RESUMEN
Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption, and visualization capability. Recently, a variety of nucleic acid amplification approaches have been developed to provide a straightforward and highly efficient way for amplifying low abundance target signals. The integration of single-molecule fluorescence imaging with nucleic acid amplification has greatly facilitated the construction of various fluorescent biosensors for in vitro and in vivo detection of DNAs, RNAs, enzymes, and live cells with high sensitivity and good selectivity. Herein, we review the advances in the development of fluorescent biosensors by integrating single-molecule fluorescence imaging with nucleic acid amplification based on enzyme (e.g., DNA polymerase, RNA polymerase, exonuclease, and endonuclease)-assisted and enzyme-free (e.g., catalytic hairpin assembly, entropy-driven DNA amplification, ligation chain reaction, and hybridization chain reaction) strategies, and summarize the principles, features, and in vitro and in vivo applications of the emerging biosensors. Moreover, we discuss the remaining challenges and future directions in this area. This review may inspire the development of new signal-amplified single-molecule biosensors and promote their practical applications in fundamental and clinical research.
Asunto(s)
Técnicas Biosensibles , Técnicas de Amplificación de Ácido Nucleico , Imagen Óptica , ADN/análisis , ADN/genética , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas/análisis , Endonucleasas/genética , Endonucleasas/metabolismo , Exonucleasas/análisis , Exonucleasas/genética , Exonucleasas/metabolismo , Humanos , ARN/análisis , ARN/genética , ARN Polimerasa Dependiente del ARN/análisis , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
By catalyzing highly specific and tightly controlled chemical reactions, enzymes are essential to maintaining normal cellular physiology. However, aberrant enzymatic activity can be linked to the pathogenesis of various diseases. Therefore, the unusual activity of particular enzymes can represent testable biomarkers for the diagnosis or screening of certain diseases. In recent years, G-quadruplex-based platforms have attracted wide attention for the monitoring of enzymatic activities. In this Personal Account, we discuss our group's works on the development of G-quadruplex-based sensing system for enzyme activities by using mainly iridium(III) complexes as luminescent label-free probes. These studies showcase the versatility of the G-quadruplex for developing assays for a variety of different enzymes.
Asunto(s)
Complejos de Coordinación/química , Pruebas de Enzimas/métodos , G-Cuádruplex , Iridio/química , Sustancias Luminiscentes/química , Mediciones Luminiscentes/métodos , Animales , Técnicas Biosensibles/métodos , Enzimas Reparadoras del ADN/análisis , Enzimas Reparadoras del ADN/metabolismo , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas/análisis , Endonucleasas/metabolismo , Exonucleasas/análisis , Exonucleasas/metabolismo , Humanos , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismoRESUMEN
Using graphene oxide (GO) as a nanoquencher, a universal sensor design strategy was developed on the basis of significantly different binding affinities of GO to single-stranded DNAs (ss-DNAs) with different lengths. The proposed sensors could be used for the activity detection of both exonucleases and restriction endonucleases. To achieve this, a single-labeled fluorescent oligonucleotide probe, which had a single-stranded structure or a hairpin structure with a long single-stranded loop, was used. Such a probe could be efficiently absorbed on the surface of GO, resulting in the quenching of the fluorescent signal. Excision of the single-stranded probe by exonucleases or site-specific cleavage at the double-stranded stem of the hairpin probe by restriction endonuclease released fluorophore-labeled nucleotide, which could not be efficiently absorbed by GO, thus leading to increase in fluorescence of the corresponding sensing system. As examples, three sensors, which were used for activity detection of the exonuclease Exo 1 and the restriction endonucleases EcoR I and Hind III, were developed. These three sensors could specifically and sensitively detect the activities of Exo 1, EcoR I and Hind III with detection limits of 0.03 U mL(-1), 0.06 U mL(-1) and 0.04 U mL(-1), respectively. Visual detection was also possible.
Asunto(s)
Técnicas Biosensibles/métodos , Enzimas de Restricción del ADN/análisis , Exonucleasas/análisis , Colorantes Fluorescentes/química , Grafito/química , Óxidos/química , Enzimas de Restricción del ADN/metabolismo , Activación Enzimática/fisiología , Exonucleasas/metabolismoRESUMEN
The 20 kDa exonuclease encoded by the interferon-stimulated gene, ISG20, can inhibit the replication of hepatitis B virus (HBV), and may represent a clinically useful prognostic marker for response to interferon-alpha (IFN-α) antiviral therapy. The present study was designed to investigate the differential expression patterns of ISG20 in liver biopsy samples from treatment-naive patients with chronic hepatitis B and non-HBV infected controls and to determine the relation between the differential expression and IFN-α treatment outcome (responders vs. non-responders). HBV infection status was determined by measuring levels of hepatitis B surface antigen (HBsAg) by chemoluminescence immunoassay and of HBV DNA by real-time quantitative (q)PCR. ISG20 protein and mRNA expressions were assessed by immunohistochemistry and qPCR, respectively. Chronic hepatitis B responders showed significantly higher levels of ISG20 protein and mRNA expressions than either the chronic hepatitis B non-responders or the controls. Moreover, increased expression of ISG20 in both the nucleus and cytoplasm was correlated with positive response to IFN-α treatment. Thus, active transcription and translation of ISG20 may represent a marker to identify chronic hepatitis B patients likely to respond to IFN-α therapy. Prognostic clinical strategies based upon this marker may include genomic screening methods and immunohistochemical staining of liver biopsies.
Asunto(s)
Biomarcadores/análisis , Exonucleasas/análisis , Expresión Génica , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Interferón-alfa/inmunología , Interferón-alfa/uso terapéutico , Adulto , Biopsia , ADN Viral/sangre , Exorribonucleasas , Femenino , Antígenos de Superficie de la Hepatitis B/sangre , Humanos , Inmunoensayo , Hígado/enzimología , Hígado/patología , Masculino , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Resultado del TratamientoRESUMEN
The distinction of ovarian granulosa cell tumors (GCTs) from other sex-cord stromal tumors may be difficult histologically. Many immunohistochemical markers have been studied for this differential diagnosis, but the available markers are not entirely specific for ovarian GCT. 14-3-3 sigma has been shown to play an anti-apoptotic role in maintaining the viability of immortalized granulosa cells. However, the potential use of this molecule as an immunohistochemical marker for the diagnosis of ovarian GCT has not been investigated. A total of 103 ovarian sex-cord stromal neoplasms were immunostained with 14-3-3 sigma. These tumors included 44 adult granulosa cell, 7 juvenile granulosa cell tumors, 12 steroid cell tumors, 3 well-differentiated Sertoli-Leydig cell tumors, 5 Sertoli cell tumors, 10 thecomas, 18 fibromas, 2 primary ovarian endometrial stromal sarcomas, and 2 unclassified sex-cord stromal tumors. Ten ovaries with cystic follicles were also included as controls. Perinuclear or cytoplasmic stain was considered to be positive. Granulosa cells within the cystic follicles were positive for 14-3-3 sigma protein. All ovarian GCT (51/51) and all steroid cell tumors (12/12) were positive for 14-3-3 sigma, and all Sertoli cell tumors, fibromas, thecomas, ovarian endometrial stromal sarcomas, and sex-cord stromal tumors, unclassified, were negative for 14-3-3 sigma. The percentage of positive cell staining is statistically significant (P<0.0001) between the above 2 groups of sex-cord stromal tumors. These findings provide the initial evidence of the overexpression of 14-3-3 sigma in granulosa cells and steroid-hormone-secreting cells. They further indicate that immunohistochemical staining of 14-3-3 sigma may be a useful marker in facilitating the diagnosis of ovarian GCT and steroid cell tumors.
Asunto(s)
Proteínas 14-3-3/análisis , Biomarcadores de Tumor/análisis , Exonucleasas/análisis , Tumor de Células de la Granulosa/diagnóstico , Neoplasias Ováricas/diagnóstico , Tumores de los Cordones Sexuales y Estroma de las Gónadas/diagnóstico , Diagnóstico Diferencial , Exorribonucleasas , Femenino , Hormonas Esteroides Gonadales/metabolismo , Tumor de Células de la Granulosa/patología , Humanos , Inmunohistoquímica , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Tumor de Células de Sertoli-Leydig/diagnóstico , Tumor de Células de Sertoli-Leydig/patología , Tumores de los Cordones Sexuales y Estroma de las Gónadas/patologíaRESUMEN
NUCLEASE SENSOR TRIO: Fluorogenic DNA sensors were developed for distinct classes of nucleases: 3'-exonucleases, 5'-exonucleases, and endonucleases. The highly selective sensors, built from very small modified DNA oligomers containing the unnatural fluorescent base pyrene, and employing thymine as a quencher, were found to function in a variety of complex biological media.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/metabolismo , Endonucleasas/análisis , Exonucleasas/análisis , Oligonucleótidos/metabolismo , Pirenos/metabolismo , ADN/química , Endonucleasas/metabolismo , Exonucleasas/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Células HeLa , Humanos , Oligonucleótidos/química , Pirenos/química , Staphylococcus/enzimologíaRESUMEN
Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.
Asunto(s)
Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas de Secreción Prostática/análisis , Proteínas de Secreción Prostática/orina , Proteínas 14-3-3/análisis , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Exonucleasas/análisis , Exorribonucleasas , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Marcaje Isotópico , Masculino , Proteínas Oncogénicas/análisis , Antígeno Prostático Específico/metabolismo , Análisis por Matrices de Proteínas , Proteína Desglicasa DJ-1 , Proteoma/análisis , Inhibidor Tisular de Metaloproteinasa-1/análisis , Transglutaminasas/análisisRESUMEN
Enzymes containing 3'-5' exonuclease activities play vital roles in maintaining genome stability. Though a wide variety of methods have been developed for detection of these enzymes, few of them can be directly applied for in situ and real-time monitoring of the secretion of these active substances by living cells. Taking advantages of the free 3'-end of stacked guanine-quenched photoinduced electron transfer fluorescent probes, here we demonstrate a novel assay capable of in situ and real-time monitoring of the 3'-5' exonucleases secreted by living cells. The detection limit of the new method achieved as low as 0.04 U/mL, allowing direct monitoring of the target enzymes in an extracellular environment without preconcentration steps. False positive signals caused by other nonspecific enzymes were easily ruled out by the use of a control probe with the 3'-end modified with exonuclease-resistant phosphorothioate guanines. Using Alexa Fluor 488 as the fluorophore, the probe is adaptable to a wide range of pH conditions. The approach was successfully applied for in situ, real-time monitoring of the 3'-5' exonucleases secreted by suspension cells of Arabidopsis thaliana. It also holds great potential for in situ and real-time detection of many other DNA end-processing enzymes produced by other types of cells.
Asunto(s)
Arabidopsis/enzimología , Técnicas Biosensibles , Exonucleasas/análisis , Oligonucleótidos Fosforotioatos/química , Exonucleasas/metabolismo , Colorantes Fluorescentes , Guanina/química , Concentración de Iones de Hidrógeno , Límite de Detección , Factores de TiempoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, largely due to metastatic disease. To better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. This poses technical challenges because of the cross-linkages induced by fixation. Using laser capture microdissection (PALM system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum, and urine resulted in a less than 30% overlap, indicating that our study has substantially expanded the current database of proteins expressed in this malignancy. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. Thus, laser capture microdissection of FFPE tissue coupled with downstream proteomic analysis is a valid approach for the investigation of metastatic PDAC. This is the first study to establish and compare the protein composition of primary PDAC and matched LN metastases, and has resulted in the identification of several potential epithelial-specific therapeutic targets, including 14-3-3 sigma and S100P.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/química , Fijadores , Formaldehído , Ganglios Linfáticos/química , Proteínas de Neoplasias/análisis , Neoplasias Pancreáticas/química , Adhesión en Parafina , Proteómica , Fijación del Tejido/métodos , Proteínas 14-3-3/análisis , Proteínas de Unión al Calcio/análisis , Carcinoma Ductal Pancreático/secundario , Exonucleasas/análisis , Exorribonucleasas , Humanos , Inmunohistoquímica , Captura por Microdisección con Láser , Ganglios Linfáticos/patología , Metástasis Linfática , Neoplasias Pancreáticas/patología , Pronóstico , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
UNLABELLED: Stratifin plays an important role in cancer biology by interfering with intracellular signalling pathways and cell-cycle checkpoints. Decreased expression of stratifin gene has been reported to be a poor prognostic indicator in a variety of human malignant tumors. AIM: To clarify the role and prognostic significance of stratifin in esophageal squamous cell carcinoma (ESCC). METHODS: The alteration of stratifin messenger RNA (mRNA) and protein was analyzed by reverse-transcription and quantitative real-time polymerase chain reaction (QRT-PCR) and Western blotting in 20 paired ESCC and nonneoplastic esophageal mucosa tissues, respectively. Then, immunohistochemistry (IHC) was used to evaluate expression of stratifin in tissues of 148 ESCC patients (including the former 20 pairs of tissues) and correlate it with clinicopathological parameters and prognosis of ESCC patients. RESULTS: The stratifin level of mRNA and protein was markedly downregulated in ESCC tissue compared with in corresponding nonneoplastic esophageal epithelium (P<0.05). Similarly, the positive rate of stratifin protein expression was lower in the esophageal cancer than in paired nonneoplastic esophageal epithelium as detected by IHC (P=0.007). Statistically, the downregulation of stratifin expression was correlated with tumor infiltration depth (P=0.003), lymph node metastasis (P=0.008), distant metastasis (P=0.013), and lymphovascular invasion (P=0.007) of ESCC. Furthermore, the reduced stratifin expression was associated with shorter 5-year survival rate of ESCC patients after curative surgery (P<0.0001). On the basis of univariate and multivariate Cox regression analysis, we found that reduced stratifin expression, T4 stage, lymph node metastasis, and distant metastasis were independent risk factors for worse prognosis in ESCC patients. CONCLUSION: The present report indicates that stratifin could be a useful indicator for prognosis of this disease, as well as a potential target for more effective therapy.
Asunto(s)
Proteínas 14-3-3/análisis , Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/química , Neoplasias Esofágicas/química , Exonucleasas/análisis , Proteínas 14-3-3/genética , Adulto , Anciano , Biomarcadores de Tumor/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/secundario , Carcinoma de Células Escamosas/cirugía , Distribución de Chi-Cuadrado , Regulación hacia Abajo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidad , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/cirugía , Esofagectomía , Exonucleasas/genética , Exorribonucleasas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
beta-catenin has emerged as a key regulator of Wnt signaling pathway, which plays an important role in the development and progression of various cancers. Its accumulation in nucleus of the esophagus squamous epithelium might be the crucial step for the carcinogenesis of esophageal squamous cell carcinoma (ESCC). To detect the proteins correlated with beta-catenin function, we used the established cell lines of pGen-3-con (Eca109 cells transfected by control vector) and pGen-3-CTNNB1 (Eca109 cells transfected by beta-catenin siRNA) as cell models for further analysis. Two-dimensional gel electrophoresis technology was performed to separate the proteins of pGen-3-con and pGen-3-CTNNB1 cell lines, respectively. The differential protein spots were analyzed by software analysis, subjected to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Consequently, 13 differentially expressed proteins between the two cell lines were identified, of which 14-3-3sigma, prohibitin, and nm23-H1 were further verified by western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction. Then, the tissue microarray and immunohistochemical analysis were employed to research their relationship in ESCC and their corresponding normal mucosa tissues. The upregulation of prohibitin or the downregulation of 14-3-3sigma and nm23-H1 proteins was significantly associated with the proliferation, invasion depth, and lymph node metastasis of ESCC. There were statistically significant correlations between the expression of beta-catenin and the three proteins. The results presented here might provide potential protein markers to elucidate the mechanism of beta-catenin-mediated biologic characteristics for ESCC.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Proteoma/análisis , beta Catenina/análisis , Proteínas 14-3-3/análisis , Proteínas 14-3-3/genética , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Neoplasias Esofágicas/genética , Esófago/citología , Exonucleasas/análisis , Exonucleasas/genética , Exorribonucleasas , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metástasis Linfática/patología , Nucleósido Difosfato Quinasas NM23/análisis , Nucleósido Difosfato Quinasas NM23/genética , Invasividad Neoplásica , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Prohibitinas , Análisis por Matrices de Proteínas , Proteínas Represoras/análisis , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , Regulación hacia Arriba , beta Catenina/genéticaRESUMEN
14-3-3 sigma (sigma) sequesters the cdc2-cyclin B1 complex in the cytoplasm resulting in G2 arrest. Inactivation and reduced expression of 14-3-3sigma have been reported in a varity of cancers. In the present study, we investigated the expression of 14-3-3sigma in a series of 297 cervical squamous cell carcinoma (SCC) to clarify the prognostic value. Using immunohistochemical methods we found high levels of 14-3-3sigma protein in cytoplasm of 143 (48.1%), in nucleus of 113 (38.0%) and in both cytoplasm and nucleus of 147 (49.5%) cases, whereas, low levels were present in cytoplasm of 154 (51.9%), in nucleus of 184 (62.0%) and in both cytoplasm and nucleus of 150 (50.5%) cases. Levels of 14-3-3sigma mRNA measured by reverse-transcription polymerase chain reaction (RT-PCR) and 14-3-3sigma protein were not significant associated. 14-3-3sigma expression in cytoplasm, nuclear and cytoplasm/nuclear were not significantly correlated to disease-specific survival or disease-free survival. In conclusion, reduced expression of 14-3-3sigma protein in the cytoplasm and shuttle of 14-3-3sigma protein into the nucleus in a relatively high number of cases indicate that 14-3-3sigma may be important in the carcinogenesis of cervical SCCs by two different mechanisms; reduction and nuclear translocation of 14-3-3sigma protein. Furthermore, the non-significant correlation between expression levels of 14-3-3sigma mRNA and protein support a post-transcriptional regulation in cervical SCCs. The protein has no prognostic value in cervical cancers.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/química , Exonucleasas/análisis , Proteínas de Neoplasias/análisis , Neoplasias del Cuello Uterino/química , Proteínas 14-3-3 , Transporte Activo de Núcleo Celular , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Núcleo Celular/química , Citoplasma/química , Supervivencia sin Enfermedad , Exonucleasas/genética , Exorribonucleasas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/terapiaRESUMEN
BACKGROUND AND OBJECTIVE: Salivary proteomics technology can be used to evaluate the disease progression of periodontitis and the systemic screening of proteomes of saliva from subjects with aggressive periodontitis has not been available. The objective of this preliminary study was to compare the proteomic profile of whole unstimulated saliva of subjects with generalized aggressive periodontitis (GAgP) with that of healthy volunteers to identify proteins, the levels of which were significantly altered between the two groups. MATERIAL AND METHODS: Whole unstimulated saliva was obtained from five subjects with GAgP and five healthy subjects, and proteins were separated using two-dimensional gel electrophoresis. Proteins, the levels of which were significantly different between the two groups, were identified by computer image analyses and subsequent electrospray ionization tandem mass spectrometry. RESULTS: Eleven proteins that exhibited a different level in the GAgP group vs. the control group were identified. Compared with whole saliva of healthy control subjects, the levels of serum albumin, immunoglobulin (Ig) gamma2 chain C region, Ig alpha2 chain C region, vitamin D-binding protein, salivary alpha-amylase and zinc-alpha2 glycoprotein were increased in whole unstimulated saliva of GAgP subjects, while those of lactotransferrin, elongation factor 2, 14-3-3 sigma, short palate, lung and nasal epithelium carcinoma-associated protein 2 precursor and carbonic anhydrase 6 were decreased. CONCLUSION: Comparison of the proteomic profile of whole unstimulated saliva of GAgP subjects with that of healthy control subjects revealed at least 11 differential proteins. The approach applied herein might be helpful to aid understanding of the etiology of GAgP.
Asunto(s)
Periodontitis Agresiva/metabolismo , Proteoma/análisis , Saliva/química , Proteínas y Péptidos Salivales/análisis , Proteínas 14-3-3 , Adipoquinas , Adulto , Biomarcadores/análisis , Biomarcadores de Tumor/análisis , Anhidrasas Carbónicas/análisis , Proteínas Portadoras/análisis , Electroforesis en Gel Bidimensional , Exonucleasas/análisis , Exorribonucleasas , Glicoproteínas/análisis , Humanos , Procesamiento de Imagen Asistido por Computador , Cadenas alfa de Inmunoglobulina/análisis , Cadenas gamma de Inmunoglobulina/análisis , Lactoferrina/análisis , Leucina Zippers , Proteínas de Neoplasias/análisis , Factor 2 de Elongación Peptídica/análisis , Fosfoproteínas/análisis , Precursores de Proteínas/análisis , alfa-Amilasas Salivales/análisis , Albúmina Sérica/análisis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Proteína de Unión a Vitamina D/análisisRESUMEN
Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain <0.5% of the endogenous unlabeled forms. Identified proteins were screened to provide a set of 15 candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the 15 candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.
Asunto(s)
Biomarcadores/análisis , Cuello del Útero/metabolismo , Cromatografía Liquida/métodos , Nacimiento Prematuro/metabolismo , Espectrometría de Masas en Tándem/métodos , Vagina/metabolismo , Proteínas 14-3-3 , Algoritmos , Secuencia de Aminoácidos , Biomarcadores de Tumor/análisis , Cuello del Útero/citología , Bases de Datos Factuales , Desmoplaquinas/análisis , Exonucleasas/análisis , Exorribonucleasas , Femenino , Humanos , Metabolómica/métodos , Datos de Secuencia Molecular , Proteínas de Neoplasias/análisis , Embarazo , Proteómica/métodos , Trombospondina 1/análisis , Vagina/citologíaRESUMEN
Eukaryotic RNA exosomes participate in 3' to 5'-processing and degradation of RNA in the nucleus and cytoplasm. RNA exosomes are multisubunit complexes composed of at least nine distinct proteins that form the exosome core. Although the eukaryotic exosome core shares structural and sequence similarity to phosphorolytic archaeal exosomes and bacterial PNPase, the eukaryotic exosome core has diverged from its archaeal and bacterial cousins and appears devoid of phosphorolytic activity. In yeast, the processive hydrolytic 3' to 5'-exoribonuclease Rrp44 associates with exosomes in the nucleus and cytoplasm. Although human Rrp44 appears homologous to yeast Rrp44, it has not yet been shown to associate with human exosomes. In the nucleus, eukaryotic exosomes interact with Rrp6, a distributive hydrolytic 3' to 5'-exoribonuclease. To facilitate analysis of eukaryotic RNA exosomes, we will describe procedures used to clone, express, purify, and reconstitute the nine-subunit human exosome and nine-, ten-, and eleven-subunit yeast exosomes. We will also discuss procedures to assess exoribonuclease activity for reconstituted exosomes.
Asunto(s)
Clonación Molecular/métodos , Exonucleasas/análisis , Exosomas/metabolismo , Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Animales , Exonucleasas/genética , Exonucleasas/aislamiento & purificación , Exonucleasas/metabolismo , Exosomas/química , Humanos , Reacción en Cadena de la Polimerasa , ARN/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Especificidad por SustratoRESUMEN
Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3varsigma for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3varsigma and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P
Asunto(s)
Biomarcadores de Tumor/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular , Células Epiteliales/citología , Exonucleasas/metabolismo , Leucocitos Mononucleares/citología , Proteínas de Neoplasias/metabolismo , Proteínas 14-3-3 , Biomarcadores de Tumor/análisis , Células de la Médula Ósea/metabolismo , Transdiferenciación Celular , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Células Epiteliales/metabolismo , Exonucleasas/análisis , Exorribonucleasas , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Queratina-5/metabolismo , Queratina-8/metabolismo , Leucocitos Mononucleares/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Proteínas de Neoplasias/análisisRESUMEN
Cyclin B1 is translocated to the nucleus from the cytoplasm, and plays an essential role in cell proliferation through promotion of mitosis. Although overexpression of cyclin B1 was previously reported in breast carcinomas, the biological significance of the intracellular localization of cyclin B1 remains unclear. Therefore, in this study, we examined cyclin B1 immunoreactivity in 109 breast carcinomas, according to the intracellular localization, that is, nucleus, cytoplasm or total (nucleus or cytoplasm). Total cyclin B1 was detected in carcinoma cells in 42% of breast carcinomas examined, whereas nuclear and cytoplasmic cyclin B1 were positive in 17 and 35% of the cases, respectively. Total or cytoplasmic cyclin B1 were positively associated with histological grade, mitosis, Ki-67, p53, c-myc or 14-3-3sigma, and inversely correlated with estrogen or progesterone receptor. Nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis, histological grade, mitosis, Ki-67 or polo-like kinase 1. Only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and multivariate analyses of disease-free and overall survival demonstrated nuclear cyclin B1 as the independent marker. A similar tendency was detected in the patients receiving adjuvant therapy after surgery. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in nuclei of breast carcinoma cells, and the nuclear translocation is regulated by polo-like kinase 1 and 14-3-3sigma. Nuclear cyclin B1-positive breast carcinoma is resistant to adjuvant therapy, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients.
Asunto(s)
Neoplasias de la Mama/patología , Ciclina B/análisis , Proteínas Nucleares/análisis , Proteínas 14-3-3 , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/análisis , Ciclina B1 , Exonucleasas/análisis , Exorribonucleasas , Femenino , Humanos , Inmunohistoquímica/estadística & datos numéricos , Estimación de Kaplan-Meier , Antígeno Ki-67/análisis , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-myc/análisis , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Proteína p53 Supresora de Tumor/análisis , Quinasa Tipo Polo 1RESUMEN
The checkpoint protein Rad9/Rad1/Hus1 heterotrimer (the 9-1-1 complex) is structurally similar to the proliferating cell nuclear antigen sliding clamp and has been proposed to sense DNA damage that leads to cell cycle arrest or apoptosis. Human (h) NEIL1 DNA glycosylase, an ortholog of bacterial Nei/Fpg, is involved in repairing oxidatively damaged DNA bases. In this study, we show that hNEIL1 interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. Residues 290-350 of hNEIL1 are important for the 9-1-1 association. A significant fraction of the hNEIL1 nuclear foci co-localize with hRad9 foci in hydrogen peroxide treated cells. Human NEIL1 DNA glycosylase activity is significantly stimulated by hHus1, hRad1, hRad9 separately and the 9-1-1 complex. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of base excision repair.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Glicosilasas/metabolismo , Exonucleasas/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/análisis , Línea Celular , ADN Glicosilasas/análisis , ADN Glicosilasas/química , Activación Enzimática , Exonucleasas/análisis , HumanosRESUMEN
To explore the significance of epigenetic mechanisms in urinary bladder carcinogenesis mediated by methylation of cytosine in CpG dinucleotides at 5' promoter regions, we analysed the methylation status of a broad panel of different genes in transitional cell carcinomas (TCC) and nonurothelial cancers, among which the 14-3-3 sigma, SYK and CAGE-1 genes were recognised as promising target genes. Using methylation-specific PCR, the rate of DNA hypermethylation proved to be related to the various histopathological cancer subtypes. The higher frequency of promoter methylation of the 14-3-3 sigma (57.1%) and SYK (64.3%) genes in high-grade, high-stage TCC in association with a reduced or even lacking immunohistochemical protein expression than in low-grade, low-stage TCC (28.6% and 42.9%, respectively), indicates that aberrant methylation of these genes plays an essential role in the progression of TCC. The importance of DNA hypermethylation in the conversion of TCC from a low to a high malignant potential was strongly supported by the finding that, unlike superficial low-grade TCC, advanced muscle invasive TCC showed a concurrent promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes. Squamous cell carcinomas revealed a peak incidence of hypermethylation of the 14-3-3 sigma gene (80%), and conversely, the lowest methylation frequency of the SYK gene (13.3%). Undifferentiated small cell carcinomas disclosed a promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in only a quarter each for the cases. Although a correlation between the methylation status and gene activity in squamous cell and undifferentiated small cell carcinomas was not observed, the underexpression of the SYK protein products in both cancer types and additionally of the 14-3-3 sigma protein in small cell carcinomas appeared to be related to the aggressive clinical behaviour of both these nonurothelial bladder carcinomas. The relevance of the high frequency of DNA hypermethylation of the CAGE-1 antigen in TCC and squamous cell carcinomas merits further study, particularly in relation to anticancer immunotherapy. The methylation status of the PTEN, COX-2, RUNX-3 and HIC-1 genes was found to be unaltered. In conclusion, the different patterns of aberrant methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in the various histopathological cancer types of the urinary bladder point to a role in tumor cell differentiation, resulting in the phenotypical conversion of TCC into nonurothelial carcinomas and in the progression of TCC to a more malignant potential.
Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Carcinoma de Células Transicionales/patología , Metilación de ADN , Exonucleasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Quinasas/genética , Neoplasias de la Vejiga Urinaria/patología , Proteínas 14-3-3 , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Ciclooxigenasa 2/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Exonucleasas/análisis , Exorribonucleasas , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/análisis , Factores de Transcripción de Tipo Kruppel , Proteínas de la Membrana/genética , Membrana Mucosa/metabolismo , Proteínas de Neoplasias/análisis , Proteínas Nucleares/análisis , Fosfohidrolasa PTEN/genética , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Proteínas Tirosina Quinasas/análisis , Quinasa Syk , Factores de Transcripción/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
PURPOSE: This study was done to explore whether the expression of a selected set of proteins could predict primary response to radiotherapy or concomitant radiotherapy and chemotherapy in patients with advanced head and neck cancer. EXPERIMENTAL DESIGN: Forty-three pretreatment tumor biopsies were taken during diagnostic panendoscopy and examined for Mcl-1, vascular endothelial growth factor (VEGF)-R2, CD9, and 14-3-3sigma expression by immunohistochemistry. Forty-three patients underwent primary radiotherapy, of which, 29 patients received concomitant chemotherapy (low dose daily cisplatin, mitomycin C bolus). The primary end-point was locoregional tumor control 6 months after completion of radiotherapy. Mcl-1, VEGF-R2, CD9, and 14-3-3sigma expression were correlated with patients' primary response to radiotherapy and chemotherapy and with established clinicopathologic variables. RESULTS: Thirty complete and 13 partial responses were observed in our patient group. High expression levels of Mcl-1 (P=0.021), VEGF-R2 (P=0.032), and 14-3-3sigma (P=0.013), but not of CD9, in tumor biopsies was correlated with complete response. Overexpression of at least two of the three aforementioned proteins in pretreatment biopsies predicted-with a likelihood of 80%-whether a patient would achieve complete response to radiotherapy and chemotherapy. However, if only one of these proteins is overexpressed, there is a likelihood of 84.6% that this patient would not completely respond to therapy. CONCLUSION: Determining the expression levels of Mcl-1, VEGF-R2, and 14-3-3sigma may be helpful in predicting the early clinical response in head and neck tumor patients receiving primary radiotherapy and chemotherapy and may further allow a pretherapeutic selection of patients.