RESUMO
BACKGROUND: Targeting DNA damage response and DNA repair proficiency of cancers is an important anticancer strategy. Kaempferol (Kae), a natural flavonoid, displays potent antitumor properties in some cancers. However, the precise underlying mechanism of Kae regulates DNA repair system are poorly understood. PURPOSE: We aim to evaluate the efficacy of Kae in the treatment of human glioma as well as the molecular mechanism regarding DNA repair. STUDY DESIGN: Effects of Kae on glioma cells were detected using CCK-8 and EdU labeling assays. The molecular mechanism of Kae on glioma was determined using RNAseq. The inhibition effects of Kae on DNA repair were verified using Immunoprecipitation, immunofluorescence, and pimEJ5-GFP report assays. For in vivo study, orthotopic xenograft models were established and treated with Kae or vehicle. Glioma development was monitored by bioluminescence imaging, Magnetic Resonance Imaging (MRI), and brain sections Hematoxylin/Eosin (HE) staining. Immunohistochemical (IHC) analysis was used to detect expression of Ku80, Ki67 and γH2AX in engrafted glioma tissue. RESULTS: We found that Kae remarkably inhibits viability of glioma cells and decreases its proliferation. Mechanistically, Kae regulates multiple functional pathways associated with cancer, including non-homologous end joining (NHEJ) repair. Further studies revealed that Kae inhibits release of Ku80 from the double-strand breaks (DSBs) sites via reducing ubiquitylation and degradation of Ku80. Therefore, Kae significantly suppresses NHEJ repair and induces accumulation of DSBs in glioma cells. Moreover, Kae displays a dramatic inhibition effects on glioma growth in an orthotopic transplantation model. These data demonstrate that Kae can induce deubiquitination of Ku80, suppress NHEJ repair and inhibit glioma growth. CONCLUSION: Our findings indicate that inhibiting release of Ku80 from the DSBs by Kae may be a potential effective approach for glioma treatment.
Assuntos
Quebras de DNA de Cadeia Dupla , Glioma , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Quempferóis/farmacologia , Reparo do DNA por Junção de Extremidades , Glioma/tratamento farmacológicoRESUMO
Selenocystine (SeC) has been identified as a novel compound with broad-spectrum anticancer activity. However, the effects of SeC on modifying DNA repair mechanism were less addressed. In this study, we demonstrated that SeC selectively induced cytotoxicity and genotoxicity against HepG2 hepatoma cell line. Comet assay revealed SeC-induced DNA damage in HepG2 cells, particularly in the form of DNA double strand breaks (DSBs), corroborated by the increase expression of the DSB marker, gamma-H2AX. We further demonstrated that SeC suppressed DNA homologous recombination repair, exacerbating DNA damage accumulation. Such effects on DNA damage and cell viability inhibition were alleviated by antioxidants, glutathione and Trolox, suggesting the involvement of reactive oxygen species (ROS). High levels of intracellular and mitochondrial ROS were detected in SeC-treated HepG2. In addition, SeC impaired the expression of antioxidant enzymes (superoxidase mutases and catalase), prompting the imbalance between antioxidant protection and excessive ROS formation and eliciting DSBs and cellular death. Decreased procaspase-3, 7, and 9 and Bcl-2 proteins and an increased Bax/Bcl-2 ratio, were observed after SeC treatment, but could be reversed by Torlox, confirming the action of SeC on ROS-induced apoptosis. In vivo, the xenograft tumor model of HepG2 cells validated the inhibition of SeC on tumor growth, and the induction of DSBs and apoptosis. In summary, SeC has the capability to induce ROS-dependent DNA damage and impeded DBS repair in HepG2 cells. Thus, SeC holds great promise as a therapeutic or adjuvant agent targeting DNA repair for cancer treatment.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Antioxidantes/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Cistina/análogos & derivados , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Compostos Organosselênicos , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reparo de DNA por RecombinaçãoRESUMO
PURPOSE: Loss of TGFß signaling increases error-prone alternative end-joining (alt-EJ) DNA repair. We previously translated this mechanistic relationship as TGFß and alt-EJ gene expression signatures, which we showed are anticorrelated across cancer types. A score representing anticorrelation, ßAlt, predicts patient outcome in response to genotoxic therapy. Here we sought to verify this biology in live specimens and additional datasets. EXPERIMENTAL DESIGN: Human head and neck squamous carcinoma (HNSC) explants were treated in vitro to test whether the signatures report TGFß signaling, indicated by SMAD2 phosphorylation, and unrepaired DNA damage, indicated by persistent 53BP1 foci after irradiation or olaparib. A custom NanoString assay was implemented to analyze the signatures' expression in explants. Each signature gene was then weighted by its association with functional responses to define a modified score, ßAltw, that was retested for association with response to genotoxic therapies in independent datasets. RESULTS: Most genes in each signature were positively correlated with the expected biological response in tumor explants. Anticorrelation of TGFß and alt-EJ signatures measured by NanoString was confirmed in explants. ßAltw was significantly (P < 0.001) better than ßAlt in predicting overall survival in response to genotoxic therapy in The Cancer Genome Atlas (TCGA) pancancer patients and in independent HNSC and ovarian cancer patient datasets. CONCLUSIONS: Association of the TGFß and alt-EJ signatures with their biological response validates TGFß competency as a key mediator of DNA repair that can be readily assayed by gene expression. The predictive value of ßAltw supports its development to assist in clinical decision making.
Assuntos
Reparo do DNA por Junção de Extremidades , Neoplasias de Cabeça e Pescoço , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fator de Crescimento Transformador beta/genéticaRESUMO
PURPOSE: Poly (ADP-ribose) polymerase inhibitors (PARPi) are known to induce radiosensitization. However, the exact mechanisms of radiosensitization remain unclear. We previously reported that PARPi may have a unique radiosensitizing effect to enhance ß-components of the linear-quadratic model. The aim of this study was to evaluate PARPi in combination with high-dose-per-fraction radiotherapy and to elucidate the underlying mechanisms of its radiosensitization. MATERIALS AND METHODS: Radiosensitizing effects of PARPi PJ34, olaparib, and veliparib were measured using a colony-forming assay in the human cancer cell lines, HCT116, NCI-H460, and HT29. Six different radiation dose fractionation schedules were examined by tumor regrowth assay using three-dimensional multicellular spheroids of HCT116, NCI-H460, SW620, and HCT15. The mechanisms of radiosensitization were analyzed by measuring DNA double-strand breaks (DSB), DNA damage responses, chromosomal translocations, cellular senescence, and cell cycle analysis. RESULTS: Olaparib and PJ34 were found to show radiosensitization preferentially at higher radiation doses per fraction. Similar results were obtained using a mouse model bearing human tumor xenografts. A kinetic analysis of DNA damage responses and repairs showed that olaparib and PJ34 reduced the homologous recombination activity. However, a neutral comet assay showed that PJ34 treatment did not affect the physical rejoining of DNA-DSBs induced by ionizing radiation. Cell cycle analysis revealed that olaparib and PJ34 strikingly increased G1 tetraploid cells following irradiation, leading to premature senescence. The C-banding analysis of metaphase spreads showed that olaparib and PJ34 significantly increased ionizing radiation-induced dicentric chromosomes. The data suggests that PARPi olaparib and PJ34 altered the choice of DNA-DSB repair pathways rather than reducing the total amount of DNA-DSB repair, which resulted in increased repair errors. Increased quadratic misrepair was one of the mechanisms of PARP-mediated radiosensitization, preferentially at the higher dose range compared to the lower dose range. CONCLUSION: PARPi may be a promising candidate to combine with stereotactic hypofractionated radiotherapy, aiming at high-dose region-directed radiosensitization.
Assuntos
Neoplasias , Radiossensibilizantes , Difosfato de Adenosina , Linhagem Celular Tumoral , DNA , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Cinética , Neoplasias/genética , Neoplasias/radioterapia , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Radiossensibilizantes/farmacologia , RiboseRESUMO
Structural maintenance of chromosome 5/6 (SMC5/6) complex is a crucial factor for preserving genome stability. Here, we show that mutants for several Arabidopsis (Arabidopsis thaliana) SMC5/6 complex subunits produce triploid offspring. This phenotype is caused by a meiotic defect leading to the production of unreduced male gametes. The SMC5/6 complex mutants show an absence of chromosome segregation during the first and/or the second meiotic division, as well as a partially disorganized microtubule network. Importantly, although the SMC5/6 complex is partly required for the repair of SPO11-induced DNA double-strand breaks, the nonreduction described here is SPO11-independent. The measured high rate of ovule abortion suggests that, if produced, such defects are maternally lethal. Upon fertilization with an unreduced pollen, the unbalanced maternal and paternal genome dosage in the endosperm most likely causes seed abortion observed in several SMC5/6 complex mutants. In conclusion, we describe the function of the SMC5/6 complex in the maintenance of gametophytic ploidy in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Segregação de Cromossomos , Pólen/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Quebras de DNA de Cadeia Dupla , Meiose , Pólen/genéticaRESUMO
DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Berberina/análogos & derivados , Berberina/farmacologia , Camptotecina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , Medicina Herbária , HumanosRESUMO
The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA/genética , Distonia/genética , Feminino , Fator C1 de Célula Hospedeira/metabolismo , Proteínas Mad2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/metabolismoRESUMO
While hyperthermia (HT) is a promising modality for cancer treatment, the knowledge on mechanisms of its effect on cells is still limited. We have investigated DNA double-strand break (DSB) and apoptosis induced by HT. Umbilical cord blood lymphocytes (UCBL) were subjected to HT at 43 °C. We have treated cells for 1 h (1 h HT), 2 h (2 h HT) and by combined HT and ice treatment (both lasting 1 h). Enumeration of DSB by 53BP1/γH2AX DNA repair focus formation and early apoptosis by γH2AX pan-staining was conducted by automated fluorescent microscopy. Apoptotic stages and viability were assessed by the annexin/propidium iodide (PI) assay using flow cytometry 0, 18, and 42 h post-treatment. HT induced either immediate (2 h HT) or postponed (1 h HT) DNA damage. The levels of 53BP1 and γH2AX foci differed under the same treatment conditions, suggesting that the ratio of co-localized γH2AX/53BP1 foci to all γH2AX and also to all 53BP1 foci could be a valuable marker. The ratio of co-localized foci increased immediately after 2 h HT regardless the way of assessment. For the first time we show, by both annexin/PI and γH2AX pan-staining assay that apoptosis can be induced during or immediately after the 2 h HT treatment. Our results suggest that HT may induce DSB in dependence on treatment duration and post-treatment time due to inhibition of DNA repair pathways and that HT-induced apoptosis might be dependent or associated with DSB formation in human lymphocytes. Assessment of γH2AX pan-staining in lymphocytes affected by HT may represent a valuable marker of HT treatment side effects.
Assuntos
Quebras de DNA de Cadeia Dupla , Sangue Fetal/citologia , Temperatura Alta/efeitos adversos , Linfócitos/efeitos da radiação , Apoptose/efeitos da radiação , Reparo do DNA , Histonas , Humanos , Hipertermia Induzida , Recém-Nascido , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Among the pleotropic roles of transforming growth factor-ß (TGFß) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFß signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFß broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFß and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFß and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFß competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFß and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFß signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFß biology.
Assuntos
Reparo do DNA por Junção de Extremidades , Neoplasias , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA/genética , Humanos , Neoplasias/genética , Fator de Crescimento Transformador betaRESUMO
Significance: Persistent oxidative stress is a common feature of cancer cells, giving a specific weapon to selectively eliminate them. Ascorbate in pharmacological concentration can contribute to the suspended formation of hydroxyl radical via the Fenton reaction; thus, it can be an important element of the oxidative stress therapy against cancer cells. Recent Advances: The main components of ascorbate-induced cell death are DNA double-strand breaks via the production of hydroxyl radical and ATP depletion due to the activation of poly (ADP-ribose) polymerase 1. Presumably, DNA damage can be the primary contributor to the anticancer activity of pharmacological ascorbate, as opposed to the rupture of bioenergetics. The caspase independency of high-dose ascorbate-induced cell death proposed the possible involvement of several types of cell death, such as ferroptosis, necroptosis, and autophagy. Critical Issues: Ascorbate can target at least two key molecular features of cancer cells as a part of the anticancer therapy: the intrinsic or acquired resistance to cell death and the dysregulated metabolism of cancer cells. It seems probable that different concentrations of ascorbate alter the nature of induced cell death. Autophagy and necroptosis may play a role at intermediate concentrations, but caspase-independent apoptosis may dominate at higher concentrations. However, ascorbate behaves as an effective inhibitor of ferroptosis that may have crucial importance in its possible clinical application. Future Directions: The elucidation of the details and the links between high-dose ascorbate-induced cancer selective cell death mechanisms may give us a tool to form and apply synergistic cancer therapies. Antioxid. Redox Signal. 34, 831-844.
Assuntos
Ácido Ascórbico/uso terapêutico , Morte Celular/efeitos dos fármacos , Neoplasias/dietoterapia , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Morte Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Humanos , Necroptose/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Genome editing (GE) technology has emerged as a multifaceted strategy that instantaneously popularised the mechanism to modify the genetic constitution of an organism. The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein-based genome editing (CRISPR/Cas) approach has huge potential for efficacious editing of genomes of numerous organisms. This framework has demonstrated to be more economical in contrast to mega-nucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) for its flexibility, versatility, and potency. The advent of sequence-specific nucleases (SSNs) allowed the precise induction of double-strand breaks (DSBs) into the genome, ensuring desired alterations through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. Researchers have utilized CRISPR/Cas-mediated genome alterations across crop varieties to generate desirable characteristics for yield enhancement, enriched nutritional quality, and stressresistance. Here, we highlighted the recent progress in the area of nutritional improvement of crops via the CRISPR/Cas-based tools for fundamental plant research and crop genetic advancements. Application of this genome editing aids in unraveling the basic biology facts in plants supplemented by the incorporation of genome-wide association studies, artificial intelligence, and various bioinformatic frameworks, thereby providing futuristic model studies and their affirmations. Strategies for reducing the 'off-target' effects and the societal approval of genome-modified crops developed via this modern biotechnological approach have been reviewed.
Assuntos
Inteligência Artificial , Sistemas CRISPR-Cas/genética , Produtos Agrícolas/genética , Edição de Genes/métodos , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Genoma de Planta/genéticaRESUMO
Plumbagin (PLB), a natural naphthoquinone constituent isolated from the roots of the medicinal plant Plumbago zeylanica L., exhibited anticancer activity against a variety of cancer cell lines including breast cancer, hepatoma, leukemia, melanoma, prostate cancer, brain tumor, tongue squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, lung cancer, kidney adenocarcinoma, cholangiocarcinoma, gastric cancer, lymphocyte carcinoma, osteosarcoma, and canine cancer. PLB played anticancer activity via many molecular mechanisms, such as targeting apoptosis, autophagy pathway, cell cycle arrest, antiangiogenesis pathway, anti-invasion, and antimetastasis pathway. Among these signaling pathways, the key regulatory genes regulated by PLB were NF-kß, STAT3, and AKT. PLB also acted as a potent inducer of reactive oxygen species (ROS), suppressor of cellular glutathione, and novel proteasome inhibitor, causing DNA double-strand break by oxidative DNA base damage. This review comprehensively summarizes the anticancer activity and mechanism of PLB.
Assuntos
Antineoplásicos/farmacologia , Naftoquinonas/farmacologia , Neoplasias/tratamento farmacológico , Extratos Vegetais/farmacologia , Plantas Medicinais/metabolismo , Superóxidos/farmacologia , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa/metabolismo , Humanos , Concentração Inibidora 50 , Lipossomos/química , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Oxidantes/química , Oxigênio/química , Inibidores de Proteassoma/farmacologia , Espécies Reativas de OxigênioRESUMO
Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.
Assuntos
Técnicas Biossensoriais , Dano ao DNA , Reparo do DNA , Endófitos/química , Garcinia/microbiologia , Extratos Vegetais/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Galinhas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fermentação , Fungos/metabolismo , Garcinia/metabolismo , Histonas/metabolismo , Recombinação Homóloga , Plantas Medicinais , Sementes/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , DNA/genética , Edição de Genes/métodos , Genoma , Mutação INDEL , Animais , Clonagem de Organismos/métodos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Técnicas de Inativação de Genes , Humanos , Camundongos , Ovinos/genética , Solanum tuberosum/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and has the highest mortality rate among cancers and high resistance to radiation and cytotoxic chemotherapy. Although some targeted therapies can partially inhibit oncogenic mutation-driven proliferation of GBM cells, therapies harnessing synthetic lethality are 'coincidental' treatments with high effectiveness in cancers with gene mutations, such as GBM, which frequently exhibits DNA-PKcs mutation. By implementing a highly efficient high-throughput screening (HTS) platform using an in-house-constructed genome-wide human microRNA inhibitor library, we demonstrated that miR-1193 inhibition sensitized GBM tumor cells with DNA-PKcs deficiency. Furthermore, we found that miR-1193 directly targets YY1AP1, leading to subsequent inhibition of FEN1, an important factor in DNA damage repair. Inhibition of miR-1193 resulted in accumulation of DNA double-strand breaks and thus increased genomic instability. RPA-coated ssDNA structures enhanced ATR checkpoint kinase activity, subsequently activating the CHK1/p53/apoptosis axis. These data provide a preclinical theory for the application of miR-1193 inhibition as a potential synthetic lethal approach targeting GBM cancer cells with DNA-PKcs deficiency.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Proteína Quinase Ativada por DNA/deficiência , Glioblastoma/enzimologia , Glioblastoma/genética , MicroRNAs/metabolismo , Mutações Sintéticas Letais/genética , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/metabolismo , Endonucleases Flap/metabolismo , Instabilidade Genômica , Humanos , MicroRNAs/genética , Modelos Biológicos , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/metabolismoRESUMO
Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteína BRCA2/genética , Cromatina/química , Proteínas de Ligação a DNA/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Reparo de DNA por Recombinação , ATPases Associadas a Diversas Atividades Celulares/deficiência , Animais , Apoptose/genética , Proteína BRCA2/deficiência , Divisão Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ciclo Celular , Sobrevivência Celular , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
Triptolide is a multi-functional natural small molecular compound extracted from a traditional Chinese medicinal herb. Triptolide and its derivatives exhibit cytotoxicity through inducing DNA damage, therefore increasing sensitivity to DNA-damage based chemotherapy or radiotherapy in different types of cells. However, the regulatory mechanism of genotoxicity by triptolide, and the loss of genome integrity induced by triptolide are not fully understood. Here, we measured the effects of triptolide on genome integrity in a human fibroblast line HCA2-hTERT using the neutral comet assay. We demonstrated that treating cells with triptolide induced genomic instability in HCA2-hTERT cells. Furthermore, we observed the accumulation of γH2AX foci in triptolide treated cells than control cells at 24 h post ionizing radiation. Further mechanistic studies indicated that triptolide inhibited the enzymatic activity of DNA-PKcs, the critical nonhomologous end joining factor. In vitro kinase activity assays showed that triptolide suppressed the kinase activity of DNA-PKcs and molecular docking also predicted a potential interaction between triptolide and DNA-PKcs. As a consequence, we found that triptolide treatment enhanced the interaction between DNA-PKcs and KU80 and hampered the following recruitment of 53BP1. Altogether, our finding provides a new perspective about the toxicity of triptolide in non-cancer cells and highlights the necessity of taking genome effects of triptolide and its derivatives into consideration in the future clinical and research applications.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Diterpenos/toxicidade , Fibroblastos/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Fenantrenos/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Compostos de Epóxi/toxicidade , Fibroblastos/enzimologia , Fibroblastos/patologia , Histonas/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Fosforilação , Telomerase/genética , Telomerase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Reparo do DNA/efeitos dos fármacos , Endófitos/química , Glioblastoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Mutações Sintéticas Letais/genética , Neoplasias Encefálicas/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaio Cometa , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Equador , Glioblastoma/genética , Humanos , Estrutura Molecular , Mutagênicos/toxicidade , Ensaio Tumoral de Célula-TroncoRESUMO
Natural compounds isolated from plants are excellent starting points in drug design and have been widely studied as anticancer agents; they hence find use in a considerable proportion of anticancer drugs. The genus Plectranthus (Lamiaceae) comprises a large and widespread group of species with various applications in traditional medicine. Therefore, the aim of the present study was to determine the effectiveness of treatment with four abietane diterpenoids isolated from P.madagascariensis and P.ecklonii, 6,7-dehydroroyleanone, 7ß,6ß-dihydroxyroyleanone, 7α-acetoxy-6ß-hydroxyroyleanone and parvifloron D, in initiating apoptosis in a glioma cell line. The pure compounds were found to exhibit anticancer effects in primary H7PX glioma cells line by inducing apoptosis G2/M cell cycle arrest and double-strand breaks, indicated by increased levels of phosphorylated H2A.X and decreasing mitochondrial membrane potential; they also influenced the expression of pro- and anti-apoptotic genes (Bax, Bcl-2, TP53, or Cas-3). Our findings indicate that these compounds may offer potential as beneficial antitumor drugs but further in vivo studies are needed.