RESUMEN
The autocrine motility factor receptor (AMFR) is a multifunctional protein involved in cellular adhesion, proliferation, motility and apoptosis. Our study showed that increased AMFR protein expression in the hippocampus of KM mice correlated with enhanced capacity for learning and memory following the shuttle-box test and was significantly elevated in the highest score group. Also, AMF and AMFR mRNA expression positively correlates with the mRNA expression of the synapse marker synaptophysin (Syp). Aging studies in the senescence-accelerated mouse strain (SAM) prone/8 (SAMP8), an animal model of Alzheimer's disease (AD), revealed significantly decreased mRNA and protein expression of AMF and AMFR in the hippocampus. This is especially true for AMFR and AMF protein expression compared with age-matched SAM resistant/1 (SAMR1) mouse strain as the control. Additionally, the low mRNA expression of AMFR could be up-regulated by the four nootropic traditional Chinese medicinal prescriptions (TCMPs): Ba-Wei-Di-Huang decoction (BW), Huang-Lian-Jie-Du decoction (HL), Dang-Gui-Shao-Yao-San (DSS) and Tiao-Xin-Fang decoction (TXF). AMFR protein expression could be up-regulated by two TCMPs, Liu-Wei-Di-Huang decoction (LW) and BW. This indicated that AMFR is involved in the process of learning and memory in the central nervous system. These results may provide useful clues for understanding the etiology of AD.
Asunto(s)
Hipocampo/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Receptores del Factor Autocrino de Motilidad/fisiología , Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Animales no Consanguíneos , Reacción de Prevención/fisiología , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glucosa-6-Fosfato Isomerasa/biosíntesis , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Actividad Motora/fisiología , Nootrópicos/farmacología , Receptores del Factor Autocrino de Motilidad/biosíntesis , Sinaptofisina/biosíntesisRESUMEN
Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) plays an important role in glycolysis and gluconeogenesis and is associated with invasion and metastasis of cancer cells. We have previously shown its role in the induction of epithelial-mesenchymal transition (EMT) in breast cancer cells, which led to increased aggressiveness; however, the molecular mechanism by which PGI/AMF regulates EMT is not known. Here we show, for the first time, that PGI/AMF overexpression led to an increase in the DNA-binding activity of NF-κB, which, in turn, led to increased expression of ZEB1/ZEB2. The microRNA-200s (miR-200s) miR-200a, miR-200b, and miR-200c are known to negatively regulate the expression of ZEB1/ZEB2, and we found that the expression of miR-200s was lost in PGI/AMF overexpressing MCF-10A cells and in highly invasive MDA-MB-231 cells, which was consistent with increased expression of ZEB1/ZEB2. Moreover, silencing of PGI/AMF expression in MDA-MB-231 cells led to overexpression of miR-200s, which was associated with reversal of EMT phenotype (i.e., mesenchymal-epithelial transition), and these findings were consistent with alterations in the relative expression of epithelial (E-cadherin) and mesenchymal (vimentin, ZEB1, ZEB2) markers and decreased aggressiveness as judged by clonogenic, motility, and invasion assays. Moreover, either reexpression of miR-200 or silencing of PGI/AMF suppressed pulmonary metastases of MDA-MB-231 cells in vivo, and anti-miR-200 treatment in vivo resulted in increased metastases. Collectively, these results suggest a role of miR-200s in PGI/AMF-induced EMT and thus approaches for upregulation of miR-200s could be a novel therapeutic strategy for the treatment of highly invasive breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Glucosa-6-Fosfato Isomerasa/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal , Femenino , Glucosa-6-Fosfato Isomerasa/genética , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , MicroARNs/genética , FN-kappa B/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Regulación hacia Arriba , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.
Asunto(s)
Beta vulgaris/enzimología , Colchicina/farmacología , Genes de Plantas , Octoxinol/farmacología , Semillas/enzimología , Alcohol Deshidrogenasa/biosíntesis , Alcohol Deshidrogenasa/genética , Beta vulgaris/efectos de los fármacos , Beta vulgaris/genética , Germinación , Glucosa-6-Fosfato Isomerasa/biosíntesis , Glucosa-6-Fosfato Isomerasa/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Malato Deshidrogenasa/biosíntesis , Malato Deshidrogenasa/genética , Semillas/efectos de los fármacos , Semillas/genéticaRESUMEN
The isozyme patterns of glucose-6-phosphate isomerase (GPI) have been analyzed in ten species of polychaetes of the genera Polydora and Dipolydora (Polychaeta: Spionidae). The GPI patterns of these species have been found to have some specific characteristics that cannot be explained in terms of the generally accepted views on the nature of isozymes. The patterns are represented by two hybridizing isozymes with different expression specificities that exhibit coordinated allozymic variation in most individuals of each species studied. Involvement of alternative splicing in the expression of the GPI gene is considered to be the most probable mechanism of the formation of the unusual GPI isozyme patterns in polydorids.
Asunto(s)
Empalme Alternativo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Glucosa-6-Fosfato Isomerasa/genética , Poliquetos/genética , Animales , Glucosa-6-Fosfato Isomerasa/biosíntesis , Isoenzimas/biosíntesis , Isoenzimas/genética , Poliquetos/enzimología , Especificidad de la EspecieRESUMEN
Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.
Asunto(s)
Senescencia Celular , Fibrosarcoma/enzimología , Regulación Neoplásica de la Expresión Génica , Glucosa-6-Fosfato Isomerasa/biosíntesis , Proteínas de Neoplasias/biosíntesis , Estrés Oxidativo , Línea Celular Tumoral , Senescencia Celular/genética , Regulación hacia Abajo/genética , Fibrosarcoma/genética , Fibrosarcoma/patología , Fibrosarcoma/terapia , Regulación Neoplásica de la Expresión Génica/genética , Gluconeogénesis/genética , Glucosa-6-Fosfato Isomerasa/genética , Glucólisis/genética , Humanos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Estrés Oxidativo/genética , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Superóxido Dismutasa/metabolismoRESUMEN
Phosphoglucose isomerase (PGI) is one of the glycolytic enzymes and is a multifunctional enzyme that functions in glucose metabolism inside the cell while acting as a cytokine outside the cell, with properties that include autocrine motility factor (AMF) regulating tumor cell motility. Although there are many studies indicating that PGI/AMF has been implicated in progression of metastasis, no direct studies of the significance of exogenous PGI/AMF on tumor progression have been reported. Here, we report on the mesenchymal-to-epithelial transition (MET), which is the reverse phenomenon of the epithelial-to-mesenchymal transition that is associated with loss of cell polarity, loss of epithelia markers, and enhancement of cell motility essential for tumor cell invasion and metastasis. Mesenchymal human fibrosarcoma HT1080 cells, which have naturally high levels of endogenous and exogenous PGI/AMF, were stably transfected with PGI/AMF small interfering RNA (siRNA). The siRNA targeting human PGI/AMF down-regulated the endogenous PGI/AMF expression and completely extinguished the secretion of PGI/AMF in a human fibrosarcoma HT1080, whereas the control siRNA showed no effects. The PGI/AMF siRNA caused cells to change shape dramatically and inhibited cell motility and invasion markedly. Suppression of PGI/AMF led to a contact-dependent inhibition of cell growth. Those PGI/AMF siRNA-transfected cells showed epithelial phenotype. Furthermore, tumor cells with PGI/AMF deficiency lost their abilities to form tumor mass. This study identifies that MET in HT1080 human lung fibrosarcoma cells was initiated by down-regulation of the housekeeping gene product/cytokine PGI/AMF, and the results depicted here suggest a novel therapeutic target/modality for mesenchymal cancers.
Asunto(s)
Fibrosarcoma/enzimología , Fibrosarcoma/patología , Glucosa-6-Fosfato Isomerasa/biosíntesis , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , ADN de Neoplasias/biosíntesis , Regulación hacia Abajo , Células Epiteliales/patología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/genética , Humanos , Mesodermo/patología , Invasividad Neoplásica , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
It has been reported that tumor infiltration is correlated with the expression of autocrine motility factor (AMF) and its receptor 78 kDa glycoprotein (gp78). The purpose of the present study was to detect AMF and gp78 mRNA expression levels and their localization in high-grade astrocytomas (glioblastoma and anaplastic astrocytoma) and to determine whether AMF and gp78 are important prognostic factors. A total of 32 formalin-fixed and paraffin-embedded glioblastomas and 23 formalin-fixed and paraffin-embedded anaplastic astrocytomas was used. The expressions of AMF and gp78 mRNA were detected using the highly sensitive in situ hybridization method. The expression of AMF mRNA was detected in 27 of 32 glioblastomas (84.4%) and 11 of 23 anaplastic astrocytomas (47.8%). The positivity of AMF mRNA was significantly higher in glioblastomas than in anaplastic astrocytomas (P = 0.0094), but gp78 mRNA was detected in most cases and no statistical significance was observed. The overall survival of patients with AMF expression was significantly shorter than patients without AMF expression (P = 0.0175). In anaplastic astrocytomas, the overall survival of patients with AMF expression was also significantly shorter than in patients without AMF expression (P = 0.0058). This study demonstrated that AMF is a poor prognostic factor in high-grade astrocytomas.
Asunto(s)
Astrocitoma/metabolismo , Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/metabolismo , Glucosa-6-Fosfato Isomerasa/biosíntesis , ARN Mensajero/análisis , Adulto , Anciano , Anciano de 80 o más Años , Astrocitoma/mortalidad , Astrocitoma/patología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Femenino , Expresión Génica , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , Pronóstico , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Tasa de Supervivencia , Ubiquitina-Proteína LigasasRESUMEN
Autocrine motility factor (AMF) stimulates, via an autocrine route, the motility of cancer cells. The current study investigated the expression of AMF and its receptor, AMFR (gp78), in breast cancer and attempted to dissect a clinical link. Breast tumor tissues (n=120) and non-neoplastic normal tissues (n=32) were studied. AMF and AMFR distribution in tissues were assessed using immunohistochemistry and their transcripts were analyzed using RT-PCR and quantitative PCR. Median follow-up of the cohort was 10 years. Normal mammary epithelial cells, but not stromal and endothelial cells, weakly stained for AMF and AMFR. However, cancer cells showed stronger staining. Both AMF and AMFR transcripts were significantly higher in tumor than in normal tissues (p=0.003 and p=0.0001, respectively). High levels of AMF and AMFR were seen in patients who died of breast cancer (p=0.049, p=0.0435) and high AMF was also seen in patients who had local recurrence (p=0.039) compared with those who remained disease free. A significant correlation was seen between long-term survival and the AMFR:CK19 ratio, in which patients with high AMFR:CK19 ratio tumors had a significantly shorter survival (101.0 months, 80.6-121.4) compared with those with low ratio (136.0 months, 123.7-148.2), p=0.0331. In conclusion, AMF and AMFR are overexpressed in human breast cancer and are negatively associated with patients' clinical outcome. This strongly indicates that the AMF-AMFR complex plays an important role in the progression of breast cancer, as well as having a prognostic role.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glucosa-6-Fosfato Isomerasa/biosíntesis , Receptores de Citocinas/biosíntesis , Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Femenino , Glucosa-6-Fosfato Isomerasa/genética , Humanos , Inmunohistoquímica , Glándulas Mamarias Humanas/metabolismo , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Reacción en Cadena de la Polimerasa/métodos , Pronóstico , ARN Mensajero/biosíntesis , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/genética , Tasa de Supervivencia , Ubiquitina-Proteína LigasasRESUMEN
Autocrine motility factor (AMF) is a cytokine known to regulate tumor cell motility. Recent studies have extended its role to many other aspects of cancer biology. In the present study, we examined the level of AMF expression and its relationship with vascular endothelial growth factor (VEGF) expression and the angiogenic phenotype in human gastric cancer and their effect on survival. The AMF and VEGF expression level and tumor microvessel density (MVD) status in archived tissue specimens from 86 resected gastric cancer cases were determined. AMF expression was significantly higher in both primary tumors and lymph node metastases than in adjacent normal gastric mucosa and normal gastric mucosa from individuals without gastric cancer. In univariate survival analyses, strong AMF expression was associated with inferior survival (P = 0.028). In a Cox proportional hazards model, strong AMF expression (P = 0.019) was independently prognostic of poor survival. Strong AMF expression in the lymph node metastases was associated with poor survival (P = 0.011). Furthermore, AMF expression in the primary tumors was directly correlated with VEGF expression and MVD status. We found the first clinical evidence that AMF expression is directly correlated with VEGF expression and MVD status and predicts clinical outcome in patients with gastric cancer, supporting the hypothesis that the AMF/AMF receptor pathway plays an important role in multiple aspects of cancer biology.
Asunto(s)
Glucosa-6-Fosfato Isomerasa/biosíntesis , Neovascularización Patológica/patología , Neoplasias Gástricas/patología , Antígenos CD34/análisis , Western Blotting , Femenino , Mucosa Gástrica/metabolismo , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neovascularización Patológica/metabolismo , Pronóstico , Neoplasias Gástricas/irrigación sanguínea , Neoplasias Gástricas/metabolismo , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser(185) by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser(185) with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucosa-6-Fosfato Isomerasa/biosíntesis , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/farmacología , Citocinas/metabolismo , Citosol/metabolismo , Dimerización , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Glucosa/metabolismo , Glucólisis , Humanos , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Metástasis de la Neoplasia , Fosforilación , Conformación Proteica , Estructura Secundaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Serina/química , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
Autocrine motility factor (AMF) stimulates cell motility in an autocrine manner and is related to tumor malignancy. AMF is a multifunctional molecule, also known as phosphoglucose isomerase and neuroleukin. Signal cascades of the AMF-stimulated motility and novel functions of this protein contributing to tumor malignancy have been presented recently. AMF stimulation activated small Rho-like GTPases and subsequently induced actin fiber rearrangement, which was removed by the C3 exoenzyme, a specific inhibitor of Rho. The expression of Jun N-terminal kinase (JNK)1, JNK2 and the Rho GDP dissociation inhibitor-beta was upregulated by AMF. The addition of AMF to culture medium stimulated the motility of the endothelial cells and the formation of tube-like structures in collagen gels. Highly AMF-expressing HT1080 cells induced aggressive angiogenesis in vivo. The expression of fms-like tyrosine kinase (Flt)-1, a vascular endothelial growth factor (VEGF) receptor, was enhanced in AMF-expressing tumors dependent on protein kinase C and phosphatidylinositol 3 kinase (PI3K) activation; meanwhile kinase insert domain-containing receptor, another receptor of VEGF, was not. Permeability of mesothelial and endothelial cell monolayers was increased by AMF, and numerous gaps were observed in the monolayers after treatment with AMF. AMF gene transfection transformed NIH3T3 cells to proliferate quickly and acquire anti-apoptosis ability induced by serum deprivation in a PI3K-dependent manner. The anti-apoptotic effect of AMF has been described by other authors who have shown that the AMF over-expressing cells were resistant to mitomycin-C-induced apoptosis showing regression of Apaf-1 and caspase-9 dependent on PI3K and MAP kinase. These novel functions of AMF makes it a likely target for cancer therapy.
Asunto(s)
Apoptosis , Movimiento Celular/efectos de los fármacos , Glucosa-6-Fosfato Isomerasa/farmacología , Neovascularización Fisiológica , Antineoplásicos/uso terapéutico , Ascitis/metabolismo , Línea Celular Tumoral , Células Endoteliales/efectos de los fármacos , Glucosa-6-Fosfato Isomerasa/biosíntesis , Glucosa-6-Fosfato Isomerasa/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic phosphoglucose isomerase, has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized by the hanging-drop method of vapour diffusion using 1.6 M sodium citrate as the precipitant at pH 6.5. Multiple-wavelength anomalous dispersive X-ray data have been collected to a maximum resolution of 1.92 A on a single selenomethionine-incorporated crystal. This crystal belongs to space group C2, with approximate unit-cell parameters a = 84.7, b = 42.4, c = 57.3 A, beta = 120.6 degrees and a monomer in the asymmetric unit.
Asunto(s)
Glucosa-6-Fosfato Isomerasa/química , Pyrococcus furiosus/enzimología , Cristalización/métodos , Cristalografía por Rayos X , Escherichia coli/metabolismo , Glucosa-6-Fosfato Isomerasa/biosíntesis , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Selenometionina/químicaRESUMEN
Intra-articular hypoxia in the inflamed rheumatoid joint is associated with increased cell proliferation, enhanced metabolism and compromised vascular perfusion. Recent clinical studies using direct measurements of hypoxia in rheumatoid joints have delineated up to 20% of soft tissue pO(2) readings as below 10mm Hg. Increased markers for glycolysis exist in rheumatoid synovial fluid and upregulation of tissue glycolytic enzymes occurs in a rat model of synovitis. Recent reports show arthritis is provoked by linked T and B cell lymphocyte recognition of the glycolytic enzyme glucose-6-phosphate isomerase (GPI). This suggests an unusual physiological feature of rheumatoid joints leads to autoimmune destruction. In this report I suggest that hypoxia, within the rheumatoid joint, leads to upregulation of the glycolytic enzyme GPI which in turn perpetuates rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/enzimología , Glucosa-6-Fosfato Isomerasa/biosíntesis , Hipoxia , Regulación hacia Arriba , Animales , División Celular , Modelos Animales de Enfermedad , Glucosa-6-Fosfato Isomerasa/genética , Glucólisis , Modelos Teóricos , Oxígeno/metabolismo , Ratas , Sinovitis/metabolismoRESUMEN
Coastal ecosystems are subjected to a wide variety of disturbances, including those due to xenobiotics of agricultural and industrial origin. These pollutants as heavy metals can modify the genetic diversity of populations by favouring or counter-selecting certain alleles or genotypes by differential mortality. In the present study, two genetic markers (phosphoglucomutase and glucosephosphate isomerase) and a protein marker (metallothionein) were monitored in order to determine the impact of heavy metals in different clam populations. Analysis of the genetic structure of the clam populations examined reveals that those inhabiting environments contaminated by heavy metals exhibit a higher allelic diversity and possess alleles at PGM loci that could be selected by the presence of heavy metals. The evaluation of metallothionein levels using a specific polyclonal antibody developed in the Pacific oyster (Crassostrea gigas) demonstrated the existence of a relationship between metallothionein concentrations and the level of metal pollution for clam populations sampled from different sites. An inter-specific difference was also detected between Ruditapes decussatus and Ruditapes philippinarum living in sympatry at the same site, suggesting a differential response of these two species upon exposure to an identical heavy metal concentration.
Asunto(s)
Adaptación Fisiológica , Bivalvos/genética , Regulación de la Expresión Génica , Genética de Población , Glucosa-6-Fosfato Isomerasa/biosíntesis , Metalotioneína/biosíntesis , Metales Pesados/efectos adversos , Fosfoglucomutasa/biosíntesis , Contaminantes del Agua/efectos adversos , Animales , Anticuerpos , Bivalvos/fisiología , Exposición a Riesgos Ambientales , Marcadores Genéticos , Glucosa-6-Fosfato Isomerasa/análisis , Metalotioneína/análisis , Fosfoglucomutasa/análisisRESUMEN
Haploid Saccharomyces cerevisiae cells growing on media lacking glucose but containing high concentrations of carbon sources such as fructose, galactose, raffinose, and ethanol exhibit enhanced agar invasion. These carbon sources also promote diploid filamentous growth in response to nitrogen starvation. The enhanced invasive and filamentous growth phenotypes are suppressed by the addition of glucose to the media and require the Snf1 kinase. Mutations in the PGI1 and GND1 genes encoding carbon source utilization enzymes confer enhanced invasive growth that is unaffected by glucose but requires active Snf1. Carbon source does not modulate FLO11 flocculin expression, but enhanced polarized bud site selection is necessary for invasion on certain carbon sources. Interestingly, deletion of SNF1 blocks invasion without affecting bud site selection. Snf1 is also required for formation of spokes and hubs in multicellular mats. To examine glucose repression of invasive growth more broadly, we performed genome-wide microarray expression analysis in wild-type cells growing on glucose and galactose, and snf1 Delta cells on galactose. SNF1 probably mediates glucose repression of multiple genes potentially involved in invasive and filamentous growth. FLO11-independent cell-cell attachment, cell wall integrity, and/or polarized growth are affected by carbon source metabolism. In addition, derepression of cell cycle genes and signalling via the cAMP-PKA pathway appears to depend upon SNF1 activity during growth on galactose.
Asunto(s)
Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Carbono/metabolismo , Adhesión Celular , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Polaridad Celular , Pared Celular/ultraestructura , Medios de Cultivo/química , Medios de Cultivo/farmacología , AMP Cíclico/fisiología , Etanol/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glucosa-6-Fosfato Isomerasa/biosíntesis , Glucosa-6-Fosfato Isomerasa/genética , Glicerol/farmacología , Hexosas/farmacología , Glicoproteínas de Membrana , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/genética , Rafinosa/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
The incidence of distant metastases is higher in the tumours with low oxygen pressure than in those with high oxygen pressure. It is well known that hypoxia induces the transcription of various genes involved in angiogenesis and anaerobic metabolism necessary for the growth of tumour cells in vivo, suggesting that hypoxia may also induce the transcription of metastasis-associated genes. We sought to identify the metastasis-associated genes differentially expressed in tumour cells under hypoxic conditions with the use of a DNA microarray system. We found that hypoxia enhanced the expression of autocrine motility factor mRNA in various cancer cells and also enhanced the random motility of pancreatic cancer cells. Autocrine motility factor inhibitors abrogated the increase of motility under hypoxic conditions. In order to explore the roles of hypoxia-inducible factor-1alpha, we established hypoxia-inducible factor-1alpha-transfectants and dominant negative hypoxia-inducible factor-1alpha-transfectants. Transfection with hypoxia-inducible factor-1alpha and dominant-negative hypoxia-inducible factor-1alpha enhanced and suppressed the expression of autocrine motility factor/phosphohexase isomerase/neuroleukin mRNA and the random motility, respectively. These results suggest that hypoxia may promote the metastatic potential of cancer cells through the enhanced autocrine motility factor/phosphohexase isomerase/neuroleukin mRNA expression and that the disruption of the hypoxia-inducible factor-1 pathway may be an effective treatment for metastasis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Hipoxia/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción , Northern Blotting , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Dominantes , Glucosa-6-Fosfato Isomerasa/biosíntesis , Secuencias Hélice-Asa-Hélice , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/genética , ARN Mensajero/metabolismo , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transfección , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas , Regulación hacia ArribaRESUMEN
Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin beta1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin beta1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin beta1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of beta1 integrins.
Asunto(s)
Membrana Basal/patología , Carcinoma Hepatocelular/patología , Glucosa-6-Fosfato Isomerasa/farmacología , Integrina beta1/fisiología , Neoplasias Hepáticas/patología , Invasividad Neoplásica , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/metabolismo , Adhesión Celular , Colágeno , Combinación de Medicamentos , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glucosa-6-Fosfato Isomerasa/análisis , Glucosa-6-Fosfato Isomerasa/biosíntesis , Humanos , Immunoblotting , Integrina beta1/análisis , Laminina , Neoplasias Hepáticas/química , Neoplasias Hepáticas/metabolismo , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Metaloproteinasas de la Matriz/biosíntesis , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Vena Porta/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteoglicanos , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/análisis , Células Tumorales Cultivadas , Ubiquitina-Proteína LigasasRESUMEN
The apicomplexan parasite Toxoplasma gondii has the ability to switch between a rapidly replicating tachyzoite and a slowly dividing encysted bradyzoite within its intermediate hosts such as humans or other warm-blooded vertebrates. It is likely that in vivo, the tachyzoites differentiate into encysted bradyzoites in response to the immune system attack during disease progression. As part of a developmental strategy and, in order to survive within infected hosts, T. gondii tachyzoites undergo profound metabolic and morphological changes by differentiating into encysted bradyzoites. Bradyzoites are characterised by their resistance to both the immune system and chemotherapy. The stimulus that triggers Toxoplasma encystation and the molecular mechanisms triggering the switch from tachyzoite to bradyzoite remain unknown. It is very important to elucidate these mechanisms since bradyzoites within tissue cysts are not only the source of infection transmitted from domestic animals to humans, but can also be converted into tachyzoites that are the cause of fatal toxoplasmic encephalitis in acquired immunodeficiency syndrome patients. In this review, I focus on recent efforts towards the characterisation of genes that encode several stage-specific isoenzymes. The picture emerging from these studies is that stage-specific expression of isoenyzmes having different biochemical properties accompanies the interconversion of tachyzoite into bradyzoite, and vice versa. It can be hypothesised that the difference found between these enzymatic activities may be instrumental in maintaining some major parasitic metabolisms such as glycolysis in pace with the stage-specific requirements of carbohydrate or polysaccharide biosynthesis.
Asunto(s)
Isoenzimas/genética , Toxoplasma/enzimología , Toxoplasma/crecimiento & desarrollo , Animales , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Glucosa-6-Fosfato Isomerasa/biosíntesis , Glucosa-6-Fosfato Isomerasa/genética , Humanos , Isoenzimas/biosíntesis , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/genética , Fosfopiruvato Hidratasa/biosíntesis , Fosfopiruvato Hidratasa/genética , Toxoplasma/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Phosphoglucose isomerase (PGI) is the second enzyme in the glycolytic pathway and catalyzes an aldose-ketose isomerization. Outside the cell, PGI has been found to function as both a cytokine and as a growth factor. The human pgi gene was cloned and the expressed enzyme was purified to homogeneity. Isomorphous crystals were obtained under two conditions and belong to the P2(1)2(1)2(1) space group, with unit-cell parameters a = 80.37, b = 107.54, c = 270.33 A. A 94.7% complete data set was obtained and processed to a limiting resolution of 2.6 A. The asymmetric unit contains two hPGI dimers according to density calculations, a self-rotation function map and molecular-replacement solution.
Asunto(s)
Glucosa-6-Fosfato Isomerasa/química , Glucosa-6-Fosfato Isomerasa/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Escherichia coli , Glucosa-6-Fosfato Isomerasa/biosíntesis , Glucosa-6-Fosfato Isomerasa/genética , Humanos , Modelos Moleculares , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Autotaxin (ATX) is a 125-kD ectonucleotide pyrophosphate/phosphodiesterase, which was initially isolated and cloned from human melanoma cells as a potent stimulator of tumour cell motility. ATX shows 44% identity to the plasma cell membrane marker PC-1. Recently, we described the decreased expression of ATX mRNA in cultured fibroblast-like synoviocytes (SFC) of patients with RA by interferon-gamma. In this study using a competitive reverse transcriptase-polymerase chain reaction, we show an increased ATX mRNA expression in SFC from patients with RA in comparison with synoviocytes from non-RA patients. The median ATX mRNA amount in SFC of RA patients (440 pg/microg total RNA) was five-fold higher than the expression in synoviocytes from non-RA patients (80 pg/microg total RNA) or foreskin fibroblasts (MRHF cells, 90 pg/microg total RNA). In contrast to the elevated ATX mRNA expression in SFC of patients with RA, we did not measure increased mRNA amounts of PC-1 in these cells. Both the ATX mRNA amount and the 5'-nucleotide phosphodiesterase (PDE) activity of SFC lysate were reduced after treatment of SFC with the cytokines IL-1beta or IL-4. IL-1beta and IL-4 induced a down-regulation of PC-1 mRNA and protein expression in SFC. In SFC treated with transforming growth factor-beta the expression of PC-1 mRNA and protein was increased, whereas no significant effect on ATX mRNA expression was detectable. Pharmacological drugs used in therapy for RA, such as dexamethasone, cyclosporin, methotrexate and indomethacin, did not show a statistically significant effect on either ATX mRNA or PC-1 mRNA expression. Only pentoxifylline suppressed ATX mRNA as well as PC-1 mRNA expression. In conclusion, we show a tight regulation of ATX and PC-1 gene expression by cytokines detectable in the inflamed tissue of RA. Further investigations will deal with the regulation of ATX protein expression as well as with the function of ATX in RA.