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CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19+ or CD19- B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19+ and CD19- relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss.
Assuntos
Receptores de Antígenos Quiméricos , Antígenos CD19 , Linfócitos B , Humanos , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Linfócitos TRESUMO
In this paper, the 4E assessment (Energetic, Exergetic, Exergoeconomic and Exergoenvironmental) of a low-temperature ORC activated by two different alternatives is presented. The first alternative (S1) contemplates the activation of the ORC through the recovery of waste heat from a flash-binary geothermal power plant. The second alternative (S2) contemplates the activation of the ORC using direct heat from a geothermal well. For both alternatives, the energetic and exergetic models were established. At the same time, the economic and environmental impact models were developed. Finally, based on the combination of the exergy concepts and the economic and ecological indicators, the exergoeconomic and exergoenvironmental performances of the ORC were obtained. The results show higher economic, exergoeconomic and exergoenvironmental profitability for S1. Besides, for the alternative S1, the ORC cycle has an acceptable economic profitability for a net power of 358.4 kW at a temperature of 110 °C, while for S2, this profitability starts being attractive for a power 2.65 times greater than S1 and with a temperature higher than 135 °C. In conclusion, the above represents an area of opportunity and a considerable advantage for the implementation of the ORC in the recovery of waste heat from flash-binary geothermal power plants.
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Methylation of lysine 4 (K4) within histone H3 has been linked to active transcription and is removed by LSD1 and the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here, we describe the deamination catalyzed by Lysyl oxidase-like 2 protein (LOXL2) as an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, LOXL2 activity is linked with the transcriptional control of CDH1 gene by regulating H3K4me3 deamination. These results reveal another H3 modification and provide a different mechanism for H3K4 modification.
Assuntos
Aminoácido Oxirredutases/fisiologia , Histonas/metabolismo , Antígenos CD , Caderinas/genética , Linhagem Celular Tumoral , Desaminação , Regulação da Expressão Gênica , Humanos , Lisina/metabolismo , MetilaçãoRESUMO
F-box proteins are the key recognition subunit of multimeric E3 ubiquitin ligase complexes that participate in the proteasome degradation of specific substrates. In the last years, a discrete number of F-box proteins have been shown to regulate the epithelial-to-mesenchymal transition (EMT), a process defined by a rapid change of cell phenotype, the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Specific EMT transcription factors (EMT-TFs), such as Snail, Slug, Twist and Zeb, control EMT induction both during development and in cancer. These EMT-TFs are short-lived proteins that are targeted to the proteasome system by specific F-box proteins, keeping them at low levels. F-box proteins also indirectly regulate the EMT process by controlling EMT inducers, such as Notch, c-Myc or mTOR. Here we summarize the role that these F-box proteins (Fbxw1, Fbxw7, Fbxl14, Fbxl5, Fbxo11 and Fbxo45) play in controlling EMT during development and cancer progression, a process dependent on post-translational modifications that govern their interaction with target proteins.
Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Animais , Proteínas Culina/química , Proteínas Culina/genética , Proteínas Culina/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos , Neoplasias/etiologia , Neoplasias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.
Assuntos
Regulação da Expressão Gênica , Fator 1 de Ligação ao Facilitador Linfoide/genética , Splicing de RNA , RNA Antissenso/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/biossíntese , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases ß-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and γ-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1.
Assuntos
Núcleo Celular/enzimologia , Proteínas F-Box/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA/metabolismo , Raios gama , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , RNA Interferente Pequeno , Fatores de Transcrição da Família Snail , Complexos Ubiquitina-Proteína LigaseRESUMO
BACKGROUND: The dismal clinical outcome of relapsed/refractory (R/R) T cell acute lymphoblastic leukemia (T-ALL) highlights the need for innovative targeted therapies. Although chimeric antigen receptor (CAR)-engineered T cells have revolutionized the treatment of B cell malignancies, their clinical implementation in T-ALL is in its infancy. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed CAR T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. Nonetheless, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients. METHODS: We carried out a comprehensive study using robust in vitro and in vivo assays comparing the efficacy of engineered T cells either expressing a second-generation CD1a-CAR or secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb). RESULTS: We show that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induce specific T cell activation. Recruitment of bystander T cells endows CD1a-STAbs with an enhanced in vitro cytotoxicity than CD1a-CAR T cells at lower effector:target ratios. CD1a-STAb T cells are as effective as CD1a-CAR T cells in cutting-edge in vivo T-ALL patient-derived xenograft models. CONCLUSIONS: Our data suggest that CD1a-STAb T cells could be an alternative to CD1a-CAR T cells in coT-ALL patients with aggressive and hyperleukocytic relapses with limited numbers of non-leukemic effector T cells.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Linfócitos T , Humanos , Imunoterapia Adotiva , Anticorpos , RecidivaRESUMO
The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase beta-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3beta. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3beta, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3beta. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.
Assuntos
Proteínas F-Box/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Western Blotting , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Proteínas F-Box/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imunoprecipitação , Camundongos , Mutação , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Transfecção , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Chemical descriptors encode the physicochemical and structural properties of small molecules, and they are at the core of chemoinformatics. The broad release of bioactivity data has prompted enriched representations of compounds, reaching beyond chemical structures and capturing their known biological properties. Unfortunately, bioactivity descriptors are not available for most small molecules, which limits their applicability to a few thousand well characterized compounds. Here we present a collection of deep neural networks able to infer bioactivity signatures for any compound of interest, even when little or no experimental information is available for them. Our signaturizers relate to bioactivities of 25 different types (including target profiles, cellular response and clinical outcomes) and can be used as drop-in replacements for chemical descriptors in day-to-day chemoinformatics tasks. Indeed, we illustrate how inferred bioactivity signatures are useful to navigate the chemical space in a biologically relevant manner, unveiling higher-order organization in natural product collections, and to enrich mostly uncharacterized chemical libraries for activity against the drug-orphan target Snail1. Moreover, we implement a battery of signature-activity relationship (SigAR) models and show a substantial improvement in performance, with respect to chemistry-based classifiers, across a series of biophysics and physiology activity prediction benchmarks.
Assuntos
Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Endothelin-converting enzyme-1 (ECE1) activates the endothelin-1 peptide, which upregulates pathways that are related to diverse hallmarks of cancer. ECE1 is expressed as four isoforms differing in their N-terminal domains. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE1c, enhancing its stability and promoting invasiveness of colorectal cancer cells. However, the specific residues in ECE1c that are phosphorylated by CK2 and how this phosphorylation promotes invasiveness was unknown. Here we demonstrate that Ser-18 and Ser-20 are the bona fide residues phosphorylated by CK2 in ECE1c. Thus, biphospho-mimetic ECE1cDD and biphospho-resistant ECE1cAA mutants were constructed and stably expressed in different colorectal cancer cells through lentiviral transduction. Biphospho-mimetic ECE1cDD displayed the highest stability in cells, even in the presence of the specific CK2 inhibitor silmitasertib. Concordantly, ECE1cDD-expressing cells showed enhanced hallmarks of cancer, such as proliferation, migration, invasiveness, and self-renewal capacities. Conversely, cells expressing the less-stable biphospho-resistant ECE1cAA showed a reduction in these features, but also displayed an important sensitization to 5-fluorouracil, an antineoplastic agent traditionally used as therapy in colorectal cancer patients. Altogether, these findings suggest that phosphorylation of ECE1c at Ser-18 and Ser-20 by CK2 promotes aggressiveness in colorectal cancer cells. Therefore, phospho-ECE1c may constitute a novel biomarker of poor prognosis and CK2 inhibition may be envisioned as a potential therapy for colorectal cancer patients.
RESUMO
Cell migration is a multifactorial/multistep process that requires the concerted action of growth and transcriptional factors, motor proteins, extracellular matrix remodeling and proteases. In this review, we focus on the role of transcription factors modulating Epithelial-to-Mesenchymal Transition (EMT-TFs), a fundamental process supporting both physiological and pathological cell migration. These EMT-TFs (Snail1/2, Twist1/2 and Zeb1/2) are labile proteins which should be stabilized to initiate EMT and provide full migratory and invasive properties. We present here a family of enzymes, the deubiquitinases (DUBs) which have a crucial role in counteracting polyubiquitination and proteasomal degradation of EMT-TFs after their induction by TGFß, inflammatory cytokines and hypoxia. We also describe the DUBs promoting the stabilization of Smads, TGFß receptors and other key proteins involved in transduction pathways controlling EMT.
Assuntos
Movimento Celular/fisiologia , Enzimas Desubiquitinantes/metabolismo , Animais , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition and fibroblast activation. As a consequence, Snail1 expression and function is regulated at multiple levels from gene transcription to protein modifications, affecting its interaction with specific cofactors. In this review, we describe the different elements that control Snail1 expression and its activity both as transcriptional repressor or activator.
RESUMO
In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFß during EMT and was required for TGFß-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFß-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição da Família Snail/química , Fator de Crescimento Transformador beta/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Estabilidade Proteica , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Most of the hypomorphic Prep1(i/i) embryos (expressing 3-10% of the Prep1 protein), die between E17.5 and P0, with profound anemia, eye malformations and angiogenic anomalies [Ferretti, E., Villaescusa, J.C., Di Rosa, P., Fernandez-Diaz, L.-C., Longobardi, E., Mazzieri, R., Miccio, A., Micali, N., Selleri, L., Ferrari G., Blasi, F. (2006). Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype. Mol. Cell. Biol. 26, 5650-5662]. We now report on the hematopoietic phenotype of these embryos. Prep1(i/i) fetal livers (FL) are hypoplastic, produce less common myeloid progenitors colonies (CFU-GEMM) in cytokine-supplemented methylcellulose and have an increased number of B-cells precursors that differentiate poorly. Prep1(i/i) FL is able to protect lethally irradiated mice only at high cell doses but the few protected mice show major anomalies in all hematopoietic lineages in both bone marrow (BM) and peripheral organs. Prep1(i/i) FL cells compete inefficiently with wild type bone marrow in competitive repopulation experiments, suggesting that the major defect lies in long-term repopulating hematopoietic stem cells (LTR-HSC). Indeed, wt embryonic expression of Prep1 in the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), cKit(+)Sca1(+)Lin(-)AA4.1(+) (KSLA) cells and B-lymphocytes precursors agrees with the observed phenotype. We therefore conclude that Prep1 is required for a correct and complete hematopoiesis.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein (LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation. DATABASE: The GEO accession number for the data referred to this paper is GSE35600.
Assuntos
Aminoácido Oxirredutases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Aminoácido Oxirredutases/genética , Antígenos CD , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Metilação , Oxirredução , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria InfravermelhoRESUMO
Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor ß1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3ß (GSK-3ß) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3ß phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the ß-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3ß inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3ß inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism.
Assuntos
Morte Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/genética , Animais , Aorta/citologia , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Camundongos , Estresse Oxidativo , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Transcrição da Família Snail , Suínos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Regulação para Cima , beta Catenina/metabolismoRESUMO
Resumen Determinar la prevalencia de parasitismo de Giardia duodenalis, en el centro de bienestar "CEIBA", del municipio de Rionegro, Colombia durante junio 2017. Se realizó un estudio descriptivo transversal, no experimental, aleatorio, fueron evaluados 112 coprológicos de caninos del centro de bienestar "CEIBA", de ambos sexos, todas las edades y alimentación comercial. Fueron sometidos a evaluación coprológica, por extracción directa de ampolla rectal y evaluada en el Laboratorio Clínico de la Corporación Universitaria Lasallista "Hermano Marco Antonio Serna f.s.c", fueron realizados dos métodos diagnósticos, flotación con solución salina saturada (Willys Molloy) y frotis directo. La presencia de Giardia duodenalis fue categorizado en escala de cruces de 0-3. Se encontraron 60 (55%) muestras con presencia de parásitos, de los cuales 10 (8,92%) estaban infestados por Giardia duodenalis, representando 16,66% de la parasitosis total, para una cruz 2 (20%), con dos cruces 5 (50%) y con tres 3 (30%). La prevalencia de Giardia duodenalis en el centro de bienestar CEIBA, es baja con respecto a datos de prevalencia en otros albergues o centros caninos, lo cual puede estar influenciado por las medidas de prevención y control de la enfermedad.
Abstract To determine the prevalence of Giardia duodenalis parasitism at the CEIBA wellness center in the municipality of Rionegro, Colombia, during June 2017. A descriptive cross-sectional, non-experimental, randomized study was carried out. 112 coprological evaluations of canines of the "CEIBA" wellness center of both sexes, all ages and commercial feeding were evaluated. They were submitted to a cochrological evaluation, by direct extraction of rectal ampulla and evaluated in the Clinical Laboratory of the University of Lasallian Hospital "Hno Marco Antonio Serna, fsc", two diagnostic methods were carried out, flotation with saturated saline solution (Willys Molloy) and direct smears. The presence of Giardia duodenalis was categorized as 0-3 crosses. A total of 60 (55%) samples were present, with 10 (8.92%) infested by Giardia duodenalis, accounting for 16.66% of the total parasite, for a cross 2 (20%), with two crosses 5 (50%) and three 3 (30%). The prevalence of Giardia duodenalis in the CEIBA well-being center is low with respect to prevalence data in other canines or hostels, which may be influenced by disease prevention and control measures.
Resumo Determinar a prevalência do parasitismo de Giardia duodenalis no centro de bem-estar CEIBA no município de Rionegro, Colômbia, em junho de 2017. Foi realizado um estudo descritivo transversal, não experimental, randomizado. Foram avaliadas as avaliações coprológicas dos caninos do centro de bem estar "CEIBA" de ambos os sexos, todasas idades e alimentação comercial. Eles foram submetidos a avaliação cochrológica, por extração direta de ampola retal e avaliada no Laboratório Clínico do Hospital Universitário das Ilhas "Hno Marco Antonio Serna, fsc", foram realizados dois métodos de diagnóstico, flotação com solução salina saturada (Willys Molloy) e esfregaços diretos. A presença de Giardia duodenalis foi categorizada como 0-3 cruzamentos. Um total de 60 (55%) amostras estavam presentes, com 10 (8,92%) infestadas por Giardia duodenalis, representando 16,66% do parasita total, para uma cruz 2 (20%), com duas cruza 5 (50%) e três 3 (30%). A prevalência de Giardia duodenalis no centro de bem-estar da CEIBA é baixa em relação aos dados de prevalência em outros caninos ou albergues, o que pode ser influenciado por medidas de prevenção e controle de doenças.
RESUMO
The HoxB cluster expression is activated by retinoic acid and transcribed in a collinear manner. The DNA-binding Pknox1-Pbx1 complex modulates Hox protein activity. Here, NT2-D1 teratocarcinoma cells -a model of Hox gene expression- were used to show that upon retinoic acid induction, Pknox1 co-localizes with polymeric nuclear actin. We have found that globular actin aggregates, polymeric actin, the elongating RNA polymerase II and THOC match euchromatic regions corresponding to nuclear speckles. Moreover, RNA polymerase II, N-WASP, and transcription/splicing factors p54(nrb) and PSF were validated as Pknox1 interactors by tandem affinity purification. PSF pulled down with THOC and nuclear actin, both of which co-localize in nuclear speckles. Although latrunculin A slightly decreases the general level of HoxB gene expression, inhibition of nuclear actin polymerization by cytochalasin D blocks the expression of HoxB transcripts in a collinear manner. Thus, our results support the hypothesis that nuclear actin polymerization is involved in the activation of HoxB gene expression by means of nuclear speckles.