RESUMEN
The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Membranas Intracelulares/metabolismo , Prostaglandinas A/farmacología , Proteínas ras/metabolismo , Animales , Calpaína/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Membranas Intracelulares/efectos de los fármacos , Ratones , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.
Asunto(s)
Proliferación Celular , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Proteína SOS1/fisiología , Animales , Antioxidantes/farmacología , Adhesión Celular , Movimiento Celular , Supervivencia Celular , Células Cultivadas , Daño del ADN , Ratones , Transducción de Señal , Proteínas Son Of Sevenless/fisiologíaRESUMEN
Background: The IL-15/NF-KappaB axis has an important role in coeliac disease (CD) and may represent a molecular target for immunomodulation. Ascorbate (vitamin C) is known to show inhibitory effects on NF-KappaB. Therefore, we studied if ascorbate supplementation to gliadin gliadin-stimulated biopsy culture could down-regulate the mucosal immune response to gliadin in CD. Methods: Duodenal biopsy explants from treated CD patients were gliadin challenged in vitro (100ìg/ml) with and without 20mM ascorbate. An extra tissue explant in basal culture was used as internal control. Secretion levels of nitrites (3h), and IFNGamma, TNFalpha, IFNalpha, IL-17, IL-13, and IL-6 (24h) were measured on the supernatants. IL-15 was assayed by western-blot on whole protein duodenal explants. Results: The addition of ascorbate to in vitro culture gliadin-challenged biopsies blocked the secretion of nitrites (p=0.013), IFNGamma (p=0.0207), TNFalpha (p=0.0099), IFNá (p=0.0375), and IL-6 (p=0.0036) compared to samples from non-ascorbate supplemented culture. Cytokine secretion was downregulated by ascorbate even to lower values than those observed in basal cultures (IFNGamma: p=0.0312; TNFalpha: p=0.0312; IFNá: p=0.0312; and IL-6: p=0.0078). Gliadin-challenge induced IL-15 production in biopsies from treated CD patients, while the addition of ascorbate to culture medium completely inhibited IL-15 production. Moreover, the inhibition of IL-15 by ascorbate took place even in the only treated CD-patient who had basal IL-15 production. Conclusions: Ascorbate decreases the mucosal inflammatory response to gluten in an intestinal biopsy culture model, so it might have a role in future supplementary therapy in CD(AU)
Asunto(s)
Humanos , Ascorbato Oxidasa/farmacocinética , Inmunidad Mucosa/fisiología , Gliadina/farmacocinética , Enfermedad Celíaca/fisiopatología , Inflamación/fisiopatología , Interleucina-15/inmunología , Ácido Ascórbico/farmacocinéticaRESUMEN
BACKGROUND: The IL-15/NF-κB axis has an important role in coeliac disease (CD) and may represent a molecular target for immunomodulation. Ascorbate (vitamin C) is known to show inhibitory effects on NF-κB. Therefore, we studied if ascorbate supplementation to gliadin gliadin-stimulated biopsy culture could down-regulate the mucosal immune response to gliadin in CD. METHODS: Duodenal biopsy explants from treated CD patients were gliadin challenged in vitro (100 µg/ml) with and without 20mM ascorbate. An extra tissue explant in basal culture was used as internal control. Secretion levels of nitrites (3h), and IFNγ, TNFα, IFNα, IL-17, IL-13, and IL-6 (24h) were measured on the supernatants. IL-15 was assayed by western-blot on whole protein duodenal explants. RESULTS: The addition of ascorbate to in vitro culture gliadin-challenged biopsies blocked the secretion of nitrites (p=0.013), IFNγ (p=0.0207), TNFα (p=0.0099), IFNα (p=0.0375), and IL-6 (p=0.0036) compared to samples from non-ascorbate supplemented culture. Cytokine secretion was downregulated by ascorbate even to lower values than those observed in basal cultures (IFNγ: p=0.0312; TNFα: p=0.0312; IFNα: p=0.0312; and IL-6: p=0.0078). Gliadin-challenge induced IL-15 production in biopsies from treated CD patients, while the addition of ascorbate to culture medium completely inhibited IL-15 production. Moreover, the inhibition of IL-15 by ascorbate took place even in the only treated CD-patient who had basal IL-15 production. CONCLUSIONS: Ascorbate decreases the mucosal inflammatory response to gluten in an intestinal biopsy culture model, so it might have a role in future supplementary therapy in CD.
Asunto(s)
Ácido Ascórbico/farmacología , Enfermedad Celíaca/tratamiento farmacológico , Gliadina/inmunología , Inflamación/prevención & control , Adulto , Anciano , Biopsia , Enfermedad Celíaca/inmunología , Citocinas/biosíntesis , Femenino , Humanos , Inmunidad Mucosa/efectos de los fármacos , Interleucina-15/antagonistas & inhibidores , Interleucina-15/fisiología , Masculino , Persona de Mediana EdadRESUMEN
Patients that receive a T-cell depleted (TCD) hematopoietic stem cell transplantation (SCT) show higher risk of graft failure/rejection and of disease relapse than those that receive unmanipulated grafts. The purpose of the present investigation was to analyze the usefulness of chimaerism quantification in bone marrow (BM), peripheral blood (PB), and leukocyte lineages such as T lymphocytes (CD3+,both CD4+ and CD8+), B lymphocytes (CD19+) and myeloid cells (CD15+), for the early detection of graft failure/rejection episodes and disease relapse after TCD-PBSCT. Two of the ten (2/10) patients included in the study showed stable complete chimaerism (CC). The other 8/10 patients showed decreasing mixed chimaerism (MC) and 7 of them had either graft failure (n = 1)/rejection (n = 3) or disease relapse (n = 3). In two patients relapsed from chronic myeloid leukemia, MC was observed in BM and PB, with higher percentages of autologous cells in BM, as well as in leukocyte lineages, with higher percentages of recipient cells in the myeloid lineage than in lymphocytes. Combined analysis of chimaerism and minimal residual disease allowed early diagnosis of relapse and successful rescue therapy with donor leukocyte infusions (DLI), before the onset of hematological relapse. Chimaerism analysis allowed early diagnosis of incipient graft rejection in 3 patients. These patients showed MC both in BM and PB, with greater percentages of recipient cells in PB. Analysis of leukocyte lineages showed higher percentages of autologous cells in T lymphocytes (mainly CD8+) than in B or myeloid cells. Two of these patients were successfully treated with DLI and recovered normal PB counts and BM cellularity, as well as CC. The graft versus recipient hemopoiesis effect harbored by the donor immunocompetent cells infused seems useful forthe treatment of graft rejection, provided that an early diagnosis is made.
Asunto(s)
Trasplante de Células Madre de Sangre Periférica , Linfocitos T/citología , Adulto , Antígenos CD19/biosíntesis , Complejo CD3/biosíntesis , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Linaje de la Célula , Femenino , Rechazo de Injerto/prevención & control , Humanos , Hibridación Fluorescente in Situ , Leucocitos/citología , Antígeno Lewis X/biosíntesis , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Quimera por TrasplanteRESUMEN
Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23-q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present inhomozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.