RESUMO
BACKGROUND: Most multiple sclerosis (MS) patients succumb to a progressive phenotype. Continued lymphocyte activity in the brain, microglia-mediated injury, iron deposition, and oxidative stress are characteristics of progressive MS. OBJECTIVE: As minocycline and hydroxychloroquine have been shown to inhibit microglia, we evaluated their effects on other outcomes relevant for progression. METHODS: Medications were evaluated in culture and in mice with acute and chronic experimental autoimmune encephalomyelitis (EAE). RESULTS: Both medications individually reduced iron neurotoxicity and a combination effect was not observed. Hydroxyl radical scavenging activity was manifested by minocycline only. Minocycline reduced T-cell proliferation more prominently than hydroxychloroquine; an aggregate effect occurred at low but not high concentrations. B-cell proliferation was mitigated to a greater extent by hydroxychloroquine and an additive effect was not evident. In EAE, suboptimal doses of minocycline and hydroxychloroquine individually delayed onset of clinical signs, while their combination suppressed clinical manifestations until treatment was stopped. In Biozzi ABH mice, a model of progressive MS, the chronic phase was beneficially altered using the combination. CONCLUSION: While minocycline and hydroxychloroquine did not manifest additive effects in most culture assays, their combination at suboptimal doses in EAE unexpectedly exceeded their individual activity. Minocycline and hydroxychloroquine combined are candidate treatments for progressive MS.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Hidroxicloroquina/farmacologia , Minociclina/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Biozzi , Camundongos Endogâmicos C57BL , Esclerose Múltipla , Neurônios/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.
Assuntos
Proteínas Cromossômicas não Histona/análise , Ensaios de Triagem em Larga Escala/economia , Histonas/análise , Técnicas Analíticas Microfluídicas/instrumentação , Peptídeos/análise , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Polarização de Fluorescência , Histonas/química , Histonas/metabolismo , Humanos , Ligantes , Técnicas Analíticas Microfluídicas/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
p21 is a potent cyclin-dependent kinase inhibitor that plays a role in promoting G1 cell cycle arrest and cellular senescence. Consistent with this role, p21 is a downstream target of several tumour suppressors and oncogenes, and it is downregulated in the majority of tumours, including breast cancer. Here, we report that protein arginine methyltransferase 6 (PRMT6), a type I PRMT known to act as a transcriptional cofactor, directly represses the p21 promoter. PRMT6 knock-down (KD) results in a p21 derepression in breast cancer cells, which is p53-independent, and leads to cell cycle arrest, cellular senescence and reduced growth in soft agar assays and in severe combined immunodeficiency (SCID) mice for all the cancer lines examined. We finally show that bypassing the p21-mediated arrest rescues PRMT6 KD cells from senescence, and it restores their ability to grow on soft agar. We conclude that PRMT6 acts as an oncogene in breast cancer cells, promoting growth and preventing senescence, making it an attractive target for cancer therapy.
Assuntos
Neoplasias da Mama/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos SCID , Proteínas Nucleares/genética , Fenótipo , Regiões Promotoras Genéticas , Proteína-Arginina N-Metiltransferases/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Matrix metalloproteinases (MMPs) are engaged in pathologies associated with infections, tumors, autoimmune disorders and neurological dysfunctions. With the identification of an upstream regulator of MMPs, EMMPRIN (Extracellular matrix metalloproteinase inducer, CD147), it is relevant to address if EMMPRIN plays a role in the pathology of central nervous system (CNS) diseases. This would enable the possibility of a more upstream and effective therapeutic target. Indeed, conditions including gliomas, Alzheimer's disease (AD), multiple sclerosis (MS), and other insults such as hypoxia/ischemia show elevated levels of EMMPRIN which correlate with MMP production. In contrast, given EMMPRIN's role in CNS homeostasis with respect to regulation of monocarboxylate transporters (MCTs) and interactions with adhesion molecules including integrins, we need to consider that EMMPRIN may also serve important regulatory or protective functions. This review summarizes the current understanding of EMMPRIN's involvement in CNS homeostasis, its possible roles in escalating or reducing neural injury, and the mechanisms of EMMPRIN including and apart from MMP induction.
Assuntos
Basigina/metabolismo , Sistema Nervoso Central/metabolismo , Metaloproteinases da Matriz/metabolismo , Basigina/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Sistema Nervoso Central/patologia , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Terapia de Alvo Molecular , Transportadores de Ácidos Monocarboxílicos/metabolismoRESUMO
EMMPRIN (CD147), originally described as an inducer of the expression of MMPs, has gained attention in its involvement in various immunologic diseases, such that anti-EMMPRIN antibodies are considered as potential therapeutic medications. Given that MMPs are involved in the pathogenesis of various disease states, it is relevant that targeting an upstream inducer would make for an effective therapeutic strategy. Additionally, EMMPRIN is now appreciated to have multiple roles apart from MMP induction, including in cellular functions, such as migration, adhesion, invasion, energy metabolism, as well as T cell activation and proliferation. Here, we review what is known about EMMPRIN in numerous immunologic/inflammatory disease conditions with a particular focus on its complex roles in T cell biology.
Assuntos
Basigina/fisiologia , Doenças do Sistema Imunitário/fisiopatologia , Linfócitos T/fisiologia , Animais , Basigina/genética , Humanos , Doenças do Sistema Imunitário/imunologia , Camundongos , Camundongos KnockoutRESUMO
While there is evidence that specific T cell populations can promote the growth of established tumors, instances where T cell activity causes neoplasms to arise de novo are infrequent. Here, we employed two conditional mutagenesis systems to delete the TGF-ß signaling pathway component Smad4 in T cells and observed the spontaneous development of massive polyps within the gastroduodenal regions of mice. The epithelial lesions contained increased levels of transcripts encoding IL-11, IL-6, TGF-ß, IL-1ß, and TNF-α, and lamina propria cells isolated from lesions contained abundant IL-17A+CD4+ T cells. Furthermore, we found that Smad4 deficiency attenuated TGF-ß-mediated in vitro polarization of FoxP3+CD4+ T cells, but not IL-17A+CD4+ T cells, suggesting that the epithelial lesions may have arisen as a consequence of unchecked Th17 cell activity. Proinflammatory cytokine production likely accounted for the raised levels of IL-11, a cytokine known to promote gastric epithelial cell survival and hyperplasia. Consistent with IL-11 having a pathogenic role in this model, we found evidence of Stat3 activation in the gastric polyps. Thus, our data indicate that a chronic increase in gut Th17 cell activity can be associated with the development of premalignant lesions of the gastroduodenal region.