Search Results (1,087)

Genomic instability caused by defective DNA double-strand break (DSB) repair is a key determinant of cellular radiosensitivity. The Long–Evans cinnamon (LEC) rat is a rare naturally occurring model with marked radiosensitivity, and a major quantitative trait locus, X-ray hypersensitivity 1 (xhs1), has been mapped to rat chromosome 4; however, the causal mechanism has remained unclear. Here, we investigated the cellular and molecular basis of xhs1-associated radiosensitivity using LEA and LEC rat-derived cells and human cultured cells. Exploratory RNA-seq of pre-hepatitic liver tissue identified a sequence variant within the Hmces transcript in LEC rats. Consistently, HMCES protein levels were markedly reduced in multiple tissues and liver-derived cell lines from LEC rats. Functional analyses showed that reduced HMCES activity prolonged γH2AX signaling after X-ray irradiation, indicating delayed DSB resolution. Clonogenic survival assays demonstrated increased radiosensitivity in HMCES-deficient cells, which was partially rescued by restoring HMCES expression in stable LEA/LEC lines. Moreover, pimEJ5GFP reporter assays revealed significantly decreased end-joining repair activity in HMCES-knockout human cells. Together, these results establish HMCES as a critical mediator of DSB repair and cellular radioresistance, identify HMCES dysfunction as a core genetic determinant underlying xhs1-associated radiosensitivity, and provide mechanistic insight into radiation response architecture in a naturally occurring radiosensitive model. Full article

Short-term hypothermic storage at 4 °C represents a promising non-freezing alternative for transporting bovine embryos and synchronizing assisted reproductive procedures. However, chilling induces oxidative stress, mitochondrial dysfunction, and apoptosis, which markedly impair post-preservation embryonic viability. Glutathione (GSH), a key intracellular antioxidant, may mitigate these damaging effects, yet its protective mechanisms during bovine blastocyst hypothermic preservation remain unclear. Here, we investigated the impact of exogenous GSH supplementation on the survival, hatching ability, cellular integrity, mitochondrial function, and developmental potential of bovine blastocysts preserved at 4 °C for seven days. Optimization experiments revealed that 4 mM GSH provided the highest post-chilling survival and hatching rates. Using DCFH-DA, TUNEL, and γ-H2AX staining, we demonstrated that 4 °C preservation significantly increased intracellular reactive oxygen species (ROS), DNA fragmentation, and apoptosis. GSH supplementation markedly alleviated oxidative injury, reduced apoptotic cell ratio, and decreased DNA double-strand breaks. MitoTracker and JC-1 staining indicated severe chilling-induced mitochondrial suppression, including decreased mitochondrial activity and membrane potential (ΔΨm), which were largely restored by GSH. Gene expression analyses further revealed that chilling downregulated antioxidant genes (SOD2, GPX1, TFAM, NRF2), pluripotency markers (POU5F1, NANOG), and IFNT, while upregulating apoptotic genes (BAX, CASP3). GSH effectively reversed these alterations and normalized the BAX/BCL2 ratio. Moreover, SOX2/CDX2 immunostaining, total cell number, and ICM/TE ratio confirmed improved embryonic structural integrity and developmental competence. Collectively, our findings demonstrate that exogenous GSH protects bovine blastocysts from chilling injury by suppressing ROS accumulation, stabilizing mitochondrial function, reducing apoptosis, and restoring developmental potential. This study provides a mechanistic foundation for improving 4 °C embryo storage strategies in bovine reproductive biotechnology. Full article

Non-homologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway that operates throughout the cell cycle to maintain the genomic stability of the cell. Unlike homologous recombination (HR), NHEJ is capable of repairing DSBs without the need for a homologous template, making it a rapid response mechanism, but potentially prone to errors. Central to NHEJ function and essential for the ligation through the recruitment and activation of additional repair factors, such as Artemis, XRCC4, and DNA ligase IV, is the DNA-dependent protein kinase (DNA-PK) complex. Dysregulation in the NHEJ pathway contributes to genomic instability, oncogenesis, and resistance to genotoxic therapies. Consequently, inhibitors of DNA-PK have emerged as promising therapeutic agents to sensitize tumor cells to radiation and DNA-damaging chemotherapeutics. Inhibiting the DNA-PK ability to recruit the protein complex needed for successful DSB repair promotes cell death through apoptosis or mitotic catastrophe. While inhibitors of DNA-PK can be used to enhance the effects of genotoxic therapies, the field still struggles to address critical problems: how to best exploit the differential DNA repair capacities among tumor subtypes, how to maximize radiosensitization of cancerous cells while sparing normal tissues, and how to translate preclinical studies into clinical benefits. Given that NHEJ constitutes the primary line of defense against radiation-induced damage, rapidly repairing the majority of double-strand breaks throughout the cell cycle, this review concentrates on targeting the DNA-PK complex, as the master regulator of this rapid-response mechanism, highlighting why its inhibition represents a strategic action to overcome intrinsic radioresistance. The implementation of DNA-PK inhibitors into medical practice can enable the stratification of oncologic patients into two categories, based on the tumors’ vulnerability to NHEJ disruptions. Thus, the therapeutic pathways of patients with NHEJ tumors could branch, combining traditional genotoxic therapies (radiation and DNA-damaging chemotherapeutics) with DNA-PK inhibitors to achieve an enhanced effect and improved survival outcomes. Full article

DNA double-strand breaks (DSBs) can be induced by cellular byproducts or genotoxic agents. Improper processing of these lesions leads to increased genome instability, which constitutes a hallmark of pathological conditions and fuels carcinogenesis. DSBs are primarily repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) and the proper balance between these two pathways is finely modulated by specific molecular events. Here, we report that the histone chaperone DAXX plays a fundamental role in the response to DSBs. Indeed, in human cells, DSBs induce ATM/ATR-dependent phosphorylation of DAXX on serine 424 and 712 and promote its binding to chromatin and the deposition of the histone variant H3.3 in proximity to DNA breaks. Enrichment of H3.3 at DSBs promotes 53BP1 recruitment to these lesions and the repair of DNA breaks by HR pathways. Moreover, H3.3-specific post translational modifications, particularly K36 tri-methylation, play a key role in these processes. Altogether, these findings indicate that DAXX and H3.3 mutations may contribute to tumorigenesis-enhancing genome instability. Full article

Background: In nuclear medicine, numerous cancer types are treated via internal irradiation with radiopharmaceuticals, including low-LET (linear energy transfer) beta-emitting radionuclides like Lu-177. In most cases, such treatments lead to low-dose exposure of organ systems with β-irradiation, which induces only few isolated DSBs (double-strand breaks) in the nuclei of hit cells, the most threatening DNA damage type. That damaging effect contrasts with the clustering of DNA damage and DSBs in nuclei traversed by high-LET particles (α particles, ions, etc.). Methods: After in-solution β-irradiation for 1 h with Lu-177 leading to an absorbed dose of about 100 mGy, we investigated the spatial nano-organization of chromatin at DSB damage sites, of repair proteins and of heterochromatin marks via single-molecule localization microscopy (SMLM) in PBMCs. For evaluation, mathematical approaches were used (Ripley distance frequency statistics, DBScan clustering, persistent homology and similarity measurements). Results: We analyzed, at the nanoscale, the distribution of the DNA damage response (DDR) proteins γH2AX, 53BP1, MRE11 and pATM in the chromatin regions surrounding a DSB. Furthermore, local changes in spatial H3K9me3 heterochromatin organization were analyzed relative to γH2AX distribution. SMLM measurements of the different fluorescent molecule tags revealed characteristic clustering of the DDR markers around one or two damage foci per PBMC cell nucleus. Ripley distance histograms suggested the concentration of MRE11 molecules inside γH2AX-clusters, while 53BP1 was present throughout the entire γH2AX clusters. Persistent homology comparisons for 53BP1, MRE11 and γH2AX by Jaccard index calculation revealed significant topological similarities for each of these markers. Since the heterochromatin organization of cell nuclei determines the identity of cell nuclei and correlates to genome activity, it also influences DNA repair. Therefore, the histone H3 tri methyl mark H3K9me3 was analyzed for its topology. In contrast to typical results obtained through photon irradiation, where γH2AX and H3K9me3 markers were well separated, the results obtained here also showed a close spatial proximity (“co-localization”) in many cases (minimum distance of markers = marker size), even with the strictest co-localization distance threshold (20 nm) for γH2AX and H3K9me3. The data support the results from the literature where only one DSB induced by low-dose low LET irradiation (<100 mGy) can remain without heterochromatin relaxation for subsequent repair. Full article

Radiotherapy is an effective treatment for cancer; however, radioresistant cancer cells result in recurrence. Therefore, elucidating the mechanisms of radioresistance is urgently needed. Super-enhancers (SEs) are clusters of enhancers occupied by a high density of master transcription factors, mediators, and bromodomain protein BRD4. Recently, we reported that ΔNp63, an oncogenic transcription factor, promotes radioresistance in human head and neck squamous cell carcinoma (HNSCC) cells. As ΔNp63 establishes SEs in HNSCC cells, SEs may be involved in radioresistance. Here, we investigated the role of the SE component BRD4 in the radiation responses of HNSCC cells using a BRD4 degrader ARV-771 or BRD4 knockdown. First, Western blotting confirmed that ARV-771 decreased BRD4 protein expression. ARV-771 treatment resulted in reduced cell proliferation and enhanced apoptosis in irradiated HNSCC cells. Moreover, colony formation assays revealed that both ARV-771 and BRD4 knockdown enhanced the radiosensitivity of HNSCC cells, suggesting BRD4 contributes to the radioresistance of HNSCC cells. Furthermore, fluorescence immunostaining revealed distinct localization patterns of γH2AX, a marker of DNA double-strand breaks, compared with BRD4 and ΔNp63 in irradiated cells. Notably, ARV-771 and BRD4 knockdown decreased ΔNp63 and BRD4 protein expression, whereas ΔNp63 knockdown had minimal impact on BRD4 expression. Taken together, these findings suggest that BRD4-dependent maintenance of ΔNp63 expression may contribute, at least in part, to the regulation of radioresistance in HNSCC cells. Full article

Protons are conventionally regarded as a low-linear energy transfer (low-LET) radiation modality with a relative biological effectiveness (RBE) of 1.1, suggesting direct mechanistic similarity to X-rays in the underpinning biological effects. However, exposure to spread-out Bragg peak (SOBP) protons reveals instructive deviations from this assumption. Indeed, proton beams have a maximum LET of ~5 keV/µm but display reduced reliance on classical non-homologous end joining (c-NHEJ) as well as an increased dependence on homologous recombination (HR) and alternative end joining (alt-EJ). These features are well described in cells exposed to high-LET radiation and typically manifest between 100 and 150 keV/µm. We hypothesized that this apparent discrepancy reflects biological consequences of proton-beam properties that remain uncharacterized. In the present study, we outline exploratory experiments aiming at uncovering such mechanisms. We begin by investigating for both entrance and SOBP protons the dose-dependent engagement of HR we recently showed for X-rays. Consistent with our previous findings with X-rays, HR engagement after exposure to both types of proton beams declined with dose, from ~80% at 0.2 Gy to less than 20% at higher doses. RAD51/γH2AX foci ratios, reflecting HR engagement, were modestly higher following proton irradiation, in line with increased HR utilization. G₂-checkpoint activation, previously linked to HR, was also stronger after exposure to protons, as was DNA end resection. Moreover, the formation of structural chromosomal abnormalities (SCAs) was higher for SOBP than entrance protons and X-rays. Collectively, our results suggest quasi-high-LET characteristics for proton beams and raise the question as to the physical proton properties that underpin them. We discuss that the commonly employed definition of LET may be insufficient for this purpose. Full article

Since 2014, a model of the individual response to ionizing radiation (IR), based on the radiation-induced nucleoshuttling of the ATM protein kinase (RIANS), has been developed by our lab: after irradiation, ATM dimers monomerize in cytoplasm and diffuse into the nucleus to trigger both recognition and repair of DNA double-strand breaks (DSB), the key-damage of IR response. Moderate radiosensitivity is generally caused by heterozygous mutations of ATM substrates (called X-proteins) that are over-expressed in cytoplasm and form complexes with ATM monomers, which reduces and/or delays the RIANS and DSB recognition. Here, we asked whether molecules, rather than X-proteins, can also influence RIANS. Caffeine was chosen as a potential “X-molecule” candidate. After incubation of cells with caffeine, cutaneous fibroblasts from an apparently healthy radioresistant donor, a patient suffering from Alzheimer’s disease (AD) and another suffering from neurofibromatosis type 1 (NF1) were exposed to X-rays. The functionality of ATM-dependent DSB repair and signaling was evaluated. We report here that caffeine molecule interaction with ATM leads to the inhibition of DSB recognition. This effect is significant in radioresistant cells. Conversely, in the AD and NF1 cells, the DSB recognition is already so low that caffeine does not provide any additional molecular effect. Full article

Polystyrene nanoplastics (PS-NPs) can cross the placenta and blood–brain barrier to accumulate in the fetal brain following inhalation or ingestion, raising concerns about PS-NPs-induced developmental neurotoxicity (DNT). However, current evidence regarding the mechanisms underlying PS-NPs-elicited DNT remains critically scarce. Given the inherent limitations of two-dimensional cell culture techniques, we employed a whole-brain organoid (WBO) model, which more faithfully recapitulates the dynamic changes and substantial alterations during the early development of the human nervous system, to investigate the PS-NPs-induced DNT. Developing WBOs were exposed to 50-nm PS-NPs at concentrations of 50 and 100 μg/mL. Additionally, we established an early developmental exposure model in neonatal rat for robust validation. The results revealed aberrant formation of the tissue architecture of neural epithelial buds in PS-NPs-exposed WBOs, accompanied by significant inflammatory responses and oxidative stress. Marked DNA damage and substantial activation of the TLR9/MyD88 pathway were observed in WBOs and in the cerebral cortex of neonatal rat, leading to significant upregulation of the excitotoxicity marker c-Fos and the excitatory synaptic marker NMDAR. In vitro assays revealed that melatonin treatment could efficiently counteract PS-NPs-mediated neuronal impairment, with both the reduced cell viability and excessive DNA damage induced by PS-NPs being restored to levels close to those of the control group. In conclusion, by establishing WBOs and early developmental exposure models in neonatal rat, we found that PS-NPs can induce DNA double-strand breaks, and activation of the TLR9 pathway mediates PS-NPs-induced excitotoxicity. Full article

Purpose: ATRIP (ATR-interacting protein) is a critical partner of ATR (ataxia telangiectasia and Rad3-related). The ATR-ATRIP heterodimer plays an essential role in initiating homologous recombination repair (HRR) during replication stress and inducing double-stranded DNA breaks following unresolved stalled replication forks. Our team recently identified ATRIP as a novel breast cancer susceptibility gene candidate through whole-exome sequencing (WES) of familial breast cancer patients and healthy controls from the Polish founder population, with subsequent validation in both Polish and British cohorts. In the present study, we report for the first time the detection of a novel deleterious mutation in ATRIP among several members of an Iranian family with clustering of breast cancer who were negative for mutations in the already known breast cancer risk genes. Methods: Six family members underwent germline DNA testing by WES, following initial negative results from multigene panel testing. Candidate variants were confirmed by Sanger sequencing and assessed according to ACMG guidelines. Results: We detected a novel ATRIP frameshift mutation (NM_130384.3:c.1033delC) in four of six family members that were tested, including two individuals affected with breast cancer. No pathogenic variants were found in other known cancer susceptibility genes. Conclusions: This is the first report of a deleterious ATRIP mutation in an Iranian family with familial breast cancer, suggesting a potential role of ATRIP in hereditary breast cancer. Further studies are required to confirm the role of ATRIP in breast cancer susceptibility, refine risk assessment, and evaluate potential personalized therapeutic strategies. In the interim, genetic counseling for ATRIP mutation carriers should proceed with caution, given current limitations in clinical interpretation. Full article

Background/Objectives: Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy, often diagnosed at advanced stages with poor survival outcomes. Homologous recombination (HR), a major DNA double-strand break (DSB) repair pathway, safeguards genomic stability via error-free repair. While HR deficiency has been well established as a driver of genomic instability and tumorigenesis in several cancer types, the role of HR in HNSCC remains relatively understudied. Methods: Here, we analyzed the expression patterns of key HR proteins in HNSCC and investigated their association with clinical parameters, DNA methylation, immune cell infiltration, and patient survival outcome. Results: Surprisingly, our results demonstrate that HR factors are consistently upregulated in HNSCC, in both HPV-positive and HPV-negative groups. Survival analysis identified many HR factors, including ATM, BRCA1, BRCA2, PALB2, LIG1, RPA1, and RPA2, as potential prognostic biomarkers for better overall survival. Interestingly, we observed a significant correlation between HR protein overexpression and immune cell infiltration in HNSCC, suggesting a potential immunomodulatory role of HR proteins. To experimentally validate this association in both HPV-positive and -negative cell lines, we showed that MRE11 and RAD51 overexpression in HNSCC cells led to increased phosphorylation of IRF3 and STAT1, indicating activation of the cGAS/STING-mediated innate immune signaling. Conclusion: Together, our findings provide a comprehensive overview of the HR pathway in HNSCC, highlighting the dual role of HR proteins in both genomic maintenance and immune regulation. The consistent upregulation of HR proteins, their association with disease progression, and potential immunogenic effects underscore their promise as diagnostic/prognostic biomarkers and therapeutic targets in HNSCC. Full article

Computed tomography (CT) is a major source of low-dose ionizing radiation exposure in medical imaging. Risk assessment at this dose level is difficult and relies on the hypothetical linear no-threshold model. To address the response to such low doses in patients undergoing CT scans, we examined radiation-induced alterations at the transcriptomic and DNA damage levels in peripheral blood cells. Peripheral whole blood of 60 patients was collected before and after CT. Post-CT samples were obtained 4–6 h after scan (n = 28, in vivo incubation) or alternatively immediately after the CT scan, followed by ex vivo incubation (n = 32). The gene expression of known radiation-responsive genes (n = 9) was quantified using qRT-PCR. DNA double-strand breaks (DSB) were assessed in 12 patients through microscopic γ-H2AX + 53BP1 DSB focus staining. The mean dose–length product (DLP) across all scans was 561.9 ± 384.6 mGy·cm. Significant differences in the median differential gene expression (DGE) were detected between in vivo and ex vivo incubation conditions, implicating that ex vivo incubation masked the true effect in low-dose settings. The median DGE of in vivo-incubated samples showed a significant upregulation of EDA2R, MIR34AHG, PHLDA3, DDB2, FDXR, and AEN (p ranging from <0.001 to 0.041). In vivo, we observed a linear dose-dependent upregulation for several genes and an explained variance of 0.66 and 0.56 for AEN and FDXR, respectively. DSB focus analysis revealed a slight, non-significant increase in the average DSB damage post-exposure, at a mean DLP of 321.0 mGy·cm. Our findings demonstrate that transcriptional biomarkers are sensitive indicators of low-dose radiation exposure in medical imaging and could prove themselves as clinically applicable biodosimetry tools. Furthermore, the results underscore the need for dose optimization. Full article

The growing production and use of silver nanoparticles continues to raise questions about their consequences for human and animal health. The method of production, particle stabilization, particle size, concentration, and duration of exposure to cells can affect their reactivity and, consequently, their toxicity. This study was conducted to determine the degree of harmfulness of colloidal silver compounds, including silver nanoparticles produced via the HVAD method, to mitotic chromosomes in chinchilla’s cells. Thanks to the sister chromatid exchange (SCE) test, chromosome damage during cell division, i.e., the actual toxic effect of the tested compounds, could be assessed. For this purpose, whole peripheral blood from chinchillas was exposed in vitro to three colloidal silver compounds (unstable AgNP-HVAD, sodium citrate-stabilized silver nanoparticles—[AgNP+C], and silver nitrate) for 3, 6, and 24 h. The toxicity of these compounds was assessed at concentrations of 5, 10, and 20 µg/L and the occurrence of sister chromatid exchanges on chromosomes, resulting from double-strand DNA breaks, was analyzed. The studies revealed a notable increase in SCEs compared to the control group, suggesting the genotoxic properties of the examined AgNPs. The highest level of chromosome damage was observed following exposure to citrate-stabilized silver nanoparticles. Further research is needed to better understand the toxicological mechanisms of AgNPs produced via the HVAD method and their effects on mammalian somatic cells. Full article

Homology search is a means through which DNA double-strand breaks (DSBs) explore the genome for sequences that enable error-free repair, known as homologous recombination. A better understanding of this search process is fundamental to the relationship between higher-order chromosome organization and DNA damage. Here, we use an entropic bead-spring polymer chain model to simulate the spatiotemporal dynamics of the yeast genome during interphase. The chromosome is organized by transient and dynamic cross-links representing structural maintenance of chromosome (SMC) complexes. DNA damage is modeled as a break in the bead-spring chain, coupled with a removal of crosslinks from beads proximal to the break site. We show that the removal of cross-links drives the exploration of genomic space by the damaged ends, while rates and densities of intact dynamic crosslinking have only a minor role. Local depletion of SMC cross-links proximal to the break site enables the damaged segment to escape the chromosome territory and enhances its ability to explore the genome. Our study reveals a foundational principle by which DSBs can encounter distant regions of sequence homology. Full article

While PARP inhibitors like Olaparib are effective against BRCA1-deficient breast cancers, their efficacy in BRCA1-proficient tumors depends on the functional status of homologous recombination (HR) repair. Here, we identify the structure-specific endonuclease SLX1 as a key regulator of HR and a determinant of Olaparib sensitivity in BRCA1-intact breast cancer. SLX1 is frequently upregulated in breast cancer and associated with poor prognosis. Functional studies revealed that SLX1 promotes RAD51-mediated HR repair of DNA double-strand breaks. Consequently, SLX1 depletion reduces HR efficiency, increases chromosomal instability, and sensitizes breast-proficient breast cancer cells to DNA-damaging agents, including camptothecin, ionizing radiation, and Olaparib. In contrast, SLX1 overexpression enhances DNA repair capacity and promotes Olaparib resistance. In vivo, SLX1 knockdown synergizes with Olaparib to suppress tumor growth in xenograft models. These findings establish SLX1 as a critical regulator of HR function in BRCA1-proficient breast cancer and a promising target for restoring PARP inhibitor sensitivity through induced HR deficiency. Full article