Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Am J Hum Genet ; 110(10): 1787-1803, 2023 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-37751738

RESUMO

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder.


Assuntos
Hérnias Diafragmáticas Congênitas , Osteoporose , Adulto , Humanos , Masculino , Animais , Camundongos , Hérnias Diafragmáticas Congênitas/genética , Actinas/genética , Mutação de Sentido Incorreto/genética , Osteoporose/genética
2.
Nature ; 471(7336): 104-9, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21368833

RESUMO

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCF(FBW7) (a SKP1-cullin-1-F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL.


Assuntos
Apoptose , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Benzenossulfonatos/farmacologia , Compostos de Bifenilo/farmacologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Niacinamida/análogos & derivados , Nitrofenóis/farmacologia , Compostos de Fenilureia , Fosforilação , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Piridinas/farmacologia , Sorafenibe , Sulfonamidas/farmacologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos
3.
Nature ; 470(7334): 359-65, 2011 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-21307849

RESUMO

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.


Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/patologia , Telômero/metabolismo , Telômero/patologia , Trifosfato de Adenosina/biossíntese , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proliferação de Células , DNA Mitocondrial/análise , Doxorrubicina/toxicidade , Gluconeogênese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Miocárdio/citologia , Miocárdio/metabolismo , RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Telomerase/deficiência , Telomerase/genética , Telômero/enzimologia , Telômero/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
4.
Proc Natl Acad Sci U S A ; 111(21): E2200-9, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24825892

RESUMO

The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains.


Assuntos
Proteínas de Transporte/genética , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estabilidade Proteica , Transdução de Sinais/fisiologia , Anfirregulina , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Família de Proteínas EGF , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Estimativa de Kaplan-Meier , Camundongos , Mutagênese , Mutação/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Cicatrização/genética
5.
Nature ; 447(7147): 966-71, 2007 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-17515920

RESUMO

Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the neoplastic transformation process. Here we engineered lymphoma-prone mice with chromosomal instability to assess the usefulness of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Along with targeted re-sequencing, our comparative oncogenomic studies identified FBXW7 and PTEN to be commonly deleted both in murine lymphomas and in human T-cell acute lymphoblastic leukaemia/lymphoma (T-ALL). The murine cancers acquire widespread recurrent amplifications and deletions targeting loci syntenic to those not only in human T-ALL but also in diverse human haematopoietic, mesenchymal and epithelial tumours. These results indicate that murine and human tumours experience common biological processes driven by orthologous genetic events in their malignant evolution. The highly concordant nature of genomic events encourages the use of genomically unstable murine cancer models in the discovery of biological driver events in the human oncogenome.


Assuntos
Instabilidade Cromossômica/genética , Aberrações Cromossômicas , Sequência Conservada/genética , Leucemia-Linfoma de Células T do Adulto/genética , Linfoma de Células T/genética , Animais , Genoma/genética , Humanos , Camundongos , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Sintenia/genética
6.
Cancer Cell ; 3(1): 4-6, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12559169

RESUMO

The analysis of compound mouse mutants for nonhomologous end-joining DNA double-strand break repair and those deficient for the p53 checkpoint pathway has provided a fascinating look at the carcinogenic consequences of the failure to properly repair DNA damage and to elicit appropriate checkpoints.


Assuntos
Transformação Celular Neoplásica/genética , Dano ao DNA , Reparo do DNA , Neoplasias/etiologia , Animais , Aberrações Cromossômicas , Genes p53/genética , Humanos , Camundongos , Camundongos Transgênicos , Modelos Animais
7.
Cancer Cell ; 2(2): 149-55, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12204535

RESUMO

Telomere dysfunction and associated fusion-breakage in the mouse encourages epithelial carcinogenesis and a more humanized genomic profile that includes nonreciprocal translocations (NRTs). Here, array comparative genomic hybridization was used to determine the pathogenic significance of NRTs and to determine whether telomere dysfunction also drives amplifications and deletions of cancer-relevant loci. Compared to tumors arising in mice with intact telomeres, tumors with telomere dysfunction possessed higher levels of genomic instability and showed numerous amplifications and deletions in regions syntenic to human cancer hotspots. These observations suggest that telomere-based crisis provides a mechanism of chromosomal instability, including regional amplifications and deletions, that drives carcinogenesis. This model provides a platform for discovery of genes responsible for the major cancers affecting aged humans.


Assuntos
Cromossomos de Mamíferos/genética , Amplificação de Genes , Deleção de Genes , Neoplasias/genética , Telômero/metabolismo , Animais , Aberrações Cromossômicas , DNA de Neoplasias/genética , Genes p53 , Genoma , Humanos , Camundongos , RNA/genética , Sintenia , Telomerase/genética , Telômero/genética
8.
Mol Cell Biol ; 27(6): 2253-65, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17145779

RESUMO

Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc(-/-) DNA-PKcs(-/-) cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)--an additional crucial NHEJ component--was also permissive for chromosome fusions in mTerc(-/-) cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc(-/-) tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.


Assuntos
Ciclo Celular , Proteína Quinase Ativada por DNA/metabolismo , Telomerase/deficiência , Telomerase/metabolismo , Telômero/metabolismo , Animais , Células da Medula Óssea/metabolismo , Domínio Catalítico , Cromossomos/genética , DNA Ligase Dependente de ATP , DNA Ligases/genética , Proteína Quinase Ativada por DNA/genética , Instabilidade Genômica/genética , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Ligação Proteica , Telomerase/genética , Testículo/citologia , Testículo/metabolismo
9.
Pain ; 160(8): 1740-1753, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31335644

RESUMO

Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.


Assuntos
Variação Genética , Genótipo , Nociceptividade/fisiologia , Dor/genética , Fenótipo , Canal de Cátion TRPA1/genética , Alelos , Animais , Camundongos de Cruzamento Colaborativo , Feminino , Formaldeído , Masculino , Camundongos , Locos de Características Quantitativas
10.
Carcinogenesis ; 29(4): 747-53, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18283039

RESUMO

Current mouse models of lung cancer recapitulate signature genetic lesions and some phenotypic features of human lung cancer. However, because mice have long telomeres, models to date do not recapitulate the aspects of lung carcinogenesis-telomere attrition and the genomic instability that ensues-believed to serve as key mechanisms driving lung tumor initiation and progression. To explore the contributions of telomere dysfunction to lung cancer progression, we combined a telomerase catalytic subunit (mTerc) mutation with the well-characterized K-rasG12D mouse lung cancer model. K-ras(G12D) mTerc(-/-) mice with telomere dysfunction but intact p53 exhibited increased lung epithelial apoptosis, delayed tumor formation and increased life span relative to K-ras(G12D) mTerc(+/-) mice with intact telomere function. This demonstrates that by itself, telomere dysfunction acts in a tumor-suppressive mechanism. Introduction of a heterozygous p53 mutation exerted a marked histopathological, biological and genomic impact. K-ras(G12D) mTerc(-/-) p53(+/-) mice developed aggressive tumors with more chromosomal instabilities and high metastatic potential, leading to decreased overall survival. Thus, we have generated a murine model that more faithfully recapitulates key aspects of the human disease. Furthermore, these findings clearly demonstrate (in an in vivo model system) the dual nature of telomere shortening as both a tumor-suppressive and tumor-promoting mechanism in lung cancer, dependent on p53 status.


Assuntos
Genes ras , Instabilidade Genômica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Telômero/genética , Proteína Supressora de Tumor p53/genética , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Mutação , Metástase Neoplásica
11.
Diabetes ; 67(5): 923-935, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29472249

RESUMO

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.ß2m-/- mice transgenically expressing human disease-associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8+ T-cell responses mediated by these human class I variants. NOD.ß2m-/--A2.1 mice were previously used to identify ß-cell autoantigens presented by this human class I variant to pathogenic CD8+ T cells and for testing therapies to attenuate such effectors. However, NOD.ß2m-/- mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-Kd and H2-Db classical class I variants either individually or in tandem (cMHCI-/-). Ablation of the H2-Ag7 class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II-/-). NOD-cMHCI-/- mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8+ T-cell development and T1D susceptibility to NOD-cMHCI-/- mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.


Assuntos
Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Antígeno HLA-A2/genética , Antígenos HLA-B/genética , Camundongos , Microglobulina beta-2/genética , Animais , Sistemas CRISPR-Cas , Diabetes Mellitus Tipo 1/terapia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Transgênicos
12.
Biol Psychiatry ; 60(5): 432-5, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16581033

RESUMO

BACKGROUND: Little is known about the biological mechanisms underlying the excess medical morbidity and mortality associated with mood disorders. Substantial evidence supports abnormalities in stress-related biological systems in depression. Accelerated telomere shortening may reflect stress-related oxidative damage to cells and accelerated aging, and severe psychosocial stress has been linked to telomere shortening. We propose that chronic stress associated with mood disorders may contribute to excess vulnerability for diseases of aging such as cardiovascular disease and possibly some cancers through accelerated organismal aging. METHODS: Telomere length was measured by Southern Analysis in 44 individuals with chronic mood disorders and 44 nonpsychiatrically ill age-matched control subjects. RESULTS: Telomere length was significantly shorter in those with mood disorders, representing as much as 10 years of accelerated aging. CONCLUSIONS: These results provide preliminary evidence that mood disorders are associated with accelerated aging and may suggest a novel mechanism for mood disorder-associated morbidity and mortality.


Assuntos
Envelhecimento/genética , Quebra Cromossômica , Transtornos do Humor/genética , Estresse Psicológico/genética , Telômero , Adulto , Ansiedade/complicações , Ansiedade/genética , Transtorno Bipolar/complicações , Transtorno Bipolar/genética , Estudos de Casos e Controles , Senescência Celular/genética , Doença Crônica , Feminino , Humanos , Masculino , Análise por Pareamento , Pessoa de Meia-Idade , Modelos Genéticos , Transtornos do Humor/complicações , Valores de Referência , Estatísticas não Paramétricas , Estresse Psicológico/etiologia
14.
Curr Mol Med ; 5(2): 145-52, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15974867

RESUMO

The telomere-based model of cell aging has proven to among been among the most enduring hypotheses in cell biology. This model, suggesting that the gradual loss of telomere sequences during the proliferation of cultured human somatic cells imposes a barrier on cellular replicative potential, has been strongly supported by recent genetic and biochemical studies. In addition, evidence implicating telomere dynamics in organismal ageing and cancer progression in vivo suggest that such a process is likely to have considerable physiological relevance in homeostasis and disease. What is the sensing mechanism for shortened telomeres and what is the molecular basis for the ensuing checkpoint response? Moreover, what is the outcome when such failsafe mechanisms are lost? Here we will review the signaling pathways that are induced by alterations in telomere length and integrity and illustrate how these processes provoke downstream effects on cell proliferation and survival. In addition, we discuss how the telomere-induced pathways intersect with the DNA damage response and document how the failure in either process results in unrestrained chromosomal instability.


Assuntos
Envelhecimento/genética , Instabilidade Genômica , Telômero/química , Telômero/metabolismo , Animais , Humanos , Camundongos , Transdução de Sinais
15.
Curr Protoc Mouse Biol ; 6(1): 39-66, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26928663

RESUMO

The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including mice. The CRISPR-Cas9 reagents can be delivered directly into the mouse zygote to derive a mutant animal carrying targeted genetic modifications. The major components of the system include the guide RNA, which provides target specificity, the Cas9 nuclease that creates the DNA double-strand break, and the donor oligonucleotide or plasmid carrying the intended mutation flanked by sequences homologous to the target site. Here we describe the general considerations and experimental protocols for creating genetically modified mice using the CRISPR-Cas9 system.


Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Genômica/métodos , Modelos Animais , Animais , Sequência de Bases , Técnicas de Genotipagem , Camundongos , Microinjeções , Oligonucleotídeos , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Zigoto
16.
DNA Repair (Amst) ; 3(8-9): 979-88, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15279784

RESUMO

DNA double strand breaks (DSBs) are repaired by an extensive network of proteins that recognize damaged DNA and catalyze its repair. By virtue of their similarity, the normal ends of linear chromosomes and internal DNA DSBs are both potential substrates for DSB repair enzymes. Thus, telomeres, specialized nucleo-protein complexes that cap chromosomal ends, serve a critical function to differentiate themselves from internal DNA strand breaks, and as a result prevent genomic instability that can result from their inappropriate involvement in repair reactions. Telomeres that become critically short due to failure of telomere maintenance mechanisms, or which become dysfunctional by loss of telomere binding proteins, elicit extensive checkpoint responses that in normal cells blocks proliferation. In this situation, the DNA DSB repair machinery plays a major role in responding to these "damaged" telomeres - creating chromosome fusions or capturing telomeres from other chromosomes in an effort to rid the cell of the perceived damage. However, a surprising aspect of telomere maintenance is that many of the same proteins that facilitate this repair of damaged telomeres are also necessary for their proper integrity. Here, we review recent work defining the roles for DSB repair machinery in telomere maintenance and in response to telomere dysfunction.


Assuntos
Dano ao DNA , Recombinação Genética , Saccharomyces cerevisiae/genética , Telômero/ultraestrutura , Reparo do DNA , Endodesoxirribonucleases/fisiologia , Exodesoxirribonucleases/fisiologia , Proteínas Fúngicas/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Biológicos , Modelos Genéticos , Mutação , Fenótipo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia
17.
J Exp Med ; 204(13): 3059-66, 2007 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-18070937

RESUMO

Recent studies have demonstrated that the MYB oncogene is frequently duplicated in human T cell acute lymphoblastic leukemia (T-ALL). We find that the human MYB locus is flanked by 257-bp Alu repeats and that the duplication is mediated somatically by homologous recombination between the flanking Alu elements on sister chromatids. Nested long-range PCR analysis indicated a low frequency of homologous recombination leading to MYB tandem duplication in the peripheral blood mononuclear cells of approximately 50% of healthy individuals, none of whom had a MYB duplication in the germline. We conclude that Alu-mediated MYB tandem duplication occurs at low frequency during normal thymocyte development and is clonally selected during the molecular pathogenesis of human T-ALL.


Assuntos
Elementos Alu/genética , Duplicação Gênica , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Proto-Oncogênicas c-myb/genética , Sequência de Bases , Humanos , Hibridização in Situ Fluorescente , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Recombinação Genética
18.
Science ; 297(5581): 565-9, 2002 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-12142527

RESUMO

Cancer is a disease of impaired genome stability. The molecular forces that maintain genome integrity and sense altered chromosome structure are invariably subverted in cancer cells. Here, we explore the contrasting contributions of telomeres in the initiation and suppression of cancer and review the evidence supporting a role for telomere dysfunction as a mechanism driving the radical chromosomal aberrations that typify cancer genomes. Recent work suggests that passage of cells through crisis in the setting of deactivated DNA damage checkpoints provides a mutational mechanism that can generate the diverse genetic alterations required for cancer initiation. A greater understanding of telomere-induced crisis and the cell's crisis management mechanisms should guide the rational development of new therapeutics for cancer and other disorders.


Assuntos
Transformação Celular Neoplásica , Neoplasias/genética , Neoplasias/fisiopatologia , Telomerase/metabolismo , Telômero/fisiologia , Animais , Ciclo Celular , Divisão Celular , Células Cultivadas , Dano ao DNA , Reparo do DNA , Progressão da Doença , Terapia Genética , Humanos , Neoplasias/patologia , Transdução de Sinais , Telomerase/antagonistas & inibidores , Telômero/ultraestrutura
19.
Genes Dev ; 17(1): 88-100, 2003 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-12514102

RESUMO

Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc(-/-) Ink4a/Arf(-/-) mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc(-/-) Ink4a/Arf(-/-) cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc(-/-) Ink4a/Arf(-/-) cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly, despite a marked increase in telomere reserve, cells derived from the ALT+ subcutaneous tumors were unable to generate lung metastases, indicating in vivo functional differences in these principal mechanisms of telomere maintenance. Together, these results are consistent with the model that although telomere dysfunction provokes chromosomal aberrations that initiate carcinogenesis, telomerase-mediated telomere maintenance enables such initiated cells to efficiently achieve a fully malignant endpoint, including metastasis.


Assuntos
Proteínas de Neoplasias/fisiologia , Telomerase/fisiologia , Telômero/fisiologia , Animais , Linhagem Celular Transformada/enzimologia , Linhagem Celular Transformada/patologia , Linhagem Celular Transformada/transplante , Aberrações Cromossômicas , Coloração Cromossômica , Cromossomos/ultraestrutura , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Embrião de Mamíferos/citologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibroblastos/transplante , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Knockout , Camundongos SCID , Transplante de Neoplasias , RNA/genética , Telomerase/deficiência , Telomerase/genética
20.
Nature ; 421(6923): 643-8, 2003 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-12540856

RESUMO

Ataxia-telangiectasia (A-T) results from the loss of ataxia-telangiectasia mutated (Atm) function and is characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, premature ageing and increased neoplasia incidence. Here we evaluate the functional interaction of Atm and telomeres in vivo. We examined the impact of Atm deficiency as a function of progressive telomere attrition at both the cellular and whole-organism level in mice doubly null for Atm and the telomerase RNA component (Terc). These compound mutants showed increased telomere erosion and genomic instability, yet they experienced a substantial elimination of T-cell lymphomas associated with Atm deficiency. A generalized proliferation defect was evident in all cell types and tissues examined, and this defect extended to tissue stem/progenitor cell compartments, thereby providing a basis for progressive multi-organ system compromise, accelerated ageing and premature death. We show that Atm deficiency and telomere dysfunction act together to impair cellular and whole-organism viability, thus supporting the view that aspects of A-T pathophysiology are linked to the functional state of telomeres and its adverse effects on stem/progenitor cell reserves.


Assuntos
Envelhecimento/genética , Homeostase/genética , Proteínas Serina-Treonina Quinases/genética , RNA/genética , Telomerase/genética , Telômero/metabolismo , Alopecia/genética , Anáfase , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Divisão Celular , Células Cultivadas , Proteínas de Ligação a DNA , Fibroblastos , Cor de Cabelo/genética , Linfoma de Células T/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência , Células-Tronco/citologia , Células-Tronco/metabolismo , Taxa de Sobrevida , Telômero/enzimologia , Telômero/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Cicatrização/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA