RESUMEN
Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.
Asunto(s)
Permeabilidad Capilar/fisiología , Neuronas/fisiología , Receptores de Trombina/metabolismo , Animales , Calcio/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Edema/inducido químicamente , Edema/metabolismo , Esófago/irrigación sanguínea , Esófago/efectos de los fármacos , Esófago/inervación , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Yeyuno/irrigación sanguínea , Yeyuno/efectos de los fármacos , Yeyuno/inervación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Oligopéptidos/farmacología , Páncreas/irrigación sanguínea , Páncreas/efectos de los fármacos , Páncreas/inervación , Piperidinas/farmacología , Quinuclidinas/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptor PAR-1 , Receptores de Trombina/agonistas , Receptores de Trombina/química , Transducción de Señal/efectos de los fármacos , Estómago/irrigación sanguínea , Estómago/efectos de los fármacos , Estómago/inervación , Trombina/farmacología , Vejiga Urinaria/irrigación sanguínea , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/inervaciónRESUMEN
Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5-10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17alpha-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme's 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor beta-chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-beta. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-beta serine phosphorylation, and three PCOS patients with increased levels of IR-beta serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17alpha-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17alpha-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells' donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 micromol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.
Asunto(s)
Síndrome del Ovario Poliquístico/enzimología , Piel/enzimología , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Adolescente , Adulto , Células Cultivadas , Femenino , Fibroblastos/enzimología , Humanos , Cinética , Fosforilación , Fosfoserina/análisis , Síndrome del Ovario Poliquístico/genética , Síndrome del Ovario Poliquístico/patología , Proteínas Recombinantes/metabolismo , Valores de Referencia , Piel/patología , TransfecciónRESUMEN
The candidate tumour-suppressor gene ING1 has been identified by using the genetic suppressor element (GSE) methodology. ING1 encodes a nuclear protein, p33ING1, overexpression of which inhibits growth of different cell lines. The properties of p33ING1 suggest its involvement in the negative regulation of cell proliferation and in the control of cellular ageing, anchorage dependence and apoptosis. These cellular functions depend largely on the activity of p53, a tumour-suppressor gene that determines the cellular response to various types of stress. Here we report that the biological effects of ING1 and p53 are interrelated and require the activity of both genes: neither of the two genes can, on its own, cause growth inhibition when the other one is suppressed. Furthermore, activation of transcription from the p21/WAF1 promoter, a key mechanism of p53-mediated growth control, depends on the expression of ING1. A physical association between p33ING1 and p53 proteins has been detected by immunoprecipitation. These results indicate that p33ING1 is a component of the p53 signalling pathway that cooperates with p53 in the negative regulation of cell proliferation by modulating p53-dependent transcriptional activation.
Asunto(s)
División Celular/fisiología , Genes Supresores de Tumor , Inhibidores de Crecimiento/fisiología , Proteínas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Proteínas de Ciclo Celular , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Inhibidores de Crecimiento/genética , Humanos , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas Nucleares , Pruebas de Precipitina , Proteínas/genética , Ratas , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de TumorRESUMEN
Death in circulation is one of the natural barriers preventing dissemination of tumor cells and formation of metastases. One of the negative factors acting in circulation is the loss of cell contact with natural substrate which can be imitated in vitro by the incubation of cells in suspension or in semi-solid media. Normal mouse fibroblasts (MEFs) stay viable in suspension and undergo p53-independent G1 growth arrest. Transformation with Ela and ras oncogenes leads to the abrogation of this arrest and to the p53-dependent apoptosis occurring in G1 phase of the cell cycle. Suppression of apoptosis by p53 gene knock-out, transduction of dominant negative p53 mutant or bcl-2 prevents death in suspension and greatly induces frequency of colony formation in semi-solid media. The ability of cells to undergo apoptosis does not correlate with their tumorigenicity in nude mice but does correlate with their ability to survive in lungs of intravenously injected mice and to form experimental metastases. We suggest that abrogation of a p53-mediated apoptosis facilitates experimental metastasis by promoting survival of tumor cells in circulation.
Asunto(s)
Adhesión Celular/fisiología , División Celular/fisiología , Metástasis de la Neoplasia/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/genética , Línea Celular Transformada , Supervivencia Celular , Ratones , Ratones Desnudos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.
Asunto(s)
Genes p53 , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos , Embrión de Mamíferos , Etopósido/toxicidad , Fibroblastos , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/biosíntesis , Ratas , Retroviridae , Supresión Genética , Transactivadores/metabolismoRESUMEN
We compared the influence of exogenous N-ras oncogene and treatment with PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Genes ras/fisiología , Proteína Quinasa C/agonistas , Acetato de Tetradecanoilforbol/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Células Cultivadas , Colchicina/farmacología , Perros , Resistencia a Múltiples Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Genes ras/genética , Vectores Genéticos/genética , Humanos , ARN Mensajero/biosíntesis , Ratas , Retroviridae/genética , Transducción de Señal/fisiología , Verapamilo/farmacologíaRESUMEN
The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)