RESUMEN
The subject of the study was the unidentified protein Ag A2/3, which is found in some cells of rat hepatoma McA RH7777 and their clones. The feature of this protein is that its expression is alternative to alfa-fetoprotein (AFP), i.e. Ag A2/3 is not found in cells and cell clones containing AFP, and vice versa. Ag A2/3 proved to be a cell stress protein--it was induced by heavy metal salts (Pb2+ and Cd2+) in the liver of adult rats and AFP+/A2/3(-) clones of hepatomas; the attenuation of AFP synthesis occurred simultaneously. This paper describes the preparation of Ag A2/3 for sequence analysis, and the scheme of Ag A2/3 purification. When trying to obtain a blot for sequencing it proved to be impossible to reveal the protein using McAb to Ag A2/3 after the transfer of separated proteins on PVDF. The reactivity of the antigen determinant was reestablished with blot processing on PVDF membrane with methanol and Twin 80.
Asunto(s)
alfa-Fetoproteínas/biosíntesis , alfa-Fetoproteínas/aislamiento & purificación , Animales , Especificidad de Anticuerpos , Hepatocitos/metabolismo , Hepatocitos/patología , Inmunohistoquímica , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Fenotipo , Ratas , alfa-Fetoproteínas/inmunologíaRESUMEN
The presented study is devoted to investigation of molecular mechanisms regulating alpha-fetoprotein (AFP) gene expression at transcriptional level. The study was carried out on AFP-positive and AFP-negative clones of rat hepatoma McA-RH 7777 that also differ in hepatocyte nuclear factors (HNF) 1 and 4 transcription levels. To examine a hypothesis of existence in AFP-non-producing clones a transcriptional factor that downregulates this gene expression, we have obtained somatic hybrids of AFP-positive and AFP-negative clones. In the obtained hybrids AFP gene expression is decreased while expression of HNF1, one of the main AFP promoter activators, is maintained. These data indicate an existence of a repressor in AFP-negative clones that determines AFP gene downregulation regardless of the HNF1 expression level.
Asunto(s)
Proteínas de Unión al ADN , Regulación de la Expresión Génica , Neoplasias Hepáticas Experimentales/genética , Proteínas Nucleares , alfa-Fetoproteínas/genética , Animales , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Células Híbridas , Ratas , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
Mutant clones of human hepatocarcinoma PLC-PRF-5 cells carrying a hepatitis B virus (HBV) genome have been obtained using selection for resistance to the toxic action of a variety of preparations to induce cell differentiation. The clones differed in various features such as expression of alpha-fetoprotein (AFP) and albumin as well as in growth rates, ability to grow in semisolid media and to be cloned in agar. Expression of the surface antigen (HBsAg) was significantly increased in mutant cells exhibiting differentiation features in contrast to the parental cells. In addition, the core antigen (HBcAg), which was silent in the original cells, was detected in some clones. There was no temporal correlation between the peak of enhanced expression of HBsAg and activation of HBcAg observed at different life periods of each clone. Evidence of cell fusion in cell culture such as premature chromatin condensation and increased numbers of binucleate cells was detected in clones with differentiation features and an increased level of viral gene expression. The approach used in this study can be used to develop cell lines of the same origin with different degrees of differentiation whilst maintaining HBV expression. This model system may be useful in the study of HBV.
Asunto(s)
Carcinoma Hepatocelular/microbiología , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/microbiología , Diferenciación Celular , División Celular , Células Clonales , Resistencia a Medicamentos , Regulación Viral de la Expresión Génica , Antígenos del Núcleo de la Hepatitis B/análisis , Antígenos de Superficie de la Hepatitis B/análisis , Humanos , Modelos Genéticos , Mutagénesis , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The extent of methylation of MspI-HpaII sites of alpha-fetoprotein (AFP) gene in DNAs from normal rat livers, rat hepatoma cell lines and their clones was compared by Southern blot hybridization. The expression of this gene was controlled by measuring the level of AFP specific RNA and by immunoperoxidase staining with antibodies to AFP. It was found that the AFP gene contained several CCGG sites in the 5'-region methylated in the nonproductive cell lines and normal adult liver and undermethylated in productive cell lines. The same phenomena was observed for productive and nonproductive hepatoma McA-RH7777 clones. These findings suggest that specific changes in the methylation pattern are associated with changes in AFP gene expression.
Asunto(s)
Neoplasias Hepáticas Experimentales/metabolismo , alfa-Fetoproteínas/biosíntesis , Animales , Línea Celular , Células Clonales , Enzimas de Restricción del ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Regulación de la Expresión Génica , Neoplasias Hepáticas Experimentales/genética , Metilación , Hibridación de Ácido Nucleico , Ratas , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMEN
McA-RH 7777 hepatoma cell line has been cloned twice at a week's interval (analytical cloning). Alpha-fetoprotein (AFP) phenotypes of primary and secondary clones from 46 primary clones were analyzed. High interclonal variability exceeded the mutation rate by several orders. The interclonal differences have been also found in the variability rates.
Asunto(s)
Variación Genética , Neoplasias Hepáticas Experimentales/patología , Animales , Línea Celular , Células Clonales/patología , Neoplasias Hepáticas Experimentales/genética , Mutación , Fenotipo , Ratas , Factores de Tiempo , alfa-Fetoproteínas/genéticaRESUMEN
A method is suggested combining the screening and cloning of hybridomas producing cytotoxic antibodies. The method is based on the Erne local hemolysis principles. The cells from the preformed hybridoma line NATF 9.9 secreted monoclonal antibodies (McAb) against murine T lymphocyte differentiation antigen Lyt-3.2. A mixture of hybridoma cells and target cells was attached to the plastic Petri dish surface pretreated with Poly-L-lysine and covered with agarose. McAb production by hybridoma cells was elicited by complement-dependent lysis of target cells. The lysis was detected by incorporation in dead cells of the fluorescent dye ethidium bromide. Subsequent studies made it possible to evaluate the clonogenic capacity of McAb production and isolate active clones.
Asunto(s)
Células Productoras de Anticuerpos/inmunología , Técnica de Placa Hemolítica , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos de Diferenciación de Linfocitos T , Antígenos Ly/inmunología , Antígenos de Superficie/inmunología , Línea Celular , Hibridomas/inmunología , Ratones , Ratones Endogámicos CBA , Linfocitos T/inmunologíaRESUMEN
Production of alpha-fetoprotein (AFP) was determined in single cells of hepatoma McA-RH7777 and in the clones of their progeny. To elucidate the heritability of this trait in a series of cell generations, a variety of local hemolysis in gel was devised. According to the method the cells and red cells conjugated with protein A were placed on the polylysine covered surface and layered with agarose gel containing antibodies. AFP production by single cells was determined from the formation of plaques--areas of red cell hemolysis. The cells forming the plaques (+AFP) and not forming them (-AFP) were distinguished and their reproduction was followed up. After 7-14 days the cells were fixed and stained by the immunoperoxidase technique with antibodies to AFP. High efficacy of the cloning has been demonstrated for both +AFP- and -AFP-cells (69 and 71%). Negative cells preserved their phenotype more frequently, producing homogenous negative clones, whereas +AFP cells gave "negative" clones in 1/3 of the cases. Both cells gave mixed clones in a small percentage of the cases. At present the AFP trait in these cells is being studied by recloning.
Asunto(s)
Neoplasias Hepáticas Experimentales/patología , alfa-Fetoproteínas/biosíntesis , Animales , Diferenciación Celular , Células Clonales/metabolismo , Células Clonales/patología , Neoplasias Hepáticas Experimentales/metabolismo , RatasRESUMEN
Clonal cell lines were isolated from rat hepatoma McA-RH7777 to study expression of alpha-fetoprotein (AFP) in these clones. AFP contained by the cells was determined by immunofluorescent and immunoperoxidase staining, while AFP secreted into the medium by aggregate-hemagglutination and immunoisotachophoresis. The existence of the clones that drastically differ in the intensity of AFP synthesis was demonstrated, with these clones being implicated in the monitoring of AFP synthesis. The differences between the clones gradually diminished in the course of long-term cultivation. It was also shown that the clones under consideration secreted, apart from AFP, at least 8 proteins of adult rat serum. One of the proteins was identified as transferrin. No albumin was detected in the cultural media tested. The level of AFP production did not correlate with the synthesis of other serum proteins and thus seems likely to be controlled by an independent mechanism.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Neoplasias Hepáticas Experimentales/metabolismo , alfa-Fetoproteínas/biosíntesis , Animales , Proteínas Sanguíneas/genética , Células Clonales , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas , Ratas , Transferrina/biosíntesis , alfa-Fetoproteínas/genéticaRESUMEN
Population aspects of specific secreted proteins (alpha-fetoprotein and serum albumin) were analyzed in the cultured human embryo hepatocytes (6 to 12 weeks' gestation). A method based on local hemolysis in gel of sheep erythrocytes conjugated with antibodies specific of proteins in question. The great majority of individual hepatocytes synthesized both proteins.
Asunto(s)
Hígado/embriología , Albúmina Sérica/biosíntesis , alfa-Fetoproteínas/biosíntesis , Células Cultivadas , HumanosRESUMEN
Alpha-fetoprotein (AFP) produced by individual hepatocytes and hepatocyte microcolonies was detected with microelectrophoresis-precipitation in polyacrylamide gel. Hepatic cells of 6--13-week-old human embryos were cultivated in vitro for 2 to 5 days. 23 of 28 individual cells, and 89 of 91 microcolonies, built up of 2--35 cells, were demonstrated to produce AFP within the range of 70--800 pg per cell.
Asunto(s)
Hígado/embriología , alfa-Fetoproteínas/biosíntesis , Células Cultivadas , Humanos , Hígado/metabolismoRESUMEN
The hepatic cells of human embryos cultivated as a monolayer retain their ability to metabolize the carcinogenic hydrocarbon benz (a) pyrene (BP). The intensity of BP metabolism is higher in the "young" cultures of hepatic cells than in fibroblast cultures obtained from the same embryo. At later terms the former becomes equal for both tissues as a result of a decreased metabolic activity of hepatic cells.
Asunto(s)
Carcinógenos/metabolismo , Fibroblastos/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Benzopirenos/metabolismo , Células Cultivadas , Embrión de Mamíferos , Femenino , Fibroblastos/enzimología , Humanos , Técnicas In Vitro , Microsomas Hepáticos/enzimología , Oxidorreductasas/metabolismo , EmbarazoRESUMEN
Ontogenetic changes of normal diploid cells and cells with genetic mutations (LHC-484 from a patient with xeroderma pigmentosum) have been studied under prolonged cultivation in a stationary phase. While investigating the dynamics of complex changes in certain cell properties and cell populations it was found that the cultivated human cells possess a particular pattern of ontogenetic changes for every culture and the dynamics of these changes depends upon the density of the cell population. Two cell subpopulations different in the degree of nucleus heterochromotinization have been discovered.
Asunto(s)
Mutación , Xerodermia Pigmentosa/genética , Células Cultivadas , Heterocromatina/ultraestructura , Humanos , Métodos , Factores de TiempoRESUMEN
A simple and reproducible method for cultivation of human embryo (7--12-week) hepatocytes in a monolayer is presented. The culture showed the capacity for limited growth, displayed a viability for 4 to 6 weeks without being subcultured, and retained some specific characteristics: synthesis of serum protein (albumin and alpha-fetoprotein), a significant amount of glycogen, a high activity of monaminooxidase, and a characteristics response to a high concentration of glucocorticoids. These properties are useful as markers for genetic experiments or for selection.