in human hepatocellular carcinoma (HCC). We report that miR-122 is specifically repressed in a subset of primary tumors that are characterized by poor prognosis. We further show that the loss of miR-122 expression in tumor cells segregates with specific gene expression profiles linked to cancer progression, namely the suppression of hepatic phenotype and the acquisition of invasive properties. We identify liver-enriched transcription factors as central regulatory molecules in the gene networks associated with loss of miR-122, and provide evidence suggesting that miR-122 is under the transcriptional control of HNF1A, HNF3A and HNF3B. We further show that loss of miR-122 results in an increase of cell migration and invasion and that restoration of miR-122 reverses this phenotype. In conclusion, miR-122 is a marker of hepatocyte-specific differentiation and an important determinant in the control of cell migration and invasion. From a clinical point of view, our study emphasizes miR-122 as a diagnostic and prognostic marker for HCC progression.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/fisiologia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , MicroRNAs/análise , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , PrognósticoRESUMO
The transforming growth factor-beta (TGF-beta) receptor complex and its downstream signaling intermediates constitute a tumor suppressor pathway. In many cancers, expression of TGF-beta type II receptor (TbetaR-II) is markedly decreased. In the present study, we show that the hepatocytes isolated from 15-day-old, but not 9-month-old, mice heterozygous for the deletion of the TbetaR-II gene are slightly less sensitive to the growth-inhibitory effect of TGF-beta when compared with wild-type littermates of same age. In addition, the proliferation index of hepatocytes as indicated by bromodeoxyuridine incorporation is mildly increased in the heterozygous mice. These subtle changes in cellular phenotype did not result in either gross or microscopic abnormality of the liver. The treatment of these mice with the chemical carcinogen, diethylnitrosamine, results in a significantly enhanced tumorigenesis in the liver when compared with the wild-type littermates. Our results demonstrate the gene-dosage effect of TbetaR-II and indicate that the reduced expression of TbetaR-II in mice increases susceptibility to tumorigenesis in the liver.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Hepáticas Experimentais/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Animais , Carcinógenos , Dietilnitrosamina , Feminino , Dosagem de Genes , Genes cdc/fisiologia , Predisposição Genética para Doença , Heterozigoto , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenobarbital/farmacologia , Gravidez , Proteínas Serina-Treonina Quinases , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1RESUMO
Mutations affecting phosphorylation sites in the beta-catenin gene have been implicated in the development of human and rodent hepatocellular carcinomas (HCCs). To further investigate the involvement of this gene in hepatocarcinogenesis, we used several transgenic mouse models of hepatic tumors induced by overexpression of c-myc in the liver either alone or in combination with transforming growth factor (TGF) alpha or TGF-beta1. Activation of beta-catenin, as judged by the presence of mutations and/or nuclear translocation of the protein, was most frequent in liver tumors from c-myc (4/17; 23.5%) and c-myc/TGF-beta1 (6/18; 33.3%) transgenic mice. However, it was very rare in faster growing and histologically more aggressive HCCs developed in c-myc/TGF-alpha mice (1/20; 5%). Administration of diethylnitrosamine, phenobarbital, or 2-amino-3,8-diethylimidazo[4,5-f]quinoxaline did not significantly affect the occurrence of beta-catenin mutations. Notably, nuclear accumulation of beta-catenin was observed only in adenomas and highly differentiated carcinomas with eosinophilic phenotype. Furthermore, preneoplastic lesions with eosinophilic phenotype frequently displayed focal nuclear positivity, colocalized with areas of high proliferation. In contrast, basophilic and clear-cell foci, as well as pseudo-glandular and poorly differentiated HCCs, exhibited a normal or reduced membranous immunoreactivity for beta-catenin. These studies suggest that nuclear translocation of beta-catenin and activation of Wingless/Wnt signaling may represent an early event in liver carcinogenesis, providing a growth advantage in a subset of hepatic tumors with a more differentiated phenotype.
Assuntos
Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Transativadores , Animais , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/metabolismo , Genes myc/genética , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-myc/biossíntese , Fator de Crescimento Transformador alfa/biossíntese , Fator de Crescimento Transformador alfa/genética , Células Tumorais Cultivadas , beta CateninaRESUMO
The liver has an extremely effective regenerative capacity. When 70% of a rat liver is removed by surgery, the liver mass regrows in 7 to 10 days by the compensatory hyperplasia of the remnant part. In case of damage to the surviving hepatocytes, the facultative stem-cell compartment is activated and the liver regenerates by means of oval-cell proliferation/differentiation. In the present study, we demonstrate that when both hepatocyte proliferation and stem-cell activation were prevented by dexamethasone (Dex) administration, the liver mass was restored in the absence of DNA synthesis. The restoration of the liver was accomplished by the preferential enlargement/hypertrophy of the periportal hepatocytes. A similar response was observed when cell proliferation was arrested by 5-fluorouracil (FU) following partial hepatectomy. Therefore, the hepatocytic hypertrophy appears to provide an alternative mechanism of liver-mass restoration. This hypertrophic condition of the liver is not stable, because following the withdrawal of Dex, the enlarged hepatocytes entered in the cell cycle and the normal liver structure and DNA content was re-established.
Assuntos
Hepatócitos/patologia , Regeneração Hepática/fisiologia , Fígado/patologia , Animais , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Dexametasona/farmacologia , Fluoruracila/farmacologia , Glucocorticoides/farmacologia , Hepatectomia/métodos , Hipertrofia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5' sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by approximately 30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases.
Assuntos
Processamento Alternativo , Variação Genética , Tiorredoxina Dissulfeto Redutase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/análise , Drosophila/enzimologia , Drosophila/genética , Éxons , Humanos , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Tiorredoxina Dissulfeto Redutase/isolamento & purificaçãoRESUMO
Deregulation of E2F transcriptional control has been implicated in oncogenic transformation. Consistent with this idea, we recently demonstrated that during hepatocarcinogenesis in c-myc/TGFalpha double transgenic mice, there is increased expression of E2F-1 and E2F-2, as well as induction of putative E2F target genes. Therefore, we generated transgenic mice expressing E2F-1 under the control of the albumin enhancer/promoter to test the hypothesis that E2F family members may contribute to liver tumor development. Overexpression of E2F-1 resulted in mild but persistent increases in cell proliferation and death during postnatal liver growth, and no increases in hepatic regenerative growth in response to partial hepatectomy. Nevertheless, from 2 months postnatally E2F-1 transgenic mice exhibited prominent hepatic histological abnormalities including preneoplastic foci adjacent to portal tracts and pericentral large cell dysplasia. From 6 to 8 months onward, there was an abrupt increase in the number of neoplastic nodules ('adenomas') with 100% incidence by 10 months. Some adenomas showed evidence of malignant transformation, and two of six mice killed at 12 months showed trabecular hepatocellular carcinoma. Endogenous c-myc was up-regulated in the early stages of E2F-1 hepatocarcinogenesis, whereas p53 was overexpressed in the tumors, suggesting that both E2F-1-mediated proliferation and apoptosis are operative but at different stages of hepatocarcinogenesis. In conclusion, E2F-1 overexpression in the liver causes dysplasia and tumors and suggests a cooperation between E2F-1 and c-myc oncogenes during liver oncogenesis.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA , Neoplasias Hepáticas Experimentais/genética , Fatores de Transcrição/fisiologia , Albuminas/genética , Animais , Apoptose/fisiologia , Divisão Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Cruzamentos Genéticos , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Elementos Facilitadores Genéticos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Regeneração Hepática/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Transforming growth factor (TGF)-beta1 functions as a tumor suppressor in vivo. Using transgenic mice, we show that hepatic TGF-beta1 overexpression inhibits abundance of the cyclin-dependent kinase activating tyrosine phosphatase cdc25A protein. The reduction in cdc25A protein levels was associated with increased binding of histone deacetylase 1 to p130 in the hepatic extracts. In cultured cells, HDAC1/p130 overexpression inhibited activity of the cdc25A promoter through an E2F site. TGF-beta1 treatment enhanced p130 binding to the cdc25A promoter E2F site assessed in chromatin immunoprecipitation assays. Hepatic proliferation induced by partial hepatectomy was associated with a decrease in the amount of HDAC1 bound to p130, without a significant decrease in p130 abundance, suggesting that HDAC1 binding to p130 may be regulated by proliferative stimuli. The induction of cdc25A abundance induced by partial hepatectomy correlated with the induction of DNA synthesis. These studies suggest that TGF-beta1 may enhance HDAC1 binding to p130 in vivo, thereby inhibiting cdc25A gene expression. TGF-beta1 regulation of HDAC1/pocket protein associations may provide a link between chromatin remodeling proteins and cdk inhibition through induction of cdc25A in vivo.
Assuntos
Histona Desacetilases/metabolismo , Proteínas , Fator de Crescimento Transformador beta/fisiologia , Proteínas Estruturais Virais/metabolismo , Animais , Divisão Celular/fisiologia , Células HeLa , Hepatectomia , Histona Desacetilase 1 , Humanos , Fígado/metabolismo , Regeneração Hepática/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Fosfoproteínas/biossíntese , Fosfoproteínas/metabolismo , Fosforilação , Testes de Precipitina , Proteína p130 Retinoblastoma-Like , Transfecção , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Proteínas Estruturais Virais/biossíntese , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/metabolismoRESUMO
Polypeptide growth factors stimulate mammalian cell proliferation by binding to specific cell surface receptors. This interaction triggers numerous biochemical responses including the activation of protein phosphorylation cascades and the enhanced expression of specific genes. We have identified several fibroblast growth factor (FGF)-inducible genes in murine NIH 3T3 cells and recently reported that one of them, the FGF-inducible 14 (Fn14) immediate-early response gene, is predicted to encode a novel, cell surface-localized type Ia transmembrane protein. Here, we report that the human Fn14 homolog is located on chromosome 16p13.3 and encodes a 129-amino acid protein with approximately 82% sequence identity to the murine protein. The human Fn14 gene, like the murine Fn14 gene, is expressed at elevated levels after FGF, calf serum or phorbol ester treatment of fibroblasts in vitro and is expressed at relatively high levels in heart and kidney in vivo. We also report that the human Fn14 gene is expressed at relatively low levels in normal liver tissue but at high levels in liver cancer cell lines and in hepatocellular carcinoma specimens. Furthermore, the murine Fn14 gene is rapidly induced during liver regeneration in vivo and is expressed at high levels in the hepatocellular carcinoma nodules that develop in the c-myc/transforming growth factor-alpha-driven and the hepatitis B virus X protein-driven transgenic mouse models of hepatocarcinogenesis. These results indicate that Fn14 may play a role in hepatocyte growth control and liver neoplasia.
Assuntos
Carcinoma Hepatocelular/genética , Regulação da Expressão Gênica , Expressão Gênica , Genes Precoces , Neoplasias Hepáticas/genética , Regeneração Hepática/genética , Proteínas de Membrana/genética , Receptores do Fator de Necrose Tumoral , Células 3T3 , Sequência de Aminoácidos/genética , Animais , Células Cultivadas , Mapeamento Cromossômico , DNA Complementar/genética , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Receptor de TWEAKRESUMO
Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c-myc and TGF-alpha transgenes during the neoplastic process. We show experimental evidence that co-expression of TGF-alpha and c-myc transgenes in mouse liver promotes overproduction of ROS and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-alpha/c-myc mouse model. Also, the role of mutagenesis in hepatocarcinogenesis induced by 2-amino-3,8-dimethylimidazo(4,5-f)-quinoxaline (MeIQx) was demonstrated in C57BL/lacZ (Muta Mice) and double transgenic c-myc/lacZ mice that carry the lacZ mutation reporter gene. The MeLQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.
Assuntos
Modelos Animais de Doenças , Neoplasias Hepáticas Experimentais/genética , Camundongos Transgênicos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Pesquisa , Fator de Crescimento Transformador alfa/genéticaRESUMO
We have previously shown that chronic activation of mitogenic signaling induced by over-expression of c-myc and transforming growth factor-alpha (TGFalpha) transgenes in mouse liver induces a state of oxidative stress. We therefore proposed that increased reactive oxygen species (ROS) generation might be responsible for the extensive chromosomal damage and acceleration of hepatocarcinogenesis characteristic for TGFalpha/c-myc mice. In this study, we show that vitamin E (VE), a potent free radical scavenging antioxidant, is able to protect liver tissue against oxidative stress and suppress tumorigenic potential of c-myc oncogene. Dietary supplementation with VE, starting from weaning, decreased ROS generation coincident with a marked inhibition of hepatocyte proliferation while increasing the chromosomal as well as mtDNA stability in the liver. Similarly, dietary VE reduced liver dysplasia and increased viability of hepatocytes. At 6 mo of age, VE treatment decreased the incidence of adenomas by 65% and prevented malignant conversion. These results indicate that ROS generated by over-expression of c-myc and TGFalpha in the liver are the primary carcinogenic agents in this animal model. Furthermore, the data demonstrate that dietary supplementation of VE can effectively inhibit liver cancer development.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Neoplasias Hepáticas/prevenção & controle , Vitamina E/análogos & derivados , alfa-Tocoferol/análogos & derivados , Animais , Dano ao DNA , DNA Mitocondrial , Suplementos Nutricionais , Modelos Animais de Doenças , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Tocoferóis , Vitamina E/metabolismo , Vitamina E/farmacologiaRESUMO
We have previously shown in transgenic mice that transforming growth factor (TGF)-alpha dramatically enhances c-myc-induced hepatocarcinogenesis by promoting proliferation and survival of hepatocellular carcinoma (HCC) cells. As transgenic livers display increased levels of mature TGF-beta1 from the early stages of hepatocarcinogenesis, we have now assessed whether impairment of TGF-beta1 signaling contributes to the deregulation of cell cycle progression and apoptosis observed during this process. Focal preneoplastic lesions lacking expression of TGF-beta receptor type II (TbetaRII) were detected in c-myc/TGF-alpha but not in c-myc livers. In c-myc/TGF-alpha mice, 40% (2/5) of adenomas and 90% (27/30) of HCCs showed down-regulation of TbetaRII expression in comparison with 11% (2/18) of adenomas and 47% (14/30) of HCCs in c-myc mice. Down-regulation of the TGF-beta1-inducible p15(INK4B) mRNA and reduced apoptotic rates in TbetaRII-negative HCCs further indicated the disruption of TGF-beta1 signaling. Furthermore, both TbetaRII-negative and -positive c-myc TGF-alpha HCCs, but not c-myc HCCs, were characterized by decreased levels of the cell cycle inhibitor p27. These results suggest 1) an inverse correlation of decreased p27 expression with the particularly strong expression of TGF-alpha in these lesions, consistent with the capacity of TGF-alpha signaling to post-transcriptionally regulate p27, and 2) the presence of alternative, downstream defects of TGF-beta1 signaling in c-myc/TGF-alpha HCCs that may impair the growth-inhibitory response to TGF-beta1. Thus, the accelerated neoplastic development in c-myc/TGF-alpha mice is associated with an early and frequent occurrence of TbetaRII-negative lesions and with reduced levels of p27 in HCC cells, indicating that disruption of TGF-beta1 responsiveness may play a crucial role in the enhancement of c-myc-induced hepatocarcinogenesis by TGF-alpha.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Transdução de Sinais/genética , Fator de Crescimento Transformador alfa/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor , Animais , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/genética , Proteínas de Transporte/metabolismo , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação para Baixo , Inibidores Enzimáticos/metabolismo , Imuno-Histoquímica , Neoplasias Hepáticas Experimentais/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , RNA Mensageiro/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/fisiologiaRESUMO
Many acute and chronic liver diseases are often associated with atypical ductular proliferation (ADP). These ADPs have gained increasing interest since a number of recent observations suggest that ADPs may represent progenies of the putative liver stem cell compartment. In this study, we show that feeding mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in persistent proliferation of primitive ductules with poorly defined lumens. Similar to oval cell proliferation in other rodent models as well as in various human liver diseases, DDC-induced ADP originated from the portal tract, spread into the hepatic lobule, and was associated closely with appearance of hepatocytes harboring an antigen (A6), which normally is expressed in biliary epithelium. Furthermore, DDC treatment severely inhibited the regenerative capacity of mice after partial hepatectomy. The development of ADP was selectively blocked in DDC-fed TGF-beta1 transgenic mice producing active TGF-beta1 in the liver and no accumulation of new hepatocytes expressing the A6 antigen was observed. Moreover, the transforming growth factor beta1 (TGF-beta1) transgenic mice did not survive beyond 3 weeks from starting the DDC-containing diet. The results suggest that persistent activation of the hepatic stem cell compartment is essential for liver regeneration in the DDC model and that active TGF-beta1 may negatively control activation of stem cells in the liver. These data further emphasize the relevance of the DDC model as an experimental tool for studying chronic liver diseases.
Assuntos
Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/patologia , Hepatopatias Alcoólicas/patologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas , Doença Crônica , Ducto Colédoco , Dicarbetoxi-Di-Hidrocolidina , Modelos Animais de Doenças , Células Epiteliais/patologia , Hepatectomia/métodos , Ligadura , Fígado/efeitos dos fármacos , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Transgênicos/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
There is strong evidence that selenium protects against certain human cancers, but the underlying mechanism is unknown. Glutathione peroxidase (GPX1) and thioredoxin reductase (TR), the most abundant antioxidant selenium-containing proteins in mammals, have been implicated in this protection. We analyzed the expression of TR and GPX1 in the following model cancer systems: (1) liver tumors in TGFalpha/c-myc transgenic mice; (2) human prostate cell lines from normal and cancer tissues; and (3) p53-induced apoptosis in a human colon cancer cell line. TR was induced while GPX1 was repressed in malignancies relative to controls in transgenic mice and prostate cell lines. In the colon cell line, p53 expression resulted in elevated GPX1, but repressed TR. The data indicate that TR and GPX1 are regulated in a contrasting manner in the cancer systems tested and reveal the p53-dependent regulation of selenoprotein expression. The data suggest that additional studies on selenoprotein regulation in different cancers are required to evaluate future implementation of selenium as a dietary supplement in individuals at risk for developing certain cancers.
Assuntos
Carcinoma Hepatocelular/enzimologia , Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/genética , Neoplasias Hepáticas/enzimologia , Neoplasias da Próstata/enzimologia , Tiorredoxina Dissulfeto Redutase/genética , Animais , Apoptose , Carcinoma Hepatocelular/genética , Linhagem Celular , Neoplasias do Colo/genética , Indução Enzimática , Células Epiteliais/enzimologia , Genes myc , Glutationa Peroxidase/biossíntese , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Tiorredoxina Dissulfeto Redutase/biossíntese , Transcrição Gênica , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/fisiologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cyclin G1 has been linked to both positive and negative growth regulation. The expression of cyclin G1 is induced by transforming growth factor beta1 and p53, as well as by multiple mitogenic stimuli in mammalian cells in culture. However, the physiological role of cyclin G1 remains unclear. To examine the cell-cycle regulation of cyclin G1 in vivo, two models of coordinated cell proliferation induced by partial hepatectomy (PH) in the presence or absence of DNA damage were used. To introduce DNA damage, mice were treated with the alkylating drug, 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin) 2 hours before PH. Cell-cycle progression was monitored by 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA, the frequency of mitoses, the expression of cell-cycle control genes, and by flow cytometry. Dipin treatment resulted in cell-cycle arrest at the G2/M boundary without affecting G0/G1 and G1/S transitions. While the hepatocytes progressively entered G2 phase arrest, the cyclin G1 mRNA and protein levels increased more than five- and eightfold, respectively. Cyclin G1 had a nuclear localization in all interphase cells with clear absence from nucleoli. In contrast, during mitosis, cyclin G1 was undetectable by immunohistochemistry. Taken together, our data provide evidence for a putative role of cyclin G1 in G2/M checkpoint control.
Assuntos
Aziridinas/farmacologia , Ciclinas/metabolismo , Dano ao DNA , Regeneração Hepática/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Ciclina G , Ciclina G1 , Cinética , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos , Distribuição TecidualRESUMO
The tightly controlled homeostatic mechanisms between cell growth and apoptosis that exist in normal liver tissue are disrupted during hepatocarcinogenesis. The TGF (transforming growth factor)-beta signaling system is a central component of the mechanisms by which cell growth and apoptosis are controlled in the liver. The recent delineation of the TGF-beta signaling pathway has provided a unique framework for analysis of the impact that disruption of individual components of this signaling pathway can have on apoptosis during hepatocarcinogenesis. Here we review recent data on involvement of the TGF-beta signaling pathway in the dysregulation of apoptosis frequently observed in hepatocellular carcinomas. The data indicate that disruption of the TGF-beta pathway at the pre-receptor, receptor, and post-receptor levels occurs in hepatocellular carcinomas and can cause dysregulation of apoptosis. Also, substantial evidence now exists that phosphatidylinositol-3-kinase (PI3K) may function as an important negative regulator of the TGF-beta 1-induced apoptosis in hepatocellular carcinomas. Taken together, the available evidence indicates that disruption of the TGF-beta 1-induced apoptosis as well as growth inhibition is an important and integral part of the multistage process of liver carcinogenesis.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Humanos , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologiaRESUMO
In previous studies we have demonstrated that transforming growth factor (TGF)-alpha/c-myc double transgenic mice exhibit an enhanced rate of cell proliferation, accumulate extensive DNA damage, and develop multiple liver tumors between 4 and 8 months of age. To clarify the biochemical events that may be responsible for the genotoxic and carcinogenic effects observed in this transgenic model, several parameters of redox homeostasis in the liver were examined prior to development of hepatic tumors. By 2 months of age, production of reactive oxygen species, determined by the peroxidation-sensitive fluorescent dye, 2',7'-dichlorofluorescin diacetate, was significantly elevated in TGF-alpha/c-myc transgenic hepatocytes versus either wild type or c-myc single transgenic cells, and occurred in parallel with an increase in lipid peroxidation. Concomitantly with a rise in oxidant levels, antioxidant defenses were decreased, including total glutathione content and the activity of glutathione peroxidase, whereas thioredoxin reductase activity was not changed. However, hepatic tumors which developed in TGF-alpha/c-myc mice exhibited an increase in thioredoxin reductase activity and a very low activity of glutathione peroxidase. Furthermore, specific deletions were detected in mtDNA as early as 5 weeks of age in the transgenic mice. These data provide experimental evidence that co-expression of TGF-alpha and c-myc transgenes in mouse liver promotes overproduction of reactive oxygen species and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-alpha/c-myc mouse model.
Assuntos
Homeostase/genética , Neoplasias Hepáticas Experimentais/fisiopatologia , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Crescimento Transformador alfa/genética , Animais , Dano ao DNA , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Camundongos , Camundongos Transgênicos , Oxirredução , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , TransgenesRESUMO
We have recently shown that overexpression of c-myc and transforming growth factor alpha (TGF-alpha) in the liver of double-transgenic mice results in severe DNA damage, aberrant hepatic growth, and development of tumors at a much younger age than that observed in c-myc single-transgenic mice. We now report that double-transgenic TGF-alpha/c-myc hepatocytes rapidly lose their ability to proliferate upon mitogenic stimulation following partial hepatectomy (PH). At 4 weeks of age, the overall rate of bromodeoxyuridine (BrdU) incorporation following PH was comparable in c-myc and TGF-alpha/c-myc livers and exceeded that seen in wild-type (WT) mice. However, by 10 weeks of age, c-myc single-transgenic hepatocytes showed proliferative advantages over the WT cells, whereas TGF-alpha/c-myc double-transgenic hepatocytes had a decreased capacity to proliferate upon mitogenic stimulation. This decreased proliferative response was accompanied by a reduction in the total fraction of proliferating hepatocytes, as well as by a decline in the induction of cyclin A, cyclin B, and cdc2 gene expression. These data show that constitutive coexpression of c-myc and TGF-alpha accelerates age-related loss in the regenerative potential following PH, and suggest that early replicative senescence of differentiated hepatocytes may have a role in providing a selective growth advantage to initiated cell populations in this model.