RESUMEN
Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.
Asunto(s)
Proteínas 14-3-3/metabolismo , Médula Renal/metabolismo , Riñón/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ratas Sprague-Dawley , Ubiquitinación , Transportadores de UreaRESUMEN
The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. Here, we report an important role of 14-3-3zeta in tumorigenesis through a mechanism that involves anoikis resistance. 14-3-3zeta is up-regulated in a number of cancer types, including lung cancer. Through an RNAi approach using human lung adenocarcinoma-derived A549 cells as a model system, we have found that knockdown of a single zeta isoform of 14-3-3 is sufficient to restore the sensitivity of cancer cells to anoikis and impair their anchorage-independent growth. Enhanced anoikis appears to be mediated in part by up-regulated BH3-only proteins, Bad and Bim, coupled with decreased Mcl-1, resulting in the subsequent activation of Bax. This study suggests a model in which anchorage-independent growth of lung cancer cells requires the presence of 14-3-3zeta. This work not only reveals a critical role of 14-3-3zeta in anoikis suppression in lung cancer cells, but also identifies and validates 14-3-3zeta as a potential molecular target for anticancer therapeutic development.
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Proteínas 14-3-3/biosíntesis , Anoicis , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Adenocarcinoma , Apoptosis , Adhesión Celular , Línea Celular Tumoral , Células Cultivadas , Humanos , Modelos Biológicos , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Retroviridae/genéticaRESUMEN
OBJECTIVE: This study aims to discover the most prevalent high-risk (hr) HPV genotypes in the regions of Loreto and La Libertad, Peru. METHODS: In 2015, cervical cell samples were collected during pelvic examinations from women in the geographically distinct regions of Loreto and La Libertad, Peru. In 2017, additional samples were collected in La Libertad. A total of 429 women between the ages of 18 and 65 years living in these regions were enrolled in the study. All samples were analyzed by polymerase chain reaction for the hrHPV genotypes 16, 18, and 35. RESULTS: Sample collection from 126 women in 2015 in Loreto revealed an hrHPV incidence of 15.9% (20 of 126), with 60% (12 of 126) of HPV infections due to hrHPV 16. Samples from La Libertad revealed an hrHPV incidence of 14.5% incidence (44 of 303) (among 303 women). Of these infections, 38% (17) were attributable to hrHPV type 35 and 20% (9) were due to hrHPV type 16. CONCLUSION: The prevalence of hrHPV genotypes in Peru may differ from those observed in North America and Europe. Loreto appears to follow the prevalence trend observed in North America, with hrHPV type 16 accounting for the majority of cases. However, hrHPV type 35 may account for a greater contribution to the cervical cancer burden in La Libertad. Further research, specifically on cervical tumor specimens, is needed.
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Alphapapillomavirus , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Adolescente , Adulto , Anciano , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Papillomaviridae/genética , Infecciones por Papillomavirus/epidemiología , Perú/epidemiología , Prevalencia , Neoplasias del Cuello Uterino/epidemiología , Adulto JovenRESUMEN
Many efforts have been taken to develop molecule target for cancer therapy. 14-3-3zeta protein has emerged as a critical regulator of diverse cellular pathways in multiple cancers. Furthermore, 14-3-3zeta expression was elevated and a predictor of poor prognosis in glioblastoma. However, there is no information to evaluate the potential effects of 14-3-3zeta RNAi in glioblastoma. The relationship between 14-3-3zeta expression and cell proliferation and apoptosis was tested in primary glioblastoma samples. Through an RNAi approach using human glioblastoma cells as a model system, we demonstrated the role of 14-3-3zeta in glioblastoma proliferation, apoptosis, invasion and tumor growth. The expression of 14-3-3zeta in glioblastoma stem cells was also investigated by immunostaining. The apoptosis was significantly higher in 14-3-3zeta-negative group than in positive group. 14-3-3zeta immunoreactivity score was negatively correlated with the apoptosis, and positively with proliferation in human specimens. 14-3-3zeta RNAi reduced cell proliferation, induced apoptosis, decreased the invasive capability and colony-formation, and impaired the growth of glioblastoma xenografts in nude mice. Moreover, 14-3-3zeta was positively expressed in glioblastoma stem cells. Our data highlight the importance of 14-3-3zeta in glioblastoma and identify 14-3-3zeta as a potential molecular target for glioblastoma treatment.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Adulto , Anciano , Animales , Apoptosis/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación hacia Abajo , Glioblastoma/genética , Glioblastoma/patología , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
To investigate the relationship of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus(ICTEV), 84 samples from patients with ICTEV were used in the study. Mutation in the coding region of HOXD13 was detected by denaturing gradinent electrophoresis. The mRNA and protein levels of HOXD13 and FHL1 were evaluated by RT-PCR and immunohistochemistry, respectively. The binding site of FHL1 to HOXD13 predicted by PMATCH software was validated by EMSA( Electrophoretic mobility shift assay,EMSA).No mutation was found in the coding region of HOXD13 in 84 samples from patients with ICTEV. Both HOXD13(33.3%) and FHL1(46.6%) were down-regulated in ICTEV muscle tissue. The result of EMSA showed that the special binding band appeared when HOXD13 existed. The results shows that HOXD13 gene mutation was not involved in outbreak in idiopathic congenital talipes equinovarus, but changes of HOXD13 and FHL1 gene expression related to the development of talipes equinovarus malformation. HOXD13 might play an role in ICTEV through regulating FHL1 expression.
Asunto(s)
Pie Equinovaro/genética , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Musculares/genética , Factores de Transcripción/genética , Niño , Pie Equinovaro/patología , ADN/genética , ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM , Masculino , Proteínas Musculares/metabolismo , Músculos/citología , Músculos/metabolismo , Mutación , Factores de Transcripción/metabolismoRESUMEN
This corrects the article DOI: 10.1038/ncomms7793.
RESUMEN
OBJECTIVE: To explore the etiology of idiopathic talipes equinovarus (ITEV) in all-trans retinoic acid (ATRA) induced clubfoot-like deformity in rat fetuses with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). METHODS: Clubfoot-like deformity model in rat fetuses was induced with ATRA (135 mg/kg) in gestation day (GD10) pregnant Wistar rats. 2-DE was applied to separate the total proteins of ankle joint tissue, ankle joint bone and spinal cord of the animal models. The Coomassie Brilliant Blue staining gels were analyzed by 2-DE software PDQuest 7.1.0. Selected differential protein spots were identified with peptide mass fingerprinting based on matrix-assisted laser adsorption/ionization time-of-flight mass spectrometry and database searching. xiap, tnnt1 and col2 alpha 1, three genes of the differential proteins, were identified furthermore. Apoptosis study was made in terminal deoxynucleotidyl transferase nick end labeling. RESULTS: There were many differential expressed proteins in the clubfoot-like deformity model. Out of the differentially expressed proteins,16 protein spots were identified to be differentially expressed in the clubfoot-like deformity model with MS. Three of the 16 protein spots, xiap, tnnt1 and col2 alpha 1 were confirmed to be significantly down-regulated by the RT-PCR, and Xiap was further confirmed to be significantly down-regulated with immunohistochemistry. Another randomly selected gene, ngfr, did not express differently in ATRA-induced clubfoot-like deformity in rat fetuses. The rates of the apoptosis in the spinal, bone of the clubfoot-like deformity fetuses was 5.4 and 10 times of those of the normal fetuses respectively. CONCLUSION: The results suggest that there are certain differently expressed proteins in ankle joint tissue, ankle joint bone and spinal cord of the ATRA-induced clubfoot-like deformity in rat fetuses, and Xiap, sTnT, and Col2 alpha 1 show a significant correlation with ITEV. Ngfr is not correlation with ITEV. Apoptosis plays a key role in the development of ITEV and related to the decreased expression of the Xiap.
Asunto(s)
Articulación del Tobillo/metabolismo , Pie Equinovaro/metabolismo , Proteómica/métodos , Médula Espinal/metabolismo , Animales , Pie Equinovaro/inducido químicamente , Pie Equinovaro/genética , Electroforesis en Gel Bidimensional , Inmunohistoquímica , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TretinoinaRESUMEN
Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.
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As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein-protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)-MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Oncogenes/efectos de los fármacos , Oncogenes/genética , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas de Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Bases de Datos de Proteínas , Genes Supresores de Tumor/efectos de los fármacos , Genes myc/genética , Genómica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oncogenes/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.
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Técnicas Biosensibles/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Mapeo de Interacción de Proteínas/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fluorescencia , Células HEK293 , Humanos , Imidazoles/farmacología , Luciferasas/metabolismo , Miniaturización , Piperazinas/farmacología , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cables1 is a candidate tumor suppressor that negatively regulates cell growth by inhibiting cyclin-dependent kinases. Cables1 expression is lost frequently in human cancer but little is known about its regulation. Here, we report that Cables1 levels are controlled by a phosphorylation and 14-3-3-dependent mechanism. Mutagenic analyses identified two residues, T44 and T150, that are specifically critical for 14-3-3 binding and that serve as substrates for phosphorylation by the cell survival kinase Akt, which by binding directly to Cables1 recruits 14-3-3 to the complex. In cells, Cables1 overexpression induced apoptosis and inhibited cell growth in part by stabilizing p21 and decreasing Cdk2 kinase activity. Ectopic expression of activated Akt (AKT1) prevented Cables1-induced apoptosis. Clinically, levels of phosphorylated Cables1 and phosphorylated Akt correlated with each other in human lung cancer specimens, consistent with pathophysiologic significance. Together, our results illuminated a dynamic regulatory system through which activated Akt and 14-3-3 work directly together to neutralize a potent tumor suppressor function of Cables1.
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Proteínas Portadoras/metabolismo , Ciclinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Apoptosis/fisiología , Sitios de Unión , Células COS , Proteínas Portadoras/genética , Muerte Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Chlorocebus aethiops , Ciclinas/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Fosfoproteínas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Genome-wide association studies (GWASs) have reproducibly associated â¼40 susceptibility loci with psoriasis. However, the missing heritability is evident and the contributions of coding variants have not yet been systematically evaluated. Here, we present a large-scale whole-exome array analysis for psoriasis consisting of 42,760 individuals. We discover 16 SNPs within 15 new genes/loci associated with psoriasis, including C1orf141, ZNF683, TMC6, AIM2, IL1RL1, CASR, SON, ZFYVE16, MTHFR, CCDC129, ZNF143, AP5B1, SYNE2, IFNGR2 and 3q26.2-q27 (P<5.00 × 10(-08)). In addition, we also replicate four known susceptibility loci TNIP1, NFKBIA, IL12B and LCE3D-LCE3E. These susceptibility variants identified in the current study collectively account for 1.9% of the psoriasis heritability. The variant within AIM2 is predicted to impact protein structure. Our findings increase the number of genetic risk factors for psoriasis and highlight new and plausible biological pathways in psoriasis.
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Pueblo Asiatico/genética , Psoriasis/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Adolescente , Adulto , Estudios de Casos y Controles , Proteínas Ricas en Prolina del Estrato Córneo/genética , Proteínas de Unión al ADN/genética , Exoma/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Proteínas I-kappa B/genética , Proteína 1 Similar al Receptor de Interleucina-1 , Subunidad p40 de la Interleucina-12/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Proteínas de Microfilamentos/genética , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Inhibidor NF-kappaB alfa , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Receptores Sensibles al Calcio/genética , Receptores de Superficie Celular/genética , Receptores de Interferón/genética , Serina Endopeptidasas/genética , Transactivadores/genética , Adulto JovenRESUMEN
8-Chloro-adenosine (8-Cl-Ado) is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt) and H1299 (p53-depleted) to 8-Cl-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-Cl-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.
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Adenosina/farmacología , Ciclo Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Mitosis/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , HumanosRESUMEN
To assess the relationship of deletion of p15 and p16 gene and EGFR gene amplification in laryngeal squamous cell carcinoma (LSCC). DNA was extracted from fresh tumor. Deletion of p15 exon 2(p15E2) and p16 exon 2(p16E2) in 30 cases of LSCC was detected by polymerase chain reaction (PCR) technique. Amplification of EGFR gene in 30 cases of LSCC was detected by FISH. The rate of p15E2 deletion in 30 cases was 13.3(4/30), and that of p16E2 was 16.7% (5/30). p15E2 and p16E2 codeletion rate was 6.7% (2/30). The rate of EGFR gene amplification in 30 cases was 30% (9/30), and was amplified 2 to 8 fold. Homozygous deletion of p16E2 and p15E2 and codeletion is related with amplification of EGFR gene (P = 0.000018), and may play an important role to oncogenesis and malignant progression in LSCC.
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Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular/genética , Receptores ErbB/genética , Eliminación de Gen , Genes p16 , Neoplasias Laríngeas/genética , Proteínas Supresoras de Tumor/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Humanos , Hibridación Fluorescente in SituRESUMEN
Loss of tumor suppressor genes and apoptosis-associated genes was common event in laryngeal squamous cell carcinoma (LSCC). Apaf-1 (apoptotic protease activating factor-1) is a key factor in cytochrome C-dependent apoptotic pathway. To investigate the effect of Apaf-1 in progression of LSCC, we analyzed Apaf-1 from DNA and RNA levels. Semi-quantitative RT-PCR analysis of Apaf-1 mRNA showed that over 40 percent of LSCCs (11/27) were low expression compared to the para-neoplastic laryngeal tissues (PNTs). The results of CGH indicated loss in 2 of 18 cases but no amplification on chromosome 12q22-23. The LOH frequencies of D12S327, D12S1657, D12S393, D12S1706, D12S346 on 12q22-23 were 18.2%, 13.6%, 18.2%, 22.2% and 16.6%, respectively in 72 matched samples of LSCCs and PNTs. Methylation-specific PCR displayed that all of 11 LSCCs, whose expression of Apaf-1 mRNA down-regulated, were methylated in promoter regions. In contrast, only 1 of 16 LSCCs with no changes in Apaf-1 mRNA levels was methylated (chi2 test, P=0.0001). The results implied that abnormal expression of Apaf-1 participates in the genesis of LSCCs and decreased expression of Apaf-1 mRNA were not mainly due to the deletion of Apaf-1 gene. The Apaf-1 DNA methylation in promoter region might contribute to the decreased transcription of Apaf-1 in LSCCs.
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Carcinoma de Células Escamosas/genética , Metilación de ADN , Neoplasias Laríngeas/genética , Regiones Promotoras Genéticas , Proteínas/genética , Factor Apoptótico 1 Activador de Proteasas , Cromosomas Humanos Par 12 , Islas de CpG , HumanosRESUMEN
OBJECTIVE: To investigate the association of p15 and p16 genes deletion, and STK15 gene overexpression with laryngeal squamous cell carcinoma (LSCC). METHODS: The cancer tissue and surrounding normal tissue were taken during operation from 50 cases of LSCC who had undergone neither chemotherapy nor radiotherapy preoperatively. DNA was extracted and PCR was used to test the homozygous deletion of p15 exon 2 (p15E2) and p16 exon 2 (p16E2). RNA was extracted, cDNA was synthesized by reverse transcription, and the expression of STK15 gene was tested by PCR with beta-actin as inner control. At the same time the expression of STK15 in human LSCC Hep-2 cell line was tested. The ratio of ADV (average density value) of STK15 gene to the ADV of beta-actin gene was calculated. RESULTS: The rate of p15E2 deletion was 12% (6/50) and that of p16E2 was 14% (7/50). The p15E2 and p16E2 codeletion rate was 6% (3/50). In 34 of the 50 cases (68%) the expression of STK15 gene in tumor tissue was higher than that of the paired surrounding normal tissue with a significant difference. The ratio of ADV of STK15 gene to ADV of beta-actin gene was 1.03 +/- 0.30 in the cancer tissue, and 0.89 +/- 0.22 in the paired normal tissue with a significant difference (t = 4.333, P < 0.01). The expression of STK15 gene was higher than that of beta-actin in Hep-2 cell line. CONCLUSION: The homozygous deletion of p15E2 and p16E2 and overexpression of STK15 gene may play a role in the oncogenesis and malignant progression of laryngeal squamous cell carcinoma.
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Proteínas de Ciclo Celular/genética , Eliminación de Gen , Genes p16 , Neoplasias Laríngeas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Aurora Quinasa A , Aurora Quinasas , Carcinoma de Células Escamosas , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Retinoic acid can induce teratogenesis of the fetus of many animals including human, and its biological activities are induced by a serious of different retinoic acid accepters and their ligands. The retinoic acid acceptor RAR plays key roles in the teratogenesis, and the ligands of RAR are strong teratogens. The intensity sequence of the relative teratogenesis is ligandalpha, ligandbeta and ligandgamma. The ligands of the retinoic acid acceptor RXR cannot induce teratogenesis, but they can enhance the teratogenesis of the RAR stimulus. The retinoic acid acceptors can also affect the development of the fetus by adjusting the expression of the other genes. The relations between the gene mutation of the retinoic acid acceptor, various retinoic acid acceptors and their ligands and teratogenesis of retinoic acid are summarized in this article. In addition, the regulations of the retinoic acid acceptors to the other genes are also discussed.
Asunto(s)
Anomalías Congénitas/etiología , Desarrollo Fetal/efectos de los fármacos , Mutación , Receptores de Ácido Retinoico/genética , Retinoides/toxicidad , Animales , Anomalías Congénitas/genética , Regulación de la Expresión Génica , Humanos , Ligandos , Receptores de Ácido Retinoico/química , Receptores X Retinoide/genética , Teratógenos/toxicidadRESUMEN
OBJECTIVE: To observe expression of Wilms' tumor-1 (WT1) gene in the different subtypes of myelodysplastic syndrome (MDS), and to explore the regularity of expression of WT1 gene in the process of MDS transforming into acute leukemia (AL). METHODS: The method of reverse transcriptase-polymerase chain reaction (RT-PCR) was used, the levels of WT1 gene's presentation in different types of montagers of MDS were analyzed, and the relationship between the level and the clinical characteristic was analyzed. At the same time, the expression of WT1 gene in AL patients, post-MDS-AL patients and normal controls were examined. RESULTS: The positive rate of WT1 gene expression in 49 patients with MDS was 22.4 percent (11/49), refractory anemia (RA) was 0(0/13), RA with excess of blast (RAEB) was 25.0 percent (6/24); RAEB in transformation (RAEB-t) was 41.7 percent (5/12). The positive rates of WT1 expression were gradually increased in three types of MDS (P<0.05 and P<0.01). The positive rates of WT1 expression were higher in AL and post-MDS-AL patients (48.5 percent, 32/66 and 50.0 percent, 4/8) than that in MDS patients (P<0.01). There was no expression of WT1 gene in normal control. CONCLUSION: There is a relatively high expression rate of WT1 gene in RAEB, RAEB-t of MDS, but relatively low expression rate in RA. The method of RT-PCR, is high sensitiveness and specificity, can trustful be used in the progression of monitoring MDS' progression and its transformation into AL.
Asunto(s)
Leucemia/etiología , Síndromes Mielodisplásicos/genética , Proteínas WT1/genética , Enfermedad Aguda , Adulto , Anemia Refractaria con Exceso de Blastos/genética , Femenino , Expresión Génica , Humanos , Masculino , Síndromes Mielodisplásicos/complicaciones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
HIP-55 (hematopoietic progenitor kinase 1 [HPK1]-interacting protein of 55 kDa) is the mammalian homologue of the yeast Abp1p. It contains a C-terminal Src homology 3 domain and an N-terminal actin depolymerization factor (ADF-H/C) domain. HIP-55 appears to be critical for organ development and immune response and is important for the regulation of the actin cytoskeleton through its interactions with F-actin and various cytoskeletal and cell signaling proteins. However, the function of HIP-55 in tumors remains unknown. Here, we found that HIP-55 is up-regulated or down-regulated in several types of tumor tissues in patients. Of these, lung cancer tissues had the highest expression of HIP-55. To gain full insight into the function of HIP-55 in lung cancer, microarray assay was performed using Affymetrix U133 Plus 2.0 expression arrays in both HIP-55 knockdown and scramble control A549 cells. The ingenuity pathway analysis tool was utilized to construct biological networks and analyze functions that might be associated with HIP-55. Functional analysis strongly suggested that HIP-55 may be involved in cancer cell survival and cell death, which was then confirmed by further experimentation. Experimental results showed that downregulation of HIP-55 decreased the viability and increased the apoptosis of A549 cells treated with the anticancer agent etoposide. Our data suggested that HIP-55 may be a newly discovered regulatory node in the growth signaling network and a new target for therapeutic interventions in proliferative disorders.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Proteínas de Microfilamentos/metabolismo , Transducción de Señal , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/metabolismo , Análisis por Matrices de Proteínas , Transducción de Señal/efectos de los fármacos , Análisis de Matrices Tisulares , Dominios Homologos srcRESUMEN
HIP-55 (HPK1-interacting protein of 55 kDa, also named DBNL, SH3P7, and mAbp1) is a multidomain adaptor protein that is critical for organ development and the immune response. Here, we report the coupling of HIP-55 to cell growth control through its 14-3-3-binding phospho-Ser/Thr-sensor sites. Using affinity chromatography, we found HIP-55 formed a complex with 14-3-3 proteins, revealing a new node in phospho-Ser/Thr-mediated signaling networks. In addition, we demonstrated that HIP-55 is required for proper cell growth control. Enforced HIP-55 expression promoted proliferation, colony formation, migration, and invasion of lung cancer cells while silencing of HIP-55 reversed these effects. Importantly, HIP-55 was found to be upregulated in lung cancer cell lines and in tumor tissues of lung cancer patients. Upregulated HIP-55 was required to promote the growth of tumors in a xenograft animal model. However, tumors with S269A/T291A-mutated HIP-55, which ablates 14-3-3 binding, exhibited significantly reduced sizes, supporting a vital role of the HIP-55/14-3-3 protein interaction node in transmitting oncogenic signals. Mechanistically, HIP-55-mediated tumorigenesis activity appears to be in part mediated by antagonizing the tumor suppressor function of HPK1. Thus, the HIP-55-mediated oncogenic pathway, through S269/T291, may be exploited for the development of new therapeutic strategies.