RESUMEN

DNA damage and ageing share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. "Omics" technologies have permitted large-scale parallel measurements covering global cellular constituents and aided the identification of specific response pathways that change during ageing and after DNA damage. We have set out to identify genes with highly conserved response patterns through meta-analysis of mRNA expression datasets collected during natural ageing and accelerated ageing caused by a Transcription-Coupled Nucleotide Excision Repair (TC-NER) defect in a diverse set of organs and tissues in mice, and from in vitro UV-induced DNA damage in a variety of murine cells. The identified set of genes that show similar expression patterns in response to organ ageing (accelerated and normal), and endogenously and exogenously induced DNA damage, consists of genes involved in anti-oxidant systems and includes the transcription factor Bach2 as one of the most consistent markers. BACH2 was originally identified as a partner of the small Maf proteins and antagonist of the NRF2 anti-oxidant defence pathway and has been implicated in B-cell differentiation and immune system homeostasis. Although BACH2 has never before been associated with UV-induced damage or ageing, it shows a strong downregulation in both conditions. We have characterized the dynamics of Bach2 expression in response to DNA damage and show that it is a highly sensitive responder to transcription-blocking DNA lesions. Gene expression profiling using Affymetrix microarray analysis after siRNA-mediated silencing of Bach2 identified cell cycle and transcription regulation as the most significantly altered processes consistent with a function as transcription factor affecting proliferation.

Asunto(s)

Envejecimiento/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Daño del ADN/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Biomarcadores , Supervivencia Celular/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Modelos Animales , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Radiación Ionizante , Rayos Ultravioleta

RESUMEN

Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and numerous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our data indicate that at least two defects are responsible for the decreased c-fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the TCF, Elk-1. In nuclei isolated from serum stimulated senescent cells the activating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the members of the extracellular-regulated kinase (ERK) family was strikingly diminished and correlated with a decrease in the abundance of activated ERK proteins. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consistently exhibited higher ERK protein abundance. Thus, the proportion of phosphorylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphorylated (active) MEK. These data indicate that in senescent cells there is a general dysregulation in the ERK signaling pathway, which results in the accumulation of inactive ERK molecules, decreased abundance of active ERK in the nucleus of senescent cells, and subsequent lack of activation of the transcription factor TCF(Elk-1). These impairments, together with the impaired DNA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dependent upon this pathway.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Factores de Transcripción , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Senescencia Celular , ADN/metabolismo , Humanos , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Microscopía Confocal , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Fenotipo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Respuesta Sérica , Factores de Tiempo , Transcripción Genética , Proteína Elk-1 con Dominio ets

RESUMEN

The EPC-1 (early population doubling level cDNA-1) gene, also known as pigment epithelium-derived factor, encodes a protein belonging to the serine protease inhibitor (serpin) superfamily that has been reported to inhibit angiogenesis and proliferation of several cell types. We have previously reported that the EPC-1 mRNA and the secreted EPC-1 protein are expressed at levels more than 100-fold higher in early passage, G(0), WI-38 cells compared to either proliferating or senescent WI-38 fibroblasts. To examine the molecular mechanisms that regulate changes in EPC-1 gene expression in WI-38 cells, we isolated and characterized the human EPC-1 gene and determined the mRNA cap site. Transcriptional assays showed no change in the transcription rates of EPC-1 between young proliferating, quiescent, and senescent WI-38 cells. These results suggest posttranscriptional regulation of the EPC-1 gene. Reverse transcriptase polymerase chain reaction measurements (of hnRNA) indicate regulation at the hnRNA level. The regulation of the EPC-1 gene at the level of hnRNA can explain the observed slow increase in the steady-state EPC-1 mRNA levels when cells become quiescent. The reduction of EPC-1 mRNA levels that occurs when cells exit G(0) and are induced to proliferate can be accounted for by a reduction of the EPC-1 mRNA stability in stimulated cells as compared to quiescent cells.

Asunto(s)

Proteínas del Ojo , Fibroblastos/efectos de los fármacos , Factores de Crecimiento Nervioso , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Serpinas/biosíntesis , Animales , Northern Blotting , Bovinos , División Celular , Línea Celular , Senescencia Celular , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero , Sangre Fetal/fisiología , Fibroblastos/metabolismo , Genes fos , Humanos , Regiones Promotoras Genéticas , Proteínas/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Nuclear Heterogéneo/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serpinas/genética

RESUMEN

The TCL1 oncogene at 14q32.1 is involved in the development of human mature T-cell leukemia. The mechanism of action of Tcl1 is unknown. Because the virus containing the v-akt oncogene causes T-cell lymphoma in mice and Akt is a key player in transduction of antiapoptotic and proliferative signals in T-cells, we investigated whether Akt and Tcl1 function in the same pathway. Coimmunoprecipitation experiments showed that endogenous Akt1 and Tcl1 physically interact in the T-cell leukemia cell line SupT11; both proteins also interact when cotransfected into 293 cells. Using several AKT1 constructs in cotransfection experiments, we determined that this interaction occurs through the pleckstrin homology domain of the Akt1 protein. We further demonstrated that, in 293 cells transfected with TCL1, the endogenous Akt1 bound to Tcl1 is 5-10 times more active compared with Akt1 not bound to Tcl1. The intracellular localization of Tcl1 and Akt1 in mouse fibroblasts was investigated by immunofluorescence. When transfected alone, Akt1 was found only in cytoplasm whereas Tcl1 was localized in the cytoplasm and in the nucleus. Interestingly, Akt1 was also found in the nucleus when AKT1 was cotransfected with TCL1, suggesting that Tcl1 promotes the transport of Akt1 to the nucleus. These findings were supported by the intracellular localization of Akt1 or Tcl1 when Tcl1 or Akt1, respectively, were confined to the specific cellular compartments. Thus, we demonstrate that Tcl1 is a cofactor of Akt1 that enhances Akt1 kinase activity and promotes its nuclear transport.

Asunto(s)

Núcleo Celular/enzimología , Proteínas de Unión al ADN/fisiología , Proteínas Proto-Oncogénicas , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de Transcripción/fisiología , Animales , Transporte Biológico , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratones , Proteína Oncogénica v-akt , Fosforilación , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética

RESUMEN

Reactive oxygen species are produced by all aerobic cells and are widely believed to play a pivotal role in aging as well as a number of degenerative diseases. The consequences of the generation of oxidants in cells does not appear to be limited to promotion of deleterious effects. Alterations in oxidative metabolism have long been known to occur during differentiation and development. Experimental perturbations in cellular redox state have been shown to exert a strong impact on these processes. The discovery of specific genes and pathways affected by oxidants led to the hypothesis that reactive oxygen species serve as subcellular messengers in gene regulatory and signal transduction pathways. Additionally, antioxidants can activate numerous genes and pathways. The burgeoning growth in the number of pathways shown to be dependent on oxidation or antioxidation has accelerated during the last decade. In the discussion presented here, we provide a tabular summary of many of the redox effects on gene expression and signaling pathways that are currently known to exist.

Asunto(s)

Regulación de la Expresión Génica , Estrés Oxidativo , Envejecimiento , Animales , Enfermedad , Humanos , Especies Reactivas de Oxígeno/fisiología

RESUMEN

The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H2O2 generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed.

Asunto(s)

Antioxidantes/metabolismo , Senescencia Celular/fisiología , Transporte de Electrón/fisiología , Pulmón/citología , Acridinas , Catalasa/genética , Catalasa/metabolismo , Células Cultivadas , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Feto/citología , Feto/enzimología , Fibroblastos/citología , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Glutatión/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Mediciones Luminiscentes , Pulmón/embriología , Pulmón/metabolismo , Mitocondrias/enzimología , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , ARN Mensajero/análisis , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1

RESUMEN

EPC-1 (early population doubling level cDNA-1) is a quiescence-specific gene expressed at high levels by early passage WI-38 fibroblasts under conditions of either density-dependent growth arrest or serum deprivation. Late passage WI-38 cells lose the ability to express EPC-1 under all conditions tested. The decline in EPC-1 mRNA is gradual during the replicative life span and correlates inversely with the population doubling level (PDL) of the cells. The objective of this study was to determine whether the decline in EPC-7 mRNA abundance observed during proliferative senescence also occurs in cultures derived from donors of different ages. To address this question, we examined the abundance of EPC-1 mRNA in 28 skin fibroblast lines established from healthy donors of different ages ranging from 12 fetal weeks to 94 years. EPC-1 expression was measured, under conditions of growth arrest, prior to the end of the replicative life span of the cultures. Despite some variability in steady-state transcript levels among the cell lines, EPC-1 expression was significantly lower in cells derived from the fetal donor group (12-20 gestational weeks) than in cells derived from adult donors. An in vitro age-dependent decline in EPC-1 expression was observed in all the skin lines examined, independent of donor age; however, no significant difference was observed between the young adult donor group (17-33 years) and the old adult donor group (78-94 years). Thus, expression of EPC-1 is linked to the replicative age of the cells and whether the cells are derived from fetal skin or adult skin. In adults, EPC-1 expression is independent of donor age.

Asunto(s)

Envejecimiento/fisiología , Senescencia Celular/fisiología , Proteínas del Ojo , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Factores de Crecimiento Nervioso , Biosíntesis de Proteínas , Serpinas/biosíntesis , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Biomarcadores , Northern Blotting , Células Cultivadas , Niño , Preescolar , Replicación del ADN , Dermis/citología , Femenino , Feto/citología , Fibroblastos/metabolismo , Edad Gestacional , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Proteínas/genética , ARN Mensajero/biosíntesis , Serpinas/genética , Donantes de Tejidos

RESUMEN

We have examined the effects of three structurally distinct antioxidants (N-acetylcysteine [NAC], Trolox C [a water-soluble vitamin E derivative], and nordihydroguaiaretic acid [NGA]) on the expression of the c-fos gene over a 2-hour period. Determination of cellular glutathione concentration (the primary determinant of the cellular redox state) over the same time-course verifies that all the compounds studied cause an increase in cellular reduction potential. The level of c-fos messenger RNA increased rapidly in response to micromolar concentrations of these compounds, reaching a peak in 30-60 minutes. Induction of c-fos expression by these antioxidants is at least partly due to an increase in transcription, as determined by nuclear run-on assay. Down regulation of protein kinase C (PKC) by pretreatment for 24 hours with 500 nm PMA prevents induction by subsequent stimulation with either PMA or NGA. NAC induction of c-fos is unaffected by PMA pretreatment, while Trolox C superinduced c-fos following PMA pretreatment. None of these treatments stimulated translocation of PKC-alpha from the cytosol to the membrane. These results suggest that increasing the intracellular reducing potential induces c-fos expression through multiple pathways.

Asunto(s)

Antioxidantes/farmacología , Pulmón/citología , Proteínas Proto-Oncogénicas c-fos/genética , Activación Transcripcional/efectos de los fármacos , Acetilcisteína/farmacología , Northern Blotting , Carcinógenos/farmacología , Cromanos/farmacología , Inhibidores Enzimáticos/farmacología , Feto/enzimología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Humanos , Masoprocol/farmacología , Naftalenos/farmacología , Proteína Quinasa C/metabolismo , ARN Mensajero/análisis , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología

Asunto(s)

Senescencia Celular , Transducción de Señal , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Senescencia Celular/efectos de los fármacos , Senescencia Celular/fisiología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Sustancias de Crecimiento/farmacología , Humanos , Masoprocol/farmacología , Modelos Biológicos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

Normal human fibroblasts exhibit a limited proliferative potential in culture. When populations are serially subcultured, they grow well initially, but ultimately reach a stage when they are no longer able to proliferate in response to mitogenic stimuli. This state is designated "replicative senescence". In addition to failure to proliferate, numerous morphological and physiological changes characterize the senescent phenotype. Both stochastic and genetic mechanisms have been postulated as causal effectors of the aging process. However, the pathway leading to cellular senescence is likely to be complex with numerous changes. In this article we provide an overview of cell and molecular changes that occur during cell aging, with special emphasis on signal transduction pathways and cell cycle proteins that are likely to play key roles in determining the limited replicative life span and the changes that occur.

Asunto(s)

Senescencia Celular/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Regulación de la Expresión Génica/fisiología , Animales , Senescencia Celular/fisiología , Regulación de la Expresión Génica/genética , Humanos

RESUMEN

The insulin-like growth factor 1 (IGF-1) receptor has been found to transform fibroblast cells when overexpressed. The removal of 108 aa from the C-terminus of the IGF-1 receptor abolishes the transforming ability of the receptor without affecting its ability to induce cell growth. The availability of this mutant receptor provides a means to examine the changes in gene expression which take place during transformation, solely in response to an increased number of IGF-1 receptors. Using differential display, we have examined differences in gene expression between cells expressing a wild-type, transforming IGF-1 receptor and cells expressing a C-terminally truncated, nontransforming IGF-1 receptor. We have cloned a novel 6. 3-kb cDNA transcript (DBI-1) which is expressed at much lower levels in cells containing the wild-type IGF-1 receptor. The predicted protein sequence of DBI-1 contains seven EGF-like repeats, which bear >90% sequence identity to the rat Notch 2 protein. The cDNA also contains a potential DEAD box in the C-terminal region. The DBI-1 message is detected at relatively high levels in cardiac tissue and at lower levels in lung, liver, and kidney. Antibodies generated to a unique region of the DBI-1 protein recognize a protein of 88 kDa, which is localized in the nucleus. Overexpression of DBI-1 in cells which contain the wild-type IGF-1 receptor diminishes the mitogenic response to IGF-1.

Asunto(s)

Genes/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Receptor IGF Tipo 1/genética , Receptores de Superficie Celular/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/química , Clonación Molecular , ARN Helicasas DEAD-box , ADN/biosíntesis , ADN Complementario/genética , Expresión Génica , Ratones , Mitógenos/farmacología , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Proteínas de Unión al ARN , Ratas , Receptor Notch2 , Receptores de Superficie Celular/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Proteínas Supresoras de Tumor

RESUMEN

The signal transduction cascade initiated by the activation of phosphoinositide 3-kinase (PI-3 kinase) is implicated in mitogenic and antiapoptotic signaling generated by growth factors in a variety of cell types. We have examined the consequences of an inhibition of this pathway in human diploid fibroblasts. We find that a specific PI-3 kinase inhibitor (LY294002) causes growth arrest in these cells accompanied by changes in gene expression that are similar to those seen during cellular senescence. A second inhibitor, PD58029, which is specific for the mitogen-activated protein kinase kinase 1 (MEK-1), also induces a growth arrest but does not induce the same spectrum of gene expression. The pattern of gene expression in the presence the MEK-1 inhibitor is similar to that seen during growth arrest induced by serum starvation. The specific phenotypic changes seen following inhibition of PI-3 kinase are: an increase in beta-galactosidase activity; a decrease in EPC-1 gene expression; and a dramatic increase in collagenase gene expression. Thus, growth arrest with a PI-3 kinase inhibitor induces a senescent-like phenotype that is not seen when cells are growth arrested by either serum starvation or a MEK-1 inhibitor.

Asunto(s)

Senescencia Celular/efectos de los fármacos , Cromonas/farmacología , Fibroblastos/efectos de los fármacos , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , División Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Inhibidores de Proteínas Quinasas , beta-Galactosidasa/antagonistas & inhibidores , beta-Galactosidasa/metabolismo

RESUMEN

We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochrome c oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of .O2- generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined.

Asunto(s)

Envejecimiento/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Desarrollo Embrionario y Fetal , NADH Deshidrogenasa/metabolismo , Piel/enzimología , Succinato Deshidrogenasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular , Niño , Transporte de Electrón , Feto , Fibroblastos/enzimología , Humanos , Recién Nacido , Sustancias Macromoleculares , ARN Mensajero/metabolismo , Piel/embriología , Piel/crecimiento & desarrollo

RESUMEN

The steady-state abundance of alpha 1(I) and alpha 1(III) procollagen mRNAs, p21Sdi1 mRNA, and beta-actin mRNA was determined in 29 skin fibroblast lines established from fetal, young and old donors. Donor ages ranged from 12 gestational weeks to nonagenarian. Adult donors were members of the Baltimore Longitudinal Study of Aging. The abundance of alpha 1(I) procollagen mRNA was decreased in cell lines from both young and old donors compared with fetal lines. Additionally, one alpha 1(I) transcript observed in the fetal lines was not detected in postnatal lines. The abundance of alpha 1(III) procollagen mRNA was decreased in postnatal lines from old donors compared with fetal lines. The abundance of beta-actin mRNA was lower in postnatal lines from both young and old donors compared to fetal lines. These results suggest that cultures of fetal skin fibroblasts exhibit a greater capacity for synthesis of procollagens and beta-actin than postnatal lines. In contrast, the abundance of p21Sdi1 mRNA was elevated in lines established from postnatal donors. These results are consistent with developmental changes in amounts of procollagen, beta-actin and p21.

Asunto(s)

Actinas/metabolismo , Envejecimiento/metabolismo , Ciclinas/metabolismo , Procolágeno/metabolismo , Actinas/genética , Adulto , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Células Epidérmicas , Epidermis/embriología , Epidermis/metabolismo , Feto/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Procolágeno/genética , ARN Mensajero/metabolismo

RESUMEN

We have examined the activities and mRNA abundance of two hydrogen peroxide metabolizing enzymes (glutathione peroxidase and catalase), glutathione concentration, and the activities of several enzymes that influence glutathione concentration, including glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PD), and gamma-glutamylcysteine synthetase (gamma-GCS), in 29 skin fibroblast lines derived from donors ranging in age from 14 gestational weeks to 94 years of age. H2O2 metabolizing enzyme activities and mRNA abundances were greater in skin fibroblast cultures established from postnatal donors than in fetally derived cultures. There were no significant differences in either of these parameters in cell lines established from postnatal donors of different ages. Total glutathione concentration decreased with age, but GR activity appeared to be unaffected by age. In order to estimate the ability of the cultures to produce NADPH (an important component of cellular redox status and a cofactor for GR), we determined glucose-6-phosphate dehydrogenase activity and mRNA abundance. We were unable to directly measure gamma-GCS activity or mRNA abundance in any of the skin lines or in fetal lung fibroblast; however, we were able to indirectly demonstrate the presence of this enzyme by stimulating fetal lung fibroblasts with H2O2 following treatment with L-buthionine-S,R-sulfoximine (BSO), an inhibitor of gamma-GCS activity. These results show that some, but not all, age-associated differences in antioxidant defense levels are maintained in a culture environment and are consistent with the hypothesis that developmental stages of life are associated with lower antioxidant defense levels than are present in postnatal phases of life.

Asunto(s)

Envejecimiento/metabolismo , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Piel/enzimología , Adolescente , Anciano , Anciano de 80 o más Años , Butionina Sulfoximina , Línea Celular , Feto/metabolismo , Fibroblastos , Regulación Enzimológica de la Expresión Génica , Glucosafosfato Deshidrogenasa/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Glutatión Reductasa/metabolismo , Humanos , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , ARN Mensajero/metabolismo

RESUMEN

The proto-oncogene c-fos (the cellular homolog of v-fos, Finkel-Biskis-Jenkins (FBJ) murine osteogenic sarcoma virus) encodes a major component of the activator protein-1 (AP-1) transcription factor. Serum stimulation as well as oxidizing treatments induce transitory increases in c-fos mRNA abundance. The induction of c-fos by serum stimulation is also known to decline during proliferative senesence. In this study, we examined the effects of two classes of antioxidants on the induction of c-fos in early and late passage human fetal lung fibroblasts (WI-38). N-acetyl cysteine (NAC) induces c-fos transcription in both early and late passage cells, while nordihydroguaiaretic acid (NGA) induced c-fos transcription in early passage cells but fails to stimulate it in late passage cells. Since we had previously observed an age-related decline in protein kinase C (PKC) translocation from the cytosol to the membrane, following its activation, and because PKC activation appears to be involved in the NGA induction of c-fos we examined the relative protein abundances of several PKC isoforms in early and late passage cells. Additionally, we examined the protein abundance of several members of the MAP kinase pathway which could play a role in c-fos induction by the PKC-dependent pathway. We were unable to detect PKC-beta or theta in early or late passage cells. Late passage cells contained a slightly greater abundance of PKC alpha, gamma and epsilon than cells at an early passage. No other differences in PKC isoforms or in members of the MAP kinase family were observed in early or late passage cells. These results clearly demonstrate that at least some pathways leading to c-fos induction remain intact in late passage cells. While we were unable to detect any decreases in PKC isoforms or MAP kinase proteins we cannot exclude the possibility that functional decrements accumulate in these proteins during senesence.

Asunto(s)

Envejecimiento/metabolismo , Antioxidantes/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Northern Blotting , Células Cultivadas , Cisteína/farmacología , Humanos , Proto-Oncogenes Mas