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1.
BMC Cell Biol ; 13: 12, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22583596

RESUMO

BACKGROUND: The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. RESULTS: We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. CONCLUSION: Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.


Assuntos
Receptor Notch1/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colágeno/química , Colágeno/metabolismo , Dipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Quinases Associadas a rho/antagonistas & inibidores
2.
Am J Physiol Lung Cell Mol Physiol ; 298(4): L584-92, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20081066

RESUMO

Airway wall remodeling comprises a broad range of structural changes including increases in the volume of airway smooth muscle (ASM) and fibrillar collagen. The impact of fibrillar collagen remodeling on ASM proliferation was investigated. Human ASM cultured on type I fibrillar collagen remodeled the collagen substrate by both degradation (collagenolysis) and formation of networks comprised of thicker reticular collagen fibrils (fibrillogenesis). In cultures maintained on fibrillar collagen, the levels of matrix metalloproteases (MMPs) -1 and -14 mRNA and active MMP-2 were higher than in cultures maintained on nonfibrillar type I collagen (gelatin) or plastic. Although there was no apparent increase in cytotoxicity or apoptosis, the number of ASM was lower on fibrillar collagen than on gelatin or plastic for control conditions. Furthermore, maintenance on fibrillar collagen attenuated basic fibroblast growth factor-stimulated increases in cell number and the percentage of cells entering S-phase. In cultures maintained on fibrillar collagen, the MMP inhibitor ilomastat (2.5 microM) 1) attenuated collagenolysis, 2) enhanced fibrillogenesis, and 3) inhibited proliferation. In contrast, knockdown of the beta1-integrin gene in ASM maintained on fibrillar collagen led to an increase in proliferation and reduced MMP-1 and -14 expression. Thus, ASM remodel the pericellular environment by degrading collagen fibrils and spinning them into larger collagen assemblies. Moreover, the collagen fibrils limit proliferation and activate autocrine MMPs in a beta-integrin-dependent manner, suggesting a potential negative feedback on modeling executed through fibrillar collagen activation of beta1-integrins.


Assuntos
Remodelação das Vias Aéreas/fisiologia , Colágenos Fibrilares/metabolismo , Músculo Liso/citologia , Músculo Liso/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Remodelação das Vias Aéreas/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Feminino , Colágenos Fibrilares/ultraestrutura , Técnicas de Silenciamento de Genes , Humanos , Ácidos Hidroxâmicos , Indóis/farmacologia , Integrina beta1/genética , Masculino , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Músculo Liso/enzimologia , Músculo Liso/ultraestrutura , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
3.
Cell Motil Cytoskeleton ; 66(6): 303-16, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19373774

RESUMO

Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodium's leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.


Assuntos
Neoplasias da Mama/patologia , Quimiotaxia , Cortactina/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiotaxia/genética , Cortactina/genética , Destrina/genética , Destrina/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , RNA Interferente Pequeno/genética , Ratos , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
4.
Am J Respir Cell Mol Biol ; 41(6): 731-41, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19329552

RESUMO

Pulmonary fibrosis is characterized by phenotypic changes to mesenchymal cells and an increase in the deposition of fibrillar collagen (fCollagen). This study investigated the effect of type I fCollagen on the phenotypic plasticity of human parenchymal fibroblasts (PFbs) in vitro. Cell numbers were 45% lower when cultured on fCollagen as compared with culture on its degradation product, monomeric collagen (mCollagen). DNA profiles indicated that fCollagen is antiproliferative, rather than proapoptotic. fCollagen suppressed basic fibroblast growth factor-stimulated increases in the levels of cyclin E and CDK2 mRNA. fCollagen also suppressed transforming growth factor-beta (100 pM)-stimulated increases in the mRNA and protein levels of alpha-smooth muscle actin (alpha-SMA), a marker of the myofibroblast phenotype. However, in cells exposed to fCollagen, the levels of matrix metalloproteinase (MMP)-1 and -14 mRNA, as well as active MMP-2 protein, were increased by between two- and fivefold. The MMP inhibitors, ilomastat (10 microM) and doxycycline (30 microM), attenuated the dissolution of collagen fibrils by fibroblasts maintained on fCollagen, with a corresponding decrease in cell number. Ilomastat also reduced alpha-SMA expression and the capacity of PFb to contract three-dimensional fCollagen gels. Thus, exposure of fibroblasts to the fibrillar form of type I collagen in vitro reduces cell proliferation, increases MMP production and activation, and attenuates differentiation of PFb into myofibroblasts. fCollagen appears to apply a phenotypic clamp on lung fibroblasts that may be partially released by autocrine MMP activity.


Assuntos
Colágeno Tipo I/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Actinas/genética , Actinas/metabolismo , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/farmacologia , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Primers do DNA/genética , Doxiciclina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ácidos Hidroxâmicos , Indóis/farmacologia , Pulmão/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Mesoderma/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Modelos Biológicos , Fenótipo , Inibidores de Proteases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/farmacologia
5.
J Cell Biol ; 166(5): 697-708, 2004 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-15337778

RESUMO

The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.


Assuntos
Carcinoma/metabolismo , Quimiotaxia/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Proteínas dos Microfilamentos/fisiologia , Metástase Neoplásica/fisiopatologia , Fosfolipases Tipo C/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina , Actinas/biossíntese , Actinas/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Carcinoma/patologia , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/genética , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Interferência de RNA , Ratos , Fosfolipases Tipo C/efeitos dos fármacos
6.
Biophys J ; 95(3): 1523-30, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18645198

RESUMO

The extracellular availability of growth factors, hormones, chemokines, and neurotransmitters under gradient conditions is required for directional cellular responses such as migration, axonal pathfinding, and tissue patterning. These responses are, in turn, important in disease and developmental processes. This article addresses critical barriers toward devising a chemotaxis assay that is broadly applicable for different kinds of cancer cells through the design of a microfluidic chamber that produces a steep gradient of chemoattractant. Photolithography was used to create microchannels for chemoattractant delivery, flow diversion barriers/conduits, and small outlets in the form of apertures. The 1-microm apertures were made at the active surface by uncapping a thin (1.5 microm) layer of AZ1518. This process also created a vertical conduit that diverted the flow such that it occurred perpendicularly to the active, experimental surface where the gradients were measured. The other side of the vertical conduit opened to underlying 20-microm deep channels that carried microfluidic flows of tracer dyes/growth factors. Modeled data using computational fluid dynamics produced gradients that were steep along the horizontal, active surface. This simulation mirrors empirically derived gradients obtained from the flow analyses of fluorescent compounds. The open chamber contains a large buffer volume, which prevents chemoattractant saturation and permits easy cell and compound manipulation. The technique obviates the use of membranes or laminar flow that may hinder imaging, rinsing steps, cell seeding, and treatment. The utility of the chamber in the study of cell protrusion, an early step during chemotaxis, was demonstrated by growing cancer cells in the chamber, inducing a chemoattractant gradient using compressed air at 0.7 bar, and performing time-lapse microscopy. Breast cancer cells responded to the rapidly developed and stable gradient of epidermal growth factor by directing centroid positions toward the gradient and by forming a leading edge at a speed of 0.45 microm/min.


Assuntos
Técnicas de Cultura de Células/instrumentação , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Fatores Quimiotáticos/administração & dosagem , Fatores Quimiotáticos/química , Análise de Injeção de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Cultura de Células/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Técnicas Analíticas Microfluídicas/métodos
7.
Microsc Res Tech ; 70(3): 252-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17279509

RESUMO

Although genetic and protein manipulations have been the cornerstone for the study and understanding of biological processes for many decades, complimentary nanoscale observations have only more recently been achieved in the live-imaging mode. It is at the nano measurement level that events such as protein-protein interactions, enzymatic conversions, and single-molecule stochastic behavior take place. Therefore, nanoscale observations allow us to reinterpret knowledge from large-scale or bulk techniques and gain new insight into molecular events that has cellular, tissue, and organismal phenotypic manifestations. This review identifies pertinent questions relating to the sensing and directional component of cancer cell chemotaxis and discusses the platforms that provide insight into the molecular events related to cell motility. The study of cell motility at the molecular imaging level often necessitates the use of devices such as microinjection, microfluidics, in vivo/intravital and in vitro chemotaxis assays, as well as fluorescence methods like uncaging and FRET. The micro- and nanofabricated devices that facilitate these techniques and their incorporation to specialized microscopes such as the multiphoton, AFM, and TIR-FM, for high-resolution imaging comprise the nanoplatforms used to explore the mechanisms of carcinogenesis. In real-time observations, within a milieu of physiological protein concentrations, true states of dynamic and kinetic fluxes can be monitored.


Assuntos
Movimento Celular , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Humanos , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nanotecnologia/métodos , Invasividade Neoplásica , Metástase Neoplásica
8.
Microsc Res Tech ; 70(3): 230-42, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17279510

RESUMO

Correlative microscopy has become increasingly important for the analysis of the structure, function, and dynamics of cells. This is largely due to the result of recent advances in light-, probe-, laser- and various electron microscopy techniques that facilitate three-dimensional studies. Furthermore, the improved understanding in the past decade of imaging cell compartments in the third dimension has resulted largely from the availability of powerful computers, fast high-resolution CCD cameras, specifically developed imaging analysis software, and various probes designed for labeling living and or fixed cells. In this paper, we review different correlative high-resolution imaging methodologies and how these microscopy techniques facilitated the accumulation of new insights in the morpho-functional and structural organization of the hepatic sieve. Various aspects of hepatic endothelial fenestrae regarding their structure, origin, dynamics, and formation will be explored throughout this paper by comparing the results of confocal laser scanning-, correlative fluorescence and scanning electron-, atomic force-, and whole-mount electron microscopy. Furthermore, the recent advances of vitrifying cells with the vitrobot in combination with the glove box for the preparation of cells for cryo-electron microscopic investigation will be discussed. Finally, the first transmission electron tomography data of the liver sieve in three-dimensions are presented. The obtained data unambiguously show the involvement of special domains in the de novo formation and disappearance of hepatic fenestrae, and focuses future research into the (supra)molecular structure of the fenestrae-forming center, defenestration center and fenestrae-, and sieve plate cytoskeleton ring by using advanced cryo-electron tomography.


Assuntos
Hepatócitos/citologia , Hepatócitos/ultraestrutura , Imageamento Tridimensional/métodos , Animais , Microscopia Crioeletrônica/métodos , Humanos , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos
9.
World J Gastroenterol ; 13(6): 821-5, 2007 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-17352008

RESUMO

Colorectal cancer (CRC) is a common malignant disease and the severe nature of cases in men and women who develop colorectal cancer makes this an important socio-economic health issue. Major challenges such as understanding and modeling colorectal cancer pathways rely on our understanding of simple models such as outlined in this paper. We discuss that the development of novel standardized approaches of multidimensional (correlative) biomolecular microscopy methods facilitates the collection of (sub) cellular tissue information in the early onset of colorectal liver metastasis and that this approach will be crucial in designing new effective strategies for CRC treatment. The application of X-ray micro-computed tomography and its potential in correlative imaging of the liver vasculature will be discussed.


Assuntos
Neoplasias Colorretais/patologia , Neoplasias Hepáticas/secundário , Fígado/patologia , Animais , Apoptose/fisiologia , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/fisiopatologia , Endotélio/irrigação sanguínea , Endotélio/patologia , Endotélio/fisiopatologia , Humanos , Imageamento Tridimensional/métodos , Fígado/irrigação sanguínea , Fígado/fisiopatologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/fisiopatologia , Fagocitose/fisiologia
10.
BMC Cancer ; 6: 151, 2006 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-16756685

RESUMO

BACKGROUND: Non-small cell lung cancer is the most common cause of early casualty from malignant disease in western countries. The heterogeneous nature of these cells has been identified by histochemical and microarray biomarker analyses. Unfortunately, the morphological, molecular and biological variation within cell lines used as models for invasion and metastasis are not well understood. In this study, we test the hypothesis that heterogeneous cancer cells exhibit variable motility responses such as chemokinesis and chemotaxis that can be characterized molecularly. METHODS: A subpopulation of H460 lung cancer cells called KINE that migrated under chemokinetic (no gradient) conditions was harvested from Boyden chambers and cultured. Time-lapsed microscopy, immunofluorescence microscopy and microarray analyses were then carried out comparing chemokinetic KINE cells with the unselected CON cell population. RESULTS: Time-lapsed microscopy and analysis showed that KINE cells moved faster but less directionally than the unselected control population (CON), confirming their chemokinetic character. Of note was that chemokinetic KINE cells also chemotaxed efficiently. KINE cells were less adhesive to substrate than CON cells and demonstrated loss of mature focal adhesions at the leading edge and the presence of non-focalized cortical actin. These characteristics are common in highly motile amoeboid cells that may favour faster motility speeds. KINE cells were also significantly more invasive compared to CON. Gene array studies and real-time PCR showed the downregulation of a gene called, ROM, in highly chemokinetic KINE compared to mainly chemotactic CON cells. ROM was also reduced in expression in a panel of lung cancer cell lines compared to normal lung cells. CONCLUSION: This study shows that cancer cells that are efficient in both chemokinesis and chemotaxis demonstrate high invasion levels. These cells possess different morphological, cytoskeletal and adhesive properties from another population that are only efficient at chemotaxis, indicating a loss in polarity. Understanding the regulation of polarity in the context of cell motility is important in order to improve control and inhibition of invasion and metastasis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Movimento Celular , Quimiotaxia , Genes Neoplásicos , Humanos , Cinética , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos
11.
Oncogene ; 21(14): 2201-11, 2002 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-11948403

RESUMO

Hepatocyte growth factor (HGF) regulates various physiological and developmental processes in concert with other growth factors, cytokines and hormones. We examined interactions between cell signaling events elicited by HGF and the cytokine interleukin (IL)-4, in the IL-3-dependent murine myeloid cell line 32D transfected with the human HGF receptor, c-Met. HGF was a potent mitogen in these cells, and prevented apoptosis in response to IL-3 withdrawal. IL-4 showed modest anti-apoptotic activity, but no significant mitogenic activity. IL-4 synergistically enhanced HGF-stimulated DNA synthesis, whereas only additive prevention of apoptosis was observed. IL-4 did not enhance HGF-dependent tyrosine phosphorylation of c-Met or Shc. In contrast, HGF-stimulated activation of MAP kinases was enhanced by IL-4, suggesting that the IL-4 and HGF signaling pathways converge upstream of these events. Although phosphatidylinositol 3-kinase (PI3K) inhibitors diminished HGF-induced mitogenesis, anti-apoptosis, and MAP kinase activation, IL-4 enhanced HGF signaling persisted even in the presence of these inhibitors. IL-4 enhancement of HGF signaling was partially blocked in 32D/c-Met cells treated with inhibitors of MEK1 or c-Src kinases, completely blocked by expression of a catalytically inactive mutant of Janus kinase 3 (Jak3), and increased in 32D/c-Met cells overexpressing STAT6. Our results suggest that the IL-4 and HGF pathways converge at multiple levels, and that IL-4-dependent Jak3 and STAT6 activities modulate signaling events independent of PI3K to enhance HGF-dependent mitogenesis in myeloid cells, and possibly other common cellular targets.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Fator de Crescimento de Hepatócito/farmacologia , Interleucina-4/farmacologia , Mitógenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Interleucina-3/farmacologia , Interleucina-4/antagonistas & inibidores , Janus Quinase 3 , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT6 , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fatores de Tempo , Transativadores/metabolismo , Transfecção , Células Tumorais Cultivadas
12.
Clin Exp Metastasis ; 29(3): 273-92, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22271313

RESUMO

Mortality in breast cancer is linked to metastasis and recurrence yet there is no acceptable biological model for cancer relapse. We hypothesise that there might exist primary tumour cells capable of escaping surgery by migration and resisting radiotherapy and chemotherapy to cause cancer recurrence. We investigated this possibility in invasive ductal carcinoma (IDC) tissue and observed the presence of solitary primary tumour cells (SPCs) in the dense collagen stroma that encapsulates intratumoural cells (ICs). In IDC tissue sections, collagen was detected with either Masson's Trichrome or by second harmonics imaging. Cytokeratin-19 (CK-19) and vimentin (VIM) antibodies were, respectively, used to identify epithelial-derived tumour cells and to indicate epithelial to mesenchymal transition (EMT). Confocal/multiphoton microscopy showed that ICs from acini were mainly CK-19(+ve) and were encapsulated by dense stromal collagen. Within the stroma, SPCs were detected by their staining for both CK-19 and VIM (confirming EMT). ICs and SPCs were subsequently isolated by laser capture microdissection followed by multiplex tandem-PCR studies. SPCs were found to be enriched for pro-migratory and anti-proliferative genes relative to ICs. In vitro experiments using collagen matrices at 20 mg/cm(3), similar in density to tumour matrices, demonstrated that SPC-like cells were highly migratory but dormant, phenotypes that recapitulated the genotypes of SPCs in clinical tissue. These data suggest that SPCs located at the breast cancer perimeter are invasive and dormant such that they may exceed surgical margins and resist local and adjuvant therapies. This study has important connotations for a role of SPCs in local recurrence.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , Movimento Celular , Colágeno Tipo II/análise , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Humanos , Imuno-Histoquímica , Queratina-19/análise , Invasividade Neoplásica , Microambiente Tumoral , Vimentina/análise , Quinases Associadas a rho/fisiologia
13.
Biophys Rev ; 2(3): 121-135, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28510069

RESUMO

To genuinely understand how complex biological structures function, we must integrate knowledge of their dynamic behavior and of their molecular machinery. The combined use of light or laser microscopy and electron microscopy has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, is often referred to as 'correlative microscopy'. Correlative imaging allows researchers to gain additional novel structure-function information, and such information provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or 'combined') microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches. In this topical review, we provide a brief historical perspective of correlative microscopy and an in-depth overview of correlative sample-preparation and imaging methods presently available, including future perspectives on the trend towards integrative microscopy and microanalysis.

14.
Cancer Res ; 68(12): 4525-30, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18559496

RESUMO

The transcriptional repressor Snail2 is overexpressed in head and neck squamous cell carcinomas (HNSCC) relative to nonmalignant head and neck mucosal epithelium, and in locally recurrent relative to nonrecurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of proinflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappaB transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin (IL)-6, IL8, or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Reexpression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.


Assuntos
Diferenciação Celular , Quimiocina CXCL1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Queratinócitos/metabolismo , Mucosa Bucal/metabolismo , Fatores de Transcrição/fisiologia , Animais , Caderinas/genética , Linhagem Celular , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Humanos , Queratinócitos/citologia , Rim/citologia , Rim/metabolismo , Luciferases/metabolismo , Mucosa Bucal/citologia , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Serina Endopeptidases/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Regulação para Cima
15.
IUBMB Life ; 59(2): 60-7, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17454296

RESUMO

The study of cancer cell chemotaxis on two-dimensional surfaces in vitro has relevance to the diverse migratory behaviours exhibited in vivo that involve a directed path. These may include translocation along collagen fibres, invasion into the basement membrane and across stroma, intravasation and extravasation to arrive at a secondary destination designated for cancer cell colonization. Chemotaxis invariably denotes the ability of cells to sense gradients, polarize, adhere and deadhere to substrate, and translocate in the right direction. Amongst these, the sensing function is perhaps the unifying aspect of different migration styles, permitting the cells to resolve its orientation and path. This review examines the decision-making processes that take place during chemotaxis and illustrates that a universal mechanism is involved. In various cell types from Dictyostelium to neutrophils, there are some unifying principles that dictate sensing and how the putative leading edge and trailing end of cells are determined. Some of these principles have recently been applied in the study of cancer cell chemotaxis albeit different pathways are substituted. In amoeboid-like cancer cells, local excitation of the EGFR/PLCgamma/cofilin pathway and parallel, global inhibition of cofilin by LIMK occur to promote the asymmetric distribution and amplification of these internal signals in response to an external EGF gradient.


Assuntos
Quimiotaxia/fisiologia , Neoplasias/patologia , Animais , Movimento Celular/fisiologia , Dictyostelium/fisiologia , Receptores ErbB/fisiologia , Neutrófilos/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfolipase C gama/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais
16.
J Electron Microsc (Tokyo) ; 55(3): 151-5, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16775217

RESUMO

In this study, X-ray micro-computed tomography (CT) was used to reconstruct the fine structure macro- and microvasculature in three dimensions in contrast-enhanced rat liver samples. The subsequent application in the experimental CC531s colorectal cancer model was concurrent with results obtained from confocal microscopy in earlier studies. The en bloc stains osmium tetroxide in combination with uranyl acetate provided an excellent contrasting result for hepatic tissue after a trial of several contrasting agents. X-ray micro-CT allowed us to image the large blood vessels together with the branching sinusoids of hepatic tissue in three dimensions. Furthermore, interruption of the microvasculature was noted when rats were injected with CC531s colorectal cancer cells indicating the presence of hepatic metastases.


Assuntos
Angiografia , Vasos Sanguíneos/ultraestrutura , Fígado/irrigação sanguínea , Microscopia Eletrônica/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Capilares/diagnóstico por imagem , Linhagem Celular Tumoral , Neoplasias do Colo/ultraestrutura , Masculino , Ratos , Ratos Endogâmicos
17.
Cell Motil Cytoskeleton ; 62(1): 27-34, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16025469

RESUMO

Direct observations of cancer cell invasion underscore the importance of chemotaxis in invasion and metastasis. Yet, there is to date, no established method for real-time imaging of cancer chemotaxis towards factors clinically correlated with metastasis. A chamber has been designed and tested, called the Soon chamber, which allows the direct observation and quantification of cancer cell chemotaxis. The premise for the design of the Soon chamber is the incorporation of a dam, which creates a steep gradient while retaining stability associated with a pressure-driven system. The design is based on the characteristics of cancer cell motility such as relatively low speeds, and slower motility responses to stimuli compared to classical amoeboid cells like neutrophils and Dictyostelium. We tested MTLn3 breast carcinoma cells in the Soon chamber in the presence of an EGF gradient, obtaining hour-long time-lapses of chemotaxis. MTLn3 cells migrated further, more linearly, and at greater speeds within an EGF gradient compared to buffer controls. Computation of the degree of orientation towards the EGF/buffer source showed that MTLn3 cells were significantly more directional toward the EGF gradient compared to buffer controls. Analysis of the time-lapse data obtained during chemotaxis demonstrated that two populations of cancer cells were present. One population exhibited oscillations in directionality occurring at average intervals of 12 min while the second population exhibited sustained high levels of directionality toward the source of EGF. This result suggests that polarized cancer cells can avoid the need for oscillatory path corrections during chemotaxis.


Assuntos
Quimiotaxia , Cultura em Câmaras de Difusão/instrumentação , Invasividade Neoplásica , Neoplasias da Mama/fisiopatologia , Carcinoma/fisiopatologia , Quimiotaxia/fisiologia , Humanos , Células Tumorais Cultivadas
18.
J Biol Chem ; 280(1): 777-86, 2005 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-15509575

RESUMO

Wnts stimulate cell migration, although the mechanisms responsible for this effect are not fully understood. To investigate the pathways that mediate Wnt-dependent cell motility, we treated Chinese hamster ovary cells with Wnt-3a-conditioned medium and monitored changes in cell shape and movement. Wnt-3a induced cell spreading, formation of protrusive structures, reorganization of stress fibers and migration. Although Wnt-3a stabilized beta-catenin, two inhibitors of the beta-catenin/canonical pathway, Dickkopf-1 and a dominant-negative T cell factor construct, did not reduce motility. The small GTPase RhoA also was activated by Wnt-3a. In contrast to beta-catenin signaling, inhibition of Rho kinase partially blocked motility. Because Dishevelled (Dvl) proteins are effectors of both canonical and noncanonical Wnt signaling, we used immunofluorescent analysis and small interference RNA technology to evaluate the role of Dvl in cell motility. Specific knock-down of Dvl-2 expression markedly reduced Wnt-3a-dependent changes in cell shape and movement, suggesting that this Dvl isoform had a predominant role in mediating Wnt-3a-dependent motility in Chinese hamster ovary cells.


Assuntos
Forma Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Proteínas/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células CHO , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/farmacologia , Proteínas Desgrenhadas , Ativação Enzimática/efeitos dos fármacos , Camundongos , Fosfoproteínas , Proteínas/metabolismo , Transdução de Sinais , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A
19.
J Biol Chem ; 278(13): 11465-70, 2003 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-12529380

RESUMO

Wnt-induced-secreted-protein-1 (WISP-1) is a cysteine-rich, secreted factor belonging to the CCN family. These proteins have been implicated in the inhibition of metastasis; however, the mechanisms involved have not been described. We demonstrated that overexpression of WISP-1 in H460 lung cancer cells inhibited lung metastasis and in vitro cell invasion and motility. We investigated the possibility that WISP-1 may regulate activation of Rac, a small GTPase important for cytoskeletal reorganizations during motility. In an indirect assay, WISP-1-expressing cells exhibited marked reduction in Rac activation compared with control cells. Blocking antibodies to alpha(v)beta(5) and alpha(1) integrins restored Rac activation in WISP-1 cells, suggesting that the inhibitory effect of WISP-1 on Rac lies downstream of integrins. Constitutively activated Rac mutant (RacG12V) was transfected into WISP-1 cells to restore Rac activation and these WISP-1/RacG12V transfectants were used for further studies. We performed microarray and real-time PCR analyses to identify genes involved in invasion that may be differentially regulated by WISP-1. Here, we showed decreased expression of metalloproteinase-1 (MMP-1) in WISP-1 cells compared with controls but increased expression in WISP-1/RacG12V cells. In an invasion assay across collagen I, an MMP-1 target matrix, WISP-1 cells were significantly less invasive compared with controls, whereas WISP-1/RacG12V cells showed elevated invasion levels. This work illustrates a negatively regulated pathway by WISP-1 involving integrins and Rac in the down-regulation of invasion.


Assuntos
Regulação para Baixo , Substâncias de Crescimento/fisiologia , Neoplasias Pulmonares/patologia , Proteínas Oncogênicas/fisiologia , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas de Sinalização Intercelular CCN , Linhagem Celular , Substâncias de Crescimento/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas , Proteínas rac de Ligação ao GTP/metabolismo
20.
Blood ; 100(3): 982-90, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12130512

RESUMO

In epithelial cells beta-catenin plays a critical role as a component of the cell-cell adhesion apparatus and as a coactivator of the TCF/LEF (T-cell transcription factor/lymphoid enhancer binding factor) family of transcription factors. Deregulation of beta-catenin has been implicated in the malignant transformation of cells of epithelial origin. However, a function for beta-catenin in hematologic malignancies has not been reported. beta-Catenin is not detectable in normal peripheral blood T cells but is expressed in T-acute lymphoblastic leukemia cells and other tumor lines of hematopoietic origin and in primary lymphoid and myeloid leukemia cells. beta-Catenin function was examined in Jurkat T-acute lymphoblastic leukemia cells. Overexpression of dominant-negative beta-catenin or dominant-negative TCF reduced beta-catenin nuclear signaling and inhibited Jurkat proliferation and clonogenicity. Similarly, these constructs inhibited proliferation of K562 and HUT-102 cells. Reduction of beta-catenin expression with beta-catenin antisense down-regulated adhesion of Jurkat cells in response to phytohemagglutinin. Incubation of Jurkat cells with anti-Fas induced caspase-dependent limited proteolysis of beta-catenin N- and C-terminal regions and rapid redistribution of beta-catenin to the detergent-insoluble cytoskeleton, concomitant with a marked decline in nuclear beta-catenin signaling. Fas-mediated apoptosis was potentiated by inhibition of beta-catenin nuclear signaling. The data suggest that beta-catenin can play a significant role in promoting leukemic cell proliferation, adhesion, and survival.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Leucemia/patologia , Ativação Linfocitária/efeitos dos fármacos , Transativadores/fisiologia , Apoptose , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/farmacologia , Citoesqueleto/metabolismo , Humanos , Leucemia/etiologia , Leucemia/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Transativadores/farmacologia , Ativação Transcricional/efeitos dos fármacos , Células Tumorais Cultivadas , beta Catenina
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