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1.
Proc Natl Acad Sci U S A ; 120(40): e2300489120, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37748077

RESUMO

Lung cancer is the leading cause of cancer deaths. Its high mortality is associated with high metastatic potential. Here, we show that the RAC1-selective guanine nucleotide exchange factor T cell invasion and metastasis-inducing protein 1 (TIAM1) promotes cell migration and invasion in the most common subtype of lung cancer, non-small-cell lung cancer (NSCLC), through an unexpected nuclear function. We show that TIAM1 interacts with TRIM28, a master regulator of gene expression, in the nucleus of NSCLC cells. We reveal that a TIAM1-TRIM28 complex promotes epithelial-to-mesenchymal transition, a phenotypic switch implicated in cell migration and invasion. This occurs through H3K9me3-induced silencing of protocadherins and by decreasing E-cadherin expression, thereby antagonizing cell-cell adhesion. Consistently, TIAM1 or TRIM28 depletion suppresses the migration of NSCLC cells, while migration is restored by the simultaneous depletion of protocadherins. Importantly, high nuclear TIAM1 in clinical specimens is associated with advanced-stage lung adenocarcinoma, decreased patient survival, and inversely correlates with E-cadherin expression.


Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Protocaderinas , Carcinoma Pulmonar de Células não Pequenas/genética , Caderinas/genética , Epigênese Genética , Proteína 28 com Motivo Tripartido , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genética
2.
Proc Natl Acad Sci U S A ; 119(3)2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35042775

RESUMO

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.


Assuntos
Citotoxicidade Imunológica , Neoplasias/metabolismo , Radioterapia , Citotoxicidade Celular Dependente de Anticorpos , Proteínas de Bactérias , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais/imunologia , Perforina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Estreptolisinas
3.
J Immunol ; 205(4): 994-1008, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32641385

RESUMO

Mucosal surfaces such as fish gills interface between the organism and the external environment and as such are major sites of foreign Ag encounter. In the gills, the balance between inflammatory responses to waterborne pathogens and regulatory responses toward commensal microbes is critical for effective barrier function and overall fish health. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune responses to pathogens and suppressing inflammation. Although considerable progress has been made in the field of fish immunology in recent years, whether the fish counterparts of these key mammalian cytokines perform similar roles is still an open question. In this study, we have generated IL-4/13A and IL-4/13B mutant zebrafish (Danio rerio) and, together with an existing IL-10 mutant line, characterized the consequences of loss of function of these cytokines. We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4-like cytokines and is essential for gill homeostasis. Thus, both IL-4/13 and IL-10 paralogs in zebrafish exhibit aspects of conserved function with their mammalian counterparts.


Assuntos
Proteínas de Peixes/imunologia , Brânquias/imunologia , Homeostase/imunologia , Inflamação/imunologia , Interleucina-10/imunologia , Interleucina-4/imunologia , Peixe-Zebra/imunologia , Animais , Imunidade/imunologia , Interleucina-13/imunologia , Mamíferos/imunologia
4.
Proc Natl Acad Sci U S A ; 115(34): 8581-8586, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-29987046

RESUMO

Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.


Assuntos
Movimento Celular , Polaridade Celular , Cromatina/metabolismo , Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/metabolismo , Cromatina/genética , Cromatina/ultraestrutura , Proteínas de Ligação a DNA , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Microscopia de Força Atômica , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética
5.
J Immunol ; 197(9): 3520-3530, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27694495

RESUMO

CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b-expressing Th2-like cells, which do not coexpress il-4/13a Additionally, we identify a contrasting population of regulatory T cell-like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.


Assuntos
Doenças dos Peixes/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Melanoma/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Células Cultivadas , Brânquias/imunologia , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Mamíferos , Sistema Fagocitário Mononuclear , Neoplasias Experimentais
6.
J Immunol ; 192(4): 1796-805, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24431232

RESUMO

The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.


Assuntos
Apoptose/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Inflamação/imunologia , Neutrófilos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Morfolinos/genética , Neutrófilos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/biossíntese , Peixe-Zebra/genética
7.
Nature ; 464(7292): 1196-200, 2010 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-20364122

RESUMO

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.


Assuntos
Aorta Torácica/embriologia , Hemodinâmica , MicroRNAs/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Animais , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , MicroRNAs/genética , Células NIH 3T3 , Fluxo Sanguíneo Regional/fisiologia , Peixe-Zebra/sangue , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
8.
Hum Mol Genet ; 21(8): 1744-59, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22210625

RESUMO

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.


Assuntos
Encéfalo/embriologia , Modelos Animais de Doenças , Síndrome Oculocerebrorrenal , Monoéster Fosfórico Hidrolases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Encéfalo/patologia , Sobrevivência Celular , Clatrina/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário , Endossomos/metabolismo , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Temperatura Alta , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Processamento de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo
10.
Proc Natl Acad Sci U S A ; 108(3): 1099-103, 2011 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-21199949

RESUMO

T2-family acidic endoribonucleases are represented in all genomes. A physiological role for RNase T2 has yet to be defined for metazoa. RNASET2 mutation in humans is linked with a leukoencephalopathy that arises in infancy characterized by cortical cysts and multifocal white matter lesions. We now show localization of RNASET2 within lysosomes. Further, we demonstrate that loss of rnaset2 in mutant zebrafish results in accumulation of undigested rRNA within lysosomes within neurons of the brain. Further, by using high field intensity magnetic resonance microimaging, we reveal white matter lesions in these animals comparable to those observed in RNASET2-deficient infants. This correlates with accumulation of Amyloid precursor protein and astrocytes at sites of neurodegeneration. Thus we conclude that familial cystic leukoencephalopathy is a lysosomal storage disorder in which rRNA is the best candidate for the noxious storage material.


Assuntos
Leucoencefalopatias/genética , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/metabolismo , Estabilidade de RNA/fisiologia , RNA Ribossômico/metabolismo , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Peixe-Zebra/genética , Animais , Encéfalo/metabolismo , Linhagem Celular , Clonagem Molecular , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Hibridização In Situ , Imageamento por Ressonância Magnética , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Neurônios/patologia , Estabilidade de RNA/genética , Ribonucleases/genética , Proteínas Supressoras de Tumor/genética
11.
PLoS Biol ; 8(12): e1000562, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21179501

RESUMO

It has not previously been possible to live image the earliest interactions between the host environment and oncogene-transformed cells as they initiate formation of cancers within an organism. Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin. Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden. Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages. We show that a major component of the signal drawing inflammatory cells to oncogenic HRAS(G12V)-transformed cells is H(2)O(2), which is also a key damage cue responsible for recruiting neutrophils to a wound. Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair. These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble non-healing wounds.


Assuntos
Transformação Celular Neoplásica/imunologia , Imunidade Inata , Melanoma/imunologia , Ferimentos e Lesões/imunologia , Peixe-Zebra/imunologia , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Larva/imunologia , Melanócitos/citologia , Microscopia Confocal , Morfolinas/imunologia , NADPH Oxidases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transgenes , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo
12.
Essays Biochem ; 67(6): 991-1002, 2023 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-37503572

RESUMO

Treatment with immune checkpoint inhibitors, widely known as immune checkpoint blockade therapy (ICBT), is now the fourth pillar in cancer treatment, offering the chance of durable remission for patients with advanced disease. However, ICBT fails to induce objective responses in most cancer patients with still others progressing after an initial response. It is necessary, therefore, to elucidate the primary and acquired resistance mechanisms to ICBT to improve its efficacy. Here, we highlight the paradoxical role of the cytokine interferon-γ (IFN-γ) in ICBT response: on the one hand induction of IFN-γ signalling in the tumour microenvironment correlates with good ICBT response as it drives the cellular immune responses required for tumour destruction; nonetheless, IFN-γ signalling is implicated in ICBT acquired resistance. We address the negative feedback and immunoregulatory effects of IFN-γ signalling that promote immune evasion and resistance to ICBT and discuss how these can be targeted pharmacologically to restore sensitivity or circumvent resistance.

13.
Nat Commun ; 14(1): 5983, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37752135

RESUMO

Resistance mechanisms to immune checkpoint blockade therapy (ICBT) limit its response duration and magnitude. Paradoxically, Interferon γ (IFNγ), a key cytokine for cellular immunity, can promote ICBT resistance. Using syngeneic mouse tumour models, we confirm that chronic IFNγ exposure confers resistance to immunotherapy targeting PD-1 (α-PD-1) in immunocompetent female mice. We observe upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in chronic IFNγ-treated cancer cell models, in patient melanoma with elevated IFNG expression, and in melanoma cell cultures from ICBT-progressing lesions characterised by elevated IFNγ signalling. Effector T cell infiltration is enhanced in tumours derived from cells pre-treated with IFNγ in immunocompetent female mice when PARP14 is pharmacologically inhibited or knocked down, while the presence of regulatory T cells is decreased, leading to restoration of α-PD-1 sensitivity. Finally, we determine that tumours which spontaneously relapse in immunocompetent female mice following α-PD-1 therapy upregulate IFNγ signalling and can also be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.


Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Feminino , Humanos , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor de Morte Celular Programada 1 , Interferon gama , Recidiva Local de Neoplasia , Modelos Animais de Doenças , Poli(ADP-Ribose) Polimerases
14.
BMC Dev Biol ; 12: 1, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22235774

RESUMO

BACKGROUND: The desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg), are the adhesion molecules of desmosomes, intercellular adhesive junctions of epithelia and cardiac muscle. Both the DCs and desmosomes have demonstrably essential roles in mammalian development. In order to initiate their study in a more tractable developmental system we have characterised zebrafish DCs and examined their roles in early zebrafish development. RESULTS: We find that zebrafish possess one Dsc, the orthologue of mammalian Dsc1, which we designate zfDsc. Unlike mammalian Dscs, zfDsc exists only as the "a" form since it lacks the alternatively-spliced mini-exon that shortens the cytoplasmic domain to produce the "b" form. Zebrafish possess two Dsgs, designated zfDsgα and zfDsgß, orthologues of mammalian Dsg2. They show 43.8% amino acid identity and the α form has a 43 amino acid glycine-rich sequence of unknown function in its extracellular domain. Both zfDsc and zfDsgα were present as maternal and zygotic transcripts whereas zfDsgß was first expressed from 8 hours post-fertilisation (hpf). All three transcripts were present throughout subsequent stages of development. Morpholino knockdown of both zfDsc and zfDsgα expression produced similar defects in epiboly, axis elongation and somite formation, associated with abnormal desmosomes or reduced desmosome numbers. CONCLUSIONS: These results demonstrate an important role for DCs and desmosomes in the early morphogenesis of the zebrafish embryo, provide a basis for more detailed analysis of their role and raise interesting questions relating to the evolution and functional significance of DC isoforms.


Assuntos
Desmocolinas/metabolismo , Desmogleínas/metabolismo , Desmossomos/metabolismo , Gastrulação , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Desmocolinas/química , Desmocolinas/genética , Desmogleínas/química , Desmogleínas/genética , Desmossomos/ultraestrutura , Éxons , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Masculino , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
15.
Curr Res Physiol ; 5: 216-223, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35637870

RESUMO

Respirometry, based on oxygen uptake, is commonly employed for measuring metabolic rate. There is a growing need for metabolic rate measurements suitable for developmental studies, particularly in Danio rerio, where many important developmental stages occur at < 4 mm. However, respirometry becomes more challenging as the size of the organism reduces. Additionally, respirometry can be costly and require significant experience and technical knowledge which may prohibit uptake in non-specialist/non-physiology labs. Thus, using equipment routine in most developmental/molecular biology laboratories, we measured glucose uptake in 96-h post fertilisation (hpf) zebrafish larvae and compared it to stop-flow respirometry measures of oxygen uptake to test whether glucose uptake was a suitable alternative measure of metabolic rate. A Passing-Bablok regression revealed that within a 95% limit of agreement, the rate of glucose uptake and the rate of oxygen uptake were equivalent as measures of metabolic rate in 96 hpf Danio rerio larvae. Thus, the methodology we outline here may be a useful alternative or a complementary method for assessing metabolic rate in small organisms.

16.
Cell Rep ; 39(12): 110995, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35732120

RESUMO

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.


Assuntos
Diacilglicerol O-Aciltransferase , Melanoma , Animais , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio , Triglicerídeos , Peixe-Zebra/metabolismo
17.
Methods Mol Biol ; 2262: 411-422, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977492

RESUMO

Hyper-activation of RAS signaling pathways causes cancer, including melanoma, and RAS signaling pathways have been successfully targeted using drugs for patient benefit. The available drugs alone cannot cure cancer, however, and so investigation continues into RAS signaling pathways, with the goal of identifying further actionable targets. The zebrafish can be used to model human malignancies, and genetic modification of zebrafish to incorporate selective disease-associated genetic alterations is practicable. The following article describes the methods we are using to genetically modify zebrafish in order to dissect oncogenic RAS signaling in melanoma development.


Assuntos
Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Melanoma/patologia , Mutação , Transgenes/genética , Proteínas ras/metabolismo , Animais , Animais Geneticamente Modificados , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Peixe-Zebra , Proteínas ras/genética
18.
Cancers (Basel) ; 13(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34359605

RESUMO

Zebrafish embryo tumor transplant models are widely utilized in cancer research. Compared with traditional murine models, the small size and transparency of zebrafish embryos combined with large clutch sizes that increase statistical power and cheap husbandry make them a cost-effective and versatile tool for in vivo drug discovery. However, the lack of a comprehensive analysis of key factors impacting the successful use of these models impedes the establishment of basic guidelines for systematic screening campaigns. Thus, we explored the following crucial factors: (i) user-independent inclusion criteria, focusing on sample homogeneity; (ii) metric definition for data analysis; (iii) tumor engraftment criteria; (iv) image analysis versus quantification of human cancer cells using qPCR (RNA and gDNA); (v) tumor implantation sites; (vi) compound distribution (intratumoral administration versus alternative inoculation sites); and (vii) efficacy (intratumoral microinjection versus compound solution in media). Based on these analyses and corresponding assessments, we propose the first roadmap for systematic drug discovery screening in zebrafish xenograft cancer models using a melanoma cell line as a case study. This study aims to help the wider cancer research community to consider the adoption of this versatile model for cancer drug screening projects.

19.
Nature ; 425(6958): 633-7, 2003 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-14534590

RESUMO

Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Valvas Cardíacas/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Divisão Celular , Linhagem da Célula , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes APC , Genótipo , Valvas Cardíacas/anormalidades , Valvas Cardíacas/citologia , Valvas Cardíacas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Mutação/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Transativadores/genética , Proteínas Wnt , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , beta Catenina
20.
Nucleus ; 10(1): 42-47, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30784352

RESUMO

Migrating cells have to cross many physical barriers and confined in 3D environments. The surrounding environment promotes mechano- and biological signals that orchestrate cellular changes, such as cytoskeletal and adhesion rearrangements and proteolytic digestion. Recent studies provide new insights into how the nucleus must alter its shape, localization and mechanical properties in order to promote nuclear deformability, chromatin compaction and gene reprogramming. It is known that the chromatin structure contributes directly to genomic and non-genomic functions, such as gene transcription and the physical properties of the nucleus. Here, we appraise paradigms and novel insights regarding the functional role of chromatin during nuclear deformation. In so doing, we review how constraint and mechanical conditions influence the structure, localization and chromatin decompaction. Finally, we highlight the emerging roles of mechanogenomics and the molecular basis of nucleoskeletal components, which open unexplored territory to understand how cells regulate their chromatin and modify the nucleus.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Epigênese Genética/genética , Humanos
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