RESUMO
Sarin is an organophosphate nerve agent that is among the most lethal chemical toxins known to mankind. Because of its vaporization properties and ease and low cost of production, sarin is the nerve agent with a strong potential for use by terrorists and rouge nations. The primary route of sarin exposure is through inhalation and, depending on the dose, sarin leads to acute respiratory failure and death. The mechanism(s) of sarin-induced respiratory failure is poorly understood. Sarin irreversibly inhibits acetylcholine esterase, leading to excessive synaptic levels of acetylcholine and, we have previously shown that sarin causes marked ventilatory changes including weakened response to hypoxia. We now show that LD50 sarin inhalation causes severe bronchoconstriction in rats, leading to airway resistance, increased hypoxia-induced factor-1α, and severe lung epithelium injury. Transferring animals into 60% oxygen chambers after sarin exposure improved the survival from about 50% to 75% at 24h; however, many animals died within hours after removal from the oxygen chambers. On the other hand, if LD50 sarin-exposed animals were administered the bronchodilator epinephrine, >90% of the animals survived. Moreover, while both epinephrine and oxygen treatments moderated cardiorespiratory parameters, the proinflammatory cytokine surge, and elevated expression of hypoxia-induced factor-1α, only epinephrine consistently reduced the sarin-induced bronchoconstriction. These data suggest that severe bronchoconstriction is a critical factor in the mortality induced by LD50 sarin inhalation, and epinephrine may limit the ventilatory, inflammatory, and lethal effects of sarin.
Assuntos
Broncoconstrição/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Epinefrina/farmacologia , Pneumopatias/tratamento farmacológico , Oxigênio/farmacologia , Sarina/toxicidade , Doença Aguda , Administração por Inalação , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Inibidores da Colinesterase/toxicidade , Relação Dose-Resposta a Droga , Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Dose Letal Mediana , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pneumopatias/induzido quimicamente , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Ratos Endogâmicos F344 , Sarina/administração & dosagemRESUMO
Tire and road wear particles (TRWP) are a component of ambient particulate matter (PM) produced from the interaction of tires with the roadway. Inhalation of PM has been associated with cardiopulmonary morbidities and mortalities thought to stem from pulmonary inflammation. To determine whether TRWP may contribute to these events, the effects of subacute inhalation of TRWP were evaluated in rats. TRWP were collected at a road simulator laboratory, aerosolized, and used to expose male and female Sprague-Dawley rats (n = 10/treatment group) at ~10, 40, or 100 µg/m³ TRWP via nose-only inhalation for 6 h/day for 28 days. Particle size distribution of the aerosolized TRWP was found to be within the respirable range for rats. Toxicity was assessed following OECD guidelines (TG 412). No TRWP-related effects were observed on survival, clinical observations, body or organ weights, gross pathology, food consumption, immune system endpoints, serum chemistry, or biochemical markers of inflammation or cytotoxicity. Rare to few focal areas of subacute inflammatory cell infiltration associated with TWRP exposure were observed in the lungs of one mid and four high exposure animals, but not the low-exposure animals. These alterations were minimal, widely scattered and considered insufficient in extent or severity to have an impact on pulmonary function. Furthermore, it is expected that these focal lesions would remain limited and may undergo resolution without long-term or progressive pulmonary alterations. Therefore, from this study we identified a no-observable-adverse-effect-level (NOAEL) of 112 µg/m³ of TRWP in rats for future use in risk assessment of TRWP.
Assuntos
Poluentes Ambientais/toxicidade , Pulmão/efeitos dos fármacos , Veículos Automotores , Material Particulado/toxicidade , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Citocinas/metabolismo , Poluentes Ambientais/química , Pulmão/metabolismo , Pulmão/patologia , Nível de Efeito Adverso não Observado , Tamanho da Partícula , Material Particulado/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade SubagudaRESUMO
An oral sulfate salt solution (OSS), under development as a bowel cleansing agent for colonoscopy in humans, is studied in rats and dogs. In rats, amaximumpractical oral OSS dose (5 g/kg/d) is compared with an oral sodium phosphate (OSP) solution, both at about 7 times the clinical dose. OSS induces the intended effects of loose stools and diarrhea. In rats, higher urine sodium and potassium accompany higher clearance rates, considered adaptive to the osmotic load of OSS. OSS for 28 days is well tolerated in rats and dogs. In contrast, OSP causes increased mortality, reduced body weight and food consumption, severe kidney tubular degeneration, and calcium phosphate deposition in rats. These are accompanied by mineralization in the stomach and aorta, along with cardiac and hepatic degeneration and necrosis. The greater safety margin of OSS over OSP at similarmultiples of the clinical dose indicates its suitability for human use.
Assuntos
Colonoscopia , Rim/efeitos dos fármacos , Fosfatos/toxicidade , Sulfatos/toxicidade , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Cálcio/sangue , Creatinina/metabolismo , Cães , Feminino , Rim/patologia , Masculino , Potássio/urina , Ratos , Sódio/sangue , Sódio/urinaRESUMO
The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1-deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27(KIP1) abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin-radixin-moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated MEC migration through paracrine signaling via induction of expression and secretion of CXCL16 and MIP1gamma. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Neoplasias da Mama/genética , Crescimento Celular , Movimento Celular , Polaridade Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Células Cultivadas , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Hospedeiro Imunocomprometido , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Transferência Adotiva , Animais , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Imunidade Celular , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-2/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , Vacinação/métodos , Vacinas Atenuadas/imunologiaRESUMO
We present a status report from the NCI Mouse Models of Human Cancers Consortium (MMHCC) Precancers Workshop held November 8 and 9, 2004. An expert panel, the Mouse Models Group (MMG) evaluated the status of mouse models of precancer emphasizing genetically engineered mouse models, especially of lining epithelium and their utilitarian value to human carcinogenesis. An outline of the background for the panel's considerations is provided with examples of past and current precancerous lesions in mice. The experimental use of oncogenic viruses and chemical carcinogens in mice led to operational definitions of initiation, promotion, and preneoplasia Preneoplastic and precancerous lesions are found in these models. In this precancer concept, most preneoplastic lesions are considered as potentially precancerous or at least an earlier stage in cancer development than typical pre-invasive epithelial lesions, which are often seen in these mouse models. Genetically engineered mice, used to test the oncogenicity of individual genes, develop precancers that are initiated by defined molecular and histopathologic changes. The mouse can be used to isolate and study precancers in detail, thereby providing a level of biological understanding not readily available in clinical disease. These studies suggest that genetically engineered mice are very useful preclinical models for chemoprevention and therapy.
Assuntos
Carcinógenos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , Infecções Tumorais por Vírus/genética , Animais , Antineoplásicos/uso terapêutico , Carcinoma in Situ/induzido quimicamente , Carcinoma in Situ/genética , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/patologia , Humanos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Oncogenes/genética , Vírus Oncogênicos/genética , Lesões Pré-Cancerosas/tratamento farmacológico , Lesões Pré-Cancerosas/prevenção & controle , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Infecções Tumorais por Vírus/patologiaRESUMO
Obstacles to the expansion of cells with proliferative potential include the induction of cell death, telomere-based senescence, and the pRb and p53 tumor suppressors. Not infrequently, the molecular pathways regulating oncogenesis recapitulate aberrations of processes governing embryogenesis. The genetic network, consisting of the dachshund (dac), eyes absent (eya), eyeless, and sine oculis (so) genes, regulates cell fate determination in metazoans, with dac serving as a cointegrator through a So DNA-binding factor. Here, DACH1 inhibited oncogene-mediated breast oncogenesis, blocking breast cancer epithelial cell DNA synthesis, colony formation, growth in Matrigel, and tumor growth in mice. Genetic deletion studies demonstrated a requirement for cyclin D1 in DACH1-mediated inhibition of DNA synthesis. DACH1 repressed cyclin D1 through a novel mechanism via a c-Jun DNA-binding partner, requiring the DACH1 alpha-helical DS domain which recruits corepressors to the local chromatin. Analysis of over 2,000 patients demonstrated increased nuclear DACH1 expression correlated inversely with cellular mitosis and predicted improved breast cancer patient survival. The cell fate determination factor, DACH1, arrests breast tumor proliferation and growth in vivo providing a new mechanistic and potential therapeutic insight into this common disease.
Assuntos
Neoplasias da Mama/patologia , Ciclina D1/metabolismo , Proteínas do Olho/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Ciclina D1/genética , DNA/biossíntese , DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Fenótipo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição AP-1/metabolismo , Ensaio Tumoral de Célula-Tronco , Proteínas ras/metabolismoRESUMO
OBJECTIVES: To perform a comparison to determine which of two methods of partial urethral ligation produces the most consistent outcome and fewest side effects. Such a study has not been previously reported. Partial urethral ligation is a means of causing reproducible bladder outlet obstruction. In the male rat model, partial urethral obstruction can be performed either by perineal incision and bulbous urethral ligation or retropubic incision and midprostatic obstruction. METHODS: Fifteen male Sprague-Dawley rats were studied. Five were selected for bulbous urethral obstruction through a perineal incision, five for midprostatic obstruction using a retropubic approach, and five for a sham operation through a perineal incision. RESULTS: The operative time was shorter and morbidity lower with the perineal approach compared with the retropubic approach. Inflammation or infection, or both, were seen in the prostate, bladder, proximal urethra, ureters, and kidneys in the rats in which a midprostatic obstruction was performed. The proximal urethra and prostate were mildly inflamed in those rats that underwent bulbous obstruction. Sham-operated rats exhibited mild prostatitis only. CONCLUSIONS: The perineal approach to the bulbous urethra is the method of choice for creating a partial urethral obstruction model of bladder outlet obstruction in the male rat.
Assuntos
Modelos Animais de Doenças , Obstrução Uretral , Obstrução do Colo da Bexiga Urinária , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Mouse mammary tumors arising during medroxyprogesterone-DMBA-mediated mammary carcinogenesis comprised three distinct phenotypes: adenocarcinoma, squamous cell carcinoma, and myoepithelial carcinoma. The molecular signature for each of the three tumor subsets was characterized by gene microarray analysis, and three distinct sets of gene expression profiles were obtained that were corroborated in part by quantitative RT-PCR and immunohistochemistry. These results suggest that this carcinogenesis and gene expression model will be useful for rapidly assessing the histopathological differences arising in mammary carcinogenesis and the effects of tumor promoting or chemoprevention agents.
Assuntos
9,10-Dimetil-1,2-benzantraceno/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Medroxiprogesterona/farmacologia , Animais , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/classificação , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid beta-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARgamma and PPARdelta agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARgamma agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARdelta agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARgamma agonist expressed estrogen receptor-alpha in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARdelta agonist exhibited increased PPARdelta levels and activated 3-phosphoinositide-dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARdelta activation. These results indicate that PPARdelta and PPARgamma agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.
Assuntos
Anticarcinógenos/farmacologia , Carcinoma Ductal/prevenção & controle , Neoplasias Mamárias Experimentais/prevenção & controle , Oxazóis/farmacologia , PPAR delta/agonistas , PPAR gama/agonistas , Tiazóis/farmacologia , Tirosina/análogos & derivados , Tirosina/farmacologia , Animais , Carcinoma Adenoescamoso/induzido quimicamente , Carcinoma Adenoescamoso/tratamento farmacológico , Carcinoma Adenoescamoso/patologia , Carcinoma Adenoescamoso/prevenção & controle , Carcinoma Ductal/induzido quimicamente , Carcinoma Ductal/tratamento farmacológico , Carcinoma Ductal/patologia , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , CamundongosRESUMO
The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen(-/-) mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and beta-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIalpha3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.
Assuntos
Adipócitos/metabolismo , Colágeno Tipo VI/metabolismo , Neoplasias Mamárias Animais/metabolismo , Animais , Colágeno Tipo VI/deficiência , Colágeno Tipo VI/genética , Ciclina D1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Imuno-Histoquímica , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Polyomavirus/metabolismo , Transativadores/metabolismo , beta CateninaRESUMO
The inhibitor of differentiation Id2 is a target of the retinoblastoma (Rb) protein during mouse embryogenesis. In Rb(+/-) mice, LOH at the wild-type Rb allele initiates pituitary adenocarcinoma, a tumor derived from embryonic melanotropes. Here we identify a critical role for Id2 in initiation, growth, and angiogenesis of pituitary tumors from Rb(+/-) mice. We show that proliferation and differentiation are intimately coupled in Rb(+/-) pituitary cells before tumor initiation. In Id2-null pituitaries, premature activation of basic helix-loop-helix-mediated transcription and expression of the cdk inhibitor p27(Kip1) impairs the proliferation of melanotropes and tumor initiation. Without Id2, Rb(+/-) mice have fewer early tumor lesions and a markedly decreased proliferation rate of the tumor foci. Expression of Id2 by pituitary tumor cells promotes growth and angiogenesis by functioning as a master regulator of vascular endothelial growth factor (VEGF). In human neuroblastoma, the N-Myc-driven expression of Id2 is sufficient and necessary for expression of VEGF. These results establish that aberrant Id2 activity directs initiation and progression of embryonal cancer.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Neovascularização Patológica/genética , Neuroblastoma/genética , Neoplasias Hipofisárias/genética , Proteínas Repressoras/fisiologia , Proteína do Retinoblastoma/genética , Fatores de Transcrição/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Ciclina G , Ciclina G1 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 2 Inibidora de Diferenciação , Camundongos , Camundongos Mutantes , Mutação/genética , Hipófise/patologia , Neoplasias Hipofisárias/irrigação sanguínea , Proteínas Repressoras/genética , Proteína do Retinoblastoma/fisiologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We report the safety and immunogenicity of a double lysine and pantothenate auxotroph of Mycobacterium tuberculosis in mice. The DeltalysA DeltapanCD mutant is completely attenuated in immunocompromised SCID and gamma interferon knockout mice yet induces short-term and long-term protection in immunocompetent and CD4-deficient mice following single-dose subcutaneous vaccination.
Assuntos
Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose , Tuberculose/prevenção & controle , Animais , Infecções por HIV/complicações , Humanos , Lisina/metabolismo , Camundongos , Mycobacterium tuberculosis/metabolismo , Ácido Pantotênico/metabolismo , Fatores de Tempo , Tuberculose/imunologia , Vacinas AtenuadasRESUMO
Oncogenic signals elevate expression of Id2 in multiple tumor types. When deregulated, Id2 inactivates the tumor suppressor proteins retinoblastoma, p107, and p130. Here, we report a novel and unexpected tumor inhibitory function of Id2 in the intestinal epithelium. First, genetic ablation of Id2 in the mouse prevents differentiation and cell cycle arrest of enterocytes at the time of formation of the crypt-villus unit. Later, these developmental abnormalities evolve toward neoplastic transformation with complete penetrance. Id2-null tumors contain severe dysplastic and metaplastic lesions and express aberrant amounts of beta-catenin. Thus, our data are the first to establish a direct requirement of basic helix-loop-helix inhibitors in driving differentiation and define an unexpected role for the retinoblastoma-binding protein Id2 in preventing tumor formation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Feminino , Proteína 2 Inibidora de Diferenciação , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF) (murine p19(ARF)). Invariant inactivation of either the p16(INK4a)-cyclin D/CDK-pRb pathway and/or p53-p14(ARF) pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16(INK4a)/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1-/- mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16(INK4a) expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16(INK4a) was observed in tissues of mice mutant for the Ink4a/Arf locus. p16(INK4a) and p19(ARF) inhibited DNA synthesis in MCF7 cells. p16(INK4a) repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16(INK4a) occurred independently of the p16(INK4a)-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19(ARF) repressed cyclin D1 through a novel distal cis-element at -1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.
Assuntos
Ciclina D1/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteína Supressora de Tumor p14ARF/genética , Animais , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclina D1/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/biossíntese , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p14ARF/biossínteseRESUMO
We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The deltaleuD deltapanCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.
Assuntos
Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose Pulmonar/prevenção & controle , Animais , Feminino , Genes Bacterianos , Cobaias , Leucina/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ácido Pantotênico/metabolismo , Vacinas contra a Tuberculose/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/farmacologiaRESUMO
PURPOSE: Caveolae are flask-shaped invaginations of the plasma membrane formed by the oligomerization of caveolins. Because only smooth muscle contains all caveolin (Cav) family members (Cav-1, 2 and 3), we examined the contribution of each caveolin to urogenital smooth muscle structure/function. MATERIALS AND METHODS: WT, Cav-1, 2, 3 and -1/3 knockout (KO) mouse bladders were characterized by Western blot, co-immunoprecipitation, immunofluorescence microscopy, electron microscopy, histochemistry and pharmacological techniques. Cystometric analysis was performed in conscious, freely moving mice. Other urogenital organs were investigated by histological analysis. RESULTS: The loss of bladder Cav-1 results in a marked decrease in Cav-2 but not Cav-3 expression. Ablation of Cav-3 fails to alter Cav-1 or Cav-2 expression. Deletion of Cav-1 results in the almost complete loss of caveolae, while Cav-2 KO and Cav-3 KO mouse smooth muscle showed a normal number of caveolae. The loss of Cav-1 generated caveolae led to significant urogenital changes in male mice (most marked by 12 months of age), namely 1) bladder weight-to-body weight ratios were increased, 2) the bladder smooth muscle layer was thickened, 3) the bladders had increased baseline, threshold and spontaneous pressures, 4) bladder strips showed a decreased contractile response to carbachol and KCl, and 5) these smooth muscle changes were accompanied by marked fluid accumulation in the prostate and seminal vesicles, with intracellular vacuolization in the kidneys. As such, male Cav-1 KO mice may be a useful animal model for studying LUTD (lower urinary tract dysfunction) that is so prevalent in aging male patients. CONCLUSIONS: The loss of Cav-1 and, thus, of most smooth muscle cell caveolae results in significant bladder dysfunction and urogenital organ changes in aged male mice.
Assuntos
Caveolinas/genética , Músculo Liso/citologia , Sistema Urogenital/citologia , Animais , Caveolina 1 , Camundongos , Camundongos KnockoutRESUMO
Adiponectin is a plasma protein expressed exclusively in adipose tissue. Adiponectin levels are linked to insulin sensitivity, but a direct effect of chronically elevated adiponectin on improved insulin sensitivity has not yet been demonstrated. We identified a dominant mutation in the collagenous domain of adiponectin that elevated circulating adiponectin values in mice by 3-fold. Adiponectinemia raised lipid clearance and lipoprotein lipase activity, and suppressed insulin-mediated endogenous glucose production. The induction of adiponectin during puberty and the sexual dimorphism in adult adiponectin values were preserved in these transgenic animals. As a result of elevated adiponectin, serum PRL values and brown adipose mass both increased. The effects on carbohydrate and lipid metabolism were associated with elevated phosphorylation of 5'-AMP-activated protein kinase in liver and elevated expression of peroxisomal proliferator-activated receptor gamma2, caveolin-1, and mitochondrial markers in white adipose tissue. These studies strongly suggest that increasing endogenous adiponectin levels has direct effects on insulin sensitivity and may induce similar physiological responses as prolonged treatment with peroxisomal proliferator-activated receptor gamma agonists.
Assuntos
Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas/genética , Proteínas/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adiponectina , Tecido Adiposo/anatomia & histologia , Tecido Adiposo/metabolismo , Animais , Composição Corporal , Calorimetria Indireta , Colágeno/genética , Ingestão de Alimentos , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnica Clamp de Glucose , Intolerância à Glucose/metabolismo , Camundongos , Camundongos Transgênicos , Prolactina/sangue , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Transcrição GênicaRESUMO
The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/fisiologia , Transdução de Sinais , Transativadores/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Proteína Smad4 , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição , TransfecçãoRESUMO
Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.