RESUMO
Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been used to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. PL inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that PL directly inhibited binding of Stat3 to its phosphotyrosyl peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as Janus kinases, Src kinase family members or receptor tyrosine kinases. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient-derived xenografts. PL's antitumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 has a role.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dioxolanos/farmacologia , Reposicionamento de Medicamentos , Fator de Transcrição STAT3/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dioxolanos/efeitos adversos , Dioxolanos/uso terapêutico , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Transdução de Sinais , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Numerical modeling of steroid hormone signaling presents an exciting challenge involving spatiotemporal coordination of multiple events. Ligand binding in cytoplasm triggers dissociation and/or association of coregulators which subsequently regulate DNA binding and transcriptional activity in nucleus. In order to develop a comprehensive multi-stage model, it is imperative to follow not only the transcriptional outcomes but also the intermediate protein complexes. Accordingly, we developed a software toolkit for simulating complex biochemical pathways as a set of non-linear differential equations in LabVIEW (Laboratory Virtual Instrumentation and Engineering Workbench, National Instruments, Austin, TX) environment. The toolkit is visual, highly modular, loosely coupled with the rest of LabVIEW, scalable and extensible. The toolkit can be used to develop and validate biochemical models and estimate model parameters from existing experimental data. We illustrate the application of the toolkit for simulation of steroid hormone response in cells, and demonstrate how the toolkit can be employed for other biological and chemical systems as well. The software module presented here can be used stand-alone as well as built into data collection and analysis applications.
Assuntos
Modelos Teóricos , Androgênios/fisiologia , Humanos , Software , Transcrição GênicaRESUMO
Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multi-copy integration of an ER-regulated promoter. Through automated single-cell analyses, we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Compostos Benzidrílicos , Western Blotting , Linhagem Celular Tumoral , Análise por Conglomerados , Disruptores Endócrinos/farmacologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Fulvestranto , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Fenóis/farmacologia , Análise de Componente Principal , Regiões Promotoras Genéticas/genética , Cloridrato de Raloxifeno/farmacologia , Reprodutibilidade dos Testes , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: The most critical factor determining the quality of colonoscopy results is the extent of bowel cleansing. AIM: This observational post-marketing study evaluated the efficacy, acceptability and safety of a range of the most commonly used bowel cleansing solutions in routine clinical practice. PATIENTS: Patients undergoing diagnostic, preventive or follow-up colonoscopy were recruited from 7 centres in Italy, Spain and Greece. METHODS: Quality of bowel preparation was assessed on a 5-point scale and included evaluation of visible bowel surface area and the amount and consistency of residual fluid. Patients evaluated ease of use and palatability. RESULTS: A total of 437 patients took part. Klean-Prep, the most commonly used preparation in this evaluation, achieved the highest score for quality of bowel cleansing and was rated as good or excellent in 72.0% of patients. In dosage-compliant patients, Klean-Prep showed better results in comparison to Fleet Phosphosoda (p < 0.05) in the maximum bowel level reached in the intestine during colonoscopy examinations. All of the bowel cleansing solutions were well tolerated. CONCLUSION: The polyethylene glycol-based preparations provided the most adequate cleansing and, of these, Klean-Prep provided the highest "good" or "excellent" level of bowel preparation.
Assuntos
Catárticos , Colonoscopia/métodos , Eletrólitos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatos , Polietilenoglicóis , Estudos Prospectivos , SoluçõesRESUMO
Aurora-A kinase, also known as STK15/BTAK kinase, is a member of a serine/threonine kinase superfamily that includes the prototypic yeast Ipl1 and Drosophila aurora kinases as well as other mammalian and non-mammalian aurora kinases involved in the regulation of centrosomes and chromosome segregation. The Aurora-A gene is amplified and overexpressed in a wide variety of human tumors. Aurora-A is centrosome-associated during interphase, and binds the poles and half-spindle during mitosis; its over-expression has been associated with centrosome amplification and multipolar spindles. GFP-Aurora-A was used to mark centrosomes and spindles, and monitor their movements in living cells. Centrosome pairs labeled with GFP-Aurora-A are motile throughout interphase undergoing oscillations and tumbling motions requiring intact microtubules and ATP. Fluorescence recovery after photobleaching (FRAP) was used to examine the relative molecular mobility of GFP-Aurora-A, and GFP-labeled alpha-tubulin, gamma-tubulin, and NuMA. GFP-Aurora-A rapidly exchanges in and out of the centrosome and mitotic spindle (t(1/2) approximately 3 sec); in contrast, both tubulins are relatively immobile indicative of a structural role. GFP-NuMA mobility was intermediate in both interphase nuclei and at the mitotic spindle (t(1/2) approximately 23-30 sec). Deletion mapping identifies a central domain of Aurora-A as essential for its centrosomal localization that is augmented by both the amino and the carboxyl terminal ends of the protein. Interestingly, amino or carboxy terminal deletion mutants that maintained centrosomal targeting exhibited significantly slower molecular exchange. Collectively, these studies contrast the relative cellular dynamics of Aurora-A with other cytoskeletal proteins that share its micro-domains, and identify essential regions required for targeting and dynamics.
Assuntos
Centrossomo/enzimologia , Neoplasias/enzimologia , Proteínas Quinases/metabolismo , Fuso Acromático/enzimologia , Antígenos Nucleares , Aurora Quinase A , Aurora Quinases , Proteínas de Ciclo Celular , Divisão Celular/genética , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes , Mutação/genética , Neoplasias/genética , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão , Tubulina (Proteína)/metabolismo , Proteínas de XenopusRESUMO
BACKGROUND: Only a few reports on the clinical features and management of childhood leiomyosarcoma are available. To contribute additional information on the management of this rare tumor, we report on a series of 16 pediatric patients treated from 1982 to 1998 by the Soft Tissue Sarcoma Italian Cooperative Group. PATIENTS AND METHODS: Primary surgery was conservative in all but two patients, and consisted of biopsy--three cases, non-radical excision--four, and radical resection--nine (involving a primary re-excision in 4 of 9). In two cases secondary radical surgery was performed after primary chemotherapy. Chemotherapy was administered to 9 of 16 patients, radiotherapy to three. RESULTS: After a median follow-up of seven years (range 3-18), the five-year event-free survival (EFS) and overall survival were 56.3% and 72.9%, respectively; 12 of 16 patients were alive (nine of them in continuos complete remission). Univariate analysis was performed to compare EFS according to different subgroups: size represented the most significant prognostic factor. CONCLUSIONS: Complete surgical resection is the mainstay of treatment for leiomyosarcoma. The role of both adjuvant chemotherapy and radiotherapy has yet to be established, and awaits cooperative multicentric studies.
Assuntos
Leiomiossarcoma/cirurgia , Adolescente , Adulto , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Itália , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/radioterapia , Masculino , Análise de SobrevidaRESUMO
BACKGROUND: Studies regarding the associations between different types of cancer in the same patient are very few and not always come to the same conclusions. Several hypothesis are suggested and particularly genetic and socioeconomical ones seem to offer an interpretation of this issue. Early detection of a second neoplasm allows to improve prognosis and survival. The knowledge of correlations between tumors help to select a population, with a high risk to develop a second cancer, to be included in a screening program. Nowadays thanks to early detection of breast cancer, ten years survival is more than 75%. Women who had breast cancer now live longer and so could have a higher risk to develop a second cancer. METHODS: From September 1998 to September 1999 in our Department 71 patients operated for breast cancer, underwent screening colonscopy. No patients refused to be included in the study. Mean age was 61 years (range 36-87). Each patient had a clinician interview in order to explain the goals of the study. RESULTS: Results show that among all patients 3 (4.2%) presented a history of colon cancer, 18.3% (13 cases) presented large bowel polyps. In 84.60% patients (11 cases) polyps were found not over 40 cm. This study shows that 93% of patients (66 cases) had a relative with cancer history. CONCLUSIONS: Our results compared with those of other authors seem to show an increased risk for breast cancer patients in developing polyps or colon cancer, so we suggest to insert sigmoidoscopy in standard follow-up of breast cancer patients.
Assuntos
Neoplasias da Mama/patologia , Pólipos do Colo/epidemiologia , Pólipos do Colo/secundário , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Programas de Rastreamento , Pessoa de Meia-IdadeRESUMO
Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor alpha (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integrated lac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP-SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP-SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP-SRC-1, while antagonist additions diminish YFP-SRC-1-CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER-SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP-SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.
Assuntos
Óperon Lac/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Receptor alfa de Estrogênio , Estrogênios/farmacologia , Genes Reporter , Histona Acetiltransferases , Ligantes , Microscopia de Fluorescência , Coativador 1 de Receptor Nuclear , Ligação Proteica/efeitos dos fármacos , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
TAK/P-TEFb is an elongation factor for RNA polymerase II-directed transcription that is thought to function by phosphorylating the C-terminal domain of the largest subunit of RNA polymerase II. TAK/P-TEFb is composed of Cdk9 and cyclin T and serves as the cellular cofactor for the human immunodeficiency virus transactivator Tat protein. In this study, we examined the subcellular distribution of Cdk9 and cyclin T1 using high resolution immunofluorescence microscopy and found that Cdk9 and cyclin T1 localized throughout the non-nucleolar nucleoplasm, with increased signal present at numerous foci. Both Cdk9 and cyclin T1 showed only limited colocalization with different phosphorylated forms of RNA polymerase II. However, significant colocalization with antibodies to several splicing factors that identify nuclear 'speckles' was observed for Cdk9 and especially for cyclin T1. The pattern of Cdk9 and cyclin T1 distribution was altered in cells treated with transcription inhibitors. Transient expression of cyclin T1 deletion mutants indicated that a region in the central portion of cyclin T1 is important for accumulation at speckles. Furthermore, cyclin T1 proteins that accumulated at speckles were capable of recruiting Cdk9 and the HIV Tat protein to this compartment in overexpression experiments. These results suggest that cyclin T1 functions to recruit its binding partners to nuclear speckles and raises the possibility that nuclear speckles are a site of TAK/P-TEFb function.
Assuntos
Núcleo Celular/ultraestrutura , Ciclinas/análise , Ciclinas/metabolismo , Matriz Nuclear/ultraestrutura , Proteínas Serina-Treonina Quinases/química , Núcleo Celular/metabolismo , Ciclina T , Ciclinas/química , Dactinomicina/administração & dosagem , Feminino , Produtos do Gene tat/química , Produtos do Gene tat/metabolismo , Células HeLa , Humanos , Fator B de Elongação Transcricional Positiva , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas , RNA Polimerase II , Spliceossomos/ultraestrutura , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-alpha (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 approximately 5-6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.
Assuntos
Transporte Biológico/genética , Cisteína Endopeptidases/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Complexos Multienzimáticos/metabolismo , Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Transcrição Gênica/fisiologia , Proteínas de Bactérias/análise , Transporte Biológico/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Dactinomicina/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Células HeLa , Histona Acetiltransferases , Humanos , Leupeptinas/farmacologia , Ligantes , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Complexos Multienzimáticos/antagonistas & inibidores , Matriz Nuclear/efeitos dos fármacos , Coativador 1 de Receptor Nuclear , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Tamoxifeno/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
We have analyzed ligand-dependent, subnuclear movements of the estrogen receptor-alpha (ERalpha) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ERalpha chimera (GFP-ERalpha), we find that 17beta-estradiol (E2) changes the normally diffuse nucleoplasmic pattern of GFP-ERalpha to a hyperspeckled distribution within 10-20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen; only a subtle effect was observed with the pure antagonist ICI 182,780. To examine the influence of ligand upon ERalpha association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM). Addition of E2, 4-hydroxytamoxifen, or ICI 182,780 causes ERalpha to partition with the NM-bound fraction on a similar time course (10-20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E2 on the redistribution and solubility of GFP-ERalpha, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ERalpha movement and NM association were coincident. Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol Ilo) and splicing domains (SRm160). Using E2 treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ERalpha pool. Experiments performed with bioluminescent derivatives of ERalpha and steroid receptor coactivator-1 (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E2 but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs. Collectively, our data suggest that ERalpha transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.
Assuntos
Núcleo Celular/ultraestrutura , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Anticorpos Monoclonais , Sítios de Ligação , Transporte Biológico , Núcleo Celular/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Deleção de Genes , Proteínas de Fluorescência Verde , Células HeLa , Histona Acetiltransferases , Humanos , Lactente , Cinética , Proteínas Luminescentes/genética , Mutagênese , Matriz Nuclear/metabolismo , Coativador 1 de Receptor Nuclear , Receptores de Estrogênio/genética , Receptores de Estrogênio/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Células Tumorais CultivadasRESUMO
Microphthalmia with linear skin defects (MLS) is an X-linked dominant, male-lethal syndrome characterized by microphthalmia, aplastic skin and agenesis of the corpus callosum, and is caused by the deletion of a 500 kb critical region in Xp22.3. Our laboratory isolated a novel rho GTPase-activating protein (rhoGAP) gene named ARHGAP6 from the MLS region. ARHGAP6 contains 14 exons encoding a 974 amino acid protein with three putative SH3-binding domains. Because exons 2-14 are deleted in all MLS patients, we hypothesized that ARHGAP6 may be responsible for some of the phenotypic features of MLS. We pursued two approaches to study the function of ARHGAP6 and its role in the pathogenesis of MLS: gene targeting of the rhoGAP domain in mouse embryonic stem cells and in vitro expression studies. Surprisingly, loss of the rhoGAP function of Arhgap6 does not cause any detectable phenotypic or behavioral abnormalities in the mutant mice. Transfected mammalian cells expressing ARHGAP6 lose their actin stress fibers, retract from the growth surface and extend thin, branching processes resembling filopodia. The ARHGAP6 protein co-localizes with actin filaments through an N-terminal domain and recruits F-actin into the growing processes. Mutation of a conserved arginine residue in the rhoGAP domain prevents the loss of stress fibers but has little effect on process outgrowth. These results suggest that ARHGAP6 has two independent functions: one as a GAP with specificity for RhoA and the other as a cytoskeletal protein that promotes actin remodeling.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Actinas/metabolismo , Actinas/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Comportamento Animal , Citoplasma/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Éxons , Feminino , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/isolamento & purificação , Humanos , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/genética , Microftalmia/patologia , Microftalmia/fisiopatologia , Dados de Sequência Molecular , Músculo Esquelético/anormalidades , Músculo Esquelético/patologia , Fragmentos de Peptídeos/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/deficiência , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/isolamento & purificaçãoRESUMO
At a simplistic level, the nucleus can be thought of as singular organelle with a nuclear envelope designed to isolate the biochemical reactions required for gene transcription and DNA replication from the cytoplasm. It has become increasingly clear, however, that many higher levels of organization exist within the nucleus. A functional consequence of this organization is that nuclear processes that include transcription, RNA processing, and DNA synthesis are isolated to specific intranuclear domains to ensure efficiency. With the advent of GFP technologies and increasingly sophisticated instrumentation, we have continued to dissect the relationship between organization and function, in particular using live cells and ligand-dependent steroid receptors as a model system. These new opportunities have provided further insight into receptor function and the dependence upon intranuclear dynamics that take place within minutes of hormone addition. J. Cell. Biochem. Suppl. 35:99-106, 2000.
Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , TransfecçãoRESUMO
Spinal and bulbar muscular atrophy (SBMA) is associated with an abnormal expansion of the (CAG)(n)repeat in the androgen receptor (AR) gene. Similar mutations have been reported in other proteins that cause neurodegenerative disorders. The CAG-coded elongated polyglutamine (polyGln) tracts induce the formation of neuronal intracellular aggregates. We have produced a model to study the effects of potentially 'neurotoxic' aggregates in SBMA using immortalized motoneuronal cells (NSC34) transfected with AR containing polyGln repeats of different sizes [(AR.Q(n = 0, 23 or 46)]. Using chimeras of AR.Q(n) and the green fluorescent protein (GFP), we have shown that aggregate formation occurs when the polyGln tract is elongated and AR is activated by androgens. In NSC34 cells co-expressing the AR with the polyGln of pathological length (AR.Q46) and the GFP we have noted the presence of several dystrophic neurites. Cell viability analyses have shown a reduced growth/survival rate in NSC34 expressing the AR.Q46, whereas testosterone treatment partially counteracted both cell death and the formation of dystrophic neurites. These observations indicate the lack of correlation between aggregate formation and cell survival, and suggest that neuronal degeneration in SBMA might be secondary to axonal/dendritic insults.
Assuntos
Neurônios Motores/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Animais , Sequência de Bases , Morte Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Proteínas de Fluorescência Verde , Células Híbridas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Dados de Sequência Molecular , Neurônios Motores/citologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Degeneração Neural/genética , Neuroblastoma/embriologia , Neuroblastoma/patologia , Peptídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a beta-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Prolactina/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores da Prolactina/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Transporte Biológico/efeitos dos fármacos , Caseínas/genética , Compartimento Celular , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Genes Reporter , Janus Quinase 2 , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT5 , Transativadores/genética , Ativação Transcricional , Quinases da Família src/metabolismoRESUMO
Spinal bulbar muscular atrophy is a neurodegenerative disorder caused by a polyglutamine expansion in the androgen receptor (AR). We show in transiently transfected HeLa cells that an AR containing 48 glutamines (ARQ48) accumulates in a hormone-dependent manner in both cytoplasmic and nuclear aggregates. Electron microscopy reveals both types of aggregates to have a similar ultrastructure. ARQ48 aggregates sequester mitochondria and steroid receptor coactivator 1 and stain positively for NEDD8, Hsp70, Hsp90 and HDJ-2/HSDJ. Co-expression of HDJ-2/HSDJ significantly represses aggregate formation. ARQ48 aggregates also label with antibodies recognizing the PA700 proteasome caps but not 20S core particles. These results suggest that ARQ48 accumulates due to protein misfolding and a breakdown in proteolytic processing. Furthermore, the homeostatic disturbances associated with aggregate formation may affect normal cell function.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Choque Térmico/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeos/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP40 , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Proteínas de Choque Térmico/genética , Histona Acetiltransferases , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mitose , Proteína NEDD8 , Coativador 1 de Receptor Nuclear , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma , Receptores Androgênicos/genética , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Subnuclear compartmentation is postulated to play an important role in many aspects of nuclear metabolism. To directly test an application of this model to transcription factor function, we examined the subnuclear partitioning behavior of Pit-1, a tissue-specific, POU-class transactivator. Biochemical and in situ assays indicate the nuclear pool of Pit-1 is normally divided between two compartments: the majority being differentially soluble in detergent, and a significant insoluble fraction (approximately 20%) bound to the nuclear matrix. Examination of Pit-1 deletion mutants and chimeric fusions reveal the highly conserved 66 amino acid POU-specific domain contains a necessary and sufficient nuclear matrix targeting signal. The nuclear partitioning behavior of several natural or engineered point mutations of Pit-1 was also examined. Surprisingly, the inactive point mutants were completely matrix-bound, irrespective of their ability to bind Pit-1 specific DNA. These results suggest that dynamic partitioning of Pit-1 is a component of its normal transactivator function that takes place upon the insoluble nuclear substructure where transcription occurs.
Assuntos
Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Anticorpos Monoclonais , Western Blotting , Núcleo Celular/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Epitopos/química , Células HeLa , Humanos , Imuno-Histoquímica , Matriz Nuclear/química , Ratos , Fator de Transcrição Pit-1 , Fatores de Transcrição/química , Fatores de Transcrição/genética , TransfecçãoRESUMO
The centromere-kinetochore complex can be divided into distinct domains based on structure and function. Previous work has used CREST auto-antibodies with various microscopic techniques to map the locations of proteins within the centromere-kinetochore complex and to analyze the maturation of prekinetochores before mitosis. Here we have focused on the centromere-specific histone Centromere Protein (CENP)-A and its spatial relationship to other histones and histone modifications found in condensed chromatin. We demonstrate that the phosphorylation of histone H3 is essentially excluded from a specific region of centromeric chromatin, defined by the presence of CENP-A. Interspersion of CENP-B with phosphorylated H3 in the inner centromere indicates that the exclusion of H3 modification is not a general property of alpha-satellite DNA. We also demonstrate that these regions are functionally distinct by fragmenting mitotic chromatin into motile centromere-kinetochore fragments that contain CENP-A with little or no phosphorylated H3 and nonmotile fragments that contain exclusively phosphorylated H3. The sequence of CENP-A diverges from H3 in a number of key residues involved in chromosome condensation and in transcription, potentially allowing a more specialized chromatin structure within centromeric heterochromatin, on which kinetochore plates may nucleate and mature. This specialized centromere subdomain would be predicted to have a very tight and static nucleosome structure as a result of the absence of H3 phosphorylation and acetylation.