RESUMEN
Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are effective in only approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers has revealed that these agents only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. Although the maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles can dramatically reduce CIN and confer resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin centromeric-associated protein-E (CENP-E) overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to antimicrotubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E. Significance: The increased incidence of polar chromosomes induced by inhibition of the mitotic kinesin CENP-E exacerbates chromosomal instability, reduces daughter cell viability, and improves sensitivity to microtubule-targeting therapies.
Asunto(s)
Neoplasias de la Mama , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona , Sinergismo Farmacológico , Microtúbulos , Humanos , Inestabilidad Cromosómica/efectos de los fármacos , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Animales , Ratones , Línea Celular Tumoral , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Mitosis/efectos de los fármacos , Antineoplásicos/farmacologíaRESUMEN
Indoor air pollution is becoming a rising public health problem and is largely resulting from the burning of solid fuels and heating in households. Burning these fuels produces harmful compounds, such as particulate matter regarded as a major health risk, particularly affecting the onset and exacerbation of respiratory diseases. As exposure to polluted indoor air can cause DNA damage including DNA sd breaks as well as chromosomal damage, in this paper, we aim to provide an overview of the impact of indoor air pollution on DNA damage and genome stability by reviewing the scientific papers that have used the comet, micronucleus, and γ-H2AX assays. These methods are valuable tools in human biomonitoring and for studying the mechanisms of action of various pollutants, and are readily used for the assessment of primary DNA damage and genome instability induced by air pollutants by measuring different aspects of DNA and chromosomal damage. Based on our search, in selected studies (in vitro, animal models, and human biomonitoring), we found generally higher levels of DNA strand breaks and chromosomal damage due to indoor air pollutants compared to matched control or unexposed groups. In summary, our systematic review reveals the importance of the comet, micronucleus, and γ-H2AX assays as sensitive tools for the evaluation of DNA and genome damaging potential of different indoor air pollutants. Additionally, research in this particular direction is warranted since little is still known about the level of indoor air pollution in households or public buildings and its impact on genetic material. Future studies should focus on research investigating the possible impact of indoor air pollutants in complex mixtures on the genome and relate pollutants to possible health outcomes.
Asunto(s)
Contaminantes Atmosféricos , Contaminación del Aire Interior , Daño del ADN , Pruebas de Micronúcleos , Contaminación del Aire Interior/efectos adversos , Contaminación del Aire Interior/análisis , Humanos , Animales , Contaminantes Atmosféricos/toxicidad , Inestabilidad Cromosómica/efectos de los fármacos , Ensayo Cometa , Material Particulado/toxicidad , Material Particulado/análisis , Histonas/metabolismo , Monitoreo del Ambiente/métodos , Inestabilidad Genómica/efectos de los fármacos , Monitoreo Biológico/métodosRESUMEN
In the department of Boyacá, Colombia, agriculture stands as one of the primary economic activities. However, the escalating utilization of pesticides within this sector has sparked concern regarding its potential correlation with elevated risks of genotoxicity, chromosomal alterations, and carcinogenesis. Furthermore, pesticides have been associated with a broad spectrum of genetic polymorphisms that impact pivotal genes involved in pesticide metabolism and DNA repair, among other processes. Nonetheless, our understanding of the genotoxic effects of pesticides on the chromosomes (as biomarkers of effect) in exposed farmers and the impact of genetic polymorphisms (as susceptibility biomarkers) on the increased risk of chromosomal damage is still limited. The aim of our study was to evaluate chromosomal alterations, chromosomal instability, and clonal heterogeneity, as well as the presence of polymorphic variants in the GSTP1 and XRCC1 genes, in peripheral blood samples of farmers occupationally exposed to pesticides in Aquitania, Colombia, and in an unexposed control group. Our results showed statistically significant differences in the frequency of numerical chromosomal alterations, chromosomal instability, and clonal heterogeneity levels between the exposed and unexposed groups. In addition, we also found a higher frequency of chromosomal instability and clonal heterogeneity in exposed individuals carrying the heterozygous GSTP1 AG and XRCC1 (exon 10) GA genotypes. The evaluation of chromosomal alterations and chromosomal instability resulting from pesticide exposure, combined with the identification of polymorphic variants in the GSTP1 and XRCC1 genes, and further research involving a larger group of individuals exposed to pesticides could enable the identification of effect and susceptibility biomarkers. Such markers could prove valuable for monitoring individuals occupationally exposed to pesticides.
Asunto(s)
Inestabilidad Cromosómica , Agricultores , Gutatión-S-Transferasa pi , Exposición Profesional , Plaguicidas , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Humanos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Gutatión-S-Transferasa pi/genética , Plaguicidas/toxicidad , Plaguicidas/efectos adversos , Exposición Profesional/efectos adversos , Masculino , Inestabilidad Cromosómica/efectos de los fármacos , Adulto , Persona de Mediana Edad , Femenino , Biomarcadores , Aberraciones Cromosómicas/inducido químicamente , Colombia , Polimorfismo de Nucleótido Simple , Predisposición Genética a la EnfermedadRESUMEN
Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the causation of urinary bladder cancer by benzo[a]pyrene (B[a]P) due to a lack of sufficient data. In this work, we focused on in-vitro DNA damage and the formation of micronuclei and chromosomal aberrations as predictors of cancer risk, applying a wide range of dosages and time periods to quantify the onset, intensity, and duration of the response. We chose two urothelial cell types to compare susceptibility and the ability to increase the malignity of a pre-existing bladder cancer: a cancer cell line (T24) and a pooled sample of primary urinary bladder epithelia cells (PUBEC) from pigs. The highest level of DNA damage assessed by comet assay was observed following 24-h treatment in both cell types, whereas PUBEC cells were clearly more susceptible. Even 4-h treatment induced DNA damage in PUBEC cells with benchmark doses of 0.0027⯵M B[a]P and 0.00023⯵M after 4-h and 24-h exposure, respectively. Nearly no effect was observed for periods of 48â¯h. The frequency of micronucleus formation increased more markedly in T24 cells, particularly with 24-h treatment. In PUBEC cells, 48-h exposure notably induced the formation of nucleoplasmic bridges and nuclear buds. Even though only one biological replicate was studied due to the sophisticated study design, our results give a strong indication of the potential of B[a]P to induce and increase malignity in human-relevant cell types.
Asunto(s)
Benzo(a)pireno , Inestabilidad Cromosómica , Daño del ADN , Urotelio , Benzo(a)pireno/toxicidad , Daño del ADN/efectos de los fármacos , Proyectos Piloto , Animales , Urotelio/efectos de los fármacos , Urotelio/patología , Inestabilidad Cromosómica/efectos de los fármacos , Humanos , Porcinos , Pruebas de Micronúcleos , Relación Dosis-Respuesta a Droga , Aberraciones Cromosómicas , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genética , Factores de Tiempo , Ensayo Cometa , Línea Celular Tumoral , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/patologíaRESUMEN
Chromosome instability, a hallmark of lung cancer, is a driving mechanism for hexavalent chromium [Cr(VI)] carcinogenesis in humans. Cr(VI) induces structural and numerical chromosome instability in human lung cells by inducing DNA double-strand breaks and inhibiting homologous recombination repair and causing spindle assembly checkpoint (SAC) bypass and centrosome amplification. Great whales are long-lived species with long-term exposures to Cr(VI) and accumulate Cr in their tissue, but exhibit a low incidence of cancer. Data show Cr(VI) induces fewer chromosome aberrations in whale cells after acute Cr(VI) exposure suggesting whale cells can evade Cr(VI)-induced chromosome instability. However, it is unknown if whales can evade Cr(VI)-induced chromosome instability. Thus, we tested the hypothesis that whale cells resist Cr(VI)-induced loss of homologous recombination repair activity and increased SAC bypass and centrosome amplification. We found Cr(VI) induces similar amounts of DNA double-strand breaks after acute (24 h) and prolonged (120 h) exposures in whale lung cells, but does not inhibit homologous recombination repair, SAC bypass, or centrosome amplification, and does not induce chromosome instability. These data indicate whale lung cells resist Cr(VI)-induced chromosome instability, the major driver for Cr(VI) carcinogenesis at a cellular level, consistent with observations that whales are resistant to cancer.
Asunto(s)
Centrosoma , Cromo , Inestabilidad Cromosómica , Roturas del ADN de Doble Cadena , Animales , Cromo/toxicidad , Inestabilidad Cromosómica/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Centrosoma/efectos de los fármacos , Centrosoma/metabolismo , Ballenas/genéticaRESUMEN
INTRODUCTION: Mono-(2-ethylhexyl) phthalate (MEHP) represents a toxicological risk for marine organisms due to its widespread presence in aquatic environments. METHODS: MEHP effects on cell viability, cell death and genotoxicity were investigated on the DLEC cell line, derived from early embryos of the European sea bass Dicentrarchus labrax L. RESULTS: A dose-dependent cytotoxic effect, with no induction of necrotic process, except at its highest concentration, was observed. Moreover, chromosomal instability was detected, both in binucleated and mononucleated cells, coupled with a minor inhibition of cell proliferation, whereas genomic instability was not revealed. To our knowledge, the overall results suggest the first evidence of a possible aneugenic effect of this compound in the DLEC cell line, that is the induction of chromosomal loss events without the induction of primary DNA damage. CONCLUSIONS: MEHP should be considered more harmful than its parent compound DEHP, because it induces genomic instability in the DLEC cell line without triggering cell death.
Asunto(s)
Organismos Acuáticos , Lubina , Inestabilidad Cromosómica , Citotoxinas , Mutágenos , Organismos Acuáticos/citología , Organismos Acuáticos/efectos de los fármacos , Organismos Acuáticos/metabolismo , Lubina/embriología , Lubina/genética , Línea Celular , Citotoxinas/toxicidad , Mutágenos/toxicidad , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Cromosómica/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Relación Dosis-Respuesta a Droga , Daño del ADNRESUMEN
Because spermatogonia transmit genetic information across generations, their DNA must be protected from environmental damages, including exposure to zinc oxide nanoparticles (ZnO NPs), which are frequently used in modern technology. Here, we used an in vitro system enriched for spermatogonia and exposed them to 10 and 20 µg/ml ZnO NPs for one/seven days. We did not detect any significant cell death, chromosomal instability, or DNA fragmentation in the spermatogonia treated with the ZnO NPs following one-day treatment with 10 or 20 µg/ml ZnO NPs. However, ZnO NPs (both 10 and 20 µg/ml) induced chromosomal instability in the spermatogonia after seven days of treatment. Moreover, one-day exposure to these NPs induced reactive oxygen species (ROS) generation and upregulation of apoptotic pathway-related genes p53, Caspase3 and Il6, as an inflammatory factor. Taken together, our study provides preliminary evidence for possible damages induced by low concentrations of ZnO NPs in spermatogonia. We should pay increased attention when using these NPs because of the silent damages in spermatogonia that can be transmitted to the next generation and cause severe effects. However, more data and validation of these results are required to determine the extent of this concern.
Asunto(s)
Nanopartículas del Metal/toxicidad , Espermatogonias/efectos de los fármacos , Óxido de Zinc/toxicidad , Animales , Proteína Quinasa CDC2/metabolismo , Caspasa 3/metabolismo , Inestabilidad Cromosómica/efectos de los fármacos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Ratones , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The ubiquitous use of phthalates in various materials and the knowledge about their potential adverse effects is of great concern for human health. Several studies have uncovered their role in carcinogenic events and suggest various phthalate-associated adverse health effects that include pulmonary diseases. However, only limited information on pulmonary toxicity is available considering inhalation of phthalates as the route of exposure. While in vitro studies are often based on submerged exposures, this study aimed to expose A549 alveolar epithelial cells at the air-liquid interface (ALI) to unravel the genotoxic and oxidative stress-inducing potential of dibutyl phthalate (DBP) with concentrations relevant at occupational settings. Within this scope, a computer modeling approach calculating alveolar deposition of DBP particles in the human lung was used to define in vitro ALI exposure conditions comparable to potential occupational DBP exposures. The deposited mass of DBP ranged from 0.03 to 20 ng/cm2 , which was comparable to results of a human lung particle deposition model using an 8 h workplace threshold limit value of 580 µg/m3 proposed by the Scientific Committee on Occupational Exposure Limits for the European Union. Comet and Micronucleus assay revealed that DBP induced genotoxicity at DNA and chromosome level in sub-cytotoxic conditions. Since genomic instability was accompanied by increased generation of the lipid peroxidation marker malondialdehyde, oxidative stress might play an important role in phthalate-induced genotoxicity. The results highlight the importance of adapting in vitro studies to exposure scenarios relevant at occupational settings and reconsidering occupational exposure limits for DBP.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Dibutil Ftalato/toxicidad , Mutágenos/toxicidad , Plastificantes/toxicidad , Células A549 , Adulto , Aire , Supervivencia Celular/efectos de los fármacos , Inestabilidad Cromosómica/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Humanos , Exposición por Inhalación , Masculino , Malondialdehído/metabolismo , Pruebas de Micronúcleos , Modelos Biológicos , Exposición Profesional , Estrés Oxidativo/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Lugar de TrabajoRESUMEN
Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum-in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues-incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.
Asunto(s)
Cloruro de Aluminio , Inestabilidad Cromosómica/efectos de los fármacos , Cromosomas de los Mamíferos/metabolismo , Roturas del ADN de Doble Cadena , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Aluminio/farmacocinética , Aluminio/toxicidad , Cloruro de Aluminio/farmacocinética , Cloruro de Aluminio/toxicidad , Animales , Línea Celular , Centrómero/metabolismo , CricetulusRESUMEN
BACKGROUND: 5-aza-2'-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages. METHODS: Human WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 µM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C). RESULTS: DNA methylation decreased significantly in 1.0 µM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 µM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 µM and 1.0 µM conditions, respectively (p = 0.001) after 4 days. CONCLUSIONS: These data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.
Asunto(s)
Daño del ADN/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Decitabina/farmacología , Telómero/efectos de los fármacos , Biomarcadores/metabolismo , Línea Celular , Inestabilidad Cromosómica/efectos de los fármacos , Citocinesis/efectos de los fármacos , Humanos , Linfocitos/efectos de los fármacos , Pruebas de Micronúcleos/métodos , Mitosis/efectos de los fármacosRESUMEN
Telomere instability is one of the main sources of genome instability and may result from chromosome end loss (due to chromosome breakage at one or both ends) or, more frequently, telomere dysfunction. Dysfunctional telomeres arise when they lose their end-capping function or become critically short, which causes chromosomal termini to behave like a DNA double-strand break. Telomere instability may occur at the chromosomal or at the molecular level, giving rise, respectively, to telomere-related chromosomal aberrations or the loss or modification of any of the components of the telomere (telomere DNA, telomere-associated proteins, or telomere RNA). Since telomeres play a fundamental role in maintaining genome stability, the study of telomere instability in cells exposed to mutagens is of great importance to understand the telomere-driven genomic instability present in those cells. In the present review, we will focus on the current knowledge about telomere instability induced by physical, chemical, and biological mutagens in human cells.
Asunto(s)
Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Mutágenos/toxicidad , Telómero/efectos de los fármacos , Animales , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Inestabilidad Cromosómica/genética , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Inestabilidad Genómica/genética , Humanos , Telómero/genéticaRESUMEN
Mycosis fungoides (MF) is the most prevalent type of skin lymphoma. In its early stages, it has a favorable prognosis. However, in its late stages, it is associated with an increased risk of mortality. This systematic review aimed to identify the transcriptomic changes involved in MF pathogenesis and progression. A literature search was conducted using the database PubMed, followed by the extraction of 2245 genes which were further filtered to 150 recurrent genes that appeared in two or more publications. Categorization of these genes identified activated pathways involved in pathways such as cell cycle and proliferation, chromosomal instability, and DNA repair. We identified 15 genes implicated in MF progression, which were involved in cell proliferation, immune checkpoints, resistance to apoptosis, and immune response. In highlighting the discrepancies in the way MF transcriptomic data is obtained, further research can focus on not only unifying their approach but also focus on the 150 pertinent genes identified in this review.
Asunto(s)
Apoptosis/genética , Inestabilidad Cromosómica/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Micosis Fungoide/genética , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Inestabilidad Cromosómica/efectos de los fármacos , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Micosis Fungoide/tratamiento farmacológico , Micosis Fungoide/metabolismo , Factores de RiesgoRESUMEN
Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.
Asunto(s)
Inestabilidad Cromosómica/efectos de los fármacos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Mitosis/efectos de los fármacos , Piruvaldehído/farmacología , Biomarcadores , Línea Celular , Cromatografía Liquida , Deleción Cromosómica , Citocinesis , Daño del ADN/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ , Linfocitos/patología , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Pruebas de Micronúcleos , Piruvaldehído/toxicidad , Espectrometría de Masas en TándemRESUMEN
R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.
Asunto(s)
Inestabilidad Cromosómica/efectos de los fármacos , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Estructuras R-Loop , Recombinasa Rad51 , Línea Celular Tumoral , Humanos , Complejo de Reconocimiento del Origen/metabolismo , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/metabolismoRESUMEN
Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ⨠3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ⨠0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ⨠0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ⨠0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.
Asunto(s)
Efecto Espectador/efectos de la radiación , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de la radiación , Mutágenos/toxicidad , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Efecto Espectador/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Cromosómica/efectos de la radiación , Daño del ADN , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Persona de Mediana Edad , Peso Molecular , Rayos XRESUMEN
Multiple sclerosis (MS), a disease in which the immune system attacks nerve cells, has been associated with both genetic and environmental risk factors. We observed increased micronucleus (MN) formation in SJL/J mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Most of these MN were due to chromosomal loss. Increased activation of MAP kinases, which leads to disruption of the mitotic spindle and improper segregation of chromosomes, is associated with MS. MAP kinase inhibitors, such as PD98059, may therefore be beneficial for MS. In the EAE model, PD98059 treatment reduced adverse effects, including MN formation, lipid peroxidation, and GSH oxidation. Interventions that mitigate chromosomal instability may have therapeutic value in MS.
Asunto(s)
Inestabilidad Cromosómica/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Flavonoides/farmacología , Proteínas Quinasas Activadas por Mitógenos/química , Esclerosis Múltiple/tratamiento farmacológico , Animales , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/patologíaRESUMEN
Caffeine, a methylxanthine analog of purine bases, is a compound that is largely consumed in beverages and medications for psychoactive and diuretic effects and plays many beneficial roles in neuronal stimulation and enhancement of anti-tumor immune responses by blocking adenosine receptors in higher organisms. In single-cell eukaryotes, however, caffeine somehow impairs cellular fitness by compromising cell wall integrity, inhibiting target of rapamycin (TOR) signaling and growth, and overriding cell cycle arrest caused by DNA damage. Among its multiple inhibitory targets, caffeine specifically interacts with phosphatidylinositol 3-kinase (PI3K)-related kinases causing radiosensitization and cytotoxicity via specialized intermediate molecules. Caffeine potentiates the lethality of cells in conjunction with several other stressors such as oxidants, irradiation, and various toxic compounds through largely unknown mechanisms. In this review, recent findings on caffeine effects and cellular detoxification schemes are highlighted and discussed with an emphasis on the inhibitory interactions between caffeine and its multiple targets in eukaryotic microorganisms such as budding and fission yeasts.
Asunto(s)
Cafeína/farmacología , Eucariontes/efectos de los fármacos , Eucariontes/genética , Inestabilidad Cromosómica/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Eucariontes/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Wnt signaling is crucial for proper development, tissue homeostasis and cell cycle regulation. A key role of Wnt signaling is the GSK3ß-mediated stabilization of ß-catenin, which mediates many of the critical roles of Wnt signaling. In addition, it was recently revealed that Wnt signaling can also act independently of ß-catenin. In fact, Wnt mediated stabilization of proteins (Wnt/STOP) that involves an LRP6-DVL-dependent signaling cascade is required for proper regulation of mitosis and for faithful chromosome segregation in human somatic cells. We show that inhibition of Wnt/LRP6 signaling causes whole chromosome missegregation and aneuploidy by triggering abnormally increased microtubule growth rates in mitotic spindles, and this is mediated by increased GSK3ß activity. We demonstrate that proper mitosis and maintenance of numerical chromosome stability requires continuous basal autocrine Wnt signaling that involves secretion of Wnts. Importantly, we identified Wnt10b as a Wnt ligand required for the maintenance of normal mitotic microtubule dynamics and for proper chromosome segregation. Thus, a self-maintaining Wnt10b-GSK3ß-driven cellular machinery ensures the proper execution of mitosis and karyotype stability in human somatic cells.
Asunto(s)
Aneuploidia , Proteínas Dishevelled/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismo , Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Cromosómica/genética , Segregación Cromosómica/efectos de los fármacos , Segregación Cromosómica/genética , Silenciador del Gen , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Mitosis/genética , Estabilidad Proteica , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Huso Acromático/metabolismo , Transfección , Proteínas Wnt/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
A novel series of benzimidazole derivatives with methoxylated aryl groups was designed and synthesized as molecules with potential cytotoxic activity. In vitro cytotoxic activity over HCT-116 cells showed that N-(benzimidazothiazolone)acetamides 11a, 11b and 11c were found to be the most cytotoxic compounds compared camptothecin (CPT). The tested compounds had a dual topoisomerase I-ß (Topo I-ß) and tubulin inhibiting activities when compared to CPT and Podophyllotoxin (Podo) where, compounds l0a, l0b, 11a and 11b exhibited a potent inhibitory activity on Topo I-ß enzyme in nano-molar concentration, on the other hand, compounds 12b and 13b exhibited the best inhibitory activity ß-tubulin polymerization. Results of the cell cycle analysis as well as the results of annexin-V on HCT-116 cells showed that benzimidazothiazoles 12b and 13b had a pro-apoptotic activity higher than CPT by 1.33- and 1.30-folds, respectively. Moreover, the concentration of p53, Bax/Bcl-2 ratio and caspase 3/7 increased in compounds l0b, 11b, l2b, 13b, especially, compounds 11b and 13b exhibited an increased level of these mediators than CPT. Finally, compound 11b regulated the radiosensitizing activity of the HCT-116 cells by modulating the chromosomal instability.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Neoplasias del Colon/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Inestabilidad Cromosómica/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacología , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Chromosomal abnormalities, as a frequent phenomenon in cultured embryonic stem cells (ESCs), is a major obstacle in the ESC application in regenerative medicine. Recent studies showed that aneuploid embryonic stem cells of humans and mice are more vulnerable to anticancer drugs, compared with normal cells. The aim of the current study was to evaluate effects of three anticancer drugs, paclitaxel, lapatinib and bortezomib, on mouse embryonic stem cells (mESCs) as a suitable and available model. To assess in vitro cell toxicity, two mESC lines were treated with the aforementioned drugs. Using G-band karyotyping and micronucleus assay, the effect of anticancer drugs in terms of reduction of chromosomal instability in the mESCs was evaluated in control and treatment groups. Also, apoptosis rate of both groups was estimated by FITC-Annexin V/Propidium Iodide (PI) double staining. In addition, the effect of these three drugs in maintaining the pluripotency was assessed through alkaline phosphatase assay and quantification of the expression of three key pluripotency genes, Nanog, Pou5f1 and Sox-2 was performed using Real Time PCR. The rate of numerical abnormalities after treatment with paclitaxel and lapatinib was lower than the control group. The expression level of pluripotency genes exhibited no significant difference between control and treatment groups. Administration of paclitaxel and lapatinib to the mESCs culture at an appropriate dose and in a timely manner could decrease chromosome stability without affecting pluripotency.