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2.
PLoS Biol ; 19(9): e3001394, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34550965

RESUMO

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.


Assuntos
Linhagem da Célula , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Células da Medula Óssea/patologia , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , RNA-Seq , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
3.
Autophagy ; : 1-23, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34530675

RESUMO

Cancer cell growth is dependent upon the sustainability of proliferative signaling and resisting cell death. Macroautophagy/autophagy promotes cancer cell growth by providing nutrients to cells and preventing cell death. This is in contrast to autophagy promoting cell death under some conditions. The mechanism regulating autophagy-mediated cancer cell growth remains unclear. Herein, we demonstrate that TSSC4 (tumor suppressing subtransferable candidate 4) is a novel tumor suppressor that suppresses cancer cell growth and tumor growth and prevents cell death induction during excessive growth by inhibiting autophagy. The oncogenic proteins ERBB2 (erb-b2 receptor tyrosine kinase 2) and the activation EGFR mutant (EGFRvIII, epidermal growth factor receptor variant III) promote cell growth and TSSC4 expression in breast cancer and glioblastoma multiforme (GBM) cells, respectively. In EGFRvIII-expressing GBM cells, TSSC4 knockout shifted the function of autophagy from a pro-cell survival role to a pro-cell death role during prolonged cell growth. Furthermore, the interaction of TSSC4 with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) via its conserved LC3-interacting region (LIR) contributes to its inhibition of autophagy. Finally, TSSC4 suppresses tumorsphere formation and tumor growth by inhibiting autophagy and maintaining cell survival in tumorspheres. Taken together, sustainable cancer cell growth can be achieved by autophagy inhibition via TSSC4 expression.ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; CQ: chloroquine; EGFRvIII: epidermal growth factor receptor variant III; ERBB2: erb-b2 receptor tyrosine kinase 2; GBM: glioblastoma multiforme; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule Associated protein 1 light chain 3; TSSC4: tumor suppressing subtransferable candidate 4.

4.
Cell Rep ; 36(8): 109618, 2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34433017

RESUMO

Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.

5.
Front Microbiol ; 12: 665041, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234758

RESUMO

An unprecedented amount of SARS-CoV-2 sequencing has been performed, however, novel bioinformatic tools to cope with and process these large datasets is needed. Here, we have devised a bioinformatic pipeline that inputs SARS-CoV-2 genome sequencing in FASTA/FASTQ format and outputs a single Variant Calling Format file that can be processed to obtain variant annotations and perform downstream population genetic testing. As proof of concept, we have analyzed over 229,000 SARS-CoV-2 viral sequences up until November 30, 2020. We have identified over 39,000 variants worldwide with increased polymorphisms, spanning the ORF3a gene as well as the 3' untranslated (UTR) regions, specifically in the conserved stem loop region of SARS-CoV-2 which is accumulating greater observed viral diversity relative to chance variation. Our analysis pipeline has also discovered the existence of SARS-CoV-2 hypermutation with low frequency (less than in 2% of genomes) likely arising through host immune responses and not due to sequencing errors. Among annotated non-sense variants with a population frequency over 1%, recurrent inactivation of the ORF8 gene was found. This was found to be present in the newly identified B.1.1.7 SARS-CoV-2 lineage that originated in the United Kingdom. Almost all VOC-containing genomes possess one stop codon in ORF8 gene (Q27∗), however, 13% of these genomes also contains another stop codon (K68∗), suggesting that ORF8 loss does not interfere with SARS-CoV-2 spread and may play a role in its increased virulence. We have developed this computational pipeline to assist researchers in the rapid analysis and characterization of SARS-CoV-2 variation.

6.
Nat Commun ; 12(1): 2622, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976180

RESUMO

Obesity is caused by an imbalance between food intake and energy expenditure (EE). Here we identify a conserved pathway that links signalling through peripheral Y1 receptors (Y1R) to the control of EE. Selective antagonism of peripheral Y1R, via the non-brain penetrable antagonist BIBO3304, leads to a significant reduction in body weight gain due to enhanced EE thereby reducing fat mass. Specifically thermogenesis in brown adipose tissue (BAT) due to elevated UCP1 is enhanced accompanied by extensive browning of white adipose tissue both in mice and humans. Importantly, selective ablation of Y1R from adipocytes protects against diet-induced obesity. Furthermore, peripheral specific Y1R antagonism also improves glucose homeostasis mainly driven by dynamic changes in Akt activity in BAT. Together, these data suggest that selective peripheral only Y1R antagonism via BIBO3304, or a functional analogue, could be developed as a safer and more effective treatment option to mitigate diet-induced obesity.


Assuntos
Arginina/análogos & derivados , Obesidade/prevenção & controle , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Termogênese/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Adulto , Animais , Arginina/farmacologia , Arginina/uso terapêutico , Biópsia , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Obesidade/etiologia , Obesidade/metabolismo , Cultura Primária de Células , Receptores de Neuropeptídeo Y/metabolismo
7.
Nat Commun ; 12(1): 84, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33398012

RESUMO

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin ß4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.


Assuntos
Isquemia/patologia , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Movimento Celular/genética , Proliferação de Células/genética , Dependovirus/metabolismo , Regulação da Expressão Gênica , Humanos , Isquemia/genética , Camundongos Knockout , Modelos Biológicos , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Neovascularização Fisiológica/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timosina/análogos & derivados , Timosina/genética , Timosina/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
8.
Stem Cell Reports ; 15(6): 1246-1259, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33296673

RESUMO

Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.


Assuntos
Loci Gênicos , Células-Tronco Embrionárias Humanas/metabolismo , Complexos Multiproteicos/metabolismo , Proteína Homeobox Nanog/metabolismo , Proteínas Correpressoras/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos
9.
Cancer Res ; 80(14): 2983-2995, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32503808

RESUMO

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.


Assuntos
Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Melanoma/patologia , Fatores de Transcrição/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Invasividade Neoplásica , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
10.
Blood ; 136(8): 957-973, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32369597

RESUMO

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.


Assuntos
Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição da Família Snail/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células HEK293 , Células HL-60 , Histona Desmetilases/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo
11.
Dis Model Mech ; 13(3)2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32005677

RESUMO

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.


Assuntos
Embrião de Mamíferos/metabolismo , Palato/embriologia , Palato/metabolismo , Fatores de Transcrição/deficiência , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Região Branquial/embriologia , Caderinas/metabolismo , Cruzamentos Genéticos , Embrião de Mamíferos/ultraestrutura , Epiderme/embriologia , Epiderme/ultraestrutura , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Epitélio/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Maxila/embriologia , Maxila/patologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Tamanho do Órgão , Fenótipo , Gravidez , Splicing de RNA/genética , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/deficiência
12.
Sci Rep ; 9(1): 10577, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332244

RESUMO

In cancer research, it remains challenging to functionally validate putative novel oncogenic drivers and to establish relevant preclinical models for evaluation of novel therapeutic strategies. Here, we describe an optimized and efficient pipeline for the generation of novel conditional overexpression mouse models in which putative oncogenes, along with an eGFP/Luciferase dual reporter, are expressed from the endogenous ROSA26 (R26) promoter. The efficiency of this approach was demonstrated by the generation and validation of novel R26 knock-in (KI) mice that allow conditional overexpression of Jarid2, Runx2, MN1 and a dominant negative allele of ETV6. As proof of concept, we confirm that MN1 overexpression in the hematopoietic lineage is sufficient to drive myeloid leukemia. In addition, we show that T-cell specific activation of MN1 in combination with loss of Pten increases tumour penetrance and stimulates the formation of Lyl1+ murine T-cell lymphoblastic leukemias or lymphomas (T-ALL/T-LBL). Finally, we demonstrate that these luciferase-positive murine AML and T-ALL/T-LBL cells are transplantable into immunocompromised mice allowing preclinical evaluation of novel anti-leukemic drugs in vivo.


Assuntos
Neoplasias Hematológicas/genética , Oncogenes/genética , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Neoplasias Hematológicas/etiologia , Humanos , Leucemia/etiologia , Leucemia/genética , Leucemia Mieloide/genética , Masculino , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Complexo Repressor Polycomb 2/genética , Transativadores/genética , Proteínas Supressoras de Tumor/genética
13.
Cancers (Basel) ; 11(5)2019 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31109112

RESUMO

Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/ß-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/ß-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-ß/axin destruction complex of ß-catenin. This results in sustained nuclear accumulation of ß-catenin, followed by ß-catenin-dependent co-transcriptional activation of Wnt/ß-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling ß-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits ß-catenin co-transcriptional activity. PKCα also phosphorylates ß-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.

14.
Cell Res ; 29(7): 579-591, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31133695

RESUMO

Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a KrasG12D-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of KrasG12D-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout KrasG12D mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.


Assuntos
Fator de Indução de Apoptose/fisiologia , Carcinogênese/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Progressão da Doença , Glicólise , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação Oxidativa
15.
Genesis ; 57(6): e23299, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30990965

RESUMO

Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front-rear polarity axis for directional movement. Although front-rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live-imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front-rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus-to-Golgi axis to each cell, which functions as a readout for cell front-rear polarity. As a proof-of-principle, we validated the efficiency of the GNrep line using an endothelial-specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large-scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front-rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.


Assuntos
Polaridade Celular/fisiologia , Engenharia de Proteínas/métodos , Animais , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Polaridade Celular/genética , Células Endoteliais , Corantes Fluorescentes , Genes Reporter , Complexo de Golgi/metabolismo , Camundongos
16.
Haematologica ; 104(8): 1608-1616, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30679322

RESUMO

ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transformação Celular Neoplásica/genética , Proteínas com Domínio LIM/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica , Hematopoese , Humanos , Imuno-Histoquímica , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Proteínas com Domínio LIM/metabolismo , Camundongos , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Timo/patologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo
17.
Microcirculation ; 26(2): e12493, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30030876

RESUMO

OBJECTIVE: Stem cell-based regenerative therapies have been intensively studied with the aim to define an ideal cell type for the treatment of myocardial infarction. We tested systemically delivered, platelet-targeted induced vascular progenitor cells (iVPCs) to study their potential to salvage damaged myocardium after ischemia-reperfusion injury. METHODS: Using a mouse model of ischemia-reperfusion injury, we tested the potential of platelet-targeted iVPCs (1 × 106 targ-iVPCs) compared to non-targ-iVPCs and a saline control. Bioluminescence imaging, echocardiography, and histological analyses were performed. RESULTS: Four weeks after ischemia-reperfusion injury, systemic delivery of targ-iVPCs led to reduced fibrosis and infarct size (PBS: 25.7 ± 3.9 vs targ-iVPC: 18.4 ± 6.6 vs non-targ-iVPC: 25.1 ± 3.7%I/LV, P < 0.05), increased neovascularization, and restored cardiac function (PBS: 44.0 ± 4.2 vs targ-iVPC: 54.3 ± 4.5 vs non-targ-iVPC: 46.4 ± 3.8%EF, P < 0.01). Cell tracking experiments revealed entrapment of intravenously injected iVPCs in the pulmonary microvasculature in both cell-treated groups. CONCLUSIONS: Systemic delivery of iVPCs after cardiac ischemia-reperfusion injury is limited by pulmonary entrapment of the cells. Nevertheless, targ-iVPCs reduced infarct size, fibrosis, increased neovascularization, and most importantly retained cardiac function. These findings contribute to the mechanistic discussion of cell-based therapy and ultimately identify activated platelet-targeted iVPCs as candidates for cell therapy and also describe cell therapy benefits without the necessity of engrafting.


Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Microvasos/citologia , Traumatismo por Reperfusão Miocárdica/terapia , Animais , Plaquetas/citologia , Comunicação Celular , Rastreamento de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Pulmão/irrigação sanguínea , Camundongos , Comunicação Parácrina , Resultado do Tratamento
18.
Cell Rep ; 23(11): 3312-3326, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29898401

RESUMO

Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Animais , Benzamidas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Hipóxia , Microscopia Intravital/métodos , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Nanopartículas/química , Nitroimidazóis/farmacologia , Nitroimidazóis/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Mostardas de Fosforamida/farmacologia , Mostardas de Fosforamida/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Microambiente Tumoral
19.
Sci Rep ; 8(1): 7642, 2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29769563

RESUMO

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.


Assuntos
Anormalidades Cardiovasculares/genética , Leucemia Prolinfocítica de Células T/genética , Neuroblastoma/genética , Sequências Repetitivas de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Dermatopatias/genética , Animais , Anormalidades Cardiovasculares/patologia , Modelos Animais de Doenças , Feminino , Humanos , Leucemia Prolinfocítica de Células T/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroblastoma/patologia , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Dermatopatias/patologia , Peixe-Zebra/metabolismo
20.
Acta Neuropathol ; 135(1): 131-148, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28780615

RESUMO

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.


Assuntos
Miopatias Distais/metabolismo , Mutação com Ganho de Função , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miopatias Congênitas Estruturais/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Atrofia/metabolismo , Atrofia/patologia , Autofagia/fisiologia , Modelos Animais de Doenças , Miopatias Distais/patologia , Feminino , Proteínas de Choque Térmico , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Chaperonas Moleculares , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia
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