Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Mol Cell ; 62(1): 34-46, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058786

RESUMO

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.


Assuntos
Melanoma/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Pirimidinas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma Experimental , Proteínas Oncogênicas/genética , Fatores de Transcrição , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
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
4.
PLoS Biol ; 5(4): e78, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17373855

RESUMO

Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.


Assuntos
Ciclo Celular/fisiologia , Ritmo Circadiano , Hidrocortisona/fisiologia , Animais , Proliferação de Células , Dados de Sequência Molecular , Mutação , Peixe-Zebra
5.
Dev Cell ; 53(5): 577-588.e7, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32516597

RESUMO

Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.


Assuntos
Orientação de Axônios , Neurônios Motores/citologia , Optogenética/métodos , Proteínas de Peixe-Zebra/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células Cultivadas , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Sinapses/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
6.
Dev Cell ; 54(3): 317-332.e9, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32652076

RESUMO

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.


Assuntos
Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Melanócitos/citologia , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Mutação , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatases/genética , Células-Tronco/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
7.
Nat Cell Biol ; 22(4): 372-379, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231306

RESUMO

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.


Assuntos
RNA Helicases DEAD-box/genética , Melanócitos/metabolismo , Crista Neural/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Receptores de Progesterona/genética , Proteínas de Peixe-Zebra/genética , Animais , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Di-Hidro-Orotato Desidrogenase , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Leflunomida/farmacologia , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Crista Neural/efeitos dos fármacos , Crista Neural/crescimento & desenvolvimento , Nucleotídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Ligação Proteica , Receptores de Progesterona/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
8.
Dev Neurosci ; 30(1-3): 65-81, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18075256

RESUMO

We used transgenic zebrafish expressing GFP or YFP in subpopulations of neurons to study the migration, homing process and axon extension of groups of CNS neurons in different regions of the zebrafish brain. We found that extensive migration takes place at all levels of the CNS and gives rise to nuclei or cell populations with specific identities. Here, we describe 4 previously unknown or only partially characterized migratory events taking place in the zebrafish telencephalon and rhombic lip, using 3 different transgenic lines, and identify the phenotypes of the cells undertaking these migrations. The migration of a subgroup of mitral cell precursors from the dorsocaudal telencephalon to the olfactory bulb, visualized in the tg(tbr1:YFP) transgenic line, is coupled with morphogenetic transformation of the dorsal telencephalon. The tg(1.4dlx5a-6a:GFP) transgenic line provides a means to analyze the migration of GABAergic interneurons from the ventral to the dorsal telencephalon, thus extending the occurrence of this migration to another vertebrate. The tg(Xeom:GFP) transgenic line provides the first demonstration of the dorsoventral migration of glutamatergic septal neurons, present in mammals and now described in fish, thus reconciling the contrasting evidence of dorsal patterning genes (tbr1, eomes) expressed in a ventral cell population. Furthermore, migration studies in the tg(1.4dlx5a-6a:GFP) and tg(Xeom:GFP) lines help determine the origin of 2 important cell populations in the fish cerebellum: projection neurons and Purkinje cells. These examples reinforce the concept that migratory events contribute to the distribution of cell types with diverse identities through the CNS and that zebrafish transgenic lines represent excellent tools to study these events.


Assuntos
Movimento Celular/fisiologia , Sistema Nervoso Central/embriologia , Morfogênese/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Linhagem da Célula/genética , Sistema Nervoso Central/citologia , Sistema Nervoso Central/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/citologia , Células-Tronco/citologia , Peixe-Zebra/fisiologia
9.
PLoS Biol ; 3(11): e351, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16176122

RESUMO

It has been well-documented that temperature influences key aspects of the circadian clock. Temperature cycles entrain the clock, while the period length of the circadian cycle is adjusted so that it remains relatively constant over a wide range of temperatures (temperature compensation). In vertebrates, the molecular basis of these properties is poorly understood. Here, using the zebrafish as an ectothermic model, we demonstrate first that in the absence of light, exposure of embryos and primary cell lines to temperature cycles entrains circadian rhythms of clock gene expression. Temperature steps drive changes in the basal expression of certain clock genes in a gene-specific manner, a mechanism potentially contributing to entrainment. In the case of the per4 gene, while E-box promoter elements mediate circadian clock regulation, they do not direct the temperature-driven changes in transcription. Second, by studying E-box-regulated transcription as a reporter of the core clock mechanism, we reveal that the zebrafish clock is temperature-compensated. In addition, temperature strongly influences the amplitude of circadian transcriptional rhythms during and following entrainment by light-dark cycles, a property that could confer temperature compensation. Finally, we show temperature-dependent changes in the expression levels, phosphorylation, and function of the clock protein, CLK. This suggests a mechanism that could account for changes in the amplitude of the E-box-directed rhythm. Together, our results imply that several key transcriptional regulatory elements at the core of the zebrafish clock respond to temperature.


Assuntos
Ritmo Circadiano , Transcrição Gênica , Animais , Temperatura Corporal , Calibragem , Linhagem Celular , Expressão Gênica , Luz , Modelos Biológicos , Dados de Sequência Molecular , Temperatura , Fatores de Transcrição/metabolismo , Peixe-Zebra
10.
Curr Biol ; 13(23): 2051-7, 2003 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-14653994

RESUMO

The timing of cell proliferation is a key factor contributing to the regulation of normal growth. Daily rhythms of cell cycle progression have been documented in a wide range of organisms. However, little is known about how environmental, humoral, and cell-autonomous factors contribute to these rhythms. Here, we demonstrate that light plays a key role in cell cycle regulation in the zebrafish. Exposure of larvae to light-dark (LD) cycles causes a range of different cell types to enter S phase predominantly at the end of the day. When larvae are raised in constant darkness (DD), a low level of arrhythmic S phase is observed. In addition, light-entrained cell cycle rhythms persist for several days after transfer to DD, both observations pointing to the involvement of the circadian clock. We show that the number of LD cycles experienced is essential for establishing this rhythm during larval development. Furthermore, we reveal that the same phenomenon exists in a zebrafish cell line. This represents the first example of a vertebrate cell culture system where circadian rhythms of the cell cycle are observed. Thus, we implicate the cell-autonomous circadian clock in the regulation of the vertebrate cell cycle by light.


Assuntos
Ciclo Celular/fisiologia , Ritmo Circadiano/fisiologia , Peixe-Zebra/fisiologia , Animais , Bromodesoxiuridina , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Larva/fisiologia , Luz , Miocárdio/citologia , Fotoperíodo , Coloração e Rotulagem
11.
Methods Mol Biol ; 362: 429-41, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17417032

RESUMO

Cell lines derived from zebrafish embryos show great potential as cell culture tools to study the regulation and function of the vertebrate circadian clock. They exhibit directly light-entrainable rhythms of clock gene expression that can be established by simply exposing cultures to light-dark cycles. Mammalian cell lines require treatments with serum or activators of signaling pathways to initiate transient, rapidly dampening clock rhythms. Furthermore, zebrafish cells grow at room temperature, are viable for long periods at confluence, and do not require a CO2-enriched atmosphere, greatly simplifying culture conditions. Here we describe detailed methods for establishing zebrafish cell cultures as well as optimizing transient and stable transfections. These protocols have been successfully used to introduce luciferase reporter constructs into the cells and thereby monitor clock gene expression in vivo. The bioluminescence assay described here lends itself particularly well to high-throughput analysis.


Assuntos
Técnicas de Cultura de Células/métodos , Peixe-Zebra , Animais , Linhagem Celular , Ritmo Circadiano/genética , Criopreservação , Eletroporação , Expressão Gênica , Luciferases/genética , Transfecção/métodos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia
12.
Science ; 351(6272): aad2197, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26823433

RESUMO

The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.


Assuntos
Carcinogênese/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Melanoma/genética , Crista Neural/metabolismo , Neoplasias Cutâneas/genética , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Melanócitos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Transcrição SOXE/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Peixe-Zebra/genética
13.
PLoS One ; 8(1): e53631, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23308265

RESUMO

The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered loci in human cancer. It is generally accepted that ARF is involved in oncogenic checkpoint pathways by sensitizing incipient cancer cells to undergo growth arrest or apoptosis through both p53-dependent and independent pathways. While intensive studies have been focused on ARF activation at the transcriptional level, only recently mechanisms governing ARF turnover have been identified. Here, we show for the first time that p14ARF is a PKC target. Prediction analysis showed many potential phosphorylation sites in PKC consensus sequences within ARF protein, and, among them, the threonine at position 8 was the most conserved. Substitution of this threonine influences both ARF stability and localization. Furthermore, a phosphomimetic ARF mutation reduces the ability to arrest cell growth although the ability to bind MDM2 and stabilize p53 result unaffected. Thus we propose that phosphorylation of ARF in both immortalized and tumor cell lines could be a mechanism to escape ARF surveillance following proliferative and oncogenic stress.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Treonina/metabolismo , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Loci Gênicos , Humanos , Fosforilação , Proteína Quinase C/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Transdução de Sinais , Treonina/genética , Transfecção , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética
14.
J Clin Invest ; 122(7): 2337-43, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22751109

RESUMO

Zebrafish have been widely used as a model system for studying developmental processes, but in the last decade, they have also emerged as a valuable system for modeling human disease. The development and function of zebrafish organs are strikingly similar to those of humans, and the ease of creating mutant or transgenic fish has facilitated the generation of disease models. Here, we highlight the use of zebrafish for defining disease pathways and for discovering new therapies.


Assuntos
Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Peixe-Zebra/genética , Animais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/genética , Oftalmopatias/genética , Feminino , Engenharia Genética , Cardiopatias/genética , Cardiopatias/patologia , Doenças Hematológicas/genética , Humanos , Nefropatias/genética , Nefropatias/patologia , Masculino , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia
15.
PLoS One ; 7(12): e51278, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23236462

RESUMO

Light serves as a key environmental signal for synchronizing the circadian clock with the day night cycle. The zebrafish represents an attractive model for exploring how light influences the vertebrate clock mechanism. Direct illumination of most fish tissues and cell lines induces expression of a broad range of genes including DNA repair, stress response and key clock genes. We have previously identified D- and E-box elements within the promoter of the zebrafish per2 gene that together direct light-induced gene expression. However, is the combined regulation by E- and D-boxes a general feature for all light-induced gene expression? We have tackled this question by examining the regulation of additional light-inducible genes. Our results demonstrate that with the exception of per2, all other genes tested are not induced by light upon blocking of de novo protein synthesis. We reveal that a single D-box serves as the principal light responsive element within the cry1a promoter. Furthermore, upon inhibition of protein synthesis D-box mediated gene expression is abolished while the E-box confers light driven activation as observed in the per2 gene. Given the existence of different photoreceptors in fish cells, our results implicate the D-box enhancer as a general convergence point for light driven signaling.


Assuntos
Ritmo Circadiano/efeitos da radiação , Criptocromos/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Luz , Proteínas Circadianas Period/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Peixe-Zebra/metabolismo , Análise de Variância , Animais , Western Blotting , Linhagem Celular , Ritmo Circadiano/fisiologia , Criptocromos/genética , Cicloeximida/farmacologia , Primers do DNA/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Luciferases , Mutagênese Sítio-Dirigida , Proteínas Circadianas Period/genética , Biossíntese de Proteínas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição AP-1/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
16.
PLoS One ; 5(12): e15170, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21170325

RESUMO

BACKGROUND: Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed. METHODOLOGY AND PRINCIPAL FINDINGS: Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period. CONCLUSIONS AND SIGNIFICANCE: This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Progressão da Doença , Genes p53 , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/genética , Modelos Genéticos , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Neoplasias Cutâneas/genética , Transgenes , Peixe-Zebra
17.
Zebrafish ; 6(4): 417-24, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20047469

RESUMO

We have established a model of melanoma progression in zebrafish through the generation of transgenic lines specifically expressing oncogenic human HRAS in the melanocytic lineage. In these tumors we have carried out quantitative expression analysis of several putative cancer genes, from known and predicted cancer gene lists. In particular, we analyzed 39 out of 101 putative cancer genes identified with a bioinformatics approach and selected for the low frequency of duplication and the high connectivity in protein networks. Data obtained by real-time polymerase chain reaction analysis from zebrafish melanoma tissue shows that the expression of many cancer genes is downregulated in zebrafish melanomas, whereas only cell cycle genes are upregulated. To understand whether this trend is due to global repression of gene expression associated to a repressive chromatin state, we investigated whether changes of histone methylation were detectable in our melanoma model. We found substantial differences in the levels of H3K9me3, H4K20me2, H3K27me3, H3K4me3, and H3R2me2a immunostaining in melanoma tissue when compared with normal skin. Thus our analysis suggests that in our model, like in human melanoma, important changes occur to the methylation status of histones. Although the outcome of these changes is still unknown, they could be responsible for the global repression of gene expression through epigenetic regulation shown in this study.


Assuntos
Modelos Animais de Doenças , Regulação para Baixo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Neoplasias Cutâneas/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Biomarcadores Tumorais/genética , Histonas/metabolismo , Humanos , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Peixe-Zebra/metabolismo
18.
Dis Model Mech ; 2(1-2): 56-67, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19132118

RESUMO

Constitutively active, 'oncogenic' H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence. Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression.


Assuntos
Senescência Celular , Genes ras/genética , Proteínas ras/genética , Proteínas ras/fisiologia , Anormalidades Múltiplas/genética , Animais , Proliferação de Células , Anormalidades Craniofaciais/genética , Modelos Animais de Doenças , Cardiopatias Congênitas/genética , Humanos , Deficiência Intelectual/genética , Camundongos , Mutação , Células NIH 3T3 , Síndrome , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra
19.
J Immunol Methods ; 346(1-2): 9-17, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19410577

RESUMO

The lack of a sufficient number of antibodies represents an obstacle in the research performed using the zebrafish (Danio rerio) as a model organism. On the other hand, high-throughput generation of antibodies, especially those suitable for immunohistochemistry, is not an established methodology. Here we present the results of an immunization experiment with a zebrafish tissue lysate that allowed for the isolation of hundreds of monoclonal antibodies suitable for labeling of a large variety of zebrafish tissue and cell structures. Some of them were further characterized in terms of detailed localization and age-dependent expression. In addition, the antigen recognized by one of them was first immunoprecipitated and then identified by mass spectrometry. Furthermore, immunofluorescence-competent recombinant antibodies were also isolated by panning large repertoire phage display libraries, in both single-chain (scFv) and single-domain (VHH) format. Such selection alternative is simpler to organize and could contribute to limit the costs of antibody screening and production.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Imunização , Biblioteca de Peptídeos , Proteínas de Peixe-Zebra/imunologia , Fatores Etários , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Western Blotting , Mapeamento de Epitopos , Hibridomas/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Proteínas Recombinantes/isolamento & purificação , Peixe-Zebra , Proteínas de Peixe-Zebra/análise
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA