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1.
J Cell Biol ; 219(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33053168

RESUMO

The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism.


Assuntos
Caveolina 1/fisiologia , Exossomos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Corpos Multivesiculares/metabolismo , Proteoma/metabolismo , Tenascina/fisiologia , Animais , Fibroblastos/citologia , Camundongos , Camundongos Knockout
3.
Cell Rep ; 25(6): 1622-1635.e6, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30404014

RESUMO

The transcriptional regulator YAP orchestrates many cellular functions, including tissue homeostasis, organ growth control, and tumorigenesis. Mechanical stimuli are a key input to YAP activity, but the mechanisms controlling this regulation remain largely uncharacterized. We show that CAV1 positively modulates the YAP mechanoresponse to substrate stiffness through actin-cytoskeleton-dependent and Hippo-kinase-independent mechanisms. RHO activity is necessary, but not sufficient, for CAV1-dependent mechanoregulation of YAP activity. Systematic quantitative interactomic studies and image-based small interfering RNA (siRNA) screens provide evidence that this actin-dependent regulation is determined by YAP interaction with the 14-3-3 protein YWHAH. Constitutive YAP activation rescued phenotypes associated with CAV1 loss, including defective extracellular matrix (ECM) remodeling. CAV1-mediated control of YAP activity was validated in vivo in a model of pancreatitis-driven acinar-to-ductal metaplasia. We propose that this CAV1-YAP mechanotransduction system controls a significant share of cell programs linked to these two pivotal regulators, with potentially broad physiological and pathological implications.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caveolina 1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mecanotransdução Celular , Proteínas 14-3-3/metabolismo , Animais , Núcleo Celular/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pancreatite/patologia , Fosfosserina/metabolismo , Polimerização , Mapeamento de Interação de Proteínas , Especificidade por Substrato , Proteínas de Sinalização YAP
4.
Sci Rep ; 5: 13047, 2015 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-26267334

RESUMO

A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been proposed, but their contribution is still debated. We tested the ability of BMDCs containing the inducible kras(V12) oncogene to initiate lung adenocarcinoma. For our experimental strategy, we reconstituted lethally irradiated wild type mice with BMDCs carrying inducible kras(V12) and subsequently induced oncogene expression by 4-OHT administration. Epithelial lung lesions, from adenoma to adenocarcinomas, appeared at successive time points. These results show that lung tumors were derived from donor BMDCs and indicate a direct involvement of bone marrow cells in the development of epithelial cancers.


Assuntos
Carcinogênese/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Transplante de Medula Óssea , Carcinogênese/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ativação Transcricional
5.
Bioconjug Chem ; 26(1): 153-60, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25494619

RESUMO

Multifunctional nanoparticles are usually produced by sequential synthesis, with long multistep protocols. Our study reports a generic modular strategy for the parallel one-step multifunctionalization of different hydrophobic nanoparticles. The method was designed and developed by taking advantage of the natural noncovalent interactions between the fatty acid binding sites of the bovine serum albumin (BSA) and the aliphatic surfactants on different inorganic nanomaterials. As a general example of the approach, three different nanoparticles-iron oxide, upconverting nanophosphors, and gold nanospheres-were nanoemulsified in water with BSA. To support specific applications, multifunctional capability was incorporated with a variety of previously modified BSA modules. These modules include different conjugated groups, such as chelating agents for (68)Ga or (89)Zr and ligand molecules for enhanced in vivo targeting. A large library of 13 multimodal contrast agents was developed with this convergent strategy. This platform allows a highly versatile and easy tailoring option for efficient incorporation of functional groups. Finally, as demonstration of this versatility, a bimodal (PET/MRI) probe including a maleimide-conjugated BSA was selectively synthesized with an RGD peptide for in vivo imaging detection of tumor angiogenesis.


Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Tomografia por Emissão de Pósitrons/métodos , Animais , Bovinos , Meios de Contraste/farmacocinética , Meios de Contraste/toxicidade , Ácidos Graxos/metabolismo , Fibroblastos/efeitos dos fármacos , Maleimidas/química , Camundongos , Modelos Moleculares , Conformação Molecular , Nanopartículas/toxicidade , Oligopeptídeos/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Distribuição Tecidual
6.
J Cell Biol ; 206(2): 307-28, 2014 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-25049275

RESUMO

The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5-dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and ß3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.


Assuntos
Neoplasias da Mama/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/fisiologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Invasividade Neoplásica/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Proteólise , Transplante Heterólogo , Proteínas rab5 de Ligação ao GTP/metabolismo
7.
Cancer Res ; 73(19): 5880-91, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23918796

RESUMO

Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.


Assuntos
Adipócitos/patologia , Tecido Adiposo Branco/patologia , Antígenos CD34/metabolismo , Neoplasias da Mama/patologia , Neoplasias Pulmonares/secundário , Neovascularização Patológica/patologia , Células-Tronco/patologia , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Feminino , Citometria de Fluxo , Imunofluorescência , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Metástase Linfática , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Células Tumorais Cultivadas
8.
Neoplasia ; 14(11): 1057-66, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23226099

RESUMO

Cell fusion plays a well-recognized physiological role during development, while its function during progression is still unclear. Here, we show that acute myeloid leukemia (AML) cells spontaneously fused with murine host cells in vivo. AML cells fused in most cases with mouse macrophages. Other targets of AML cell fusion were dendritic and endothelial cells. Cytogenetic and molecular analysis revealed that successive recipients conserved detectable amounts of parental DNA. Moreover, in a mouse AML1-ETO model where female AML1-ETO-leukemic cells, expressing CD45.2, were injected in congenic CD45.1 male mice AML cells, we found hybrid cells expressing both allelic types of CD45 and XXY set of sexual chromosomes. More importantly, the fusion protein AML1-ETO was transferred in the hybrid cells. When sorted hybrid cells were reinjected in a secondary recipient, they gave rise to leukemia with 100% penetrance and similar time of onset of leukemic cells. Our data indicate that in vivo fusion of cancer cells with host cells may be a mechanism of gene transfer for cancer dissemination and suggest that fused cells may be used to identify still unrecognized leukemogenic genes that are conserved in hybrid cells and able to perpetuate leukemia in vivo.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Macrófagos/metabolismo , Fusão de Membrana , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD34/metabolismo , Antígeno CD47/imunologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Humanos , Receptores de Hialuronatos/imunologia , Células Híbridas/metabolismo , Leucemia Linfoide/genética , Leucemia Linfoide/metabolismo , Leucemia Mieloide Aguda/genética , Masculino , Fusão de Membrana/efeitos dos fármacos , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante Heterólogo
9.
PLoS One ; 7(12): e51109, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226561

RESUMO

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45-) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.


Assuntos
Antígenos CD34/metabolismo , Sangue Fetal/citologia , Hemangioblastos/citologia , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Hemangioblastos/efeitos dos fármacos , Hemangioblastos/metabolismo , Hematopoese/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Imunofenotipagem , Recém-Nascido , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Fenótipo
10.
Lab Invest ; 92(7): 952-66, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22546866

RESUMO

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related deaths. Currently available chemotherapeutic options are not curative due in part to tumor resistance to conventional therapies. We generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of human alpha-feto protein (hAFP)- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. In this model, low-dose metronomic administration of cyclophosphamide (LDM-CTX) caused complete regression of the tumor mass. A significant increase in survival (P<0.0001), reduced aberrant angiogenesis and hyperproliferation, and decrease in the number of circulating tumor cells were found in LDM-CTX-treated animals, in comparison with untreated mice. Co-administration of LDM-CTX with anti-VEGF therapy further improved the therapeutic efficacy. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, discontinuation of therapy resulted in tumor regrowth. Moreover, in-vitro LDM-CTX treatment reduced hepatosphere formation in both number and size, and the resulting spheres were enriched in CD13+ cells indicating that these cells were particularly resistant to therapy. Co-treatment of the CD13-targeting drug, bestatin, with LDM-CTX leads to slower tumor growth and a decreased tumor volume. Therefore, combining a CD13 inhibitor, which targets the CSC-like population, with LDM-CTX chemotherapy may be used to eradicate minimal residual disease and improve the treatment of liver cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologia , Células-Tronco Neoplásicas/patologia , Administração Metronômica , Inibidores da Angiogênese/administração & dosagem , Animais , Antineoplásicos Alquilantes/administração & dosagem , Antígenos CD13/antagonistas & inibidores , Linhagem Celular Tumoral , Ciclofosfamida/administração & dosagem , Sinergismo Farmacológico , Humanos , Leucina/administração & dosagem , Leucina/análogos & derivados , Neoplasias Hepáticas Experimentais/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasia Residual/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/fisiologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Res ; 72(1): 325-34, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22052460

RESUMO

Previous studies have suggested a "catalytic role" in neoplastic angiogenesis and cancer progression for bone marrow-derived endothelial progenitor cells (EPC). However, preclinical and clinical studies have shown that the quantitative role of marrow-derived EPCs in cancer vascularization is extremely variable. We have found that human and murine white adipose tissue (WAT) is a very rich reservoir of CD45-CD34(+) EPCs with endothelial differentiation potential, containing a mean of 263 times more CD45-CD34(+) cells/mL than bone marrow. Compared with marrow-derived CD34(+) cells mobilized in blood by granulocyte colony-stimulating factor, purified WAT-CD34(+) cells expressed similar levels of stemness-related genes, significantly increased levels of angiogenesis-related genes, and increased levels of FAP-α, a crucial suppressor of antitumor immunity. In vitro, WAT-CD34(+) cells generated mature endothelial cells and capillary tubes as efficiently as mature mesenchymal cells. The coinjection of human WAT-CD34(+) cells from lipotransfer procedures contributed to tumor vascularization and significantly increased tumor growth and metastases in several orthotopic models of human breast cancer in immunodeficient mice. Endothelial cells derived from human WAT-CD34(+) cells lined the lumen of cancer vessels. These data indicate that CD34(+) WAT cells can promote cancer progression and metastases. Our results highlight the importance of gaining a better understanding of the role of different WAT-derived cells used in lipotransfer for breast reconstruction in patients with breast cancer.


Assuntos
Tecido Adiposo/citologia , Antígenos CD34/imunologia , Neoplasias/patologia , Células-Tronco/imunologia , Tecido Adiposo/imunologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Citometria de Fluxo , Humanos , Camundongos , Microscopia Confocal , Neoplasias/imunologia , Reação em Cadeia da Polimerase em Tempo Real
12.
Clin Cancer Res ; 17(19): 6163-73, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21856771

RESUMO

PURPOSE: Blastic natural killer (NK) cell lymphoma/blastic plasmacytoid dendritic cell neoplasm (BNKL) is a rare and aggressive neoplasia characterized by infiltration of blast CD4(+)/CD56(+) cells in the skin, the bone marrow, and peripheral blood. Currently, more efforts are required to better define molecular and biological mechanisms associated with this pathology. To the best of our knowledge, no mouse model recapitulated human BNKL so far. EXPERIMENTAL DESIGN: Primary bone marrow cells from a BNKL patient were injected in nonobese diabetes/severe combined immunodeficient interleukin (IL) 2rγ(-/-) mice with the intent to generate the first BNKL orthotopic mouse model. Moreover, because of the lack of efficient treatments for BNKL, we treated mice with lenalidomide, an immunomodulatory and antiangiogenic drug. RESULTS: We generated in mice a fatal disease resembling human BNKL. After lenalidomide treatment, we observed a significant reduction in the number of peripheral blood, bone marrow, and spleen BNKL cells. Tumor reduction parallels with a significant decrease in the number of circulating endothelial and progenitor cells and CD31(+) murine endothelial cells. In mice treated with lenalidomide, BNKL levels of active caspase-3 were significantly augmented, thus showing proapoptotic and cytotoxic effects of this drug in vivo. An opposite result was found for proliferating cell nuclear antigen, a proliferation marker. CONCLUSIONS: Our BNKL model might better define the cellular and molecular mechanisms involved in this disease, and lenalidomide might be considered for the future therapy of BNKL patients.


Assuntos
Antineoplásicos/uso terapêutico , Células Dendríticas , Modelos Animais de Doenças , Células Matadoras Naturais , Linfoma/tratamento farmacológico , Talidomida/análogos & derivados , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Lenalidomida , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Talidomida/uso terapêutico , Transplante Heterólogo
13.
Drug Discov Today ; 16(23-24): 1052-60, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21875682

RESUMO

The anti-angiogenic class of drugs is one of the few where representatives have gained international approval for clinical use in oncology during the past decade. Most of the biological and clinical activity of the currently available generation of anti-angiogenic drugs targets vascular endothelial growth factor (VEGF) and its related pathways. However, the clinical benefits associated with the use of these drugs have, so far, been limited. There is, therefore, an unmet need for biomarkers that can be used to identify patients who are most likely to benefit therapeutically and also to predict the best schedule and dosage for these drugs. Here, we discuss some of the emerging new combination strategies involving the approved anti-angiogenic drugs, some of the emerging targets associated with neoplastic angiogenesis and some novel agents used as a paradigm of the next generation of anti-angiogenic drugs.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
PLoS One ; 6(6): e21369, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21731718

RESUMO

BACKGROUND AND AIMS: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC). METHODS: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice. RESULTS: The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Clonais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Genoma Humano/genética , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Microscopia de Fluorescência , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Pirróis/farmacologia , Pirróis/uso terapêutico , Sunitinibe , Fatores de Tempo , Células Tumorais Cultivadas
15.
Int J Cancer ; 129(6): 1344-50, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21128230

RESUMO

Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells (ECs) and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumors. Similarly to what we have done with circulating ECs and progenitors, we developed a flow cytometry procedure for the enumeration of circulating PPCs and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their morphology was studied by electron microscopy, PPC specificity confirmed by reverse trascription-PCR (RT-PCR) expression of CD140b mRNA, and viability assessed by Syto16 and 7AAD. In preclinical marrow transplantation studies, 9 ± 4% of circulating PPCs were derived from the marrow donor. PPCs were increased in cancer-bearing mice and in patients affected by some types of cancer. At variance with the kinetic of circulating endothelial progenitors, high-dose cyclophosphamide reduced the number of viable PPCs. The administration of sunitinib, a drug known to inhibit PDGFR, was associated in murine models and in cancer patients with an increase of apoptotic/necrotic circulating PPC, suggesting a direct targeting of these cells. PPC enumeration might be studied as a tool for the definition of the optimal biologic dose of anti-PDGFR drugs and investigated clinically as a possible predictive/prognostic tool in patients receiving anti-PDGFR drugs.


Assuntos
Neoplasias/sangue , Pericitos/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células-Tronco/fisiologia , Inibidores da Angiogênese , Animais , Células da Medula Óssea/citologia , Contagem de Células , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos , Pericitos/citologia , Pericitos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pirróis/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Células-Tronco/citologia , Células-Tronco/metabolismo , Sunitinibe
17.
PLoS One ; 5(8): e12015, 2010 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-20700488

RESUMO

BACKGROUND: Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. METHODOLOGY/PRINCIPAL FINDINGS: We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G-->T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34(+) cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. CONCLUSIONS/SIGNIFICANCE: Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.


Assuntos
Antígenos CD34/metabolismo , Diferenciação Celular , Células Endoteliais/patologia , Policitemia/genética , Policitemia/patologia , Receptores da Eritropoetina/metabolismo , Adolescente , Adulto , Sequência de Bases , Estudos de Casos e Controles , Membrana Celular/patologia , Proliferação de Células , Pré-Escolar , Citoplasma/metabolismo , Células Endoteliais/metabolismo , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Feminino , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Fosforilação , Policitemia/sangue , Policitemia/metabolismo , Estrutura Terciária de Proteína , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Fator de Transcrição STAT5/metabolismo , Adulto Jovem
18.
Blood ; 114(25): 5191-200, 2009 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-19855080

RESUMO

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin(-)CD34(-)) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34(-) cells are leukemic. CML Lin(-)CD34(-) cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin(-)CD34(-) cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34(+) cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin(-)CD34(-) cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin(-)CD34(-) cells in vitro. Moreover, leukemic CD34(-) cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34(-) leukemic stem cell subset in CML with peculiar molecular and functional characteristics.


Assuntos
Antígenos CD34/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Benzamidas , Células da Medula Óssea/metabolismo , Células Cultivadas , Análise por Conglomerados , Citometria de Fluxo , Proteínas de Fusão bcr-abl/genética , Perfilação da Expressão Gênica , Humanos , Mesilato de Imatinib , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Cariotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/transplante , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Microglobulina beta-2/deficiência , Microglobulina beta-2/genética
19.
Front Biosci (Schol Ed) ; 1(1): 304-18, 2009 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-19482704

RESUMO

An increased number of circulating endothelial cells (CECs) and endothelial progenitor cells (CEPs) has been reported in cancer patients. CEPs are derived from the bone marrow and will, during angiogenesis, differentiate into endothelial cells. CECs are mature endothelial cells (ECs) released from the vessel intima during physiological endothelial turnover or as a result of tumor treatment. Preclinical studies have shown that during tumor progression, the amount of circulating CECs correlates with angiogenesis. Moreover, there is growing evidence suggesting that CECs and CEPs viability and kinetics correlate with the patient responses to anti-angiogenic therapies. Thus, circulating CECs and CEPs may act as surrogate markers to test putative therapeutic efficacy. Moreover measuring CECs and CEPs may be useful to assess effects of antiangiogenic therapy.


Assuntos
Biomarcadores Tumorais , Células da Medula Óssea/citologia , Endotélio Vascular/citologia , Neovascularização Patológica , Células da Medula Óssea/metabolismo , Endotélio Vascular/metabolismo , Instabilidade Genômica , Humanos
20.
Endothelium ; 15(5-6): 276-87, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19065319

RESUMO

Previous studies showed that p66(Shc-/-) mice on a very-high-fat diet (HFD) had reduced oxidative stress, foam cell, and early atherosclerotic lesion formation. Here, the authors have used hypercholesterolemic apolipoprotein E (ApoE(-/-)) mice to investigate the role of p66Shc deletion in advanced atheroma. The authors generated mice deficient of both ApoE and p66Shc genes (ApoE(-/-) /p66(Shc-/-)). They used microsatellite polymerase chain reaction (PCR) analysis to analyze the genetic background and considered only animals with a constant percentages of C57B6L and 129SV background strands (it was obtained the 50.3% +/- 6.4% of C57B6L background). Computer-assisted analysis revealed that advanced atherosclerotic lesions in ApoE(-/-)/p66(Shc+/+) were significantly larger than those observed in ApoE(-/-)/p66(Shc-/-). Accordingly, the lipid-laden macrophage foam cells and oxidation-specific epitopes in ApoE(-/-)/p66(shc+/+) HFD-treated groups were higher than those observed in normal diet (ND)-treated groups. Thus, p66(Shc-/-) plays an important protective role also against advanced atherosclerotic lesion formation. Finally, the authors have used microarray to investigate major changes in gene expression in aortas of mice with ApoE(-/-)/p66(Shc-/-) background treated with a very HFD in comparison to ApoE(-/-)/p66(Shc+/+) (these data have been confirmed by by real-time PCR and immunohistochemistry). DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis revealed that CD36 antigen (CD36), tissue inhibitor of metalloproteinase 2 (TIMP2), apolipoprotein E (ApoE), acetyl-coenzyme A acetyltransferase 1 (ACAT1), and thrombospondin 1 (THBS1) can be involved in p66 deletion-dependent vascular protection through the adipocytokine/lipid signaling pathway.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Adipocinas/genética , Adipocinas/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Apolipoproteínas E/metabolismo , Aterosclerose/fisiopatologia , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Regulação para Baixo/genética , Células Espumosas/metabolismo , Células Espumosas/patologia , Alimentos Formulados/efeitos adversos , Deleção de Genes , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Genótipo , Hipercolesterolemia/fisiopatologia , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
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