Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
Biochem Biophys Res Commun ; 524(4): 847-852, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32046857

RESUMO

Homeostasis of metal micronutrients such as copper is tightly regulated to ensure deficiency does not occur while restricting damage resulting from excess accumulation. Using LC-MS the effect on the proteome of intestinal Caco-2 cells of exposure to the chelator triethylenetetramine (TETA) was investigated. Continuous exposure of TETA at 25 µM to Caco-2 cells caused decreased cell yields and morphological changes. These effects were reversed when cells were no longer exposed to TETA. Quantitative proteomic analysis identified 957 mostly low-fold differentially expressed proteins, 41 of these returned towards control Caco-2 expression following recovery. Proteins exhibiting this "reciprocal" behaviour included upregulated deoxyhypusine hydroxylase (DOHH, 15.69- fold), a protein essential for eIF-5A factor hypsuination, a post translational modification responsible for eIF-5A maturation, which in turn is responsible for translation elongation. Exposure to TETA also resulted in 87 proteins, the expression of which was stable and remained differentially expressed following recovery. This study helps to elucidate the stable and transient proteomic effects of TETA exposure in intestinal cells.


Assuntos
Quelantes/farmacologia , Biologia Computacional/métodos , Cobre/metabolismo , Trientina/farmacologia , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Anotação de Sequência Molecular , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A
2.
Prenat Diagn ; 38(3): 184-189, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29315677

RESUMO

OBJECTIVE: The American College of Obstetricians and Gynecologists (ACOG) and Society for Maternal-Fetal Medicine (SMFM) recommend chromosomal microarray analysis (CMA) for prenatal diagnosis in cases with 1 or more fetal structural abnormalities. For patients who elect prenatal diagnosis and have a structurally normal fetus, either microarray or karyotype is recommended. This study evaluates the frequency of clinically significant chromosomal abnormalities (CSCA) that would have been missed if all patients offered the choice between CMA and karyotyping chose karyotyping. METHODS: A total of 3223 prenatal samples undergoing CMA were evaluated. Cases were categorized into 2 groups: those that met ACOG guidelines for CMA versus those that met ACOG guidelines for either CMA or karyotype. RESULTS: Of the 3223 cases, 1475 (45.8%) met ACOG recommendations for CMA, and 1748 (54.2%) met recommendations for either CMA or karyotype. In patients who could have elected either CMA or karyotype, 2.5% had CSCA that would have been missed if the patient had elected to pursue karyotype. CONCLUSION: This study suggests that 2.5% of patients will have a CSCA that may be missed if the guidelines continue to suggest that CMA and karyotyping have equivalent diagnostic value for patients without a fetal structural abnormality.


Assuntos
Aberrações Cromossômicas , Cariotipagem , Análise de Sequência com Séries de Oligonucleotídeos , Diagnóstico Pré-Natal/normas , Feminino , Fidelidade a Diretrizes , Humanos , Guias de Prática Clínica como Assunto , Gravidez , Estudos Retrospectivos , Sociedades Médicas
3.
Genet Med ; 19(1): 83-89, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27337029

RESUMO

PURPOSE: Chromosomal microarray analysis (CMA) is currently considered first-tier testing in pediatric care and prenatal diagnosis owing to its high diagnostic sensitivity for chromosomal imbalances. The aim of this study was to determine the efficacy and diagnostic power of CMA in both fresh and formalin-fixed paraffin-embedded (FFPE) samples of products of conception (POCs). METHODS: Over a 44-month period, 8,118 consecutive samples were received by our laboratory for CMA analysis. This included both fresh (76.4%) and FFPE samples (22.4%), most of which were ascertained for recurrent pregnancy loss and/or spontaneous abortion (83%). The majority of samples were evaluated by a whole-genome single-nucleotide polymorphism (SNP)-based array (81.6%); the remaining samples were evaluated by array-comparative genomic hybridization (CGH). RESULTS: A successful result was obtained in 7,396 of 8,118 (91.1%), with 92.4% of fresh tissue samples and 86.4% of FFPE samples successfully analyzed. Clinically significant abnormalities were identified in 53.7% of specimens (3,975 of 7,396), 94% of which were considered causative. CONCLUSION: Analysis of POC specimens by karyotyping fails in 20-40% of cases. SNP-based CMA is a robust platform, with successful results obtained in >90% of cases. SNP-based CMA can identify aneuploidy, polyploidy, whole-genome homozygosity, segmental genomic imbalances, and maternal cell contamination, thus maximizing sensitivity and decreasing false-negative results. Understanding the etiology of fetal loss enables clarification of recurrence risk and assists in determining appropriate management for future family planning.Genet Med 19 1, 83-89.


Assuntos
Aborto Espontâneo/genética , Hibridização Genômica Comparativa/métodos , Testes Genéticos , Diagnóstico Pré-Natal , Aborto Espontâneo/diagnóstico , Adulto , Fatores Etários , Aneuploidia , Aberrações Cromossômicas , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem/métodos , Pessoa de Meia-Idade , Inclusão em Parafina , Polimorfismo de Nucleotídeo Único , Gravidez
4.
In Vitro Cell Dev Biol Anim ; 57(3): 359-371, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33559028

RESUMO

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 µM CuSO4 (the early inflection point) was more toxic than 25 µM CuSO4 in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon.The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 µM CuSO4 but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 µM CuSO4 but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 µM CuSO4 compared to 3.125 µM CuSO4 at 48 h. These results suggest that 3.125 µM CuSO4 at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.


Assuntos
Cobre/toxicidade , Intestinos/patologia , Células CACO-2 , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Desdobramento de Proteína/efeitos dos fármacos , Proteômica , Reprodutibilidade dos Testes , Fatores de Tempo
5.
Toxicol In Vitro ; 65: 104773, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31981602

RESUMO

Studies in hepatic systems identify multiple factors involved in the generation of copper resistance. As the intestine is the route of exposure to dietary copper, we wanted to understand how intestinal cells overcome the toxic effects of high copper and what mechanisms of resistance develop. Using the intestinal cell line Caco-2, resistance was developed by serial subculture in 50 µM copper in inorganic (CuSO4) or organic (Cu proteinate) forms. Caco-2 variants exhibited resistance to copper and retained the non-monotonic dose response while displaying stable phenotypes following repeated subculture in the absence of copper. Phenotypic changes on exposure to copper in parental Caco-2 cells included significantly increased total protein yield, ROS, SOD, metallothionein expression, GSH and total glutathione. These phenotypic changes were not replicated in resistant variants on a per cell basis. Quantitative label-free LC-MS/MS proteomic analysis identified 1113 differentially expressed proteins (DEPs) between parental Caco-2 and resistant cells. With some exceptions, most of the DEPs were overexpressed to a low level around 2-fold suggesting resistance was supported by multiple small changes in protein expression. These variants may be a useful tool in studying the toxicity of stress responses in further Cu-related studies.


Assuntos
Cobre/toxicidade , Proteoma/efeitos dos fármacos , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Tolerância a Medicamentos , Glutationa/metabolismo , Humanos , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
6.
Metallomics ; 12(10): 1521-1529, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32760989

RESUMO

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper. The clones show considerable homogeneity in response to Cu-induced toxicity and heterogeneity in morphology with variations in level of cross-resistance to other metals and doxorubicin. Population growth was reduced for Cu-resistant clones In2 and Or3 in selective pressure relative to parental Caco-2 cells. Gene expression analysis identified 4026 total (2102 unique and 1924 shared) differentially expressed genes including those involved in the MAP Kinase and Rap1 signalling pathways, and in the focal adhesion and ECM-receptor contact pathways. Gene expression changes common to all clones included upregulation of ANXA13 and GPx2. Our analysis additionally identified differential expression of multiple genes specific to copper proteinate exposure (including overexpressed UPK1B) in isolated clones Or1, Or2 and Or3 and CuSO4 exposure (including decreased AIFM2 expression) in isolated clones In1 and In2. The adaptive transcriptional responses established in this study indicate a cohort of genes, which may be involved in copper resistance regulation and chronic copper exposure.


Assuntos
Cobre/metabolismo , Células Epiteliais/metabolismo , Transcriptoma , Células CACO-2 , Cobre/toxicidade , Sulfato de Cobre/metabolismo , Sulfato de Cobre/toxicidade , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Transcriptoma/efeitos dos fármacos
7.
In Vitro Cell Dev Biol Anim ; 55(4): 221-225, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30798514

RESUMO

Copper is an essential dietary micronutrient in humans for proper cell function; however, in excess, it is toxic. The human cell line Caco-2 is popular as an in vitro model for intestinal absorption and toxicology. This study investigated the response of exponentially growing Caco-2 cells to prolonged copper exposure (120 h). An unexpected non-monotonic dose-response profile was observed in Caco-2 cells. Exposure to media supplemented with 3.125 µM CuSO4 resulted in decreased cell yield vs. untreated. However, toxicity was progressively reduced from 90% at 3.125 µM to 60% at 25 µM. This effect was documented between 48 and 120 h continuous exposure (p < 0.05). This triphasic toxicity curve was observed to be specific to copper in Caco-2 cells, as iron, manganese and zinc displayed monotonic dose-response profiles. Two inorganic copper forms, copper sulphate and copper chloride, were shown to conserve the non-monotonic dose-response curve. The triphasic effect was shown to be specific to Caco-2 cells. These results have implications for research investigating the effect of copper and other micronutrients using Caco-2 cells.


Assuntos
Cobre/farmacologia , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Sulfato de Cobre/farmacologia , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA