RESUMO
In this study, gastric myoelectric activity in patients with acute cerebral infarction was investigated using electrogastrography. The patients were divided into four groups; those with mild brainstem infarction(group A, n=13, men:8, women:5, 75±2 years old), severe brainstem infarction(group B, n=6, men:4, women:2, 79±4 years old), mild non-brainstem infarction(group C, n=14, men:7, women:7, 76±3 years old), and severe non-brainstem infarction(group D, n=9, men:3, women:6, 87±2 years old). In group B, the% ratio of normogastria(2.4-3.6 cycles per minute)was significantly lower in the fasting period. The dominant power(DP)significantly increased after the meal in group C, but did not in group A, compared to before the meal. The DP increased in all patients in group C after the meal, whereas it increased in only five of ten patients in group A. The possibility of gastric dysfunction should be considered in patients with brainstem infarction.
Assuntos
Infarto Cerebral/fisiopatologia , Gastropatias/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Eletrodos , Fenômenos Eletrofisiológicos , Feminino , Humanos , Masculino , Músculo Esquelético/fisiopatologia , Gastropatias/diagnósticoRESUMO
Small integral membrane protein of the lysosome/late endosome (SIMPLE) is a 161-amino acid cellular protein that contains a characteristic C-terminal domain known as the SIMPLE-like domain (SLD), which is well conserved among species. Several studies have demonstrated that SIMPLE localizes to the trans-Golgi network (TGN), early endosomes, lysosomes, multivesicular bodies, aggresomes and the plasma membrane. However, the amino acid regions responsible for its subcellular localization have not yet been identified. The SLD resembles the FYVE domain, which binds phosphatidylinositol (3)-phosphate (PI3P) and determines the subcellular localization of FYVE domain-containing proteins. In the present study, we have found that SIMPLE binds specifically to PI4P through its SLD. SIMPLE co-localized with PI4P and Rab11, a marker for recycling endosomes (REs, organelles enriched in PI4P) in both the IMS32 mouse Schwann cell line and Hela cells. Sucrose density-gradient centrifugation revealed that SIMPLE co-fractionated with syntaxin-6 (a TGN marker) and Rab11. We have also found that SIMPLE knockdown impeded recycling of transferrin and of transferrin receptor. Our overall results indicate that SIMPLE may regulate protein trafficking physiologically by localizing to the TGN and/or REs by binding PI4P.
Assuntos
Endossomos/metabolismo , Proteínas Nucleares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Células de Schwann/metabolismo , Fatores de Transcrição/metabolismo , Rede trans-Golgi/metabolismo , Animais , Linhagem Celular Transformada , Proteínas de Ligação a DNA , Endossomos/genética , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Fosfatos de Fosfatidilinositol/genética , Domínios Proteicos , Fatores de Transcrição/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/genéticaRESUMO
Responses of the liver to chronic injury include inflammation, regeneration and fibrosis, which finally lead to cirrhosis. The cause of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage, and subsequent accumulation of extracellular matrix produced by hepatic stellate cells (HSCs). Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in hepatocyte proliferation and hepatofibrogenesis, respectively. However, sequential analyses of the intrahepatic expression of EGF and TGF-beta1 in the course of cirrhosis development have not been examined fully. In the present study, liver cirrhosis was produced in rats by intraperitoneal administration of dimethylnitrosamine (DMN), and intrahepatic mRNA expression levels of proliferating cell nuclear antigen (PCNA), EGF and TGF-beta1 were quantitatively estimated by a real-time reverse transcription-polymerase chain reaction method. Histological and semiquantitative densitometric examination of liver sections revealed that the accumulation of extracellular matrix components was increased according to the period of DMN treatment. Histological examination of liver sections of rats treated with DMN for 4 and 6 weeks revealed pre-cirrhosis and cirrhosis, respectively. Intrahepatic mRNA expression levels of PCNA and EGF correlated well. Expression levels of both molecules were increased significantly during the course of cirrhosis development, but decreased significantly at the time of complete cirrhosis manifestation. In contrast, intrahepatic TGF-beta1 expression was increased significantly according to the period of DMN treatment, and reached a peak at the time of cirrhosis manifestation. These results suggest that proliferative capability of hepatocytes was impaired by continuous liver damage due, in part, to the decrease of a hepatocyte mitogen EGF, and that increased intrahepatic TGF-beta1 activated HSCs to retrieve space lost by hepatocyte destruction, resulting in complete cirrhosis manifestation.
Assuntos
Fator de Crescimento Epidérmico/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fator de Crescimento Transformador beta1/genética , Animais , Colágeno/metabolismo , Dimetilnitrosamina/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Masculino , Antígeno Nuclear de Célula em Proliferação/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND/AIMS: Recent genome-wide analyses have provided strong evidence concerning adverse events caused by thiopurine drugs such as azathioprine (AZA) and 6-mercaptopurine. The strong associations identified between NUDT15 p.Arg139Cys and thiopurine-induced leukopenia and severe hair loss have been studied and confirmed over the last 2 years. However, other coding variants, including NUDT15 p.Val18_Val19insGlyVal, NUDT15 p.Val18Ile, and FTO p.Ala134Thr, and a noncoding variation in RUNX1 (rs2834826) remain to be examined in detail in this respect. Therefore, we investigated the correlation between these adverse events and the 5 recently identified variants mentioned above among Japanese patients with inflammatory bowel diseases (IBD). METHODS: One hundred sixty thiopurine-treated patients with IBD were enrolled. Genotyping was performed using TaqMan SNP Genotyping Assays or Sanger sequencing. RESULTS: None of the 5 variants were associated with gastrointestinal intolerance to AZA. However, NUDT15 p.Arg139Cys was significantly associated with the interval between initiation and discontinuation of AZA among patients with gastrointestinal intolerance. This variant was strongly associated with early (<8 weeks) and late (≥8 weeks) leukopenia and severe hair loss. Moreover, it correlated with the interval between initiation of thiopurine therapy and leukopenia occurrence, and average thiopurine dose. NUDT15 p.Val18_Val19insGlyVal, NUDT15 p.Val18Ile, FTO p.Ala134Thr, and RUNX1 rs2834826 exhibited no significant relationship with the adverse events examined. CONCLUSIONS: Of the 5 variants investigated, NUDT15 p.Arg139Cys had the strongest impact on thiopurine-induced leukopenia and severe hair loss; therefore, its genotyping should be prioritized over that of other variants in efforts to predict these adverse events in Japanese patients with IBD.
RESUMO
Liver cirrhosis is the fatal end stage of various chronic liver diseases. One of the most important causes of liver cirrhosis appears to be an impaired proliferative capability of hepatocytes caused by continuous hepatic damage. Hepatocyte growth factor (HGF) and its specific receptor, c-Met, play a pivotal role in hepatocyte proliferation. However, the relationship between the proliferative capability of hepatocytes and the intrahepatic expression of HGF and c-Met during the course of cirrhosis development has not been studied fully. In the present study, liver cirrhosis was produced in rats by intraperitoneally administering dimethylnitrosamine (DMN) and intrahepatic expression levels of HGF and c-Met were quantitatively estimated using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Histological examination of liver sections and biochemical estimation of serum levels of transaminase revealed that the degree of hepatocyte destruction was elevated gradually during cirrhosis development in the DMN-induced rat cirrhosis model. The proliferative capability of hepatocytes estimated immunohistochemically by proliferating cell nuclear antigen staining was markedly increased at an early stage of cirrhosis development. However, it was gradually decreased thereafter and suppressed substantially at the time of cirrhosis manifestation. Intrahepatic HGF expression was also increased significantly during the course of cirrhosis development but decreased significantly at the time of cirrhosis manifestation. Conversely, there was a tendency for the intrahepatic expression of c-Met to decrease from an early stage of cirrhosis development and intrahepatic c-Met expression was decreased significantly at the time of cirrhosis manifestation. These results suggest that the highly proliferative capability of hepatocytes at an early stage of cirrhosis development is induced by increased intrahepatic HGF expression. However, both HGF and c-Met expression levels in the liver are decreased significantly there-after. Accordingly, the proliferative capability of hepatocytes is severely impaired and extracellular matrix components are deposited to retrieve space lost by the destruction of hepatic parenchyma, resulting in the establishment of liver cirrhosis.
Assuntos
Proliferação de Células , Expressão Gênica/genética , Fator de Crescimento de Hepatócito/genética , Hepatócitos/metabolismo , Cirrose Hepática/genética , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Dimetilnitrosamina/administração & dosagem , Dimetilnitrosamina/toxicidade , Hepatócitos/citologia , Imuno-Histoquímica , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/induzido quimicamente , Masculino , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Proto-Oncogênicas c-met/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
A number of studies have shown that various K vitamins, specifically vitamins K2 and K3, possess antitumor activity on various types of rodent- and human-derived neoplastic cell lines. In the present study, we examined the antitumor effects of vitamins K1, K2 and K3 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of these vitamins in vitro and in vivo. Although vitamin K1 did not inhibit proliferation of PLC/PRF/5 cells at a 90-microM concentration (the highest tested), vitamins K2 and K3 suppressed proliferation of the cells at concentrations of 90 and 9 microM, respectively. By flow cytometric analysis, it was shown that not only vitamin K1, but also vitamin K2 did not induce apoptosis or cell cycle arrest on PLC/PRF/5 cells. In contrast, vitamin K3 induced G1 arrest, but not apoptosis on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that both vitamins K2 and K3 markedly suppressed the growth of HCC tumors to similar extent. Protein expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4), but not p16INK4a Cdk inhibitor in the tumor was significantly reduced by vitamin K2 or K3 treatment, indicating that vitamins K2 and K3 may induce G1 arrest of cell cycle on PLC/PRF/5 cells in vivo. Taken collectively, vitamins K2 and K3 were able to induce potent antitumor effects on HCC in vitro and in vivo, at least in part, by inducing G1 arrest of the cell cycle. The results indicate that vitamins K2 and K3 may be useful agents for the treatment of patients with HCC.
Assuntos
Antifibrinolíticos/farmacologia , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Vitamina K 1/farmacologia , Vitamina K 2/farmacologia , Vitamina K 3/farmacologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Humanos , Camundongos , Camundongos Nus , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent substrate for protein kinase C (PKC) in a variety of cells. The aim of this study was not only to evaluate the expression and localization of MARCKS in various pathological liver tissues, including HCC, but also to analyze the difference in MARCKS expression between hepatitis virus-induced HCC and cirrhosis. The level of MARCKS and its phosphorylated proteins, as well as its localization, were determined using Western blot and/or immunohistochemistry in HCC and other pathological liver tissues. We also analyzed the change of MARCKS localization on the influence of MARCKS phosphorylation in the HLF cancer cell line by phosphorylation study. In addition, the relationship between MARCKS expression and proliferative activity was studied in HCC. In the immunohistochemical study, a very small amount of MARCKS protein was found along the contour of the hepatocellular membrane in normal liver and in cases of chronic hepatitis. MARCKS was up-regulated in liver cirrhosis tissue and was localized in the cytoplasm of hepatocytes. The expression of MARCKS was down-regulated in HCC tissues, as compared with non-tumorous liver cirrhosis tissues from the same patients. Furthermore, MARCKS was serine-phosphorylated in liver cirrhosis and HCC, and phosphorylated MARCKS was detected in a cytosolic fraction of these tissues. In a phosphorylation study using the HLF HCC cell line, MARCKS was displaced from the plasma membrane to the cytosol following the activation of protein kinase C (PKC) by phorbol 12-myristrate 13-acetate (PMA). Furthermore, the activity of cyclin D1 and cyclin E kinases was found to be higher in HCCs with low MARCKS expression than in HCCs with high MARCKS expression. These results suggest that up-regulation of MARCKS might be essential in the generation of cirrhotic nodules through chronic hepatitis from normal liver, and that the phosphorylation and/or down-regulation of MARCKS might play an important role in the development and progression of HCC from liver cirrhosis.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Hepatite C Crônica/genética , Hepatite C Crônica/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas de Membrana/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Regulação para Baixo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Substrato Quinase C Rico em Alanina Miristoilada , Proteína Quinase C , Regulação para CimaRESUMO
BACKGROUND: In endoscopic biliary stenting against malignant biliary obstruction, stent blockage remains as an important problem. Stent blockage occurs as a result of bacterial adherence to the inner wall of the stent. We evaluated the stent placement above the intact sphincter of Oddi to retain the function of the sphincter of Oddi as a bacteriological barrier. METHODS: Sixteen patients with malignant biliary obstruction were assessed as the patients with the stent above the intact sphincter of Oddi. Sixteen patients with malignant biliary obstruction were assessed as the patients with the conventional stent placement across the sphincter of Oddi. Tannenbaum 10-Fr. stents were used in both the groups. RESULTS: The median patency periods of the stent were 255 days (25th to 75th percentiles, 212-454 days; range, 39-454 days) for the group of the stents placed above the sphincter of Oddi and 82 days (25th to 75th percentiles, 48-131 days; range, 22-196 days) for the group of the stents placed across the sphincter of Oddi, respectively, with significant difference (P = 0.0001). The occlusion rates of stents placed above and across the sphincter of Oddi were 37.5% and 93.8%, respectively, with significant difference (P = 0.0008). The dislocation rates of the stent were 0% and 6.3%, respectively (not significant). CONCLUSIONS: Placement of the stent above the intact sphincter of Oddi was associated with longer stent patency and lower occlusion rate.
Assuntos
Colestase/cirurgia , Implantação de Prótese/instrumentação , Stents , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/complicações , Colangiopancreatografia Retrógrada Endoscópica , Colestase/diagnóstico por imagem , Colestase/etiologia , Endoscopia do Sistema Digestório , Feminino , Seguimentos , Neoplasias da Vesícula Biliar/complicações , Humanos , Masculino , Neoplasias Pancreáticas/complicações , Falha de Prótese , Implantação de Prótese/métodos , Estudos Retrospectivos , Esfíncter da Ampola Hepatopancreática , Resultado do TratamentoRESUMO
It has been shown that a variety of cell cycle-related proteins play important roles in the process of carcinogenesis including hepatocarcinogenesis. In the present study, we evaluated mRNA and protein expression of G1 phase-related cell cycle molecules in the process of hepatocarcinogenesis, using Long-Evans Cinnamon (LEC) rats, an animal model of hepatocellular carcinoma (HCC). The expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6 was measured quantitatively by real-time polymerase chain reaction. Cyclin D1 mRNA expression was increased significantly in chronic hepatitis liver compared with normal liver, and then decreased in HCC and the surrounding precancerous liver of LEC rats. Levels of Cdk4 mRNA were increased significantly in HCC compared to precancerous and chronic hepatitis livers. In contrast, mRNA levels of Cdk6 did not change significantly during hepatocarcinogenesis. We also evaluated the protein levels of these G1 phase-related cell cycle molecules by Western blot analyses and confirmed similar results. Total amounts of retinoblastoma protein (pRb) in the liver did not change significantly in the process of hepatocarcinogenesis in LEC rats. However, levels of phosphorylated pRb were increased markedly in the process of hepatocarcinogenesis, and the highest in HCC compared to precancerous, chronic hepatitis and normal livers. These results indicate that cyclin D1 may be involved in the regeneration of hepatocytes rather than hepatocarcinogenesis, while Cdk4 but not Cdk6 may play an important role in the development of HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fase G1 , Neoplasias Hepáticas Experimentais/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Hepatite Animal/metabolismo , Hepatite Animal/patologia , Hepatite Crônica/metabolismo , Hepatite Crônica/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas Experimentais/patologia , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos LEC , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismoRESUMO
alpha-fetoprotein (AFP) is an important marker for the diagnosis of hepatocellular carcinoma (HCC) and has been widely used in clinical settings. Recently, the importance of lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) has been indicated. However, the clinical significance of the level of AFP-L3 protein in relation to the characteristics of HCC has not been fully evaluated. In the present study, both the ratio of AFP-L3 (AFP-L3%) and the absolute value of AFP-L3 (AFP-L3-AV) were examined in 80 patients with HCC, and evaluated with respect to characteristics of HCC such as grade of differentiation, size of tumor and morphological findings. Among HCC-specific tumor markers, AFP, AFP-L3% and protein induced in vitamin K absence (PIVKA-II), AFP showed the highest positive rate in patients with HCC, while AFP-L3% showed the lowest rate. AFP-L3% and AFP-L3-AV were, however, most significantly correlated with the grade of HCC differentiation, while AFP showed the least significant correlation. Furthermore, AFP-L3% was most significantly correlated with the size of HCC in patients with solitary HCC. Conversely, neither AFP-L3-AV nor PIVKA-II showed a significant correlation with the size of HCC. In relation to morphological differences of HCC, although AFP-L3%, AFP-L3-AV and PIVKA-II were significantly higher in the diffuse type of HCC than in the nodular type of HCC, AFP was most significantly correlated with the morphological differences of HCC. These results indicate that tumor markers for HCC, such as AFP, AFP-L3%, AFP-L3-AV and PIVKA-II, may play different roles in predicting the characteristics of HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Biomarcadores , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Lectinas/metabolismo , Lectinas de Plantas , alfa-Fetoproteínas/metabolismo , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Humanos , Precursores de Proteínas/sangue , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , RadioimunoensaioRESUMO
The efficacy of radiofrequency ablation (RFA) and RFA with concurrent ethanol injection (EI-RFA) was compared. RFA (3-cm-electrode) was applied to bovine liver using three types of RFA equipment; Radionics, RITA and Radio Therapeutics Corporation (RTC). For EI-RFA, 5 ml of 99.5% ethanol was injected around the electrode. A total number of 40 RFA and EI-RFA treatments were performed. We compared RFA with EI-RFA by examining the size, shape of ablation zone, treatment time, power, and needle tip temperature. Liver specimens were examined for pathological changes. EI-RFA produced a larger zone of ablation than RFA alone using Radionics and RITA (Radionics, 35.3+/-7.4 cm(3) vs 23.2+/-7.7 cm(3), p<0.05; RITA, 30.7+/-10.3 cm(3) vs 19.7+/-4.7 cm(3), p<0.05), corresponding to shortest diameters of coagulation zone (Radionics, 3.7+/-0.4 cm vs 3.0+/-0.4 cm, p<0.05; RITA, 3.8+/-0.4 cm vs 3.1+/-0.3 cm, p<0.01). However, a larger ablation zone was not seen with the RTC device. The ablated volume per energy and the ablated volume per current density administered were greater with EI-RFA than with RFA using Radionics (p<0.05). The shape of the ablated zone changed from ellipsoid to spherical with EI-RFA using Radionics. No pathological differences between RFA and EI-RFA samples were detected. For a given amount of energy and current administered, ethanol injection caused a better ablation effect, in terms of the size and shape of the ablated zone, than RFA with Radionics and RITA equipment.
Assuntos
Ablação por Cateter/métodos , Etanol/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/efeitos da radiação , Animais , Anti-Infecciosos Locais/farmacologia , Bovinos , Eletrodos , Temperatura Alta , Fígado/metabolismo , Temperatura , Fatores de TempoRESUMO
The adaptor molecule Shc is a proto-oncogene product, and it is known to be associated with cell proliferation. However, the role of Shc in the proliferation and regeneration of hepatocytes remains unknown. In the present study, we report that p46 Shc is specifically expressed in the nuclei of proliferative (or regenerative) hepatocytes, suggesting that p46 Shc protein plays a role in hepatocellular proliferation. The expression of Shc was analyzed in liver tissue after partial hepatectomy (PH) or sham operation in Wistar rats by using immunohistochemistry and/or Western blot analysis. In addition, the expression of various cell cycle-related proteins, such as Cdk4, cyclin D1, PCNA, and Cdk1 was analyzed in the tissues of regenerating rat liver. Furthermore, the tyrosine phosphorylation of Shc was studied in liver tissue after PH or sham operation by immunoprecipitation using a monoclonal phosphotyrosine antibody. Although the protein levels of p52 Shc were unchanged in liver tissues after PH or sham operation, tyrosine phosphorylation was detected only in the regenerating rat liver after PH. The levels of p46 Shc protein were markedly increased in liver tissues during the liver regenerative process. In contrast, p66 Shc was not detected in the liver tissues after PH or sham operation. Western blotting and immunohistochemistry showed that the main location of p46 Shc was in the nuclei of proliferating hepatocytes after PH. These data suggest that p46 Shc expressed in hepatocellular nuclei may be closely related to the proliferation of hepatocytes. Therefore, it is suggested that p46 Shc expressed in hepatocellular nuclei may be a useful marker for detecting hepatocytes with high proliferative activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Regeneração Hepática/fisiologia , Animais , Western Blotting , Proteína Quinase CDC2/metabolismo , Divisão Celular , Fracionamento Celular , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Citosol/metabolismo , Hepatectomia , Imuno-Histoquímica , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Wistar , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Tirosina/metabolismoRESUMO
Liver cirrhosis is the end stage of various chronic liver diseases and its prognosis is very poor. One of the most important causes of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage. Cell cycle-related molecules have been shown to play essential roles in cell proliferation. Specifically, G1-related cell cycle molecules are important, because they are requisite for the entry into the cell cycle from the quiescent state. However, the role of these cell cycle molecules during the development of liver cirrhosis remains to be examined. In the present study, liver cirrhosis was produced in rats by intraperitoneally administering dimethylnitrosamine (DMN). Proliferative capability of hepatocytes estimated immunohistochemically by proliferating cell nuclear antigen staining was markedly increased at an early stage of cirrhosis development. However, it was gradually decreased thereafter and suppressed substantially at the time of cirrhosis manifestation. Cyclin D1 expression estimated by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method was also increased markedly at an early stage of cirrhosis development but decreased substantially thereafter. mRNA levels of catalytic subunits of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6, did not show significant changes during the development of liver cirrhosis. Among G1-specific Cdk inhibitors, expression of p15INK4b and p16INK4a estimated by an RT-PCR method was increased according to the progression of cirrhosis and reached a peak at the time of cirrhosis manifestation. Conversely, p18INK4c expression did not change significantly during the development of liver cirrhosis. These results suggest that cyclin D1 plays an essential role in hepatocyte proliferation in response to hepatic damage. However, with the decrease of cyclin D1 expression and increase of p15INK4b and p16INK4a expression, proliferative capability of hepatocytes is severely impaired and extracellular matrix components are deposited to retrieve space lost by the destruction of hepatic parenchyma, resulting in establishment of liver cirrhosis.
Assuntos
Ciclina D1/metabolismo , Fase G1/fisiologia , Hepatócitos/patologia , Cirrose Hepática/metabolismo , Fígado/patologia , Animais , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p18 , Quinases Ciclina-Dependentes/metabolismo , Dimetilnitrosamina/toxicidade , Matriz Extracelular/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/etiologia , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Supressoras de Tumor/metabolismoRESUMO
The Bcl-2 family proteins are important regulators of apoptosis and have been implicated in the occurrence of autoimmune diseases. There have been reports that Bcl-2-positive lymphocytes play important roles in pathogenesis of at least some types of autoimmune diseases, including autoimmune hepatitis. In this study, we examined the role of Bcl-2-positive lymphocytes in the development of primary biliary cirrhosis (PBC). The expression of Bcl-2 in lymphocytes infiltrating into the liver was evaluated from liver biopsy specimens of 25 patients with PBC (stage I/II/III/IV, 11/3/8/3) and 24 controls with chronic hepatitis B (CH-B) or chronic hepatitis C (CH-C). Bcl-2 expression in lymphocytes infiltrating into the liver was investigated by immunohistochemistry using a monoclonal antibody to Bcl-2 with an avidin-biotin complex system. Significant overexpression of Bcl-2 in lymphocytes infiltrating into the liver was observed in the early stage of PBC, especially in areas of destructed bile ducts. Most of Bcl-2-positive cells were CD45RO-positive helper T-cells visualized by the double staining method. These results suggest that Bcl-2-positive helper T-cells may have important roles in the pathogenesis of PBC.
Assuntos
Cirrose Hepática Biliar/metabolismo , Fígado/metabolismo , Linfócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Adulto , Idoso , Anticorpos Monoclonais/metabolismo , Antígenos CD20/biossíntese , Antígenos CD8/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Antígenos Comuns de Leucócito/biossíntese , Masculino , Pessoa de Meia-IdadeRESUMO
Immunopathological differences between autoimmune hepatitis (AIH) and chronic hepatitis C (CH-C) have not been well investigated. Therefore, we immunohistochemically examined the expression of various cytokeratins (CKs) not only in liver tissues of AIH but also in those of CH-C at the active stage. Furthermore, to evaluate the immune surveillance system and the susceptibility to apoptosis, immunohistochemical staining of human leukocyte antigen (HLA)-DRalpha, cathepsin D, B cell leukemia-2 (bcl-2), bcl-2-associated X protein (bax) and caspase 3 was also performed. Heterogeneous expression of CK 8 and CK 18 was observed in hepatocytes of AIH, while homogeneous expression was observed in hepatocytes of CH-C. Aberrant expression of CK 7 and CK 19 was observed in hepatocytes of AIH, while it was not in hepatocytes of CH-C. Expression of HLA-DRalpha was observed in hepatocytes of AIH but not in those of CH-C. Furthermore, expression of cathepsin D, bax and caspase 3 was much stronger in hepatocytes of AIH than in those of CH-C. These results indicate that cytoskeletal alterations of hepatocytes in AIH may increase the susceptibility to apoptosis and induce hepatocyte destruction.
RESUMO
Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18INK4C, a member of the INK4 family of CdkIs, is a potential tumor-suppressor gene product. However, the expression of p18INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18INK4C- positivity. Further, the kinase activity of Cdk4 was found to be higher in p18INK4C-negative HCCs than in p18INK4C- positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18INK4C- positive HCCs, p18INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18INK4C - negative than in p18INK4C - positive HCCs. In conclusion, the loss of p18INK4C expression may play a role in the differentiation and development of HCC through the up-regulation of Cdk4 activity.