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2.
Cancer Cell ; 33(2): 322-336.e8, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29438700

RESUMO

Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.


Assuntos
Histona Desmetilases/genética , Histona Desmetilases com o Domínio Jumonji/genética , Melanoma/genética , Animais , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Camundongos Nus , Regiões Promotoras Genéticas/genética
3.
Nephron ; 138(4): 310-323, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29342457

RESUMO

BACKGROUND: von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene. METHODS: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. RESULTS: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. CONCLUSIONS: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.


Assuntos
Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Desenvolvimento Embrionário/genética , Glomérulos Renais/anormalidades , Glomérulos Renais/crescimento & desenvolvimento , Túbulos Renais Proximais/anormalidades , Túbulos Renais Proximais/crescimento & desenvolvimento , Larva , Mutação , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
4.
Zebrafish ; 14(4): 379-382, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28557653

RESUMO

The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.


Assuntos
Vetores Genéticos/administração & dosagem , Lentivirus/genética , Melanoma/genética , Retroviridae/genética , Transdução Genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Melanoma/patologia , Glicoproteínas de Membrana/genética , Células Tumorais Cultivadas , Proteínas do Envelope Viral/genética , Peixe-Zebra/crescimento & desenvolvimento
5.
Value Health ; 20(4): 627-636, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28408005

RESUMO

OBJECTIVES: The aim of this article was to provide practical guidance in setting up patient registries to facilitate real-world data collection for health care decision making. METHODS: This guidance was based on our experiences and involvement in setting up patient registries in oncology in the Netherlands. All aspects were structured according to 1) mission and goals ("the Why"), 2) stakeholders and funding ("the Who"), 3) type and content ("the What"), and 4) identification and recruitment of patients, data handling, and pharmacovigilance ("the How"). RESULTS: The mission of most patient registries is improving patient health by improving the quality of patient care; monitoring and evaluating patient care is often the primary goal ("the Why"). It is important to align the objectives of the registry and agree on a clear and functional governance structure with all stakeholders ("the Who"). There is often a trade off between reliability, validity, and specificity of data elements and feasibility of data collection ("the What"). Patient privacy should be carefully protected, and address (inter-)national and local regulations. Patient registries can reveal unique safety information, but it can be challenging to comply with pharmacovigilance guidelines ("the How"). CONCLUSIONS: It is crucial to set up an efficient patient registry that serves its aims by collecting the right data of the right patient in the right way. It can be expected that patient registries will become the new standard alongside randomized controlled trials due to their unique value.


Assuntos
Coleta de Dados/métodos , Tomada de Decisões , Pesquisa sobre Serviços de Saúde/métodos , Oncologia/métodos , Formulação de Políticas , Sistema de Registros , Confidencialidade , Confiabilidade dos Dados , Coleta de Dados/economia , Coleta de Dados/normas , Fidelidade a Diretrizes , Guias como Assunto , Pesquisa sobre Serviços de Saúde/economia , Pesquisa sobre Serviços de Saúde/normas , Humanos , Oncologia/economia , Oncologia/normas , Países Baixos , Objetivos Organizacionais , Farmacovigilância , Sistema de Registros/normas , Reprodutibilidade dos Testes , Apoio à Pesquisa como Assunto
6.
Pigment Cell Melanoma Res ; 30(4): 402-412, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28379616

RESUMO

Melanoma is the most aggressive and deadliest form of skin cancer. A detailed knowledge of the cellular, molecular, and genetic events underlying melanoma progression is highly relevant to diagnosis, prognosis and risk stratification, and the development of new therapies. In the last decade, zebrafish have emerged as a valuable model system for the study of melanoma. Pathway conservation, coupled with the availability of robust genetic, transgenic, and chemical tools, has made the zebrafish a powerful model for identifying novel disease genes, visualizing cancer initiation, interrogating tumor-microenvironment interactions, and discovering new therapeutics that regulate melanocyte and melanoma development. In this review, we will give an overview of these studies, and highlight recent advancements that will help unravel melanoma pathogenesis and impact human disease.


Assuntos
Melanoma/patologia , Melanoma/terapia , Pesquisa Médica Translacional , Peixe-Zebra/fisiologia , Animais , Carcinogênese/patologia , Modelos Animais de Doenças , Humanos , Melanócitos/patologia
7.
J Exp Med ; 214(3): 623-637, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28148688

RESUMO

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.


Assuntos
Doenças do Desenvolvimento Ósseo/etiologia , Deficiências do Desenvolvimento/etiologia , Síndromes de Imunodeficiência/etiologia , Mutação , N-Acetilglucosaminiltransferases/genética , Animais , Pré-Escolar , Feminino , Heparitina Sulfato/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lactente , Linfócitos/fisiologia , Peixe-Zebra
8.
Dev Neurorehabil ; 20(3): 173-178, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27055081

RESUMO

OBJECTIVE: Determine healthcare costs of upper-extremity surgical correction in children with spastic cerebral palsy (CP). METHOD: This cohort study included 39 children with spastic CP who had surgery for their upper extremity at a Dutch hospital. A retrospective cost analysis was performed including both hospital and rehabilitation costs. Hospital costs were determined using microcosting methodology. Rehabilitation costs were estimated using reference prices. RESULTS: Hospital costs averaged €6813 per child. Labor (50%), overheads (29%), and medical aids (15%) were important cost drivers. Rehabilitation costs were estimated at €3599 per child. CONCLUSIONS: Surgery of the upper extremity is an important contributor to the healthcare costs of children with CP. Our study shows that labor is the most important cost driver for hospital costs, owing to the multidisciplinary approach and patient-specific treatment plan. A remarkable finding was the substantial amount of rehabilitation costs.


Assuntos
Paralisia Cerebral/economia , Paralisia Cerebral/cirurgia , Custos e Análise de Custo/economia , Extremidade Superior/cirurgia , Adolescente , Criança , Feminino , Humanos , Masculino , Estudos Retrospectivos
9.
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 , Transcrição Genética , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
10.
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
11.
Cancer Res ; 75(20): 4272-4282, 2015 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-26282170

RESUMO

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.


Assuntos
Modelos Biológicos , Neoplasias/patologia , Peixe-Zebra , Algoritmos , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Mutação , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Transcriptoma
12.
J Clin Invest ; 125(5): 1987-97, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25866969

RESUMO

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl(-/-) mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl(-/-) embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Hidrazonas/uso terapêutico , Sulfonas/uso terapêutico , Doença de von Hippel-Lindau/tratamento farmacológico , Regiões 5' não Traduzidas , Aminoácidos Dicarboxílicos/toxicidade , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/irrigação sanguínea , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Rim/patologia , Fígado/patologia , Contração Miocárdica/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Fenótipo , Policitemia/tratamento farmacológico , Policitemia/genética , Vasos Retinianos/patologia , Sulfonas/farmacologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/patologia , Doença de von Hippel-Lindau/fisiopatologia
13.
J Pathol ; 231(1): 117-29, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23744542

RESUMO

Biallelic mutations of the von Hippel-Lindau (VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma (RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product (pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer (AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC.


Assuntos
Carcinoma de Células Renais/mortalidade , Fator de Transcrição E2F1/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Renais/mortalidade , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Animais , Western Blotting , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Senescência Celular , Modelos Animais de Doenças , Fator de Transcrição E2F1/metabolismo , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Organismos Geneticamente Modificados , Plasmídeos , Prognóstico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Taxa de Sobrevida , Transfecção , Células Tumorais Cultivadas , Peixe-Zebra
14.
PLoS Genet ; 9(4): e1003384, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23599692

RESUMO

Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.


Assuntos
Seminoma , Peixe-Zebra , Animais , Genes Supressores de Tumor , Genótipo , Humanos , Mutação , Peixe-Zebra/genética
15.
Cancer Res ; 72(16): 4017-27, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22665266

RESUMO

Hypoxic signaling is a central modulator of cellular physiology in cancer. Core members of oxygen-sensing pathway including the von Hippel-Lindau tumor suppressor protein (pVHL) and the hypoxia inducible factor (HIF) transcription factors have been intensively studied, but improved organismal models might speed advances for both pathobiologic understanding and therapeutic modulation. To study HIF signaling during tumorigenesis and development in zebrafish, we developed a unique in vivo reporter for hypoxia, expressing EGFP driven by prolyl hydroxylase 3 (phd3) promoter/regulatory elements. Modulation of HIF pathway in Tg(phd3::EGFP) embryos showed a specific role for hypoxic signaling in the transgene activation. Zebrafish vhl mutants display a systemic hypoxia response, reflected by strong and ubiquitous transgene expression. In contrast to human VHL patients, heterozygous Vhl mice and vhl zebrafish are not predisposed to cancer. However, upon exposure to dimethylbenzanthracene (DMBA), the vhl heterozygous fish showed an increase in the occurrence of hepatic and intestinal tumors, a subset of which exhibited strong transgene expression, suggesting loss of Vhl function in these tumor cells. Compared with control fish, DMBA-treated vhl heterozygous fish also showed an increase in proliferating cell nuclear antigen-positive renal tubules. Taken together, our findings establish Vhl as a genuine tumor suppressor in zebrafish and offer this model as a tool to noninvasively study VHL and HIF signaling during tumorigenesis and development.


Assuntos
Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Neoplasias Intestinais/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Transformação Celular Neoplásica/induzido quimicamente , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Intestinais/induzido quimicamente , Neoplasias Hepáticas Experimentais/induzido quimicamente , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo
16.
Methods Cell Biol ; 105: 163-90, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21951530

RESUMO

The von Hippel-Lindau (VHL) tumor suppressor gene encodes an adaptor protein that regulates an array of transcription-dependent and -independent cellular and physiological processes. Mutations in this gene cause VHL disease, congenital polycythemia, and several sporadic tumor types. The last 15 years of fundamental and clinical research have helped define the phenotypic spectrum of VHL-associated diseases and have introduced new cellular functions for pVHL. Here, we review the current knowledge of VHL function, and the different animal models for VHL disease, with a particular focus on the zebrafish. Zebrafish vhl mutants develop key aspects of the human disease condition, including activation of the hypoxia-inducible factor (HIF) signaling pathway, polycythemia, excessive neovascularization, macular edema, and pronephric abnormalities. The zebrafish vhl model offers a platform for the identification of genetic pathways, modifiers, and interactors involved in the development of VHL-associated neoplasms. Vhl mutants represent a unique and clinically relevant in vivo model for studying genotype-phenotype correlations and the identification of prognostic biomarkers. The amenability of zebrafish for chemical genetic screens will not only be helpful to identify novel therapeutic agents but may also reveal novel processes that require regulation by VHL.


Assuntos
Teste de Complementação Genética/métodos , Ensaios de Triagem em Larga Escala , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/genética , Policitemia/genética , Proteínas Supressoras de Tumor , Proteínas de Peixe-Zebra , Peixe-Zebra/metabolismo , Doença de von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Estudos de Associação Genética , Humanos , Fator 1 Induzível por Hipóxia/genética , Dados de Sequência Molecular , Mutação , Policitemia/patologia , Alinhamento de Sequência , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Doença de von Hippel-Lindau/patologia
17.
Proc Natl Acad Sci U S A ; 108(11): 4358-63, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21368212

RESUMO

Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel-Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells.


Assuntos
Metabolismo Energético , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Peixe-Zebra/metabolismo , Animais , Metabolismo Basal , Polaridade Celular , Embrião não Mamífero/enzimologia , Embrião não Mamífero/patologia , Privação de Alimentos , Fator de Crescimento Insulin-Like I/metabolismo , Intestinos/patologia , Larva/citologia , Larva/metabolismo , Mutação/genética , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
18.
Dis Model Mech ; 3(5-6): 343-53, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20335444

RESUMO

Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. We show that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, we show, by tetramethyl rhodamine-dextran angiography, that vascular abnormalities in the vhl(-/-) retina lead to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl(-/-)-induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl(-/-) angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since we show that the vhl(-/-) angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments.


Assuntos
Hipóxia/complicações , Hipóxia/patologia , Mutação/genética , Neovascularização Retiniana/complicações , Neovascularização Retiniana/patologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Modelos Animais de Doenças , Edema/complicações , Edema/patologia , Humanos , Macula Lutea/metabolismo , Macula Lutea/patologia , Proteínas Mutantes/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Descolamento Retiniano/complicações , Descolamento Retiniano/patologia , Neovascularização Retiniana/enzimologia , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genética
19.
Hum Mutat ; 31(5): 521-37, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20151405

RESUMO

Mutations in the von Hippel-Lindau (VHL) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types as well. Reports of VHL mutations are dispersed throughout original articles and databases that have not been recently updated. We compiled a comprehensive mutation table of 1,548 germline and somatic VHL mutations, derived from this protein of only 213 amino acids. We describe detailed phenotype and gene mutation information for 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). These data represent the most extensive catalog of germline VHL mutations to date. We also review VHL disease, known and theorized pathogenesis of common VHL manifestations, and genotype-phenotype correlations. Analysis of all VHL families, excluding germline mutations resulting in congenital polycythemias, describes the spectrum of mutation types: 52% missense, 13% frameshift, 11% nonsense, 6% in-frame deletions/insertions, 11% large/complete deletions, and 7% splice mutations. This easy-to-use compilation of VHL mutations is intended to facilitate research and function as a necessary adjunct for physicians when providing patient information.


Assuntos
Estudos de Associação Genética , Doença de von Hippel-Lindau/diagnóstico , Doença de von Hippel-Lindau/genética , Mutação da Fase de Leitura , Mutação em Linhagem Germinativa , Humanos , Neoplasias Renais/genética , Mutação , Linhagem , Doença de von Hippel-Lindau/patologia
20.
Blood ; 113(25): 6449-60, 2009 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-19304954

RESUMO

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response. The vhl mutants develop polycythemia with concomitantly increased epo/epor mRNA levels and erythropoietin signaling. In situ hybridizations reveal global up-regulation of both red and white hematopoietic lineages. Hematopoietic tissues are highly proliferative, with enlarged populations of c-myb(+) hematopoietic stem cells and circulating erythroid precursors. Chemical activation of hypoxia-inducible factor signaling recapitulated aspects of the vhl(-/-) phenotype. Furthermore, microarray expression analysis confirms the hypoxic response and hematopoietic phenotype observed in vhl(-/-) embryos. We conclude that VHL participates in regulating hematopoiesis and erythroid differentiation. Injections with human VHLp30 and R200W mutant mRNA demonstrate functional conservation of VHL between mammals and zebrafish at the amino acid level, indicating that vhl mutants are a powerful new tool to study genotype-phenotype correlations in human disease. Zebrafish vhl mutants are the first congenital embryonic viable systemic vertebrate animal model for VHL, representing the most accurate model for VHL-associated polycythemia to date. They will contribute to our understanding of hypoxic signaling, hematopoiesis, and VHL-associated disease progression.


Assuntos
Modelos Animais de Doenças , Hipóxia/genética , Policitemia/genética , Proteínas Supressoras de Tumor/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem da Célula , Sequência Conservada , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa , Hematopoese/genética , Humanos , Hipóxia/fisiopatologia , Dados de Sequência Molecular , Mutação Puntual , Policitemia/fisiopatologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sintenia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
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