RESUMO
Interferon-gamma/transforming growth factor-beta (IFN-gamma/TGF-beta) pathways have opposite effects on diverse cellular functions. However, little is known about interactions between IFN-alpha/TGF-beta. In previous studies, we showed that IFN-alpha2b increases TGF-beta(1) production and secretion in hepatocytes from preneoplastic rat livers. Here, the interaction between IFN-alpha/TGF-beta(1) pathways was explored. We observed a positive cross-talk between IFN-alpha and TGF-beta(1) signaling, with activation of both pathways. p300 protein levels in hepatocytes from preneoplastic livers were enough to interact with both activated Stat1 and Smad2/3. Besides, Smad7 was not directly related with TGF-beta(1) and IFN-alpha signals. Interestingly, we reported the novel finding that the autocrine TGF-beta(1) up-regulates TGF-betaRII at protein and mRNA levels. In conclusion, the intracellular signals triggered by IFN-alpha2b and by autocrine TGF-beta(1) are integrated at the nuclear level, where activated Stat1 and Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Interferon-alfa/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Interferon alfa-2 , Fígado/citologia , Fígado/metabolismo , Fígado/fisiopatologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Masculino , Lesões Pré-Cancerosas/fisiopatologia , Ratos , Ratos Wistar , Proteínas Recombinantes , Fator de Transcrição STAT1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.
Assuntos
Apoptose , Interferon-alfa/farmacologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Lesões Pré-Cancerosas/fisiopatologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultura/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Interferon alfa-2 , Fígado/metabolismo , Masculino , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes , Proteína Smad2 , Proteína Smad3 , Transativadores/metabolismo , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta1RESUMO
The influences of fasting on DEN-initiation and of intermittent fasting (IF) on the rat liver chemical carcinogenesis process were evaluated in a 52-week long assay. Three groups of adult male Wistar rats were used: Groups 1 to 3 were treated with a single i.p. injection of 200 mg/kg of diethylnitrosamine (DEN). Group 2 was submitted to 48 h fasting prior to DEN treatment. After the 4th week, Group 3 was submitted to IF, established as 48 h weekly fasting during 48 weeks, while Groups 1 and 2 were fed ad libitum until the 52nd week. All animals were submitted to 70% partial hepatectomy and sacrificed at the 3rd and 52nd weeks, respectively. Fasting prior to DEN-initiation did not influence the development of altered foci of hepatocytes (AFHs) and of hepatic nodules (Group 2 vs. Group G1). IF inhibited the development of preneoplastic lesions, since this dietary regimen decreased the number and the size of glutathione S-transferase (GST-P) positive foci and the number and size of liver nodules (Group G3 vs. Group G1). The inhibitory effect of IF was also reflected in the development of clear and basophilic cell foci. These results indicate that long-term IF regimen exerts an anti-promoting effect on rat hepatocarcinogenesis induced by DEN.
Assuntos
Alquilantes/toxicidade , Carcinoma Hepatocelular/fisiopatologia , Dietilnitrosamina/toxicidade , Jejum/fisiologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Animais , Basófilos/patologia , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Progressão da Doença , Glutationa Transferase/metabolismo , Técnicas Imunoenzimáticas , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
In the present experiments we studied the effect of extracts from intact liver (LE), ES2 tumor extract (TE), plasmas from intact mice (PI), and from tumor bearing animals (PT) on different phases of hepatocytes and renocytes cell cycles. C3HS 28-day-old male mice, standardized for periodicity analysis, were injected at 16:00 hours and killed every 4 hours during a circadian cycle at 20:00/04; 00:00/08; 04:00/12; 08:00/16; 12:00/20 and 16:00/24 (time of day/hours post treatment). Colchicine (2 microg/g) was injected 4 hours before killing them. Samples of livers and kidneys were processed for histology and mitotic index determinations. The results were expressed as colchicine arrested metaphases per 1000 nuclei. The TE, LE and PI had a promoting effect on the mitotic activity of hepatocytes during the first 12 hours post treatment. During the subsequent 12 hours, not only these treatments but also the PI had an inhibiting effect on the mitotic activity of the same cell population. Also the TE and the PT had a promoting effect on the mitotic activity of the renocytes during the first 12 hours while the effect of all treatments showed a clear inhibition of the mitotic activity studied during the last 12 hours. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.
Assuntos
Ciclo Celular/fisiologia , Rim/citologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Fígado/citologia , Fígado/fisiologia , Extratos de Tecidos/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ritmo Circadiano , Colchicina/farmacologia , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/sangue , Masculino , Metáfase/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Índice Mitótico , Plasma , Fatores de TempoRESUMO
Un extracto del hepatocarcinoma probablemente diferenciado de crecimiento rápido ss1k y el plasma de animales portadores de tumor se inyectaron por vía intraperitoneal a las 16.00/oo (hora del día/horas postinyección) en ratones machos dela cepa C3HS de 21-28 días. Los animales control fueron inyectados con solución fisiológica. Cuatro horas antes del sacrificio se les inyectó Colcemid. Se les sacrificó a las 08:00, 12:00 y 16:00 durante cuatro días consecutivos. La actividad mitótica se expresó como metafases colchicínicas por 1000 núcleos. Se observó inhibición de la actividad mitótica de hepatocitos y células litorales durante los tres primeros días, más intensa en el 2 día, y una onda compensadora de la actividad mitótica de ambas poblaciones celulares al cuarto día. Este efecto se ejerce probablemente sobre el pasaje G1-S del ciclo celular.