RESUMO
The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Membrana Basal , Citoplasma , Células Epiteliais , Fatores de Transcrição , Proteínas de Sinalização YAP , Animais , Humanos , Camundongos , Células Epiteliais/metabolismo , Proteínas de Sinalização YAP/metabolismo , Feminino , Citoplasma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Membrana Basal/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/citologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/citologia , Núcleo Celular/metabolismo , Transdução de SinaisRESUMO
Amyotrophic lateral sclerosis damages proteostasis, affecting spinal and upper motor neurons earlier than a subset of cranial motor neurons. To aid disease understanding, we exposed induced cranial and spinal motor neurons (iCrMNs and iSpMNs) to proteotoxic stress, under which iCrMNs showed superior survival, quantifying the transcriptome and proteome for >8,200 genes at 0, 12, and 36 h. Two-thirds of the proteome showed cell-type differences. iSpMN-enriched proteins related to DNA/RNA metabolism, and iCrMN-enriched proteins acted in the endoplasmic reticulum (ER)/ER chaperone complex, tRNA aminoacylation, mitochondria, and the plasma/synaptic membrane, suggesting that iCrMNs expressed higher levels of proteins supporting proteostasis and neuronal function. When investigating the increased proteasome levels in iCrMNs, we showed that the activity of the 26S proteasome, but not of the 20S proteasome, was higher in iCrMNs than in iSpMNs, even after a stress-induced decrease. We identified Ublcp1 as an iCrMN-specific regulator of the nuclear 26S activity.
Assuntos
Esclerose Lateral Amiotrófica , Proteostase , Humanos , Proteostase/fisiologia , Proteoma/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo EndoplasmáticoRESUMO
Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines. Key features ⢠Protocol for fractionation of active nuclear and cytoplasmic proteasome complexes. ⢠Native electrophoresis and fluorescence-based in-gel activity assay, which allows the visualization and quantification of active complexes within the acrylamide gel matrix. ⢠In-gel protein denaturation followed by transfer of complexes to PVDF membrane, which allows the analysis of complexes' abundance using antibodies.
RESUMO
The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFßTrCP E3 ubiquitin ligase and silencing ßTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that ßTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.
Assuntos
Serina-Treonina Quinase 3 , Ubiquitina-Proteína Ligases , Proteínas Contendo Repetições de beta-Transducina , Humanos , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Matriz Extracelular/metabolismo , Fosforilação , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Serina-Treonina Quinase 3/metabolismoRESUMO
Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.
Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Neoplasias da Mama/metabolismo , Feminino , Humanos , Integrina alfaV/genética , Integrina alfaV/metabolismo , Prognóstico , RNA Mensageiro/genéticaRESUMO
In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting â¼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.
Assuntos
Células-Tronco Embrionárias , Células-Tronco Pluripotentes , Animais , Humanos , Camundongos , Diferenciação Celular/genética , Sistemas CRISPR-Cas , Células-Tronco Embrionárias/metabolismo , Camadas Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In steroid-producing cells, cholesterol transport from the outer to the inner mitochondrial membrane is the first and rate-limiting step for the synthesis of all steroid hormones. Cholesterol can be transported into mitochondria by specific mitochondrial protein carriers like the steroidogenic acute regulatory protein (StAR). StAR is phosphorylated by mitochondrial ERK in a cAMP-dependent transduction pathway to achieve maximal steroid production. Mitochondria are highly dynamic organelles that undergo replication, mitophagy and morphology changes, all processes allowed by mitochondrial fusion and fission, known as mitochondrial dynamics. Mitofusin (Mfn) 1 and 2 are GTPases involved in the regulation of fusion, while dynamin-related protein 1 (Drp1) is the major regulator of mitochondrial fission. Despite the role of mitochondrial dynamics in neurological and endocrine disorders, little is known about fusion/fission in steroidogenic tissues. In this context, the present work aimed to study the role of angiotensin II (Ang II) in protein subcellular compartmentalization, mitochondrial dynamics and the involvement of this process in the regulation of aldosterone synthesis. We demonstrate here that Ang II stimulation promoted the recruitment and activation of PKCε, ERK and its upstream kinase MEK to the mitochondria, all of them essential for steroid synthesis. Moreover, Ang II prompted a shift from punctate to tubular/elongated (fusion) mitochondrial shape, in line with the observation of hormone-dependent upregulation of Mfn2 levels. Concomitantly, mitochondrial Drp1 was diminished, driving mitochondria toward fusion. Moreover, Mfn2 expression is required for StAR, ERK and MEK mitochondrial localization and ultimately for aldosterone synthesis. Collectively, this study provides fresh insights into the importance of hormonal regulation in mitochondrial dynamics as a novel mechanism involved in aldosterone production.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Carcinoma Adrenocortical/metabolismo , Angiotensina II/farmacologia , Colesterol/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Quinases/metabolismo , Vasoconstritores/farmacologia , Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Neoplasias das Glândulas Suprarrenais/patologia , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/patologia , Transporte Biológico , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
Tissue architecture and cell-extracellular matrix (cell-ECM) interaction determine the organ specificity; however, the influences of these factors on anticancer drugs preclinical studies are highly neglected. For considering such aspects, three-dimensional (3D) cell culture models are relevant tools for accurate analysis of cellular responses to chemotherapy. Here we compared the MCF-7 breast cancer cells responses to cisplatin in traditional two-dimensional (2D) and in 3D-reconstituted basement membrane (3D-rBM) cell culture models. The results showed a substantial increase of cisplatin resistance mediated by 3D microenvironment. This phenotype was independent of p53 status and autophagy activity and was also observed for other cellular models, including lung cancer cells. Such strong decrease on cellular sensitivity was not due to differences on drug-induced DNA damage, since similar levels of γ-H2AX and cisplatin-DNA adducts were detected under both conditions. However, the processing of these cisplatin-induced DNA lesions was very different in 2D and 3D cultures. Unlike cells in monolayer, cisplatin-induced DNA damage is persistent in 3D-cultured cells, which, consequently, led to high senescence induction. Moreover, only 3D-cultured cells were able to progress through S cell cycle phase, with unaffected replication fork progression, due to the upregulation of translesion (TLS) DNA polymerase expression and activation of the ATR-Chk1 pathway. Co-treatment with VE-821, a pharmacological inhibitor of ATR, blocked the 3D-mediated changes on cisplatin response, including low sensitivity and high TLS capacity. In addition, ATR inhibition also reverted induction of REV3L by cisplatin treatment. By using REV3L-deficient cells, we showed that this TLS DNA polymerase is essential for the cisplatin sensitization effect mediated by VE-821. Altogether, our results demonstrate that 3D-cell architecture-associated resistance to cisplatin is due to an efficient induction of REV3L and TLS, dependent of ATR. Thus co-treatment with ATR inhibitors might be a promising strategy for enhancement of cisplatin treatment efficiency in breast cancer patients.
Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias da Mama/tratamento farmacológico , Microambiente Celular/efeitos dos fármacos , Cisplatino/farmacologia , Células A549 , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células/métodos , Senescência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cisplatino/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Histonas/metabolismo , Humanos , Células MCF-7 , Pirazinas/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sulfonas/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cells from prokaryota to the more complex metazoans cease proliferating at some point in their lives and enter a reversible, proliferative-dormant state termed quiescence. The appearance of quiescence in the course of evolution was essential to the acquisition of multicellular specialization and compartmentalization and is also a central aspect of tissue function and homeostasis. But what makes a cell cease proliferating even in the presence of nutrients, growth factors, and mitogens? And what makes some cells "wake up" when they should not, as is the case in cancer? Here, we summarize and discuss evidence showing how microenvironmental cues such as those originating from metabolism, extracellular matrix (ECM) composition and arrangement, neighboring cells and tissue architecture control the cellular proliferation-quiescence decision, and how this complex regulation is corrupted in cancer.
RESUMO
Nuclear actin (N-actin) is known to participate in the regulation of gene expression. We showed previously that N-actin levels mediate the growth and quiescence of mouse epithelial cells in response to laminin-111 (LN1), a component of the mammary basement membrane (BM). We know that BM is defective in malignant cells, and we show here that it is the LN1/N-actin pathway that is aberrant in human breast cancer cells, leading to continuous growth. Photobleaching assays revealed that N-actin exit in nonmalignant cells begins as early as 30 min after LN1 treatment. LN1 attenuates the PI3K pathway leading to upregulation of exportin-6 (XPO6) activity and shuttles actin out of the nucleus. Silencing XPO6 prevents quiescence. Malignant cells are impervious to LN1 signaling. These results shed light on the crucial role of LN1 in quiescence and differentiation and how defects in the LN1/PI3K/XPO6/N-actin axis explain the loss of tissue homeostasis and growth control that contributes to malignant progression.
Assuntos
Actinas/metabolismo , Neoplasias da Mama/metabolismo , Carioferinas/metabolismo , Laminina/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Actinas/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Carioferinas/genética , Laminina/genética , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Throughout postnatal development, the gastric epithelium expresses Transforming Growth Factor beta1 (TGFß1), but it is also exposed to luminal peptides that are part of milk. During suckling period, fasting promotes the withdrawal of milk-born molecules while it stimulates gastric epithelial cell proliferation. Such response can be reversed by exogenous TGFß1, as it directly affects cell cycle through the regulation of p27 levels. We used fasting condition to induce the hyperproliferation of gastric epithelial cells in 14-day-old Wistar rats, and evaluated the effects of TGFß1 gavage on p27 expression, phosphorylation at threonine 187 (phospho-p27Thr187) and degradation. p27 protein level was reduced during fasting when compared to suckling counterparts, while phospho-p27Thr187/p27 ratio was increased. TGFß1 gavage reversed this response, which was confirmed through immunostaining. By using a neutralizing antibody against TGFß1, we found that it restored the p27 and phosphorylation levels detected during fasting, indicating the specific role of the growth factor. We noted that neither fasting nor TGFß1 changed p27 expression, but after cycloheximide administration, we observed that protein synthesis was influenced by TGFß1. Next, we evaluated the capacity of the gastric mucosa to degrade p27 and we recorded a higher concentration of the remaining protein in pups treated with TGFß1, suggesting augmented stability under this condition. Thus, we showed for the first time that luminal TGFß1 increased p27 levels in the rat gastric mucosa by up- regulating translation and reducing protein degradation. We concluded that such mechanisms might be used by rapidly proliferating cells to respond to milk-born TGFß1 and food restriction.
Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Western Blotting , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/genética , Masculino , Fosforilação , Proteólise , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/genéticaRESUMO
OBJECTIVE: To assess whether the month of birth in different latitudes of South America might influence the presence or severity of multiple sclerosis (MS) later in life. METHODS: Neurologists in four South American countries working at MS units collected data on their patients' month of birth, gender, age, and disease progression. RESULTS: Analysis of data from 1207 MS patients and 1207 control subjects did not show any significant variation in the month of birth regarding the prevalence of MS in four latitude bands (0-10; 11-20; 21-30; and 31-40 degrees). There was no relationship between the month of birth and the severity of disease in each latitude band. CONCLUSION: The results from this study show that MS patients born to mothers who were pregnant at different Southern latitudes do not follow the seasonal pattern observed at high Northern latitudes.
Assuntos
Progressão da Doença , Esclerose Múltipla/epidemiologia , Parto , Adulto , Métodos Epidemiológicos , Feminino , Humanos , Masculino , Esclerose Múltipla/etiologia , Estações do Ano , América do Sul/epidemiologia , Topografia MédicaRESUMO
Objective To assess whether the month of birth in different latitudes of South America might influence the presence or severity of multiple sclerosis (MS) later in life. Methods Neurologists in four South American countries working at MS units collected data on their patients' month of birth, gender, age, and disease progression. Results Analysis of data from 1207 MS patients and 1207 control subjects did not show any significant variation in the month of birth regarding the prevalence of MS in four latitude bands (0–10; 11–20; 21–30; and 31–40 degrees). There was no relationship between the month of birth and the severity of disease in each latitude band. Conclusion The results from this study show that MS patients born to mothers who were pregnant at different Southern latitudes do not follow the seasonal pattern observed at high Northern latitudes. .
Objetivo Avaliar se o mês de nascimento em diferentes latitudes da América do Sul pode influenciar a presença ou gravidade da esclerose múltipla (EM) na vida. Método Neurologistas de quatro países da América do Sul trabalhando em unidades de EM coletaram os dados de seus pacientes com referência ao mês de nascimento, gênero, idade e progressão da doença. Resultados A análise dos dados mostrou que, para 1207 pacientes com EM e 1207 controles, não havia diferença significativa no mês de nascimento com relação à prevalência de EM em quatro zonas de latitude (0–10; 11–20; 21–30; e 31–40 graus). Não houve relação entre o mês de nascimento e a gravidade da doença em nenhuma destas zonas. Conclusão Os resultados deste estudo mostram que pacientes com EM nascidos de mães grávidas em diferentes latitudes sul não seguem o padrão dos resultados sazonais encontrados nas latitudes norte. .
Assuntos
Adulto , Feminino , Humanos , Masculino , Progressão da Doença , Esclerose Múltipla/epidemiologia , Parto , Métodos Epidemiológicos , Esclerose Múltipla/etiologia , Estações do Ano , América do Sul/epidemiologia , Topografia MédicaAssuntos
Declaração de Nascimento , Clima , Esclerose Múltipla/epidemiologia , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-beta/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-beta and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-beta and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-beta/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.
Assuntos
Ativinas/fisiologia , Proteínas Smad Reguladas por Receptor/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Adenoma/metabolismo , Carcinoma Papilar, Variante Folicular/metabolismo , Bócio Nodular/metabolismo , Humanos , Transdução de Sinais/fisiologia , Proteína Smad2/análise , Proteína Smad3/análise , Proteína Smad4/análise , Proteína Smad7/análiseRESUMO
OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.
OBJETIVO: Investigar a expressão de proteínas SMAD em tecidos de tiroide humana desde que a inativação dos componentes da sinalização de TGF-β/activina é relatada em diversos tipos de câncer. SMAD 2 e SMAD3 fosforilados (pSMAD2/3) associados com SMAD4 induzem a transmissão do sinal gerado por TGF-β e activina, enquanto SMAD7 inibe essa sinalização intracelular. Embora TGF-β e activina exerçam efeitos antiproliferativos nas células foliculares da tiroide, tumores de tiroide expressam altos níveis dessas proteínas. MATERIAIS E MÉTODOS: A expressão proteica de SMADs foi avaliada em bócio multinodular, adenoma folicular, carcinomas papilífero e folicular por imuno-histoquímica. RESULTADOS: A expressão de pSMAD2/3, SMAD4 e SMAD7 foi observada tanto em tumores benignos como malignos da tiroide. Embora pSMAD2/3, SMAD4 e SMAD7 exibissem alta positividade citoplasmática em carcinomas, a positividade nuclear de pSMAD2/3 não foi diferente entre lesões benignas e malignas da tiroide. CONCLUSÕES: O achado da expressão de SMADs em células tiroidianas e a presença das proteínas pSMAD2/3 e SMAD4 no núcleo de células tumorais indicam propagação da sinalização TGF-β/activina. Contudo, a alta expressão de SMAD7 inibitório, principalmente em tumores malignos, poderia contribuir para atenuação da sinalização antiproliferativa de SMADs em carcinomas de tiroide.
Assuntos
Humanos , Ativinas/fisiologia , Proteínas Smad Reguladas por Receptor/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Adenoma/metabolismo , Carcinoma Papilar, Variante Folicular/metabolismo , Bócio Nodular/metabolismo , Transdução de Sinais/fisiologia , /análise , /análise , /análise , /análiseRESUMO
BACKGROUND: Papillary thyroid carcinoma (PTC) is frequently associated with a RET gene rearrangement that generates a RET/PTC oncogene. RET/PTC is a fusion of the tyrosine kinase domain of RET to the 5' portion of a different gene. This fusion results in a constitutively active MAPK pathway, which plays a key role in PTC development. The RET/PTC3 fusion is primarily associated with radiation-related PTC. Epidemiological studies show a lower incidence of PTC in radiation-exposed regions that are associated with an iodine-rich diet. Since the influence of excess iodine on the development of thyroid cancer is still unclear, the aim of this study is to evaluate the effect of high iodine concentrations on RET/PTC3-activated thyroid cells. METHODS: PTC3-5 cells, a rat thyroid cell lineage harboring doxycycline-inducible RET/PTC3, were treated with 10(-3) M NaI. Cell growth was analyzed by cell counting and the MTT assay. The expression and phosphorylation state of MAPK pathway-related (Braf, Erk, pErk, and pRet) and thyroid-specific (natrium-iodide symporter [Nis] and thyroid-stimulating hormone receptor [Tshr]) proteins were analyzed by Western blotting. Thyroid-specific gene expression was further analyzed by quantitative reverse transcription (RT)-polymerase chain reaction. RESULTS: A significant inhibition of proliferation was observed, along with no significant variation in cell death rate, in the iodine-treated cells. Further, iodine treatment attenuated the loss of Nis and Tshr gene and protein expression induced by RET/PTC3 oncogene induction. Finally, iodine treatment reduced Ret and Erk phosphorylation, without altering Braf and Erk expression. CONCLUSION: Our results indicate an antioncogenic role for excess iodine during thyroid oncogenic activation. These findings contribute to a better understanding of the effect of iodine on thyroid follicular cells, particularly how it may play a protective role during RET/PTC3 oncogene activation.
Assuntos
Iodo/administração & dosagem , Coativadores de Receptor Nuclear/genética , Fusão Oncogênica/efeitos dos fármacos , Oncogenes/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret/genética , Glândula Tireoide/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Contagem de Células , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Coativadores de Receptor Nuclear/metabolismo , Fusão Oncogênica/genética , Oncogenes/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Fatores de TempoRESUMO
Multiple sclerosis is a chronic disease characterized by demyelination and neurodegeneration of the central nervous system. Immunomodulatory treatment is possible at an early stage of the disease, and consists of injections of either beta-interferon or glatiramer acetate. The drugs are not curative, and the need for frequent injections may give rise to a serious problem regarding adherence to treatment. The present study analyzed the database of all Brazilian patients using glatiramer acetate between June 2003 and December 2006 who had enrolled in the patient program run by the pharmaceutical company commercializing the drug. The rate of treatment discontinuation was 10% over this period, and the main reason for suspending the drug was medical decisions (47% of all discontinuations), rather than side effects or the patient's choice. The present work did not take into consideration the regularity of injections and the main objective was to assess discontinuation. It was concluded that adequate healthcare, education, and a specific program for patients were the factors responsible for this 90% adherence to glatiramer acetate treatment in Brazil.
RESUMO
OBJECTIVE: To register multiple sclerosis (MS) patients residing in the coastal region of the State of São Paulo (CEREM Litoral Paulista). METHOD: Individual interviews with identified cases of MS. RESULTS: 81 individuals with diagnosis of MS agreed to come for registration (62 females [76.5%], 19 males [23.5%]). 65% of all patients were residents of the city of Santos. The mean age of these patients was 43 years (14 to 74 years), and the Expanded Disability Status Scale (EDSS) was < or = 5.5 in 76.5% of the cases. 82.7% of the assessed patients presented the relapsing/remitting form of MS. 81.5% of all patients were undergoing treatment with immunomodulators. CONCLUSION: Due to their clinical profile, patients seem to come to CEREM Litoral Paulista for prescription of immunomodulators. There is a clear need to identify other cases in the region and to allow other forms of treatment to be put into practice.
Assuntos
Esclerose Múltipla/epidemiologia , Adolescente , Adulto , Idoso , Brasil/epidemiologia , Censos , Feminino , Seguimentos , Humanos , Fatores Imunológicos/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Satisfação do Paciente , Prevalência , Estudos RetrospectivosRESUMO
AIM: To assess tolerability, adverse events and compliance to treatment with glatiramer acetate in multiple sclerosis. METHOD: Review of patient records and individual interviews. RESULTS: 30 individuals residing in the coastal region of the State of São Paulo who had been in use of glatiramer acetate for at least 6 months were identified. From this group, 28 individuals came to regular consultations and were individually assessed, their complaints being noted down in confidential records. Ten patients reported systemic reactions to the drug. Four of them stopped the medication due to such reactions. Eight patients reported local reactions to the injections. Compliance with injections was achieved, although three patients reported forgetting the injection on a few days. CONCLUSION: We noticed a higher level of systemic adverse events in our patients than in reports in the literature.