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1.
BMC Cancer ; 12: 605, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23244248

RESUMO

BACKGROUND: Neoplastic transformation of cultured cells by a number of oncogenes such as src suppresses gap junctional, intercellular communication (GJIC); however, the role of Src and its effector Signal transducer and activator of transcription-3 (Stat3) upon GJIC in non small cell lung cancer (NSCLC) has not been defined. Immunohistochemical analysis revealed high Src activity in NSCLC biopsy samples compared to normal tissues. Here we explored the potential effect of Src and Stat3 upon GJIC, by assessing the levels of tyr418-phosphorylated Src and tyr705-phosphorylated Stat3, respectively, in a panel of NSCLC cell lines. METHODS: Gap junctional communication was examined by electroporating the fluorescent dye Lucifer yellow into cells grown on a transparent electrode, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. RESULTS: An inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity GJIC was absent, while two lines with extensive GJIC (QU-DB and SK-LuCi6) had low Src levels, similar to a non-transformed, immortalised lung epithelial cell line. Interestingly, examination of the mechanism indicated that Stat3 inhibition in any of the NSCLC lines expressing high endogenous Src activity levels, or in cells where Src was exogenously transduced, did not restore GJIC. On the contrary, Stat3 downregulation in immortalised lung epithelial cells or in the NSCLC lines displaying extensive GJIC actually suppressed junctional permeability. CONCLUSIONS: Our findings demonstrate that although Stat3 is generally growth promoting and in an activated form it can act as an oncogene, it is actually required for gap junctional communication both in nontransformed lung epithelial cells and in certain lung cancer lines that retain extensive GJIC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/fisiologia , Adesão Celular , Conexinas/metabolismo , Regulação para Baixo , Humanos , Imuno-Histoquímica , Fosforilação , Fator de Transcrição STAT3/antagonistas & inibidores , Células Tumorais Cultivadas , Quinases da Família src/metabolismo
2.
Methods Mol Biol ; 423: 173-89, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18370198

RESUMO

Cultured adherent cells can be electroporated in situ, as they grow on a glass slide coated with electrically conductive, optically transparent indium-tin oxide (ITO). Although the introduction of DNA is a common use, the technique of electroporation in situ is valuable for studying many aspects of signal transduction. This is because, under the appropriate conditions, in situ electroporation can be remarkably nontraumatic, while a large variety of molecules, such as peptides, oligonucleotides, or drugs, are introduced instantly and into essentially 100% of the cells, making this technique especially suitable for kinetic studies of effector activation. Following the introduction of the material, the cells can be either extracted or biochemically analyzed, or their morphology and gene expression can be examined by immunocytochemistry. In this chapter, we describe the introduction of a peptide blocking the Src-homology 2 domain of the adaptor Grb2 to inhibit the activation of the downstream effector Erk1/2 by EGF. The setup includes nonelectroporated, control cells growing side by side with the electroporated ones on the same type of ITO-coated surface. In a modified version, this assembly can be used very effectively for studying intercellular, junctional communication: cells are grown on a glass slide half of which is ITO-coated. An electric pulse is applied in the presence of the fluorescent dye lucifer yellow, causing its penetration into the cells growing on the conductive part of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.


Assuntos
Comunicação Celular/fisiologia , Eletroporação/métodos , Junções Comunicantes/fisiologia , Transdução de Sinais/fisiologia , Animais , Adesão Celular , Comunicação Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis , Eletrodos , Eletroporação/instrumentação , Corantes Fluorescentes , Proteína Adaptadora GRB2/antagonistas & inibidores , Proteína Adaptadora GRB2/química , Junções Comunicantes/efeitos dos fármacos , Isoquinolinas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Compostos de Estanho , Domínios de Homologia de src
3.
Curr Mol Med ; 4(5): 529-38, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15267224

RESUMO

In recent years, peptide aptamers have emerged as novel molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to and inactivate a given target protein at the intracellular level, they provide a new experimental strategy for functional protein analyses, both in vitro and in vivo. In addition, by using peptide aptamers as "pertubagens", they can be employed for genetic analyses, in order to identify biochemical pathways, and their components, that are associated with the induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be developed into diagnostic tools for the detection of a given target protein or for the generation of high-throughput protein arrays. Finally, the peptide aptamer technology has direct therapeutic implications. Peptide aptamers can be used in order to validate therapeutic targets at the intracellular level. Moreover, the peptide aptamer molecules themselves should possess therapeutic potential, both as lead structures for drug design and as a basis for the development of protein drugs.


Assuntos
Fenômenos Fisiológicos Celulares , Vírus da Hepatite B/fisiologia , Oligopeptídeos/metabolismo , Papillomaviridae/fisiologia , Transdução de Sinais/fisiologia , Animais , Desenho de Fármacos , Humanos , Oligopeptídeos/genética , Oligopeptídeos/uso terapêutico , Análise Serial de Proteínas , Ligação Proteica/genética , Técnicas do Sistema de Duplo-Híbrido , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
4.
DNA Cell Biol ; 22(5): 339-46, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12941161

RESUMO

We previously developed a technique, termed in situ electroporation, where nonpermeant molecules are introduced through an electrical pulse into adherent cells, while they grow on electrically conductive, optically transparent, indium-tin oxide (ITO). Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Electroporation of radioactive material is faced with two important considerations: (1) potential for exposure of personnel to irradiation, and (2) the requirement for electroporation of a large number of cells. In this report, we describe a modification in the geometry of the slides and electrodes which permits the use of inexpensive ITO-coated glass of lower conductivity that can be discarded after use, to electroporate large numbers of cells using a minimum volume of radioactive nucleotide solution. The results demonstrate that, using this assembly, the determination of the Ras-bound GTP/GTP+GDP ratios through electroporation of [alpha32P]GTP can be conducted using approximately five times lower amounts of isotope than in previous designs. Moreover, this assembly permits efficient upscaling, which makes the determination of Ras-GTP binding in cells which are deficient in Ras activity possible. In addition, we demonstrate the labeling of two viral phosphoproteins--the Simian Virus 40 Large Tumor antigen, and Adenovirus E1A--through [gamma32P]ATP electroporation using this setup. In both cases, electroporation of the nucleotide can achieve a great increase in the efficiency and specificity of labeling compared to the addition of [32P]-orthophosphate to the culture medium, presumably because the immediate phosphate donor nucleotide itself is introduced, which can directly bind to the target proteins.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Guanosina Trifosfato/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Radioisótopos de Fósforo/metabolismo , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroporação/métodos , Fibroblastos/metabolismo , Guanosina Difosfato/metabolismo , Camundongos , Proteínas Virais/metabolismo
5.
DNA Cell Biol ; 22(1): 33-40, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12590735

RESUMO

Studies using normal or neoplastically transformed established mouse lung epithelial cell lines revealed a reduction in gap junctional, intercellular communication (GJIC) with transformation. To determine the stage in tumor development at which GJIC is interrupted, we used the well-established model of lung tumors induced in strain A/J mice by urethane. In this system, tumor development follows a well-characterized pattern; hyperplasias, adenomas, and carcinomas are manifested at approximately 8, 16, and 40 weeks after urethane treatment, respectively. GJIC levels were examined using a novel technique where cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse that opens transient pores on the plasma membrane is applied in the presence of the fluorescent dye, Lucifer yellow, causing dye penetration into cells growing on the conductive part of the slide. Migration of the dye through gap junctions to nonelectroporated cells growing on the nonconductive area is then microscopically observed under fluorescence illumination. Unexpectedly, primary cells cultured from urethane-induced tumors, even late stage carcinomas, possessed extensive GJIC immediately upon isolation. Upon passage for several months however, these cells lost GJIC. These results suggest that the molecular changes that lead to the formation of the tumor in vivo are not sufficient to interrupt gap junctions. Propagation of tumor cells in culture induces additional alterations that can lead to gap junction closure.


Assuntos
Eletroporação/métodos , Junções Comunicantes/metabolismo , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/fisiologia , Animais , Corantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Camundongos
6.
J Biol Chem ; 281(30): 21345-21352, 2006 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-16717089

RESUMO

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.


Assuntos
Aptâmeros de Peptídeos/química , Vírus da Hepatite B/genética , Proteínas/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/farmacologia , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Vimentina/química , Replicação Viral
7.
Mol Carcinog ; 43(1): 38-50, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15768386

RESUMO

To investigate the functional relationship between the ability of the adenovirus-5 E1A oncogene product to transform with its ability to block adipocytic differentiation and induce apoptosis, we expressed E1A in the 3T3 L1 preadipocytic cell line. The results demonstrate a dramatic, quantitative reciprocal regulation of differentiation and several transformation-associated properties in response to graded levels of E1A expression, with the suppression of differentiative capacity, focus formation, and anchorage-independent proliferation requiring increasing levels of E1A. Progressively higher E1A levels were accompanied by apoptosis induction. The effect of E1A upon adipocytic differentiation as well as transformation and apoptosis required binding to the retinoblastoma-susceptibility gene product. These data reveal a dissociation between E1A signals leading to transformation, suppression of differentiation and induction of apoptosis, based on levels of expression.


Assuntos
Proteínas E1A de Adenovirus/fisiologia , Adipócitos/citologia , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Células 3T3-L1 , Proteínas E1A de Adenovirus/genética , Animais , Camundongos , Mutação
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