RESUMEN
Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Western Blotting , División Celular/efectos de los fármacos , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Femenino , Fase G1/efectos de los fármacos , Humanos , Mutación/genética , Proteínas Serina-Treonina Quinasas , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales CultivadasRESUMEN
We review the hepatocarcinogenic effects of fungal cultures of Fusarium verticillioides(= Fusarium moniliforme) strain MRC 826 in male BD IX rats. Subsequent chemical analyses of the fumonisin B (FB) mycotoxin content in the culture material used and long-term carcinogenesis studies with purified FB1 provide information about dose-response effects, relevance of hepatotoxicity during FB1-induced carcinogenesis, and the existence of a no-effect threshold. Fumonisin intake levels of between 0.08 and 0.16 mg FB/100 g body weight (bw)/day over approximately 2 years produce liver cancer in male BD IX rats. Exposure levels < 0.08 mg FB/100 g bw/day fail to induce cancer, although mild toxic and preneoplastic lesions are induced. The nutritional status of the diets used in the long-term experiments was marginally deficient in lipotropes and vitamins and could have played an important modulating role in fumonisin-induced hepatocarcinogenesis. Short-term studies in a cancer initiation/promotion model in rat liver provided important information about the possible mechanisms involved during the initial stages of cancer development by this apparently nongenotoxic mycotoxin. These studies supported the findings of long-term investigations indicating that a cytotoxic/proliferative response is required for cancer induction and that a no-effect threshold exists for cancer induction. The mechanisms proposed for cancer induction are highlighted and include the possible role of oxidative damage during initiation and the disruption of lipid metabolism, integrity of cellular membranes, and altered growth-regulatory responses as important events during promotion.
Asunto(s)
Ácidos Carboxílicos/toxicidad , Carcinógenos Ambientales/toxicidad , Fumonisinas , Neoplasias Hepáticas Experimentales/inducido químicamente , Micotoxinas/toxicidad , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Peso Corporal/efectos de los fármacos , Ácidos Carboxílicos/aislamiento & purificación , Carcinógenos Ambientales/aislamiento & purificación , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ácidos Grasos Insaturados/metabolismo , Fusarium/química , Fusarium/clasificación , Lípidos/biosíntesis , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Micotoxinas/aislamiento & purificación , Fosfolípidos/metabolismo , RatasRESUMEN
Growth factors bind to membrane receptor tyrosine kinases, resulting in autophosphorylation and subsequent binding to proteins with SH2 domains, including growth factor receptor-bound protein 2 (Grb2). Grb2 bridges receptors to tyrosine kinase substrates such as SHC and SOS, which in turn facilitate the activation of downstream signaling pathways, including Ras and mitogen-activated protein kinase (MAPK). Overexpression of Grb2 has been demonstrated in several types of neoplasia but has not been investigated in liver tumorigenesis. Here we investigated Grb2 expression in liver lesions in N-nitrosodimethylamine (NDMA)-treated Helicobacter hepaticus-infected and -noninfected A/J mice at 1 year of age. Previously, we reported (6) that infection promotes the development of these NDMA-initiated tumors. In controls, Grb2 immunostaining was absent from normal hepatic tissues, whereas the inflammatory lesions in infected livers were positive for cytoplasmic Grb2 in both hepatocytes and infiltrating leukocytes. All preneoplastic foci (7 of 7), 15 of 27 adenomas, and 3 of 7 carcinomas were positive for Grb2 by immunostaining in both infected and noninfected NDMA-initiated livers. Involvement of Grb2 was confirmed by immunoblotting of similarly infected mice at 9 to 18 months of age, showing a 2.5- to 3.3-fold increase in Grb2 protein in infected livers (p < 0.05 compared with uninfected controls) as well as in preneoplastic foci, adenomas, and carcinomas. These livers also showed a 2.5- to 2.8-fold increase in total Ras protein. The results suggest that upregulation of Grb2 is an early event in liver carcinogenesis, whether caused by the bacterial infection or by NDMA. Concomitant upregulation of Ras p21 would ensure transmission of amplified signal from growth factors via Grb2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Receptores ErbB/biosíntesis , Infecciones por Helicobacter/metabolismo , Hepatitis A/metabolismo , Inflamación/metabolismo , Neoplasias/metabolismo , Lesiones Precancerosas/metabolismo , Biosíntesis de Proteínas , Animales , Animales Recién Nacidos , Dimetilnitrosamina/metabolismo , Proteína Adaptadora GRB2 , Infecciones por Helicobacter/patología , Hepatitis A/patología , Immunoblotting , Inmunohistoquímica , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos A , Neoplasias/patología , Proteína Oncogénica p21(ras)/biosíntesis , Proteína Oncogénica p21(ras)/genética , Lesiones Precancerosas/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Ácidos Carboxílicos/toxicidad , Carcinógenos Ambientales/toxicidad , Ciclina D1/metabolismo , Fumonisinas , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/metabolismo , Micotoxinas/toxicidad , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclina D1/biosíntesis , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Activadores de Enzimas/toxicidad , Fase G1/efectos de los fármacos , Fase G1/fisiología , Regulación Neoplásica de la Expresión Génica , Genes ras/genética , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/enzimología , Mutación , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/enzimología , Lesiones Precancerosas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Endogámicas F344 , Fase S/efectos de los fármacos , Fase S/fisiologíaRESUMEN
Helicobacter hepaticus is a new bacterial species that is homologous to Helicobacter pylori, a human gastric carcinogen. H. hepaticus causes chronic active hepatitis, with progression to hepatocellular tumors. We hypothesized that chronic up-regulation of epidermal growth factor (EGF), transforming growth factor-alpha, and nuclear oncogenes (cyclin D1 and c-Myc), all known to transform by overexpression, might contribute to tumorigenesis. Livers from mice that were 6-18 months old were analyzed, including nonneoplastic and preneoplastic tissues and tumors, along with age-matched controls, by immunohistochemistry and immunoblotting. EGF and transforming growth factor-alpha were increased at the earliest stage, with a further increase in EGF in tumors. Cyclin D1, cyclin-dependent kinase 4, and c-Myc were strongly increased in all infected livers, with even greater increases in tumors. An increase in cyclin D1/cyclin-dependent kinase 4 complex was also demonstrated in tumors, and its functionality was confirmed by an increase in the hyperphosphorylated:hypophosphorylated retinoblastoma protein ratio. Our findings suggest a possible cooperation of growth factors, cell cycle proteins, and transcription factors during the development of H. hepaticus-associated liver tumors and may have relevance to human cancers associated with bacterial, viral, or parasitic infections.
Asunto(s)
Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Infecciones por Helicobacter/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas , Factor de Crecimiento Transformador alfa/metabolismo , Animales , Quinasa 4 Dependiente de la Ciclina , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Hepatitis Animal/complicaciones , Hepatitis Animal/metabolismo , Hepatitis Animal/microbiología , Neoplasias Hepáticas Experimentales/microbiología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos A , Fosforilación , Proteína de Retinoblastoma/metabolismoRESUMEN
A new murine Helicobacter species, Helicobacter hepaticus, infects the livers of mice, causing a progressive chronic active hepatitis culminating in hepatocellular tumors. To examine the role of chronic H. hepaticus infection in carcinogenesis, H. hepaticus-infected male infant mice of A/JCr strain were given a single i.p. dose of N-nitrosodimethylamine (NDMA). Noninfected A/J mice similarly treated with NDMA served as controls. The effect of hepatitis induced by H. hepaticus was studied for 64 wk. At 31-36 wk, the incidence of hepatocellular adenomas in infected mice was significantly higher than in noninfected mice (82 vs 52%; p = 0.05). The multiplicity of hepatocellular tumors was also significantly higher in infected mice compared to noninfected mice (3.2 +/- 0.09 vs 0.09 +/- 0.2; p = 0.03). At 51-64 wk, many (10/18) infected mice developed hepatocellular carcinomas while only 2 of 19 control mice developed such tumors (p = 0.005). Overexpression of cyclin D was observed in hepatocytes as well as adenomas induced by NDMA in H. hepaticus-infected mice, suggesting its role in inflammation, abnormal cell growth, and early neoplasia. High molecular weight keratins were highly expressed in hyperplastic oval cells in hepatitis and in liver tumors in mice with hepatitis, establishing a reliable marker for oval cells in formalin-fixed, paraffin-embedded tissue. Thus, chronic H. hepaticus infection significantly stimulated cyclin D expression, accelerated the development of liver tumors, increased the multiplicity of such lesions, and enhanced the progression of benign to malignant tumors.
Asunto(s)
Carcinógenos/toxicidad , Cocarcinogénesis , Infecciones por Helicobacter/complicaciones , Hepatitis Animal/complicaciones , Neoplasias Hepáticas Experimentales/complicaciones , Neoplasias Hepáticas Experimentales/microbiología , Compuestos Nitrosos/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Inmunohistoquímica , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/microbiología , Linfoma/inducido químicamente , Linfoma/microbiología , Masculino , Ratones , Ratones Endogámicos ARESUMEN
Glyceryl trinitrate (GTN) was previously reported to induce hepatocellular carcinoma (HCC) in rats after prolonged feeding. The present experiments were undertaken to evaluate the histogenesis and molecular biology of these tumors and the possible role of nitric oxide (NO), a GTN metabolite, in their development. Male F344 rats received a single i.g. intubation of GTN (1.2 g/kg) at 6 weeks of age and/or a diet containing 1% GTN from 8 weeks of age until necropsy, i.e. for up to 78 weeks. Some animals were subjected to 2/3 partial hepatectomy (PH) at 9 weeks of age. Five sequential sacrifices (14, 32, 52, 78 and 84 weeks of age) were performed. No liver tumors developed in control rats or in rats that received GTN only by a single i.g. intubation, even when intubation was followed by PH. Preneoplastic foci, mainly of clear cell and mixed cell type (identified as positive for glutathione S-transferase placental form) were found from 14 weeks of age in rats receiving GTN in the diet. Focal eosinophilic areas (atypical foci) composed of atypical hepatocytes that often extended into the veins were observed beginning at 52 weeks of age. Some mixed hepatocholangiocellular adenomas and carcinomas arose in eosinophilic lesions. HCCs were seen beginning at 78 weeks of age, but only in rats receiving dietary GTN. Incidence of HCC in the latter animals was 50-75%. Most HCCs were well differentiated. The carcinogenic effect of GTN given in the diet was not affected by prior intubation of a large single dose followed by PH. No p53 mutations were found in 18 tumors but K-ras point mutations, all within codon 12, were found in 8/18 tumors, mostly those with cholangiocellular elements. These were first or second position G-->T transversions or second position G-->A transitions. While these mutation types have also been commonly seen in bacteria after NO-related DNA damage, the fact that tumors arose only on prolonged feeding of this potently bioactive agent at massive doses seems consistent with a more complex mechanism involving multiple (i.e. genetic and/or epigenetic) factors in carcinogenesis by GTN.