RESUMO
Androgens have key roles in normal physiology and in male sexual differentiation as well as in pathological conditions such as prostate cancer. Androgens act through the androgen receptor (AR), which is a ligand-modulated transcription factor. Antiandrogens block AR function and are widely used in disease states, but little is known about their mechanism of action in vivo. Here, we describe a rapid differential interaction of AR with target genomic sites in living cells in the presence of agonists which coincides with the recruitment of BRM ATPase complex and chromatin remodeling, resulting in transcriptional activation. In contrast, the interaction of antagonist-bound or mutant AR with its target was found to be kinetically different: it was dramatically faster, occurred without chromatin remodeling, and resulted in the lack of transcriptional inhibition. Fluorescent resonance energy transfer analysis of wild-type AR and a transcriptionally compromised mutant at the hormone response element showed that intramolecular interactions between the N and C termini of AR play a key functional role in vivo compared to intermolecular interactions between two neighboring ARs. These data provide a kinetic and mechanistic basis for regulation of gene expression by androgens and antiandrogens in living cells.
Assuntos
Receptores Androgênicos/metabolismo , Elementos de Resposta/fisiologia , Adenocarcinoma/patologia , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Anilidas/farmacologia , Animais , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Acetato de Ciproterona/farmacologia , Di-Hidrotestosterona/farmacologia , Feminino , Recuperação de Fluorescência Após Fotodegradação , Flutamida/análogos & derivados , Flutamida/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Hibridização in Situ Fluorescente , Ligantes , Luciferases/metabolismo , Neoplasias Mamárias Animais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Metribolona/farmacologia , Camundongos , Microscopia de Vídeo , Mifepristona/farmacologia , Modelos Biológicos , Nitrilas/farmacologia , Plasmídeos , Regiões Promotoras Genéticas , Receptores Androgênicos/efeitos dos fármacos , Testosterona/farmacologia , Compostos de Tosil/farmacologia , Transcrição GênicaRESUMO
We have identified a novel gene, six transmembrane protein of prostate 2 (STAMP2), named for its high sequence similarity to the recently identified STAMP1 gene. STAMP2 displays a tissue-restricted expression with highest expression levels in placenta, lung, heart, and prostate and is predicted to code for a 459-amino acid six transmembrane protein. Using a form of STAMP2 labeled with green flourescent protein (GFP) in quantitative time-lapse and immunofluorescence confocal microscopy, we show that STAMP2 is primarily localized to the Golgi complex, trans-Golgi network, and the plasma membrane. STAMP2 also localizes to vesicular-tubular structures in the cytosol and colocalizes with the Early Endosome Antigen1 (EEA1) suggesting that it may be involved in the secretory/endocytic pathways. STAMP2 expression is exquisitely androgen regulated in the androgen-sensitive, androgen receptor-positive prostate cancer cell line LNCaP, but not in androgen receptor-negative prostate cancer cell lines PC-3 and DU145. Analysis of STAMP2 expression in matched normal and tumor samples microdissected from prostate cancer specimens indicates that STAMP2 is overexpressed in prostate cancer cells compared with normal prostate epithelial cells. Furthermore, ectopic expression of STAMP2 in prostate cancer cells significantly increases cell growth and colony formation suggesting that STAMP2 may have a role in cell proliferation. Taken together, these data suggest that STAMP2 may contribute to the normal biology of the prostate cell, as well as prostate cancer progression.
Assuntos
Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Adenocarcinoma/genética , Sequência de Aminoácidos , Divisão Celular , Linhagem Celular Tumoral , Clonagem Molecular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Oxirredutases , Plasmídeos/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Kallikreins (KLKs) are highly conserved serine proteases that play key roles in a variety of physiological and pathological processes. KLKs are secreted proteins that have extracellular substrates and function. For example, prostate-specific antigen (or KLK3) is a secreted protein that is widely used as a diagnostic marker for prostate cancer. KLK4 is a recently identified member of the kallikrein family that is regulated by androgens and is highly specific to prostate for expression. Here, we show that the gene product of KLK4, hK4, is the first member of the KLK family that is intracellularly localized. We provide strong evidence that the previously assigned first exon that was predicted to code for a signal peptide that would target hK4 for secretion is not part of the physiologically relevant form of KLK4 mRNA. In addition to detailed mapping of the KLK4 mRNA 5' end by RT-PCR, this conclusion is supported by predominantly nuclear localization of the hK4 protein in the cell, documented by both immunofluorescence and cell fractionation experiments. Furthermore, in addition to androgens, hK4 expression is regulated by estrogen and progesterone in prostate cancer cells. Finally, in situ hybridization on normal and hyperplastic prostate samples in tissue microarrays indicate that KLK4 is predominantly expressed in the basal cells of the normal prostate gland and overexpressed in prostate cancer. These data suggest that KLK4 has a unique structure and function compared with other members of the KLK family and may have a role in the biology and characterization of prostate cancer.
Assuntos
Calicreínas/biossíntese , Proteínas Nucleares/biossíntese , Neoplasias da Próstata/enzimologia , Animais , Células COS , Fracionamento Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Chlorocebus aethiops , Éxons , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hiperplasia Prostática/enzimologia , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The effect of magnesium deficiency on antioxidant defence system was studied in RBC of mice suffering from hypomagnesemia. The animals were kept for 8, 15 and 22 days on magnesium-deficient diet with consequent reduction of magnesium level in plasma by 38% at the first 8 days and by 64% after 22 days of experiment. The activities of the most important antioxidant enzymes, catalase, glutathione peroxidase, superoxide dismutase, glutathione reductase, glutahione S-transferase were assayed in hemolysates. The level of reduced glutathione in erythrocytes was measured as well. Apart from catalase, the activities of antioxidant enzymes were decreasing. The activity of superoxide dismutase decreased gradually during the experiment and on the 15th and 22nd day of experiment was significantly (P < 0, 05) lowered by 30 and 32% respectively. The catalase activity was increased on each point of the experiment with the peak value up to 149% on 15th day, and by 32% on 22nd day. Glutathione peroxidase activity was insignificantly reduced. The reduction of Glutatione reductase and Glutathione S-transferase activities by 24 and 21%, respectively, were observed after 8 days of the experiment with a further downward tendency. The reduced glutathione was significantly depleted after 8 days by 33% and was kept on that level in the course of the study. These findings support previous reports on the hypomagnesemia--induced alteration in endogenous enzyme antioxidant defences and glutathione redox cycle of mice.