RESUMEN
DEAD-box helicase 5 (DDX5) has been shown to promote tumorigenesis and cancer progression. However, the relationship between DDX5 and recurrence in breast cancer (BC) patients remains unknown. The objective of the present study was to evaluate the correlation of DDX5 with recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) in patients with BC. The expression of DDX5 was examined by immunohistochemical analysis. RFS was calculated by Kaplan-Meier survival analysis. Univariate and multivariable associations were assessed by Cox proportional hazards models. In the present study, a total of 868 BC patients were analysed, and we found that DDX5 protein was significantly overexpressed in BC tissues compared to adjacent normal tissues. Elevated DDX5 was associated with an aggressive phenotype in BC patients. Moreover, DDX5 protein was upregulated in recurrent patients compared with nonrecurrent patients, and DDX5 protein levels were positively associated with worse RFS and BCSS in BC patients. High DDX5 expressing BC patients with age more than 50 year, advanced clinical stage or histological grade had a significantly increased risk of recurrence and shorter survival. Our findings highlight the significance of DDX5 in the recurrence and clinical outcome of BC patients and suggest that DDX5 may be a potential predictive biomarker for patients with BC.
Asunto(s)
Neoplasias de la Mama/química , Neoplasias de la Mama/metabolismo , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/biosíntesis , Recurrencia Local de Neoplasia/química , Neoplasias de la Mama/mortalidad , Correlación de Datos , Femenino , Humanos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Pronóstico , Tasa de Supervivencia , Regulación hacia ArribaRESUMEN
Fetal adenocarcinoma of the lung (FLAC) is a rare lung tumor classified into low-grade fetal adenocarcinoma of the lung (LG-FLAC) and high-grade fetal adenocarcinoma of the lung (HG-FLAC). It remains debatable whether HG-FLAC is a subset of FLAC or a distinct subtype of the conventional lung adenocarcinoma (CLA). In this study, samples of 4 LG-FLAC and 2 HG-FLAC cases were examined, and the clinicopathologic, immunohistochemical (IHC), and mutational differences between the 2 subtypes were analyzed using literature review. Morphologically, LG-FLACs had a pure pattern with complex glandular architecture composed of cells with subnuclear and supranuclear vacuoles, mimicking a developing fetal lung. In contrast, HG-FLACs contained both fetal lung-like (FLL) and CLA components. With regard to IHC markers, ß-catenin exhibited a nuclear/cytoplasmic staining pattern in LG-FLACs but a membranous staining pattern in HG-FLACs. Furthermore, p53 was expressed diffusely and strongly in HG-FLACs, whereas in LG-FLACs, p53 staining was completely absent. Using next-generation sequencing targeting a 1021-gene panel, mutations of CTNNB1 and DICER1 were detected in all 4 LG-FLAC samples, and a novel mutation, MYCN P44L, was discovered in 2 LG-FLAC samples. DNA samples of the FLL and CLA components of HG-FLACs were separately extracted and sequenced. The FLL component harbored no CTNNB1, DICER1, or MYCN mutations; moreover, the FLL genetic profile largely overlapped with that of the CLA component. The morphologic, IHC, and genetic features of HG-FLAC indicate that it is a variant of CLA rather than a subset of FLAC. Thus, HG-FLAC should be treated differently from LG-FLAC.
Asunto(s)
Adenocarcinoma del Pulmón/diagnóstico , Biomarcadores de Tumor , Análisis Mutacional de ADN , Inmunohistoquímica , Neoplasias Pulmonares/diagnóstico , Mutación , Adenocarcinoma del Pulmón/química , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Niño , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteína Proto-Oncogénica N-Myc/análisis , Proteína Proto-Oncogénica N-Myc/genética , Clasificación del Tumor , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Ribonucleasa III/análisis , Ribonucleasa III/genética , Adulto Joven , beta Catenina/análisis , beta Catenina/genéticaRESUMEN
Male breast cancer (MBC) is a rare disease. Due to its rarity, treatment is still directed by data mainly extrapolated from female breast cancer (FBC) treatment, despite the fact that it has recently become clear that MBC has its own molecular characteristics. DDX3 is a RNA helicase with tumor suppressor and oncogenic potential that was described as a prognosticator in FBC and can be targeted by small molecule inhibitors of DDX3. The aim of this study was to evaluate if DDX3 is a useful prognosticator for MBC patients. Nuclear as well as cytoplasmic DDX3 expression was studied by immunohistochemistry in a Dutch retrospective cohort of 106 MBC patients. Differences in 10-year survival by DDX3 expression were analyzed using log-rank test. The association between clinicopathologic variables, DDX3 expression, and survival was tested in uni- and multivariate Cox-regression analysis. High cytoplasmic DDX3 was associated with high androgen receptor (AR) expression while low nuclear DDX3 was associated with negative lymph node status. Nuclear and cytoplasmic DDX3 were not associated with each other. In a univariate analysis, high cytoplasmic DDX3 (p = 0.045) was significantly associated with better 10-year overall survival. In multivariate analyses, cytoplasmic DDX3 had independent prognostic value (p = 0.017). In conclusion, cytoplasmic DDX3 expression seems to be a useful prognosticator in MBC, as high cytoplasmic DDX3 indicated better 10-year survival.
Asunto(s)
Neoplasias de la Mama Masculina/genética , Neoplasias de la Mama Masculina/metabolismo , ARN Helicasas DEAD-box/metabolismo , Adulto , Anciano , Neoplasias de la Mama/genética , Neoplasias de la Mama Masculina/fisiopatología , Núcleo Celular/metabolismo , Estudios de Cohortes , Citoplasma/metabolismo , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/genética , Supervivencia sin Enfermedad , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Países Bajos , Oncogenes/genética , Pronóstico , Supervivencia sin Progresión , Receptores Androgénicos/metabolismo , Estudios Retrospectivos , Transcriptoma/genéticaRESUMEN
AIMS: Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies. Hence, there is a need for new markers and treatment strategies. P68/DEAD box protein 5 (DDX5) is an ATP-dependent RNA helicase of the DEAD box protein family. It is a prognostic marker for several cancers. In this study, we aimed to evaluate the expression and clinical relevance of DDX5 in PDAC. METHODS: DDX5 expression in tissue microarray blocks containing 230 PDAC samples was examined using immunohistochemical analysis. DDX5 expression was considered high when more than 50% of the cells were stained and low when less than 50% of the cells were stained. We investigated the association between DDX5 expression and clinicopathological parameters, including patient survival. RESULTS: The nuclei of normal pancreatic ducts, normal acinar cells and PDAC cells were stained positive for DDX5 although the intensity and distribution of DDX5 expression varied. Islet cells showed strong and diffuse staining of DDX5. DDX5 expression was low and high in 148 (64.3%) and 82 cases (35.7%), respectively. Low DDX5 expression was significantly associated with an advanced pT factor (pT2-pT3: tumour size,>20 mm), lymphatic involvement, advanced tumour-node-metastasis (TNM) stage (stages IIB, III, and IV), and venous involvement. In addition, the multivariate analysis revealed that DDX5 expression is an independent prognostic factor for PDAC. CONCLUSION: These results suggest that DDX5 plays an important role in tumour invasiveness and PDAC prognosis.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/fisiopatología , ARN Helicasas DEAD-box/análisis , Biomarcadores de Tumor/metabolismo , ARN Helicasas DEAD-box/metabolismo , Humanos , Estadificación de Neoplasias , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/fisiopatología , Pronóstico , Tasa de SupervivenciaRESUMEN
DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X) is demonstrated to have crucial functions in the antiviral immune response. To our knowledge, little information focuses on the function of duck DDX3X. In this study, duck DDX3X (duDDX3X) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated using duck embryo fibroblast (DEF) cells. Full-length duDDX3X cDNA encodes 652 amino acid residues and contains a DEADc domain and a HELICc domain. According to tissue distribution analysis, duDDX3X mRNA was widely expressed in different tissues, especially the spleen and the liver. Overexpression of duDDX3X in DEF cells induced IFN-ß by activating transcription factors IRF1 and NF-κB. Knockdown of duDDX3X in DEF cells with siRNA significantly reduced IFN-ß expression induced by poly(I:C), a double-stranded RNA (dsRNA) analog. Our results demonstrated that duck DDX3X was involved in the dsRNA-mediated type I IFN signaling pathway in DEF cells.
Asunto(s)
Proteínas Aviares/genética , ARN Helicasas DEAD-box/genética , Patos/genética , Secuencia de Aminoácidos , Animales , Proteínas Aviares/análisis , Proteínas Aviares/inmunología , Clonación Molecular , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/inmunología , ADN Complementario/genética , Patos/inmunología , Inmunidad Innata , Interferón beta/inmunología , Modelos Moleculares , FN-kappa B/inmunología , Filogenia , Transducción de SeñalRESUMEN
In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen to chimeric embryos containing germ cells derived from recombinant ES cells. DDX6-null germ cells exhibited both similar and distinct defects from those observed in NANOS2-null germ cells. These results demonstrate that NANOS2 function is carried out via both P-body-dependent and -independent mechanisms. RNA-seq analyses further supported the phenotypic differences between DDX6-null and NANOS2-null germ cells, and indicated distinct molecular cascades involved in NANOS2-mediated gene regulation.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Células Germinativas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular , Quimera/genética , Quimera/metabolismo , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Masculino , Ratones , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/genética , Espermatogénesis , TranscriptomaRESUMEN
BACKGROUND: Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure. RESULTS: Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (Kd) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured kcat (7.2 ± 0.5 min- 1) and KM (1.2 ± 0.3 µM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. CONCLUSIONS: The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher kcat and KM values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.
Asunto(s)
ARN Helicasas DEAD-box/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr/genética , Células HEK293/metabolismo , Ribonucleasa III/biosíntesis , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/genética , Ensayo de Cambio de Movilidad Electroforética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Regulación de la Expresión Génica , Humanos , Ribonucleasa III/análisis , Ribonucleasa III/genética , TransfecciónRESUMEN
The DEAD-box-protein DDX5 is an ATP-dependent RNA helicase and also acts as co-activator that contributes to progression and metastasis of various tumours. However, its expression as well as prognostic roles of DDX5 in hepatocellular carcinoma (HCC) remain elusive. In this study, we investigated clinical significance and biological functions of DDX5 in HCC. Our results suggested that DDX5 showed overexpression at both transcriptional and translational levels in HCC tissues compared with adjacent normal tissues. Moreover, DDX5 expression was demonstrated to be correlated with tumor size (pâ¯<â¯0.001), N stage (pâ¯=â¯0.013), M stage (pâ¯=â¯0.006), tumor differentiation (pâ¯<â¯0.001) and American Joint Committee on Cancer (AJCC) stage (pâ¯=â¯0.001). Simultaneously, high DDX5 expression was found to be significantly correlated to worse outcome including Disease free survival (DFS) (pâ¯=â¯0.016) and overall survival (OS) (pâ¯=â¯0.032) according to Kaplan-Meier survival analysis. In vitro studies, it suggested that knockdown of DDX5 suppressed HCC cells migration, invasion and epithelial -to- mesenchymal transition (EMT) process. Depletion of DDX5 could promote HCC cells growth. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that PI3K/Akt signaling pathway obtained the highest enrichment. Furthermore, we found that knockdown of DDX5 decreased Akt as well as p-Akt (S473) expressions. Collectively, these findings suggested that DDX5 facilitated HCC cells growth via Akt signaling pathway. DDX5 played a crucial role in HCC proliferation and tumorigenesis and may be a novel prognostic marker and potential therapeutic target for HCC.
Asunto(s)
Carcinoma Hepatocelular/etiología , ARN Helicasas DEAD-box/metabolismo , Neoplasias Hepáticas/etiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Carcinogénesis , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Proliferación Celular , Células Cultivadas , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/genética , Humanos , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Análisis de SupervivenciaRESUMEN
The chromatoid body is a cytoplasmic male germ cell structure that plays a role in the regulation of mRNA transcription during spermatogenesis. A proteomic analysis of this structure has identified the presence of its classic molecular markers (MVH and MIWI), as well as a significant number of transient proteins. Circadian locomotor output cycles protein kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), which are molecular components of the circadian clock, are likely located in the chromatoid body in a transient fashion. This study sought to determine whether aging produces morphological changes in the chromatoid bodies of round spermatids similar to those previously observed in BMAL1 knockout mice. A sample of 30 male mice was divided into three groups: juvenile mice (45 days old), adult mice (120 days old), and old mice (+180 days old). Aging was confirmed by viability and sperm count analyses and testosterone dosage. Squash slides prepared with fragments of seminiferous tubules were immunostained for MVH, MIWI, BMAL1, and CLOCK detection. In juvenile and adult specimens, single round chromatoid bodies were observed using MVH/BMAL1 and MIWI/CLOCK immunostaining. In old specimens, many chromatoid bodies displayed changes in number and morphology, as well as an increase in the interactions between MVH and BMAL1; MIWI and CLOCK. Changes in chromatoid body morphology increased interactions between the proteins analyzed herein, and decreased amounts of these proteins in seminiferous tubules of older mice may indicate that aging influences the assembly and physiology of chromatoid bodies, which may, in turn, affect fertility. Impact statement The results discussed in this paper indicate that aging compromises the structure and physiology of chromatoid bodies (CBs) in post-meiotic male cells. Since CB is a fundamental structure for the differentiation of the mature male germ cell it is possible that this imbalance in CB physiology may play a role in the reduction of fertility in older men. It is important to note that not only the classic CB markers (such as the MIWI and MVH proteins) were used to showcase the structural changes in the CBs but also the main components of circadian cycle control (the CLOCK and BMAL1 proteins), indicating that the reduction of circadian control in aged males may contribute to these changes in CBs as well. Therefore, it is intriguing to evaluate the hypothesis that controlling these physiological/structural changes in CBs may be a way of delaying the effects of aging in males.
Asunto(s)
Envejecimiento/patología , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Espermátides/patología , Factores de Transcripción ARNTL/análisis , Factores de Edad , Animales , Proteínas Argonautas/análisis , Proteínas CLOCK/análisis , ARN Helicasas DEAD-box/análisis , Masculino , Ratones , Microscopía Fluorescente , Nucleoproteínas/metabolismoRESUMEN
The existence of a population of putative stem cells with germline developmental potential (oogonial stem cells: OSCs) in the adult mammalian ovary has been marked by controversy over isolation methodology and potential for in-vitro transformation, particularly where cell sorting has been based on expression of DEAD box polypeptide 4 (DDX4). This study describes a refined tissue dissociation/fluorescence-activated cell sorting (FACS) protocol for the ovaries of adult women which results in increased cell viability and yield of putative OSCs. A FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) demonstrates the existence of two sub-populations of small DDX4-positive cells (approx. 7 µm diameter) with ALDH1 activity, distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. These may indicate stages of differentiation from a progenitor population and provide a likely explanation for the expression disparities reported previously. These findings provide a robust basis for the further characterisation of these cells, and exploration of their potential physiological roles and therapeutic application.
Asunto(s)
ARN Helicasas DEAD-box/análisis , Isoenzimas/análisis , Células Madre Oogoniales/citología , Ovario/citología , Retinal-Deshidrogenasa/análisis , Familia de Aldehído Deshidrogenasa 1 , Separación Celular/métodos , Células Cultivadas , ARN Helicasas DEAD-box/genética , Femenino , Citometría de Flujo/métodos , Expresión Génica , Humanos , Células Madre Oogoniales/metabolismo , Ovario/metabolismo , Adulto JovenRESUMEN
Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3-deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT-PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3-knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild-type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.
Asunto(s)
Células Germinativas/metabolismo , Ovario/metabolismo , Proteínas de Unión al ARN/genética , Ranidae/embriología , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas , Clonación Molecular , ARN Helicasas DEAD-box/análisis , Femenino , Imagenología Tridimensional , Masculino , Ratones , Ovario/citología , ARN Mensajero/genética , Testículo/citologíaRESUMEN
This paper aims to determine the expression and clinical significance of DDX43 in lung adenocarcinoma. Expression of DDX43 gene and protein of lung adenocarcinoma tissue and para-carcinoma tissues was observed in 27 cases by RT-PCR and immunohistochemistry. These patients were diagnosed as lung adenocarcinoma in the Huaihe Hospital of Henan University from February 2015 to December 2015. The relative ratio of DDX43 mRNA expression in lung adenocarcinoma and para-carcinoma tissues was 0.87±0.62 versus 0.21±0.77 and the difference between the two groups was statistically significant (P<0.01). The expression of DDX43 in normal lung tissues and lung adenocarcinoma tissues was different. The positive rate of DDX43 expression in lung adenocarcinoma tissues was significantly higher than that in normal lung tissues, and the difference was statistically significant (P<0.05). The analysis of clinical pathological characteristics showed that the increase of protein expression was related to the stage and metastasis of lung adenocarcinoma. DDX43 is highly expressed in lung adenocarcinoma, and the expression level is related to the stage and metastasis of lung adenocarcinoma, suggesting that DDX43 is closely related to the occurrence and development of lung adenocarcinoma, and may be a molecular marker for early diagnosis of lung adenocarcinoma.
Asunto(s)
Adenocarcinoma del Pulmón/enzimología , Biomarcadores de Tumor/análisis , ARN Helicasas DEAD-box/análisis , Neoplasias Pulmonares/enzimología , Proteínas de Neoplasias/análisis , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/secundario , Anciano , Biomarcadores de Tumor/genética , ARN Helicasas DEAD-box/genética , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.
Asunto(s)
Cromatografía de Afinidad/métodos , ARN Helicasas DEAD-box/aislamiento & purificación , ADN/química , G-Cuádruplex , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/genética , Replicación del ADN , Células HeLa , Humanos , Cinética , ARN/química , Telómero/metabolismoRESUMEN
Alternative cleavage and polyadenylation (APA) is involved in several important biological processes in animals, e.g. cell growth and development, and cancer progression. The increasing data show that cancer cells are inclined to produce mRNA isoforms with a shortened 3'UTR undergoing APA. For example, the Dicer1 isoform with a shorter 3'untranslated region (3'UTR) was found to be overexpressed in some cancer cells, which may be used as a potential novel prognostic biomarker for cancer. In the present work, a novel electrochemical biosensor for ultrasensitive determination of Dicer1 was designed by using gold nanoparticles and p-sulfonated calix[6]arene functionalized reduced graphene oxide (Au@SCX6-rGO) as nanocarriers. The results showed that the expressions of the shorter 3'UTR (Dicer1-S) both in BT474 and SKBR3 were obviously higher than those of the longer Dicer1 (Dicer1-L) by the constructed biosensor, which agreed well with the result analyzed by the RT-qPCR method. The detection ranges of Dicer1-S and Dicer1-L were 10-14-10-9 M and 10-15-10-10 M. The LODs were 3.5 and 0.53 fM. The specificity of the proposed biosensor was also very high. For the first time, the expressional analysis of different 3'UTRs caused by APA was studied by an electrochemical method. Moreover, the use of a macrocyclic host for constructing an electrochemical/biosensing platform has rarely been reported. The proposed electrochemical sensing strategy is thus expected to provide a new method for determination of novel biomarkers and a novel method for fast and cheap analysis of APA.
Asunto(s)
Regiones no Traducidas 3' , Técnicas Biosensibles , ARN Helicasas DEAD-box/análisis , Oro , Nanopartículas del Metal , Ribonucleasa III/análisis , Animales , Línea Celular Tumoral , Técnicas Electroquímicas , Humanos , PoliadenilaciónRESUMEN
Spermatogenesis in testes requires precise spermatogonia differentiation. Spermatocytes lacking the Rad9a gene are arrested in pachytene prophase, implying a possible role for RAD9A in spermatogonia differentiation. However, numerous RAD9A-positive pachytene spermatocytes are still observed in mouse testes following Rad9a excision using the Stra8-Cre system, and it is unclear whether Rad9a deletion in spermatogonia interrupts differentiation. Here, we generated a mouse model in which Rad9a was specifically deleted in spermatogonial stem cells (SSCs) using Cre recombinase expression driven by the germ cell-specific Vasa promoter. Adult Rad9a-null male mice were infertile as a result of completely blocked spermatogonia differentiation. No early spermatocytes were detected in mutant testicular cords of 9-day-old mice. Mutant spermatogonia were prone to apoptosis, although proliferation rates were unaffected. Rad9a deletion also resulted in malformation of seminiferous tubules, in which cells assembled irregularly into clusters, and malformation led to testicular cord disruption. Our findings suggest that Rad9a is indispensable for spermatogonia differentiation and testicular development in mice.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Espermatogonias/citología , Animales , Apoptosis , Diferenciación Celular , ARN Helicasas DEAD-box/análisis , Proteínas de Unión al ADN/análisis , Infertilidad Masculina/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Factores de Transcripción/análisisRESUMEN
Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G1 phase cell-cycle arrest. Conversely, the low DDX3-expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer. Cancer Res; 76(21); 6340-50. ©2016 AACR.
Asunto(s)
Azepinas/farmacología , ARN Helicasas DEAD-box/antagonistas & inhibidores , Imidazoles/farmacología , Neoplasias de la Próstata/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ARN Helicasas DEAD-box/análisis , ARN Helicasas DEAD-box/fisiología , Daño del ADN , Histonas/análisis , Humanos , Masculino , Ratones , Neoplasias de la Próstata/química , Neoplasias de la Próstata/patologíaRESUMEN
Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.
Asunto(s)
ARN Helicasas DEAD-box/análisis , Células Germinativas/química , Células Germinativas/fisiología , Células Madre Oogoniales/química , Células Madre Oogoniales/fisiología , Ovario/citología , Animales , Femenino , Citometría de Flujo , Ratones Endogámicos C57BLRESUMEN
Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.
Asunto(s)
Enfermedad de Alexander , Agregado de Proteínas , Proteoma/análisis , Animales , Astrocitos , Ciclina D2/análisis , ARN Helicasas DEAD-box/análisis , Proteínas de Unión al GTP/análisis , Humanos , Inmunohistoquímica , Ratones , Proteínas de Neoplasias/análisis , Neuropéptidos/análisis , Agregación Patológica de Proteínas , ARN Helicasas/análisis , Receptores de Cinasa C Activada , Receptores de Superficie Celular/análisisRESUMEN
Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Gránulos Citoplasmáticos/química , Proteoma/análisis , Ribonucleoproteínas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/análisis , Línea Celular Tumoral , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/análisis , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Proteoma/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Represoras/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Azida Sódica/farmacología , Levaduras/citologíaRESUMEN
BACKGROUND: Malignant potential in unfavorable neuroblastoma (NB) is dependent on an undifferentiated status. The aim of this study was to identify a novel biomarker associated with the undifferentiated status of NB in vitro and to evaluate its prognostic implication. PROCEDURE: Shotgun proteomic analysis was performed in undifferentiated and all trans-retinoic acid induced differentiated NB cells in vitro. An identified protein was verified by multiple reaction monitoring (MRM) and evaluated by Western blot analysis. Immunohistochemistry (IHC) was used to examine the expression of the identified protein in 33 primary NB tissues. RESULTS: Twelve proteins, including ATP-dependent RNA helicase (DDX39A), were only detected in undifferentiated NB cells. A peptide of DDX39A was detected at a significantly higher level in undifferentiated IMR-32 (P = 0.002) and LA-N-1 (P < 0.001) cells by MRM. Western blot analysis revealed that DDX39A expression was significantly higher in undifferentiated IMR-32 (P = 0.02) and LA-N-1 (P = 0.025) cells. IHC demonstrated that DDX39A was highly expressed in the primary tumor tissues of patients with poor prognosis, and univariate and multivariate survival analyses showed that DDX39A expression could be an independent unfavorable prognostic factor (P = 0.027). CONCLUSIONS: DDX39A is a potential biomarker for unfavorable NB using a proteomic approach. Evaluation of DDX39A protein expression in NB tumor tissues may provide complementary prognostic information for further subclassification of these tumors.