RESUMEN
The reasons for the increase of pituitary tumor-transforming gene (PTTG) transcripts in about 90% of pituitary adenomas are still not fully understood, although upregulation by basic fibroblast growth factor (bFGF) has been discussed as a potential cause. A possible influence of the Insulin like Growth Factor 1 (IGF-1) might be of interest, since this protein is also synthesized in most pituitary adenomas. Moreover, the principal regulation of the PTTG gene by IGF-1 and Insulin has been demonstrated in astrocytoma and breast cancer cells. We analyzed a large group (103 patients) of unselected clinical pituitary adenoma samples. From total RNA of frozen tumor samples (all subtypes) cDNA ( COMPLEMENTARY DNA) was synthesized and transcripts of PTTG, bFGF, IGF-1 were measured by Real-Time-PCR. Not only mRNA ( MESSENGER RNA) levels of bFGF, but also of IGF-1, correlated strongly with PTTG transcripts. This result was obtained, when all pituitary adenoma samples were included in the statistical calculations, irrespective of their subclassification. Our study suggests a connection between PTTG and IGF-1 in pituitary adenomas.
Asunto(s)
Adenoma/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Factor I del Crecimiento Similar a la Insulina/genética , Proteínas de Neoplasias/genética , Hipófisis/metabolismo , Neoplasias Hipofisarias/genética , Adenoma/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Hipofisarias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SecurinaRESUMEN
The mechanism of retinal ganglion cell loss in Leber's hereditary optic neuropathy (LHON) is still uncertain, and a role of enhanced superoxide production by the mutant mitochondrial complex I has been hypothesized. In the present study, it was shown that LHON cybrids, carrying the np11778 mutation, became selectively more H(2)O(2) sensitive compared with the parental cell line only following short-term retinoic acid differentiation. They contained a decreased cellular glutathione pool (49%, p< or =0.05), despite 1.5-fold enhanced expression of the regulatory subunit of gamma-glutamylcysteine synthetase (p< or =0.05). This points to a reduction of the capacity to detoxify H(2)O(2) and to changes in thiol redox potential. The activity of the H(2)O(2) degrading enzyme glutathione peroxidase (GPx) and the activities of glutathione reductase (GR) and superoxide dismutase (SOD) were unaffected.
Asunto(s)
Antioxidantes/metabolismo , Complejo I de Transporte de Electrón/genética , Glutatión/metabolismo , Atrofia Óptica Hereditaria de Leber/genética , Atrofia Óptica Hereditaria de Leber/metabolismo , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Complejo I de Transporte de Electrón/metabolismo , Genotipo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Mitocondrias/enzimología , Mitocondrias/genética , Atrofia Óptica Hereditaria de Leber/patología , Oxidantes/farmacología , Mutación Puntual , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Teratoma , Tretinoina/farmacologíaRESUMEN
We investigated the effects of 2-methoxyestradiol (2-ME), a promising new antitumor agent, on viable cell number and nuclear morphology of malignant glioma cells (three human and one rat glioma cell lines) and analyzed the controversial role of death recepor 5 (DR5) upregulation in 2-ME induced apoptosis. Microtiter-tetrazolium (MTT) assays showed a significant reduction of viable cells after incubation with 2 microM and 20 microM 2-ME for 48 and 72 hours in all cultures. In the 20 microM concentration, there were even significant effects in the majority of shorter incubation periods. Hoechst 33258 stains showed a substantial amount of cells with nuclear fragmentation indicating a late stage of apoptosis after 20 microM 2-ME treatments of 24 hours and more. The role of the DR5-mediated extrinsic apoptotic pathway was further studied in the three human glioma cell lines; 50 ng/ml of the DR5 ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and 2 microM 2-ME showed no synergism, as determined by MTT assays. Real-time PCR revealed no significantly increased amount of DR5 mRNA, suggesting that receptor upregulation does not play a major role for 2-ME-induced apoptosis in glioma cells, in contrast to data for a breast cancer cell line in the literature.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Estradiol/análogos & derivados , Glioma/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/metabolismo , 2-Metoxiestradiol , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Estradiol/uso terapéutico , Glioma/metabolismo , Humanos , Glicoproteínas de Membrana/uso terapéutico , ARN Mensajero/análisis , Ratas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/genética , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/uso terapéutico , Regulación hacia ArribaRESUMEN
The purpose of this study was to investigate the potential effects of 2-methoxyestradiol, a natural mammalian steroid, in glioma cells, since antiproliferative effects of this compound had been shown earlier in several leukemia and carcinoma cell lines. The effects of 0.2, 2 and 20 microM concentrations of 2-methoxyestradiol were measured in three malignant human glioma cell lines (U87MG, U138MG, LN405) and one malignant rat glioma cell line (RG-2) using a microtiter-tetrazolium (MTT) assay. In all cell lines, a significant reduction of the viable cell number by more then 75% occurred ( P < 0.05) for concentrations of 2 and 20 microM 2-methoxyestradiol after 6 days. A concentration of 0.2 microM had smaller effects (10-40% cell reduction), which were significant in two of the cell lines tested. The apoptotic nature of cell death was further analyzed in U87MG and RG-2 cells. Caspase-3 activity was significantly induced to levels between 3.4- and 23-fold after 4 days for the two higher 2-methoxyestradiol concentrations (P < 0.05). In the cell line RG-2 nuclear fragmentation was visible in many nuclei, following stains with Hoechst H33258. A round cell morphology occurred in most treated cells, which was not accompanied by a complete destruction of the microtubule network, as it can be observed with other microtubule targeting drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Núcleo Celular/patología , Congéneres del Estradiol/administración & dosificación , Estradiol/análogos & derivados , Estradiol/administración & dosificación , Glioma/patología , Microtúbulos/patología , 2-Metoxiestradiol , Análisis de Varianza , Animales , Antineoplásicos Hormonales/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Caspasa 3 , Caspasas/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/patología , Relación Dosis-Respuesta a Droga , Glioma/tratamiento farmacológico , Glioma/metabolismo , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , RatasRESUMEN
Dalpha3 is a functional alpha-subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here, we produced Dalpha3-specific antibodies to study which other nAChR subunits can co-assemble with Dalpha3 in receptor complexes of the Drosophila nervous system. Immunohistochemical studies revealed that Dalpha3 is co-distributed with the beta-subunit ARD in synaptic neuropil regions of the optic lobe. Both subunits can be co-purified by alpha-bungarotoxin affinity chromatography. Dalpha3 antibodies co-immunoprecipitate Dalpha3 and ARD proteins and, vice versa, anti-ARD antibodies co-precipitate ARD and Dalpha3. These data demonstrate that one type of fly nAChRs includes these two subunits as integral components.
Asunto(s)
Drosophila melanogaster/metabolismo , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Anticuerpos/inmunología , Pruebas de Precipitina , Receptores Nicotínicos/inmunología , Distribución TisularRESUMEN
Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.