RESUMO
A modulatory role of neurotrophins (NTs) in activity-dependent neuronal plasticity by pre- and postsynaptic mechanisms is now well established. In this context, it is important to identify the storage compartments and to localize the precise site(s) and mechanism of NT secretion in order to deduce the spatial and temporal availability of NTs. We approached these questions at the ultrastructural level, exploiting the unique property of NT6 to bind tightly to heparan sulfate proteoglycans at the neuronal surface (R. Götz et al., 1994, Nature 372, 266-269), permitting the localization of secretion sites excluding diffusion artifacts. The myc tagging of NT6 permitted glutaraldehyde fixation and hence good preservation of the membrane structure, permitting immunogold labeling of NT6myc at the neuronal surface. NT6myc is preferentially secreted from neurites compared to neuronal cell bodies. In agreement with light-microscopic observations, the ultrastructural localization of NT6myc by postembedding procedures showed a predominant localization in ER-like membrane-confined compartments, partially associated with microtubules.
Assuntos
Hipocampo/citologia , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Compartimento Celular , Membrana Celular/ultraestrutura , Fixadores , Glutaral , Glicosilação , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/farmacologia , Hipocampo/metabolismo , Imuno-Histoquímica , Microtúbulos/ultraestrutura , Modelos Moleculares , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/genética , Plasticidade Neuronal , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Células PC12 , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/química , Fixação de Tecidos/métodos , TransfecçãoRESUMO
Prostate cancer is characterized by elevated serum levels of prostate-specific antigen (PSA). PSA gene expression is controlled by an androgen-responsive transcriptional enhancer. Our study suggests that formation of a nucleoprotein complex, encompassing 170 base pairs of enhancer DNA, mediates androgen-responsive PSA enhancer activity. The complex is assembled by cooperative binding of androgen receptor to at least four tandem, nonconsensus androgen response elements (AREs). Systematic mutagenesis of the AREs demonstrated that they act synergistically to stimulate androgen receptor-responsive gene expression. We discuss a mechanism whereby a combination of high androgen receptor levels in the prostate and low affinity AREs contribute to the cell type specificity and activity of the enhancer.
Assuntos
Regulação da Expressão Gênica , Nucleoproteínas/genética , Antígeno Prostático Específico/genética , Receptores Androgênicos/genética , Células HeLa , Humanos , Mutagênese , Nucleoproteínas/metabolismo , Antígeno Prostático Específico/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/genética , TransfecçãoRESUMO
Prostate cancer progresses from a hormone-sensitive, androgen-dependent stage to a hormone-refractory, androgen-independent tumor. The androgen receptor pathway functions in these androgen-independent tumors despite anti-androgen therapy. In our LAPC-4 prostate cancer model, androgen-independent sublines expressed higher levels of the HER-2/neu receptor tyrosine kinase than their androgen-dependent counterparts. Forced overexpression of HER-2/neu in androgen-dependent prostate cancer cells allowed ligand-independent growth. HER-2/neu activated the androgen receptor pathway in the absence of ligand and synergized with low levels of androgen to 'superactivate' the pathway. By modulating the response to low doses of androgen, a tyrosine kinase receptor can restore androgen receptor function to prostate cancer cells, a finding directly related to the clinical progression of prostate cancer.