RESUMEN

In the present study, the expression of the epidermal growth factor receptor (EGFR) was investigated in putative preneoplastic and neoplastic acinar cell lesions induced in the rat pancreas by azaserine, using Northern blotting, in situ hybridisation (ISH) and immunohistochemistry. EGFR protein levels were decreased in putative preneoplastic eosinophilic acinar cell lesions (atypical acinar cell nodules, AACN) in comparison with normal acinar cells of the pancreas. However, EGFR mRNA expression correlated positively with the volume of AACN in pancreatic homogenates and ISH showed equal or stronger EGFR mRNA expression in AACN than in the surrounding normal acinar cells. Neither EGFR protein nor EGFR mRNA was detected in more advanced lesions such as acinar adenocarcinomas (in situ). Moreover, EGFR protein expression showed an inverse relationship with the mitotic rate of the acinar cells. These findings suggest that down-regulation of EGFR at the protein level may abrogate negative constraints on cell growth, which may stimulate the development of putative preneoplastic AACN to more advanced lesions and, ultimately, acinar adenocarcinomas.

Asunto(s)

Azaserina , Carcinógenos , Receptores ErbB/análisis , Proteínas de Neoplasias/análisis , Neoplasias Pancreáticas/inducido químicamente , Neoplasias Pancreáticas/ultraestructura , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/ultraestructura , Animales , Northern Blotting , Inmunohistoquímica , Hibridación in Situ , Lesiones Precancerosas/patología , Antígeno Nuclear de Célula en Proliferación/análisis , ARN Mensajero/análisis , Ratas , Ratas Wistar

RESUMEN

Using immunohistochemistry, Northern blotting and a semi-quantitative PCR technique, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) expression were studied in the pancreas of N-nitrosobis(2-oxopropyl)-amine (BOP)-treated hamsters. After initiation pancreatic carcinogenesis was modulated by a high fat diet or by injections with the cholecystokinin analogue caerulein. Autopsies were performed 6 and 12 months after the last injection with BOP. Immunohistochemistry revealed a weak expression of TGF-alpha in nomal acinar cells and a stronger expression in ductular and centro-acinar cells. Over-expression of TGF-alpha was observed in advanced putative preneoplastic lesions (classified as borderline lesions) and in ductular adenocarcinomas. EGFR immunoreactivity was present only in ductular adenocarcinomas. EGF peptide expression was observed both in acinar and ductular normal and tumorous cells and the level of expression did not change significantly during carcinogenesis. Moreover, the post-initiation treatments did not cause differences in EGF, TGF-alpha or EGFR peptide or mRNA levels, except for a significantly lower expression of TGF-alpha mRNA in hamsters fed a high fat diet when compared with those fed a low fat diet. TGF-alpha mRNA levels increased, whereas EGF mRNA levels decreased significantly in total pancreatic homogenates of BOP-treated hamsters in comparison with untreated controls. Also, in ductular adenocarcinomas TGF-alpha and EGFR (but not EGF) mRNA levels were significantly higher than in normal pancreatic homogenates. In pancreatic homogenates obtained 6 months after the last BOP injection, these differences were less pronounced in comparison with those obtained after 12 months. The present results indicate that TGF-alpha (but not EGF) might act in a paracrine or autocrine manner in pancreatic tumours in BOP-treated hamsters via simultaneously expressed EGFR. However, TGF-alpha, EGF and EGFR do not seem to be involved in the modulating effects of a high fat diet or caerulein treatment on pancreatic carcinogenesis in BOP-treated hamsters.

Asunto(s)

Adenocarcinoma/genética , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Neoplasias Pancreáticas/genética , Factor de Crecimiento Transformador alfa/genética , Adenocarcinoma/inducido químicamente , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Northern Blotting , Peso Corporal , Carcinógenos , Cricetinae , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Inmunohistoquímica , Mesocricetus , Nitrosaminas , Tamaño de los Órganos , Páncreas/patología , Neoplasias Pancreáticas/inducido químicamente , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador alfa/metabolismo

RESUMEN

Expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) was studied in normal pancreatic tissue and in (pre)neoplastic pancreatic lesions of azaserine-treated rats. They were given either a low fat, high fiber (low caloric) diet, to inhibit carcinogenesis, or a low fat diet combined with injections of the cholecystokinin analog caerulein to enhance carcinogenesis. The control groups, maintained on a low fat diet, were injected with azaserine or were not treated at all. Autopsy was performed at 6 and 15 months after the last azaserine injection. After both 6 and 15 months immunohistochemistry revealed a weak expression of EGF and TGF-alpha peptides in the acinar cells, and a stronger expression in the ductular and centroacinar cells. TGF-alpha peptide expression was reduced in both putative preneoplastic and neoplastic acinar cell lesions, but no differences in EGF peptide expression were observed between the various stages of exocrine pancreatic carcinogenesis. After 16 months an increase in TGF-alpha mRNA due to treatment with azaserine was detected by semi-quantitative PCR in total pancreatic homogenates, whereas EGF mRNA expression had decreased. TGF-alpha mRNA levels in macroscopically isolated tumors were significantly lower, but EGF mRNA levels were significantly higher, than in total pancreatic homogenates from azaserine-treated rats. Furthermore, EGF and TGF-alpha mRNA levels in isolated tumors did not differ significantly from mRNA levels in non-carcinogen-treated rats. Neither with immunohistochemistry nor with PCR were differences in EGF or TGF-alpha expression observed due to either inhibition or stimulation of carcinogenesis. It is concluded that putative preneoplastic acinar cell lesions induced in rat pancreas by azaserine may develop into acinar adenocarcinomas independently of TGF-alpha and EGF. The results suggest involvement of these growth factors at the early stage of the carcinogenic process, during the initiation of normal acinar cells into putative preneoplastic cells. However, modulation of azaserine-induced pancreatic carcinogenesis by cholecystokinin or a low fat, high fiber (caloric restricted) diet appeared not to be regulated by EGF or TGF-alpha.

Asunto(s)

Cocarcinogénesis , Dieta con Restricción de Grasas , Fibras de la Dieta/uso terapéutico , Factor de Crecimiento Epidérmico/biosíntesis , Páncreas/efectos de los fármacos , Factor de Crecimiento Transformador alfa/biosíntesis , Animales , Azaserina/toxicidad , Secuencia de Bases , Peso Corporal/efectos de los fármacos , Carcinógenos/toxicidad , Ceruletida/toxicidad , Sinergismo Farmacológico , Ingestión de Energía , Factor de Crecimiento Epidérmico/genética , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Tamaño de los Órganos/efectos de los fármacos , Páncreas/anatomía & histología , Páncreas/metabolismo , Neoplasias Pancreáticas/inducido químicamente , Neoplasias Pancreáticas/dietoterapia , Neoplasias Pancreáticas/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factor de Crecimiento Transformador alfa/genética

RESUMEN

Earlier data from experiments in rats have shown that administration of retinyl esters (vitamin A) strongly influences the effects of CCl4 on the liver. The accumulation of collagen was inhibited, but an increase in CCl4-toxicity with high mortality was observed. The present study was conducted to examine the effects of beta-carotene (provitamin A) on CCl4-related general and hepatic toxicity in rats. Oral administration of beta-carotene during CCl4-treatment resulted, biochemically, in a significantly lower increase in the hydroxyproline liver content and, histopathologically, in less severe liver fibrosis as compared with the liver of rats not treated with beta-carotene. The study also showed that beta-carotene administration could prevent the long-term loss of retinoids from the CCl4-injured liver. No significant toxic effects of beta-carotene, as previously found with retinyl esters (vitamin A), were observed. This experimental study suggests that beta-carotene has the therapeutic potential to decrease the severity of liver fibrosis without marked toxicity.

Asunto(s)

Intoxicación por Tetracloruro de Carbono/patología , Carotenoides/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cirrosis Hepática Experimental/patología , Animales , Colágeno/metabolismo , Femenino , Hidroxiprolina/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Pruebas de Función Hepática , Ratas , Ratas Endogámicas BN , beta Caroteno

RESUMEN

This study on the appearance, distribution and kinetics of fibroblast-like cells (fat-storing cells, transitional cells, myofibroblasts and fibroblasts) after CCl4-treatment was undertaken to delineate further the respective roles of these cell types in liver fibrogenesis. The different cell types were distinguished on the basis of their immunophenotypic pattern with a combination of marker antibodies and on the basis of ultrastructural characteristics. Combined staining for alpha-smooth muscle actin (sma) and desmin (Des) revealed perisinusoidal fat-storing cells (FSC) as d+ sma- and myofibroblasts around the central veins of the normal rat liver as d+ sma+. During the initial phase of CCl4-induced hepatic fibrosis (week 1 and 2), the number of d+ sma+ cells increased in the degenerating area around the central veins and d+ sma+ cells appeared in the very thin fibrotic septa at week 2. Ultrastructural examination of the affected central areas showed the presence of myofibroblasts. These sma+ cells proliferated, as shown by double staining for bromodeoxyuridine (BrdU) and sma. In degenerating parenchymal areas, d+ sma- FSC were present. The FSC in the perisinusoidal space of areas which were not affected by CCl4 intoxification, remained d+ sma-. These immunostaining findings support the electron microscopical results, which show the presence of cells with the typical ultrastructural characteristics of FSC in both the degenerating areas and the perisinusoidal space of unaffected areas. After one week of CCl4-treatment, enhanced deposition of procollagen type III was observed around the central veins. Enhanced deposition of collagen type IV was seen subendothelially along the sinusoids, notably in degenerating parenchymal areas where the septa were later formed. FSC appear to be the principal source of collagen type IV during fibrogenesis. These observations further support and specify the role of FSC in early fibrogenesis. With the progression of the CCl4-induced fibrosis, d+ sma+ myofibroblasts remained localized in the fibrotic septa, but now along their outer edge. The majority of the cells in the septa were formed by d- sma- cells indicating a prominent role of fibroblasts in the septal formation. Septal fibroblasts are not only likely to produce matrix components, but also were shown to degrade collagen, as evidenced by the increased number of collagen-containing vacuoles during the course of fibrosis. In conclusion, myofibroblasts and FSC appear to be the main cell types involved in the initial phase of liver fibrogenesis induced by CCl4. Both myofibroblasts and FSC divide and transform.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Cirrosis Hepática Experimental/patología , Hígado/ultraestructura , Animales , Tetracloruro de Carbono , División Celular , Colágeno/metabolismo , Femenino , Fibroblastos/patología , Fibroblastos/ultraestructura , Técnicas para Inmunoenzimas , Hígado/metabolismo , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Microscopía Electrónica , Ratas , Ratas Endogámicas BN

RESUMEN

Earlier studies have shown that retinoid administration suppresses the generation of hepatic fibrosis and stimulates its regression in normal (i.e., vitamin A-sufficient) carbon tetrachloride-treated rats. This study focuses on the possible role of a marginal or deficient vitamin A status on carbon tetrachloride-induced fibrosis. This experimental study in rats shows that vitamin A status, reflected by hepatic retinoid content (retinol and retinyl esters), modulates the development of hepatic fibrosis induced by carbon tetrachloride. In rats with low hepatic retinoid levels (12 +/- 0.9 micrograms/gm liver), carbon tetrachloride-induced liver fibrosis was more pronounced than in rats with sufficient hepatic retinoid levels (1,065 +/- 327 micrograms/gm liver). Enhanced liver fibrogenesis was confirmed both morphologically and by a higher hydroxyproline content of the liver. It was associated with a reduced liver weight and the development of parenchymal regeneration nodules. Furthermore, carbon tetrachloride treatment itself reduced the hepatic retinoid content in rats independently of the liver vitamin A status before treatment and increased serum retinol levels in vitamin A-sufficient rats. The results show that the vitamin A status of the liver plays an important role in hepatic fibrogenesis. Low hepatic vitamin A levels, which can be the result not only of low dietary intake but also of interference with vitamin A metabolism by agents such as ethanol and carbon tetrachloride, may be a risk factor for the development of liver fibrosis. We suggest that retinoids modulate collagen synthesis and deposition irrespective of the degree of hepatocellular necrosis induced by carbon tetrachloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Cirrosis Hepática Experimental/metabolismo , Deficiencia de Vitamina A/complicaciones , Animales , Tetracloruro de Carbono , Colágeno/metabolismo , Femenino , Hidroxiprolina/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , Ratas , Ratas Endogámicas , Retinoides/metabolismo , Organismos Libres de Patógenos Específicos , Vitamina A/sangre , Deficiencia de Vitamina A/metabolismo

RESUMEN

The uptake characteristics of both the retinol and retinol-binding protein (RBP) moieties of the retinol-RBP complex by liver parenchymal cells (PC) in vitro were studied to assess whether retinol uptake is mediated by a cell-surface receptor for RBP. At 37 degrees C as well as 4 degrees C, [3H]retinol uptake from [3H]retinol-RBP showed a time-dependent increase, and was not saturable at concentrations exceeding the physiological concentration by more than a factor of 2 (3 microM). Uptake of [3H]retinol was not inhibited by a 10-fold molar excess of unlabeled retinol-RBP. Cell association of 125I-RBP at 37 and 4 degrees C was low and showed no time dependence. In addition, the association of 125I-RBP was not saturable at concentrations up to 3 microM. These data do not support the existence of a cell-surface receptor for RBP on rat liver PC. The uptake of [3H]retinol from RBP was also compared to the uptake of retinol from cellular retinol-binding protein (CRBP) and lactoglobulin. Uptake characteristics of [3H]retinol from CRBP and lactoglobulin were similar to that of [3H]retinol from RBP. Furthermore, a similar percentage of the [3H]retinol taken up by PC was metabolized into retinyl esters, irrespective of its carrier. These data suggest that the uptake of retinol and its subsequent metabolic processing do not depend on binding to RBP. The low level of cell association of 125I-binding proteins was not due to uptake, degradation, and secretion of ligand by PC. This suggests that retinol is dissociated from its binding protein before uptake by PC.

Asunto(s)

Hígado/metabolismo , Proteínas de Unión al Retinol/metabolismo , Vitamina A/metabolismo , Animales , Unión Competitiva , Células Cultivadas , Precipitación Química , Femenino , Cinética , Lactoglobulinas/metabolismo , Ratas , Ratas Endogámicas BN , Proteínas Celulares de Unión al Retinol , Temperatura , Ácido Tricloroacético , Tritio