DNA Double-strand Break Repair Research Articles

Understanding the Human RECQ5 Helicase-Connecting the Dots from DNA to Clinics.

RECQ5, a member of the conserved RECQ helicase family, is the sole human RECQ homolog that has not been linked to a hereditary developmental syndrome. Nonetheless, dysregulation of RECQ5 has emerged as a significant clinical concern, being linked to cancer predisposition, cardiovascular disease, and inflammation. In cells, RECQ5 assumes a crucial role in the regulation of DNA repair pathways, particularly in the repair of DNA double-strand breaks and inter-strand DNA crosslinks. Moreover, RECQ5 exhibits a capacity to modulate gene expression by interacting with transcription machineries and their co-regulatory proteins, thus safeguarding against transcription-induced DNA damage. This review aims to provide an overview of the multifaceted functions of RECQ5 and its implications in maintaining genomic stability. We will discuss the potential effects of clinical variants of RECQ5 on its cellular functions and their underlying mechanisms in the pathogenesis of cancer and cardiovascular disease. We will review the impact of RECQ5 variants in the field of pharmacogenomics, specifically their influence on drug responses, which may pave the way for novel therapeutic interventions targeting RECQ5 in human diseases.

RBM14 promotes DNA end resection during homologous recombination repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) is crucial for the maintenance of genome stability and integrity. In this study, we aim to identify novel RNA binding proteins (RBPs) involved in HR repair because little is known about RBP function in HR. For this purpose, we carry out pulldown assays using a synthetic ssDNA/dsDNA structure coated with replication protein A (RPA) to mimic resected DNA, a crucial intermediate in HR-mediated DSB repair. Using this approach, we identify RNA-binding motif protein 14 (RBM14) as a potential binding partner. We further show that RBM14 interacts with an essential HR repair factor, CtIP. RBM14 is crucial for CtIP recruitment to DSB sites and for subsequent RPA coating and RAD51 replacement, facilitating efficient HR repair. Moreover, inhibition of RBM14 expression sensitizes cancer cells to X-ray irradiation. Together, our results demonstrate that RBM14 promotes DNA end resection to ensure HR repair and may serve as a potential target for cancer therapy.

Arsenic affects homologous recombination and single-strand annealing but not end-joining pathways during DNA double-strand break repair.

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double-strand breaks (DSBs) have been implicated in arsenic-induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2 O3 ) on four different DSB repair pathways using the human pre-B cell line Nalm-6. Random integration analysis showed that As2 O3 does not negatively affect non-homologous end joining or polymerase theta-mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2 O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single-strand annealing. Consistent with this finding, As2 O3 decreased gene-targeting efficiency, owing to a significant reduction in the frequency of HR-mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one-ended DSBs requiring HR for precise repair. Intriguingly, we found that As2 O3 significantly enhances sensitivity to those anticancer agents in HR-proficient cells. Our results suggest that arsenic-induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic-associated carcinogenesis and therapeutic options.

Recombinase-independent chromosomal rearrangements between dispersed inverted repeats in Saccharomyces cerevisiae meiosis.

DNA double-strand break (DSB) repair by homologous recombination (HR) uses a DNA template with similar sequence to restore genetic identity. Allelic DNA repair templates can be found on the sister chromatid or homologous chromosome. During meiotic recombination, DSBs preferentially repair from the homologous chromosome, with a proportion of HR events generating crossovers. Nevertheless, regions of similar DNA sequence exist throughout the genome, providing potential DNA repair templates. When DSB repair occurs at these non-allelic loci (termed ectopic recombination), chromosomal duplications, deletions and rearrangements can arise. Here, we characterize in detail ectopic recombination arising between a dispersed pair of inverted repeats in wild-type Saccharomyces cerevisiae at both a local and a chromosomal scale-the latter identified via gross chromosomal acentric and dicentric chromosome rearrangements. Mutation of the DNA damage checkpoint clamp loader Rad24 and the RecQ helicase Sgs1 causes an increase in ectopic recombination. Unexpectedly, additional mutation of the RecA orthologues Rad51 and Dmc1 alters-but does not abolish-the type of ectopic recombinants generated, revealing a novel class of inverted chromosomal rearrangement driven by the single-strand annealing pathway. These data provide important insights into the role of key DNA repair proteins in regulating DNA repair pathway and template choice during meiosis.

Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype.

This study aimed to investigate the aggressive behavior of triple-negative breast cancer (TNBC) cells that had survived ionizing radiation and explore the potential targets of TNBC combination treatment. Consistent with the previous literature, Axl was highly expressed in TNBC and closely associated with the degree of malignancy based on immunohistochemical staining. Using a gradient irradiation method, the ionizing radiation-resistant mouse TNBC cell line 4T-1/IRR was established. It was found that Axl expression was upregulated in 4T-1/IRR cells. After irradiation by X-ray, the cell viability and colony formation ability of 4T-1/IRR cells were significantly increased when compared with the 4T-1 cells. Combined radiotherapy with Axl inhibition by treatment with R428 and small interfering RNA lentivirus targeting Axl infection significantly reduced cell viability, colony formation ability, DNA double-stranded break repair, and the invasive and migratory ability of 4T-1/IRR cells. In vivo, the small animal radiation research platform was applied to precisely administer radiotherapy of the tumor-bearing mice. R428 treatment combined with 6 Gy X-ray significantly inhibited the growth of 4T-1/IRR cells-derived xenograft tumors in the BALB/c mouse. The results of western blotting showed that the critical molecular mechanism involved in the radioresistance of TNBC cells was the PI3K/Akt/mTOR signaling pathway induced by Axl activation. Thus, it is hypothesized that targeted Axl therapy combined with radiotherapy may have significant potential for the treatment of TNBC.

Preomic profile of BxPC-3 cells after treatment with BRC4

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SignificanceRAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

Antioxidant Nanoparticles Restore Cisplatin-Induced Male Fertility Defects by Promoting MDC1-53bp1-Associated Non-Homologous DNA Repair Mechanism and Sperm Intracellular Calcium Influx.

Cisplatin, a commonly used anticancer compound, exhibits severe off-target organ toxicity. Due to its wide application in cancer treatment, the reduction of its damage to normal tissue is an imminent clinical need. Cisplatin-induced testicular oxidative stress and damage lead to male sub- or infertility. Despite earlier studies showing that the natural polyphenol extracts honokiol serve as the free radical scavenger that reduces the accumulation of intracellular free radicals, whether honokiol exhibits direct effects on the testis and sperm is unclear. Thus, the aim of the current study is to investigate the direct effects of honokiol on testicular recovery and sperm physiology. We encapsulated this polyphenol antioxidation compound into liposome-based nanoparticles (nHNK) and gave intraperitoneally to mice at a dosage of 5 mg/kg body mass every other day for consecutive 6 weeks. We showed that nHNK promotes MDC1-53bp1-associated non-homologous DNA double-strand break repair signaling pathway that minimizes cisplatin-induced DNA damage. This positive effect restores spermatogenesis and allows the restructuring of the multi-spermatogenic layers in the testis. By reducing mitochondrial oxidative damage, nHNK also protects sperm mitochondrial structure and maintains both testicular and sperm ATP production. By a yet-to-identify mechanism, nHNK restores sperm calcium influx at the sperm midpiece and tail, which is essential for sperm hypermotility and their interaction with the oocyte. Taken together, the nanoparticulated antioxidant counteracts cisplatin-induced male fertility defects and benefits patients undertaking cisplatin-based chemotherapy. These data may allow the reintroduction of cisplatin for systemic applications in patients at clinics with reduced testicular toxicity.

ADNP is associated with immune infiltration and radiosensitivity in hepatocellular carcinoma for predicting the prognosis

BackgroundHepatocellular carcinoma (HCC) is one of the most lethal diseases due to its high faculty of invasiveness and metastasis. Activity-dependent neuroprotective protein (ADNP) has been regarded as an oncogene in bladder cancer and ovarian cancer. However, the role of ADNP in the regulation of tumor immune response, development, and treatment resistance in HCC remains unknown and is worth exploring.MethodsThe correlation between ADNP and prognosis, immune cell infiltration, immune checkpoints, chemokines, tumor mutation burden, microsatellite instability, and genomic mutation of pan-cancer cohorts in The Cancer Genome Atlas was analyzed. ADNP expression in HCC cell lines, HCC and the adjacent normal tissues was measured by western blotting and immunochemistry. Nomogram was constructed to predict the survival of patients with HCC based on the ADNP expression and significant clinical characteristics. The potential biological functions and impacts on radiotherapy of ADNP in HCC cell lines were verified by vitro experiments.ResultsADNP was upregulated in most cancers and patients with elevated ADNP expression were related to poor survival in several types of cancers including HCC. Functional enrichment analysis showed ADNP participated in the pathways correlated with coagulation cascades and DNA double strand break repair. Further, ADNP exhibited a negative correlation with the immune score, stromal score, estimated score, and chemokines, and a positive correlation with cancer-associated fibroblasts, myeloid-derived suppressor cells, neutrophils, regulatory T cells, and endothelial cells. Immunochemistry and western blotting results demonstrated ADNP was up-regulated in HCC. Vitro experiments verified that suppressing the ADNP expression significantly inhibited the proliferation, invasion and migration and elevated the radiosensitivity via decreasing DNA damage repair in HCC.ConclusionADNP might play an oncogene and immunosuppression role in tumor immune infiltration and response, thus influencing the prognosis. Its downregulation could attenuate the proliferation, invasion, migration, radioresistance of HCC. Our results indicated the potential of ADNP as a promising biomarker to predict the survival of HCC patients, providing a theoretical basis for novel integrative strategies.

Structure and mechanism in non-homologous end joining

DNA double-stranded breaks (DSBs) are a particularly challenging form of DNA damage to repair because the damaged DNA must not only undergo the chemical reactions responsible for returning it to its original state, but, additionally, the two free ends can become physically separated in the nucleus and must be bridged prior to repair. In nonhomologous end joining (NHEJ), one of the major pathways of DSB repair, repair is carried out by a number of repair factors capable of binding to and directly joining DNA ends. It has been unclear how these processes are carried out at a molecular level, owing in part to the lack of structural evidence describing the coordination of the NHEJ factors with each other and a DNA substrate. Advances in cryo-Electron Microscopy (cryo-EM), allowing for the structural characterization of large protein complexes that would be intractable using other techniques, have led to the visualization several key steps of the NHEJ process, which support a model of sequential assembly of repair factors at the DSB, followed by end-bridging mediated by protein-protein complexes and transition to full synapsis. Here we examine the structural evidence for these models, devoting particular attention to recent work identifying a new NHEJ intermediate state and incorporating new NHEJ factors into the general mechanism. We also discuss the evolving understanding of end-bridging mechanisms in NHEJ and DNA-PKcs’s role in mediating DSB repair.

The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair.

Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.

Templated insertions-DNA repair gets acrobatic.

Deletions associated with the repair of DNA double-strand breaks is a source of genetic alternation and a recognized source of disease-causing mutagenesis. Theta-mediated end joining is a DNA repair mechanism, which guarantees deletions by its employment of microhomology (MH) alignment to facilitate end joining. A lesser-characterized templated insertion ability of this pathway, on the other hand, is associated with both deletion and insertion. This mechanism is characterized by at least one round of polymerase θ-mediated synthesis, which does not result in successful repair, followed by a subsequent round of polymerase engagement and synthesis that does lead to repair. Here we focus on the mechanisms by which polymerase θ introduces these insertions-direct, inverse, and a new class which we have termed strand switching. We observe this new class of templated insertions at multiple loci and across multiple species, often at a comparable frequency to those previously characterized. Templated insertion mutations are often enriched in cancer genomes and repeat expansion disorders. This repair mechanism thus contributes to disease-associated mutagenesis, and may plausibly even promote disease. Characterization of the types of polymerase θ-dependent insertions can provide new insight into these diseases and clinical promise for treatment.

Lost in the bloom: DNA-PKcs in green plants.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a protein encoded by the PRKDC gene in humans and plays a crucial role in repairing DNA double-strand breaks (DSBs). Recent studies have revealed that DNA-PKcs has additional functions in the cell beyond DSB repair, including transcriptional regulation, telomere protection and capping, preserving chromosomal integrity, and regulating senescence, apoptosis, and autophagy. Moreover, DNA-PKcs has also been implicated in regulating the innate immune response, and dysregulation of DNA-PKcs has been commonly observed in various types of cancers. Until recently it was believed that DNA-PKcs is not present in plants in general. However, DNA-PKcs is conserved in green plants ranging from microscopic green algae such as Ostreococcus of the chlorophytes to the tallest living trees on earth, Sequoia of the gymnosperms. Interestingly, DNA-PKcs has not been detected in angiosperms, or in basal angiosperms which are considered sister groups to all other flowering plants. The long polypeptide and gene length of DNA-PKcs coupled with errors in genome assembly, annotation, and gene prediction, have contributed to the challenges in detecting and extracting DNA-PKcs sequences in plant lineages. Sequence alignment showed that several amino acids throughout the length of DNA-PKcs are conserved between plants and human, and all the typical domains identified in human DNA-PKcs are also found in DNA-PKcs from green plants suggesting possible structural and functional conservation. Given the highly conserved nature of DNA repair pathways between mammals and plants further highlights the potential significance of DNA-PKcs in plant biology.

RHINO and Polθ Mediate DNA Double-Strand Break Repair during Mitosis.

Microhomology-mediated end-joining (MMEJ) is essential for mitotic double-strand break (DSB) repair.

FIGL1 coordinates with dosage-sensitive BRCA2 in modulating meiotic recombination in maize.

Meiotic crossover (CO) formation between homologous chromosomes ensures their subsequent proper segregation and generates genetic diversity among offspring. In maize, however, the mechanisms that modulate crossover formation remain poorly characterized. Here, we found that both maize BREAST CANCER SUSCEPTIBILITY PROTEIN 2 (BRCA2) and AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) act as positive factors of crossover formation by controlling the assembly or/and stability of two conserved DNA recombinases RAD51 and DMC1 filaments. Our results revealed that ZmBRCA2 not only is involved in repair of DNA double-strand breaks (DSBs), but also regulates crossover formation in a dosage-dependent manner. In addition, ZmFIGL1 interacts with RAD51 and DMC1, and Zmfigl1 mutants had a significantly reduced number of RAD51/DMC1 foci and COs. Further, simultaneous loss of ZmFIGL1 and ZmBRCA2 abolished RAD51/DMC1 foci and exacerbated meiotic defects compared with the single mutant Zmbrca2 or Zmfigl1. Together, our data demonstrate that ZmBRCA2 and ZmFIGL1 act coordinately to regulate the dynamics of RAD51/DMC1-dependent DSB repair to promote crossover formation in maize. This conclusion is surprisingly different from the antagonistic roles of BRCA2 and FIGL1 in Arabidopsis, implying that although key factors that control crossover formation are evolutionarily conserved, specific characteristics have been adopted in diverse plant species. This article is protected by copyright. All rights reserved.

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

Dynamic viral integration patterns actively participate in the progression of BK polyomavirus-associated diseases after renal transplantation

The classical lytic infection theory along with large T antigen-mediated oncogenesis cannot explain the BK polyomavirus (BKPyV)-associated tumor secondary to BKPyV-associated nephropathy (BKVAN), viremia/DNAemia, and viruria after renal transplantation. This study performed virome capture sequencing and pathological examination on regularly collected urine sediment and peripheral blood samples, and BKVAN and tumor biopsy tissues of 20 patients with BKPyV-associated diseases of different stages. In the early noncancerous stages, well-amplified integration sites were visualized by in situ polymerase chain reaction, simultaneously with BKPyV inclusion bodies and capsid protein expression. The integration intensity, the proportion of microhomology-mediated end-joining integration, and host PARP-1 and POLQ gene expression levels increased with disease progression. Furthermore, multiomics analysis was performed on BKPyV-associated urothelial carcinoma tissues, identifying tandem-like structures of BKPyV integration using long-read genome sequencing. The carcinogenicity of BKPyV integration was proven to disturb host gene expression and increase viral oncoprotein expression. Fallible DNA double-strand break repair pathways were significantly activated in the parenchyma of BKPyV-associated tumors. Olaparib showed an antitumor activity dose-response effect in the tumor organoids without BRCA1/2 genes mutation. In conclusion, the dynamic viral integration patterns actively participate in the progression of BKPyV-associated diseases and thus could be a potential target for disease monitoring and intervention.

Photoexcited cryptochromes interact with ADA2b and SMC5 to promote the repair of DNA double-strand breaks in Arabidopsis.

Cryptochromes (CRYs) act as blue-light photoreceptors that regulate development and circadian rhythms in plants and animals and as navigating magnetoreceptors in migratory birds. DNA double-strand breaks (DSBs) are the most serious type of DNA damage and threaten genome stability in all organisms. Although CRYs have been shown to respond to DNA damage, whether and how they participate in DSB repair is not well understood. Here we report that Arabidopsis CRYs promote the repair of DSBs through direct interactions with ADA2b and SMC5 in a blue-light-dependent manner to enhance their interaction. Mutations in CRYs and in ADA2b lead to similar enhanced DNA damage accumulation. In response to DNA damage, CRYs are localized at DSBs, and the recruitment of SMC5 to DSBs is dependent on CRYs. These results suggest that CRY-enhanced ADA2b-SMC5 interaction promotes ADA2b-mediated recruitment of SMC5 to DSBs, leading to DSB repair.

Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

Fatty acid oxidation facilitates DNA double-strand break repair by promoting PARP1 acetylation

DNA repair is a tightly coordinated stress response to DNA damage, which is critical for preserving genome integrity. Accruing evidence suggests that metabolic pathways have been correlated with cellular response to DNA damage. Here, we show that fatty acid oxidation (FAO) is a crucial regulator of DNA double-strand break repair, particularly homologous recombination repair. Mechanistically, FAO contributes to DNA repair by activating poly(ADP-ribose) polymerase 1 (PARP1), an enzyme that detects DNA breaks and promotes DNA repair pathway. Upon DNA damage, FAO facilitates PARP1 acetylation by providing acetyl-CoA, which is required for proper PARP1 activity. Indeed, cells reconstituted with PARP1 acetylation mutants display impaired DNA repair and enhanced sensitivity to DNA damage. Consequently, FAO inhibition reduces PARP1 activity, leading to increased genomic instability and decreased cell viability upon DNA damage. Finally, our data indicate that FAO serves as an important participant of cellular response to DNA damage, supporting DNA repair and genome stability.

Superparamagnetic iron oxide nanoparticles induce persistent large foci of DNA damage in human melanoma cells post-irradiation.

The synergy of superparamagnetic iron oxide nanoparticles (SPIONs) and ionizing radiation (IR), attributed to reactive oxygen species (ROS) and DNA double-strand breaks (DSBs) increase, was widely investigated in different cancers, but scarcely in melanoma. Herein, SPIONs were evaluated as radiosensitizers in A-375 human melanoma cells. Moreover, the effect of the combined treatment of SPIONs and gamma irradiation (SPIONs-IR) was assessed at the DNA level, where DSBs induction and their repair capacity were studied. SPIONs were synthesized, stabilized by poly(ethylene glycol) methyl ether and physicochemically characterized by high resolution-transmission electron microscopy (HR-TEM), X-ray diffraction and magnetometry and dynamic light scattering. The obtained nanoparticles showing superparamagnetic behavior and low dispersion in shape and sizes were tested in A-375 cells. The intracellular internalization of SPIONs was verified by HR-TEM and quantified by inductively coupled plasma atomic emission spectroscopy. Cells treated with SPIONs exhibited high ROS levels without associated cytotoxicity. Next, a significant radiosensitization in SPIONs-IR vs. control (IR) cells was demonstrated at 1Gy of gamma radiation. Furthermore, a decreased DSBs repair capacity in SPIONs-IR vs. IR-treated cells was evidenced by the size increase of persistent phosphorylated H2AX foci at 24h post-irradiation. In conclusion, these nanoparticles show the potential to radiosensitize melanoma cells by the induction of unrepairable DNA damage.