RESUMEN
Inducible nitric oxide synthase (iNOS) may be a key mediator of intestinal injury, which varies with developmental age. One member of the mitogen-activated protein kinase (MAPK) family, p38, is involved in the lipopolysaccharide (LPS)-mediated iNOS induction. The involvement of p38 MAPK in basal and LPS-induced iNOS expression was examined in the rat intestine at two developmental ages. Neonatal (4 days postnatal) and adolescent (15 days postnatal) rats were injected with LPS (5 microg/g ip), a selective p38 inhibitor (SB 203580), or both. Tissue was removed after 4 h and 6 h for mRNA and protein analysis. iNOS mRNA and protein were markedly upregulated in the adolescent female following LPS exposure, whereas males had an attenuated response. Neonates had a minimal response. SB 203580 suppressed LPS-induced iNOS mRNA and protein in the ileum, more so in females than in males. Adolescent ileal p38 activation was constitutively high and nonresponsive to LPS. Basal and post-LPS p38 phosphorylation was low in neonatal ileum. We conclude that ileal iNOS expression is developmentally regulated and influenced by gender and that p38 is permissive for LPS effect. The developmental regulation of p38 may contribute to age-dependent variations of intestinal injury.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Íleon/crecimiento & desarrollo , Íleon/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Animales , Animales Recién Nacidos , Inhibidores Enzimáticos/farmacología , Femenino , Imidazoles/farmacología , Lipopolisacáridos/farmacología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Piridinas/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The confluence of two distinct but related activities in the past 10 years has dramatically accelerated efforts towards the discovery and development of novel drugs to treat cancer. The first is a rapidly emerging understanding that a number of distinct tyrosine kinases play roles in diverse but fundamentally important aspects of tumor progression (growth, survival, metastasis and angiogenesis). The second is the discovery that small molecule compounds have the capacity to potently and selectively inhibit the biochemical function of tyrosine kinases by competing for ATP binding at the enzyme catalytic site. These observations have been conjoined in major efforts to bring forward into clinical development novel cancer drugs with the potential to provide both clinical efficacy and improved tolerability. The focus of this review is on the development of small molecule tyrosine kinase inhibitors, and does not extend to other approaches that could be applied to disrupt the same pathways in clinical tumors (receptor and/or ligand-competitive antibodies, intrabodies, antisense ribonucleotides, ribozymes, phosphatase inhibitors or SH2/SH3-directed agents). Selected tyrosine kinase inhibitors, known or believed to be in development in cancer treatment trials, are summarized as are some of the key issues that must be addressed if these compounds are to be developed into clinically useful cancer chemotherapeutic agents.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Diseño de Fármacos , Inhibidores Enzimáticos/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Ensayos Clínicos como Asunto , Receptores ErbB/antagonistas & inhibidores , Humanos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidoresRESUMEN
Phosphorylation of tyrosine residues on the epidermal growth factor (EGF) receptor (EGFr) is an important early event in signal transduction, leading to cell replication for major human carcinomas. CP-358,774 is a potent and selective inhibitor of the EGFr tyrosine kinase and produces selective inhibition of EGF-mediated tumor cell mitogenesis. To assess the pharmacodynamic aspects of EGFr inhibition, we devised an ex vivo enzyme-linked immunosorbent assay for quantification of EGFr-specific tyrosine phosphorylation in human tumor tissue specimens obtained from xenografts growing s.c. in athymic mice. When coupled with pharmacokinetic analyses, this measurement can be used to describe the extent and duration of kinase inhibition in vivo. CP-358,774 is an effective, orally active inhibitor of EGFr-specific tyrosine phosphorylation (ED(50) = 10 mg/kg, single dose). It has a significant duration of action, producing, on average, a 70% reduction in EGFr-associated phosphotyrosine over a 24-h period after a single 100 mg/kg dose. Inhibition of EGFr phosphotyrosine in an ex vivo assay format effectively estimates the potency and degree of inhibition of EGFr-dependent human LICR-LON-HN5 head and neck carcinoma tumor growth. Substantial growth inhibition of human tumor xenografts was achieved with p.o. doses of the compound (ED(50) = 10 mg/kg q.d. for 20 days). Combination chemotherapy with cisplatin produced a significant response above that of cisplatin alone with no detectable effects on body weight or lethal toxicity. Taken together, these observations suggest that CP-358,774 may be useful for the treatment of EGFr-driven human carcinomas.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Quinazolinas/farmacología , Tirosina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , División Celular/efectos de los fármacos , Cisplatino/toxicidad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Clorhidrato de Erlotinib , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Ratones Desnudos , Fosforilación , Fosfotirosina/metabolismo , Polifarmacia , Quinazolinas/sangre , Factores de Tiempo , Trasplante Heterólogo/fisiología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Impairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon gamma (IFN-gamma) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells. AIMS: To examine iNOS induction by IFN-gamma in vitro as a function of enterocyte differentiation. METHODS: Preconfluent and postconfluent Caco-2 cells were treated with IFN-gamma in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-gamma receptor mRNA and protein levels were determined. RESULTS: iNOS mRNA induction by IFN-gamma occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-kappaB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation-that is, in postconfluent Caco-2 cells. This increased IFN-gamma responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-gamma receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-gamma receptor levels as a function of postconfluent growth. CONCLUSIONS: With regard to iNOS mRNA induction, IFN-gamma responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-gamma receptors.
Asunto(s)
Interferón gamma/farmacología , Mucosa Intestinal/enzimología , Óxido Nítrico Sintasa/biosíntesis , Northern Blotting , Western Blotting , Células CACO-2/efectos de los fármacos , Diferenciación Celular/fisiología , Neoplasias del Colon/enzimología , Inducción Enzimática , Expresión Génica , Humanos , Mucosa Intestinal/citología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/genética , Receptores de Interferón/biosíntesis , Receptores de Interferón/genética , Proteínas Recombinantes , Receptor de Interferón gammaRESUMEN
Compounds that stabilize the DNA binding domain of p53 in the active conformation were identified. These small synthetic molecules not only promoted the stability of wild-type p53 but also allowed mutant p53 to maintain an active conformation. A prototype compound caused the accumulation of conformationally active p53 in cells with mutant p53, enabling it to activate transcription and to slow tumor growth in mice. With further work aimed at improving potency, this class of compounds may be developed into anticancer drugs of broad utility.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Pirimidinas/farmacología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , ADN/metabolismo , Epítopos , Genes p53 , Humanos , Ratones , Mutación , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Pirimidinas/química , Pirimidinas/uso terapéutico , Temperatura , Transcripción Genética , Transfección , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
OBJECTIVES: To characterize the mechanisms leading to excessive production of nitric oxide within the gut as a consequence of endotoxemia. We sought to: a) determine the time course of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in the intestine after challenging rats with lipopolysaccharide (LPS); and b) investigate whether there is differential expression of iNOS in enterocytes along the longitudinal or crypt-villus axes of the intestine in rats after LPS administration. DESIGN: Prospective, randomized, unblinded study. SETTING: Research laboratories at a large university-affiliated medical center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: At T = 0 hr, rats were injected with O111:B4 Escherichia coli LPS (5 mg/kg) or a similar volume of the saline vehicle. At various time points thereafter, samples of duodenum, jejunum, ileum, colon, and liver were harvested for subsequent extraction of RNA. In some cases, populations of enterocytes enriched in either crypt or villus cells were harvested from the ileum. In some studies, rats were injected with cycloheximide (25 mg i.p.) 15 mins before being challenged with LPS or dexamethasone (2 mg i.p.) 30 mins before being injected with LPS. MEASUREMENTS AND MAIN RESULTS: iNOS mRNA was undetectable in ileal tissue from rats under basal conditions, but was evident by T = 1 hr and was maximal at T = 2 hrs after injection of LPS. Thereafter, ileal iNOS mRNA concentrations decreased and were undetectable again at T = 24 hrs. At T = 2 hrs after LPS injection, there was marked expression of iNOS mRNA in the ileum, whereas much lower concentrations of iNOS mRNA were detected in the jejunum and colon, and no iNOS mRNA was detected in the duodenum. At T = 3 hrs after LPS injection, expression of iNOS mRNA was up-regulated in both villus and crypt cells, although LPS-induced iNOS mRNA was more prominent in the former than the latter cell type. Pretreatment of rats with dexamethasone virtually abrogated the expression of iNOS mRNA in ileal samples obtained 3 hrs after the injection of LPS. Prior treatment of rats with the protein synthesis inhibitor, cycloheximide, also blunted LPS-induced iNOS mRNA expression. CONCLUSIONS: LPS-induced iNOS expression is differentially regulated along both the longitudinal and crypt villus axes of the intestinal mucosa, being most prominent in the villus cells of the ileum. LPS-induced iNOS expression is blunted by pretreating rats with dexamethasone or cycloheximide. The latter finding suggests that LPS-induced expression of iNOS mRNA in the gut requires new protein synthesis. Differential regulation of nitric oxide production along the longitudinal and crypt-villus axes of the gut may be a determinant of the pattern of sepsis-induced intestinal damage.
Asunto(s)
Endotoxemia/enzimología , Regulación Enzimológica de la Expresión Génica , Mucosa Intestinal/metabolismo , Óxido Nítrico Sintasa/biosíntesis , ARN Mensajero/metabolismo , Animales , Endotoxemia/inducido químicamente , Inducción Enzimática , Mucosa Intestinal/ultraestructura , Lipopolisacáridos , Masculino , Microvellosidades/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/genética , Ciclo Celular/genética , División Celular/efectos de los fármacos , Fragmentación del ADN , ADN de Neoplasias/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: The permeability of intestinal epithelial monolayers increases after exposure to nitric oxide. The aim of this study was to investigate the role of excessive NO production on intestinal barrier function in rats injected with lipopolysaccharide (LPS). METHODS: Rats were injected with saline or LPS (5 mg/ kg). Bacterial translocation to mesenteric lymph nodes, liver, and spleen was assessed 24 hours after LPS injection. Mucosal permeability was determined by loading fluorescein-labeled dextran (mol wt, 4000 daltons) into an intestinal segment and measuring its appearance in plasma. Intestinal mucosal mitochondrial respiration was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. RESULTS: Intestinal tissue from LPS-challenged rats showed upregulation of inducible NO synthase (iNOS) messenger RNA expression and subsequent up-regulation of iNOS enzymatic activity. Plasma concentrations of nitrite plus nitrate (NO2-/NO3-) were increased for at least 24 hours after injection of LPS. Treatment with the selective iNOS inhibitor, aminoguanidine, inhibited iNOS enzymatic activity and overproduction of NO2-/NO3-. LPS-induced bacterial translocation was reduced by aminoguanidine. LPS-induced intestinal hyperpermeability was ameliorated by both aminoguanidine and another selective iNOS inhibitor, S-methylisothiourea. LPS depressed intestinal mucosal mitochondrial function, and this effect was ameliorated by aminoguanidine. CONCLUSIONS: Overproduction of NO may contribute to intestinal barrier dysfunction in LPS challenged rats, possibly by interfering with mitochondrial oxidative metabolism.
Asunto(s)
Bacterias , Endotoxinas/farmacología , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Mucosa Intestinal/microbiología , Mucosa Intestinal/fisiología , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa/biosíntesis , Transcripción Genética/efectos de los fármacos , Animales , Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Presión Sanguínea , Mucosa Intestinal/efectos de los fármacos , Hígado/microbiología , Ganglios Linfáticos/microbiología , Masculino , Mitocondrias/metabolismo , Nitratos/sangre , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/sangre , Consumo de Oxígeno/efectos de los fármacos , Permeabilidad , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Bazo/microbiologíaRESUMEN
DNA-based immunizations have been used to analyze the ability of DNA-expressed hemagglutinin (HA) and nucleoprotein (NP) to protect BALB/c mice against a homologous influenza virus, A/PR/8/34 (H1N1), challenge. The HA DNA, but not the NP DNA, protected mice against the lethal viral challenge. For the HA DNA, single gene gun inoculations of 0.04 microg and boosted inoculations of 0.004 microg of DNA raised complete protection. For the NP DNA, boosted gene gun immunizations of 0.4 microg of DNA and boosted intradermal or intramuscular injections of 50 microg of DNA failed to protect. The protection elicited by the HA DNA vaccine correlated with the titers of neutralizing antibody.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Vacunas contra la Influenza/inmunología , Nucleoproteínas/genética , Proteínas de Unión al ARN , Vacunas de ADN/inmunología , Proteínas del Núcleo Viral/genética , Animales , Anticuerpos Antivirales/sangre , Biolística , Femenino , Inmunización , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
Reovirus type 3 Dearing (T3D) causes a prominent neutrophil influx, substantially greater than seen with reovirus type 1 Lang (T1L) in a rat model of viral pneumonia. We sought to measure reovirus-mediated increases in chemokine mRNA expression in pulmonary cells. We found that the neutrophilia induced by T1L and T3D infection in vivo correlated directly with increased levels of chemokine mRNA expression in T3D-infected compared with those of T1IL-infected lungs. In vitro, reovirus-infected normal alveolar macrophages (AMs) and the rat AM cell line NR8383 expressed greater levels of macrophage inflammatory protein 2, KC, and tumor necrosis factor alpha mRNA. A synergism between reovirus and lipopolysaccharide was also detected for macrophage inflammatory protein 2 and KC mRNA expression. Tumor necrosis factor protein secretion was also increased to a greater extent by T3D than by T1L in primary rat AMs and the NR8383 cells. We conclude that the virus-mediated inflammatory cytokine induction suggests a role for these cytokines in the neutrophil influx observed in the rat reovirus pneumonia model.
Asunto(s)
Citocinas/biosíntesis , Neumonía Viral/virología , ARN Mensajero/biosíntesis , Reoviridae , Animales , Células Cultivadas , Citocinas/genética , Femenino , Neutrófilos/metabolismo , Neutrófilos/patología , Neumonía Viral/metabolismo , Neumonía Viral/patología , Ratas , Ratas Sprague-Dawley , Reoviridae/clasificación , SerotipificaciónRESUMEN
We undertook the present study to elucidate the pathogenesis of the pathologic response to reovirus infection in the lungs and further understand the interactions of reoviruses with pulmonary cells. We found that reoviruses were capable of causing acute pneumonia in 25- to 28-day-old Sprague-Dawley rats following intratracheal inoculation with the reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The onset of the pneumonia was rapid, marked by type I alveolar epithelial cell degeneration, type II alveolar epithelial cell hyperplasia, and the infiltration of leukocytes into the alveolar spaces. More neutrophils were recruited into the lungs during T3D infection than during T1L infection, and the serotype difference in the neutrophil response was mapped to the S1 gene of reovirus. Viral replication in the lungs was required for the development of pneumonia due to T1L and T3D infections, and replication occurred in type I alveolar epithelial cells. T1L grew to higher titers in the lungs than did either T3D or type 3 clone 9, and the S1 gene was found to play a role in determining the level of viral replication. We propose that experimental reovirus infection in the lungs can serve as a model for the pathogenesis of viral pneumonia in which pulmonary inflammation results following direct infection of lung epithelial cells.
Asunto(s)
Proteínas de la Cápside , Orthoreovirus Mamífero 3/fisiología , Orthoreovirus/fisiología , Neumonía Viral/virología , Proteínas de Unión al ARN , Infecciones por Reoviridae/virología , Animales , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , Genes Virales , Macrófagos Alveolares/virología , Orthoreovirus Mamífero 3/inmunología , Neutrófilos/metabolismo , Orthoreovirus/inmunología , Neumonía Viral/inmunología , Neumonía Viral/patología , Ratas , Ratas Sprague-Dawley , Virus Reordenados/inmunología , Virus Reordenados/fisiología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/patología , Proteínas Virales/genética , Proteínas Virales/fisiologíaRESUMEN
Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.
Asunto(s)
Linfocitos B/inmunología , ADN Viral/inmunología , Hemaglutininas Virales/genética , Virus de la Influenza A/inmunología , Vacunas contra la Influenza , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Formación de Anticuerpos , Médula Ósea/inmunología , Linfocitos T CD4-Positivos , ADN Viral/metabolismo , ADN Viral/toxicidad , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/biosíntesis , Hemaglutininas Virales/inmunología , Inmunización Secundaria , Memoria Inmunológica , Vacunas contra la Influenza/toxicidad , Dosificación Letal Mediana , Ganglios Linfáticos/inmunología , Depleción Linfocítica , Ratones , Bazo/inmunología , Factores de TiempoRESUMEN
Many microorganisms gain access to the systemic circulation after entering the respiratory tract. The precise pathways used to cross the mucosal barriers of the lungs have not been clearly described. We have used the mammalian reoviruses in order to determine the pathway that a systemic virus uses to penetrate the mucosal barrier and enter the systemic circulation after entering the airways of the lungs. Reoviruses enter through pulmonary M cells, which overlie bronchus-associated lymphoid tissue, and subsequently spread to regional lymph nodes. Thus, the pathway through M cells represents a strategy by which viruses and probably other microorganisms can penetrate the mucosal surface of the respiratory tract and thereby enter the systemic circulation.
Asunto(s)
Pulmón/microbiología , Reoviridae/patogenicidad , Animales , Pulmón/citología , Tejido Linfoide/microbiología , Ratas , Ratas Sprague-DawleyRESUMEN
Human colon adenocarcinoma cells, treated with deoxycholate for 24 h prior to exposure to 1 mM butyrate, exhibited dose-dependent increases in the activities of three markers of colonic differentiation (alkaline phosphatase, lactase and CEA). Treatment with deoxycholate alone, for 24 h or longer, did not increase the secretion of CEA or the activities of either of the brush border-associated enzyme activities. Increases in differentiation markers were found to be bile acid-specific. Pretreatment with either dehydrocholic acid or cholic acid, even at cytotoxic concentrations, led to no significant butyrate-induced increases in brush-border associated hydrolase activities. The addition of a bacterial superoxide dismutase decreased the short-term cytotoxicity of deoxycholate and increased the maturation-potentiating effects of the bile acid in HCT-116 DO cells. The results of these studies demonstrate that bile acids, which are commonly thought to have tumor promoting activities in vivo, may also have physiological effects which serve to limit carcinogenic processes in the human colon by potentiating tumor cell differentiation.
Asunto(s)
Adenocarcinoma/patología , Fosfatasa Alcalina/biosíntesis , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Ácido Desoxicólico/farmacología , beta-Galactosidasa/biosíntesis , Adenocarcinoma/inducido químicamente , Adenocarcinoma/enzimología , Ácido Butírico , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/enzimología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inducción Enzimática/efectos de los fármacos , Humanos , Lactasa , Superóxido Dismutasa/farmacologíaRESUMEN
In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Proteína Quinasa C/genética , Acetato de Tetradecanoilforbol/farmacología , Animales , Northern Blotting , Western Blotting , Catálisis , Cromatografía Liquida , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Fenotipo , Poli A/metabolismo , Proteína Quinasa C/metabolismo , ARN/metabolismo , ARN Mensajero/genética , Ratas , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Several clonal sublines of HCT-116 human colon adenocarcinoma cells were isolated and characterized on the basis of their growth characteristics, intrinsic enterocyte-like differentiation (as assessed by alkaline phosphatase and lactase activities), and responses to butyrate, an inducer of colon tumor cell maturation. The HCT-116 sublines were found to be heterogeneous and several phenotypically distinct clones were identified. Further characterization of these clones indicated that the effects of butyrate on cell growth, alkaline phosphatase activity, and lactase activity were distinct and separable. The growth of all of the clones were inhibited by butyrate (IC50 values varied from 0.44 to 1.5 mM), but the effects of this agent on alkaline phosphatase and lactase activities varied widely. In several sublines butyrate had no effect on either enzyme while in others one or both activities were induced. Additionally, the binding of 125I-epidermal growth factor (EGF) to cell surface receptors was found to be proportional to the expression of lactase activity in the cell. The D3 clone and other sublines with intrinsic lactase activities greater than 100 nmol/mg/min expressed a class of high-affinity EGF receptors (e.g., D3 cells had 3.48 X 10(4) EGF receptors/cell with a kd of 0.61 nM). Other clones with less lactase activity had undetectable levels of 125I-EGF binding. In clones which exhibited greater than twofold increases in lactase activity in response to butyrate, the expression of a large number of low-affinity EGF receptors was also induced. In one such clone, the P1 subline, lactase activity was increased from 70 nmol/mg/min to 230 nmol/mg/min after 96 h in 2 mM butyrate, and the expression of EGF receptors was increased from undetectable levels to 1.18 X 10(5) EGF receptors/cell (kd of 3.2 nM). Northern blot analysis indicated that the increased 125I-EGF binding after butyrate treatment may have been due, in part, to a greater than twofold accumulation of EGF receptor mRNA. In addition, the expression of the messages for transforming growth factor alpha (TGF-alpha) and transforming growth factor beta (TGF-beta) was examined in butyrate-treated cells. While TGF-alpha mRNA levels were found to correlate with EGF receptor message levels in the HCT-116 clones, TGF-beta mRNA expression was not found to correlate with the butyrate-induced growth inhibition or with increases in EGF receptor expression, alkaline phosphatase activity, or lactase activity in these cells.
Asunto(s)
Butiratos/farmacología , Neoplasias del Colon/metabolismo , Receptores ErbB/biosíntesis , Fosfatasa Alcalina/metabolismo , Ácido Butírico , Diferenciación Celular/efectos de los fármacos , División Celular , Células Clonales , Neoplasias del Colon/enzimología , Receptores ErbB/genética , Humanos , Poli A/análisis , ARN Mensajero/análisis , Factores de Crecimiento Transformadores/biosíntesis , Factores de Crecimiento Transformadores/genética , Células Tumorales Cultivadas , beta-Galactosidasa/metabolismoRESUMEN
There is significant evidence to suggest that protein kinase C and DNA topoisomerases are functionally linked in signal transduction pathways. Much of this is based on the observation that phosphorylation of topoisomerase II by protein kinase C may lead to its activation in vitro and that inhibitors of topoisomerase II block phorbol diester-induced differentiation in HL-60 cells. In the present study, the activities of the DNA topoisomerases I and II have been quantitated to examine their regulation in phorbol diester-treated HL-60 cells undergoing differentiation. The activity of topoisomerase I increased rapidly after treatment with phorbol myristate acetate (PMA); it increased maximally (150% of control activity) at 3 hr post-treatment and remained elevated for at least 24 hr. Conversely, from the onset of exposure to PMA through 12 hr, there was no measurable alteration in topoisomerase II activity in PMA-treated cells. Moreover, there was a measurable decrease in topoisomerase II activity at the later time points, a result that occurred concomitantly with the loss of proliferative potential in differentiating HL-60 cells. Similar results were obtained when the activities of both enzymes were measured in nuclear extracts. The apparent increase in topoisomerase I activity was not due to an increase in the mass of the enzyme after PMA treatment, as measured by both western blotting and by the formation of camptothecin-dependent, topoisomerase I-DNA complexes. Taken together, these data suggest that the activities of the topoisomerases I and II may have been regulated independently in PMA-treated HL-60 cells, that the activity of topoisomerase II was not increased under conditions in which protein kinase C was activated in vivo, and that an increase in the activity of topoisomerase I may have had a role in the mechanism through which HL-60 cells underwent monocytic maturation in response to phorbol diesters.