RESUMEN
There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivo footprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ?898/9, at a putative partial heat shock element (HSE) and at -893/4, a site bordering an E-box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E-box and a partial HSE within the iNOS enhancer. Binding to the E-box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heeat shock factor-1 (HSF-1). Mutations at the HSE revealed the importance of HSF-1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever-associated gram-negative infection.
Asunto(s)
Proteínas de Unión al ADN , Respuesta al Choque Térmico/genética , Óxido Nítrico Sintasa/genética , Animales , Sitios de Unión , Huella de ADN , Lipopolisacáridos/farmacología , Ratones , Modelos Genéticos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Activación Transcripcional , Factores Estimuladores hacia 5'RESUMEN
Nitric oxide (NO) is generated by a family of isoenzymes named nitric oxide synthases (NOS) which includes a cytokine-inducible form, NOSII. NO is a free radical known to inhibit cell proliferation, to induce apoptosis, and to be a mediator of macrophage cytostatic and cytotoxic effects. We investigated NOS in 40 human breast carcinomas and 8 benign breast lesions. NOSII was localized in tumor cells by immunohistochemistry. NOS activity, measured with the citrulline assay, was detected in 27 of 40 tumors. Neither immunohistologic labeling nor NOS activity was detected in benign samples. NOS labeling and activity were significantly related (p < 0.02). For the first time, a significant negative relationship between NOS activity and tumor cell proliferation (p < 0.002) was found. We also showed that tumors with high NOS activity expressed progesterone receptors (p < 0.04). These results are consistent with the observation of high NOS activity in tumors with low grade (p < 0.05). These in vivo observations were related to in vitro data: cytokines (IL-1beta, IFN-gamma, and TNF-alpha) induced NOSII expression in human MCF-7 breast cancer cells, and NO inhibited their proliferation. Thus, we show herein that tumors with high NOS activity have low proliferation rate and low grade, which correlates with the in vitro observation of the inhibition of proliferation of human breast cancer cells by NO. These results may have future therapeutic implications.
Asunto(s)
Neoplasias de la Mama/enzimología , Óxido Nítrico Sintasa/análisis , Receptores de Progesterona/análisis , Neoplasias de la Mama/patología , Diferenciación Celular/fisiología , División Celular/fisiología , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Invasividad Neoplásica , Óxido Nítrico Sintasa de Tipo II , Pronóstico , Células Tumorales CultivadasRESUMEN
It is well documented that nitric oxide (NO) is an effector molecule of macrophage-mediated tumor cell toxicity in vitro; however, little is known about the role of NO in the antitumor immune response in vivo. We have developed a treatment protocol using lipid A. We have investigated the effects of lipid A on inducible NO synthase (NOS II) expression and evolution inside tumors during the course of treatment. Lipid A (OM-174) treatment induced tumor regression in rats bearing established colon tumors. Furthermore, NO was synthesized and secreted inside the tumors of lipid A-treated rats, as demonstrated by the increase of NOS II mRNA and NOS II content in the tumors, as well as of NOS II activity and NO production. During treatment, NOS II was localized in tumor cells only. Lipid A had no direct effect on tumor cells in vitro, while the combination of interferon gamma (IFN-gamma) plus interleukin-1 beta (IL-1beta) induced production of NO by tumor cells which was cytostatic. The content of IFN-gamma and IL-1beta in tumors was enhanced during lipid A treatment; this is in agreement with an indirect effect of lipid A in vivo via the IFN-gamma and IL-1beta pathways.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Lípido A/uso terapéutico , Óxido Nítrico Sintasa/biosíntesis , Adenocarcinoma/metabolismo , Animales , División Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Femenino , Interferón gamma/metabolismo , Interferón gamma/farmacología , Interleucina-1/metabolismo , Interleucina-1/farmacología , Masculino , Trasplante de Neoplasias , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Células Tumorales CultivadasRESUMEN
Repeated exposure to bacterial endotoxin causes a diminished response by the host to further exposure. One important feature of this hyporesponsiveness is a reduced macrophage production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) pathway. Using a murine macrophage model, we observed that hyporesponsiveness was accompanied by a decrease in the levels of NO release (measured as nitrite), iNOS protein and iNOS gene transcription. The expression of the putative lipopolysaccharide (LPS) receptor, CD14, was not altered. In vivo genomic footprinting showed that the same binding sites are occupied in the iNOS promoter and enhancer of desensitized macrophages and of LPS-responsive macrophages, yet the composition of NF-kappaB in the nuclei of these cells was found to be altered. The transcriptionally inactive homodimer p50-p50 represented the predominant binding activity in nuclei from LPS-pretreated cells before and after stimulation. Nuclei from cells which had not been pretreated but were stimulated contained more of the transcriptionally active p50-p65 heterodimer than their pretreated counterparts. Consistent with this, the cytosolic steady-state level of an inhibitor of NF-kappaB activity, I-kappaBalpha, was decreased in normal cells but not in pretreated cells. We propose that the presence of an overwhelming excess of transcriptionally inactive p50 homodimers on their kappaB sites in the iNOS control region in pretreated cells may block kappaB site binding by p50-p65, thereby reducing the activity of the protein complex governing iNOS transcription.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/inmunología , Lipopolisacáridos/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , FN-kappa B/inmunología , Óxido Nítrico Sintasa/inmunología , Animales , Línea Celular , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Ratones , FN-kappa B/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo IIRESUMEN
Cancer cells often resist Fas-mediated apoptosis even when the Fas receptor is expressed at the cell surface. We show here that human and rat colon cancer cells undergo massive apoptosis when they are exposed to soluble Fas ligand in the presence of sodium butyrate, an agent that induces by itself only a low rate of apoptosis. Sodium butyrate potentiates Fas-dependent apoptosis in seven out of eight colon cancer cell lines. Sodium butyrate does not increase Fas receptor cell surface expression and does not modify cell levels of Bcl-2, Bcl-xL, Bcl-xS and Bax. Sodium butyrate also induces tumor cell sensitization to the apoptotic effect of the combination of TNF-alpha and IFN-gamma, but it does not modify the level of the FADD/Mort1 adaptator molecule, at the connection between Fas- and TNF-dependent apoptosis pathways. Because the clinical toxicity of butyrate is low, its ability to enhance Fas-signal delivery in cancer cells could be of therapeutic interest.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/efectos de los fármacos , Ácido Butírico/farmacología , Glicoproteínas de Membrana/genética , Animales , Proteínas Portadoras/metabolismo , Neoplasias del Colon/metabolismo , Proteína Ligando Fas , Proteína de Dominio de Muerte Asociada a Fas , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Interferón gamma/farmacología , Microscopía de Contraste de Fase , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Although PROb colonic tumor cells are immunosuppressive, the immunomodulator OM 163 induced the disappearance of macroscopic peritoneal nodules in 50% of rats bearing a peritoneal carcinomatosis (p.c.) induced by PROb cells. When the p.c. developed, the number of T lymphocytes was low and the expression of interferon (IFN)-gamma mRNA in tumor nodules decreased very rapidly. The TGF-beta 1 secreted by PROb cells could be responsible for the immunosuppression, because the PROb cell supernatant inhibited IFN-gamma production and T lymphocyte proliferation. When the rats were treated with OM 163, an infiltration of T lymphocytes was observed in tumor nodules, as well as a high expression of the IFN-gamma mRNA. The antitumor efficiency of the immunomodulator OM 163 could be explained in part by a direct effect of OM 163 on T lymphocytes, because in vitro it stimulated the proliferation and the secretion of IFN-gamma of T lymphocytes. OM 163 could also act at the TGF-beta 1 level. Although OM 163 alone or in combination with IFN-gamma did not modify the TGF-beta 1 secretion by PROb cells in vitro, the expression of the TGF-beta 1 mRNA and the TGF-beta 1 protein content was decreased in vivo in treated tumor nodules.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antineoplásicos/farmacología , Regulación hacia Abajo/efectos de los fármacos , Linfocitos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Relación Dosis-Respuesta a Droga , Escherichia coli , Expresión Génica/efectos de los fármacos , Terapia de Inmunosupresión , ARN Mensajero/metabolismo , RatasRESUMEN
A wide variety of cells usefully but sometimes destructively produce nitric oxide via inducible nitric oxide synthase (iNOS). Data obtained by gel shift analysis and reporter assays have linked murine iNOS gene induction by cytokines and bacterial products with the binding of a number of proteins to a proximal promoter, as well as to a distal enhancer of the iNOS gene. Nevertheless, these techniques do not necessarily reflect protein occupation of sites in vivo. To address this, we have used dimethyl sulphate in vivo footprinting to determine binding events in the two murine iNOS transcription control regions, using a classical lipopolysaccharide induction of RAW 264.7 macrophages. Protein-DNA interactions are absent before activation. Exposure to lipopolysaccharide induces protection at a NF-kappaB site and hypersensitivity at a shared gamma-activated site/interferon-stimulated response element within the enhancer. Protections are seen at a NF-IL6, and an Oct site within the promoter. We also observe modulations in guanine methylation at two regions which do not correspond to any known putative binding elements. Furthermore, we confirm the probable involvement of interferon regulatory factor-1 (binding to its -901 to -913 site) and the binding of NF-kappaB to its proximal site. Our data demonstrate an abundance of hitherto-unrecognised protein-DNA binding events upon simple lipopolysaccharide activation of the iNOS gene and suggests a role for protein-protein interactions in its transcriptional induction.