Modulating DNA damage response in uveal melanoma through embryonic stem cell microenvironment.

BACKGROUND: Uveal melanoma (UVM) is the most common primary intraocular tumor in adults, with a median survival of 4-5 months following metastasis. DNA damage response (DDR) upregulation in UVM, which could be linked to its frequent activation of the PI3K/AKT pathway, contributes to its treatment resistance. We have reported that embryonic stem cell microenvironments (ESCMe) can revert cancer cells to less aggressive states through downregulation of the PI3K signaling, showing promise in modulating the DDR of UVM. METHODS: Since nonhomologous end joining (NHEJ) is the main DNA repair mechanism in UVM, this study utilized gene expression analysis and survival prognosis analysis to investigate the role of NHEJ-related genes in UVM based on public databases. Xenograft mouse models were established to assess the therapeutic potential of ESC transplantation and exposure to ESC-conditioned medium (ESC-CM) on key DNA repair pathways in UVM. Quantitative PCR and immunohistochemistry were used to analyze NHEJ pathway-related gene expression in UVM and surrounding normal tissues. Apoptosis in UVM tissues was evaluated using the TUNEL assay. RESULTS: PRKDC, KU70, XRCC5, LIG4 and PARP1 showed significant correlations with UM progression. High expression of PRKDC and XRCC5 predicted poorer overall survival, while low PARP1 and XRCC6 expression predicted better disease-free survival in UVM patients. ESCMe treatment significantly inhibited the NHEJ pathway transcriptionally and translationally and promoted apoptosis in tumor tissues in mice bearing UVM. Furthermore, ESC transplantation enhanced DDR activities in surrounding normal cells, potentially mitigating the side effects of cancer therapy. Notably, direct cell-to-cell contact with ESCs was more effective than their secreted factors in regulating the NHEJ pathway. CONCLUSIONS: Our results suggest that NHEJ-related genes might serve as prognostic markers and therapeutic targets in UVM. These findings support the therapeutic potential of ESC-based therapy in enhancing UVM sensitivity to radiochemotherapy and improving treatment outcomes while minimizing damage to healthy cells.

The impact of developmental stage, tissue type, and sex on DNA double-strand break repair in Drosophila melanogaster.

Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR-white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR-white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms.

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

CircUGGT2 facilitates progression and cisplatin resistance of bladder cancer through nonhomologous end-joining pathway.

The development of resistance to cisplatin (CDDP) in bladder cancer presents a notable obstacle, with indications pointing to the substantial role of circular RNAs (circRNAs) in this resistance. Nevertheless, the precise mechanisms through which circRNAs govern resistance are not yet fully understood. Our findings demonstrate that circUGGT2 is significantly upregulated in bladder cancer, facilitating cancer cell migration and invasion. Additionally, our analysis of eighty patient outcomes revealed a negative correlation between circUGGT2 expression levels and prognosis. Using circRNA pull-down assays, mass spectrometry analyses, and RNA Immunoprecipitation (RIP), it was shown that circUGGT2 interacts with the KU heterodimer, consisting of KU70 and KU80. Both KU70 and KU80 are critical components of the non-homologous end joining (NHEJ) pathway, which plays a role in CDDP resistance. Flow cytometry was utilized in this study to illustrate the impact of circUGGT2 on the sensitivity of bladder cancer cell lines to CDDP through its interaction with KU70 and KU80. Additionally, a reduction in the levels of DNA repair factors associated with the NHEJ pathway, such as KU70, KU80, DNA-PKcs, and XRCC4, was observed in chromatin of bladder cancer cells following circUGGT2 knockdown post-CDDP treatment, while the levels of DNA repair factors in total cellular proteins remained constant. Thus, the promotion of CDDP resistance by circUGGT2 is attributed to its facilitation of repair factor recruitment to DNA breaks via interaction with the KU heterodimer. Furthermore, our study demonstrated that knockdown of circUGGT2 resulted in reduced levels of γH2AX, a marker of DNA damage response, in CDDP-treated bladder cancer cells, implicating circUGGT2 in the NHEJ pathway for DNA repair.

Changes in repair pathways of radiation-induced DNA double-strand breaks at the midblastula transition in Xenopus embryo.

Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis, serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos.

High expression of PPP1CC promotes NHEJ-mediated DNA repair leading to radioresistance and poor prognosis in nasopharyngeal carcinoma.

Protein phosphatase 1 catalytic subunit gamma (PPP1CC) promotes DNA repair and tumor development and progression, however, its underlying mechanisms remain unclear. This study investigated the molecular mechanism of PPP1CC's involvement in DNA repair and the potential clinical implications. High expression of PPP1CC was significantly correlated with radioresistance and poor prognosis in human nasopharyngeal carcinoma (NPC) patients. The mechanistic study revealed that PPP1CC bound to Ku70/Ku80 heterodimers and activated DNA-PKcs by promoting DNA-PK holoenzyme formation, which enhanced nonhomologous end junction (NHEJ) -mediated DNA repair and led to radioresistance. Importantly, BRCA1-BRCA2-containing complex subunit 3 (BRCC3) interacted with PPP1CC to enhance its stability by removing the K48-linked polyubiquitin chain at Lys234 to prevent PPP1CC degradation. Therefore, BRCC3 helped the overexpressed PPP1CC to maintain its high protein level, thereby sustaining the elevation of DNA repair capacity and radioresistance. Our study identified the molecular mechanism by which PPP1CC promotes NHEJ-mediated DNA repair and radioresistance, suggesting that the BRCC3-PPP1CC-Ku70 axis is a potential therapeutic target to improve the efficacy of radiotherapy.

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair.

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

LC3B drives transcription-associated homologous recombination via direct interaction with R-loops.

Exploring the connection between ubiquitin-like modifiers (ULMs) and the DNA damage response (DDR), we employed several advanced DNA damage and repair assay techniques and identified a crucial role for LC3B. Notably, its RNA recognition motif (RRM) plays a pivotal role in the context of transcription-associated homologous recombination (HR) repair (TA-HRR), a particular subset of HRR pathways. Surprisingly, independent of autophagy flux, LC3B interacts directly with R-loops at DNA lesions within transcriptionally active sites via its RRM, promoting TA-HRR. Using native RNA immunoprecipitation (nRIP) coupled with high-throughput sequencing (nRIP-seq), we discovered that LC3B also directly interacts with the 3'UTR AU-rich elements (AREs) of BRCA1 via its RRM, influencing its stability. This suggests that LC3B regulates TA-HRR both proximal to and distal from DNA lesions. Data from our LC3B depletion experiments showed that LC3B knockdown disrupts end-resection for TA-HRR, redirecting it towards the non-homologous end joining (NHEJ) pathway and leading to chromosomal instability, as evidenced by alterations in sister chromatid exchange (SCE) and interchromosomal fusion (ICF). Thus, our findings unveil autophagy-independent functions of LC3B in DNA damage and repair pathways, highlighting its importance. This could reshape our understanding of TA-HRR and the interaction between autophagy and DDR.

The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK.

BRCA1-associated protein 1 (BAP1) has emerged as a major tumor suppressor gene in diverse cancer types, notably in malignant pleural mesothelioma (DPM), and has also been identified as a germline cancer predisposition gene for DPM and other select cancers. However, its role in the response to DNA damage has remained unclear. Here, we show that BAP1 inactivation is associated with increased DNA damage both in Met-5A human mesothelial cells and human DPM cell lines. Through proteomic analyses, we identified PRKDC as an interaction partner of BAP1 protein complexes in DPM cells and 293 T human embryonic kidney cells. PRKDC encodes the catalytic subunit of DNA protein kinase (DNA-PKcs) which functions in the nonhomologous end-joining (NHEJ) pathway of DNA repair. Double-stranded DNA damage resulted in prominent nuclear expression of BAP1 in DPM cells and phosphorylation of BAP1 at serine 395. A plasmid-based NHEJ assay confirmed a significant effect of BAP1 knockdown on cellular NHEJ activity. Combination treatment with X-ray irradiation and gemcitabine (as a radiosensitizer) strongly suppressed the growth of BAP1-deficient cells. Our results suggest reciprocal positive interactions between BAP1 and DNA-PKcs, based on phosphorylation of BAP1 by the latter and deubiquitination of DNA-PKcs by BAP1. Thus, functional interaction of BAP1 with DNA-PKcs supports a role for BAP1 in NHEJ DNA repair and may provide the basis for new therapeutic strategies and new insights into its role as a tumor suppressor.

New Discoveries on Protein Recruitment and Regulation during the Early Stages of the DNA Damage Response Pathways.

Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR). When these pathways do not properly repair damaged DNA, genomic stability is compromised and can contribute to diseases such as cancer. It is essential that the causes of DNA damage and the consequent repair pathways are fully understood, yet the initial recruitment and regulation of DNA damage response proteins remains unclear. In this review, the causes of DNA damage, the various mechanisms of DNA damage repair, and the current research regarding the early steps of each major pathway were investigated.

Exploring DNA repair deficient CHO cell response to low dose rate radiation.

INTRODUCTION: DNA double-strand breaks (DSBs) induced by ionizing radiation pose a significant threat to genome integrity, necessitating robust repair mechanisms. This study explores the responses of repair-deficient cells to low dose rate (LDR) radiation. Non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways play pivotal roles in maintaining genomic stability. The hypothesis posits distinct cellular outcomes under LDR exposure compared to acute radiation, impacting DNA repair mechanisms and cell survival. MATERIALS AND METHODS: Chinese hamster ovary (CHO) cells, featuring deficiencies in NHEJ, HR, Fanconi Anemia, and PARP pathways, were systematically studied. Clonogenic assays for acute and LDR gamma-ray exposures, cell growth inhibition analyses, and γ-H2AX foci assays were conducted, encompassing varied dose rates to comprehensively assess cellular responses. RESULTS: NHEJ mutants exhibited an unexpected inverse dose rate effect, challenging conventional expectations. HR mutants displayed unique radiosensitivity patterns, aligning with responses to major DNA-damaging agents. LDR exposure induced cell cycle alterations, growth delays, and giant cell formation, revealing context-dependent sensitivities. γ-H2AX foci assays indicated DSB accumulation during LDR exposure. DISCUSSION: These findings challenge established paradigms, emphasizing the intricate interplay between repair pathways and dose rates. The study offers comprehensive insights into repair-deficient cell responses, urging a reevaluation of conventional dose-response models and providing potential avenues for targeted therapeutic strategies in diverse radiation scenarios.

TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) stands as the fifth most prevalent malignant tumor on a global scale and presents as the second leading cause of cancer-related mortality. DNA damage-based radiotherapy (RT) plays a pivotal role in the treatment of HCC. Nevertheless, radioresistance remains a primary factor contributing to the failure of radiation therapy in HCC patients. In this study, we investigated the functional role of transketolase (TKT) in the repair of DNA double-strand breaks (DSBs) in HCC. Our research unveiled that TKT is involved in DSB repair, and its depletion significantly reduces both non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair. Mechanistically, TKT interacts with PARP1 in a DNA damage-dependent manner. Furthermore, TKT undergoes PARylation by PARP1, resulting in the inhibition of its enzymatic activity, and TKT can enhance the auto-PARylation of PARP1 in response to DSBs in HCC. The depletion of TKT effectively mitigates the radioresistance of HCC, both in vitro and in mouse xenograft models. Moreover, high TKT expression confers resistance of RT in clinical HCC patients, establishing TKT as a marker for assessing the response of HCC patients who received cancer RT. In summary, our findings reveal a novel mechanism by which TKT contributes to the radioresistance of HCC. Overall, we identify the TKT-PARP1 axis as a promising potential therapeutic target for improving RT outcomes in HCC.

PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

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.

PKCδ Regulates Chromatin Remodeling and DNA Repair through SIRT6.

Irradiation (IR) is a highly effective cancer therapy; however, IR damage to tumor-adjacent healthy tissues can result in significant comorbidities and potentially limit the course of therapy. We have previously shown that protein kinase C delta (PKCδ) is required for IR-induced apoptosis and that inhibition of PKCδ activity provides radioprotection in vivo. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double-stranded break (DSB) repair through a mechanism that requires Sirtuin 6 (SIRT6). Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via nonhomologous end joining (NHEJ) and homologous recombination (HR) as evidenced by increased formation of DNA damage foci, increased expression of DNA repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis reveals increased chromatin associated H3K36me2 in PKCδ-depleted cells which is accompanied by chromatin disassociation of KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased SIRT6 expression, and depletion of SIRT6 reverses changes in chromatin accessibility, histone modification and DSB repair in PKCδ-depleted cells. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to regulate DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ. IMPLICATIONS: PKCδ controls sensitivity to irradiation by regulating DNA repair.

MiR-223-3p promotes genomic stability of hematopoietic progenitors after radiation.

When hematopoietic cells are overwhelmed with ionizing radiation (IR) DNA damage, the alternative non-homologous end-joining (aNHEJ) repair pathway is activated to repair stressed replication forks. While aNHEJ can rescue cells overwhelmed with DNA damage, it can also mediate chromosomal deletions and fusions, which can cause mis-segregation in mitosis and resultant aneuploidy. We previously reported that a hematopoietic microRNA, miR-223-3p, normally represses aNHEJ. We found that miR-223-/- mice have increased survival of hematopoietic stem and progenitor cells (HSPCs) after sublethal IR. However, this came at the cost of significantly more genomic aberrancies, with miR-223-/- hematopoietic progenitors having increased metaphase aberrancies, including chromothripsis, and increased sequence abnormalities, especially deletions, which is consistent with aNHEJ. These data imply that when an HSPC is faced with substantial DNA damage, it may trade genomic damage for its own survival by choosing the aNHEJ repair pathway, and this choice is regulated in part by miR-223-3p.

Decline of DNA damage response along with myogenic differentiation.

DNA integrity is incessantly confronted to agents inducing DNA lesions. All organisms are equipped with a network of DNA damage response mechanisms that will repair DNA lesions and restore proper cellular activities. Despite DNA repair mechanisms have been revealed in replicating cells, still little is known about how DNA lesions are repaired in postmitotic cells. Muscle fibers are highly specialized postmitotic cells organized in syncytia and they are vulnerable to age-related degeneration and atrophy after radiotherapy treatment. We have studied the DNA repair capacity of muscle fiber nuclei and compared it with the one measured in proliferative myoblasts here. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair, the nonhomologous end joining, and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For base excision repair and HR, this decline can be linked to reduced steady-state levels of key proteins involved in these processes.

Palmitoyl ascorbic acid glucoside enhanced cell survival with post irradiation treatment.

Radiation exposure poses a significant threat to cellular integrity by inducing DNA damage through the generation of free radicals and reactive oxygen species. Ascorbic acid, particularly its derivative Palmitoyl Ascorbic Acid 2-Glucoside (PA2G), has demonstrated remarkable radioprotective properties. While previous research focused on its pre-irradiation application, this study explores the post-irradiation radiomitigation potential of PA2G. Our findings reveal that post-irradiation treatment with PA2G enhances cell survival and accelerates DNA repair processes, particularly the non-homologous end-joining (NHEJ) repair pathway. Notably, PA2G treatment reduces the frequency of lethal chromosomal aberrations and micronuclei formation, indicating its ability to enhance the repair of complex DNA lesions. Furthermore, PA2G is shown to play a role in potentially lethal damage repair (PLDR). These radioprotective effects are specific to NHEJ and ATM pathways, as cells deficient in these mechanisms do not benefit from PA2G treatment. This study highlights PA2G as a versatile radioprotector, both pre- and post-irradiation, with significant potential for applications in radiation therapy and protection, offering new insights into its mechanism of action. Further research is required to elucidate the precise molecular mechanisms underlying PA2G's radiomitigation effects and its potential clinical applications.