RESUMO
Thymic epithelial cells (TECs) play a central role in T-cell development by presenting self-antigens on MHC proteins. Double-positive (DP) thymocytes that fail to interact with TEC via their TCR die by 'Death by Neglect'. We demonstrated a role for TEC-derived glucocorticoids (GCs) in this process. In a previous study, we used an in vitro system recapitulating Death by Neglect, to demonstrate the involvement of nitric oxide (NO) and inducible NO synthase (iNOS) in this process. In this study, we show that NO synergizes with GCs to induce apoptosis of DP thymocytes in a fetal thymic organ culture. Also, DP thymocytes from iNOSâ»/â» mice are less sensitive to GC-induced apoptosis. Furthermore, the number of DP thymocytes in iNOSâ»/â» mice is higher than in wild-type mice, suggesting a role for NO in Death by Neglect. This phenomenon effects T-cell function profoundly: iNOSâ»/â» T cells do not respond to TCR-mediated activation signals, measured by up-regulation of CD69, IL-2R and IFNγ secretion. This failure to activate is a result of TCR incompetence because iNOâ»/â» T cells respond to TCR-independent stimuli (phorbol myristate acetate and calcium ionophore). This study suggests that NO and GCs synergize to execute TEC-induced death of DP thymocytes.
Assuntos
Apoptose , Células Epiteliais/efeitos dos fármacos , Glucocorticoides/farmacologia , Óxido Nítrico/metabolismo , Células Precursoras de Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Timo/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Autoantígenos/imunologia , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Seleção Clonal Mediada por Antígeno/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Células Precursoras de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Linfócitos T/imunologiaRESUMO
The mechanisms by which glucocorticoid receptor (GR) mediates glucocorticoid (GC)-induced apoptosis are unknown. We studied the role of mitochondrial GR in this process. Dexamethasone induces GR translocation to the mitochondria in GC-sensitive, but not in GC-resistant, T cell lines. In contrast, nuclear GR translocation occurs in all cell types. Thymic epithelial cells, which cause apoptosis of the PD1.6 T cell line in a GR-dependent manner, induce GR translocation to the mitochondria, but not to the nucleus, suggesting a role for mitochondrial GR in eliciting apoptosis. This hypothesis is corroborated by the finding that a GR variant exclusively expressed in the mitochondria elicits apoptosis of several cancer cell lines. A putative mitochondrial localization signal was defined to amino acids 558-580 of human GR, which lies within the NH2-terminal part of the ligand-binding domain. Altogether, our data show that mitochondrial and nuclear translocations of GR are differentially regulated, and that mitochondrial GR translocation correlates with susceptibility to GC-induced apoptosis.
Assuntos
Apoptose , Glucocorticoides/metabolismo , Mitocôndrias/metabolismo , Receptores de Glucocorticoides/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Dexametasona , Células Epiteliais/metabolismo , Humanos , TimoRESUMO
In addition to variations on the spatial scale, short- and long-term temporal variations, too, can impose intense selection on the overall genetic diversity and composition of a population. We hypothesized that the allelic composition in populations of the eastern spadefoot toad (Pelobates syriacus) would change among successive years in accordance with the short-term changes in environmental conditions. Surprisingly, the effect of short-term climate fluctuations on genetic composition have rarely been addressed in the literature, and to our knowledge the effect of annual climatic fluctuations have not been considered meaningful. Our findings show that climatic variation among successive years, primarily the amount of rainfall and rainy days, can significantly alter both microsatellite allelic composition and diversity. We suggest that environmental (i.e. fluctuating) selection is differential across the globe, and that its intensity is expected to be greatest in regions where short-term climatic conditions are least stable.
Assuntos
Alelos , Anuros/genética , Clima , Variação Genética , Repetições de Microssatélites , AnimaisRESUMO
T cell development in the thymus is controlled by thymic epithelial cells (TE). While it is accepted that TE interact with maturing T cells, the mechanisms by which they trigger 'death by neglect' of double-positive (DP) thymocytes are poorly understood. We and others have demonstrated a role for TE-derived glucocorticoids (GCs) in this process. We have studied TE-induced apoptosis using an in vitro system based on co-culturing a thymic epithelial cell line (TEC) with DP thymic lymphoma cells or thymocytes (DP thymic cells). Here, we demonstrate that nitric oxide (NO*) is also involved in this death process. The inducible nitric oxide synthase (iNOS) inhibitors N(G)-methyl-L-arginine and 1,4-PBIT attenuated TEC-induced apoptosis of DP thymic cells. Co-cultivation of TEC with DP thymic cells increased the expression of iNOS in TEC. A concomitant increase in NO* was detected by staining with DAF-FM diacetate. Moreover, the iNOS-regulating cytokines IL-1alpha, IL-1beta and IFNgamma were up-regulated upon interaction of TEC with DP thymic cells. Neutralizing IL-1R or IFNgamma reduced TEC-induced apoptosis of DP thymic cells. Cardinally, NO* synergizes with GCs in eliciting apoptosis of DP thymic cells. Our data indicate that a cross-talk between DP thymic cells and TEC is required for proper induction of iNOS-up-regulating cytokines with a subsequent increase in iNOS expression and NO* production in TEC. NO*, in turn, cooperates with GCs in promoting death by neglect. We suggest that NO* together with GCs fine-tune the T cell selection process.
Assuntos
Apoptose/imunologia , Células Epiteliais/imunologia , Glucocorticoides/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico/imunologia , Timo/imunologia , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glucocorticoides/metabolismo , Antagonistas de Hormônios/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-1alfa/imunologia , Interleucina-1alfa/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mifepristona/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/imunologia , Receptores de Glucocorticoides/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologia , Timo/efeitos dos fármacos , Timo/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , ômega-N-Metilarginina/farmacologiaRESUMO
Glucocorticoids (GCs) are commonly used in the treatment of hematopoietic malignancies owing to their ability to induce apoptosis of these cancerous cells. Whereas some types of lymphoma and leukemia respond well to this drug, others are resistant. Also, GC-resistance gradually develops upon repeated treatments ultimately leading to refractory relapsed disease. Understanding the mechanisms regulating GC-induced apoptosis is therefore uttermost important for designing novel treatment strategies that overcome GC-resistance. This review discusses updated data describing the complex regulation of the cell's susceptibility to apoptosis triggered by GCs. We address both the genomic and nongenomic effects involved in promoting the apoptotic signals as well as the resistance mechanisms opposing these signals. Eventually we address potential strategies of clinical relevance that sensitize GC-resistant lymphoma and leukemia cells to this drug. The major target is the nongenomic signal transduction machinery where the interplay between protein kinases determines the cell fate. Shifting the balance of the kinome towards a state where Glycogen synthase kinase 3alpha (GSK3alpha) is kept active, favors an apoptotic response. Accumulating data show that it is possible to therapeutically modulate GC-resistance in patients, thereby improving the response to GC therapy.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Glucocorticoides/metabolismo , Neoplasias Hematológicas/metabolismo , Leucemia/metabolismo , Linfoma/metabolismo , Animais , Linhagem da Célula , Resistencia a Medicamentos Antineoplásicos , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Imunossupressores/metabolismo , Camundongos , Receptores de Glucocorticoides/metabolismoRESUMO
Amphibian declines are seen as an indicator of the onset of a sixth mass extinction of life on earth. Because of a combination of factors such as habitat destruction, emerging pathogens and pollutants, over 156 amphibian species have not been seen for several decades, and 34 of these were listed as extinct by 2004. Here we report the rediscovery of the Hula painted frog, the first amphibian to have been declared extinct. We provide evidence that not only has this species survived undetected in its type locality for almost 60 years but also that it is a surviving member of an otherwise extinct genus of alytid frogs, Latonia, known only as fossils from Oligocene to Pleistocene in Europe. The survival of this living fossil is a striking example of resilience to severe habitat degradation during the past century by an amphibian.
Assuntos
Anuros/anatomia & histologia , Fósseis , Animais , Osso e Ossos/anatomia & histologia , Europa (Continente) , Feminino , Israel , Funções Verossimilhança , Masculino , Dados de Sequência Molecular , FilogeniaRESUMO
Glucocorticoids (GCs) are integral components in the treatment protocols of acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin lymphoma owing to their ability to induce apoptosis of these malignant cells. Resistance to GC therapy is associated with poor prognosis. Although they have been used in clinics for decades, the signal transduction pathways involved in GC-induced apoptosis have only partly been resolved. Accumulating evidence shows that this cell death process is mediated by a communication between nuclear GR affecting gene transcription of pro-apoptotic genes such as Bim, mitochondrial GR affecting the physiology of the mitochondria, and the protein kinase glycogen synthase kinase-3 (GSK3), which interacts with Bim following exposure to GCs. Prevention of Bim up-regulation, mitochondrial GR translocation, and/or GSK3 activation are common causes leading to GC therapy failure. Various protein kinases positively regulating the pro-survival Src-PI3K-Akt-mTOR and Raf-Ras-MEK-ERK signal cascades have been shown to be activated in malignant leukemic cells and antagonize GC-induced apoptosis by inhibiting GSK3 activation and Bim expression. Targeting these protein kinases has proven effective in sensitizing GR-positive malignant lymphoid cells to GC-induced apoptosis. Thus, intervening with the pro-survival kinase network in GC-resistant cells should be a good means of improving GC therapy of hematopoietic malignancies.
Assuntos
Apoptose , Glucocorticoides/farmacologia , Neoplasias Hematológicas/patologia , Proteínas Quinases/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Redes Reguladoras de Genes/fisiologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/terapia , Humanos , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Recent data cast new light on the mechanisms by which glucocorticoids (GCs) elicit apoptosis of thymocytes and leukemia cells. Here we attempt to integrate recent studies by others and us, which provide a novel insight to this apoptotic process. In the last few years it was made clear that there is a tight cooperation between genomic and non-genomic effects exerted by GC receptors (GRs). GC invokes major alterations in the gene expression profile through GR-mediated transactivation and transrepression, which ultimately tip the balance between pro-survival and pro-apoptotic proteins. Although essential in shaping the cell's proteome, these genomic effects are insufficient to elicit apoptotic death and additional signals are required for activating the pro-apoptotic proteins. Several non-genomic effects have been described that occur immediately following exposure to GC, which are imperative for the induction of apoptosis. We have recently observed that GC induces instant GR translocation to the mitochondria in GC-sensitive, but not in GC-resistant, T lymphoid cells. This response contrasts the nuclear translocation of GR occurring in both cell types. We propose that the sustained elevation of GR in the mitochondria following GC exposure is crucial for triggering apoptosis.