Asunto(s)
Ácido Gástrico/metabolismo , Úlcera Péptica/tratamiento farmacológico , Inhibidores de la Bomba de Protones , Receptores Histamínicos/efectos de los fármacos , Bencimidazoles/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Hormonas , Humanos , Transducción de SeñalAsunto(s)
Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Antagonistas de los Receptores H2 de la Histamina/farmacología , Receptores Histamínicos H2/efectos de los fármacos , Cimetidina/farmacología , Famotidina/farmacología , Mucosa Gástrica/efectos de los fármacos , Humanos , Ranitidina/farmacología , Receptores Histamínicos/fisiología , Receptores Histamínicos H2/fisiologíaRESUMEN
The permanent SLC-11 and -41 intestinal epithelial cells respectively immortalized by the E1A and large T oncogenes and their clonal derivatives showed a cytogenetic heterogeneity characterized by a near diploidy in SLC-11 and -12 cells and a generalized polyploidy in SLC-41 and -44 cells. Persistence of chromosome translocations and trisomy 3 were observed. The expression of the E1A oncogene in immortalized SLC-11 cells is associated with a strong repression of c-fos transcription during the exponential growth, as compared to the resting phase or to control rat fetal intestinal epithelial cells. The transcription of c-myc was also reduced in SLC-11 cells, especially in confluent cells. A complex relationship between the levels and size of the c-fos, c-myc mRNAs and the expression of the E1A oncogene was therefore observed in SLC-11 cells. Immortalized SLC-11 and -41 cells showed a remarkable growth inhibition in response to recombinant rat gamma-IFN. Neoplastic transformation by activated human Ha-ras in SLC-12T and -44 T cells confer resistance to the antigrowth effects of IFN. The combination of culture conditions using defined medium, membrane matrix (laminin, collagen, proteoglycans) and intestinal mesenchyme revealed the persistence of the undifferentiated phenotype of the E1A, large T-immortalized and Ha-ras-transformed SLC cells in vitro or in the nude mice. In association with the intestinal chick endoderm, SLC-11 cells possess some inductive properties on the differentiation of villi projections arising from the chick endoderm in vivo. In contrast, SLC-41 cells were induced to differentiate in enterocyte-like cells by the intestinal chick mesenchyme. The immortalized and Ha-ras-transformed SLC cells therefore constitute new models in the sequential analysis of the molecular and genetic mechanisms involved in the proliferation, differentiation and oncogene-mediated neoplastic transformation in gut. Further attempts in SLC cell differentiation have to be accomplished using chemical inducers for prolonged periods of time, or by transfection of intestinal epithelial cells using temperature- or glucocorticoid-inducible vectors.
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Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Interferón gamma/farmacología , Intestinos/citología , Proto-Oncogenes/genética , Animales , Northern Blotting , División Celular , Línea Celular , Regulación Neoplásica de la Expresión Génica/genética , Técnicas In Vitro , Cariotipificación , Ratones , Ratones Desnudos , Proteínas Oncogénicas/genética , Oncogenes/genética , Proto-Oncogenes Mas , Ratas , Proteínas RecombinantesRESUMEN
Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.
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Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Serina Endopeptidasas/genética , Animales , Antígenos CD34/inmunología , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Mieloblastina , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Serina Endopeptidasas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras and pp60c-src in human colonic Caco-2 cells is associated with specific alterations of syndecan-1, a membrane-anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated (Val 12) human Ha-ras gene or with the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines (1) contained smaller amounts of membrane-anchored PGs; (2) exhibited decreased syndecan-1 expression at the protein but not the mRNA level; (3) synthesized 35S-labelled syndecan-1 with decreased specific activity; (4) produced a syndecan-1 ectodomain with a lower molecular mass and reduced GAG chain size and sulphation; and (5) expressed heparanase degradative activity. These results show that the dramatic activation of the tumorigenic potential induced by oncogenic p21ras or Py-MT/pp60c-src in Caco-2 cells is associated with marked alterations of syndecan-1 expression at the translational and post-translational levels.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Genes ras , Glucuronidasa , Glicoproteínas de Membrana/biosíntesis , Proteoglicanos/biosíntesis , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Células CACO-2 , Sulfatos de Condroitina/análisis , Glicósido Hidrolasas/metabolismo , Heparitina Sulfato/análisis , Humanos , Glicoproteínas de Membrana/genética , Proteoglicanos/genética , ARN Mensajero/análisis , Sindecano-1 , SindecanosRESUMEN
BACKGROUND: Activation of the p21ras and pp60c-src oncoproteins occurred at high incidence in the early stage of human colorectal carcinogenesis. Our study aimed to investigate the role of these signal-transduction pathways in the process of initiation and promotion of the malignant phenotype in intestinal cells. METHODS: The human Ha-ras and the polyoma middle T (Py-MT) viral oncogenes were transferred into large T oncogene of simian virus 40 immortalized rat intestinal epithelial SLC-44 cells and human colonic adenocarcinoma Caco-2 cells. RESULTS: These transfers conferred the tumorigenic and invasive phenotypes on immortalized SLC-44 cells and potentiated the tumorigenicity of Caco-2 cells and markedly repressed the terminal differentiation of this cell line. In SLC-44T cells, induction of the invasive phenotype by the activated Ha-ras oncogene correlated with weak expression of E-cadherin and reduced accumulation of the transcripts encoding the basement membrane components alpha 1 (IV) collagen, nidogen, and BM40, which might result partly from the inactivation of the transforming growth factor beta signaling pathway. The down-regulation of the alpha 1 (IV) collagen messenger RNA in SLC-44T cells was not due to the protein kinase C-dependent pathways or the secretion of autocrine factor(s). CONCLUSIONS: These results suggest that the activation of the p21ras and Py-MT/pp60c-src oncogenic pathways are critical effectors at different stages of colorectal carcinogenesis and in Caco-2 cells interferes with the program of enterocyte differentiation.
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Antígenos Transformadores de Poliomavirus/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/genética , Genes ras , Oncogenes , Transfección , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Colágeno/genética , Neoplasias Colorrectales/etiología , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , ARN Mensajero/análisis , RatasRESUMEN
Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro.
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Antígenos Transformadores de Poliomavirus/genética , Transformación Celular Viral , Fibrosis Quística/patología , Mucosa Intestinal/patología , Secuencia de Bases , Línea Celular , Canales de Cloruro , Colforsina/farmacología , AMP Cíclico/biosíntesis , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Expresión Génica , Humanos , Técnicas In Vitro , Mucosa Intestinal/fisiopatología , Ionomicina/farmacología , Isoproterenol/farmacología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Neuropéptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Canales de Potasio/efectos de los fármacos , Transfección , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Little is known about the the signalling pathways driving the adenoma-to-carcinoma sequence in human colonic epithelial cells. Accumulation and activation of the src tyrosine kinase in colon cancer suggest a potential role of this oncogene in this early progression. Therefore, we introduced either activated src (m-src), polyoma-MT alone or combined with normal c-src in the adenoma PC/AA/C1 cell line (PC) to define the function and phenotypic transformations induced by these oncogenes in familial adenomatous polyposis (FAP) colonic epithelial cells. Functional expression of these oncoproteins induced the adenoma-to-carcinoma conversion, overexpression of the hepatocyte growth factor (HGF) receptor Met, but failed to confer invasiveness in vivo and in vitro, or to produce alterations in cell proliferation and differentiation. In contrast, PC-msrc cells became susceptible to the HGF-induced invasion of collagen gels and exhibited sustained activation of the pp60src tyrosine kinase and Tyr phosphorylation of the 120-kDa E-cadherin, which was further increased by HGF Transcripts of HGF were clearly identified by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot in the parental and transformed PC cells, suggesting an autocrine mechanism. Taken together, the data indicate that: (1) experimental activation of src and PyMT pathways directly induces tumorigenicity and Met upregulation in a colon adenoma cell line; (2) HGF-activated Met and src cooperate in inducing invasion; (3) in view of the molecular associations between catenins and cadherin or the tumour-suppressor gene product APC, the cell adhesion molecule E-cadherin may constitute a downstream effector of src and Met.
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Poliposis Adenomatosa del Colon/genética , Antígenos Transformadores de Poliomavirus/genética , Invasividad Neoplásica , Poliposis Adenomatosa del Colon/patología , Proteína de la Poliposis Adenomatosa del Colon , Animales , Cadherinas/metabolismo , Cateninas , Moléculas de Adhesión Celular/metabolismo , Pollos , Colágeno , Proteínas del Citoesqueleto/metabolismo , ADN de Neoplasias/genética , Geles , Genes src , Factor de Crecimiento de Hepatocito/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Oncogenes , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Proteínas Proto-Oncogénicas c-met , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Transfección , Trasplante Heterólogo , alfa Catenina , Catenina deltaRESUMEN
The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.