RESUMEN
The transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase (pac) gene was studied by modifying DNA sequences responsible for promoter activation by cyclic AMP receptor protein (CRP). The nucleotide sequence of the 5'-flanking region of the pac gene contains putative tandem CRP binding sites positioned at -69/-70 and at -111/-112 with respect to the transcriptional start site. Our results obtained with either point mutations or insertion or deletion mutants (each of which rotated the helix structure at the CRP binding site one-half turn) showed significant decrease of penicillin amidase (PA) activity, suggesting the CRP as a major activator. In this study, the evidence for the importance of spacing between tandem binding sites for CRP as well as for their location related to the promoter core sequence has been provided. Involvement of integration host factor (IHF) as an additional regulatory protein in the pac gene transcription regulation was also analyzed. It is shown that activation of the pac gene transcription is elevated by IHF.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Penicilina Amidasa/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , Escherichia coli/genética , Genes Reporteros , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Penicilina Amidasa/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: The notion that a deficit in immune cell functions permits tumor growth has received experimental support with the discovery of several different biochemical defects in T lymphocytes that infiltrate cancers. Decreased levels of enzymes involved with T-cell signal transduction have been reported by several laboratories, suggesting that tumors or host cells recruited to the tumor site actively down-regulate antitumor T-cell immune response. This permits tumor escape from immune-mediated killing. The possibility that defects in T-cell signal transduction can be reversed, which would potentially permit successful vaccination or adoptive immunotherapy, motivates renewed interest in the field. Summarizing the literature concerning tumor-induced T-cell dysfunction, we focus on the end stage of immune response to human cancer, that of defective cytotoxic T lymphocyte killing function. Based on the data from several laboratories, we hypothesize a biochemical mechanism that accounts for the unusual phenotype of antitumor T-cell accumulation in tumors, but with defective killing function.
Asunto(s)
Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos de Neoplasias , Apoptosis/inmunología , Citotoxicidad Inmunológica , Femenino , Humanos , Tolerancia Inmunológica , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Masculino , Modelos Biológicos , Neoplasias/patología , Transducción de Señal , Linfocitos T Citotóxicos/patologíaRESUMEN
We have investigated the ability of different cells present in murine tumors to induce apoptosis of activated CD8(+) T cells in vitro. Tumor cells do not induce apoptosis of T cells; however, macrophages that infiltrate tumors are potent inducers of apoptosis. Tumor macrophages express cell surface-associated TNF, TNF type I (CD120a) and II (CD120b) receptors, and, upon contact with T cells which induces release of IFN-gamma from T cells, secrete nitric oxide. Killing of T cells in vitro is blocked by Abs to IFN-gamma, TNF, CD120a, or CD120b, or N-methyl-L-arginine. In concert with that finding, tumor macrophages isolated from either TNF type I or type II receptor -/- mice are not proapoptotic and do not produce nitric oxide upon contact with activated T cells. Control macrophages do not express TNF receptors or release nitric oxide. Tumor cells or tumor-derived macrophages do not express FasL, and blocking Abs to either Fas or FasL have no effect on macrophage-mediated T cell killing. These results demonstrate that macrophages which infiltrate tumors are highly proapoptotic and may be responsible for elimination of activated antitumor T cells within the tumor bed.
Asunto(s)
Apoptosis , Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Macrófagos/inmunología , Neoplasias Experimentales/inmunología , Óxido Nítrico/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Anticuerpos/inmunología , Adhesión Celular , Células Cultivadas , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Inmunológicos , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN-gamma mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-gamma. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-gamma, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G(1), do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Experimentales/inmunología , Receptor fas/fisiología , Animales , Linfocitos T CD8-positivos/patología , Ciclo Celular/inmunología , Movimiento Celular/inmunología , Separación Celular , Células Cultivadas , Sueros Inmunes/administración & dosificación , Inmunofenotipificación , Etiquetado Corte-Fin in Situ , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Interleucina-2/administración & dosificación , Linfocitos Infiltrantes de Tumor/patología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Neoplasias Experimentales/patología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Células Tumorales CultivadasRESUMEN
Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8(+) TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-gamma is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Gránulos Citoplasmáticos/fisiología , Citotoxicidad Inmunológica , Exocitosis , Linfocitos Infiltrantes de Tumor/inmunología , Glicoproteínas de Membrana/fisiología , Centro Organizador de los Microtúbulos/fisiología , Animales , Calcio/metabolismo , Citocinas/biosíntesis , Quinasa 2 de Adhesión Focal , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/fisiología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal , Sinapsis/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTPRESUMEN
Immune suppression in tumor-bearing hosts is considered to be one factor causally associated with the growth of antigenic tumors. Support for this hypothesis has come from reports that spleen T cells in tumor-bearing mice are deficient in either priming or effector phase functions. We have reexamined this hypothesis in detail using multiple murine tumor models, including transplantable adenocarcinoma, melanoma, sarcoma, and thymoma, and also a transgenic model of spontaneous breast carcinoma. In both in vitro and in vivo assays of T cell function (proliferation, cytokine production, induction of CD8+ alloreactive CTL, and development of anti-keyhole limpet hemocyanin CD4+ T cells, rejection of allogeneic or syngeneic regressor tumors, respectively) we show that mice bearing sizable tumor burdens are not systemically suppressed and do not have diminished T cell functions. Therefore, if immune suppression is a causal function in the growth of antigenic tumor, the basis for escape from immune destruction is likely to be dependent upon tumor-induced T cell dysfunction at the site of tumor growth.
Asunto(s)
Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Linfocitos T/inmunología , Animales , División Celular/inmunología , Separación Celular , Células Cultivadas , Citocinas/análisis , Femenino , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Hemocianinas/administración & dosificación , Hemocianinas/inmunología , Inyecciones Subcutáneas , Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neutrófilos/patología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Esplenomegalia/inmunología , Esplenomegalia/patología , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Antígenos Thy-1/biosíntesisRESUMEN
Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.