RESUMEN
In pituitary tumors, P27(CDKN1B) is underexpressed. We aimed to clarify whether translational regulation underlies this phenomenon. This study evaluated the expression of P27/CDKN1B, its targets (CCNE1, CDK2) and translational regulators (DKC1, RPS13, miR221, miR222) and screened for DKC1 variants in sporadic pituitary adenomas. Samples were obtained during transsphenoidal surgery from 48 patients with pituitary adenomas: 10 ACTH-, 17 GH-secreting, and 21 nonfunctioning (NFPA). The control group comprised 7 normal pituitaries (NP) obtained during autopsies. Gene expression was assessed by RT-PCR and protein expression by immunohistochemistry. The 15 exons of DKC1 were sequenced. P27 protein underexpression was observed in all adenomas subtypes (p=0.001). CCNE1 mRNA (p=0.01) overexpression, but not protein, was observed in NFPA. No differential gene expression among groups was observed in CDKN1B regulators RPS13 (p=0.23) and DKC1 (p=0.34). The expression of miR221 and miR222 was similar among tumors and NP. Frequent DKC1 variants (SNPs) were found in exon 14 and in the 3'-UTR in similar frequency to NCBI-dsSNP databases. We also observed rare DKC1 variants in 11% of the studied tumor samples, indicating a high prevalence in pituitary adenomas, however, in silico studies failed to indicate deleterious effects. The high frequency of DKC1 variants may influence, in some extent, pituitary tumors development, without clear role in its tumorigenesis. Our data reinforce the P27 underexpression in pituitary adenomas and provide further evidence of the post-translational machinery involvement, although this phenomenon cannot be explained either by mis-expression of P27 translational regulators - DKC1, RPS13, miR221, miR222 - or directly by DKC1 mutations.
Asunto(s)
Carcinogénesis/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias Hipofisarias/metabolismo , Biosíntesis de Proteínas , Secuencia de Bases , Carcinogénesis/patología , Proteínas de Ciclo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Hipófisis/metabolismo , Hipófisis/patología , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Estándares de ReferenciaRESUMEN
PURPOSE: Telomere dysfunction and telomerase activation underlie cancer transformation. This study aims to investigate the contribution of telomere biology to pituitary tumor behavior. SUBJECTS AND METHODS: Samples from 50 patients with pituitary tumors (11 ACTH-secreting, 18 GH-secreting, and 21 non-secreting tumors) and 7 subjects without pituitary lesions were collected. The expressions of telomerase essential components TERT and TERC and tumor telomere content were measured by quantitative PCR techniques. RESULTS: Telomerase (TERT) expression was detected in 36% of tumors. No correlation was observed between TERT and TERC expression level and tumor size in any tumor type. There was no association between gene expression and clinical findings. Telomere content (T/S ratio) was similar between pituitary adenomas (0.39 ± 0.16) and normal pituitaries (0.47 ± 0.12; p = 0.24) and also was between the different adenoma types: ACTH-secreting (0.43 ± 0.08), GH-secreting (0.31 ± 0.12), and non-secreting (0.42 ± 0.20; p = 0.10) tumors. CONCLUSIONS: The telomere content and expression of telomerase components are comparable between normal pituitary glands and tumor tissues, suggesting that telomere biology does not play an important role in pituitary tumor development.
Asunto(s)
Expresión Génica/fisiología , Neoplasias Hipofisarias/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Adulto , Humanos , Persona de Mediana Edad , Neoplasias Hipofisarias/enzimología , ARN/metabolismoRESUMEN
Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98 th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.
Asunto(s)
Cerebelo/metabolismo , Miosina Tipo V/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Cadáver , Niño , Preescolar , Electroforesis en Gel de Agar , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Adulto JovenRESUMEN
Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.
Asunto(s)
Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Adulto Joven , Cerebelo/metabolismo , Miosina Tipo V/metabolismo , Factores de Edad , Cadáver , Electroforesis en Gel de Agar , Immunoblotting , InmunohistoquímicaRESUMEN
Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.
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Neoplasias de la Corteza Suprarrenal/genética , Adolescente , Neoplasias de la Corteza Suprarrenal/patología , Niño , Preescolar , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Factor II del Crecimiento Similar a la Insulina/genética , MasculinoRESUMEN
Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.
Asunto(s)
Colorantes/farmacología , Técnicas de Preparación Histocitológica/métodos , Amoníaco , Compuestos Azo , Encéfalo , Fluorescencia , Técnica del Anticuerpo Fluorescente , Técnica del Anticuerpo Fluorescente Directa , Formaldehído , Secciones por Congelación , Técnicas Histológicas , Humanos , Indicadores y Reactivos , Naftalenos , Hibridación de Ácido Nucleico , Adhesión en Parafina , FotoblanqueoRESUMEN
Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.
Asunto(s)
Cadherinas/genética , Perfilación de la Expresión Génica , Neoplasias Neuroepiteliales/genética , Adolescente , Adulto , Encéfalo/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Neoplasias Neuroepiteliales/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.
Asunto(s)
Humanos , Adolescente , Adulto , Persona de Mediana Edad , Cadherinas/genética , Perfilación de la Expresión Génica , Neoplasias Neuroepiteliales/genética , Cerebro/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Neuroepiteliales/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
We describe the relative frequency, clinical features, neuroimaging and pathological results, and outcome after pharmacological or surgical intervention for a series of pediatric patients with temporal lobe epilepsy (TLE) from an epilepsy center in Brazil. The medical records of children younger than 12 years with features strongly suggestive of TLE were reviewed from January 1999 to June 1999. Selected children were evaluated regarding clinical, EEG, and magnetic resonance imaging (MRI) investigation and divided into three groups according to MRI: group 1 (G1, N = 9), patients with hippocampal atrophy; group 2 (G2, N = 10), patients with normal MRI, and group 3 (G3, N = 12), patients with other specific temporal lesions. A review of 1732 records of children with epilepsy revealed 31 cases with TLE (relative frequency of 1.79 percent). However, when the investigation was narrowed to cases with intractable seizures that needed video-EEG monitoring (N = 68) or epilepsy surgery (N = 32), the relative frequency of TLE increased to 19.11 (13/68) and 31.25 percent (10/32), respectively. At the beginning of the study, 25 of 31 patients had a high seizure frequency (80.6 percent), which declined to 11 of 31 (35.5 percent) at the conclusion of the study, as a consequence of pharmacological and/or surgical therapy. This improvement in seizure control was significant in G1 (P < 0.05) and G3 (P < 0.01) mainly due to good postsurgical outcome, and was not significant in G2 (P > 0.1, McNemar's test). These results indicate that the relative frequency of TLE in children was low, but increased considerably among cases with pharmacoresistant seizures. Patients with specific lesions were likely to undergo surgery, with good postoperative outcomes.
Asunto(s)
Humanos , Masculino , Femenino , Preescolar , Niño , Epilepsia del Lóbulo Temporal/cirugía , Hipocampo/patología , Lóbulo Temporal/patología , Atrofia , Electroencefalografía/métodos , Imagen por Resonancia Magnética , Periodo Posoperatorio , Estudios Retrospectivos , Resultado del Tratamiento , Grabación en VideoRESUMEN
We describe the relative frequency, clinical features, neuroimaging and pathological results, and outcome after pharmacological or surgical intervention for a series of pediatric patients with temporal lobe epilepsy (TLE) from an epilepsy center in Brazil. The medical records of children younger than 12 years with features strongly suggestive of TLE were reviewed from January 1999 to June 1999. Selected children were evaluated regarding clinical, EEG, and magnetic resonance imaging (MRI) investigation and divided into three groups according to MRI: group 1 (G1, N = 9), patients with hippocampal atrophy; group 2 (G2, N = 10), patients with normal MRI, and group 3 (G3, N = 12), patients with other specific temporal lesions. A review of 1732 records of children with epilepsy revealed 31 cases with TLE (relative frequency of 1.79%). However, when the investigation was narrowed to cases with intractable seizures that needed video-EEG monitoring (N = 68) or epilepsy surgery (N = 32), the relative frequency of TLE increased to 19.11 (13/68) and 31.25% (10/32), respectively. At the beginning of the study, 25 of 31 patients had a high seizure frequency (80.6%), which declined to 11 of 31 (35.5%) at the conclusion of the study, as a consequence of pharmacological and/or surgical therapy. This improvement in seizure control was significant in G1 (P < 0.05) and G3 (P < 0.01) mainly due to good postsurgical outcome, and was not significant in G2 (P > 0.1, McNemar's test). These results indicate that the relative frequency of TLE in children was low, but increased considerably among cases with pharmacoresistant seizures. Patients with specific lesions were likely to undergo surgery, with good postoperative outcomes.
Asunto(s)
Epilepsia del Lóbulo Temporal/patología , Hipocampo/patología , Lóbulo Temporal/patología , Atrofia , Niño , Preescolar , Electroencefalografía/métodos , Epilepsia del Lóbulo Temporal/cirugía , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Periodo Posoperatorio , Estudios Retrospectivos , Resultado del Tratamiento , Grabación en VideoRESUMEN
The objective of the present study was to assess the effectiveness of a combined protocol of muscle stretching and strengthening after immobilization of the hindlimb. Thirty female Wistar rats were divided into 6 groups: group immobilized for 14 days to cause full plantar flexion by cast (GI, n = 6); group immobilized/stretched (GIS, n = 6): submitted to the same immobilization and to 10 days of passive stretching; group immobilized/electrically stimulated (GIES, n = 6): similarly immobilized and submitted to 10 days of low frequency electrical stimulation (ES); group immobilized/stretched/electrically stimulated (GISES, n = 6): similarly immobilized, submitted to 10 days of stretching and ES application; group immobilized/free (GIF, n = 3): similarly immobilized and then left with free limbs for 10 days; control group (CG, n = 3). The middle portion of the soleus muscle was frozen and sections were stained with HE or mATPase. Morphological analysis revealed high cellular reactivity in the GISES, GIES and GIS groups. The lesser diameter and proportion of type I fibers (TIF) and type II fibers (TIIF) (at pH 9.4) and connective area (at HE stain) were measured with an image analyzer and the data obtained were analyzed statistically by the unpaired Student t-test (p < or = 0.05). The results indicated that: a) immobilization generated atrophy of both fiber types (p < 0.05); b) joint application of ES and stretching was not efficient in reestablishing the size of the two fiber types compared to CG (p < 0.05); c) the ES protocol reestablished only the size of TIIF, which showed values similar to those detected in CG (p < 0.05); d) the stretch increased the proliferation of the perimysium connective tissue (p < 0.05). Thus, we conclude that, in the model applied here to female rats, a stretching protocol may limit the volume protein gain of soleus muscle fibers and increase the connective interstitial tissue.
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Músculo Esquelético/patología , Animales , Proliferación Celular , Estimulación Eléctrica , Femenino , Concentración de Iones de Hidrógeno , Procesamiento de Imagen Asistido por Computador , Inmovilización , Modelos Estadísticos , Contracción Muscular , Fibras Musculares Esqueléticas/patología , Atrofia Muscular , Ratas , Ratas WistarRESUMEN
Costicosteroides sistemicos em altas doses podem causar miopatia metabolica. O Objetivo deste estudo foi valiar, por meio de ensaios de tracao, os efeitos da miopatia induzida por corticosteroides nas propriedades mecanicas do musculo gastrocnemio medial de coelhos. Foram estudados dois grupos de 15 coelhas da raca Nova Zelandia: grupo experimental (GE), que recebeu injecoes subcutaneas de metil-prednisolona (2mg/kg/dia), e grupo-controle (GC), que recebeu solucao fisiologica por via subcutanea. Os grupos foram tratados por 21 dias. Foram feitos ensaios de tracao nos musculos gastrocnemios mediais esquerdos. Resultados: o peso final dos animais do GE foi 3,6+-0,1kg e do GC, 40+-0,1kg. O peso final do gastrocnemio do GE foi 5,6+-1,0g e do GC, 7,0+-1,3g. Os valores de area, largura e espessura do gastrocnemio...
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Corticoesteroides , Fenómenos BiomecánicosRESUMEN
BACKGROUND: Although neuroimage and surgical techniques have improved substantially, the prognosis of patients with astrocytic tumors remains unchanged. The purpose of this study was to evaluate the proliferative activity in astrocytic tumors in different grades of malignancy and correlate it to other clinical features. PATIENTS AND METHODS: From archival paraffin-embedded surgical specimens of 40 patients of the Ribeirão Preto Medical School with World Health Organization grade II (n = 10), grade III (n = 5) and grade IV astrocytomas (n = 25), the MIB-1 labeling index (LI) was determined using at least a half of the blocks per case. The results were correlated to the biological behavior of the tumors. The aims of this study were to determine the level of MIB-1 LI values (cut-off) that reflect differences in biological behavior of these tumors, the impact on survival of clinical features such as age, tumor location or extension of surgical removal as well as the adjuvant therapy. RESULTS AND CONCLUSIONS: As expected, a wide range of MIB-1 LI values was disclosed (mean of 2.35% in grade II astrocytomas to 12.28% in glioblastomas). A close relationship was found between MIB-1 LI and survival of patients with astrocytomas according to the histological grade. All but 1 recurrent tumor presented higher MIB-1 LI in the second biopsy, and the mean MIB-1 LI of the patients who died in the immediate postoperative period (n = 7) was higher in comparison to the MIB-1 LI of the respective grade. Postoperative radiation therapy was an important factor that affected the survival of patients with high-grade astrocytomas (p = 0.006). MIB-1 LI cut-off of 3% divided the astrocytomas in 2 groups with significantly different survival (p < 0.001): median survival time of 12 months (low-grade) versus 45 months (high-grade). On the other hand, univariate analysis did not show any correlation between survival and extension of surgical resection (radical versus partial), tumor's location or patient's age at surgery.
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Astrocitoma/metabolismo , Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/metabolismo , Antígeno Ki-67/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Astrocitoma/mortalidad , Astrocitoma/patología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Niño , Preescolar , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pronóstico , Valores de Referencia , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
Castleman's disease is an atypical lymphoproliferative disorder that may present as a localized or multicentric form. The involvement of the central nervous system is rare. We describe here a case of Castleman's disease with involvement of the hypothalamus and meninges, presenting as hypopituitarism. Radiological and clinical pathological features are emphasized and a review of the literature is presented.