RESUMEN
Nephroblastoma (Wilms tumor) is a kidney neoplasia, predominately occurring at very young age, resulting from the malignant transformation of renal stem cells. The Ras proto-oncogenes and B-Raf are members of an intracellular cascade pathway, which regulates cell growth and differentiation, and ultimately cancer development. Our objective was to determine the mutation rate and to measure the mRNA levels of the three Ras genes and of B-Raf in formalin-fixed paraffin-embedded tissue samples from 32 patients with nephroblastoma and 10 controls. No mutations were detected in the four studied genes among our Wilms tumors cases, while Ras and B-Raf expression was higher in malignant samples versus controls. Statistical analysis revealed a positive correlation of K-Ras (p < 0.001) and B-Raf (p = 0.006) with tumor size, a negative correlation of K-Ras (p = 0.041) and H-Ras (p = 0.033) with the percentage of tissue necrosis, and an association of N-Ras (p = 0.047) and B-Raf (p = 0.044) with tissue histology. From the above, we deduce that although Ras and B-Raf mutations are rare events in Wilms tumors, their expression pattern suggests that they play an important role in the development and progression of this malignancy.
Asunto(s)
Neoplasias Renales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Tumor de Wilms/genética , Estudios de Casos y Controles , Procesos de Crecimiento Celular/genética , Niño , Preescolar , Femenino , GTP Fosfohidrolasas/biosíntesis , GTP Fosfohidrolasas/genética , Humanos , Neoplasias Renales/enzimología , Neoplasias Renales/patología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Adhesión en Parafina , Proteínas Proto-Oncogénicas B-raf/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tumor de Wilms/enzimología , Tumor de Wilms/patologíaRESUMEN
BACKGROUND: YKL-40 is an extracellular matrix glycoprotein with a significant role in tissue inflammation and remodeling. MIP-1a has chemotactic and pro-inflammatory properties, and is induced by YKL-40 in several lung disorders. The aim of this study was to determine the levels of YKL-40 and MIP-1a in blood serum and pleural fluids of various pulmonary diseases, and to evaluate their potential role as differential diagnosis biomarkers. METHODS: We recruited 60 patients (age: 62.5 ± 20.6 years) with pleural effusions: 49 exudates and 11 transudates (T). Exudates were further classified based on the underlying disease: ten with tuberculosis (TB), 13 with lung cancer (LCa), 15 with metastatic cancer (MCa) of non-lung origin and 11 with parapneumonic (PN) effusions. YKL-40 and MIP-1a levels were measured by ELISA. RESULTS: Pleural YKL-40 levels (ng/ml) were similar among all patient groups (TB: 399 ± 36, LCa: 401 ± 112, MCa: 416 ± 34, PN: 401 ± 50, T: 399 ± 42, p = 0.92). On the contrary, YKL-40 was significantly lower in the serum of TB patients (TB: 58 ± 22, LCa: 212 ± 106, MCa: 254 ± 140, PN: 265 ± 140, T: 229 ± 123, p < 0.001). Pleural MIP-1a protein levels (ng/ml) were statistically lower only in patients with LCa (TB: 25.0 ± 20.2, LCa: 7.3 ± 6.0, MCa: 16.1 ± 14.9, PN: 25.4 ± 27.9, T: 18.5 ± 7.9, p = 0.012), a finding also observed in serum MIP-1a levels (TB: 17.1 ± 7.6, LCa: 9.4 ± 7.0, MCa: 28.7 ± 28.7, PN: 33.3 ± 24.0, T: 22.9 ± 8.7, p = 0.003). CONCLUSIONS: Our data suggest that both YKL-40 and MIP-1a, particularly in serum, could prove useful for the differentiation of pleural effusions in clinical practice, especially of TB or LCa origin. However, large-scale studies are needed to validate these findings.
Asunto(s)
Adipoquinas/metabolismo , Quimiocina CCL3/metabolismo , Exudados y Transudados/metabolismo , Lectinas/metabolismo , Neoplasias Pulmonares/diagnóstico , Derrame Pleural/metabolismo , Neumonía/diagnóstico , Tuberculosis Pulmonar/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Proteína 1 Similar a Quitinasa-3 , Diagnóstico Diferencial , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Masculino , Persona de Mediana Edad , Proyectos Piloto , Derrame Pleural/etiología , Neumonía/complicaciones , Neumonía/metabolismo , Estudios Retrospectivos , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/metabolismoRESUMEN
Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3-5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p = 0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p = 0.029), of NICD3 protein levels (p = 0.028) and DLL3 mRNA levels (p = 0.041) with birth weight centile, and of HEY2 transcript levels with parity (p = 0.034) and mode of delivery (p = 0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE.
Asunto(s)
Placenta/metabolismo , Preeclampsia/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Adulto , Análisis por Conglomerados , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Recién Nacido , Masculino , Preeclampsia/genética , Embarazo , Nacimiento Prematuro , Nacimiento a Término , Transcriptoma , Adulto JovenRESUMEN
INTRODUCTION: The placental anticoagulant protein Annexin A5 (ANXA5) is a multifunctional protein that is highly expressed on the apical surfaces of syncytiotrophoblasts, and plays an important role in haemostatic regulations, maintaining blood fluidity of the placenta. The aim of this study was to investigate the expression of ANXA5 in pregnancies complicated by preeclampsia (PE). MATERIALS AND METHODS: Placental tissue samples were collected from 23 pregnancies with PE and 34 normal pregnancies. ANXA5 mRNA levels were measured by quantitative Real-Time PCR (qPCR), while ANXA5 protein expression was measured by Western Blot (WB) and immunohistochemistry. RESULTS: ANXA5 mRNA expression in PE samples was lower than 1% of its expression in normal samples (mean ± SD: 0.002 ± 0.004 vs. 0.55 ± 0.38, p < 0.001), while ANXA5 protein levels in PE samples were approximately at 65% of the average normal expression (mean ± SD: 0.53 ± 0.30 vs. 0.81 ± 0.25, p=0.001). Immunohistochemical analysis also verified the above results, since PE placentas tended to have low labelling indexes (LIs), in contrast to controls which demonstrated high LIs (p=0.020). Statistical analysis of the WB data revealed that ANXA5 protein expression was increased in PE smokers vs. PE non-smokers (mean ± SD: 0.64 ± 0.23 vs. 0.41 ± 0.33, p=0.027). CONCLUSIONS: These results suggest that ANXA5 downregulation could be part of the pathophysiology of PE and the possible impairment in coagulation processes, which are seen in pregnancies that demonstrate PE. Further studies may investigate whether ANXA5 could be used as a biomarker for the early detection of PE and for the prediction of its severity.
Asunto(s)
Anexina A5/biosíntesis , Preeclampsia/metabolismo , ARN Mensajero/biosíntesis , Adulto , Anexina A5/genética , Biomarcadores/análisis , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Placenta/metabolismo , Preeclampsia/genética , Embarazo , ARN Mensajero/genética , Adulto JovenRESUMEN
p53 is a tumour suppressor gene with an established role in the majority of human neoplasias. Its homologues-p63 and p73-cannot be classified as tumour suppressors, since they encode isoforms with oncogenic properties as well. p63 plays a crucial role in epithelial cell differentiation and p73 is essential for neuronal cell development. The p63 and p73 expressions have been investigated in a variety of human tumours including bladder carcinomas; yet, this is the first study to simultaneously analyse the transcriptional levels of all p53 family members in bladder cancer. Using quantitative real-time polymerase chain reaction, we measured the mRNA expression of p53, p63 and p73 in 30 bladder tumours, each paired with adjacent normal tissue. All three studied genes were up-regulated in malignant specimens, p53 by 1.9-fold, p63 by threefold and p73 by twofold, respectively. Further analysis suggested that p63 and p73 act independently of p53 in the malignant bladder epithelium. Statistical analysis revealed that p63 overexpression was more frequent in recurrent bladder tumours (p = 0.045) and in older patients (p = 0.022). Papillary tumours also exhibited abnormal p63 expression (p = 0.026). Finally, p73 was up-regulated in Grade III one-site tumours (p = 0.040). Our results indicate that all p53 family members are abnormally expressed in bladder cancer but do not act synergistically. High levels of p63 correlate with non-muscle invasive tumours with frequent relapses, whereas p73 overexpression is associated with a more aggressive tumour phenotype.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de la Membrana/biosíntesis , Recurrencia Local de Neoplasia/genética , Proteínas Nucleares/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Neoplasias de la Vejiga Urinaria/genética , Anciano , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Femenino , Humanos , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Growth factors mediate various cellular responses to environmental stimuli. Specifically, exposure of lung epithelium to oxidative stress induced by cigarette smoke stimulates aberrant epidermal growth factor receptor (ERBB) family activation. This study's objective was to evaluate the expression of ERBB1-4 receptors in the lung tissue of smokers with or without chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: ERBBs expression was measured by microarray analysis in lung tissue samples from five patients with COPD and five non-COPD smokers, and by quantitative real-time PCR in additional 20 patients with COPD (GOLD stage II), 15 non-COPD smokers and 10 nonsmoker controls. RESULTS: Microarray data analysis revealed that ERBB receptors expression was elevated in patients with COPD compared to non-COPD smokers, ranging from 1·62- to 2·45-fold, (P < 0·01). Real-time qPCR verified that patients with COPD had higher ERBB1-3 expression levels compared with non-COPD smokers (PERBB1 < 0·001; PERBB2 = 0·003; PERBB3 = 0·003) and nonsmokers (PERBB1 = 0·019; PERBB2 = 0·005; PERBB3 = 0·011). On the other hand, ERBB4 mRNA levels gradually increased from nonsmokers (0·74 ± 0·19) to non-COPD smokers (1·11 ± 0·05) to patients with COPD (1·57 ± 0·28) and were correlated with the degree of airflow obstruction (PFEV1 < 0·001). DISCUSSION: These data suggest that ERBB1-3 overexpression is not related only to smoking exposure but probably to epithelial remodelling and mucociliary system distortion, characterizing COPD. Additionally, the inverse correlation of ERBB4 with FEV1 exhibits a possible link between ERBB4 and COPD severity.
Asunto(s)
Obstrucción de las Vías Aéreas/metabolismo , Receptores ErbB/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ADN Complementario/biosíntesis , Volumen Espiratorio Forzado/fisiología , Humanos , Pulmón/metabolismo , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Fumar/metabolismo , Capacidad Vital/fisiologíaRESUMEN
BACKGROUND: Lung cytotoxic mechanisms trigger the release of perforin and granzymes, causing oxidative DNA damage that ultimately leads to apoptosis. These effects, although demonstrated in COPD, have not been investigated in patients with asthma and in particular in patients with asthma who smoke. Our aim was to measure perforin, granzyme A, granzyme B, and 8-OHdG expression in sputum from smoking and nonsmoking patients with asthma, compared with smoking and nonsmoking control subjects. METHODS: Perforin, granzyme A, granzyme B, and 8-OHdG expression levels were detected by enzyme-linked immunosorbent assays in induced sputum specimens. RESULTS: Perforin expression was increased in 40% of smokers and 45% of smoking patients with asthma and in only 7% of nonsmoking patients with asthma (P = .004), compared with control subjects' values. In contrast, granzymes A and B levels were increased in > 40% of patients in all three groups vs control subjects. Finally, 8-OHdG levels were elevated in 35% of smoking patients with asthma, in 20% of smokers, and in only 10% of nonsmoking patients with asthma. Statistical analysis revealed a positive correlation between granzyme A (P < .001) and granzyme B (P = .006) expression levels and the number of pack-years in smoking patients with asthma. CONCLUSIONS: Asthma cytotoxic immune response is mainly represented by granzymes A and B, whereas in smoking patients with asthma perforin and 8-OHdG are additionally involved, resembling the immune response in COPD.
Asunto(s)
Asma/genética , Asma/metabolismo , Daño del ADN , Desoxiguanosina/análogos & derivados , Granzimas/biosíntesis , Perforina/biosíntesis , Fumar/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Asma/complicaciones , Desoxiguanosina/biosíntesis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxidación-ReducciónRESUMEN
BACKGROUND: The mechanical stress that the human diaphragm is exposed to during mechanical ventilation affects a variety of processes, including signal transduction, gene expression, and angiogenesis. OBJECTIVES: The study aim was to assess the change in the production of major angiogenic regulators [vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF2), and transforming growth factor beta 1 (TGFB1)] on the human diaphragm before and after contraction/relaxation cycles during mechanical ventilation. METHODS: This observational study investigates the diaphragmatic mRNA expression of VEGF, FGF2, and TGFB1 in surgical patients receiving general anesthesia with controlled mechanical ventilation (CMV) with muscle relaxation (group A, n = 13), CMV without muscle relaxation (group B, n = 10), and pressure support of spontaneous breathing (group C, n = 9). Diaphragmatic samples were obtained from each patient at two time points: 30 min after the induction of anesthesia (t1) and 90 min after the first specimen collection (t2). RESULTS: No significant changes in the mRNA expression of VEGF, FGF2, and TGFB1 were documented in groups A and C between time points t1 and t2. In contrast, in group B, the mRNA levels of the above angiogenic factors were increased in time point t2 compared to t1, a finding which was statistically significant (pVEGF = 0.003, pFGF2 = 0.028, pTGFB1 = 0.001). CONCLUSIONS: These findings suggest that the molecular response of the human diaphragm before and after application of diverse modes of mechanical ventilation is different. Angiogenesis via the expression of VEGF, FGF2, and TGFB1 was only promoted in CMV without muscle relaxation, and this may have important clinical implications.
Asunto(s)
Diafragma/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Respiración Artificial , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto , Anestesia General , Femenino , Humanos , Persona de Mediana Edad , Relajación Muscular , Neovascularización FisiológicaRESUMEN
B-Raf, a member of the Raf serine/threonine kinase family, is an intermediate molecule in the mitogen-activated protein kinase pathway, which relays extracellular signals from the cell membrane to the nucleus via a cascade of phosphorylation events, ultimately promoting cancer development. This pathway is usually activated in human neoplasias. The purpose of this study was to investigate the role of B-Raf in thyroid pathology. We scanned for the presence of mutations at codon 600 (V â E) of the B-Raf gene, using a PCR-RFLP assay. In tumors with no mutation (32 benign and malignant thyroid tumors) and in their adjacent normal tissue, we measured the expression levels of B-Raf gene, using a quantitative real-time PCR (qPCR) assay. B-Raf expression in V600E-negative tumors deviated from the normal pattern, since it was overexpressed in 42 % of benign samples and downregulated in 54 % of malignant specimens. Hashimoto's thyroiditis also seemed to play an important role, since benign specimens with Hashimoto's thyroiditis had a 2.2-fold higher B-Raf expression than samples without thyroiditis (1.71 ± 0.63 vs. 0.78 ± 0.13). Statistical analysis revealed that B-Raf deregulation postponed disease onset by more than 10 years in both benign and malignant thyroid (benign: 55.6 ± 3.9 vs. 45.3 ± 3.3, p = 0.049; malignant: 52.2 ± 3.5 vs. 33.0 ± 7.9, p = 0.020). From the above results, we deduce that in the absence of mutation activation, B-Raf overexpression or downregulation is a protective event, since it delays the development of both malignant and benign thyroid tumors.
Asunto(s)
Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/genética , Edad de Inicio , Femenino , Enfermedad de Hashimoto/complicaciones , Enfermedad de Hashimoto/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Proto-Oncogénicas B-raf/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias de la Tiroides/complicaciones , Neoplasias de la Tiroides/metabolismo , TranscriptomaRESUMEN
Genetic, immune and environmental interactions are key elements for the development of COPD. Cigarette smoking is considered the primary risk factor initiating inflammatory cascades in genetically susceptible individuals. The "danger signals" elicited by the injured cells of non-specific immunity induce the downstream activation of proinflammatory cascades and antigen-specific adaptive immune responses. The produced oxidative stress further damages the lung leading to acquired genetic changes (histone deacetylation, microsatellite DNA instability, DNA methylation, telomere shortening, miRNA alterations) due to an inefficient DNA repair machinery. On the other hand, augmented apoptosis, impaired efferocytosis and abnormal tissue remodeling contribute to the chronic inflammatory response and tissue destruction in COPD. This review focuses on the role of genetic, epigenetic and immune mechanisms in the development of COPD in order to put forward possible prognostic and therapeutic targets.
Asunto(s)
Inflamación/fisiopatología , Terapia Molecular Dirigida , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Inmunidad Adaptativa , Animales , Apoptosis , Epigénesis Genética , Predisposición Genética a la Enfermedad , Humanos , Inflamación/etiología , Inflamación/terapia , Estrés Oxidativo , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/terapia , Factores de Riesgo , Fumar/efectos adversosRESUMEN
Heparansulfate proteoglycans (HSPG) play an important role in cell-cell and cell-matrix interactions and signaling, and one of the key enzymes in heparansulfate biosynthesis is d-glucuronyl C5-epimerase (GLCE). A tumor suppressor function has been demonstrated for GLCE in breast and lung carcinogenesis; however, no data are available as to the expression and regulation of the gene in prostate cancer. In this study, decreased GLCE expression was observed in 10% of benign prostate hyperplasia (BPH) tissues and 53% of prostate tumors, and increased GLCE mRNA levels were detected in 49% of BPH tissues and 21% of tumors. Statistical analysis showed a positive correlation between increased GLCE expression and Gleason score, TNM staging, and prostate-specific antigen (PSA) level in the prostate tumors (Pearson correlation coefficients GLCE/Gleason = 0.56, P < 0.05; GLCE/TNM = 0.62, P < 0.05; and GLCE/PSA = 0.88, P < 0.01), suggesting GLCE as a candidate molecular marker for advanced prostate cancer. Immunohistochemical analysis revealed an intratumoral heterogeneity of GLCE protein levels both in BPH and prostate cancer cells, resulting in a mixed population of GLCE-expressing and nonexpressing epithelial cells in vivo. A model experiment on normal (PNT2) and prostate cancer (LNCaP, PC3, DU145) cell lines in vitro showed a 1.5- to 2.5-fold difference in GLCE expression levels between the cancer cell lines and an overall decrease in GLCE expression in cancer cells. Methyl-specific polymerase chain reaction (PCR), bisulfite sequencing, and deoxy-azacytidin (aza-dC) treatment identified differential GLCE promoter methylation (LNCaP 70-72%, PC3 32-35%, DU145, and PNT2 no methylation), which seems to contribute to heterogeneous GLCE expression in prostate tumors. The obtained results reveal the complex deregulation of GLCE expression in prostatic diseases compared with normal prostate tissue and suggest that GLCE may be used as a potential model to study the functional role of intratumor cell heterogeneity in prostate cancer progression.
Asunto(s)
Carbohidrato Epimerasas/biosíntesis , Epigénesis Genética/genética , Neoplasias de la Próstata/genética , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carbohidrato Epimerasas/genética , Metilación de ADN , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Masculino , Estadificación de Neoplasias , Regiones Promotoras Genéticas , Antígeno Prostático Específico/metabolismo , Hiperplasia Prostática/enzimología , Hiperplasia Prostática/genética , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales CultivadasRESUMEN
Prohibitins (PHB1 and PHB2) are versatile proteins located at the inner mitochondrial membrane, maintaining normal mitochondrial function and morphology. They interact with the NADH dehydrogenase protein complex, which is essential for oxidoreductase activity within cells. However, their expression in lung epithelium, especially in smokers and patients with inflammatory lung diseases associated with increased oxidative stress, such as COPD, is unknown. Lung tissue specimens from 45 male subjects were studied: 20 COPD patients [age: 65.7 ± 5.8 years, smoking: 84.6 ± 33.6 pack-years, FEV(1) (%pred.): 58.7 ± 14.6, FEV(1)/FVC (%): 63.8 ± 9.4], 15 non-COPD smokers [age: 59.0 ± 12.1 years, smoking: 52.5 ± 20.8 pack-years, FEV(1) (%pred.): 85.5 ± 14.2, FEV(1)/FVC (%): 78.5 ± 4.7] and 10 non-smokers. Quantitative real-time PCR experiments were carried out for PHB1 and PHB2, using ß-actin as internal control. Non-COPD smokers exhibited lower PHB1 mRNA levels when compared to non-smokers (0.55 ± 0.06 vs. 0.90 ± 0.06, P = 0.043), while PHB1 expression was even further decreased in COPD patients (0.32 ± 0.02), a statistically significant finding vs. both non-COPD smokers (P = 0.040) and non-smokers (P < 0.001). By contrast, PHB2 levels were similar among the three study groups. Western blot analysis for the PHB1 protein verified the qPCR results (non-smokers: 1.77 ± 0.13; non-COPD smokers: 0.97 ± 0.08; COPD patients: 0.59 ± 0.10, P = 0.007). Further analysis revealed that PHB1 downregulation in COPD patients cannot be attributed solely to smoking, and that PHB1 expression levels are associated with the degree of airway obstruction [FEV(1) (P(mRNA) = 0.004, P(protein) = 0.014)]. The significant downregulation of PHB1 in COPD and non-COPD smokers in comparison to non-smokers possibly reflects a distorted mitochondrial function due to decreased mitochondrial stability, especially in the mitochondria of COPD patients.
Asunto(s)
Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Proteínas Represoras/metabolismo , Anciano , Western Blotting , ADN Complementario/metabolismo , Regulación hacia Abajo , Volumen Espiratorio Forzado/fisiología , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Estrés Oxidativo/fisiología , Prohibitinas , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Fumar/metabolismo , Capacidad Vital/fisiologíaRESUMEN
PURPOSE: p63 plays an important role in several intracellular processes such as transcription activation and apoptosis. p63 has two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of the p53 family and is considered oncogenic. Although p63 and its isoforms are overexpressed in a wide variety of human malignancies such as cervical, head and neck, and lung cancer, their role in endometrial carcinoma has not been investigated. METHODS: We measured by quantitative real-time polymerase chain reaction the mRNA expression of TAp63 and ΔNp63 in a series of 20 endometrioid adenocarcinomas paired with adjacent normal tissue. RESULTS: TAp63 isoform exhibited 1.8-fold overexpression in malignant samples, while ΔNp63 was 4.3-fold overexpressed in cancer specimens. Further analysis revealed that the ΔN/TA isoform ratio shifted from 0.5 in normal samples to 1.2 in tumor specimens. Statistical analysis also revealed an association of TAp63 expression with high body mass index (p = 0.034), late menopause (p = 0.020), and lower tumor grade (p = 0.034). ΔNp63 was also correlated with grade I/II tumors (p = 0.044). CONCLUSIONS: These results indicate that both p63 isoforms and especially ΔNp63 play an important role in the development and progression of grade I/II endometrial adenocarcinoma, especially in obese and late-menopause women.
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Adenocarcinoma/genética , Neoplasias Endometriales/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Endometriales/patología , Femenino , Humanos , Menopausia , Persona de Mediana Edad , Clasificación del Tumor , Obesidad/patología , Isoformas de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia Arriba/genéticaRESUMEN
Abnormal apoptotic events in chronic obstructive pulmonary disease (COPD) subvert cellular homeostasis and may play a primary role in its pathogenesis. However, studies in human subjects are limited. p53 and bcl2 protein expression was measured by western blot on lung tissue specimens from 43 subjects (23 COPD smokers and 20 non-COPD smokers), using beta-actin as internal control. Additionally, p53 and bcl2 expression patterns were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded lung tissue sections from the same individuals. Western blot analysis showed statistically significant increased p53 protein levels in COPD smokers in comparison with non-COPD smokers (p = 0.038), while bcl2 protein levels were not statistically different between the two groups. Lung immunohistochemistry showed increased ratio of positive p53-stained type II pneumocytes/total type II pneumocytes in COPD smokers compared to non-COPD smokers (p = 0.01), whereas the p53 staining ratio in alveolar macrophages and in lymphocyte-like cells did not differ statistically between the two groups. On the other hand, bcl2 expression did not differ between the two groups in all three cell types. The increased expression of pro-apoptotic p53 in type II pneumocytes of COPD patients not counterbalanced by the anti-apoptotic bcl2 could reflect increased apoptosis in the alveolar epithelium of COPD patients. Our results confirm previous experiments and support the hypothesis of a disturbance in the balance between the pro- and anti-apoptotic mediators in COPD.
Asunto(s)
Apoptosis , Pulmón/química , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Proteína p53 Supresora de Tumor/análisis , Anciano , Células Epiteliales Alveolares/química , Células Epiteliales Alveolares/patología , Western Blotting , Estudios de Casos y Controles , Humanos , Inmunohistoquímica , Pulmón/patología , Linfocitos/química , Linfocitos/patología , Macrófagos Alveolares/química , Macrófagos Alveolares/patología , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/patología , Transducción de Señal , Fumar , Regulación hacia ArribaRESUMEN
BACKGROUND: The placental anticoagulant protein Annexin A5 (ANXA5) is highly expressed on the apical surfaces of syncytiotrophoblasts and plays an important role in maintaining blood fluidity in the placental circulation. We investigated the mRNA and protein expression of ANXA5 in placentas from pregnancies complicated by fetal growth restriction (FGR) compared with uncomplicated pregnancies. MATERIALS AND METHODS: Placental tissue was collected from 18 pregnancies complicated by FGR and 16 pregnancies with a normal outcome. ANXA5 mRNA expression was quantified by Real-Time PCR (RT-PCR), and protein concentrations were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: A decreased ANXA5 mRNA expression was observed in placenta samples from FGR-affected pregnancies compared to those from uncomplicated pregnancies. However, similar ANXA5 protein levels were measured in both specimen groups. No correlation was observed between ANXA5 mRNA and protein levels. CONCLUSIONS: Transcriptional ANXA5 down-regulation was demonstrated in FGR-affected pregnancies, although protein levels were similar in FGR-related placentas and controls. We can speculate that either recruitment of the protein from the bloodstream or increased apoptosis or post-transcriptional modifications occur, which affect ANXA5 protein levels in FGR-related placentas. Further studies are required to reveal the role of ANXA5 in FGR pathology.
Asunto(s)
Anexina A5/metabolismo , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Placenta/metabolismo , ARN Mensajero/metabolismo , Animales , Anexina A5/genética , Apoptosis/genética , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Femenino , Mamíferos/genética , Mamíferos/metabolismo , Procedimientos Quirúrgicos Obstétricos , Circulación Placentaria/genética , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/metabolismoRESUMEN
In this study we examined by QRT-PCR the mRNA expression of TGF-beta 1, IGF-1, EGF, FGF-2 and YY1 in human brain tumors. Our findings introduce YY1, for the first time, as a novel gene implicated in brain gliomatogenesis and meningioma establishment. We present a positive correlation between the autocrine expression of YY1 and TGF-beta 1, IGF-1 and FGF-2, known to be involved in the progression of gliomas and meningiomas. We suggest that mRNA profiling of the above genes in the early stages of disease development could be useful for prognostic purposes, and these genes can be considered as potential targets for therapeutic approaches against brain tumors.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Glioma/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Meningioma/metabolismo , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta1/genética , Factor de Transcripción YY1/genética , Adulto , Anciano , Neoplasias Encefálicas/etiología , Femenino , Glioma/etiología , Humanos , Masculino , Meningioma/etiología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Peptide growth factors play an important role in several intracellular processes, such as cellular growth and differentiation, angiogenesis and apoptosis, as well as in carcinogenesis, since they contribute significantly to the malignant transformation. The prostate gland is abundant in growth factors. The two most known prostatic diseases, prostate cancer (PCa) and benign prostatic hyperplasia (BPH), are among the most common diseases that affect elderly men. This study was conducted using a quantitative real-time RT-PCR method in order to determine mRNA expression levels of peptide growth factors VEGF, FGF2, TGFB1, EGF, and IGF1 in tissue specimens from 42 patients with PCa, 42 with BPH, and 10 normal prostate samples obtained post-mortem from young individuals, in order to examine their association with prostatic hyperplasia and neoplasia. Our results show that in PCa, growth factors VEGF, EGF and FGF2 are overexpressed, while TGFB1 and IGF1 have reduced mRNA levels. In BPH, transcript levels of FGF2 and EGF are normal, while VEGF, TGFB1 and IGF1 exhibit downregulation. Further statistical analysis revealed that PCa patients with high levels of PSA blood levels have decreased FGF2 expression (p=0.016). Additionally, cancer patients with low Gleason score (<7) have increased EGF (p=0.035) and IGF1 (p=0.031) mRNA levels. IGF1 levels are also elevated in tumors with TNM stages T1-T2 (p=0.030). In BPH, older patients have reduced EGF expression (p=0.018), while IGF1 is overexpressed in younger patients (p=0.041). Additionally, the co-expression pattern of the five studied growth factors differs significantly among normal, benign and malignant prostate. These results implicate VEGF, FGF2, TGFB1, EGF and IGF1 in the development of both PCa and BPH, rendering them potential targets for disease detection, monitoring and therapy.
Asunto(s)
Factor de Crecimiento Epidérmico/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Regulación Neoplásica de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Hiperplasia Prostática/patología , Neoplasias de la Próstata/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: The deleted in liver cancer-1 (DLC-1) gene that encodes a Rho GTPase-activating protein with tumor suppressor function is located on chromosome 8p21-22, a region frequently deleted in prostate carcinomas. This study was designed to determine whether DLC-1 is deregulated in prostate carcinomas and to assess the contribution of DLC-1 alterations to prostate carcinogenesis. EXPERIMENTAL DESIGN: Primary prostate carcinomas, prostate carcinoma cell lines, benign prostatic hyperplasias, and normal prostatic tissues were examined for detection of functional and structural alterations of the DLC-1 gene by real-time PCR, methylation-specific PCR, and Southern and Western blots. RESULTS: Down-regulation or loss of DCL-1 mRNA expression was detected in 10 of 27 (37%) prostate carcinomas, 3 of 5 (60%) prostate carcinoma cell lines, and 5 of 21 (24%) benign prostatic hyperplasias. DLC-1 promoter methylation was identified in 13 of 27 (48%) prostate carcinomas and 2 matching normal tissues and in 15 of 21 (71%) benign prostatic hyperplasias but was absent in 10 normal prostatic tissues from noncancerous individuals. Genomic deletions were found in only 3 prostate carcinomas and 1 benign prostatic hyperplasia. DLC-1 protein was not detected in 8 of 27 (30%) prostate carcinomas and 11 of 21 (52%) benign prostatic hyperplasias. Methylation of DLC-1 correlated with age in prostate carcinoma patients (P = 0.006) and with prostate-specific antigen blood levels in benign prostatic hyperplasia patients (P = 0.029). Treatment of the three prostate carcinoma cell lines (PC-3, LNCaP, and 22Rv1) expressing a low level of DLC-1 transcripts with inhibitors of DNA methyltransferase or histone deacetylase increased DLC-1 expression. CONCLUSIONS: These results show that the transcriptional silencing of DLC-1 by two epigenetic mechanisms is common and may be involved in the pathogenesis of prostate carcinomas and benign prostatic hyperplasias and could have potential clinical application in the early detection and gene therapy of prostate cancer.
Asunto(s)
Metilación de ADN , Eliminación de Gen , Neoplasias de la Próstata/genética , Proteínas Supresoras de Tumor/genética , Acetilación , Adulto , Anciano , Anciano de 80 o más Años , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Southern Blotting , Western Blotting , Inmunoprecipitación de Cromatina , Decitabina , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Proteínas Activadoras de GTPasa , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Próstata/metabolismo , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
p73, a p53 homologue important for growth suppression, differentiation and induction of apoptosis, utilizes different promoters and undergoes alternative splicing to produce several isoforms differing in their ability to overlap p53 functions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) to assess the mRNA levels of p53, p73 (total and isoforms specific for exons 2 and 13), MDM2, CDKN1A and beta2-microglobulin as internal control, we analyzed 35 prostate carcinomas and 44 benign prostate hyperplasias (BPH) compared to 14 normal prostates. Shift of p73 isoform mRNA levels from exon 13 lacking to exon 13 containing copies was observed in 80% of prostate cancer cases and in 52.3% of BPH specimens, and from exon 2 containing to exon 2 lacking (p73Deltaexon2) transcripts in 45.7% of cancer cases, but only in 9.1% of BPH samples. From these findings we deduce that p73 isoform balance is disrupted in prostate cancer and BPH, suggesting that this disequilibrium could play an important role in both prostate hyperplasia and malignancy.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/genética , Genes Supresores de Tumor , Humanos , Masculino , Estadificación de Neoplasias , Proteínas Nucleares/genética , Próstata/metabolismo , Próstata/patología , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Isoformas de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
p53 codon 72 Arg homozygosity has been associated with increased risk of developing cervical cancer. This association has been tested in various human cancers with controversial results. In the present study we investigated the impact of this polymorphism in a population-based case-control study of bladder cancer. Using allele-specific polymerase chain reaction to detect the p53 codon 72 polymorphism, we tested peripheral blood samples from 50 patients with bladder cancer and 99 healthy individuals of similar age and from the same geographical region. Tumor specimens from all bladder cancer patients were examined for the presence of human papilloma virus (HPV). The distribution of p53 alleles in bladder cancer patients and in controls was statistically significant (P<0.002; odds ratio, 2.67; 95% confidence interval, 1.38-5.20), and homozygosity for arginine at residue 72 was associated with an increased risk for bladder cancer (P<0.00002; odds ratio, 4.69; 95% confidence interval, 2.13-10.41). The presence of HPV was found in six of the 50 patients (12%). This is the first study correlating p53 codon 72 polymorphism with bladder cancer. Our results provide evidence that this p53 polymorphism is implicated in bladder carcinogenesis and that individuals harboring the Arg/Arg genotype have an increased risk of developing bladder cancer.