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BACKGROUND: Citrin deficiency (CD), a disorder caused by mutations in the SLC25A13 gene, may result in neonatal intrahepatic cholestasis. This study was purposely to explore the mutation spectrum of SLC25A13 gene in Vietnamese CD patients. METHODS: The 292 unrelated CD patients were first screened for four high-frequency mutations by PCR/PCR-RFLP. Then, Sanger sequencing was performed directly for heterozygous or undetected patients. Novel mutations identified would need to be confirmed by their parents. RESULTS: 12 pathogenic SLC25A13 mutations were identified in all probands, including three deletions c.851_854del (p.R284Rfs*3), c.70-63_133del (p.Y24_72Ifs*10), and c.[1956C>A;1962del] (p.[N652K;F654Lfs*45]), two splice-site mutations (IVS6+5G>A and IVS11+1G>A), one nonsense mutations c.1399C>T (p.R467*), one duplication mutation c.1638_1660dup (p.A554fs*570), one insertion IVSl6ins3kb (p.A584fs*585), and four missense mutation c.2T>C (p.M1T), c.1231G>A (p.V411M), c.1763G>A (p.R588Q), and c.135G>C (p.L45F). Among them, c.851_854del (mut I) was the most identified mutant allele (91.78%) with a total of 247 homozygous and 42 heterozygous genotypes of carriers. Interestingly, two novel mutations were identified: c.70-63_133del (p.Y24_72Ifs*10) and c.[1956C>A;1962del] (p.[N652K;F654Lfs*45]). CONCLUSION: The SLC25A13 mutation spectrum related to intrahepatic cholestasis infants in Vietnam revealed a quite similar pattern to Asian countries' reports. This finding supports the use of targeted SLC25A13 mutation for CD screening in Vietnam and contributed to the SLC25A13 mutation spectra worldwide. It also helps emphasize the role of DNA analysis in treatment, genetic counseling, and prenatal diagnosis.
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Colestase Intra-Hepática , Citrulinemia , Proteínas de Transporte da Membrana Mitocondrial , Feminino , Humanos , Lactente , Recém-Nascido , Gravidez , Colestase Intra-Hepática/genética , Citrulinemia/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Mutação , População do Sudeste Asiático , VietnãRESUMO
INTRODUCTION: The aim of the current study was to assess the effect of a 600-µm corneal pre-cut on wound architecture and its impact on surgically induced astigmatism. The images were acquired intraoperatively and postoperatively with high-resolution spectral-domain optical coherence tomography (SD-OCT). METHODS: This study included patients scheduled for cataract surgery. Preoperatively, optical biometry and corneal topography were performed (IOL Master 500 and Atlas 9000, both Carl Zeiss Meditec AG, Germany). The first eye randomly received a 600-µm corneal pre-cut during cataract surgery, or a single-plane stab-incision and the second eye received the other incision technique. Incision architecture was assessed intraoperatively using a continuous intraoperative optical coherence tomography (iOCT) device (ReScan 700, Carl Zeiss Meditec AG, Germany) at three time points: after the incision, after irrigation/aspiration and after intraocular lens (IOL) implantation. Additionally, OCT (Spectralis, Heidelberg Engineering, Germany) measurements were performed 1 h, 1 week and 1 month postoperatively. RESULTS: Forty eight eyes of 24 patients were analysed. The pre-cut group and the stab-incision group had a significant decrease in wound thickness from the 1-h to the 1-week measurement (p = 0.022 and p = 0.001). Corneal astigmatism showed a vector difference from preoperatively to the 1-week measurement of 0.48 D (SD, ± 0.27) in the stab incision group and 0.49 D (SD, ± 0.24) in the stab incision group. No significant differences were found between the groups. CONCLUSION: To our knowledge, this was the first study which compared the wound alterations in pre-cut and stab-incision groups. TRIAL REGISTRATION: NCT02155270.
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The leaching of chemicals by materials has been integrated into risk management procedures of many sectors where hygiene and safety are important, including food, medical, pharmaceutical, and biotechnological applications. The approaches focus on direct contact and do not usually address the risk of cross-mass transfer of chemicals from one item or object to another and finally to the contacting phase (e.g., culture medium, biological fluids). Overpackaging systems, as well as secondary or ternary containers, are potentially large reservoirs of non-intentionally added substances (NIAS), which can affect the final risk of contamination. This study provides a comprehensive description of the cross-mass transfer phenomena for single-use bags along the chain of value and the methodology to evaluate them numerically on laminated and assembled systems. The methodology is validated on the risk of migration i) of ϵ-caprolactam originating from the polyamide 6 internal layer of the overpackaging and ii) of nine surrogate migrants with various volatilities and polarities. The effects of imperfect contacts between items and of an air gap between them are particularly discussed and interpreted as a cutoff distance depending on the considered substance. A probabilistic description is suggested to define conservative safety-margins required to manage cross-contamination and NIAS in routine.
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Modelos Teóricos , Plásticos/química , Algoritmos , HumanosRESUMO
Sirtuin 2 (SIRT2) plays a major role in aging, carcinogenesis and neurodegeneration. While it has been shown that SIRT2 is a mediator of stress-induced cell death, the mechanism remains unclear. In this study, we report the role of SIRT2 in mediating radiation-induced cell death and DNA damage using mouse embryonic fibroblasts (MEFs), progenitor cells and tissues from Sirt2 wild-type and genomic knockout mice, and human tumor and primary cell lines as models. The presence of Sirt2 in cells and tissues significantly enhanced the cell's sensitivity to radiation-induced cytotoxicity by delaying the dispersion of radiation-induced γ-H2AX and 53BP1 foci. This enhanced cellular radiosensitivity correlated with reduced expression of pro-survival and DNA repair proteins, and decreased DNA repair capacities involving both homologous repair and non-homologous end joining DNA repair mechanisms compared to those in Sirt2 knockout (KO) and knockdown (KD) phenotypes. Together, these data suggest SIRT2 plays a critical role in mediating the radiation-induced DNA damage response, thus regulating radiation-induced cell death and survival.
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Lesões Experimentais por Radiação/metabolismo , Sirtuína 2/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Cognição/efeitos da radiação , Dano ao DNA , Fibroblastos/efeitos da radiação , Recombinação Homóloga/efeitos da radiação , Camundongos , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/fisiopatologia , Tolerância a RadiaçãoRESUMO
Migration due to indirect contact with packaging caused several major sanitary crises, including the spread contamination of dry food by mineral oils and printing ink constituents from cardboard. The issues are still not fully resolved because the mechanisms have been insufficiently described and the relationship between design, contamination level, type of contaminant, and conditions of storage (time and temperature) are poorly understood. This study proposes a forensic analysis of these phenomena when food is separated from cardboard by a plastic layer. Practical relationships and advanced simulation scenarios were devised and validated against the long-term migration between 20 and 60°C of 15 substances. They were chosen to be representative of the main contaminants of cardboard: aliphatic and aromatic mineral oils, photo-initiators and plasticisers. Data were summarised as iso-contamination curves and iso-contamination times up to 2 years. Simple rules are illustrated to extrapolate the results to arbitrary conditions in order to identify critical substances and to estimate the plastic film's thickness to keep the contamination within acceptable limits. Recommendations for the risk management of contamination routes without contact are finally drafted.
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Análise de Alimentos , Contaminação de Alimentos/análise , Embalagem de Alimentos , Óleo Mineral/química , Tinta , PapelRESUMO
We argue that thanks to molecular modeling approaches, many thermodynamic properties required in Food Science and Food Engineering will be calculable within a few hours from first principles in a near future. These new possibilities will enable to bridge via multiscale modeling composition, process and storage effects to reach global optimization, innovative concepts for food or its packaging. An outlook of techniques and a series of examples are given in this perspective. We emphasize solute chemical potentials in polymers, liquids and their mixtures as they cannot be understood and estimated without theory. The presented atomistic and coarse-grained methods offer a natural framework to their conceptualization in polynary systems, entangled or crosslinked homo- or heteropolymers.
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Whole brain radiotherapy (WBRT) produces unwanted sequelae, albeit via unknown mechanisms. A deacetylase expressed in the central nervous system, Sirtuin 2 (SIRT2), has been linked to neurodegeneration. Therefore, we sought to challenge the notion that a single disease pathway is responsible for radiation-induced brain injury in Sirt2 wild-type (WT) and knockout (KO) mice at the proteomic level. We utilized isobaric tag for relative and absolute quantitation to analyze brain homogenates from Sirt2 WT and KO mice with and without WBRT. Selected proteins were independently verified, followed by ingenuity pathway analysis. Canonical pathways for Huntington's, Parkinson's, and Alzheimer's were acutely affected by radiation within 72 h of treatment. Although loss of Sirt2 preferentially affected both Huntington's and Parkinson's pathways, WBRT most significantly affected Huntington's-related proteins in the absence of Sirt2. Identical protein expression patterns were identified in Mog following WBRT in both Sirt2 WT and KO mice, revealing a proteomic radiation signature; however, long-term radiation effects were found to be associated with altered levels of a small number of key neurodegeneration-related proteins, identified as Mapt, Mog, Snap25, and Dnm1. Together, these data demonstrate the principle that the presence of Sirt2 can have significant effects on the brain proteome and its response to ionizing radiation.
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Encéfalo/efeitos da radiação , Raios gama , Redes e Vias Metabólicas/efeitos da radiação , Proteoma/genética , Sirtuína 2/genética , Animais , Encéfalo/metabolismo , Química Encefálica , Modelos Animais de Doenças , Dinamina I/genética , Dinamina I/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Anotação de Sequência Molecular , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteoma/metabolismo , Sirtuína 2/deficiência , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
UNLABELLED: Wnt signaling is critical to maintaining cellular homeostasis via regulation of cell division, mitigation of cell stress, and degradation. Aberrations in Wnt signaling contribute to carcinogenesis and metastasis, whereas sirtuins have purported roles in carcinogenesis, aging, and neurodegeneration. Therefore, the hypothesis that sirtuin 2 (SIRT2) directly interacts with ß-catenin and whether this interaction alters the expression of Wnt target genes to produce an altered cellular phenotype was tested. Coimmunoprecipitation studies, using mouse embryonic fibroblasts (MEF) from Sirt2 wild-type and genomic knockout mice, demonstrate that ß-catenin directly binds SIRT2. Moreover, this interaction increases in response to oxidative stress induced by ionizing radiation. In addition, this association inhibits the expression of important Wnt target genes such as survivin (BIRC5), cyclin D1 (CCND1), and c-myc (MYC). In Sirt2 null MEFs, an upregulation of matrix metalloproteinase 9 (MMP9) and decreased E-cadherin (CDH1) expression is observed that produces increased cellular migration and invasion. Together, these data demonstrate that SIRT2, a tumor suppressor lost in multiple cancers, inhibits the Wnt signaling pathway in nonmalignant cells by binding to ß-catenin and that SIRT2 plays a critical role in the response to oxidative stress from radiation. IMPLICATIONS: Disruption of the SIRT2-ß-catenin interaction represents an endogenous therapeutic target to prevent transformation and preserve the integrity of aging cells against exogenous stressors such as reactive oxygen species.
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Estresse Oxidativo/efeitos da radiação , Sirtuína 2/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Animais , Proliferação de Células/efeitos da radiação , Ciclina D1/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Radiação , Transdução de Sinais , Sirtuína 2/genética , Via de Sinalização Wnt/efeitos da radiação , beta Catenina/genéticaRESUMO
SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. The long isoform is generated via intra-exon splicing creating a frame-shift to expose a novel upstream translation start site. Mitochondrial localization is significantly more robust following transfection of the long compared with the short isoform. Insertion of this alternatively spliced novel 5' sequence upstream of a GFP-reporter plasmid shows greater than 80% enrichment in mitochondria, confirming this region as a legitimate mitochondrial localization sequence. Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.
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Mitocôndrias/enzimologia , Sinais Direcionadores de Proteínas , Sirtuína 3/química , Sirtuína 3/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Sirtuína 3/genética , Frações Subcelulares/metabolismo , Transcrição GênicaRESUMO
The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.
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Envelhecimento/fisiologia , Transformação Celular Neoplásica/genética , Genes Supressores de Tumor , Mitocôndrias/metabolismo , Sirtuína 3/genética , Estresse Fisiológico/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Estresse Oxidativo/fisiologia , Sirtuína 3/metabolismo , Superóxidos/metabolismoRESUMO
To identify pathways controlling prostate cancer metastasis we performed differential display analysis of the human prostate carcinoma cell line PC-3 and its highly metastatic derivative PC-3M. This revealed that a 78-kDa interferon-inducible GTPase, MxA, was expressed in PC-3 but not in PC-3M cells. The gene encoding MxA, MX1, is located in the region of chromosome 21 deleted as a consequence of fusion of TMPRSS2 and ERG, which has been associated with aggressive, invasive prostate cancer. Stable exogenous MxA expression inhibited in vitro motility and invasiveness of PC-3M cells. In vivo exogenous MxA expression decreased the number of hepatic metastases following intrasplenic injection. Exogenous MxA also reduced motility and invasiveness of highly metastatic LOX melanoma cells. A mutation in MxA that inactivated its GTPase reversed inhibition of motility and invasion in both tumor cell lines. Co-immunoprecipitation studies demonstrated that MxA associated with tubulin, but the GTPase-inactivating mutation blocked this association. Because MxA is a highly inducible gene, an MxA-targeted drug discovery screen was initiated by placing the MxA promoter upstream of a luciferase reporter. Examination of the NCI diversity set of small molecules revealed three hits that activated the promoter. In PC-3M cells, these drugs induced MxA protein and inhibited motility. These data demonstrate that MxA inhibits tumor cell motility and invasion, and that MxA expression can be induced by small molecules, potentially offering a new approach to the prevention and treatment of metastasis.
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Movimento Celular/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Interferon-alfa/farmacologia , Neoplasias/patologia , Substituição de Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/genética , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/enzimologia , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas de Resistência a Myxovirus , Invasividade Neoplásica , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/análise , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
Chromatin status is characterized in part by covalent posttranslational modifications of histones that regulate chromatin dynamics and direct gene expression. BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein that is thought to play a role in chromatin organization and gene expression. BORIS is a cancer-germ line gene; these are genes normally present in male germ cells (testis) that are also expressed in cancer cell lines as well as primary tumors. This work identifies SET1A, an H3K4 methyltransferase, and BAT3, a cochaperone recruiter, as binding partners for BORIS, and these proteins bind to the upstream promoter regions of two well-characterized procarcinogenic genes, Myc and BRCA1. RNA interference (RNAi) knockdown of BAT3, as well as SET1A, decreased Myc and BRCA1 gene expression but did not affect the binding properties of BORIS, but RNAi knockdown of BORIS prevented the assembly of BAT3 and SET1A at the Myc and BRCA1 promoters. Finally, chromatin analysis suggested that BORIS and BAT3 exert their effects on gene expression by recruiting proteins such as SET1A that are linked to changes in H3K4 dimethylation. Thus, we propose that BORIS acts as a platform upon which BAT3 and SET1A assemble and exert effects upon chromatin structure and gene expression.
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Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas/metabolismo , Animais , Proteína BRCA1/genética , Células COS , Chlorocebus aethiops , Células HCT116 , Humanos , Metilação , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/genética , Fatores de Transcrição/metabolismoRESUMO
Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.
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Fatores de Transcrição Forkhead/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Sirtuínas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Dissulfeto de Glutationa/metabolismo , Células HCT116 , Humanos , Proteínas Mitocondriais/genética , Ligação Proteica , Sirtuína 3 , Sirtuínas/genética , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , TransfecçãoRESUMO
The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele.
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Metilação de DNA , Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Metilação , RNA não Traduzido/química , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Pai , Feminino , Humanos , Masculino , Camundongos , RNA Longo não Codificante , TransgenesRESUMO
In a previous genomic analysis, using somatic methyltransferase (DNMT) knockout cells, we showed that hypomethylation decreased the expression of as many genes as were observed to increase, suggesting a previously unknown mechanism for epigenetic regulation. To address this idea, the expression of the BAG family genes was used as a model. These genes were used because their expression was decreased in DNMT1(-/-), DNMT3B(-/-), and double knockout cells and increased in DNMT1-overexpressing and DNMT3B-overexpressing cells. Chromatin immunoprecipitation analysis of the BAG-1 promoter in DNMT1-overexpressing or DNMT3B-overexpressing cells showed a permissive dimethyl-H3-K4/dimethyl-H3-K9 chromatin status associated with DNA-binding of CTCFL/BORIS, as well as increased BAG-1 expression. In contrast, a nonpermissive dimethyl-H3-K4/dimethyl-H3-K9 chromatin status was associated with CTCF DNA-binding and decreased BAG-1 expression in the single and double DNMT knockout cells. BORIS short hairpin RNA knockdown decreased both promoter DNA-binding, as well as BAG-1 expression, and changed the dimethyl-H3-K4/dimethyl-H3-K9 ratio to that characteristic of a nonpermissive chromatin state. These results suggest that DNMT1 and DNMT3B regulate BAG-1 expression via insulator protein DNA-binding and chromatin dynamics by regulating histone dimethylation.
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DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Cromatina , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , Primers do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Histona Metiltransferases , Humanos , Immunoblotting , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Metiltransferases , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Transfecção , DNA Metiltransferase 3BRESUMO
We have previously shown that hydrogen peroxide-resistant permanent (OC-14) cells are resistant to the cytotoxicity of several exogenous oxidative and anticancer agents including H(2)O(2), etoposide, and cisplatin; and we refer to this process as an oxidative multimodality-resistant phenotype (MMRP). Furthermore, OC-14 cells contain increased activator protein 1 activity, and inhibition of activator protein 1 reversed the MMRP. In this study, we show that permanent Rat-1 cell lines genetically altered to overexpress c-Fos also displayed a similar MMRP to H(2)O(2), etoposide, and cisplatin as OC-14 cells. Gene expression analysis of the OC-14 cells and c-Fos-overexpressing cells showed increased DNMT1 expression. Where OC-14 and c-Fos-overexpressing cells were exposed to 5-aza-2'-deoxycytidine, which inhibits DNMT activity, a significant but incomplete reversal of the MMRP was observed. Thus, it seems logical to suggest that DNMT1 might be at least one target in the MMRP. Rat-1 cells genetically altered to overexpress DNMT1 were also shown to be resistant to the cytotoxicity of H(2)O(2), etoposide, and cisplatin. Finally, somatic HCT116 knockout cells that do not express either DNMT1 (DNMT1(-/-)) or DNMT3B (DNMT3B(-/-)) were shown to be more sensitive to the cytotoxicity of H(2)O(2), etoposide, and cisplatin compared with control HCT116 cells. This work is the first example of a role for the epigenome in tumor cell resistance to the cytotoxicity of exogenous oxidative (H(2)O(2)) or systemic (etoposide and cisplatin) agents and highlights a potential role for DNMT1 as a potential molecular target in cancer therapy.
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DNA (Citosina-5-)-Metiltransferases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias/enzimologia , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Azacitidina/farmacologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , DNA Metiltransferase 3BRESUMO
PURPOSE: There is a growing awareness that radiation-induced normal tissue injury in late-responding organs, such as the brain, kidney, and lung, involves complex and dynamic responses between multiple cell types that not only lead to targeted cell death but also acute and chronic alterations in cell function. The specific genes involved in the acute and chronic responses of these late-responding normal tissues remain ill defined; understanding these changes is critical to understanding the mechanism of organ damage. As such, the aim of the present study was to identify candidate genes involved in the development of radiation injury in the murine kidney and brain using microarray analysis. EXPERIMENTAL DESIGN: A multimodality experimental approach combined with a comprehensive expression analysis was done to determine changes in normal murine tissue gene expression at 8 and 24 hours after irradiation. RESULTS: A comparison of the gene expression patterns in normal mouse kidney and brain was strikingly different. This observation was surprising because it has been long assumed that the changes in irradiation-induced gene expression in normal tissues are preprogrammed genetic changes that are not affected by tissue-specific origin. CONCLUSIONS: This study shows the potential of microarray analysis to identify gene expression changes in irradiated normal tissue cells and suggests how normal cells respond to the damaging effects of ionizing radiation is complex and markedly different in cells of differing origin.
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Encéfalo/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Rim/efeitos da radiação , Animais , Encéfalo/fisiologia , Ciclo Celular/efeitos da radiação , Integrinas/metabolismo , Integrinas/efeitos da radiação , Rim/fisiologia , Pulmão/fisiologia , Pulmão/efeitos da radiação , Metabolismo/efeitos da radiação , Camundongos , Dobramento de Proteína , Transporte Proteico/efeitos da radiação , Radiação IonizanteRESUMO
Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. The understanding of these complex mechanisms should lead to the identification of potential targets for therapeutic intervention. Redox-sensitive signaling factors also regulate multiple cellular processes including proliferation, cell cycle, and pro-survival signaling cascades, suggesting their potential as molecular targets for anticancer agents. These observations suggest that redox-sensitive signaling factors may be potential novel molecular markers. We hypothesized that thioredoxin reductase-1 (TR), a component of several redox-regulated pathways, may represent a potential molecular target candidate in response to agents that induce oxidative stress. There have been numerous biological studies over the last decade investigating the cell biological, biochemical, and genetic properties of TR both in culture and in in vivo models. In addition, using a series of permanent cell lines that express either a wild-type TR or a dominant mutant TR gene or a chemical agent that inhibits TR we demonstrated that TR meets most criteria that would identify a molecular target. Based on these results we believe TR is a potential molecular target and discuss potential clinical possibilities.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/enzimologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Radicais Livres/metabolismo , Humanos , Neoplasias/patologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tiorredoxina Redutase 1 , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
HMGN1 is a nuclear protein that binds to nucleosomes and alters the accessibility of regulatory factors to their chromatin targets. To elucidate its biological function and identify specific HMGN1 target genes, we generated Hmgn1-/- mice. DNA microarray analysis of Hmgn1+/+ and Hmgn1-/- embryonic fibroblasts identified N-cadherin as a potential HMGN1 gene target. RT-PCR and western blot analysis confirmed a linkage between HMGN1 expression and N-cadherin levels. In both transformed and primary mouse embryonic fibroblasts (MEFs), HMGN1 acted as negative regulator of N-cadherin expression. Likewise, the N-cadherin levels in early embryos of Hmgn1-/- mice were higher than those of their Hmgn1+/+ littermates. Loss of HMGN1 increased the adhesiveness, motility and aggregation potential of Hmgn1-/- MEFs, a phenotype consistent with increased levels of N-cadherin protein. Re-expression of wild-type HMGN1, but not of the mutant HMGN1 protein that does not bind to chromatin, in Hmgn1-/- MEFs, decreased the levels of N-cadherin and restored the Hmgn1+/+ phenotype. These studies demonstrate a role for HMGN1 in the regulation of specific gene expression. We suggest that in MEFs, and during early mouse development, the interaction of HMGN1 with chromatin down-regulates the expression of N-cadherin.
Assuntos
Caderinas/metabolismo , Cromossomos/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína HMGN1/metabolismo , Animais , Western Blotting , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular Transformada , Movimento Celular , Células Cultivadas , Cromatina/metabolismo , Regulação para Baixo , Embrião de Mamíferos , Fibroblastos/citologia , Marcação de Genes , Proteína HMGN1/genética , Camundongos , Camundongos Knockout , Mutação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/metabolismoRESUMO
Tumor cells undergoing proliferation, de-differentiation and progression depend on a complex set of respiratory pathways to generate the necessary energy. The metabolites from these pathways produce significant oxidative stress and must be buffered to prevent permanent cell damage and cell death. It is now clear that, in order to cope with and defend against the detrimental effects of oxidative stress, a series of redox-sensitive, pro-survival signaling pathways and factors regulate a complex intracellular redox buffering network. This review develops the hypothesis that tumor cells use these redox-sensitive, pro-survival signaling pathways and factors - up-regulated due to increased tumor cell respiration - to evade the damaging and cytotoxic effects of specific anticancer agents. It further suggests that redox-sensitive, signaling factors may be potential novel targets for drug discovery.