RESUMEN
Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.
Asunto(s)
Apoptosis , Núcleo Celular/enzimología , Fragmentación del ADN , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/metabolismo , Retículo Endoplásmico/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Emparejamiento Base , Calcio/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Proteasas de Cisteína/metabolismo , Fragmentación del ADN/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Etopósido , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Ratones , Nucleosomas/efectos de los fármacos , Nucleosomas/metabolismo , Inhibidores de Proteasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Serina Proteasas/metabolismoRESUMEN
PARP-1, the best studied isoform and most abundantly expressed member of the PARP family of 18 proteins, catalyzes the poly(ADP-ribosyl)ation (PARylation) of various nuclear proteins and play key roles in DNA repair, genome maintenance, DNA replication, recombination, apoptosis, gene expression, and regulation of chromatin function. PARylation modulates the functions of target proteins, mainly PARP-1 itself. A multifunctional enzyme, PARP-1 has been localized within DNA replication, repair, recombination, and transcription complexes, and modifies and regulates the functions of specific components of these complexes. PARylation can regulate the activities of replicative enzymes, such as DNA polymerases α, δ, and ε, topo I and II, primase, RPA, and PCNA in isolated enzymes or within DNA replication complexes (DNA synthesome). PARP-1 and PARylation may (1) play dual roles in nuclear processes, depending on the levels of the substrate NAD and the presence of PARP-activating DNA breaks, (2) recruit acceptor proteins to certain sites or complexes through direct association or through binding to PAR and PAR-binding proteins, and (3) alters the nucleosomal structure of DNA by PARylation of nucleosomal proteins, such as histone H1 to destabilize higher order chromatin structures and promote access of DNA repair and replication enzymes as well as transcription factors to these sites. Here, we describe biochemical approaches that have been utilized in our laboratory for the purification and characterization of PARylated DNA replicative complexes. These methods can be modified for the purification of complexes involved in other nuclear processes. This chapter also briefly discusses current methods by which new PARylated complexes are being identified and studied. Identification, evaluation, and characterization of new complexes could aid in the elucidation of the molecular mechanisms by which PARylation and PARP mediates its pleiotropic roles in various nuclear processes.
Asunto(s)
ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Animales , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Ratones , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Subunidad p50 de NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Animales , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Subunidad p50 de NF-kappa B/genética , Proteínas Nucleares/genética , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Elementos de Respuesta/fisiología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
BACKGROUND: Phenoxodiol, a synthetic analog of Genistein, is being assessed in several clinical studies against a range of cancer types and was shown to have a good efficacy and safety profile. In this study we tested the effects of Phenoxodiol against prostate cancer cell lines. METHODS: Cell-cycle analysis, plasmatic membrane damage, clonogenic assay, comet assay, and Western blot methodologies were employed to assess the effects of Phenoxodiol on prostate cancer cell lines. An in vivo model confirmed the potential therapeutic efficacy of Phenoxodiol when administered orally to tumor bearing mice. RESULTS: Phenoxodiol treatment promoted a marked inhibition of proliferation and loss of colony formation in LNCaP cells in a dose- and time-dependent manner. Similar effects were also observed in the metastatic prostate cell lines PC3 and DU145. Activation of poly(ADP ribose) polymerase 1 (PARP-1) clearly indicates the induction of DNA damage by Phenoxodiol. Oral administration of Phenoxodiol induced a considerable growth inhibition of malignant tumors generated by inoculation of LNCaP cells into Balb/c nu/nu athymic mice. CONCLUSIONS: These data demonstrated that Phenoxodiol promotes apoptosis, as determined by PARP-1 degradation, via mitochondrial depolarization and G1/S cell-cycle arrest thereby confirming that it is active against androgen-dependent and independent prostate cancer cells. Although a precise target for Phenoxodiol has not been identified, these data contribute to our understanding of the mechanism by which this drug promotes cell death in prostate cancer cells, and warrants the continued clinical development of Phenoxodiol as a therapeutic for the treatment of metastatic prostate cancer.
Asunto(s)
Isoflavonas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Poly(ADP-ribose) polymerase-1 (PARP-1) is the most abundant and the best-studied isoform of a family of enzymes that catalyze the polymerization of ADP-ribose from NAD(+) onto target proteins. PARP-1 is well known to involve in DNA repair, genomic stability maintenance, transcription regulation, apoptosis, and necrosis. Polyubiquitylation targets proteins towards degradation and regulates cell cycle progression, transcription, and apoptosis. Here we report polyubiquitylation of PARP-1 in mouse fibroblasts in the presence of proteasome inhibitor and in full-length recombinant PARP-1 in vitro under standard ubiquitylation assay conditions by immunoprecipitation and immunoblotting. Mutation of ubiquitin K48R but not ubiquitin K63R abolishes polyubiquitylation of PARP-1, indicating that K48 of ubiquitin was used in the formation of polyubiquitin chain and that ubiquitylated PARP-1 is likely destined for degradation. Full-length PARP-1 was ubiquitylated most likely at the N-terminal 24 kDa domain of PARP-1 as suggested by the inhibition of ubiquitylation by activated DNA and the absence of polyubiquitin in the C-terminal 89 kDa PARP-1 derived from caspase-catalyzed cleavage. NAD(+) inhibited ubiquitylation of PARP-1, while dipeptides ArgAla and LeuAla enhanced ubiquitylation of PARP-1. ATP inhibited the synthesis of poly(ADP-ribose) by PARP-1 and affinity purified polyubiquitylated PARP-1 was active in PAR synthesis. The results suggest polyubiquitylation of PARP-1 could regulate poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 and consequently apoptosis and PARP-1 regulated cellular processes through ubiquitin-dependent degradation pathways.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Ubiquitina/metabolismo , Animales , Células Cultivadas , ADN/farmacología , Dipéptidos/farmacología , Fibroblastos/metabolismo , Ratones , Mutación Missense , NAD/farmacología , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Procesamiento Proteico-Postraduccional , Ubiquitina/genética , UbiquitinaciónRESUMEN
Environmental pollutants inducing oxidative stress stimulate chronic inflammatory responses in the lung leading to pulmonary tissue dysfunction. In response to oxidative stress, alveolar macrophages produce both reactive oxygen species and reactive nitrogen species, which induce the expression of a wide variety of immune-response genes. We found that a prolonged exposure of alveolar macrophages to a nonlethal dose (8 microg/ml) of JP-8, the kerosene-based hydrocarbon jet fuel, induced the persistent expression of IL-1, iNOS, and COX-2, as well as cell adhesion molecules (ICAM-1 and VCAM-1). Because poly(ADP-ribose) polymerase (PARP-1), a coactivator of NF-kappaB, regulates inflammatory responses and associated disorders in the airways, we determined whether JP-8 induces the poly(ADP-ribosyl)ation automodification of PARP-1 in alveolar macrophages. We observed that PARP-1 is activated in a time-dependent manner, which was temporally coincident with the prolonged activation of NF-kappaB and with the augmented expression of the proinflammatory factors described above. The 4 microg/ml dilution of JP-8 also increased the activity of PARP-1 as well as the expression of iNOS and COX-2, indicating that lower doses of JP-8 also affect the regulation of proinflammatory factors in pulmonary macrophages. Together, these results demonstrate that an extensive induction of PARP-1 might coordinate the persistent expression of proinflammatory mediators in alveolar macrophages activated by aromatic hydrocarbons that can result in lung injury from occupational exposure.
Asunto(s)
Citocinas/genética , Contaminantes Ambientales , Hidrocarburos/toxicidad , Macrófagos Alveolares/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Línea Celular , Medios de Cultivo , Combustibles Fósiles/toxicidad , Glutatión/metabolismo , Modelos Animales , Exposición Profesional , Ácido Peroxinitroso/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lung epithelial cells are critical in the regulation of airway inflammation in response to environmental pollutants. Altered activation of NF-kappaB is associated with expression of several proinflammatory factors in respiratory epithelial cells in response to an insult. Here we show that a low threshold dose (8 microg/ml) of the jet fuel JP-8 induces in a rat alveolar epithelial cell line (RLE-6TN) a prolonged activation of NF-kappaB as well as the increased expression of the proinflammatory cytokines TNF-alpha and IL-8, which are regulated by NF-kappaB. The up-regulation of IL-6 mRNA in cells exposed to JP-8 appears to be a reaction of RLE-6TN cells to reduce the enhancement of proinflammatory mediators in response to the fuel. Moreover, lung tissues from rats exposed to occupational levels of JP-8 by nasal aerosol also showed dysregulated expression of TNF-alpha, IL-8, and IL-6, confirming the in vitro data. The poly(ADP-ribosyl)ation of PARP-1, a coactivator of NF-kappaB, was coincident with the prolonged activation of NF-kappaB during JP-8 treatment. These results evidenced that a persistent exposure of the airway epithelium to aromatic hydrocarbons may have deleterious effects on pulmonary function.
Asunto(s)
Hidrocarburos/efectos adversos , Pulmón/metabolismo , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Aerosoles , Animales , Línea Celular , Supervivencia Celular , Expresión Génica , Proteínas I-kappa B , Interleucina-6/metabolismo , Pulmón/efectos de los fármacos , FN-kappa B/fisiología , Estrés Oxidativo , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/fisiología , Procesamiento Proteico-Postraduccional , Ratas , Ratas Endogámicas F344 , Factores de TiempoRESUMEN
Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1's catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1's catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.
Asunto(s)
Repeticiones de Microsatélite , Proteínas del Tejido Nervioso/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Alelos , Secuencia de Bases , Benzamidas/farmacología , Sitios de Unión/genética , Línea Celular , ADN/genética , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Humanos , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Polimorfismo Genético , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sinucleínas , alfa-SinucleínaRESUMEN
Many military personnel are at risk of lung damage or systemic toxicity as a result of exposure to the jet fuel JP-8. We have now used microarray analysis to characterize changes in the gene expression profile of lung tissue induced by exposure of rats to JP-8 at a concentration of 171 or 352 mg/m(3) for 1 h/d for 7 d, with the higher dose estimated to mimic the level of occupational exposure in humans. The expression of 56 genes was significantly affected by a factor of = 0.6 or >/= 1.5 by JP-8 at the low dose. Eighty-six percent of these genes were downregulated by JP-8. The expression of 66 genes was similarly affected by JP-8 at the higher dose, with the expression of 42% of these genes being upregulated. Prominent among the latter genes was that for the centrosome-associated protein gamma-synuclein, whose expression was consistently increased. The expression of various genes related to antioxidant responses and detoxification, including those for glutathione S-transferases and cytochrome P450 proteins, were also upregulated. The microarray data were confirmed by quantitative RT-PCR analysis. Our extensive data set may thus provide important insight into the pulmonary response to occupational exposure to JP-8 in humans.
Asunto(s)
Contaminantes Ocupacionales del Aire/farmacología , Expresión Génica/efectos de los fármacos , Hidrocarburos/farmacología , Proteínas del Tejido Nervioso/genética , Animales , Perfilación de la Expresión Génica , Inmunohistoquímica , Pulmón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinucleínas , gamma-SinucleínaRESUMEN
The skin is the principal barrier against any environmental insult. Therefore, there is a high risk for a large number of military and civilian personnel exposed to jet fuel JP-8 to suffer percutaneous absorption of this fuel. This paper reports the use of cDNA microarray to identify the gene expression profile in normal human epidermal keratinocytes exposed to JP-8 for 24-h and 7-day periods. The effects of JP-8 exposure on keratinocytes at these two different periods induced a set of genes with altered expression in response to this type of insult. Microarray data were visualized using a novel algorithm based on simple statistical analyses to reduce data dimensionality and identify subsets of discriminant genes. Predictive neural networks were built using a multiplayer perceptron to carry out a proper classification task in microarray data in the untreated versus JP-8-treated samples. The pattern of expressions in response to JP-8 provides evidences that detoxificant-related and cell growth regulator genes with the most variability in the level of expression may be useful genetic markers in adverse health effects of personnel exposed to JP-8. The approaches in our analysis provide a simple, safe, novel, and effective method that is reliable in identifying and analyzing gene expression in samples treated with JP-8 or over potential toxic agents. Gene expression data from these studies can be used to build accurate predictive models that separate different molecular profiles. The data establish the use and effectiveness of these approaches for future prospective studies.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hidrocarburos/toxicidad , Queratinocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Hidrocarburos/farmacocinética , Queratinocitos/metabolismo , Queratinocitos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Absorción Cutánea , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
1. The mechanism of toxicity of sulphur mustard was investigated by examining the biochemical effects of the analog 2-chloroethylethyl sulphide (CEES) in both human Jurkat cells as well as normal human lymphocytes. 2. Exposure of both types of cells to CEES resulted in a marked decrease in the intracellular concentration of the reduced form of glutathione (GSH), and CEES-induced cell death was potentiated by l-buthionine sulphoximine, an inhibitor of GSH synthesis. 3. CEES increased the endogenous production of reactive oxygen species (ROS) in Jurkat cells, and CEES-induced cell death was potentiated by hydrogen peroxide. 4. CEES induced various hallmarks of apoptosis, including collapse of the mitochondrial membrane potential, proteolytic processing and activation of procaspase-3, and cleavage of poly (ADP-ribose) polymerase. 5. The effects of CEES on the accumulation of ROS, the intracellular concentration of GSH, the mitochondrial membrane potential, and caspase-3 activity were all inhibited by pretreatment of cells with the GSH precursor N-acetyl cysteine or with GSH-ethyl ester. Furthermore, CEES-induced cell death was also prevented by these antioxidants. 6. CEES toxicity appears to be mediated, at least in part, by the generation of ROS and consequent depletion of GSH. Given that sulphur mustard is still a potential biohazard, the protective effects of antioxidants against CEES toxicity demonstrated in Jurkat cells and normal human lymphocytes may provide the basis for the development of a therapeutic strategy to counteract exposure to this chemical weapon.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Linfocitos/efectos de los fármacos , Gas Mostaza/análogos & derivados , Gas Mostaza/toxicidad , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Células Jurkat , Linfocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The transcription factor E2F-1 is implicated in the activation of S-phase genes as well as induction of apoptosis, and is regulated by interactions with Rb and by cell cycle-dependent alterations in E2F-1 abundance. We earlier demonstrated a pivotal role for poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of E2F-1 expression and promoter activity during S-phase re-entry when quiescent cells re-enter the cell cycle. We now investigate the putative mechanism(s) by which PARP-1 may upregulate E2F-1 promoter activity during S-phase re-entry. DNase-1 footprint assays with purified PARP-1 showed that PARP-1 did not directly bind the E2F-1 promoter in a sequence-specific manner. In contrast to p53, a positive acceptor in poly(ADP-ribosyl)ation reactions, E2F-1 was not poly(ADP-ribosyl)ated by wild-type PARP-1 in vitro, indicating that PARP-1 does not exert a dual effect on E2F-1 transcriptional activation. Protein-binding reactions and coimmunoprecipitation experiments with purified PARP-1 and E2F-1, however, revealed that PARP-1 binds to E2F-1 in vitro. More significantly, physical association of PARP-1 and E2F-1 in vivo also occurred in wild-type fibroblasts 5 h after re-entry into S phase, coincident with the increase in E2F-1 promoter activity and expression of E2F-1-responsive S-phase genes cyclin A and c-Myc. Mapping of the interaction domains revealed that full-length PARP-1 as well as PARP-1 mutants lacking either the catalytic active site or the DNA-binding domain equally bind E2F-1, whereas a PARP-1 mutant lacking the automodification domain does not, suggesting that the protein interaction site is located in this central domain. Finally, gel shift analysis with end-blocked E2F-1 promoter sequence probes verified that the binding of PARP-1 to E2F-1 enhances binding to the E2F-1 promoter, indicating that PARP-1 acts as a positive cofactor of E2F-1-mediated transcription.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Fase S/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Sitios de Unión , Ciclina A/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes myc/fisiología , Humanos , Pruebas de PrecipitinaRESUMEN
The jet fuel JP-8 is widely used and a large number of military and civilian personnel is, therefore, exposed to it. Treatment of several cell lines, including human Jurkat cells, with JP-8 induces cell death that exhibits various biochemical and morphological characteristics of apoptosis. The molecular mechanism of JP-8 cytotoxicity, however, has remained unclear. The effects of exposure of Jurkat cells to JP-8 (1/10,000 dilution) for 4 h on gene expression have now been examined by cDNA macroarray analysis. We had previously shown in these cells that under the above conditions, JP-8 causes significant apoptosis, based upon the observation that caspase-3 activation occurs at approximately 4 h and consequently most of the other classical apoptotic biochemical and morphological alterations progress until apoptotic cell death at 24 h. Of the 439 apoptosis- or stress response-related genes examined, the expression of 16 genes was up-regulated and that of ten genes was down-regulated by a factor of > or =2. The changes in the expression of 11 of these 26 genes were confirmed by reverse transcription and polymerase chain reaction analysis. These results provide insight into the mechanism of JP-8 toxicity and the associated induction of apoptosis.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hidrocarburos/toxicidad , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Airway inflammation is a central feature of asthma and chronic obstructive pulmonary disease. Reactive oxygen species (ROS) contribute to inflammation by damaging DNA, which, in turn, results in the activation of poly(ADP-ribose) polymerase-1 (PARP-1) and depletion of its substrate, nicotinamide adenine dinucleotide. Here we show that prevention of PARP-1 activation protects against both ROS-induced airway epithelial cell injury in vitro and airway inflammation in vivo. H(2)O(2) induced the generation of ROS, PARP-1 activation and concomitant nicotinamide adenine dinucleotide depletion, and release of lactate dehydrogenase in A549 human airway epithelial cells. These effects were blocked by the PARP-1 inhibitor 3-aminobenzamide (3-AB). Furthermore, 3-AB inhibited both activation of the proinflammatory transcription factor nuclear factor-kappaB and expression of the interleukin-8 gene induced by H(2)O(2) in these cells. In a murine model of allergen-induced asthma, 3-AB prevented airway inflammation elicited by ovalbumin. Moreover, PARP-1 knockout mice were resistant to such ovalbumin-induced inflammation. These protective effects were associated with an inhibition of expression of the inducible nitric oxide synthase. These results implicate PARP-1 activation in airway inflammation, and suggest this enzyme as a potential target for the development of new therapeutic strategies in the treatment of asthma as well as other respiratory disorders such as chronic obstructive pulmonary disease.
Asunto(s)
Asma/prevención & control , Pulmón/patología , Poli(ADP-Ribosa) Polimerasas/fisiología , Alérgenos/administración & dosificación , Alérgenos/inmunología , Animales , Benzamidas/farmacología , Línea Celular , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Inflamación/inducido químicamente , Inflamación/prevención & control , Interleucina-8/metabolismo , L-Lactato Deshidrogenasa/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Oxidantes/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
Sporadic Parkinson's disease (PD) affects primarily dopaminergic neurons of the substantia nigra pars compacta. There is evidence of necrotic and apoptotic neuronal death in PD, but the mechanisms behind selected dopaminergic neuronal death remain unknown. The tumor suppressor protein p53 functions to selectively destroy stressed or abnormal cells during life and development by means of necrosis and apoptosis. Activation of p53 leads to death in a variety of cells including neurons. p53 is a target of the nuclear enzyme Poly(ADP-ribose)polymerase (PARP), and PARP is activated following DNA damage that occurs following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP is the favored in vivo model of PD, and reproduces the pathophysiology, anatomy and biochemistry of PD. p53 protein normally exhibits a fleeting half-life, and regulation of p53 stability and activation is achieved mainly by post-translational modification. We find that p53 is heavily poly(ADP-ribosyl)ated by PARP-1 following MPTP intoxication. This post-translational modification serves to stabilize p53 and alters its transactivation of downstream genes. These influences of PARP-1 on p53 may underlie the mechanisms of MPTP-induced parkinsonism and other models of neuronal death.
Asunto(s)
Neuronas/metabolismo , Trastornos Parkinsonianos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , ADN/metabolismo , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Immunoblotting , Masculino , Mesencéfalo/química , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/inducido químicamente , Poli Adenosina Difosfato Ribosa/metabolismo , Pruebas de Precipitina , Unión Proteica/fisiologíaRESUMEN
The cytotoxic effect of the chemotherapeutic drug etoposide (VP-16) is thought to result from its ability to induce DNA damage and thereby to trigger apoptosis. Internucleosomal DNA fragmentation occurs late during apoptosis in many cell types. However, whereas human osteosarcoma cells undergo internucleosomal DNA fragmentation during staurosporine-induced apoptosis, they fail to do so in response to VP-16. Recently, we showed that these cells also do not express the poly(ADP-ribosyl)ation-regulated Ca(2+)- and Mg(2+)-dependent endonuclease DNAS1L3. The possibility that this deficiency underlies the failure of these cells to undergo internucleosomal DNA fragmentation in response to VP-16 was investigated. The proteolytic processing and consequent activation of procaspase-3, cleavage of the inhibitory subunit of DNA fragmentation factor, and the degradation of DNA into 50-kb fragments occurred similarly in osteosarcoma cells exposed to either staurosporine or VP-16. However, the additional processing of the 50-kb DNA fragments to oligonucleosomal fragments was not apparent in the VP-16-treated cells. Ectopic expression of DNAS1L3 conferred on osteosarcoma cells the ability to undergo VP-16-induced internucleosomal DNA fragmentation. Furthermore, expression of DNAS1L3 markedly potentiated the cytotoxic effect of VP-16 in these cells. Both DNAS1L3-mediated and staurosporine-induced internucleosomal DNA fragmentation were Ca(2+) dependent, but only the DNAS1L3-mediated DNA cleavage was blocked by expression of a caspase-3-resistant mutant of poly(ADP-ribose) polymerase-1. The present work results suggest a direct relation between the activity of a chemotherapeutic drug (VP-16) and a specific endonuclease (DNAS1L3). They also indicate that internucleosomal DNA fragmentation plays an active role in apoptosis and that the failure of cancer cells to undergo such DNA degradation may contribute to the development of resistance to chemotherapeutic drugs.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias Óseas/enzimología , Fragmentación del ADN/efectos de los fármacos , Endodesoxirribonucleasas/fisiología , Etopósido/farmacología , Osteosarcoma/enzimología , Poli(ADP-Ribosa) Polimerasas/fisiología , Antineoplásicos Fitogénicos/toxicidad , Proteínas Reguladoras de la Apoptosis , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Calcio/metabolismo , Calcio/fisiología , Caspasa 3 , Caspasas/metabolismo , Fragmentación del ADN/fisiología , Endodesoxirribonucleasas/biosíntesis , Endodesoxirribonucleasas/genética , Activación Enzimática , Etopósido/toxicidad , Humanos , Nucleosomas/efectos de los fármacos , Nucleosomas/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Estaurosporina/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P-450-generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK-Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time-dependent manner. Staining of cells with annexin-V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, 8, and 9, cleavage of poly(ADP-ribose) polymerase, and degradation of lamin B1 and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK-Hep1 and primary lymphocytes, DNA was only degraded to 50-kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl-XL prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase-8 activation was a postmictochondrial event and occurred in a Fas-independent manner. These results demonstrate that acetaminophen induces caspases-dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Linfocitos/efectos de los fármacos , Caspasas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN , Activación Enzimática/efectos de los fármacos , Humanos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The toxic jet fuel JP-8 induces morphological and biochemical changes characteristic of apoptosis in rat lung epithelial (RLE-6TN) cells. The mechanism of JP-8 toxicity in these cells was further investigated in an attempt to identify potential therapeutic interventions. Given that oxidative stress and changes in the concentrations of endogenous antioxidants, such as glutathione (GSH), have been associated with the cellular damage elicited by numerous toxicants, the possibility that JP-8 induces cellular oxidative stress was investigated. Experimentally induced depletion of intracellular GSH or exposure of cells to a low concentration of H(2)O(2) markedly enhanced JP-8-induced cell death. A significant reduction in intracellular concentrations of GSH was noted in RLE-6TN cells shortly after exposure to JP-8. Furthermore, JP-8 induced the generation of reactive oxygen species (ROS) in RLE-6TN cells. Consistent with the notion that JP-8 toxicity is mediated by generation of ROS and depletion of intracellular GSH, JP-8-induced cell death was inhibited by exogenous GSH or the thiol-containing antioxidant N-acetyl-cysteine. This protective effect was associated with marked inhibition of both the activation of caspase-3 and the loss of the mitochondrial membrane potential induced by JP-8. Inhibition of the JP-8-induced activation of poly(ADP-ribose) polymerase by 3-aminobenzamide did not protect cells against JP-8 toxicity. Together, these results indicate that thiol antioxidants are highly effective in rescuing cells from JP-8-induced cell death and that they may provide a basis for new therapeutic approaches to counteract JP-8 toxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Glutatión/metabolismo , Hidrocarburos/toxicidad , Teratógenos/toxicidad , Acetilcisteína/farmacología , Animales , Benzamidas/farmacología , Butionina Sulfoximina/farmacología , Caspasa 3 , Caspasas/análisis , Caspasas/biosíntesis , Células Epiteliales , Depuradores de Radicales Libres/farmacología , Glutatión/deficiencia , Hidrocarburos/metabolismo , Hidrocarburos/farmacología , Peróxido de Hidrógeno/farmacología , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Ratas , Teratógenos/metabolismo , Teratógenos/farmacologíaRESUMEN
Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme that is activated by DNA strand breaks and catalyzes the transfer of ADP-ribose groups from NAD to itself and other nuclear proteins. Although caspase-mediated PARP-1 cleavage occurs during almost all forms of apoptosis, the contribution of PARP-1 activation and cleavage to this cell death process remains unclear. Using immortalized fibroblasts from wild-type (PARP-1(+/+)) and PARP-1 knockout (PARP-1(-/-)) mice, and a mouse neuroblastoma cell line (N18), the role that poly(ADP-ribosyl)ation plays in Sindbis virus (SV)-induced apoptosis was examined. Robust PARP-1 activation occurred in SV-infected cells prior to morphologic changes associated with apoptotic cell death and PARP-1 activity ceased simultaneously with caspase-3 activation and PARP-1 proteolysis. PARP-1 activity was maximal before detectable DNA fragmentation, but was absent when DNA damage was most intense. SV and staurosporine-induced cell death was delayed in fibroblasts lacking PARP-1 activity, suggesting that PARP-1 activation contributes to apoptotic cell death induced by these stimuli. SV replication was not affected by lack of PARP-1 activity, but DNA fragmentation and caspase-3 activation were delayed and occurred at lower levels in PARP-1-deficient fibroblasts. Early virus-induced PARP-1 activation may represent a novel way by which cells signal to the nucleus to regulate protein function by poly(ADP-ribosyl)ation in response to virus infection.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Poli(ADP-Ribosa) Polimerasas/fisiología , Virus Sindbis/fisiología , Estaurosporina/farmacología , Animales , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Fragmentación del ADN , Activación Enzimática , Ratones , Replicación ViralRESUMEN
DNA fragmentation factor (DFF) comprises DFF45 and DFF40 subunits, the former of which acts as an inhibitor of the latter (the catalytic subunit) and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks the generation of 50-kb DNA fragments and confers resistance to apoptosis. We recently suggested that the early fragmentation of DNA by DFF and the consequent activation of poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial dysfunction, and activation of caspase-3 contribute to an amplification loop in the apoptotic process. To verify the existence of such a loop, we have now examined the effects of restoring DFF expression in DFF45-deficient fibroblasts. Co-transfection of mouse DFF45(-/-) fibroblasts with plasmids encoding human DFF40 and DFF45 reversed the apoptosis resistance normally observed in these cells. The DFF45(-/-) cells regained the ability to fragment their DNA into 50-kb pieces in response to TNF, which resulted in a marked activation of PARP-1 and a concomitant depletion of intracellular NAD. DFF expression also resulted in an increase both in cytochrome c release into the cytosol and in caspase-3 activation triggered by TNF. These results support the importance of DFF, PARP-1, mitochondria, and caspase-3 in an amplification phase of TNF-induced apoptosis.