RESUMO
Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão bcr-abl/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína SOS1/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Camundongos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tirosina/metabolismo , Proteínas rac de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
PURPOSE: The NKX-3.1gene is an androgen regulated prostate specific homeobox gene that is believed to have a vital role in normal prostate development. In mice the homologue NKx3.1 is exclusively expressed in prostate epithelium. In humans NKX3.1 expression is also restricted to the prostate but to our knowledge the cellular location has not been described. Furthermore, since NKX3.1 maps to chromosomal band 8p21, a region with high loss of heterozygosity in prostate cancer, the gene has been proposed to have tumor suppressor function. In this study we demonstrate that in human prostates NKX3.1 is expressed exclusively in secretory epithelial cells and the level of NKX3.1 expression remains invariant in normal tissue and in tissue showing various grades of prostate cancer. In the 19 cases examined the DNA sequences of the NKX3.1 gene were identical and no mutation was detected. MATERIALS AND METHODS: Frozen tissue from patients who underwent radical prostatectomy was used for this study. For in situ hybridization experiments a 377 bp fragment corresponding to a portion of the 3' untranslated region of the NKX3.1 gene was amplified by polymerase chain reaction and cloned into the pCRII plasmid vector Invitrogen. Antisense or sense [33P] uridine triphosphate labeled RNA probes were generated with SP6 or T7 RNA polymerase and hybridized to the tissue sections. Slides were exposed to photographic emulsion and visualized on autoradiography. Laser capture microdissection was performed to procure pure populations of malignant epithelium. DNA was isolated by digesting samples in proteinase K buffer. Polymerase chain reaction and direct sequencing was performed using standard protocols. RESULTS: In vitro hybridization showed that NKX3.1 expression was restricted to secretory epithelial cells within benign prostate glands. No expression was detected in stroma or infiltrating lymphocytes. NKX3.1 was expressed in all grades of malignant epithelium in all 25 cases examined. Direct sequencing of the coding region of NKX3.1 revealed the wild-type sequence in all 18 microdissected cancers analyzed. CONCLUSIONS: Based on our studies we propose that NKX3.1 gene expression is restricted to benign and malignant secretory epithelium within the prostate but NKX3.1 does not appear to be a classic tumor suppressor gene responsible for prostate cancer initiation. These findings are consistent with the role of NKX3.1 in the development of normal prostate epithelium and maintenance of normal secretory function. Thus, NKX3.1 may represent a useful molecular marker for benign and malignant prostate epithelium.
Assuntos
Análise Mutacional de DNA , Expressão Gênica , Genes Homeobox/genética , Genes Supressores de Tumor/genética , Proteínas de Homeodomínio/genética , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/genética , Epitélio , Humanos , Hibridização In Situ , Masculino , Regiões Promotoras GenéticasRESUMO
Sebox is a mouse paired-like homeobox gene, previously named OG-9. Sebox genomic DNA and cDNA were cloned and sequenced. In addition, rat and human Sebox genomic DNAs were cloned and sequenced, and the predicted amino acid sequences were compared. The mouse Sebox gene was mapped to chromosome 11 near the Evi 2 locus. The mouse Sebox gene is expressed in brain, skin, ovary, and liver of mice. In the brain, the Sebox gene is expressed in the cerebral cortex and CA areas of the hippocampus, pontine nuclei, choroid plexus, and the cerebellum. Northern analysis and RNase protection assays revealed low levels of Sebox RNA in 12-day mouse embryos and higher levels in 18- and 19-day embryos. In late embryos and newborn mice, Sebox expression is localized in the epidermis. In adult mice, Sebox RNA was found in maturing oocytes and in fertilized eggs; however, the abundance of Sebox RNA is decreased in the two-cell embryo, and little or none was detected in the four-cell embryo. Hence, Sebox is a maternally expressed homeobox gene.
Assuntos
Encéfalo/metabolismo , Fase de Clivagem do Zigoto/metabolismo , Genes Homeobox , Proteínas de Homeodomínio/genética , Oócitos/metabolismo , Pele/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Mapeamento Cromossômico , DNA Complementar , Desenvolvimento Embrionário , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Gravidez , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
A mouse homeobox gene, Nkx-1.2, (previously termed Sax-1) that is closely related to the Drosophila NK-1/S59 gene was cloned, and genomic DNA and cDNA were sequenced. Nine Nkx-1.2 cDNA clones were found that correspond to three species of Nkx-1.2 mRNA that are formed by alternative splicing at conventional 5' donor and 3' acceptor splice sites; however, seven cDNA clones were found that correspond to three species of Nkx-1.2 mRNA from testes that have novel TG/AC 5' and 3' splice sites. The consensus splice sequences are: 5' donor, CC downward arrowTGGAAG; 3' acceptor, ACTTAC downward arrow. Predicted amino acid sequences suggest that some transcripts may be translated into proteins that lack part or all of the homeodomain. At least three bands of Nkx-1.2 mRNA were found in RNA from the testes. Nkx-1.2 mRNA was shown to be present in postmeiotic germ cells of the testis and in mature spermatozoa. Nkx-1.2 mRNA also was found in regions of the adult cerebral cortex, hippocampus, diencephalon, pons/medulla, and cerebellum. Nkx-1.2 mRNA was found in embryos in highest abundance in 10-day embryos; the mRNA levels decrease during further development. Nkx-1.2 mRNA also was found in discrete zones of the embryonic mesencephalon and myelencephalon.
Assuntos
Processamento Alternativo , Proteínas de Homeodomínio/genética , Proteínas Nucleares , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/patologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro , Homologia de Sequência de Aminoácidos , EspermatozoidesRESUMO
This study was designed to determine whether psychological stress alters the function of the GABAergic synapse, examined as biochemical changes of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding, in unwashed membranes of rat cerebral cortex. Psychological stress increased the number of [35S]TBPS binding sites by 22%. This enhancement was very similar to that after acute foot shock (24%). Psychological stress was induced very rapidly, because only 1 day after previous foot shock exposure, [35S]TBPS binding was increased by 23%. Diazepam [3 mg/kg intraperitoneally (subcutaneously)] and ipsapirone (5 mg/kg subcutaneously), injected 30 min before psychological stress, antagonized the enhancement of [35S]TBPS binding. This result suggests that psychological stress is a good animal model for investigating the various biochemical changes related to stress, avoiding the physical components associated with most of the normally used stressors and mimicking only emotional state alterations.
Assuntos
Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Convulsivantes/metabolismo , Estresse Fisiológico/metabolismo , Animais , Sítios de Ligação , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The NMB human neuronal cell line, transfected with a newly prepared plasmid expressing rat serotonin transporter (NMB-rSERT), shows specific [3H]5-HT uptake which is blocked by citalopram and fenfluramine (F) stereoisomers with IC50 values (1 nM. 0.5 microM (dF) and and 5 microM (IF), respectively) which are similar to those found in rat brain synaptosomes. d-Fenfluramine (0.5 and 10 microM) also stimulates tritium release from NMB-rSERT cells preloaded with [3H-]-5-HT. The d-fenfluramine-induced [3H-]5-HT release is blocked by 0.3 microM citalopram and is dependent on the density of SERT expressed per cell, but is not affected by removal of Ca++ ions from the incubation medium. Manipulation of the Na+ gradient across the plasma membrane (replacing 60 mM NaCl with an equimolar concentration of KCl or choline) also induced [3H-]5-HT release from NMB-rSERT cells, which was inhibited by 0.3 microM citalopram. These results, together with the finding that NMB-rSERT cells preloaded with 500 nM unlabelled 5-HT take up [3H-]d-fenfluramine, make NMB-rSERT cells a valuable tool for studying the transporter-mediated exchange release induced by amphetamine derivatives.
Assuntos
Proteínas de Transporte/genética , Fenfluramina/farmacologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Neuroblastoma/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/metabolismo , Animais , Cálcio/farmacologia , Citalopram/farmacologia , Humanos , Técnicas In Vitro , Neuroblastoma/genética , Potássio/farmacologia , Ratos , Proteínas da Membrana Plasmática de Transporte de Serotonina , Transfecção , Células Tumorais CultivadasRESUMO
The effect of polymyxin B and KN-62 on the [3H]5-HT release induced by depolarization and by 0.5 microM D-fenfluramine (dF) from superfused rat hippocampal synaptosomes was examined. Polymyxin B and KN-62 were initially characterized as inhibitors, respectively, of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (Ca/CaM-KII), although both compounds were subsequently described as inhibitors of the depolarization-induced Ca2+ influx through voltage-operated Ca2+ channels, at concentrations similar to those interacting with the CaM systems. Three micromolar KN-62 significantly inhibited the dF- and the depolarization-induced [3H]5-HT release, by 25% and 33%. Polymyxin B, tested at concentrations from 30 to 1000 IU ml-1, dose-dependently inhibited both the dF- and the depolarization-induced [3H]5-HT release with similar potency, with complete inhibition at the highest concentration tested. These compounds did not significantly alter 5-HT transporter function. Moreover dF had no direct effect on Ca/CaM-KII activity. These results further support the suggestion that the [3H]5-HT release induced by low concentrations of dF (0.5 microM), previously found to be Ca(2+)-dependent, actually involves a dF-induced Ca2+ influx into the nerve terminal and the subsequent exocytosis.
Assuntos
Cálcio/fisiologia , Exocitose/efeitos dos fármacos , Fenfluramina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Antibacterianos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Polimixina B/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Flunarizine (35 mg/kg), but not haloperidol and trifluperazine, counteracted the initial indole depletion induced by D-fenfluramine (dF) in vivo (5 mg/kg), without affecting ex vivo [3H]-serotonin (5-HT) uptake by synaptosomes or changing the brain concentrations of the parent drug and its main active metabolite, D-norfenfluramine (dNF). The long-term indole depletion induced by repeated doses of dF (5 mg/kg, b.i.d. for 4 days) was also reversed by flunarizine pretreatment. Flunarizine, methiothepin, and trifluperazine, but not haloperidol, reduced in vitro the Ca(2+)-dependent [3H]5-HT release stimulated by 0.5 microM dF and dNF from superfused synaptosomes. At the concentrations used in release experiments the drugs were not active on [3H]5-HT uptake nor on the calcium-calmodulin protein kinase activity, thus excluding an effect on the uptake carrier or on phosphorylation of synaptic proteins involved in exocytosis, respectively. The drugs did not consistently affect [3H]5-HT release induced by depolarization, or dNF-induced [3H]dopamine release in vitro. The fact that flunarizine, as methiothepin and 5-HT uptake inhibitors, counteract dF-induced indole depletion in vivo suggests a relation between the reduction of the Ca(2+)-dependent release of [3H]5-HT induced by dF in vitro and the protective effect on the short- and long-lasting depletion of indoles induced in vivo by high doses of dF.
Assuntos
Fenfluramina/antagonistas & inibidores , Fenfluramina/farmacologia , Flunarizina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Dopamina/metabolismo , Interações Medicamentosas , Ácido Hidroxi-Indolacético/metabolismo , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Serotonina/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/enzimologia , Sinaptossomos/metabolismoRESUMO
The involvement of protein kinase C (PKC)-dependent processes in adaptive and plastic changes underlying neuronal plasticity was tested in an in vivo animal model characterized by targeted cellular ablation of cortical and hippocampal neurons, cognitive impairment and lack of induction of long-term potentiation. [3H]Phorbol ester binding performed on brain slices revealed a 67.4 and 35.0% increase in membrane-bound protein kinase C in the cortex and hippocampus respectively of rats treated with methylazoxy-methanol acetate compared with saline-treated control rats, and there was no modification in the expression of mRNAs of different protein kinase C isozymes. In situ phosphorylation experiments performed with 32Pi-labelled synaptosomes from the affected areas demonstrated that the phosphorylation of the nervous tissue-specific presynaptic membrane-associated protein kinase C substrate B-50/GAP-43 was increased by 51.4 and 44.8% in cortex and hippocampus respectively. Western blot analysis of protein kinase C in synaptosomal cytosol and membrane fractions prepared from cortex and hippocampus showed an increased proportion of protein kinase C in the membrane compartment in treated animals, but no change in the total synaptosomal protein kinase C activity. Our data are consistent with increased activity of presynaptic protein kinase C and predict a sustained increase in glutamate release in methylazoxy-methanol-treated rats.
Assuntos
Acetato de Metilazoximetanol/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Animais , Autorradiografia , Córtex Cerebral/fisiologia , Feminino , Proteína GAP-43 , Hipocampo/fisiologia , Hibridização In Situ , Glicoproteínas de Membrana/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We describe here integrated studies conducted in an animal model of brain malformation induced by prenatal treatment with a potent antimitotic agent, methylazoxymethanol acetate (MAM). When given at gestational day 15, MAM induces a marked and dose-dependent hypoplasia of cortex and hippocampus. The alteration of specific neurotransmitter systems in these brain areas reflect the specificity of the damage induced by MAM administration at this particular stage of brain development. These animals, when adult, show impairments in learning and memory performance, without gross alterations of spontaneous behavior. The impairment in cognitive functions is correlated with changes, both in cortex and hippocampus, of the phosphorylation state of the neuron-specific protein B-50, a substrate of Protein Kinase C, known to play a key role in synaptic plasticity. Moreover, Long-Term Potentiation (LTP), a cellular model for studying synaptic plasticity associated with learning and memory, is impaired in the hippocampal subfields affected by MAM treatment. All these results--obtained with anatomical, behavioral, neurochemical and electrophysiological studies-point to the usefulness of this animal model to understand the long-lasting consequences of the interference of neurotoxic compounds with the developing CNS.