RESUMEN
53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.
Asunto(s)
Reparación del ADN , Complejos Multiproteicos/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/genética , Femenino , Genes BRCA1 , Humanos , Cambio de Clase de Inmunoglobulina/genética , Ratones , Modelos Biológicos , Complejos Multiproteicos/química , Complejos Multiproteicos/deficiencia , Complejos Multiproteicos/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de Unión a Telómeros/metabolismo , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of the stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation (γH2AX) and is not reduced by DNA polymerase θ (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies γH2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations, and warrants further exploration in vivo to determine if the phenomenon has implications for radiation risk assessment.
Asunto(s)
Histonas/metabolismo , Mutagénesis Insercional/efectos de la radiación , Radiación Ionizante , Animales , Línea Celular , Células Cultivadas , Roturas del ADN de Doble Cadena , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Ratones , Reparación del ADN por Recombinación , ADN Polimerasa thetaRESUMEN
The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that ectopic production of HSF2BP, a BRCA2-interacting protein required for meiotic HR during mouse spermatogenesis, in non-germline human cells acutely sensitize them to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP compromises HR by triggering the removal of BRCA2 from the ICL site and thereby preventing the loading of RAD51. This establishes ectopic expression of a wild-type meiotic protein in the absence of any other protein-coding mutations as a new mechanism that can lead to an FA-like cellular phenotype. Naturally occurring elevated production of HSF2BP in tumors may be a source of cancer-promoting genomic instability and also a targetable vulnerability.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN , Proteínas de Choque Térmico/metabolismo , Recombinación Homóloga , Animales , Proteína BRCA2/metabolismo , Línea Celular , Daño del ADN , Anemia de Fanconi/genética , Humanos , Ratones , Unión Proteica , Proteolisis , Recombinasa Rad51/metabolismo , XenopusRESUMEN
Cobalt metal and cobalt sulfate are carcinogenic in rodents following inhalation exposure. The pre-carcinogenic effects associated with exposure to these cobalt substances include oxidative stress and genotoxicity. Some, but not all, cobalt substances induce in vitro clastogenicity or an increase in micronuclei. As a result, these substances are classified genotoxic carcinogens, having major impacts on their risk assessment, e.g. assumption of a non-thresholded dose response. Here, we investigated the potential of nine cobalt substances to cause genotoxicity and oxidative stress using the ToxTracker assay, with an extension to measure biomarkers of hypoxia. None of the nine tested substances activated the DNA damage markers in ToxTracker, and five substances activated the oxidative stress response reporters. The same five substances also activated the expression of several hypoxia target genes. Consistent with the lower tier of testing found in the preceding paper of this series, these compounds can be grouped based on their ability to release bioavailable cobalt ion and to trigger subsequent key events.
Asunto(s)
Carcinógenos/química , Carcinógenos/farmacología , Cobalto/química , Cobalto/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Administración por Inhalación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Genotipo , Pruebas de Mutagenicidad , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Tamaño de la PartículaRESUMEN
In vitro (geno)toxicity assessment of electronic vapour products (EVPs), relative to conventional cigarette, currently uses assays, including the micronucleus and Ames tests. Whilst informative on induction of a finite endpoint and relative risk posed by test articles, such assays could benefit from mechanistic supplementation. The ToxTracker and Aneugen Clastogen Evaluation analysis can indicate the activation of reporters associated with (geno)toxicity, including DNA damage, oxidative stress, the p53-related stress response and protein damage. Here, we tested for the different effects of a selection of neat e-liquids, EVP aerosols and Kentucky reference 1R6F cigarette smoke samples in the ToxTracker assay. The assay was initially validated to assess whether a mixture of e-liquid base components, propylene glycol (PG) and vegetable glycerine (VG) had interfering effects within the system. This was achieved by spiking three positive controls into the system with neat PG/VG or phosphate-buffered saline bubbled (bPBS) PG/VG aerosol (nicotine and flavour free). PG/VG did not greatly affect responses induced by the compounds. Next, when compared to cigarette smoke samples, neat e-liquids and bPBS aerosols (tobacco flavour; 1.6% freebase nicotine, 1.6% nicotine salt or 0% nicotine) exhibited reduced and less complex responses. Tested up to a 10% concentration, EVP aerosol bPBS did not induce any ToxTracker reporters. Neat e-liquids, tested up to 1%, induced oxidative stress reporters, thought to be due to their effects on osmolarity in vitro. E-liquid nicotine content did not affect responses induced. Additionally, spiking nicotine alone only induced an oxidative stress response at a supraphysiological level. In conclusion, the ToxTracker assay is a quick, informative screen for genotoxic potential and mechanisms of a variety of (compositionally complex) samples, derived from cigarettes and EVPs. This assay has the potential for future application in the assessment battery for next-generation (smoking alternative) products, including EVPs.
Asunto(s)
Aneugénicos/toxicidad , Sistemas Electrónicos de Liberación de Nicotina , Glicerol/toxicidad , Pruebas de Mutagenicidad/métodos , Nicotiana/toxicidad , Nicotina/toxicidad , Propilenglicol/toxicidad , Aerosoles/efectos adversos , Aerosoles/análisis , Animales , Fumar Cigarrillos/efectos adversos , Daño del ADN , Glicerol/análisis , Humanos , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones , Mutágenos/toxicidad , Nicotina/análisis , Estrés Oxidativo , Propilenglicol/análisis , Medición de Riesgo , Humo/efectos adversos , Fumar/efectos adversosRESUMEN
Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas Mad2/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Recombinación , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Humanos , Cambio de Clase de Inmunoglobulina/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mad2/deficiencia , Proteínas Mad2/genética , Ratones , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system. METHODS: We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities. RESULTS: Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations. CONCLUSIONS: Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Citidina Desaminasa/genética , Daño del ADN/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Menor/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Western Blotting , Sistemas CRISPR-Cas , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa de Punto de Control 2/antagonistas & inhibidores , Cisplatino/farmacología , Citidina Desaminasa/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Mutación , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Interferencia de ARN , Uracilo/metabolismoRESUMEN
ToxTracker is an in vitro mammalian stem cell-based reporter assay that detects activation of specific cellular signaling pathways (DNA damage, oxidative stress, and/or protein damage) upon chemical exposure using flow cytometry. Here we used quantitative methods to empirically analyze historical control data, and dose-response data across a wide range of reference chemicals. First, we analyzed historical control data to define a fold-change threshold for identification of a significant positive response. Next, we used the benchmark dose (BMD) combined-covariate approach for potency ranking of a set of more than 120 compounds; the BMD values were used for comparative identification of the most potent inducers of each reporter. Lastly, we used principal component analysis (PCA) to investigate functional and statistical relationships between the ToxTracker reporters. The PCA results, based on the BMD results for all substances, indicated that the DNA damage (Rtkn, Bscl2) and p53 (Btg2) reporters are functionally complementary and indicative of genotoxic stress. The oxidative stress (Srxn1 and Blvrb) and protein stress (Ddit3) reporters are independent indicators of cellular stress, and essential for toxicological profiling using the ToxTracker assay. Overall, dose-response modeling of multivariate ToxTracker data can be used for potency ranking and mode-of-action determination. In the future, IVIVE (in vitro to in vivo extrapolation) methods can be employed to determine in vivo AED (administered equivalent dose) values that can in turn be used for human health risk assessment.
Asunto(s)
Daño del ADN , Estrés Oxidativo , Pruebas de Toxicidad , Animales , Humanos , Mamíferos/genética , Pruebas de Mutagenicidad/métodos , Medición de Riesgo , Proteínas Supresoras de Tumor/genética , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/estadística & datos numéricosRESUMEN
To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2-octen-4-one, lauric aldehyde, veratraldehyde, and p-methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow-up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal-based follow-up testing with appropriate animal alternative assay(s).
Asunto(s)
Daño del ADN , Odorantes , Animales , Humanos , Pruebas de Micronúcleos/métodos , Piel , Pruebas de Mutagenicidad/métodosRESUMEN
Nucleoside analogues have long been designed and tested in cancer treatment and against viral infections. However, several early compounds were shown to have mutagenic properties as a consequence of their mode-of-action. This limited their use, and several have been discontinued for lengthy treatments or altogether. Nonetheless, nucleoside analogues remain an attractive modality for virally driven diseases, of which many still are without proper treatment options. To quantitatively assess the genotoxic mode-of-action of a panel of nucleoside analogues, we applied the ToxTracker® reporter assay. Many of the early nucleoside analogues showed a genotoxic response. The more recently developed nucleoside analogues, Remdesivir and Molnupiravir that are currently being repurposed for Covid-19 treatment, had a different profile in ToxTracker and did not induce the genotoxicity reporters. Our analyses support the metabolite GS-441524 over the parent analogue Remdesivir. In contrast, Molnupiravir was devoid of clear cellular toxicity while its active metabolite (EIDD-1931) was cytotoxic and induced several biomarkers. Nucleoside analogues continue to be attractive treatment options upon viral infections. ToxTracker readily distinguished between the genotoxic analogues and those with different profiles and provides a basis for clustering and potency ranking, offering a comprehensive tool to assess the toxicity of nucleoside analogues.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Mutágenos , Daño del ADN , Humanos , Mutágenos/toxicidad , Nucleósidos/toxicidadRESUMEN
Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment.
Asunto(s)
Aneugénicos , Venenos , Aneugénicos/toxicidad , División Celular , Pruebas de Micronúcleos/métodos , Microtúbulos , Mutágenos/farmacología , Venenos/farmacología , Tubulina (Proteína)RESUMEN
BACKGROUND: Testing for developmental toxicity according to the current regulatory guidelines requires large numbers of animals, making these tests very resource intensive, time-consuming, and ethically debatable. Over the past decades, several alternative in vitro assays have been developed, but these often suffered from low predictability and the inability to provide a mechanistic understanding of developmental toxicity. METHODS: To identify embryotoxic compounds, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay. The assay is based on the differentiation of hiPSCs into functional cardiomyocytes and hepatocytes. Proper stem cell differentiation is investigated by morphological profiling and assessment of time-dependent expression patterns of cell-specific biomarkers. In this system, a decrease in the expression of the biomarker genes and morphology disruption of the differentiated cells following compound treatment indicated teratogenicity. RESULTS: The hiPSCs-based biomarker assay was validated with 21 well-established in vivo animal teratogenic and non-teratogenic compounds during cardiomyocyte and hepatocyte differentiation. The in vivo teratogenic compounds (e.g., thalidomide and valproic acid) markedly disrupted morphology, functionality, and the expression pattern of the biomarker genes in either one or both cell types. Non-teratogenic chemicals generally had no effect on the morphology of differentiated cells, nor on the expression of the biomarker genes. Compared to the in vivo classification, the assay achieved high accuracy (91%), sensitivity (91%), and specificity (90%). CONCLUSION: The assay, which we named ReproTracker®, is a state-of-the-art in vitro method that can identify the teratogenicity potential of new pharmaceuticals and chemicals and signify the outcome of in vivo test systems.
Asunto(s)
Células Madre Pluripotentes Inducidas , Teratogénesis , Animales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Pruebas de Toxicidad/métodos , Teratógenos/farmacología , Diferenciación Celular , Biomarcadores/metabolismoRESUMEN
The toxicity of co-formulants present in glyphosate-based herbicides (GBHs) has been widely discussed leading to the European Union banning the polyoxyethylene tallow amine (POEA). We identified the most commonly used POEA, known as POE-15 tallow amine (POE-15), in the widely used US GBH RangerPro. Cytotoxicity assays using human intestinal epithelial Caco-2 and hepatocyte HepG2 cell lines showed that RangerPro and POE-15 are far more cytotoxic than glyphosate alone. RangerPro and POE-15 but not glyphosate caused cell necrosis in both cell lines, and that glyphosate and RangerPro but not POE-15 caused oxidative stress in HepG2 cells. We further tested these pesticide ingredients in the ToxTracker assay, a system used to evaluate a compound's carcinogenic potential, to assess their capability for inducing DNA damage, oxidative stress and an unfolded protein response (endoplasmic reticulum, ER stress). RangerPro and POE-15 but not glyphosate gave rise to ER stress. We conclude that the toxicity resulting from RangerPro exposure is thus multifactorial involving ER stress caused by POE-15 along with oxidative stress caused by glyphosate. Our observations reinforce the need to test both co-formulants and active ingredients of commercial pesticides to inform the enactment of more appropriate regulation and thus better public and environmental protection.
Asunto(s)
Herbicidas , Aminas/toxicidad , Células CACO-2 , Excipientes , Grasas , Herbicidas/toxicidad , Humanos , Necrosis/inducido químicamente , Polietilenglicoles , Tensoactivos/toxicidadRESUMEN
Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remain controversial. As GBHs are more cytotoxic than glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (United Kingdom), and MON 76207 (United States) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased miR-30, whereas miR-10 levels were increased. DNA methylation profiling of liver revealed 5727 and 4496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals, respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate.
Asunto(s)
Herbicidas , MicroARNs , Animales , Daño del ADN , Femenino , Glicina/análogos & derivados , Herbicidas/toxicidad , Mamíferos , Ratas , Ratas Sprague-Dawley , Células Madre , Toxicogenética , GlifosatoRESUMEN
The current generation of carcinogenicity tests is often insufficient to predict cancer outcomes from pesticide exposures. In order to facilitate health risk assessment, The International Agency for Research on Cancer identified 10 key characteristics which are commonly exhibited by human carcinogens. The ToxTracker panel of six validated GFP-based mouse embryonic stem reporter cell lines is designed to measure a number of these carcinogenic properties namely DNA damage, oxidative stress and the unfolded protein response. Here we present an evaluation of the carcinogenic potential of the herbicides glyphosate, 2,4-D and dicamba either alone or in combination, using the ToxTracker assay system. The pesticide 2,4-D was found to be a strong inducer of oxidative stress and an unfolded protein response. Dicamba induced a mild oxidative stress response, whilst glyphosate did not elicit a positive outcome in any of the assays. The results from a mixture of the three herbicides was primarily an oxidative stress response, which was most likely due to 2,4-D with dicamba or glyphosate only playing a minor role. These findings provide initial information regarding the risk assessment of carcinogenic effects arising from exposure to a mixture of these herbicides.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/toxicidad , Daño del ADN/efectos de los fármacos , Dicamba/toxicidad , Glicina/análogos & derivados , Herbicidas/toxicidad , Pruebas de Mutagenicidad , Estrés Oxidativo/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , Ácido 2,4-Diclorofenoxiacético/administración & dosificación , Animales , Dicamba/administración & dosificación , Relación Dosis-Respuesta a Droga , Glicina/administración & dosificación , Glicina/toxicidad , Herbicidas/administración & dosificación , Humanos , Ratones , Pruebas de Mutagenicidad/métodos , Ratas , GlifosatoRESUMEN
Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of 6 different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress, and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of 2 independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here, we show that the differential activation of these 2 genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic MOA of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established MOA. Furthermore, indirect genotoxicity related to the production of reactive oxygen species was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different reactive oxygen species scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or nongenotoxic and could discriminate between DNA-reactive compounds, aneugens, and indirect genotoxicity caused by oxidative stress.
Asunto(s)
Aneugénicos , Mutágenos , Aneugénicos/toxicidad , Biomarcadores/metabolismo , Daño del ADN , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Estrés OxidativoRESUMEN
The DNA damage response (DDR) is a designation for a number of pathways that protects our DNA from various damaging agents. In normal cells, the DDR is extremely important for maintaining genome integrity, but in cancer cells these mechanisms counteract therapy-induced DNA damage. Inhibition of the DDR could therefore be used to increase the efficacy of anti-cancer treatments. Hyperthermia is an example of such a treatment-it inhibits a sub-pathway of the DDR, called homologous recombination (HR). It does so by inducing proteasomal degradation of BRCA2 -one of the key HR factors. Understanding the precise mechanism that mediates this degradation is important for our understanding of how hyperthermia affects therapy and how homologous recombination and BRCA2 itself function. In addition, mechanistic insight into the process of hyperthermia-induced BRCA2 degradation can yield new therapeutic strategies to enhance the effects of local hyperthermia or to inhibit HR. Here, we investigate the mechanisms driving hyperthermia-induced BRCA2 degradation. We find that BRCA2 degradation is evolutionarily conserved, that BRCA2 stability is dependent on HSP90, that ubiquitin might not be involved in directly targeting BRCA2 for protein degradation via the proteasome, and that BRCA2 degradation might be modulated by oxidative stress and radical scavengers.
RESUMEN
The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2.
Asunto(s)
Proteína BRCA2/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Espermatogénesis , Animales , Proteína BRCA2/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Proteínas de Choque Térmico/genética , Humanos , Ratones , Mutación Missense , Dominios ProteicosRESUMEN
Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.
Asunto(s)
Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasa-1/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Anciano , Animales , Proteína BRCA1/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Embrionarias de Ratones , Mutagénesis , Neoplasias/genética , Neoplasias/patología , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Mutación Puntual , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Medicina de Precisión/métodos , Secuenciación Completa del Genoma/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Dedos de Zinc/genéticaRESUMEN
INTRODUCTION: Defects in the DNA damage response (DDR) drive the development of cancer by fostering DNA mutation but also provide cancer-specific vulnerabilities that can be exploited therapeutically. The recent approval of three different PARP inhibitors for the treatment of ovarian cancer provides the impetus for further developing targeted inhibitors of many of the kinases involved in the DDR, including inhibitors of ATR, ATM, CHEK1, CHEK2, DNAPK and WEE1. Areas covered: We summarise the current stage of development of these novel DDR kinase inhibitors, and describe which predictive biomarkers might be exploited to direct their clinical use. Expert opinion: Novel DDR inhibitors present promising candidates in cancer treatment and have the potential to elicit synthetic lethal effects. In order to fully exploit their potential and maximize their utility, identifying highly penetrant predictive biomarkers of single agent and combinatorial DDR inhibitor sensitivity are critical. Identifying the optimal drug combination regimens that could used with DDR inhibitors is also a key objective.