RESUMEN
The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , ATPasas de Translocación de Protón Vacuolares/genética , Alelos , Animales , Encéfalo/anomalías , Ciclo Celular , Centrosoma/metabolismo , Endosomas/metabolismo , Fibroblastos/metabolismo , Genómica , Células HEK293 , Células HeLa , Humanos , Ratones , Neuronas/metabolismo , Dominios Proteicos , Transporte de Proteínas , Huso Acromático/metabolismoRESUMEN
An association between proper chromosome segregation and intact mitochondria has been extensively reported. This could be related to the effects on the progression of cell division of altered energy production, increased oxidative stress, and deregulated calcium homeostasis. However, evidence for a direct relationship is still lacking. The present study was aimed at investigating the possible effect of mitochondrial dysfunction on chromosomal instability as detected in primary human cells treated with the mitochondrial poison carbonyl cyanide 3-chlorophenyl hydrazone (CCCP). Chromosome instability was analyzed in anaphase and interphase cells to follow the fate of chromosome damage during the progression of mitosis and the subsequent cell cycle. Through the combination of cytogenetic approaches and molecular analyses, i.e. morphological cell analysis, formation and characterization of micronucleus content, Comet assay, and gene expression, it was demonstrated that the prevalent DNA damage associated with CCCP treatment was the induction of chromosome loss, while primary DNA damage was not detected. No alterations in the shape of anaphase cells were observed nor induction of multipolar spindles. The proper activation of mitotic checkpoint was maintained. A linear dose-response curve characterizing the CCCP effects suggested that multiple cellular targets could be affected by the CCCP-induced mitochondrial dysfunctions triggering aneuploidy. Conversely, a steep increase was induced by the positive control vinblastine, known to have tubulin as a unique target. In addition, the effect of CCCP on mitochondrial function was demonstrated by changes in mitochondrial DNA copy number and in the expression of genes involved in mitochondrial maintenance. Overall, these results indicate that the mitochondrial poison CCCP may induce aneugenic effects.
Asunto(s)
Hidrazonas , Venenos , Humanos , Carbonil Cianuro m-Clorofenil Hidrazona/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Hidrazonas/metabolismo , Hidrazonas/farmacología , Aneugénicos/metabolismo , Venenos/metabolismo , Venenos/farmacología , Mitocondrias , Fibroblastos , ADN/metabolismoRESUMEN
PFAS (perfluoroalkyl substances) are considered non-genotoxic. However, PFAS exposure has been associated with the induction of oxidative stress in vitro and in vivo, and the possible induction of indirect genotoxic effects under sustained PFAS exposure has not been investigated. In order to shed light on this aspect, in this study a comprehensive assessment of genotoxicity was carried out in mice administered with perfluorooctanoic acid (PFOA, 0.1, 1 and 5â¯mg/kg body weight) and its C4 analogue perfluorobutyric acid (PFBA, 5â¯mg/kg body weight) for five weeks through drinking water. Markers of cell toxicity, oxidative stress and DNA strand breaks were measured in liver, the main target of toxicity of PFOA in rodents; systemic genotoxicity was also assessed by the analysis of micronuclei in reticulocytes and spleen lymphocytes, and germ cell effects by the Comet assay on testis cells. PFOA administration at the highest dose (5â¯mg/kg body weight) induced marked liver hypertrophy with signs of cell injury (elevated ALT and AST), with no concurrent evidence of lipid peroxidation and oxidative stress (decreased antioxidant capacity). Only mild liver hypertrophy, with no other signs of toxicity, was determined by PFBA administration. No evidence of treatment related genotoxicity was observed in any experimental group. Overall, data indicate that under the experimental conditions of this study, severe liver toxicity induced by PFOA administration is not associated with oxidative stress. Accordingly, no genotoxic effect is observed in liver and in the other tissues examined. Milder evidence of liver toxicity, with no genotoxicity, and a lower tendency to bioaccumulation were observed in PFBA treated mice.
Asunto(s)
Caprilatos/administración & dosificación , Caprilatos/toxicidad , Fluorocarburos/administración & dosificación , Fluorocarburos/toxicidad , Pruebas de Mutagenicidad , Administración Oral , Animales , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.
Asunto(s)
Daño del ADN/efectos de la radiación , Exposición a Riesgos Ambientales/efectos adversos , Fumar/efectos adversos , Apoptosis/efectos de la radiación , Ensayo Cometa , Estudios Transversales , Reparación del ADN/efectos de la radiación , Determinación de Punto Final , Femenino , Ácido Fólico/sangre , Rayos gamma , Histonas/genética , Histonas/metabolismo , Homocisteína/sangre , Humanos , Cinética , Modelos Lineales , Linfocitos/metabolismo , Linfocitos/efectos de la radiación , Masculino , Fosforilación , Tolerancia a Radiación , Gemelos Monocigóticos , Vitamina B 12/sangreRESUMEN
BACKGROUND: The Italian National Institute of Health (Istituto Superiore di Sanità) funded a 30-month project (July 2021-January 2024) to conduct a twin study of the relationships between Positive Mental Health (PMH) and cellular longevity. Only a few previous studies have focused on the biomarkers of aging in relation to psychological well-being, and none of them exploited the potential of the twin design. METHOD: In this project, following the standard procedures of the Italian Twin Registry (ITR), we aim to recruit 200 adult twin pairs enrolled in the ITR. They are requested to complete a self-report questionnaire battery on PMH and to undergo a blood withdrawal for the assessment of aging biomarkers, i.e., telomere length and mitochondrial DNA functionality. The association between psychological and aging biomarker measures will be assessed, controlling for genetic and familial confounding effects using the twin study design. RESULTS AND CONCLUSIONS: Biomarker assays are underway. Once data are available for the total study sample, statistical analyses will be performed. The project's results may shed light on new mechanisms underlying the mind-body connection and may prove helpful to promote psychological well-being in conjunction with biological functioning.
RESUMEN
Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.
Asunto(s)
Inestabilidad Cromosómica , Dieta , Acortamiento del Telómero , Antioxidantes/administración & dosificación , Biomarcadores/análisis , Femenino , Genotipo , Humanos , Estilo de Vida , Linfocitos/patología , Masculino , Pruebas de Micronúcleos , Micronutrientes/administración & dosificación , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Análisis de Secuencia de ADN , Fumar/efectos adversos , Encuestas y Cuestionarios , Telómero/patología , Verduras , beta Caroteno/administración & dosificaciónRESUMEN
hMTH1 protects against mutation during oxidative stress. It degrades 8-oxodGTP to exclude potentially mutagenic oxidized guanine from DNA. hMTH1 expression is linked to ageing. Its downregulation in cultured cells accelerates RAS-induced senescence, and its overexpression in hMTH1-Tg mice extends lifespan. In this study, we analysed the effects of a brief (5 weeks) high-fat diet challenge (HFD) in young (2 months old) and adult (7 months old) wild-type (WT) and hMTH1-Tg mice. We report that at 2 months, hMTH1 overexpression ameliorated HFD-induced weight gain, changes in liver metabolism related to mitochondrial dysfunction and oxidative stress. It prevented DNA damage as quantified by a comet assay. At 7 months old, these HFD-induced effects were less severe and hMTH1-Tg and WT mice responded similarly. hMTH1 overexpression conferred lifelong protection against micronucleus induction, however. Since the canonical activity of hMTH1 is mutation prevention, we conclude that hMTH1 protects young mice against HFD by reducing genome instability during the early period of rapid growth and maximal gene expression. hMTH1 protection is redundant in the largely non-growing, differentiated tissues of adult mice. In hMTH1-Tg mice, expression of a less heavily mutated genome throughout life provides a plausible explanation for their extended longevity.
Asunto(s)
Grasas de la Dieta , Longevidad , Animales , Dieta Alta en Grasa , Grasas de la Dieta/farmacología , Longevidad/genética , Ratones , Ratones Transgénicos , Estrés Oxidativo , Estrés FisiológicoRESUMEN
In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.
Asunto(s)
Furanos/administración & dosificación , Furanos/toxicidad , Hígado/efectos de los fármacos , Hígado/patología , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Proliferación Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Metilación de ADN/efectos de los fármacos , Citometría de Flujo , Fluoresceína-5-Isotiocianato/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Pruebas de Mutagenicidad , Tamaño de los Órganos/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
Gastric cancer (GC) shows a familiar predisposition which is largely unexplained. In this study the hypothesis that radiation sensitivity is implicated in the familiar predisposition to GC was investigated by means of the cytokinesis-block micronucleus assay. Data indicate that a family history of GC is not associated with any of the biomarkers investigated and does not interact with the demographic variables considered. When study subjects were dichotomized around the median age, a significant prevalence of micronuclei was observed in older subjects. Age and both spontaneous and radiation-induced micronuclei were linearly correlated. The effect of age was not modified by gender or smoking habits.
Asunto(s)
Inestabilidad Cromosómica/efectos de la radiación , Neoplasias Gástricas/genética , Factores de Edad , Anciano , Biomarcadores/análisis , Salud de la Familia , Femenino , Humanos , Masculino , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Pruebas de Micronúcleos , Persona de Mediana Edad , Fumar/genética , Neoplasias Gástricas/ultraestructuraRESUMEN
The trace element vanadium interacts with living cells, in which it exerts a variety of biological effects depending on its chemical form and oxidation state. Tetravalent vanadium was shown to affect several genotoxicity end-points in vitro, but its genotoxic potential in vivo is not elucidated. In this study, the genotoxic effects induced in vivo by subacute oral exposure to vanadyl sulphate (VOSO4), a tetravalent vanadium salt, were investigated. To this aim male CD1 mice were administered with VOSO4 in drinking water over the dose range 2-1000 mg/l for 5 weeks. The incidence of micronucleated blood reticulocytes was measured along treatment period. At the end of treatment, micronuclei in both blood reticulocytes and bone marrow polychromatic erythrocytes were determined; in addition, DNA lesions detectable by comet assay were assessed in marrow and testicular cells. Tissue distribution of vanadium at sacrifice was determined by atomic absorption spectrometry. Comet assays and the analysis of micronuclei in polychromatic erythrocytes did not reveal treatment related effects. A slight increase in micronucleated reticulocytes, with no relationship with the administered dose, was observed in some treated groups. The determination of vanadium content in kidney, liver, spleen, bone, stomach, small intestine and testis highlighted low internal exposure, especially in soft tissues. Overall, data indicate scarce bioavailability for orally administered tetravalent vanadium, and lack of significant genotoxic potential in vivo.
Asunto(s)
Micronúcleos con Defecto Cromosómico/inducido químicamente , Mutágenos/toxicidad , Compuestos de Vanadio/toxicidad , Administración Oral , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Ensayo Cometa , Masculino , Ratones , Ratones Endogámicos , Mutágenos/farmacocinética , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Factores de Tiempo , Distribución Tisular , Compuestos de Vanadio/farmacocinéticaRESUMEN
Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter-individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co-twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co-twins were contrasted, selecting as variables the intra-twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke-induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551-559, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Metilación de ADN/genética , Neoplasias Pulmonares/genética , Fumar/genética , Telomerasa/genética , Adulto , Epigénesis Genética , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas , Fumar/efectos adversos , Fumar/patología , Telómero/genética , Homeostasis del Telómero/genética , Gemelos MonocigóticosRESUMEN
Vanadium compounds are able to interact with living cells exerting a variety of biological effects. The pentavalent form is the most stable and toxic form of the element. In systems in vitro pentavalent vanadium is an effective genotoxic agent, inducing DNA damage and chromosome malsegregation at low doses. On the other hand, no adequate in vivo data are available for the characterization of the genotoxic hazard following oral intake, the most relevant route of human exposure. In this study, the genotoxic effects produced by the oral intake of sodium ortho-vanadate (Na(3)VO(4)) were investigated. Male CD-1 mice were treated for 5 weeks with a range of concentrations of Na(3)VO(4) in drinking water (0.75-1500 mg/l). Both micronuclei and primary DNA lesions as detected by comet assay were assessed in several tissues. Statistically significant increases of micronuclei in bone marrow were observed in mice receiving the two highest concentrations of Na(3)VO(4) (750 and 1500 mg/l). A significant increase of comet tail length was observed in splenocytes of mice receiving Na(3)VO(4) at 1500 mg/l, whereas no effect was observed in bone marrow and testis cells. No treatment-related effect on sperm chromatin structure or on testis cell population was observed. The determination of vanadium content in mouse tissues at the end of treatment highlighted a very low internal exposure, especially in soft tissues. Overall, the results obtained indicate that the genotoxic activity of pentavalent vanadium is expressed in vivo only following high dose exposure, possibly as a consequence of the poor bioavailability of the element.
Asunto(s)
Daño del ADN , ADN/efectos de los fármacos , Mutágenos/toxicidad , Vanadatos/toxicidad , Administración Oral , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Huesos/efectos de los fármacos , Huesos/metabolismo , Ensayo Cometa , ADN/análisis , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos , Micronúcleos con Defecto Cromosómico/inducido químicamente , Pruebas de Micronúcleos , Tamaño de los Órganos/efectos de los fármacos , Reticulocitos/efectos de los fármacos , Reticulocitos/patología , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Bazo/efectos de los fármacos , Bazo/patología , Testículo/efectos de los fármacos , Testículo/patología , Vanadatos/farmacocinética , Vanadio/análisis , Vanadio/metabolismo , Abastecimiento de AguaRESUMEN
Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.
Asunto(s)
Epigénesis Genética , Hábitos , Fumar/genética , Gemelos Monocigóticos/genética , Biología Computacional , Metilación de ADN/genética , Ontología de Genes , Sitios Genéticos , Humanos , Internet , Programas InformáticosRESUMEN
The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p= 1.23 × 10(-7)), with no significant difference related to smoking habits (p = 0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p = 0.021) and plasma folic acid level (p = 0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile.
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Metilación de ADN , Reparación del ADN , Regiones Promotoras Genéticas , Fumar/genética , Adulto , Estudios Transversales , Femenino , Ácido Fólico/sangre , Homocisteína/sangre , Humanos , Estilo de Vida , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Gemelos Monocigóticos , Vitamina B 12/sangre , Adulto JovenRESUMEN
Genes and molecular pathways involved in familial clustering of gastric cancer have not yet been identified. The purpose of the present study was to investigate gene expression changes in response to a cellular stress, and its link with a positive family history for this neoplasia. To this aim leukocytes of healthy first-degree relatives of gastric cancer patients and controls were challenged in vitro with ionizing radiation and gene expression evaluated 4 h later on microarrays with 1,800 cancer-related genes. Eight genes, mainly involved in signal transduction and cell cycle regulation, were differentially expressed in healthy relatives of gastric cancer cases. Functional class scoring by Gene Ontology classification highlighted two G-protein related pathways, implicated in the proliferation of neoplastic tissue, which were differentially expressed in healthy subjects with positive family history of gastric cancer. The relative expression of 84 genes related to these pathways was examined using the SYBR green-based quantitative real-time PCR. The results confirmed the indication of an involvement of G-protein coupled receptor pathways in GC familiarity provided by microarray analysis. This study indicates a possible association between familiarity for gastric cancer and altered transcriptional response to ionizing radiation.
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Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Leucocitos/efectos de la radiación , Radiación Ionizante , Neoplasias Gástricas/genética , Anciano , Estudios de Casos y Controles , Línea Celular Tumoral , Salud de la Familia , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leucocitos/fisiología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Folic acid plays a key role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for DNA methylation. Besides dietary habits, folic acid metabolism is influenced by genetic polymorphism. The C677T polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene is associated with a reduction of catalytic activity and is suggested to modify cancer risk differently depending on folate status. In this work the effect of folic acid deficiency on genome stability and radiosensitivity has been investigated in cultured lymphocytes of 12 subjects with different MTHFR genotype (four for each genotype). Cells were grown for 9 days with 12, 24 and 120 nM folic acid and analyzed in a comprehensive micronucleus test coupled with centromere characterization by CREST immunostaining. In other experiments, cells were grown with various folic acid concentrations, irradiated with 0.5 Gy of gamma rays and analyzed in the micronucleus test. The results obtained indicate that folic acid deficiency induces to a comparable extent chromosome loss and breakage, irrespective of the MTHFR genotype. The effect of folic acid was highly significant (P < 0.001) and explained >50% of variance of both types of micronuclei. Also nucleoplasmic bridges and buds were significantly increased under low folate supply; the increase in bridges was mainly observed in TT cells, highlighting a significant effect of the MTHFR genotype (P = 0.006) on this biomarker. Folic acid concentration significantly affected radiation-induced micronuclei (P < 0.001): the increased incidence of radiation-induced micronuclei with low folic acid was mainly accounted for by carriers of the variant MTHFR allele (both homozygotes and heterozygotes), but the overall effect of genotype did not attain statistical significance. Treatment with ionizing radiations also increased the frequency of nucleoplasmic bridges. The effect of folic acid level on this end-point was modulated by the MTHFR genotype (P for interaction = 0.02), with TT cells grown at low folic acid concentration apparently resistant to the induction of radiation-induced bridges. Finally, the effect of in vitro folate deprivation on global DNA methylation was evaluated in lymphocytes of six homozygous subjects (three CC and three TT). The results obtained suggest that, under the conditions of this work, folic acid deprivation is associated with global DNA hypermethylation.
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Deficiencia de Ácido Fólico/complicaciones , Linfocitos/efectos de la radiación , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Pruebas de Micronúcleos , Polimorfismo Genético , Adulto , Citocinesis/fisiología , Metilación de ADN , Femenino , Genotipo , Humanos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Pruebas de Micronúcleos/métodos , Proyectos de Investigación , Coloración y Etiquetado/métodosRESUMEN
Adrenal adenomas and carcinomas are mostly monoclonal, suggesting that a genetic alteration in a progenitor cell may contribute to their development. However, the molecular pathogenesis of these tumors still remains unclear. It has been already excluded that activating mutations of the ACTH receptor or of G protein stimulator alpha sub-units, affecting cAMP pathway, is involved in the tumorigenesis. Therefore, this work has been focused on post-transductional (ACTH) signal alterations and in particular on the mutational analysis of the Steroid Acute Regulatory protein (StAR) gene to verify whether somatic mutations or genomic polymorphisms of this gene may be correlated with adrenal tumorigenesis. Tissue DNA was extracted from 40 functional and non-functional adrenocortical tumors that were removed from patients aged between 17 and 72 years (mean 43 +/- 4). Blood DNA was obtained from 24 patients (aged between 26 and 70 years) affected by adrenal tumors and from 100 healthy subjects without radiological and clinical evidence of adrenal masses, aged between 25-35 years (90 Caucasians and 10 Africans). The DNA was used as the template for the amplification of the StAR gene using the polymerase chain reaction. The amplified DNA of each exon of the StAR gene was purified and sequenced in automatic sequenciator. With the exception of exon 5 showing in codon 203 an homozygous missense mutation, the sequence of the other exons of the StAR gene resulted normal in all tumors studied. The same homozygous mutation (Asp203Ala) was observed in the sequence of exon 5 performed on genomic DNA of the 24 affected patients and in the control subjects. The homozygosity of the mutation observed in all patients (either in tissue or blood samples) and in control subjects, independently of their ethnic origin, led us to suggest that the Asp203Ala cannot be considered as mutation or as polymorphism, but that it must be considered as a mistake in the sequence entered in the Genbank, which needs to be modified accordingly. These data, and those up to now reported in the literature, allow us to suggest that mutations of the gene coding for the protein involved in the initial step of the steroidogenesis could not be considered as a possible cause for the development of adrenal tumors.