RESUMO
Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of beta-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for beta-catenin-negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Carcinoma Hepatocelular/genética , Transformação Celular Neoplásica/genética , Neoplasias Hepáticas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/patologia , Ciclina D1/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Hepatócitos/metabolismo , Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , beta Catenina/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Transformação Celular Neoplásica/genética , Neoplasias Hepáticas Experimentais/genética , Lesões Pré-Cancerosas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Antioxidantes/metabolismo , Ciclo Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Doença Crônica , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Inflamação/imunologia , Metabolismo dos Lipídeos , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Knockout , Oncogenes , Estresse Oxidativo , Fosfatidilcolinas/metabolismo , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Dietary antioxidants and selenium compounds were shown to have a therapeutic effect against hepatocellular carcinoma in several mouse models. We tested the effects of tannic acid and selenomethionine on hepatocellular carcinoma development in Mdr2 knockout (Mdr2-KO) mice. Mdr2-KO and age-matched Mdr2 heterozygous control mice were fed with tannic acid or selenomethionine during the first 3 months of life. Then, several mice from each group were sacrificed, and liver tissue samples were removed for analysis. The remaining mice were fed a regular diet until the age of 16 months, at which time the number and size of liver tumors were determined. Liver tissue samples of 3-month-old mice were subjected to gene expression profiling analysis using cDNA macroarrays containing probes for 240 genes that regulate responses to oxidative stress and inflammation or lipid metabolism. Both tannic acid and selenomethionine had partial chemopreventive effect on development of hepatocellular carcinoma in Mdr2-KO mice: they reduced the incidence of large tumor nodules (diameter >1 cm) at age 16 months. Both agents inhibited gene expression and reversed up-regulation of many genes that control inflammation or response to oxidative stress in Mdr2-KO livers at age 3 months. This inhibitory effect on gene expression correlated with the ability of agents to reduce incidence of large tumors: selenomethionine was more active than tannic acid in both aspects. Understanding the molecular mechanism of chemoprevention effect could improve our therapeutic modalities while using these agents.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Lesões Pré-Cancerosas/patologia , Selenometionina/farmacologia , Taninos/farmacologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/prevenção & controle , Quimioprevenção , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/prevenção & controle , Camundongos , Camundongos Knockout , Lesões Pré-Cancerosas/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Selenometionina/uso terapêutico , Taninos/uso terapêutico , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
To overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken beta-globin 5'HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver-specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders.
Assuntos
Regulação da Expressão Gênica/fisiologia , Processamento de Imagem Assistida por Computador , Elementos Isolantes/fisiologia , Luciferases/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transgenes/fisiologia , Animais , Galinhas , Clonagem Molecular , Metilação de DNA , Globinas/genética , Globinas/metabolismo , Luciferases/genética , Camundongos , Camundongos Transgênicos , Microscopia de Vídeo , Especificidade de Órgãos/fisiologiaRESUMO
BACKGROUND: The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. CONCLUSION: These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner.