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1.
Mol Biol Rep ; 47(2): 1381-1391, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31833031

RESUMO

The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1f/f). Nomo1f/f mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1f/f mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1f/f mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.

2.
BMC Cancer ; 10: 454, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20735813

RESUMO

BACKGROUND: Gliomas are the most common type of primary brain tumours, and in this group glioblastomas (GBMs) are the higher-grade gliomas with fast progression and unfortunate prognosis. Two major aspects of glioma biology that contributes to its awful prognosis are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells. Despite of advances, two-year survival for GBM patients with optimal therapy is less than 30%. Even in those patients with low-grade gliomas, that imply a moderately good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells with characteristics of neural stem cells which are able to grow in vitro forming neurospheres and that can be isolated in vivo using surface markers such as CD133. The aim of this study was to define the molecular signature of GBM cells expressing CD133 in comparison with non expressing CD133 cells. This molecular classification could lead to the finding of new potential therapeutic targets for the rationale treatment of high grade GBM. METHODS: Eight fresh, primary and non cultured GBMs were used in order to study the gene expression signatures from its CD133 positive and negative populations isolated by FACS-sorting. Dataset was generated with Affymetrix U133 Plus 2 arrays and analysed using the software of the Affymetrix Expression Console. In addition, genomic analysis of these tumours was carried out by CGH arrays, FISH studies and MLPA; RESULTS: Gene expression analysis of CD133+ vs. CD133- cell population from each tumour showed that CD133+ cells presented common characteristics in all glioblastoma samples (up-regulation of genes involved in angiogenesis, permeability and down-regulation of genes implicated in cell assembly, neural cell organization and neurological disorders). Furthermore, unsupervised clustering of gene expression led us to distinguish between two groups of samples: those discriminated by tumour location and, the most importantly, the group discriminated by their proliferative potential; CONCLUSIONS: Primary glioblastomas could be sub-classified according to the properties of their CD133+ cells. The molecular characterization of these potential stem cell populations could be critical to find new therapeutic targets and to develop an effective therapy for these tumours with very dismal prognosis.


Assuntos
Antígenos CD/genética , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glicoproteínas/genética , Peptídeos/genética , Antígeno AC133 , Idoso , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Hibridização Genômica Comparativa , Feminino , Citometria de Fluxo , Glioblastoma/metabolismo , Glicoproteínas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Cell Cycle ; 8(9): 1314-8, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19279406

RESUMO

A cancer dogma states that inactivation of oncogene(s) can cause cancer remission, implying that oncogenes are the Achilles' heel of cancers. This current "hands on" model of cancer has kept oncogenes firmly in focus as therapeutic targets and is in agreement with the fact that in human cancers all cancerous cells, with independence of the cellular heterogeneity existing within the tumor, carry the same oncogenic genetic lesions. This rule has now been broken in a study of the effect of the BCR-ABL oncogene in cancer development in a mouse model in which oncogene expression is restricted to the stem cell compartment. BCR-ABL is linked to chronic myeloid leukemia (CML) disease in humans, and this study shows that by limiting the oncogene expression to Sca1(+) cells CML arises, indicating that maintenance of oncogene expression is not critical for the generation of differentiated tumor cells and showing a "hands off" role for BCR-ABL in regulating cancer formation. Here we provide an update on the use of this system for modeling human cancer and its potential application for therapeutic targeting of cancer stem cells (CSCs) and the hands-off function of oncogenes.


Assuntos
Células-Tronco Neoplásicas/patologia , Lesões Pré-Cancerosas/patologia , Animais , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos
4.
EMBO J ; 28(1): 8-20, 2009 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-19037256

RESUMO

In human cancers, all cancerous cells carry the oncogenic genetic lesions. However, to elucidate whether cancer is a stem cell-driven tissue, we have developed a strategy to limit oncogene expression to the stem cell compartment in a transgenic mouse setting. Here, we focus on the effects of the BCR-ABLp210 oncogene, associated with chronic myeloid leukaemia (CML) in humans. We show that CML phenotype and biology can be established in mice by restricting BCR-ABLp210 expression to stem cell antigen 1 (Sca1)(+) cells. The course of the disease in Sca1-BCR-ABLp210 mice was not modified on STI571 treatment. However, BCR-ABLp210-induced CML is reversible through the unique elimination of the cancer stem cells (CSCs). Overall, our data show that oncogene expression in Sca1(+) cells is all that is required to fully reprogramme it, giving rise to a full-blown, oncogene-specified tumour with all its mature cellular diversity, and that elimination of the CSCs is enough to eradicate the whole tumour.


Assuntos
Expressão Gênica , Genes abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco , Animais , Ataxina-1 , Ataxinas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/análise , Proteínas Nucleares/análise , Análise de Sobrevida
5.
Annu Rev Genet ; 41: 41-61, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17550342

RESUMO

Elucidation of the molecular mechanisms that underlie disease development is still a tremendous challenge for basic science, and a prerequisite to the development of new and disease-specific targeted therapies. This review focuses on the function of SNAI2, a member of the Snail family of zinc-finger transcription factors, and discusses its possible role in disease development. SNAI2 has been implicated in diseases of melanocyte development and cancer in humans. Many malignancies arise from a rare population of cells that alone have the ability to self-renew and sustain the tumor (i.e., cancer stem cells). SNAI2 controls key aspects of stem cell function in mouse and human, suggesting that similar mechanisms control normal development and cancer stem cell properties. These insights are expected to contribute significantly to the genetics of cancer and to the development of both cancer therapy and new methods for assessing treatment efficacy.


Assuntos
Neoplasias Experimentais/fisiopatologia , Fatores de Transcrição/fisiologia , Dedos de Zinco , Animais , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética
6.
Curr Med Chem ; 13(15): 1719-25, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16787215

RESUMO

Can cancer be cured or will it have to be controlled as a chronic disease? Despite a better understanding of the biology of tumour cells, the treatment of most cancers has not significantly changed for the past three decades. Are current cancer drugs targeted at the wrong kind of cells? Accumulating evidence has implicated that cancer is a disease of stem cells. In this context, a small fraction of cancer cells adopt the properties of stem cells. In some cases, the cancer stem cells (CSC) could be the close derivative of normal tissue stem cells. In either situation, the net result will be the same, in that CSC are the cells to be used as targets in the development of molecular and pharmaceutical therapies to treat and prevent human cancer. This could be a paradigm shift in the treatment of cancer, away from targeting the blast cells and towards the targeting of the CSC. A challenge to this approach will be to find a way to specifically target CSC without toxicity to normal cells. In this article, we propose how CSC can be used in therapy programs (target identification, drug discovery, etc.). Therefore, in the future, it might be possible to rid a patient of all his/her cancer cells, including the cancer stem cells.


Assuntos
Neoplasias/patologia , Células-Tronco/efeitos dos fármacos , Animais , Biomarcadores Tumorais , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Camundongos , Neoplasias/terapia , Células-Tronco/citologia
7.
Genomics ; 87(1): 113-8, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16311016

RESUMO

There is a need to reveal mechanisms that account for maintenance of the mesenchymal phenotype in normal development and cancer. Slug (approved gene symbol Snai2), a member of the Snail gene family of zinc-finger transcription factors, is believed to function in the maintenance of the nonepithelial phenotype. This study identified candidate Slug target genes linked to Slug gene suppression in primary mouse embryonic fibroblasts. Expression analyses were performed with a mouse cDNA microarray (Mousechip-CNIO) containing 15,000 clones. A total of 15 novel Slug target species were validated by real-time PCR or Western analyses. These included self-renewal genes (Bmi1, Nanog, Gfi1), epithelial-mesenchymal genes (Tcfe2a, Ctnb1, Sin3a, Hdac1, Hdac2, Muc1, Cldn11), survival genes (Bcl2, Bbc3), and cell cycle/damage genes (Cdkn1a, Rbl1, Mdm2). Expression patterns were studied in wild-type MEFs and Slug-deficient MEFs. Slug-complementation studies recovered aberrant gene expression in cells lacking Slug, indicating that these genes were regulated directly by Slug. These results highlight their potential roles in mediating Slug function in mesenchymal cells and may help to identify novel therapeutic biomarkers in cancers linked to Slug.


Assuntos
Embrião de Mamíferos/fisiologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/genética , Animais , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Dano ao DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genes cdc/fisiologia , Teste de Complementação Genética/métodos , Camundongos , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo
8.
Hum Mol Genet ; 14(22): 3449-61, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16207734

RESUMO

The zinc-finger transcription factor Snail is believed to trigger epithelial-mesenchymal transitions (EMTs) during cancer progression. This idea is supported by analysis of Snail knockout mice, which uncovered crucial role of Snail in gastrulation, and of individuals with cancer, in whom Snail expression is frequently upregulated. However, these results have not shown a direct link between Snail and the pathogenesis of cancer. Here we show that mice carrying hypomorphic tetracycline-repressible Snail transgenes, that increase Snail expression to 20% above normal levels, exhibit no morphological alterations and develop both epithelial and mesenchymal tumours (leukaemias). Suppression of the Snail transgene did not rescue the malignant phenotype, indicating that alterations induced by Snail are irreversible. CombitTA-Snail murine embryonic fibroblasts show similar migratory ability to that of control mouse embryonic fibroblasts (MEFs). However, CombitTA-Snail-MEFs induce tumour formation in nude mice. CombitTA-Snail expression results in increased radioprotection in vivo, although it does not affect p53 regulation in response to DNA damage. In concert with these results, Snail expression is repressed following DNA damage. This regulation of Snail by DNA damage is p53-independent. Our results connect DNA damage with the requirement of a critical level of an EMT regulator and provide genetic evidence that Snail plays essential roles in cancer development in mammals and thereby influences cell fate in the genotoxic stress response.


Assuntos
Neoplasias/genética , Neoplasias/metabolismo , Fatores de Transcrição/genética , Animais , Células COS , Linhagem Celular , Células Cultivadas , Raios gama , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Neoplasias/patologia , Neoplasias/radioterapia , RNA Mensageiro , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo
9.
Oncogene ; 24(19): 3073-82, 2005 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-15735690

RESUMO

The SNAIL-related zinc-finger transcription factor, SLUG (SNAI2), is critical for the normal development of neural crest-derived cells and loss-of-function SLUG mutations have been proven to contribute to piebaldism and Waardenburg syndrome type 2 in a dose-dependent fashion. While aberrant induction of SLUG has been documented in cancer cells, relatively little is known about the consequences of SLUG overexpression in malignancy. To investigate the potential role of SLUG overexpression in development and in cancer, we generated mice carrying a tetracycline-repressible Slug transgene. These mice were morphologically normal at birth, and developed mesenchymal tumours (leukaemia and sarcomas) in almost all cases examined. Suppression of the Slug transgene did not rescue the malignant phenotype. Furthermore, the BCR-ABL oncogene, which induces Slug expression in leukaemic cells, did not induce leukaemia in Slug-deficient mice, implicating Slug in BCR-ABL leukaemogenesis in vivo. Overall, the findings indicate that while Slug overexpression is not sufficient to cause overt morphogenetic defects in mice, they demonstrate a specific and critical role for Slug in the pathogenesis of mesenchymal tumours.


Assuntos
Neoplasias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Complementar/metabolismo , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Heterozigoto , Homozigoto , Humanos , Células K562 , Leucemia/etiologia , Leucemia/genética , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Mutação , Transplante de Neoplasias , Neoplasias/etiologia , Neoplasias Experimentais/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Tempo , Transfecção , Transgenes , Células U937
10.
Oncogene ; 22(27): 4205-11, 2003 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-12833143

RESUMO

Radiation-induced destruction of the hematopoietic system is the primary cause of death based on the findings that transfer of normal bone marrow cells prevents death from lethal irradiation. The stem cell factor-c-kit signaling pathway (SCF/c-kit) has been previously implicated in the hematopoietic recovery which prevents death from lethal irradiation, but the molecular mechanisms that mediate this biological effect are unknown. Since mutations on SCF, c-kit and Slug genes have a similar phenotype in mice, we examined if Slug could complement the radiosensitivity of kit-deficient mice. In this report, we show that Slug acts as a radioprotection agent as lack of Slug results in increased radiosensitivity. This effect cannot be recovered by activating SCF/c-kit in lethally irradiated Slug-deficient mice, as SCF-treated mice did not demonstrate stimulation of hematopoietic recovery leading to survival of the Slug-deficient mice. We found that we could complement the hematopoietic failure in lethally irradiated c-kit-deficient mice by transducing them with a TAT-Slug protein. We conclude that the zinc-finger transcription factor Slug is absolutely necessary for survival from lethal irradiation and identify Slug as the molecular target that mediates the radioprotection through SCF/c-kit. These results indicate that Slug may be a molecular component conferring radioresistance to cancer cells.


Assuntos
Proteínas Proto-Oncogênicas c-kit/fisiologia , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/fisiologia , Animais , Células da Medula Óssea/metabolismo , Sobrevivência Celular , DNA/metabolismo , Dano ao DNA , Raios gama , Genótipo , Heterozigoto , Homozigoto , Immunoblotting , Camundongos , Microscopia de Fluorescência , Mutação , Proteínas Oncogênicas/metabolismo , Fases de Leitura Aberta , Fenótipo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco
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