RESUMO
Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.
Assuntos
Antígeno B7-H1 , Núcleo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku , Humanos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Animais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/patologia , Camundongos , Glicosilação , Radiação Ionizante , Sistemas CRISPR-CasRESUMO
Objective: Ionizing radiation generates genomic instability by promoting the accumulation of chromosomal rearrangements. The oncogenic translocation RET/PTC1 is present in more than 70% of radiation-induced thyroid cancers. Both RET and CCDC6, the genes implicated in RET/PTC1, are found within common fragile sites - chromosomal regions prone to DNA breakage during slight replication stress. Given that irradiated cells become more susceptible to genomic destabilization due to the accumulation of replication-stress-related double-strand breaks (DSBs), we explored whether RET and CCDC6 exhibit DNA breakage under replicative stress several days post-irradiation of thyroid cells. Methods: We analyzed the dynamic of DNA replication in human thyroid epithelial cells (HThy-ori-3.1) 4 days post a 5-Gy exposure using molecular DNA combing. The DNA replication schedule was evaluated through replication-timing experiments. We implemented a ChIP-qPCR assay to determine whether the RET and CCDC6 genes break following irradiation. Results: Our study indicates that replicative stress, occurring several days post-irradiation in thyroid cells, primarily causes DSBs in the RET gene. We discovered that both the RET and CCDC6 genes undergo late replication in thyroid cells. However, only RET's replication rate is notably delayed after irradiation. Conclusion: The findings suggest that post-irradiation in the RET gene causes a breakage in the replication fork, which could potentially invade another genomic area, including CCDC6. As a result, this could greatly contribute to the high prevalence of chromosomal RET/PTC rearrangements seen in patients exposed to external radiation.
Assuntos
Replicação do DNA , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Glândula Tireoide , Humanos , Replicação do DNA/efeitos da radiação , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Glândula Tireoide/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Linhagem Celular , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/radioterapia , Células Epiteliais/efeitos da radiação , Células Epiteliais/metabolismo , Proteínas do CitoesqueletoRESUMO
BACKGROUND/AIM: Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. MATERIALS AND METHODS: The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. RESULTS: Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. CONCLUSION: The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.
Assuntos
Reparo do DNA , Radioterapia com Íons Pesados , Melatonina , Tolerância a Radiação , Radiossensibilizantes , Melatonina/farmacologia , Linhagem Celular Tumoral , Tolerância a Radiação/efeitos dos fármacos , Camundongos , Animais , Humanos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Radiossensibilizantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Melanoma Experimental/radioterapia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiaçãoRESUMO
Radiation cytogenetics has a rich history seldom appreciated by those outside the field. Early radiobiology was dominated by physics and biophysical concepts that borrowed heavily from the study of radiation-induced chromosome aberrations. From such studies, quantitative relationships between biological effect and changes in absorbed dose, dose rate and ionization density were codified into key concepts of radiobiological theory that have persisted for nearly a century. This review aims to provide a historical perspective of some of these concepts, including evidence supporting the contention that chromosome aberrations underlie development of many, if not most, of the biological effects of concern for humans exposed to ionizing radiations including cancer induction, on the one hand, and tumor eradication on the other. The significance of discoveries originating from these studies has widened and extended far beyond their original scope. Chromosome structural rearrangements viewed in mitotic cells were first attributed to the production of breaks by the radiations during interphase, followed by the rejoining or mis-rejoining among ends of other nearby breaks. These relatively modest beginnings eventually led to the discovery and characterization of DNA repair of double-strand breaks by non-homologous end joining, whose importance to various biological processes is now widely appreciated. Two examples, among many, are V(D)J recombination and speciation. Rapid technological advancements in cytogenetics, the burgeoning fields of molecular radiobiology and third-generation sequencing served as a point of confluence between the old and new. As a result, the emergent field of "cytogenomics" now becomes uniquely positioned for the purpose of more fully understanding mechanisms underlying the biological effects of ionizing radiation exposure.
Assuntos
Aberrações Cromossômicas , Citogenética , Radiobiologia , Humanos , Aberrações Cromossômicas/efeitos da radiação , Animais , Reparo do DNA/efeitos da radiação , Radiação Ionizante , História do Século XX , Quebras de DNA de Cadeia Dupla/efeitos da radiaçãoRESUMO
Rhabdomyosarcoma (RMS) represents one of the most lethal soft-tissue sarcomas in children. The toxic trace element arsenic has been reported to function as a radiosensitizer in sarcomas. To investigate the role of arsenic sulfide (As4S4) in enhancing radiation sensitization in RMS, this study was conducted to elucidate its underlying mechanism in radiotherapy. The combination of As4S4 and radiotherapy showed significant inhibition in RMS cells, as demonstrated by the cell counting kit-8 (CCK-8) assay and flow cytometry. Subsequently, we demonstrated for the first time that As4S4, as well as the knockdown of NFATc3 led to double-strand break (DSB) through increased expression of RAG1. In vivo experiment confirmed that co-treatment efficiently inhibited RMS growth. Furthermore, survival analysis of a clinical cohort consisting of 59 patients revealed a correlation between NFATc3 and RAG1 expression and overall survival (OS). Cox regression analysis also confirmed the independent prognostic significance of NFATc3 and RAG1.Taken together, As4S4 enhances radiosensitivity in RMS via activating NFATc3-RAG1 mediated DSB. NFATc3 and RAG1 are potential therapeutic targets. As4S4 will hopefully serve as a prospective radio-sensitizing agent for RMS.
Assuntos
Arsenicais , Quebras de DNA de Cadeia Dupla , Fatores de Transcrição NFATC , Tolerância a Radiação , Rabdomiossarcoma , Sulfetos , Humanos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Sulfetos/farmacologia , Sulfetos/uso terapêutico , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/radioterapia , Rabdomiossarcoma/patologia , Rabdomiossarcoma/genética , Linhagem Celular Tumoral , Masculino , Feminino , Arsenicais/farmacologia , Arsenicais/uso terapêutico , Animais , Tolerância a Radiação/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Camundongos , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Camundongos Nus , Criança , Radiossensibilizantes/farmacologia , Radiossensibilizantes/uso terapêutico , Camundongos Endogâmicos BALB CRESUMO
PURPOSE: Interdisciplinary scientific communities have shown large interest to achieve a mechanistic description of radiation-induced biological damage, aiming to predict biological results produced by different radiation quality exposures. Monte Carlo track-structure simulations are suitable and reliable for the study of early DNA damage induction used as input for assessing DNA damage. This study presents the most recent improvements of a Geant4-DNA simulation tool named "dsbandrepair". METHODS: "dsbandrepair" is a Monte Carlo simulation tool based on a previous code (FullSim) that estimates the induction of early DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). It uses DNA geometries generated by the DNAFabric computational tool for simulating the induction of early single-strand breaks (SSBs) and double-strand breaks (DSBs). Moreover, the new tool includes some published radiobiological models for survival fraction and un-rejoined DSB. Its application for a human fibroblast cell and human umbilical vein endothelial cell containing both heterochromatin and euchromatin was conducted. In addition, this new version offers the possibility of using the new IRT-syn method for computing the chemical stage. RESULTS: The direct and indirect strand breaks, SSBs, DSBs, and damage complexity obtained in this work are equivalent to those obtained with the previously published simulation tool when using the same configuration in the physical and chemical stages. Simulation results on survival fraction and un-rejoined DSB are in reasonable agreement with experimental data. CONCLUSIONS: "dsbandrepair" is a tool for simulating DNA damage and repair, benchmarked against experimental data. It has been released as an advanced example in Geant4.11.2.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Método de Monte Carlo , Humanos , Reparo do DNA/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA , DNA/efeitos da radiação , Simulação por Computador , Células Endoteliais da Veia Umbilical Humana , Quebras de DNA de Cadeia Simples/efeitos da radiação , SoftwareRESUMO
PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.
Assuntos
Proteína-Arginina N-Metiltransferases , Tolerância a Radiação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Animais , Humanos , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Linhagem Celular Tumoral , Camundongos , Reparo do DNA , Proliferação de Células/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Camundongos NusRESUMO
Radiation delivery at ultrahigh dose rates (UHDRs) has potential for use as a new anticancer therapeutic strategy. The FLASH effect induced by UHDR irradiation has been shown to maintain antitumour efficacy with a reduction in normal tissue toxicity; however, the FLASH effect has been difficult to demonstrate in vitro. The objective to demonstrate the FLASH effect in vitro is challenging, aiming to reveal a differential response between cancer and normal cells to further identify cell molecular mechanisms. New high-intensity petawatt laser-driven accelerators can deliver very high-energy electrons (VHEEs) at dose rates as high as 1013 Gy/s in very short pulses (10-13 s). Here, we present the first in vitro experiments carried out on cancer cells and normal non-transformed cells concurrently exposed to laser-plasma accelerated (LPA) electrons. Specifically, melanoma cancer cells and normal melanocyte co-cultures grown on chamber slides were simultaneously irradiated with LPA electrons. A non-uniform dose distribution on the cell cultures was revealed by Gafchromic films placed behind the chamber slide supporting the cells. In parallel experiments, cell co-cultures were exposed to pulsed X-ray irradiation, which served as positive controls for radiation-induced nuclear DNA double-strand breaks. By measuring the impact on discrete areas of the cell monolayers, the greatest proportion of the damaged DNA-containing nuclei was attained by the LPA electrons at a cumulative dose one order of magnitude lower than the dose obtained by pulsed X-ray irradiation. Interestingly, in certain discrete areas, we observed that LPA electron exposure had a different effect on the DNA damage in healthy normal human epidermal melanocyte (NHEM) cells than in A375 melanoma cells; here, the normal cells were less affected by the LPA exposure than cancer cells. This result is the first in vitro demonstration of a differential response of tumour and normal cells exposed to FLASH irradiation and may contribute to the development of new cell culture strategies to explore fundamental understanding of FLASH-induced cell effect.
Assuntos
Técnicas de Cocultura , Elétrons , Lasers , Humanos , Técnicas de Cocultura/métodos , Linhagem Celular Tumoral , Melanócitos/efeitos da radiação , Dano ao DNA , Melanoma/radioterapia , Melanoma/patologia , Quebras de DNA de Cadeia Dupla/efeitos da radiaçãoRESUMO
Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1ß leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.
Assuntos
Autofagia , Hipóxia Celular , Tolerância a Radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Autofagia/efeitos da radiação , Autofagia/genética , Tolerância a Radiação/genética , Linhagem Celular Tumoral , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína Beclina-1/metabolismo , Proteína Beclina-1/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Reparo do DNA/efeitos da radiação , Reparo do DNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Raios X , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas Supressoras de TumorRESUMO
Background. Radiation-induced DNA damages such as Single Strand Break (SSB), Double Strand Break (DSB) and Complex DSB (cDSB) are critical aspects of radiobiology with implications in radiotherapy and radiation protection applications.Materials and Methods. This study presents a thorough investigation into the effects of protons (0.1-100 MeV/u), helium ions (0.13-100 MeV/u) and carbon ions (0.5-480 MeV/u) on DNA of human fibroblast cells using Geant4-DNA track structure code coupled with DBSCAN algorithm and Monte Carlo Damage Simulations (MCDS) code. Geant4-DNA-based simulations consider 1µm × 1µm × 0.5µm water box as the target to calculate energy deposition on event-by-event basis and the three-dimensional coordinates of the interaction location, and then DBSCAN algorithm is used to calculate yields of SSB, DSB and cDSB in human fibroblast cell. The study investigated the influence of Linear Energy Transfer (LET) of protons, helium ions and carbon ions on the yields of DNA damages. Influence of cellular oxygenation on DNA damage patterns is investigated using MCDS code.Results. The study shows that DSB and SSB yields are influenced by the LET of the particles, with distinct trends observed for different particles. The cellular oxygenation is a key factor, with anoxic cells exhibiting reduced SSB and DSB yields, underscoring the intricate relationship between cellular oxygen levels and DNA damage. The study introduced DSB/SSB ratio as an informative metric for evaluating the severity of radiation-induced DNA damage, particularly in higher LET regions.Conclusions. The study highlights the importance of considering particle type, LET, and cellular oxygenation in assessing the biological effects of ionizing radiation.
Assuntos
Algoritmos , Carbono , Dano ao DNA , DNA , Fibroblastos , Hélio , Transferência Linear de Energia , Método de Monte Carlo , Prótons , Humanos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Carbono/química , Íons , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Simulação por Computador , Quebras de DNA de Cadeia Simples/efeitos da radiaçãoRESUMO
Exposure to UV affects the development and growth of a wide range of organisms. Nowadays, researchers are focusing on the impact of UV radiation and its underlying molecular mechanisms, as well as devising strategies to mitigate its harmful effects. Different forms of UV radiation, their typical exposure effects, the impact of UV on DNA integrity, and the deterioration of genetic material are discussed in this review; furthermore, we also review the effects of UV radiation that affect the biological functions of the organisms. Subsequently, we address the processes that aid organisms in navigating the damage in genetic material, neuroinflammation, and neurodegeneration brought on by UV-mediated double-strand breaks. To emphasize the molecular pathways, we conclude the review by going over the animal model studies that highlight the genes and proteins that are impacted by UV radiation.
Assuntos
Quebras de DNA de Cadeia Dupla , Doenças Neurodegenerativas , Doenças Neuroinflamatórias , Raios Ultravioleta , Raios Ultravioleta/efeitos adversos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/genética , Animais , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Humanos , Doenças Neuroinflamatórias/etiologia , Reparo do DNA/genética , Dano ao DNA/efeitos da radiaçãoRESUMO
Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.
Assuntos
Astronautas , Radiação Cósmica , MicroRNAs , Voo Espacial , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Radiação Cósmica/efeitos adversos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Lesões por Radiação/genética , Lesões por Radiação/prevenção & controle , Masculino , Mitocôndrias/efeitos da radiação , Mitocôndrias/metabolismo , Mitocôndrias/genética , Feminino , AdultoRESUMO
Small fractions of patients suffer from radiotherapy late severe adverse events (AEs Grade ≥ 3), which are usually irreversible and badly affect their quality of life. A novel functional DNA repair assay characterizing several steps of double-strand break (DSB) repair mechanisms was used. DNA repair activities of peripheral blood mononuclear cells were monitored for 1 week using NEXT-SPOT assay in 177 breast and prostate cancer patients. Only seven patients had Grade ≥ 3 AEs, 6 months after radiotherapy initiation. The machine learning method established the importance of variables among demographic, clinical and DNA repair data. The most relevant ones, all related to DNA repair, were employed to build a predictor. Predictors constructed with random forest and minimum bounding sphere predicted late Grade ≥ 3 AEs with a sensitivity of 100% and specificity of 77.17 and 86.22%, respectively. This multiplex functional approach strongly supports a dominant role for DSB repair in the development of chronic AEs. It also showed that affected patients share specific features related to functional aspects of DSB repair. This strategy may be suitable for routine clinical analysis and paves the way for modelling DSB repair associated with severe AEs induced by radiotherapy.
Assuntos
Algoritmos , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Masculino , Feminino , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Idoso , Pessoa de Meia-Idade , Radioterapia/efeitos adversos , Neoplasias da Próstata/radioterapia , Neoplasias da Mama/radioterapia , Leucócitos Mononucleares/efeitos da radiação , Aprendizado de Máquina , Lesões por Radiação/etiologiaRESUMO
DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.
Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA , Reparo do DNA , Exodesoxirribonucleases , Humanos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , DNA/metabolismo , DNA/genética , Ubiquitinação , Proteínas de Ciclo CelularRESUMO
Diffusing alpha-emitters radiation therapy (Alpha-DaRT) is a unique method, in which interstitial sources carrying 224Ra release a chain of short-lived daughter atoms from their surface. Although DNA damage response (DDR) is crucial to inducing cell death after irradiation, how the DDR occurs during Alpha-DaRT treatment has not yet been explored. In this study, we temporo-spatially characterized DDR such as kinetics of DNA double-strand breaks (DSBs) and cell cycle, in two-dimensional (2D) culture conditions qualitatively mimicking Alpha-DaRT treatments, by employing HeLa cells expressing the Fucci cell cycle-visualizing system. The distribution of the alpha-particle pits detected by a plastic nuclear track detector, CR-39, strongly correlated with γH2AX staining, a marker of DSBs, around the 224Ra source, but the area of G2 arrested cells was more widely spread 24 h from the start of the exposure. Thereafter, close time-lapse observation revealed varying cell cycle kinetics, depending on the distance from the source. A medium containing daughter nuclides prepared from 224Ra sources allowed us to estimate the radiation dose after 24 h of exposure, and determine surviving fractions. The present experimental model revealed for the first time temporo-spatial information of DDR occurring around the source in its early stages.
Assuntos
Partículas alfa , Quebras de DNA de Cadeia Dupla , Humanos , Células HeLa , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Ciclo Celular/efeitos da radiação , Histonas/metabolismo , Técnicas de Cultura de Células/métodosRESUMO
Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.
Assuntos
Metilases de Modificação do DNA , Enzimas Reparadoras do DNA , Glioblastoma , Melanoma , Tolerância a Radiação , Proteínas Supressoras de Tumor , Humanos , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma/radioterapia , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Linhagem Celular Tumoral , Tolerância a Radiação/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos da radiação , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas , Metilação de DNA , Reparo do DNA , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Temozolomida/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , PurinasRESUMO
DNA double-strand breaks (DSBs) yield highly determines radiotherapy efficacy. However, improving the inherent radiosensitivity of tumor DNA to promote radiation-induced DSBs remains a challenge. Using theoretical and experimental models, the underexplored impact of Z-DNA conformations on radiosensitivity, yielding higher DSBs than other DNA conformations, is discovered. Thereout, a radiosensitization strategy focused on inducing Z-DNA conformation, utilizing CBL@HfO2 nanocapsules loaded with a Z-DNA inducer CBL0137, is proposed. A hollow mesoporous HfO2 (HM-HfO2) acts as a delivery and an energy depositor to promote Z-DNA breakage. The nanocapsule permits the smart DSBs accelerator that triggers its radiosensitization with irradiation stimulation. Impressively, the CBL@HfO2 facilitates the B-Z DNA conformational transition, augmenting DSBs about threefold stronger than irradiation alone, generating significant tumor suppression with a 30% cure rate. The approach enables DSBs augmentation by improving the inherent radiosensitivity of DNA. As such, it opens up an era of Z-DNA conformation manipulation in radiotherapy.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Forma Z , Conformação de Ácido Nucleico , DNA Forma Z/química , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Humanos , Animais , Camundongos , Nanocápsulas/química , Linhagem Celular Tumoral , Tolerância a Radiação , Radiossensibilizantes/química , Neoplasias/radioterapiaRESUMO
This work reports on a model that describes patient-specific absorbed dose-dependent DNA damage response in peripheral blood mononuclear cells of thyroid cancer patients during radioiodine therapy and compares the results with the ex vivo DNA damage response in these patients. Blood samples of 18 patients (nine time points up to 168 h post-administration) were analyzed for radiation-induced γ-H2AX + 53BP1 DNA double-strand break foci (RIF). A linear one-compartment model described the absorbed dose-dependent time course of RIF (Parameters: c characterizes DSB damage induction; k1 and k2 are rate constants describing fast and slow repair). The rate constants were compared to ex vivo repair rates. A total of 14 patient datasets could be analyzed; c ranged from 0.012 to 0.109 mGy-1, k2 from 0 to 0.04 h-1. On average, 96% of the damage is repaired quickly with k1 (range: 0.19-3.03 h-1). Two patient subgroups were distinguished by k1-values (n = 6, k1 > 1.1 h-1; n = 8, k1 < 0.6 h-1). A weak correlation with patient age was observed. While induction of RIF was similar among ex vivo and in vivo, the respective repair rates failed to correlate. The lack of correlation between in vivo and ex vivo repair rates and the applicability of the model to other therapies will be addressed in further studies.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/radioterapia , Neoplasias da Glândula Tireoide/sangue , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Pessoa de Meia-Idade , Masculino , Feminino , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Adulto , Idoso , Dano ao DNA , Radioisótopos do Iodo/uso terapêutico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Histonas/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Modelos BiológicosRESUMO
PURPOSE: Diffusing alpha-emitters radiation therapy (DaRT) is a brachytherapy technique using α-particles to treat solid tumours. The high linear energy transfer (LET) and short range of α-particles make them good candidates for the targeted treatment of cancer. Treatment planning of DaRT requires a good understanding of the dose from α-particles and the other particles released in the 224Ra decay chain. METHODS: The Geant4 Monte Carlo toolkit has been used to simulate a DaRT seed to better understand the dose contribution from all particles and simulate the DNA damage due to this treatment. RESULTS: Close to the seed α-particles deliver the majority of dose, however at radial distances greater than 4 mm, the contribution of ß-particles is greater. The RBE has been estimated as a function of number of double strand breaks (DSBs) and complex DSBs. A maximum seed spacing of 5.5 mm and 6.5 mm was found to deliver at least 20 Gy RBE weighted dose between the seeds for RBEDSB and RBEcDSB respectively. CONCLUSIONS: The DNA damage changes with radial distance from the seed and has been found to become less complex with distance, which is potentially easier for the cell to repair. Close to the seed α-particles contribute the majority of dose, however the contribution from other particles cannot be neglected and may influence the choice of seed spacing.
Assuntos
Partículas alfa , Dano ao DNA , Método de Monte Carlo , Partículas alfa/uso terapêutico , Dosagem Radioterapêutica , Doses de Radiação , Eficiência Biológica Relativa , Difusão , Braquiterapia/métodos , Humanos , Transferência Linear de Energia , Planejamento da Radioterapia Assistida por Computador/métodos , Quebras de DNA de Cadeia Dupla/efeitos da radiaçãoRESUMO
Without the protective shielding of Earth's atmosphere, astronauts face higher doses of ionizing radiation in space, causing serious health concerns. Highly charged and high energy (HZE) particles are particularly effective in causing complex and difficult-to-repair DNA double-strand breaks compared to low linear energy transfer. Additionally, chronic cortisol exposure during spaceflight raises further concerns, although its specific impact on DNA damage and repair remains unknown. This study explorers the effect of different radiation qualities (photons, protons, carbon, and iron ions) on the DNA damage and repair of cortisol-conditioned primary human dermal fibroblasts. Besides, we introduce a new measure, the Foci-Integrated Damage Complexity Score (FIDCS), to assess DNA damage complexity by analyzing focus area and fluorescent intensity. Our results show that the FIDCS captured the DNA damage induced by different radiation qualities better than counting the number of foci, as traditionally done. Besides, using this measure, we were able to identify differences in DNA damage between cortisol-exposed cells and controls. This suggests that, besides measuring the total number of foci, considering the complexity of the DNA damage by means of the FIDCS can provide additional and, in our case, improved information when comparing different radiation qualities.