RESUMO
Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA. We re-examined the role of TFIID by rapid depletion of S. cerevisiae TFIID subunits and measurement of changes in nascent transcription. We find that transcription of nearly all mRNAs is strongly dependent on TFIID function. Degron-dependent depletion of Taf1, Taf2, Taf7, Taf11, and Taf13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing, and TATA-less gene classes. The magnitude of TFIID dependence varies with growth conditions, although this variation is similar genome-wide. Many studies have suggested differences in gene-regulatory mechanisms between TATA and TATA-less genes, and these differences have been attributed in part to differential dependence on SAGA or TFIID. Our work indicates that TFIID participates in expression of nearly all yeast mRNAs and that differences in regulation between these two gene categories is due to other properties.
Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Transativadores/química , Transcrição Gênica , Deleção de Genes , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição TFIID/deficiência , Fator de Transcrição TFIID/genéticaRESUMO
Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.
Assuntos
Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Transativadores/genética , Fatores de Transcrição/genética , Deleção de Genes , Histona Acetiltransferases/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Negative symptoms reflect a currently much-untreated loss of normal functioning and are frequently found in psychotic disorders. We present the first translation of the Brief Negative Symptom Scale (BNSS) to European Portuguese and evaluate its validity in a sample of Portuguese male patients with a psychotic spectrum disorder. The Portuguese BNSS showed excellent internal consistency, high convergent validity (i.e., strong correlation with the PANSS negative factor), and high discriminant validity (i.e., a lack of association with the PANSS positive factor). In sum, the present European Portuguese BNSS has shown to be reliable, thus extending this instrument's clinical availability worldwide.
Assuntos
Esquizofrenia , Humanos , Masculino , Esquizofrenia/diagnóstico , Escalas de Graduação Psiquiátrica , Portugal , Psicometria , Reprodutibilidade dos TestesRESUMO
The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.
Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Regulação da Expressão Gênica , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Perfilação da Expressão Gênica , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , UbiquitinaçãoRESUMO
Statoliths are nonskeletal calcified structures included in most invertebrates' gravireceptors. They have been identified and characterized in several gastropod and cephalopod molluscs and have proved to be very useful for age estimation, growth studies, and connectivity analysis, among other applications. Beyond the scarce available records on their occurrence in Class Bivalvia, statoliths are yet to be documented in the grooved carpet shell, Ruditapes decussatus, a species of high ecological and commercial value. An easy method for the extraction and processing of R. decussatus statoliths is described herein. The statolith growth was followed from the initial shell length (SL) of 2.5-3.5 mm (seed commercial size T1.5) for a period of 6 months in a nursery facility located in the Ria de Aveiro (an estuarine system in NW Portugal). The relationship between statolith diameter (StD) and SL follows the function StD=14.305 SL0.254 (N=173; r=0.855, p<0.001). All statoliths observed showed similar morphostructure and general chemistry: hard, translucent spheres of crystalline calcium oxalate (whewellite), with a central nucleus delimited by a growth check of 6.7±1.0 µm in diameter, possibly as a result of growth arrest during metamorphosis, a metamorphic ring, as described for their gastropod counterparts. Subsequent studies should validate this and will involve a search for the occurrence of additional checks that may potentially be present in older specimens and if they are, would open a new range of most promising applications for bivalve statoliths.
Assuntos
Bivalves/fisiologia , Membrana dos Otólitos/anatomia & histologia , Animais , Sensação Gravitacional , Membrana dos Otólitos/crescimento & desenvolvimento , PortugalRESUMO
Deregulated expression of histone deacetylases (HDACs) has been implicated in tumorigenesis. Herein, we investigated class I HDACs expression in bladder urothelial cell carcinoma (BUCC), its prognostic value and biological significance. Significantly increased transcript levels of all HDACs were found in BUCC compared to 20 normal mucosas, and these were higher in lower grade and stage tumors. Increased HDAC3 levels were associated with improved patient survival. SiRNA experiments showed decrease cell viability and motility, and increased apoptosis. We concluded that class I HDACs play an important role in bladder carcinogenesis through deregulation of proliferation, migration and apoptosis, constituting putative therapeutic targets.
Assuntos
Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno/genética , Bexiga Urinária/metabolismoRESUMO
Epigenetic alterations are common in prostate cancer (PCa) and seem to contribute decisively to its initiation and progression. Moreover, aberrant promoter methylation is a promising biomarker for non-invasive screening. Herein, we sought to characterize EFEMP1 as biomarker for PCa, unveiling its biological relevance in prostate carcinogenesis. Microarray analyses of treated PCa cell lines and primary tissues enabled the selection of differentially methylated genes, among which EFEMP1 was further validated by MSP and bisulfite sequencing. Assessment of biomarker performance was accomplished by qMSP. Expression analysis of EFEMP1 and characterization of histone marks were performed in tissue samples and cancer cell lines to determine the impact of epigenetic mechanisms on EFEMP1 transcriptional regulation. Phenotypic assays, using transfected cell lines, permitted the evaluation of EFEMP1's role in PCa development. EFEMP1 methylation assay discriminated PCa from normal prostate tissue (NPT; P < 0.001, Kruskall-Wallis test) and renal and bladder cancers (96% sensitivity and 98% specificity). EFEMP1 transcription levels inversely correlated with promoter methylation and histone deacetylation, suggesting that both epigenetic mechanisms are involved in gene regulation. Phenotypic assays showed that EFEMP1 de novo expression reduces malignant phenotype of PCa cells. EFEMP1 promoter methylation is prevalent in PCa and accurately discriminates PCa from non-cancerous prostate tissues and other urological neoplasms. This epigenetic alteration occurs early in prostate carcinogenesis and, in association with histone deacetylation, progressively leads to gene down-regulation, fostering cell proliferation, invasion and evasion of apoptosis.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA/genética , Epigênese Genética/genética , Proteínas da Matriz Extracelular/genética , Neoplasias da Próstata/genética , Apoptose/genética , Biomarcadores Tumorais/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/genética , Ilhas de CpG , Proteínas da Matriz Extracelular/biossíntese , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Masculino , Invasividade Neoplásica/genética , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Abnormal hemoglobin (Hb) levels lead to poorer outcomes in ischemic stroke, though the mechanisms remain elusive. We aimed to study the role of Hb on imaging and clinical outcomes, namely on collaterals as it is a known mediator of infarct growth. METHODS: Retrospective cohort study of patients with large vessel occlusion ischemic stroke admitted to our center. Demographics, clinical and imaging variables were collected, particularly baseline hemoglobin, presence of anemia and collateral score. Collaterals were scored from 0 to 3 and defined as poor if 0-1. Multivariable analyses were performed for collateral score and clinical outcomes (3-month mortality and good prognosis). RESULTS: We included 811 patients, 215 (26.5 %) with anemia. Patients with anemia were older, had more comorbidities and more severe strokes. Hemoglobin levels and anemia were not associated with collateral score (OR 0.97, 95 % CI 0.89-1.05, p = 0.414 and OR 0.89, 95 % CI 0.64-1.24, p = 0.487, respectively) nor with poor collaterals (OR 0.96, 95 % CI 0.88-1.05, p = 0.398 and OR 0.86, 95 % CI 0.60-1.23, p = 0.406, respectively). Hb levels were associated with 3-month mortality (OR 0.85, 95 % CI 0.76-0.96, p = 0.008). CONCLUSION: Hemoglobin or anemia were not found to be associated with collateral status. Our results raise further questions regarding the pathophysiology of anemia and outcomes in ischemic stroke, highlighting the need for future research.
Assuntos
Anemia , Arteriopatias Oclusivas , Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Isquemia Encefálica/complicações , Isquemia Encefálica/diagnóstico por imagem , Estudos Retrospectivos , Acidente Vascular Cerebral/diagnóstico por imagem , Hemoglobinas , Anemia/complicações , Circulação Colateral/fisiologia , Angiografia Cerebral/métodos , Resultado do TratamentoRESUMO
BACKGROUND: Multidrug resistance 1 (MDR1) gene encodes for an ATP binding cassette transporter--P-glycoprotein (P-gp)-- involved in chemoresistance to taxanes. MDR1 promoter methylation is frequent in prostate carcinoma (PCa), suggesting an epigenetic regulation but no functional correlation has been established. We aimed to elucidate the epigenetic mechanisms involved in MDR1 deregulation in PCa. RESULTS: MDR1 promoter methylation and P-gp expression were assessed in 121 PCa, 39 high-grade prostatic intraepithelial neoplasia (HGPIN), 28 benign prostatic hyperplasia (BPH) and 10 morphologically normal prostate tissue (NPT) samples, using quantitative methylation specific PCR and immunohistochemistry, respectively. PCa cell lines were exposed to a DNA methyltransferases inhibitor 5-aza-2'deoxycytidine (DAC) and histone deacetylases inhibitor trichostatin A (TSA). Methylation and histone posttranscriptional modifications status were characterized and correlated with mRNA and protein expression. MDR1 promoter methylation levels and frequency significantly increased from NPTs, to HGPIN and to PCa. Conversely, decreased or absent P-gp immunoexpression was observed in HGPIN and PCa, inversely correlating with methylation levels. Exposure to DAC alone did not alter significantly methylation levels, although increased expression was apparent. However, P-gp mRNA and protein re-expression were higher in cell lines exposed to TSA alone or combined with DAC. Accordingly, histone active marks H3Ac, H3K4me2, H3K4me3, H3K9Ac, and H4Ac were increased at the MDR1 promoter after exposure to TSA alone or combined with DAC. CONCLUSION: Our data suggests that, in prostate carcinogenesis, MDR1 downregulation is mainly due to histone post-translational modifications. This occurs concomitantly with aberrant promoter methylation, substantiating the association with P-gp decreased expression.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/farmacologia , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Próstata/metabolismo , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Coccydynia has many causes, including fracture, subluxation, and hypermobility of sacrococcygeal segments. Existing treatments are limited in their effectiveness. Coccygeoplasty (CP) is a relatively new, minimally invasive treatment that appears to address this difficult clinical challenge. OBJECTIVE: To describe clinical results at the time of the procedure and at 3- and 12-months' follow-up of patients with coccydynia related to subluxation and coccyx hypermobility treated with the CP technique. Additionally, to determine if there is any correlation between the final imaging and clinical results at 3- and 12-months' follow-up. METHODS: A prospectively maintained database was used, and all patients who underwent CP for chronic coccydynia between January 2005 and October 2018 were retrospectively reviewed. All the patients had painful hypermobility (greater than 25°) with anterior flexion confirmed on radiological imaging. Alternative causes of coccydynia were excluded using CT and MRI. Procedures were performed under local anesthesia with combined fluoroscopic and CT guidance. Clinical follow-up was performed at two time points: 3 and 12 months after treatment using the Visual Analogue Scale (VAS). RESULTS: Twelve patients were treated in a single center. No procedural complications occurred. At 3- and 12-months' follow-up, the majority (75%) of patients had significantly lower VAS scores than at baseline, with mean changes of 3.5 and 4.9, respectively. There was no pain recurrence at 12 months and just one patient had no improvement of the pain. Follow-up CT images confirmed fixation of the sacrococcygeal bone segments in nine patients; however, no correlation was found between final imaging results and clinical outcome (p=0.1). CONCLUSIONS: Patients with refractory painful coccyx subluxation and hypermobility undergoing CP have a favorable clinical response at 3- and 12-months' follow-up. Further studies are required to validate this technique and to identify predictors of treatment response. Coccygeoplasty may be considered a reasonable alternative to coccygectomy.
Assuntos
Cóccix , Região Sacrococcígea , Humanos , Cóccix/diagnóstico por imagem , Cóccix/cirurgia , Estudos Retrospectivos , Resultado do Tratamento , Região Sacrococcígea/cirurgia , Medição da Dor/métodos , DorRESUMO
INTRODUCTION: Since the publication of endovascular treatment trials and European Stroke Guidelines, Portugal has re-organized stroke healthcare. The nine centers performing endovascular treatment are not equally distributed within the country, which may lead to differential access to endovascular treatment. Our main aim was to perform a descriptive analysis of the main treatment metrics regarding endovascular treatment in mainland Portugal and its administrative districts. MATERIAL AND METHODS: A retrospective national multicentric cohort study was conducted, including all ischemic stroke patients treated with endovascular treatment in mainland Portugal over two years (July 2015 to June 2017). All endovascular treatment centers contributed to an anonymized database. Demographic, stroke-related and procedure-related variables were collected. Crude endovascular treatment rates were calculated per 100 000 inhabitants for mainland Portugal, and each district and endovascular treatment standardized ratios (indirect age-sex standardization) were also calculated. Patient time metrics were computed as the median time between stroke onset, first-door, and puncture. RESULTS: A total of 1625 endovascular treatment procedures were registered. The endovascular treatment rate was 8.27/100 000 inhabitants/year. We found regional heterogeneity in endovascular treatment rates (1.58 to 16.53/100 000/year), with higher rates in districts closer to endovascular treatment centers. When analyzed by district, the median time from stroke onset to puncture ranged from 212 to 432 minutes, reflecting regional heterogeneity. DISCUSSION: Overall endovascular treatment rates and procedural times in Portugal are comparable to other international registries. We found geographic heterogeneity, with lower endovascular treatment rates and longer onset-to-puncture time in southern and inner regions. CONCLUSION: The overall national rate of EVT in the first two years after the organization of EVT-capable centers is one of the highest among European countries, however, significant regional disparities were documented. Moreover, stroke-onset-to-first-door times and in-hospital procedural times in the EVT centers were comparable to those reported in the randomized controlled trials performed in high-volume tertiary hospitals.
Introdução: A aprovação do tratamento endovascular para o acidente vascular cerebral isquémico obrigou à reorganização dos cuidados de saúde em Portugal. Os nove centros que realizam tratamento endovascular não estão distribuídos equitativamente pelo território, o que poderá causar acesso diferencial a tratamento. O principal objetivo deste estudo é realizar uma análise descritiva da frequência e métricas temporais do tratamento endovascular em Portugal continental e seus distritos. Material e Métodos: Estudo de coorte nacional multicêntrico, incluindo todos os doentes com acidente vascular cerebral isquémico submetidos a tratamento endovascular em Portugal continental durante um período de dois anos (julho 2015 a junho 2017). Foram colhidos dados demográficos, relacionados com o acidente vascular cerebral e variáveis do procedimento. Taxas de tratamento endovascular brutas e ajustadas (ajuste indireto a idade e sexo) foram calculadas por 100 000 habitantes/ano para Portugal continental e cada distrito. Métricas de procedimento como tempo entre instalação, primeira porta e punção foram também analisadas. Resultados: Foram registados 1625 tratamentos endovasculares, indicando uma taxa bruta nacional de tratamento endovascular de 8,27/100 000 habitantes/ano. As taxas de tratamento endovascular entre distritos variaram entre 1,58 e 16,53/100 000/ano, com taxas mais elevadas nos distritos próximos a hospitais com tratamento endovascular. O tempo entre sintomas e punção femural entre distritos variou entre 212 e 432 minutos. Discussão: A análise nacional a taxas de tratamento endovascular e tempos de atuação é comparável a outros registos internacionais. Verificaram-se heterogeneidades geográficas, com taxas de tratamento endovascular menores e maior tempo para tratamento nos distritos do sul e interior. Conclusão: Portugal continental apresenta uma taxa nacional de tratamento endovascular elevada, apresentando, contudo, assimetrias regionais no acesso. As métricas temporais foram comparáveis com as observadas nos ensaios clínicos piloto.
Assuntos
Isquemia Encefálica , Procedimentos Endovasculares , AVC Isquêmico , Acidente Vascular Cerebral , Isquemia Encefálica/terapia , Estudos de Coortes , Humanos , Portugal , Estudos Retrospectivos , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/terapia , Resultado do TratamentoRESUMO
Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.
Assuntos
RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Tiouracila/análogos & derivados , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Tiouracila/químicaRESUMO
General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.
Assuntos
Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID/metabolismo , Cristalografia por Raios X , DNA/metabolismo , Histona Acetiltransferases/metabolismo , Humanos , Espectrometria de Massas , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Fator de Transcrição TFIID/químicaRESUMO
Regulation of gene expression includes the replacement of canonical histones for non-allelic histone variants, as well as their multiple targeting by postranslational modifications. H2A variants are highly conserved between species suggesting they execute important functions that cannot be accomplished by canonical histones. Altered expression of many H2A variants is associated to cancer. MacroH2A variants are enriched in heterocromatic foci and necessary for chromatin condensation. MacroH2A1.1 and macroH2A1.2 are two mutually exclusive isoforms. MacroH2A1.1 and macroH2A2 inhibit proliferation and are associated with better cancer prognosis; while macroH2A1.2 is associated to cancer progression. H2AX variant functions as a sensor of DNA damage and defines the cellular response towards DNA repair or apoptosis; therefore, screening approaches and therapeutic options targeting H2AX have been proposed. H2A.Z is enriched in euchromatin, acting as a proto-oncogene with established roles in hormone responsive cancers and overexpressed in endocrine-resistant disease. Other H2A family members have also been found altered in cancer, but their function remains unknown. Substantial progress has been made to understand histone H2A variants, their contribution to normal cellular function and to cancer development and progression. Yet, implementation of high resolution mass spectrometry is needed to further our knowledge on highly homologous H2A variants expression and function.
Assuntos
Histonas/metabolismo , Neoplasias/metabolismo , Animais , Expressão Gênica , Histonas/biossíntese , Histonas/genética , Humanos , Neoplasias/genética , Proto-Oncogene MasRESUMO
BACKGROUND: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. METHODS: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-ß2, by quantitative methylation-specific PCR and RT-PCR, respectively. RESULTS: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-ß2 promoter hypermethylation levels, although mRNA reexpression was only attained for GSTP1 and APC. CONCLUSIONS: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA , Ftalimidas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Triptofano/análogos & derivados , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Masculino , Ftalimidas/administração & dosagem , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Triptofano/administração & dosagem , Triptofano/farmacologiaRESUMO
Prostate cancer (PCa) is one of the most incident malignancies worldwide. Although efficient therapy is available for early-stage PCa, treatment of advanced disease is mainly ineffective and remains a clinical challenge. microRNA (miRNA) dysregulation is associated with PCa development and progression. In fact, several studies have reported a widespread downregulation of miRNAs in PCa, which highlights the importance of studying compounds capable of restoring the global miRNA expression. The main aim of this study was to define the usefulness of enoxacin as an anti-tumoral agent in PCa, due to its ability to induce miRNA biogenesis in a TRBP-mediated manner. Using a panel of five PCa cell lines, we observed that all of them were wild type for the TARBP2 gene and expressed TRBP protein. Furthermore, primary prostate carcinomas displayed normal levels of TRBP protein. Remarkably, enoxacin was able to decrease cell viability, induce apoptosis, cause cell cycle arrest, and inhibit the invasiveness of cell lines. Enoxacin was also effective in restoring the global expression of miRNAs. This study is the first to show that PCa cells are highly responsive to the anti-tumoral effects of enoxacin. Therefore, enoxacin constitutes a promising therapeutic agent for PCa.
Assuntos
Enoxacino/farmacologia , MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Processamento Pós-Transcricional do RNA/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Enoxacino/uso terapêutico , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , MicroRNAs/genética , Mutação/genética , Invasividade Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismoRESUMO
Histone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes' upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2'-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patients.
Assuntos
Histonas/genética , Sirtuína 1/genética , Idoso , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Histonas/biossíntese , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Sirtuína 1/metabolismoRESUMO
Endoluminal reconstruction with flow diverter devices represents an innovative technique in the treatment of intracranial aneurysms.These new stents, self-expandable and of low porosity, are released through the microcatherization of the parent artery. The main goal of these systems is thereby rebuilding the vessel wall and excluding the aneurysmal formation of the arterial circulation.We show the preliminary results in treating 10 patients at Hospital de São José, Lisbon. These patients, with wide-necked (> 4 mm)or unfavorable dome/neck ratios (> 1.5) aneurysms, were treated with the PIPELINE® system, and angiographic control were made at three and six months. New control will be done at 12 months. The mean age of enrolled patients is 54.3 years; eight patients were female and two male. Aneurysms were incidentally discovered in two patients. The remaining patients were diagnosed during imaging investigation for headache (n = 3), visual field defect (n = 1), vertigo(n = 1) and at least one cranial palsy (n = 2). Only two patients had had prior subarachnoid hemorrhage and two patients underwent prior endovascular treatment with coils. The locations of aneurysms treated were the proximal segment of the middle cerebral artery(n = 1) and the paraophthalmic (n = 6), ophthalmic (n = 2) and cavernous (n = 4) internal carotid artery segments. Thirteen intracranial aneurysms were treated as three patients had multiple aneurysms. Control studies were conducted and shown an average degree of occlusion at three months of 74% and at six months of 86%. There was no reduction in size of one paraophthalmic artery aneurysm.The experience of this department is favorable to the use of flow-diverter devices to treat selected aneurysms. High occlusion rates were obtained given the existing challenges in the treatment of such aneurysms.