RESUMO
This study evaluates the effects of insulin-like growth factor (IGF)-1 receptor (IGF-1R) down-regulation in stimulated T lymphocytes by investigating the expression of early activation proteins CD69, CD25, and interleukin (IL)-2. We found that IGF-1 does not modify CD69 expression but increases transcription and protein synthesis of CD25 and IL-2. The lowest level of IGF-1R detected after 15 min of activation suggested that the effects of IGF-1 occur at the initiation of cell activation. The activation of IGF-1R was confirmed by IGF-1R phosphorylation and increased phosphorylation of microtubule-associated protein kinase. We also detected the alternative IGF-1 transcripts Ea, with paracrine/autocrine regulation, and Eb, with endocrine regulation, in Jurkat cells and in quiescent T lymphocytes, and we detected IGF-1 protein in the culture medium after stimulation. These data suggest that the proliferative effects of IGF-1 on T lymphocytes include both autocrine/paracrine and endocrine processes.
Assuntos
Proteínas Imediatamente Precoces/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Ativação Linfocitária , Linfócitos T/efeitos dos fármacos , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Regulação para Baixo , Humanos , Fator de Crescimento Insulin-Like I/genética , Interleucina-2/metabolismo , Células Jurkat , Cinética , Lectinas Tipo C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores de Interleucina-2/efeitos dos fármacos , Receptores de Interleucina-2/metabolismo , Linfócitos T/imunologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.
Assuntos
Comunicação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Genes APC , Proteína da Polipose Adenomatosa do Colo , Anticorpos , Anticorpos Monoclonais , Núcleo Celular/metabolismo , Neoplasias do Colo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/genética , Biblioteca Gênica , Humanos , Junções Intercelulares/fisiologia , Microscopia Confocal , Oligodesoxirribonucleotídeos/química , Transporte Proteico , Moldes Genéticos , Células Tumorais CultivadasRESUMO
Oligonucleotide aptamer(s), obtained by using the SELEX procedure, has been used as reagents to recognize different molecules with high affinity and specificity. However, until recently, it was not possible to obtain oligonucleotide-based reagents able to recognize proteins with high specificity in assays typical of antibodies, such as immunohistochemistry, Western blotting and immunoprecipitations. Here, we show the results obtained by applying the strategy of "target switching" to obtain specific polyclonal and monoclonal oligobodies against the protein ERK2. We were able to develop highly specific polyclonal oligobodies by using only one selection step with a temporary target and one selection step with the final target (ERK2). Since only two selection steps were required, these results demonstrate that it is possible to obtain specific reagents against a protein without a need for an "in vitro evolution" using many selection steps, or error-prone polymerases. After one additional selection step, the polyclonal oligobodies were cloned to obtain a highly specific monoclonal oligobody.
Assuntos
Anticorpos Monoclonais/imunologia , Proteína Quinase 1 Ativada por Mitógeno/análise , Sequência de Aminoácidos , DNA de Cadeia Simples/imunologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Dados de Sequência Molecular , Desnaturação Proteica , Células Tumorais CultivadasRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/síntese química , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Imuno-Histoquímica , Camundongos , Oligonucleotídeos/imunologia , Oligonucleotídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Testes de Precipitina , CoelhosRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.
RESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.
Assuntos
Animais , Coelhos , Ratos , Oligonucleotídeos/síntese química , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/imunologia , Testes de Precipitina , Imuno-Histoquímica , Western Blotting , Reação em Cadeia da Polimerase , Anticorpos Monoclonais/imunologiaRESUMO
Although it is known that IGF-1 increases T lymphocyte proliferation, the regulation of its receptor expression during the process has not been defined. Consequently the regulation of IGF-1R expression by IGF-1 and the activation events in human blood T lymphocytes and the Jurkat T cell line were now investigated. IGF-1R expression in nonstimulated Jurkat cells was confirmed and downregulation by IGF-1 was demonstrated. In addition, both cell types showed a time-dependent reduction in the number of IGF-1R-positive cells following activation, which was increased by IGF-1. In Jurkat cells the negative regulation of IGF-1R levels was correlated with the appearance and continuous increase in IL-2R. In T lymphocytes the decrease in IGF-1R expression was faster, reaching its plateau after 3 h of activation. Our findings suggest that loss of the IGF-1R is one of the initial events of the activation process not regulated by IL-2.
Assuntos
Ativação Linfocitária , Receptor IGF Tipo 1/biossíntese , Linfócitos T/imunologia , Regulação para Baixo , Citometria de Fluxo , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Células Jurkat , Linfócitos T/efeitos dos fármacosRESUMO
Recently we demonstrated that IGF-1 has the same effect on DNA synthesis and G1-phase length during human lymphocyte proliferation as IL-2. In order to determine the link between IGF-1 and IL-2 on lymphocyte proliferation, we tested the ability of these cytokines, alone or in combination, to induce DNA synthesis and increase the number of cells expressing IL-2-alpha chain receptors. Our results showed that the increase in DNA synthesis produced by the addition of IGF-1 to cells cultured with saturable concentrations of IL-2, correlated well with an increase in the number of cells expressing IL-2-alpha chain receptor. The effect on both DNA synthesis and IL-2-alpha chain receptor expression was greater at lower concentrations of PHA. This study suggests that the proliferative effect of IGF-1 might be due to separate stimulation of different cell populations and/or by activation of a tyrosine-kinase signal transduction pathway complementary to the IL-2 activation signal.