RESUMO

A poly(thiophene acetic acid)/Au/poly(methylene blue) nanostructured interface was electrochemically assembled step-by-step on screen-printed carbon electrodes (SPCE) for label-free detection of p53 protein. The initial electrical conductive properties of the polymeric interface were increased with an additional layer of poly(methylene blue) electropolymerized in the presence of gold nanoparticles. The nano-immunosensing architecture was prepared by covalent immobilization of anti-p53 antibodies as bioreceptors through the poly(thiophene acetic acid) moieties. The nano-immunosensor assembly was extensively characterized by ultraviolet-visible spectrophotometry, dynamic and electrophoretic light scattering, scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, atomic force microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Under optimal conditions, p53 was specifically and selectively detected by square wave voltammetry in a linear range between 1 and 100 ng mL-1 with a limit of detection of 0.65 ng mL-1. In addition, the electrochemical nano-immunosensor detected p53 in spiked human serum samples and colorectal cancer cell lysates, and the results were validated with a standard spectrophotometric method using a paired samples t test, which did not exhibit significant differences between both methods. The resultant p53 nano-immunosensor is simple to assemble, robust, and has the potential for point-of-care biomarker detection applications.

Assuntos

RESUMO

Background: Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

RESUMO

Background: Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: β-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

RESUMO

Background: Endogenous phospholipases A2 (PLA2 ) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM médium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.(AU)

Assuntos

RESUMO

In Southern and Southeastern Brazil, there is a germline pathogenic variant with incomplete penetrance located in the oligomerization domain of TP53, c.1010G>A (p.Arg337His). Due to a founder effect, the variant is present in 0.3% of the general population of the region. Recently, this variant was identified in 4.4 and 8.9% of two apparently unselected, single center case series of Brazilian lung adenocarcinoma (LUAD) patients from the Southeastern and Central regions of the country, respectively. In the present study, our aim was to examine TP53 c.1010G>A allele and genotype frequencies in LUAD samples obtained from patients diagnosed in Southern Brazil. A total of 586 LUAD samples (tumor DNA) recruited from multiple centers in the region were tested, and the mutant allele was identified using TaqMan® assays in seven cases (7/586, 1.2%) which were submitted to next generation sequencing analyses for confirmation. Somatic EGFR mutations were more frequent in TP53 c.1010G>A carriers than in non-carriers (57.1 vs. 17.6%, respectively). Further studies are needed to confirm if TP53 c.1010G>A is a driver in LUAD carcinogenesis and to verify if there is a combined effect of EGFR and germline TP53 c.1010G>A. Although variant frequency was higher than observed in the general population, it is less than previously reported in LUAD patients from other Brazilian regions. Additional data, producing regional allele frequency information in larger series of patients and including cost-effectiveness analyses, are necessary to determine if TP53 c.1010G>A screening in all Brazilian LUAD patients is justified.

RESUMO

The tumor suppressor protein p53 is often called "the genome guardian" and controls the cell cycle and the integrity of DNA, as well as other important cellular functions. Its main function is to trigger the process of apoptosis in tumor cells, and approximately 50% of all cancers are related to the inactivation of the p53 protein through mutations in the TP53 gene. Due to the association of mutant p53 with cancer therapy resistance, different forms of restoration of p53 have been subject of intense research in recent years. In this sense, this review focus on the main currently adopted approaches for activation and reactivation of p53 tumor suppressor function, focusing on the synthetic approaches that are involved in the development and preparation of such small molecules.

Assuntos

RESUMO

BACKGROUND/AIM: Although risk stratification using the Prognostic Scores Systems (IPSS, WPSS and IPSS-R) incorporate key information about prognosis of patients with Myelodysplastic syndromes (MDS), patients classified as low-risk may evolve rapidly and aggressively, despite a "favorable" prognostic stratification. The aim of this study was to identify biomarkers for predicting prognosis, and for better stratification and management of these patients. MATERIALS AND METHODS: Expression of CD34 and p53 in megakaryocytes was examined by immunohistochemistry in 71 MDS patients classified as low-risk. RESULTS: CD34 staining in megakaryocytes was associated with p53 expression (p=0.0166). CD34 and p53 expression were associated to worse overall survival in patients (p=0.0281). CONCLUSION: The presence of CD34 in megakaryocytes is associated with p53 expression and an adverse prognosis for MDS patients.

Assuntos

RESUMO

OBJECTIVE: To evaluate the prognostic significance of microvessel density and p53 expression in pancreatic cancer. METHODS: Between 2008 and 2012, 49 patients with pancreatic adenocarcinoma underwent resection with curative intention. The resected specimens were immunohistochemically stained with anti-p53 and anti-CD34 antibodies. Microvessel density was assessed by counting vessels within ten areas of each tumoral section a highpower microscope. RESULTS: The microvessel density ranged from 21.2 to 54.2 vessels/mm2. Positive nuclear staining for p53 was found in 20 patients (40.6%). The overall median survival rate after resection was 24.1 months and there were no differences in survival rates related to microvessel density or p53 positivity. Microvessel density was associated with tumor diameter greater than 3.0 cm and with R0 resection failure. CONCLUSIONS: Microvessel density was associated with R1 resection and with larger tumors. p53 expression was not correlated with intratumoral microvessel density in pancreatic adenocarcinoma.

Assuntos

RESUMO

El Ameloblastoma Acantomatoso es una variante histopatológica donde la parte central de las células Epiteliales Odontogénicas sufren una metaplasia escamosa. Este cambio biológico podría darle a esta forma de ameloblastoma características de mayor agresividad y recidiva. El objetivo del presente trabajo fue determinar la expresión de las proteínas p53 y Ki-67 en ameloblastomas acantomatosos. Metodología: 12 especímenes diagnosticados histopatológicamente como ameloblastoma acantomatoso fueron examinados y la expresión de p53 y Ki-67 determinada inmunohistoquímicamente usando los anticuerpos anti-humanos clones DO-7 y MIB-1 respectivamente (DAKO®). La expresión de ambas proteínas fue evaluada mediante microscopio de luz y la intensidad y número de núcleos positivos semicuantificados y analizados mediante estadística descriptiva. Resultados: La expresión de p53 fue observada en el 50% de los casos tanto en las células basales como centrales de las islas tumorales. La expresión de Ki-67 fue observada en el 66,7% de los casos, en su mayoría tanto en las células basales como tumorales y en su totalidad de fuerte intensidad. Conclusiones: La expresión de p53 y Ki-67 en los ameloblastomas acantomatosos sugiere una proliferación celular acelerada y por ende una mayor capacidad de crecimiento. Estas proteínas podrían conformar una herramienta para la selección de tratamientos más radicales en ameloblastomas que las expresen

Acanthomatous Ameloblastoma is a histopathological variant where the central portions of odontogenic epithelial cells undergo squamous metaplasia. This biological change could confer more aggressive features and recurrence to the tumour. The aim of this study was to determine the expression of p53 and Ki-67 in acanthomatous ameloblastoma. Methods: 12 specimens histologically diagnosed as acanthomatous ameloblastomas were examined and the expression of p53 and Ki-67 determined immunohistochemically using anti-human antibody clone DO-7 and MIB-1 respectively (DAKO ®). The expression of both proteins was assessed by light microscopy and the intensity and number of positive cells semi-quantified and analyzed using descriptive statistics. Results: The expression of p53 was observed in 50% of the cases both in the basal cells as tumor central islands. The Ki-67 expression was observed in intensely in 66.7% of cases, mainly on basal cell as central. Conclusions: The expression of p53 and Ki-67 in acanthomatous ameloblastomas suggests accelerated cell proliferation and therefore increased capacity for growth. Expression of these proteins may constitute a tool for prognosis and treatment selection

Assuntos

RESUMO

INTRODUÇÃO E OBJETIVO: O carcinoma de células renais (CCR) é umas das doenças urológicas de maior malignidade, correspondendo a 1 por cento-3 por cento de todos os tumores sólidos dos adultos. As lesões pré-malignas do CCR ainda não foram totalmente descritas. Achados de displasia intraepitelial tubular renal (DIETR) em áreas próximas ao tumor, livres de neoplasia, sugerem que esta alteração possa ser a precursora biológica de alguns tipos de CCR. O objetivo deste estudo é avaliar a possibilidade do uso de tissue microarray (TMA) no estudo da DIETR e do CCR e os parâmetros para sua utilização imuno-histoquímica (IHQ) utilizando os anticorpos anti-Ki-67 e p53 para confirmar a evidência de DIETR como precursora biológica do CCR. MATERIAL E MÉTODOS: O estudo foi realizado em 10 peças cirúrgicas de nefrectomias realizadas em pacientes com diagnóstico de CCR selecionadas no período de 2000 a 2005, em material arquivado no Instituto Nacional de Câncer (INCA). DISCUSSÃO: A utilização da técnica de TMA permite amostragem de várias áreas e realização da IHQ utilizando apenas duas lâminas, uma para cada marcador. Como não há na literatura descrição de utilização de TMA para estudo da DIETR, foi necessário inicialmente realizar estudo de validação para saber se haveria representatividade de lesões focais no TMA. RESULTADOS: A análise estatística não mostrou diferença entre a utilização de TMA com dois ou quatro cilindros de tecido ou o corte inteiro. CONCLUSÃO: O teste de validação de técnica artesanal de TMA autorizou o uso de dois cilindros de 1 mm² por caso.

INTRODUCTION AND OBJECTIVE: Renal cell carcinoma is one of the most malignant urological diseases, corresponding to 1 percent-3 percent of solid tumors in adults. The finding of intratubular epithelial dysplasia (IED) next to tumor areas suggests that this can be the biological precursor of some types of renal cell carcinoma (RCC). The objective of this study is to evaluate the use of tissue microarray (TMA) to study IED and RCC by immunohistochemistry (IHC) with Ki-67 and p53 protein to confirm the evidence that IED is the biological precursor of RCC. MATERIAL AND METHOD: This study was performed in archival material from ten nephrectomy surgical specimens from Instituto Nacional de Câncer, between 2000 and 2005. DISCUSSION: TMA technique allows to sample and study several different areas from many patients in the same slide, using just one slide for each IHC marker. Once there was no previous study on IED by TMA, a validation test was needed first. RESULTS: There was no statistical difference between 2 or 4 tissue cores and the whole section analysis. CONCLUSION: TMA validation assay demonstrated that the use of two tissue cores with 1 mm² each was suitable to evaluate RCC and IED.

RESUMO

Due to its prominent role in major excretory pathways, the kidney is particularly sensitive especially to toxicity for antimicrobials drugs. Storage of these drugs in the renal cortex, their effect on renal cells, have consequences on the renal function, and then reabsorbed by renal tubules induce nephrotixicity. Our objective was to show the renal morphopatological alterations induced by gentamicin through the histochemical methods of routine periodic acid de Schiff (PAS) staining and imunohistochemical staining for the expression of the protein P53, which is considered as a marker for cellular apoptosis. This allows the early detection of tubular lesions. The renal morphopathologic findings were cell apoptosis, basal membrane interruption, mesangial proliferation cells, decreased Bowman's space. This result clearly shows that gentamicin administration induces renal morphopatological alterations.

Debido a su importante rol en la función de excreción mayor, el riñon es especialmente propenso a la toxicidad por los antibióticos bactericidas. La acumulación de los antibióticos aminoglicosidos en la corteza renal tiene como consecuencia efectos en las células renales y en la función renal y cuando son reabsorbidos por los túbulos renales, pueden conducir a toxicidad renal. Nuestro objetivo fue mostrar alteraciones morfopatológicas renales causadas por la administración de gentamicina, a través de métodos histoquímicos de rutina con ácido periódico de Schiff (PAS) y tinción inmunohistoquímica para la expresión de la proteína P53, la cual es considerada como un marcador para la apoptosis celular, permitiendo la detección precoz de lesiones tubulares. Los resultados morfopatológicos renales fueron apoptosis celular, interrupción de la membrana basal, proliferación de células mesangiales y disminución del espacio de Bowman. Los resultados mostraron claramente que la administración de gentamicina induce alteraciones morfopatológicas renales.

Assuntos

RESUMO

Los meningiomas son neoplasias primarias frecuentes del sistema nervioso central, usualmente benignas y susceptibles de curación mediante cirugía. El grado histológico de la Organización Mundial de la Salud y la extensión de la resección quirúrgica inicial son factores pronósticos determinantes en estos tumores. Sin embargo, una tasa de recidiva cercana al 20 por ciento en meningiomas benignos totalmente resecados plantea la necesidad de considerar nuevos factores pronósticos. Un total de 93 casos fueron seleccionados para el estudio inmunohistoquímico de la proteína p53 en relación con el grado histológico y riesgo de recidivas en los meningiomas. El índice de marcaje inmunohistoquímico de la proteína p53 se incrementó con la progresión del grado histológico (promedios de 37,15 por ciento para el grado I, 44,49 por ciento para el II y 57,81 por ciento para el III) y fue significativamente superior en meningiomas recidivantes (46,19 por ciento vs 32,29 por ciento de los no recidivantes). El punto de corte de 40 por ciento resultó estadísticamente útil para separar a los meningiomas benignos (grado I) de los no benignos (grados II-III) y a los tumores recidivantes de los no recidivantes. De esta manera, un índice de marcaje de la proteína p53 igual o mayor de 40 por ciento puede indicar la posibilidad de un grado más alto en casos histológicamente ambiguos o un riesgo mayor para desarrollar recidivas en meningiomas de un grado histológico en particular (especialmente en tumores benignos)

Meningiomas are frequent primary neoplasms of the central nervous system, usually benign and susceptible to healing through surgery. The histological grade of the World Health Organization and the extension of the initial surgical resection, are determining prognostic factors in these tumors. Nevertheless, a recurrence rate close to 20 percent in benign meningiomas completely resected arises the need of considering new prognostic factors. A total of 93 cases were selected for the immunohistochemical study of p53 protein in relation to the histological grade and the risk of recurrences in meningiomas. The immunohistochemical labeling index of p53 protein increased with the progression of the histological grade (means of 37,15 percent for grade I, 44,49 percent for grade II, and 57,81 percent for grade III) and was significantly superior in recurrent meningiomas (46,19 percent vs 32,29 percent of non recurrent). The cut off of 40 percent became statistically useful to separate the benign meningiomas (grade I) from the non benign (grade II and III) and the recurrent tumors from the non recurrent. In this way, a labelling index of p53 protein equal or superior to 40 percent can indicate the possibility of a higher grade in histologically ambiguous cases or a higher risk to develop recurrencies in meningiomas of a particular histological grade (specially in benign tumors)

Assuntos

RESUMO

Glioblastoma é um dos tumores primários mais letais do sistema nervoso central (SNC). Apesar dos significativos progressos, há poucas análises em crianças. Com o objetivo de avaliar localização, idade, sexo, sobrevida e imunoistoquímica para proteína p53, foram coletados casos de glioblastomas pediátricos do "Banco de Tumores do SNC de Curitiba", durante 1987-2003 e do Hospital Municipal Jesus, Rio de Janeiro, de 1970 a 1988. Doze preencheram os critérios de inclusão. A idade variou até 12 anos (média 7), sendo sete do sexo feminino e cinco do masculino. A sobrevida média foi 7,9 meses. Localizavam-se em hemisférios cerebrais (58,4 por cento), mesencéfalo e tronco (33,3 por cento) e um no cerebelo. A imunoistoquímica demonstrou p53 positivo em 9 (75 por cento). Em conclusão, glioblastoma tem comportamento semelhante entre crianças e adultos, sendo nestas menos freqüentes. Acomete hemisférios cerebrais com maior freqüência que estruturas infratentoriais, mostrando alta sensitividade com a imunomarcação para proteína p53, sendo nestes casos mais agressivos, com menor sobrevida.

Glioblastoma is one of the most lethal central nervous system (CNS) primary tumor. Although significant progress, only few analysis have been made in pediatric glioblastoma, which are less common and have worse prognosis than in adults. To evaluate gender, site, age, survival, and immunohistochemistry to p53, we selected cases of pediatric glioblastoma of "CNS Tumors Database in Curitiba", 1987-2003 and of the Hospital Municipal Jesus, Rio de Janeiro, 1970-1988. Twelve tumors were included. The age ranged from up to 12 years (median 7). There were 7 females and 5 males. The median survival was 7.9 months. Location was: cerebral hemispheres (58.4 percent), mesencephalon and brainstem (33.3 percent) and one case in the cerebellum. Immunostained to p53 in 9 (75 percent) cases. In conclusion, glioblastoma behaves similarly in children and adults. It is rare in children, affects both cerebral hemispheres more than brainstem and cerebellum and shows strong immunohistochemistry to p53.

Assuntos

RESUMO

Objetivo: Os carcinomas epidermóides do trato aerodigestivo superior são tumores de comportamento biológico heterogêneo. O objetivo deste trabalho é verificar se a expressão imuno-histoquímica dos marcadores Ki67, PCNA e P53 apresenta correlações com parâmetros prognósticos clínico-patológicos. Métodos: Determinação da expressão imuno-histoquímica dos antígenos Ki67, PCNA e P53 em espécimes tumorais fixados e embebidos em parafina de 53 pacientes com carcinoma epidermóide em diferentes sítios primários do trato aerodigestivo superior. Resultados: Os marcadores tiveram altos índices de expressão imuno-histoquímica, sendo 46,5% para o Ki67, 66,5% para o PCNA e 36,5% para o P53. Não houve correlação da expressão do Ki67 e do PCNA com o estadiamento TNM (AJCC), nem com o grau de malignidade. Aexpressão do Ki67 apresentou correlação positiva com a expressão do PCNA (p = 0,037). O mesmo aconteceupara o PCNA e o número de mitoses por campo (p = 0,001). Conclusões: De acordo com estes resultados, concluiu-se que a determinação da imunorreatividade dos marcadores Ki67 e PCNA é um método objetivo e quantificável para avaliar proliferação celular que pode subsidiar as informações prognósticas.

Background: Squamous cell carcinomas of upper aerodigestive tract are tumors with heterogeneous biological behaviour. The aim of present study is to assess whether any correlation exists between immunohistochemical expression of Ki67, PCNA and P53 markers and clinicopathologic parameters. Methods: Immunohistochemical determination of Ki67, PCNA and P53 antigens in fixated and paraffinembedded tumour especimens from 53 patients with squamous cell carcinoma in different primary locations of upper aerodigestive tract. Results: The markers had high evidence average rates with 46,5% forKi67, 66,5% for PCNA and 36,5% for P53. There was neither correlation of Ki67 and PCNA evidence nor with TNM staging nor with malignancy degree. The expression of Ki67 demonstrated correlation with the expression of PCNA (p = 0,037). The same ocurred to PCNA and the number of mitoses (p = 0,001). Conclusions: According to these results, it has been concluded that the determination of immunoreactivityof Ki67 and PCNA proliferation markers is an accurate and quantitative method which may provide prognostic information.