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1.
Cancer Res ; 71(7): 2541-9, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21447735

RESUMO

It has been challenging to engineer lung adenocarcinoma models via oncogene-mediated transformation of primary cultured normal human cells. Although viral oncoprotein-mediated malignant transformation has been reported, xenografts derived from such transformed cells generally represent poorly differentiated cancers. Here, we demonstrate that the combined expression of multiple cellular factors induces malignant transformation in normal human lung epithelial cells. Although a combination of four genetic alterations, including hTERT overexpression, inactivation of the pRB and p53 pathways, and KRAS activation, is insufficient for normal human small airway epithelial cells to be fully transformed, expression of one additional oncogene induces malignant transformation. Notably, we have succeeded in reproducing human lung adenocarcinoma phenotypes in the flanks of nude mice by introducing an active form of PIK3CA, CYCLIN-D1, or a dominant-negative form of LKB1 in combination with the four genetic alterations above. Besides differentiated lung cancer, poorly differentiated cancer models can also be engineered by employing c-MYC as one of the genetic elements, indicating that histologic features and degree of differentiation of xenografts are controllable to some extent by changing the combination of genetic elements introduced. This is the first study reporting malignant transformation of normal lung epithelial cells in the absence of viral oncoproteins. We propose that our model system would be useful to identify the minimal and most crucial set of changes required for lung tumorigenesis, and that it would provide a broadly applicable approach for discovering attractive therapeutic targets.


Assuntos
Transformação Celular Viral/fisiologia , Retroviridae/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/virologia , Adenocarcinoma de Pulmão , Adulto , Animais , Diferenciação Celular/fisiologia , Transformação Celular Viral/genética , Quinase 4 Dependente de Ciclina/biossíntese , Quinase 4 Dependente de Ciclina/genética , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fenótipo , Retroviridae/genética , Telomerase/biossíntese , Telomerase/genética , Transplante Heterólogo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Adulto Jovem
2.
J Cell Biol ; 189(5): 901-17, 2010 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-20513769

RESUMO

LL5beta has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)-bound microtubule plus ends to the cell cortex. In this study, we show that LL5beta and its homologue LL5alpha (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins alpha3beta1 and alpha6beta4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin alpha3 at the basal cell cortex. Activation of integrin alpha3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin-integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Laminina/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Transporte/genética , Adesão Celular , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Feminino , Humanos , Integrina alfa3/genética , Integrina alfa3/metabolismo , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina/genética , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica/fisiologia , RNA Interferente Pequeno/genética , Receptores de Laminina/genética , Receptores de Laminina/metabolismo
3.
J Med Invest ; 54(3-4): 370-4, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17878690

RESUMO

Quercetin is a major dietary flavonoid found in onions and other vegetables. It is known that dietary quercetin is metabolized in the intestinal mucosa and the liver and is present as its glucuronide/sulfate conjugates with or without methylation. Although quercetin is known to possess strong antioxidant activity, there are only limited reports on the antioxidant activity of its metabolites. In this study, the antioxidant capacity of quercetin metabolites under physiological conditions was investigated. After consumption of cooked onion, more than 80% of quercetin metabolites were localized in the human plasma fraction containing concentrated serum albumin. Other lipoprotein fractions contained only small amounts of quercetin metabolites. Addition of quercetin 3-O-beta-glucuronide to the lipoprotein-eliminated plasma fraction generated antioxidant activity against LDL oxidation in a dose-dependent manner. However, onion consumption failed to enhance the antioxidant activity of the lipoprotein-eliminated plasma fraction against LDL oxidation, probably because the amount of quercetin metabolites bound to albumin was less than the effective level in an ex vivo study. The physiological role of plasma albumin in retaining quercetin metabolites needs to be further clarified.


Assuntos
Antioxidantes/metabolismo , Cebolas/química , Quercetina/metabolismo , Adulto , Antioxidantes/administração & dosagem , Humanos , Técnicas In Vitro , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Oxirredução , Quercetina/administração & dosagem , Quercetina/sangue , Albumina Sérica/metabolismo
5.
J Cell Biol ; 172(4): 497-504, 2006 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-16461359

RESUMO

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Proteína 7 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Ratos , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Raios Ultravioleta
6.
Mol Biol Cell ; 16(1): 32-9, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15509652

RESUMO

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.


Assuntos
Amidoidrolases/genética , Amidoidrolases/fisiologia , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/fisiologia , Actinas/metabolismo , Animais , Células COS , Proliferação de Células , Citocalasina D/farmacologia , Hipocampo/metabolismo , Hidrolases , Immunoblotting , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Semaforina-3A/metabolismo , Transdução de Sinais
7.
J Biol Chem ; 278(49): 49129-33, 2003 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-14551201

RESUMO

We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. Here we report a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice displayed an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain.


Assuntos
Proteínas de Fusão gag-onc/fisiologia , Microtúbulos/fisiologia , Proteínas Tirosina Quinases/fisiologia , Animais , Sequência de Bases , Células COS , Primers do DNA , Camundongos , Microscopia Imunoeletrônica , Mutagênese Sítio-Dirigida
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