RESUMEN

The anti-oxidant enzyme system protects cellular macromolecules against damage from reactive oxygen species. One component of this system, manganese superoxide dismutase (MnSOD), has also been shown to display tumor suppressor gene-like activity. The purpose of this study was to examine changes in MnSOD expression during hamster cheek pouch carcinogenesis, and the effects of MnSOD overexpression using an adenoviral vector. Tumor induction was carried out using 7,12-dimethylbenz[alpha]anthracene. Animals were killed at periodic intervals, and cheek pouch tissues were excised and examined for MnSOD expression by immunohistochemistry and digital image analysis. We observed a reduction in MnSOD expression as early as 2 weeks after the start of carcinogen application. Low MnSOD expression persisted until the end of the 23-week experimental period. Solid hamster cheek pouch carcinoma xenografts were then established in nude mice. An adenoviral vector encoding the human MnSOD gene was delivered to the xenografts by direct injection. We observed high, immediate expression of MnSOD in the xenografts that persisted for 10 days following cessation of viral construct delivery. Delivery of the MnSOD construct resulted in a maximal 50% reduction in tumor growth compared with untreated controls. Our results suggest that MnSOD may be a tumor suppressor gene in the hamster cheek pouch model system.

Asunto(s)

Adenoviridae/genética , Depuradores de Radicales Libres/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Neoplasias de la Boca/enzimología , Superóxido Dismutasa/genética , 9,10-Dimetil-1,2-benzantraceno/efectos adversos , Análisis de Varianza , Animales , Anticarcinógenos/farmacología , Antioxidantes/farmacología , Carcinógenos/efectos adversos , Mejilla , Cricetinae , Depuradores de Radicales Libres/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Mesocricetus , Ratones , Ratones Desnudos , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Trasplante de Neoplasias , Superóxido Dismutasa/fisiología , Trasplante Heterólogo

RESUMEN

We hypothesized that the cytotoxic effect of GLA observed in glioma but not normal glial cells reflects differences in GLA metabolism and/or antioxidant enzyme levels between these cells. The PUFA content of unsupplemented glioma cells was approximately 50% of that seen in unsupplemented astrocytes. Supplementation with 20 microM GLA for 24 h led to a 230 and 22% increase in glioma and astrocyte PUFA content, respectively, such that both supplemented cell types contained similar levels of PUFA. No major differences were seen in terms of GLA metabolites retained in the cells or secreted into the media following incubation with [(3)H]-GLA. No significant differences were observed in activity of MnSOD or CuZn-SOD between the cells. However, CAT and GPx activity in the glioma cells was significantly higher and lower, respectively, than observed in normal astrocytes. GLA supplementation resulted in a significant increase in CAT activity in normal astrocytes; glioma CAT activity was unchanged. No significant change was seen in the other antioxidant enzymes following GLA supplementation. These results suggest that the cytotoxic effect of GLA on glioma cells reflects both increased PUFA content and an inability to upregulate CAT.

Asunto(s)

Antioxidantes/metabolismo , Glioma/enzimología , Ácido gammalinolénico/toxicidad , Animales , Astrocitos/metabolismo , Radioisótopos de Carbono/metabolismo , Catalasa/biosíntesis , Catalasa/metabolismo , Catalasa/fisiología , Medios de Cultivo Condicionados , Ácidos Grasos Insaturados/análisis , Glioma/metabolismo , Glutatión Reductasa/biosíntesis , Glutatión Reductasa/metabolismo , Glutatión Reductasa/fisiología , Metabolismo de los Lípidos , Ratas , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/fisiología , Células Tumorales Cultivadas , Ácido gammalinolénico/metabolismo

RESUMEN

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).

Asunto(s)

Apoptosis/efectos de los fármacos , Células HL-60/metabolismo , Células HL-60/patología , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Peroxidasa/metabolismo , Activación Enzimática , Humanos , Células K562

RESUMEN

The 17-kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication-defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double-stranded oligonucleotide probe containing the consensus binding sequence for NF-kappaB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF-kappaB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen-activated protein kinase inhibitor SB-203580 also suppressed the GMF-activated NF-kappaB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate activated NF-kappaB whether or not GMF was overexpressed. Along with NF-kappaB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF-kappaB nuclear translocation was blocked by its specific decoy DNA, implicating NF-kappaB as an upstream mediator of this antioxidant enzyme. The p38 inhibitor SB-203580 also blocked the GMF-activated SOD. As NF-kappaB and SOD are both pro-survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.

Asunto(s)

Factor de Maduración de la Glia/fisiología , FN-kappa B/fisiología , Animales , Catalasa/genética , Catalasa/fisiología , Factor de Maduración de la Glia/genética , Glioma/metabolismo , Humanos , FN-kappa B/efectos de los fármacos , Ácido Pirúvico/farmacología , ARN Mensajero/metabolismo , Ratas , Superóxido Dismutasa/genética , Transfección , Células Tumorales Cultivadas

RESUMEN

The p16 INK4A tumor suppressor gene participates in establishing and maintaining the malignant phenotype of a variety of cancer cell lines and primary tumors. Recently it has been observed that p16 expression is lost in oral cavity cancer cell lines in the presence of a normal intact gene. To examine the role of DNA methylation as an explanation for these findings, we analyzed the DNA methylation patterns of the p16 INK4A promoter in DNA isolated from primary cultures of normal human oral keratinocytes and squamous cell carcinoma (SCC-15) oral cancer cells using bisulfite genomic sequencing. Our results demonstrated striking differences in the methylation status of the 5' CpG island of the p16 gene between normal and cancer cells. Normal human oral keratinocytes showed practically no methylation of the p16 INK4A promoter, while SCC-15 oral cancer cells showed almost complete methylation in this region. These data implicate DNA methylation as a mechanism for transcriptional silencing of the p16 INK4A gene in oral cancer cells.

Asunto(s)

Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN , Genes p16 , Neoplasias de la Boca/genética , Humanos , Queratinocitos/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Reacción en Cadena de la Polimerasa/métodos , Células Tumorales Cultivadas

RESUMEN

In order to evaluate the intracellular function of glia maturation factor (GMF), we overexpressed GMF in C6 rat glioma cells using two methods: stable transfection using the pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation as shown by the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls, and express the mature astrocytic marker glial fibrillary acidic protein. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent superoxide dismutase (CuZnSOD) activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an induction of the enzyme. In view of recent findings that reactive oxygen species (ROS) and the antioxidant enzymes are intricately involved in key physiologic processes such as proliferation, differentiation and apoptosis, the study raises the possibility that CuZnSOD may be a mediator of GMF function.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/fisiología , Glioma/metabolismo , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Superóxido Dismutasa/metabolismo , Adenovirus Humanos/genética , Animales , Factor de Maduración de la Glia , Humanos , Ratones , Ratones Desnudos , Ratas , Transfección , Células Tumorales Cultivadas

RESUMEN

Liver transplantation is the only therapeutic strategy for many inherited and acquired diseases. The formation of reactive oxygen species following ischemia/reperfusion is a cause of hepatocellular injury during transplantation. This report describes the therapeutic application of mitochondrial superoxide dismutase gene transfer to the liver for acute ischemia/reperfusion injury. Recombinant adenoviral expression of mitochondrial superoxide dismutase in mouse liver prior to lobar ischemia/reperfusion significantly reduced acute liver damage and associated redox activation of both NF-kappaB and AP1. These immediate early transcription factors represent common pathways by which cells respond to environmental stress. This work provides the foundation for redox-mediated gene therapies directed at ameliorating ischemia/reperfusion injury and associated acute rejection in orthotopic liver transplantation.

Asunto(s)

Terapia Genética , Hígado/irrigación sanguínea , FN-kappa B/metabolismo , Daño por Reperfusión/terapia , Superóxido Dismutasa/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción , Animales , Expresión Génica/genética , Hígado/enzimología , Masculino , Ratones , Ratones Desnudos , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/genética , FN-kappa B/antagonistas & inhibidores , Oxidación-Reducción , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/genética , Daño por Reperfusión/genética , Superóxido Dismutasa/análisis , Superóxido Dismutasa/fisiología , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción ReIB , Activación Transcripcional/genética , Activación Transcripcional/fisiología , Transgenes/genética

RESUMEN

This study examined the effect of ambient temperature on perirenal adipose tissue development and thermoregulation over the first month of postnatal life in lambs treated with a drug that reduces thyroid hormone synthesis (methimazole; at a dose of 50 mg day-1 (kg body weight)-1). Twin lambs were hand-reared at a fixed level of nutrition in either a warm (WR; 25 degrees C) or cool (CR; 10-15 degrees C) ambient temperature. Oxygen consumption, heart rate and colonic temperature were measured during non-rapid eye movement sleep in different ambient temperatures (6, 15, 25 and 34-36 degrees C) at 7, 14 and 28 days of age. Plasma thyroid hormone concentrations decreased with postnatal age and were higher in CR than in WR lambs. All lambs increased plasma thyroid hormone concentrations and O2 consumption during cold exposure but this was associated with a mean increase in colonic temperature in WR lambs at 7 or 28 days. Colonic temperature increased with the onset of panting at all ages when lambs were exposed to 34-36 degrees C, a response that was greatest at 28 days in CR lambs. An increase in colonic temperature with age was observed in CR but not WR lambs. Heart rate declined with age only in the WR group. At 8 and 29 days there were no significant differences between WR and CR groups in the occurrence of shivering or in the thermogenic activity (i.e. GDP binding to mitochondria) of perirenal adipose tissue. At 29 days WR lambs possessed more adipose tissue with a higher lipid and DNA content. It is concluded that modest changes in rearing temperature can have a large influence on the control of body temperature and thyroid hormone response to methimazole treatment. CR lambs are able to maintain higher plasma thyroid hormone concentrations and exhibit improved thermoregulation compared with WR lambs without any detectable differences in brown adipose tissue function.

Asunto(s)

Tejido Adiposo/crecimiento & desarrollo , Animales Recién Nacidos/crecimiento & desarrollo , Antitiroideos/farmacología , Regulación de la Temperatura Corporal/efectos de los fármacos , Metimazol/farmacología , Ovinos/crecimiento & desarrollo , Temperatura , Animales , Peso Corporal , Sueño REM , Hormonas Tiroideas/sangre

RESUMEN

This study examined the effects of modest changes in ambient temperature in hand-reared lambs (experiment one) and in ewe-reared lambs (experiment two). Lambs were killed at either 8 or 31 days of age and perirenal adipose tissue was identified as being brown adipose tissue (BAT) from measurements of thermogenic activity (i.e. GDP binding to uncoupling protein in isolated mitochondria) or thermogenic capacity (i.e. detection of uncoupling protein by immunoblotting). In addition, type I and II iodothyronine 5' monodeiodonase (5'MDI) activities were assayed in perirenal adipose tissue, plus type I 5'MDI activity in liver. Plasma samples were also taken for measurements of glucose, lactate, insulin, triiodothyronine (T3) and thyroxine (T4) concentrations. In experiment one, lambs were hand-reared at either warm (WR; 25 degrees C) or cool (CR; 10-15 degrees C) ambient temperatures. Mean growth rate over the first 8 days of life in CR lambs was 88 g/day and increased to 128 g/day over the first month of life. Growth rate in WR lambs was constant at 141 g/day. Thermogenic activity of BAT was significantly higher in CR than WR lambs, but total weight and tissue lipid content of perirenal adipose tissue were significantly lower in the CR group. In both WR and CR lambs, the thermogenic activity of BAT fell by an average of 71% between 8 and 31 days. At 31 days of age, uncoupling protein in mitochondria could be detected only by immunoblotting in adipose tissue sampled from CR lambs. There was no effect of ambient temperature on type I or type II 5'MDI activity in BAT or liver; it decreased in adipose but not liver tissue between 8 and 31 days of age. The plasma concentrations of glucose, insulin and T3 tended to decline with age in CR but not in WR lambs. In ewe-reared lambs perirenal adipose tissue weight and tissue lipid content more than doubled between 8 and 31 days of age, but the level of GDP binding decreased from 85 to 5 pmol/mg mitochondrial protein over this period. Liver weight increased by 55% between 8 and 31 days of age, but hepatic 5'MDI activity remained unchanged. The plasma concentrations of T3, T4 and lactate, but not glucose or insulin, increased between 8 and 31 days of age. It is concluded that hand-rearing lambs at a cool ambient temperature significantly delays postnatal development, to the extent that BAT characteristics are retained. Ewe-rearing lambs enhances the rate at which BAT adopts the characteristics of white adipose tissue, and it prevents the postnatal decline in plasma concentrations of thyroid hormones.

Asunto(s)

Tejido Adiposo Pardo/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Conducta Materna/fisiología , Animales , Glucemia/metabolismo , Hormonas/sangre , Yoduro Peroxidasa/metabolismo , Riñón/crecimiento & desarrollo , Ácido Láctico/metabolismo , Ovinos , Temperatura , Aumento de Peso/fisiología

RESUMEN

This study examines the effect of hand-rearing developing lambs in a warm (WR; 25 degrees C) or cool (CR; 10-15 degrees C) ambient temperature on the control of thermoregulation and breathing patterns, when maintained at a fixed level of nutrition over the first month of postnatal life. Measurements were made during non-rapid eye movement sleep whilst lambs were maintained for at least 1 h at warm (28-19 degrees C) and cold (14-5 degrees C) ambient temperatures at 1, 7, 14 and 30 days of age. All lambs were able to maintain normal body temperature, but oxygen consumption was higher in CR lambs at 14 and 30 days of age. At 1 day of age shivering was rarely observed in any lambs, but at 7 and 14 days of age more WR than CR lambs responded to cold exposure via shivering. Plasma concentrations of triiodothyronine were higher at 7 and 14 days of age in CR lambs. Breathing frequencies were similar in WR and CR lambs, and from 7 days of age the incidence of expiratory laryngeal braking was higher in warm compared with cold study temperatures. By 30 days of age the recruitment of this mechanism was greater in CR lambs. Mean growth rate was slower over the first week of postnatal life in CR compared with WR lambs. This difference decreased over the first month of life, as growth rate increased from 83 to 130 g day-1 in the CR group but remained constant at approximately 150 g day-1 in the WR lambs. Total weight of the lungs and heart, but not the liver, were lower at 1 month but not at 1 week of postnatal life in CR lambs. It is concluded that a modest decrease in the ambient temperature in which postnatal lambs are reared, when on a fixed feed intake, alters lung size, the recruitment of laryngeal braking and the control of body temperature.

Asunto(s)

Animales Recién Nacidos/fisiología , Regulación de la Temperatura Corporal/fisiología , Mecánica Respiratoria/fisiología , Ovinos/fisiología , Temperatura , Animales , Dióxido de Carbono/metabolismo , Dieta , Crecimiento/fisiología , Hígado/crecimiento & desarrollo , Hígado/fisiología , Pulmón/crecimiento & desarrollo , Pulmón/fisiología , Tamaño de los Órganos/fisiología , Consumo de Oxígeno/fisiología , Glándula Tiroides/crecimiento & desarrollo , Glándula Tiroides/fisiología , Hormonas Tiroideas/sangre

RESUMEN

We examined the effect of delivering near-term twin lambs by cesarean section into a warm (30 degrees C) or cool (15 degrees C) ambient temperature on the control of thermoregulation. Heat production was measured 20-30 h after birth during non-rapid-eye-movement sleep at 29 and 14 degrees C. At 29 degrees C there was no difference in heat production between groups, but at 14 degrees C cool-delivered (CD) lambs exhibited a 62% greater metabolic response. Irrespective of delivery temperature, 15 of the 18 lambs used shivering thermogenesis during cold exposure, indicating a reduction in the ability to use nonshivering thermogenesis in brown adipose tissue (BAT). Mean plasma concentrations of thyroxine and triiodothyronine were 35 and 45% greater, respectively, in CD lambs than in warm-delivered lambs. The level of guanosine 5'-diphosphate binding in BAT was lower than in normally delivered lambs and was not different between CD and warm-delivered lambs. Cesarean section delivery prevents the rise in BAT thermogenic activity, which results in an increased reliance on shivering thermogenesis to maintain colonic temperature. Under these conditions, delivery into a cool environment increases the plasma concentration of thyroid hormones, which benefits the neonate by enabling a greater thermogenic response via shivering.

Asunto(s)

Animales Recién Nacidos/fisiología , Regulación de la Temperatura Corporal/fisiología , Cesárea , Tejido Adiposo Pardo/metabolismo , Animales , Peso Corporal/fisiología , Dióxido de Carbono/sangre , Dieta , Femenino , Guanosina Difosfato/metabolismo , Yoduro Peroxidasa/metabolismo , Mitocondrias/enzimología , Mitocondrias/metabolismo , Consumo de Oxígeno/fisiología , Embarazo , Ovinos , Tiritona/fisiología , Sueño/fisiología , Temperatura , Hormonas Tiroideas/sangre

RESUMEN

1. This study examines the effect of chronic cold exposure during pregnancy, induced by winter shearing twin-bearing ewes 4 weeks before predicted lambing date, on O2 consumption and CO2 production during non-rapid-eye-movement (REM) sleep in lambs maintained for at least 1 h at warm (28-18 degrees C) and cold (14-5 degrees C) ambient temperatures at 1, 4, 14 and 30 days of age. This was combined with measurement of the thermogenic activity (GDP binding to uncoupling protein in mitochondrial preparations) of perirenal adipose tissue from lambs immediately after birth and at 33 days of age. 2. Lambs born from shorn (cold-exposed) ewes were 15% heavier (P < 0.01) and possessed 21% (P < 0.01) more perirenal adipose tissue that contained 40% more protein and mitochondrial protein than unshorn (P < 0.05) controls. Total GDP binding in perirenal adipose tissue was 40% greater (P < 0.05) in lambs born from shorn ewes but there was no difference in lipid content of this tissue between the two groups. 3. At 1 day of age, lambs born from shorn ewes exhibited a 16% higher (P < 0.05) rate of O2 consumption (per kilogram bodyweight) at the warm temperature and a 40% greater metabolic response to the cold ambient temperature. All lambs born from shorn ewes responded to cold exposure without shivering (i.e. via non-shivering thermogenesis) whilst shivering was measured in four out of seven lambs in the unshorn group. These differences had disappeared by 4 days of age as a result of a 25% increased (P < 0.01) rate of O2 consumption in the warm in lambs born from unshorn ewes and a 20% decrease (P < 0.05) in the response to the cold in lambs from shorn ewes. Shivering during cold exposure was measured in six out of nine lambs born from shorn ewes indicating a rapid alteration in thermoregulatory responses to cold during the first few days of life. 4. The levels of GDP binding and mitochondrial protein in perirenal adipose tissue fell by one-third in both groups of lambs during the first 33 days of life whereas lipid content either increased or was unchanged. This indicated that brown adipose tissue (BAT) was developing the characteristics of white adipose tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Tejido Adiposo/metabolismo , Regulación de la Temperatura Corporal/fisiología , Frío , Ovinos/fisiología , Animales , Animales Recién Nacidos , Temperatura Corporal , Peso Corporal , Femenino , Guanosina Difosfato/metabolismo , Consumo de Oxígeno , Embarazo , Efectos Tardíos de la Exposición Prenatal