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Abstract Background RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear. Methods To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomics approach. Results Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion. Conclusions Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

RAD51 loading onto chromatin is a key step during the homologous recombination (HR) repair. We recently reported a new mode of RAD51 regulation, which is mediated by TOPORS E3 SUMO ligase and RAD51 SUMOylation. ATM/ATR-induced phosphorylation of TOPORS is necessary for this event, revealing a new role of these master DNA damage response kinases in HR repair.

Macrobrachium rosenbergii is an important cultured shrimp worldwide, and its precocious puberty has led to serious economic losses. Although reproductive sterilization can avoid this problem, little is known about the molecular mechanisms underlying gonadal development and gametogenesis in M. rosenbergii. Here, we conducted transcriptome analysis of the ovaries, testes, and male/female brain tissues of M. rosenbergii to discover germ cell development-related genes. A total of 60,607 unigenes were identified, of which 20,963 unigenes could be functionally annotated. Eighteen candidate genes were identified by Venn diagram analysis, keyword, and known marker search, followed by elimination of low-expression and repetitive genes. Subsequent real-time quantitative reverse transcription polymerase chain reaction and in situ hybridization identified five genes (RAD51, vasa, SPDS, MRR, and Fem-1C) associated with germ cell development—RAD51, vasa, and SPDS were expressed in both male and female gonads, whereas Fem-1C was specifically expressed in the ovary and MRR in the testis. In the ovary, vasa, SPDS, and Fem-1C were mainly expressed in stage 1–3 oocytes, while RAD51 was expressed in stage 2–3 oocytes. In testis, vasa were significantly expressed in spermatogonia and primary spermatocytes, whereas RAD51 and SPDS were only enriched in spermatogonia and MRR in vas deferens. Our research indicates that these five genes are important germ cell development-related genes, of which RAD51, SPDS, and Fem-1C were proven to function in germ cells of this species for the first time. The discovery of these genes could help develop molecular breeding techniques to overcome precocious puberty in M. rosenbergii.

PARP inhibitors (PARPi) are currently used as first-line therapy for advanced and recurrent ovarian cancer, but the clinical efficacy is limited by drug resistance. We aimed to investigate the role of KIAA1529 in PARPi resistance in ovarian cancer. The expression of KIAA1529 was determined in ovarian cancer cells using qRT‒PCR and western blotting. Immunohistochemistry was used to examine the expression of KIAA1529 in primary ovarian cancer and recurrent ovarian cancer tissues. The effects of KIAA1529 on PARPi resistance were evaluated by knocking down KIAA1529 expression in ovarian cancer cells and assessing cell viability by CCK8 assays, apoptosis by flow cytometry, and homologous recombination (HR) repair by immunofluorescence analysis. The interaction between KIAA1529 and RAD51 was examined by western blotting. KIAA1529 was confirmed to be expressed in all ovarian cancer cell lines, and high expression of KIAA1529 was observed in recurrent ovarian cancer tissues. Inhibiting KIAA1529 expression increased the sensitivity of ovarian cancer cells to PARPi treatment. Furthermore, KIAA1529 increased the expression of the downstream effector RAD51 via Aurora-A, and HR was restored in ovarian cancer cells. This study demonstrates that KIAA1529 regulates RAD51 expression through Aurora-A to restore HR, which confers resistance to PARPi in ovarian cancer cells. These findings could provide a novel therapeutic target to overcome PARPi resistance in ovarian cancer.

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.

DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS-1 pretreatment could increase the survival and regeneration of irradiated intestinal organoids in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6 Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms. • RAD51-stimulatory compound RS-1 protect intestinal tissue against lethal irradiation. • Protective effect of RS-1 against irradiation on hematopoietic tissue is limited. • Transient HR enhancement can protect intestinal stem cells from mitotic death. • Transient HR enhancement can facilitate the survival of intestinal progenitor cells. [ABSTRACT FROM AUTHOR]

DNA replication stress is one of the primary causes of genome instability. In response to replication stress, cells can employ replication restart mechanisms that rely on homologous recombination to resume replication fork progression and preserve genome integrity. In this review, we provide an overview of various methods that have been developed to induce site-specific replication fork stalling or collapse in eukaryotic cells. In particular, we highlight recent studies of mechanisms of replication-associated recombination resulting from site-specific protein-DNA barriers and single-strand breaks, and we discuss the contributions of these findings to our understanding of the consequences of these forms of stress on genome stability. • Homologous recombination is required tomaintain genome integrity in response to replication stress. • Site-specific, protein-induced replicationfork barriers induce local Rad51-dependent recombination. • Replication fork collision with a nick canproduce one-ended or two-ended double-strand breaks. • Replication-dependent DSBs are repaired byhomologous recombination with the sister chromatid. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer is the most aggressive subtype of mammary carcinoma. In the early stage, neoadjuvant chemotherapy (NAC) is the standard of care for prognostic stratification and the best adjuvant treatment strategy. A 30-year-old female presented in the emergency room because of a gigantic right breast associated with an ulcerated lump at the upper quadrants. The right axillary nodes were palpable. An ultrasound was performed, showing the ulcerated neoformation with enlarged right axillary lymph nodes observed to level III. A core biopsy of the breast lesion was performed, and the pathological examination revealed a nonspecial type, grade 3, invasive, triple-negative breast cancer. No distant disease was found in the PET-CT scan. A germline genetic panel by next-generation sequencing identified a likely pathogenic variant in RAD51D (c.898C>T). Assessment of the functionality of the DNA homologous recombination repair pathway by RAD51 foci in the tumor revealed a profile of homologous recombination deficiency. NAC consisting of weekly carboplatin and paclitaxel followed by dose-dense doxorubicin/cyclophosphamide was performed with a complete metabolic response achieved in the PET-CT scan. The patient underwent a modified radical mastectomy plus axillary lymphadenectomy with a pathological complete response in the breast and axilla and remains disease-free after 2 years of follow-up. We report a young female with a triple-negative breast cancer stage cT4bN3M0 and a hereditary pathogenic mutation in RAD51D. The tumor was highly proliferative and homologous recombination-deficient by RAD51. The patient received platinum-based NAC, achieving a pathologic complete response. More effort should be made to identify predictive functional biomarkers of treatment response, such as RAD51 foci, for platinum sensitivity. [ABSTRACT FROM AUTHOR]

Ultraviolet-induced DNA lesions impede DNA replication and transcription and are therefore a potential source of genome instability. Here, we performed serial transfer experiments on nucleotide excision repair-deficient (rad14D) yeast cells in the presence of chronic low-dose ultraviolet irradiation, focusing on the mechanisms underlying adaptive responses to chronic low-dose ultraviolet irradiation. Our results show that the entire haploid rad14D population rapidly becomes diploid during chronic low-dose ultraviolet exposure, and the evolved diploid rad14D cells were more chronic low-dose ultraviolet-resistant than haploid cells. Strikingly, single-stranded DNA, but not pyrimidine dimer, accumulation is associated with diploid-dependent fitness in response to chronic low-dose ultraviolet stress, suggesting that efficient repair of single-stranded DNA tracts is beneficial for chronic low-dose ultraviolet tolerance. Consistent with this hypothesis, homologous recombination is essential for the rapid evolutionary adaptation of diploidy, and rad14D cells lacking Rad51 recombinase, a key player in homologous recombination, exhibited abnormal cell morphology characterized by multiple RPA-yellow fluorescent protein foci after chronic low-dose ultraviolet exposure. Furthermore, interhomolog recombination is increased in chronic low-dose ultravioletexposed rad14D diploids, which causes frequent loss of heterozygosity. Thus, our results highlight the importance of homologous recombination in the survival and genomic stability of cells with unrepaired lesions. [ABSTRACT FROM AUTHOR]

Radiation therapy induces double-stranded DNA breaks in tumor cells, which leads to their death. A fraction of glioblastoma cells repair such breaks and reinitiate tumor growth. It was necessary to identify the relationship between high radiation doses and the proliferative activity of glioblastoma cells, and to evaluate the contribution of DNA repair pathways, homologous recombination (HR), and nonhomologous end joining (NHEJ) to tumor-cell recovery. We demonstrated that the GO1 culture derived from glioblastoma cells from Patient G, who had previously been irradiated, proved to be less sensitive to radiation than the Sus\fP2 glioblastoma culture was from Patient S, who had not been exposed to radiation before. GO1 cell proliferation decreased with radiation dose, and MTT decreased to 35% after a single exposure to 125 Gg. The proliferative potential of glioblastoma culture Sus\fP2 decreased to 35% after exposure to 5 Gg. At low radiation doses, cell proliferation and the expression of RAD51 were decreased; at high doses, cell proliferation was correlated with Ku70 protein expression. Therefore, HR and NHEJ are involved in DNA break repair after exposure to different radiation doses. Low doses induce HR, while higher doses induce the faster but less accurate NHEJ pathway of double-stranded DNA break repair. [ABSTRACT FROM AUTHOR]

Defects in DNA repair pathways are emerging hallmarks of cancer. Accurate DNA repairs and replications are essential for genomic stability. Cancer cells require residual DNA repair capabilities to repair the damage from replication stress and genotoxic anti-tumor agents. Defective DNA repair also promotes the accumulation of genomic changes that eventually lead to tumorigenesis, tumor progression, and therapeutic resistance to DNA-damaging anti-tumor agents. Rad51 recombinase is a critical effector of homologous recombination, which is an essential DNA repair mechanism for double-strand breaks. Rad51 has been found to be upregulated in many malignant solid tumors, and is correlated with poor prognosis. In multiple tumor types, Rad51 is critical for tumor metabolism, metastasis and drug resistance. Herein, we initially introduced the structure, expression pattern of Rad51 and key Rad51 mediators involved in homologous recombination. Additionally, we primarily discussed the role of Rad51 in tumor metabolism, metastasis, resistance to chemotherapeutic agents and poly-ADP ribose polymerase inhibitors. [ABSTRACT FROM AUTHOR]

The recurrent mutation (S34F) in splicing factor U2AF1 is frequently observed in lung adenocarcinoma, but its function remains largely unknown. To determine the mechanistic basis and consequences of U2AF1 mutations, we established non–small cell lung carcinoma A549 cell lines with exogenous expression of wildtype (U2AF1‐WT) or mutant (U2AF1‐S34F). Splicing analysis revealed that U2AF1‐S34F mainly caused aberrant exon usage and affected splicing of numerous DNA damage repair genes. Compared to A549 cells expressing U2AF1‐WT, cells expressing U2AF1‐S34F showed enhanced DNA damage and cell death in response to ATR inhibitors (ATRi). Mechanistically, U2AF1‐S34F induced mis‐splicing and downregulation of a key homologous recombination protein RAD51. Overexpression of RAD51 could largely rescue the defective DNA damage response in cells expressing U2AF1‐S34F. Moreover, A549 cells expressing U2AF1‐S34F, but not U2AF1‐WT, were highly sensitive to treatment even with low dose of RAD51 inhibitor on ATRi‐induced DNA damage. Our results suggest that U2AF1‐S34F causes mis‐splicing of DNA damage repair factors in lung cancer and sensitizes cells to RAD51 inhibition. [ABSTRACT FROM AUTHOR]

Abstract Background Ovarian cancer has greatly endangered and deteriorated female health conditions worldwide. Refinement of predictive biomarkers could enable patient stratification and help optimize disease management. Methods RAD51 expression profile, target-disease associations, and fitness scores of RAD51 were analyzed in ovarian cancer using bioinformatic analysis. To further identify its role, gene enrichment analysis was performed, and a regulatory network was constructed. Survival analysis and drug sensitivity assay were performed to evaluate the effect of RAD51 expression on ovarian cancer prognosis. The predictive value of RAD51 was then confirmed in a validation cohort immunohistochemically. Results Ovarian cancer expressed more RAD51 than normal ovary. RAD51 conferred ovarian cancer dependency and was associated with ovarian cancer. RAD51 had extensive target-disease associations with various diseases, including ovarian cancer. Genes that correlate with and interact with RAD51 were involved in DNA damage repair and drug responsiveness. High RAD51 expression indicated unfavorable survival outcomes and resistance to platinum, taxane, and PARP inhibitors in ovarian cancer. In the validation cohort (126 patients), high RAD51 expression indicated platinum resistance, and platinum-resistant patients expressed more RAD51. Patients with high RAD51 expression had shorter OS (HR = 2.968, P

Triple-negative breast cancer is the most aggressive subtype of mammary carcinoma. In the early stage, neoadjuvant chemotherapy (NAC) is the standard of care for prognostic stratification and the best adjuvant treatment strategy. A 30-year-old female presented in the emergency room because of a gigantic right breast associated with an ulcerated lump at the upper quadrants. The right axillary nodes were palpable. An ultrasound was performed, showing the ulcerated neoformation with enlarged right axillary lymph nodes observed to level III. A core biopsy of the breast lesion was performed, and the pathological examination revealed a nonspecial type, grade 3, invasive, triple-negative breast cancer. No distant disease was found in the PET-CT scan. A germline genetic panel by next-generation sequencing identified a likely pathogenic variant in RAD51D (c.898C>T). Assessment of the functionality of the DNA homologous recombination repair pathway by RAD51 foci in the tumor revealed a profile of homologous recombination deficiency. NAC consisting of weekly carboplatin and paclitaxel followed by dose-dense doxorubicin/cyclophosphamide was performed with a complete metabolic response achieved in the PET-CT scan. The patient underwent a modified radical mastectomy plus axillary lymphadenectomy with a pathological complete response in the breast and axilla and remains disease-free after 2 years of follow-up. We report a young female with a triple-negative breast cancer stage cT4bN3M0 and a hereditary pathogenic mutation in RAD51D. The tumor was highly proliferative and homologous recombination-deficient by RAD51. The patient received platinum-based NAC, achieving a pathologic complete response. More effort should be made to identify predictive functional biomarkers of treatment response, such as RAD51 foci, for platinum sensitivity.

Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic; however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele, whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

Radiation therapy induces double-stranded DNA breaks in tumor cells, which leads to their death. A fraction of glioblastoma cells repair such breaks and reinitiate tumor growth. It was necessary to identify the relationship between high radiation doses and the proliferative activity of glioblastoma cells, and to evaluate the contribution of DNA repair pathways, homologous recombination (HR), and nonhomologous end joining (NHEJ) to tumor-cell recovery. We demonstrated that the GO1 culture derived from glioblastoma cells from Patient G, who had previously been irradiated, proved to be less sensitive to radiation than the Sus\fP2 glioblastoma culture was from Patient S, who had not been exposed to radiation before. GO1 cell proliferation decreased with radiation dose, and MTT decreased to 35% after a single exposure to 125 Gγ. The proliferative potential of glioblastoma culture Sus\fP2 decreased to 35% after exposure to 5 Gγ. At low radiation doses, cell proliferation and the expression of RAD51 were decreased; at high doses, cell proliferation was correlated with Ku70 protein expression. Therefore, HR and NHEJ are involved in DNA break repair after exposure to different radiation doses. Low doses induce HR, while higher doses induce the faster but less accurate NHEJ pathway of double-stranded DNA break repair.

Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.

Cellular fate decisions are influenced by their topographical location in the adult body. For instance, tissue repair and neoplastic growth are greater in anterior than in posterior regions of adult animals. However, the molecular underpinnings of these regional differences are unknown. We identified a regional switch in the adult planarian body upon systemic disruption of homologous recombination with RNA-interference of Rad51 Rad51 knockdown increases DNA double-strand breaks (DSBs) throughout the body, but stem cells react differently depending on their location along the anteroposterior axis. In the presence of extensive DSBs, cells in the anterior part of the body resist death, whereas cells in the posterior region undergo apoptosis. Furthermore, we found that proliferation of cells with DNA damage is induced in the presence of brain tissue and that the retinoblastoma pathway enables overproliferation of cells with DSBs while attending to the demands of tissue growth and repair. Our results implicate both autonomous and non-autonomous mechanisms as key mediators of regional cell behavior and cellular transformation in the adult body.

Abstract Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A‐priori identification of platinum resistance is therefore crucial to improve on standard first‐line carboplatin–paclitaxel treatment. The DNA repair pathway homologous recombination (HR) repairs platinum‐induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK‐compliant study of pre‐treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein expression in situ. In a discovery cohort (n = 284), RAD51‐High tumours had shorter progression‐free and overall survival compared to RAD51‐Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin monotherapy SCOTROC4 clinical trial cohort (n = 264) and was predominantly noted in HR‐proficient cancers (Myriad HRDscore

The DNA damage response (DDR) is a complex signal transduction network that is activated when endogenous or exogenous genotoxins damage or interfere with the replication of genomic DNA. Under such conditions, the DDR promotes DNA repair and ensures accurate replication and division of the genome. High levels of genomic instability are frequently observed in cancers and can stem from germline loss‐of‐function mutations in certain DDR genes, such as BRCA1, BRCA2, and p53, that form the basis of human cancer predisposition syndromes. In addition, mutation and/or aberrant expression of multiple DDR genes are frequently observed in sporadic human cancers. As a result, the DDR is considered to represent a viable target for cancer therapy in humans and a variety of strategies are under investigation. Cancer is also a significant cause of mortality in dogs, a species that offers certain advantages for experimental oncology. Domestic dogs present numerous inbred lines, many of which display predisposition to specific forms of cancer and the study of which may provide insight into the biological basis of this susceptibility. In addition, clinical trials are possible in dogs and may lead to therapeutic insights that could ultimately be extended to humans. Here we review what is known specifically about the DDR in dogs and discuss how this knowledge could be extended and exploited to advance experimental oncology in this species. [ABSTRACT FROM AUTHOR]

Background: Limb regeneration in the axolotl is achieved by epimorphosis, thus depending on the blastema formation, a mass of progenitor cells capable of proliferating and differentiating to recover all lost structures functionally. During regeneration, the blastema cells accelerate the cell cycle and duplicate its genome, which is inherently difficult to replicate because of its length and composition, thus being prone to suffer double‐strand breaks. Results: We identified and characterized two remarkable components of the homologous recombination repair pathway (Amex.RAD51 and Amex.MRE11), which were heterologously expressed, biochemically characterized, and inhibited by specific chemicals. These same inhibitors were applied at different time points after amputation to study their effects during limb regeneration. We observed an increase in cellular senescent accompanied by a slight delay in regeneration at 28 days postamputation regenerated tissues; moreover, inhibitors caused a rise in the double‐strand break signaling as a response to the inhibition of the repair mechanisms. Conclusions: We confirmed the participation and importance of homologous recombination during limb regeneration. The chemical inhibition induces double‐strand breaks that lead to DNA damage associated senescence, or in an alternatively way, this damage could be possibly repaired by a different DNA repair pathway, permitting proper regeneration and avoiding senescence. Key Findings: A. mexicanum possesses true homologs for Rad51 and Mre11.B02 and Mirin chemicals inhibit Amex.RAD51 and Amex.MRE11 respectively.B02 and Mirin provoke an increase in DNA damage during regeneration.B02 produces an increase in the number of senescent cells during regeneration. [ABSTRACT FROM AUTHOR]

The BRCA2 germline missense variant, R3052W, resides in the DNA binding domain and has been previously classified as a pathogenic allele. In this study, we sought to determine how R3052W alters the cellular functions of BRCA2 in the DNA damage response. The BRCA2 R3052W mutated protein exacerbates genome instability, is unable to rescue homology-directed repair, and fails to complement cell survival following exposure to PARP inhibitors and crosslinking drugs. Surprisingly, despite anticipated defects in DNA binding or RAD51-mediated DNA strand exchange, the BRCA2 R3052W protein mislocalizes to the cytoplasm precluding its ability to perform any DNA repair functions. Rather than acting as a simple loss-of-function mutation, R3052W behaves as a dominant negative allele, likely by sequestering RAD51 in the cytoplasm. [ABSTRACT FROM AUTHOR]

Simple Summary: Recent studies re-classified the c.5339T->C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as "likely pathogenic" in ACMG classification, which shows a high prevalence in the Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC). BRCA1 L1780P BRCT domain mutation has been recognized as a pathogenic mutation in patients with breast cancer. However, the molecular significance of this mutation has not yet been studied in triple-negative breast cancer (TNBC) cells in vitro. We established MDA-MB 231, HCC1937, and HCC1395 TNBC cell lines expressing BRCA1 L1780P mutant. BRCA1 L1780P mutant TNBC cells showed increased migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type cells. BRCA1 L1780P mutant TNBC cells showed decreased RAD51 expression and reduced nuclear RAD51 foci formation following carboplatin and olaparib treatment. The molecular interaction between p-ATM and BRCA1 was abrogated following introduction of BRCA1 L1780P mutant plasmid in TNBC cells, suggesting that the BRCA1 L1780P mutation disrupts the p-ATM-BRCA1 protein–protein interaction. We established an olaparib-resistant BRCA1 L1780P mutant TNBC cell line by chronic drug treatment. Olaparib-resistant cell lines showed upregulation of RAD51 expression upon olaparib treatment, and reduction in RAD51 expression in olaparib-resistant cells restored olaparib sensitivity. Collectively, these results suggest that the BRCA1 L1780P mutation impairs RAD51 recruitment by disrupting p-ATM-BRCA1 interaction, which is a crucial molecular factor in homologous recombination and olaparib sensitivity. Further therapeutic targeting of RAD51 in BRCA1 L1780P mutant breast cancer is warranted. [ABSTRACT FROM AUTHOR]

Purpose: Although the DNA repair mechanism is important in preventing carcinogenesis, its activation in established cancer cells may support their proliferation and aggravate cancer progression. RAD51 cooperates with BRCA2 and is essential in the homologous recombination of DNA repair. To this end, we hypothesized that RAD51 gene expression is associated with cancer cell proliferation and poor prognosis of breast cancer (BC) patients. Methods: A total of 8515 primary BC patients with transcriptome and clinical data from 17 independent cohorts were analyzed. The median value was used to divide each cohort into high and low RAD51 expression groups. Results: High RAD51 expression enriched the DNA repair gene set and was correlated with DNA repair-related genes. Nottingham histological grade, Ki67 expression and cell proliferation-related gene sets (E2F Targets, G2M Checkpoint and Myc Targets) were all significantly associated with the high RAD51 BC group. RAD51 expression was positively correlated with Homologous Recombination Deficiency, as well as both mutational burden and neoantigens that accompanied a higher infiltration of immune cells. Primary BC with lymph node metastases was associated with high expression of RAD51 in two cohorts. There was no strong correlation between RAD51 expression and drug sensitivity in cell lines, and RAD51 expression was lower after the neoadjuvant chemotherapy compared to before the treatment. High RAD51 BC was associated with poor prognosis consistently in three independent cohorts. Conclusion: RAD51 gene expression is associated with aggressive cancer biology, cancer cell proliferation, and poor survival in breast cancer. [ABSTRACT FROM AUTHOR]

Objective: Hypersensitivity towards proton versus photon irradiation was demonstrated in homologous recombination repair (HRR)-deficient cell lines. Hence, combined treatment concepts targeting HRR provide a rational for potential pharmaceutical exploitation. The HSP90 inhibitor ganetespib (STA-9090) downregulates a multitude of HRR-associated proteins and sensitizes for certain chemotherapeutics. Thus, the radiosensitizing effect of HSP90-inhibiting ganetespib was investigated for reference photon irradiation and proton irradiation at a proximal and distal position in a spread-out Bragg peak (SOBP).Methods: A549 and FaDu cells were treated with low-dose (2 nM resp. 1 nM) ganetespib and irradiated with 200 kV photons. Proton irradiation was performed at a proximal and a distal position within a SOBP, with corresponding dose-averaged linear-energy transfer (LETD) values of 2.1 and 4.5 keV/µm, respectively. Cellular survival data was fitted to the linear-quadratic model to calculate relative biological effectiveness (RBE) and the dose-modifying factor (DMF). Additionally, A549 cells were treated with increasing doses of ganetespib and investigated by flow cytometry, immunoblotting, and immunofluorescence microscopy to investigate cell cycle distribution, Rad51 protein levels, and γH2AX foci, respectively.Results: Low-dosed ganetespib significantly sensitized both cancer cell lines exclusively for proton irradiation at both investigated LETD, resulting in increased RBE values of 10-40%. In comparison to photon irradiation, the fraction of cells in S/G2/M phase was elevated in response to proton irradiation with 10 nM ganetespib consistently reducing this population. No changes in cell cycle distribution were detected in unirradiated cells by ganetespib alone. Protein levels of Rad51 are downregulated in irradiated A549 cells by 10 nM and also 2 nM ganetespib within 24 h. Immunofluorescence staining demonstrated similar induction and removal of γH2AX foci, irrespective of irradiation type or ganetespib administration.Conclusion: Our findings illustrate a proton-specific sensitizing effect of low-dosed ganetespib in both employed cell lines and at both investigated SOBP positions. We provide additional experimental data on cellular response and a rational for future combinatorial approaches with proton radiotherapy. [ABSTRACT FROM AUTHOR]

Background: This study aimed to evaluate for the first time whether certain genetic and clinical factors could serve as minimally invasive predictors of survival and toxicity to platinum-based chemotherapy in advanced lung adenocarcinoma. Methods: The study included 121 advanced lung adenocarcinoma patients treated with platinum-based dublets until progression or unacceptable toxicity. Response was evaluated using standard radiological methods and toxicity graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Genotyping was performed using PCR-RFLP. Statistical significance was set at P <.05. Results: No significant influence of the examined polymorphisms on the occurrence of high-grade toxicity was detected. However, TP53 72Pro allele carriers were more prone to nausea (P =.037) and thrombocytopenia (P =.051). Anemia and neuropathy occurred more frequently in XRCC1 399Arg allele carriers (Pearson χ2 test, P =.025 and P =.004 respectively). RAD51 135CC carriers were significantly more prone to neutropenia (P =.027). Conclusions: A set of easily determined genetic and clinical predictors of survival and specific toxicity profiles of platinum-based chemotherapy in advanced lung adenocarcinoma were determined in this study, which might be useful for the construction of population-specific, time- and cost-efficient prognostic and predictive algorithms. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study is to identify biomarkers that predict efficacy of preoperative therapy and survival for esophageal squamous cell carcinoma (ESCC). Background: It is essential to improve the accuracy of preoperative molecular diagnostics to identify specific patients who will benefit from the treatment; thus, this issue should be resolved with a large-cohort, retrospective observational study. Methods: A total of 656 patients with ESCC who received surgery after preoperative CDDP + 5-FU therapy, docetaxel + CDDP + 5-FU therapy or chemoradiotherapy (CRT) were enrolled. Immunohistochemical analysis of TP53, CDKN1A, RAD51, MutT-homolog 1, and programmed death-ligand 1 was performed with biopsy samples obtained before preoperative therapy, and expression was measured by immunohistochemistry. Results: In all therapy groups, overall survival was statistically separated by pathological effect (grade 3 > grade 2 > grade 0, 1, P < 0.0001). There was no correlation between TP53, CDKN1A, MutT-homolog 1, programmed death-ligand 1 expression, and pathological effect, whereas the proportion of positive RAD51 expression (>=50%) in cases with grade 3 was lower than that with grade 0, 1, and 2 (P = 0.022). In the CRT group, the survival of patients with RAD51-positive tumor was significantly worse than RAD51-negative expressors (P = 0.0119). Subgroup analysis of overall survival with respect to positive RAD51 expression indicated preoperative chemotherapy (CDDP + 5-FU or docetaxel + CDDP + 5-FU) was superior to CRT. Conclusions: In ESCC, positive RAD51 expression was identified as a useful biomarker to predict resistance to preoperative therapy and poor prognosis in patients who received preoperative CRT. Administration of preoperative chemotherapy may be warranted for patients with positive RAD51 expression. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) infection is the strongest known risk factor for the development of gastric cancer. DNA damage response (DDR) and autophagy play key roles in tumorigenic transformation. However, it remains unclear how H. pylori modulate DDR and autophagy in gastric carcinogenesis. Here we report that H. pylori infection promotes DNA damage via suppression of Rad51 expression through inhibition of autophagy and accumulation of p62 in gastric carcinogenesis. We find that H. pylori activated DNA damage pathway in concert with downregulation of repair protein Rad51 in gastric cells, C57BL/6 mice and Mongolian gerbils. In addition, autophagy was increased early and then decreased gradually during the duration of H. pylori infection in vitro in a CagA-dependent manner. Moreover, loss of autophagy led to promotion of DNA damage in H. pylori-infected cells. Furthermore, knockdown of autophagic substrate p62 upregulated Rad51 expression, and p62 promoted Rad51 ubiquitination via the direct interaction of its UBA domain. Finally, H. pylori infection was associated with elevated levels of p62 in gastric intestinal metaplasia and decreased levels of Rad51 in dysplasia compared to their H. pylori- counterparts. Our findings provide a novel mechanism into the linkage of H. pylori infection, autophagy, DNA damage and gastric tumorigenesis.

Objective: Chemoresistance is one of the major barriers in chemotherapy-based hepatocellular carcinoma (HCC) intervention. 5-Fluorouracil (5-Fu) is a widely used as an anticancer drug. Liver cancer stem cells (LCSCs) are considered the origin of tumor recurrence and resistance. CCND1 (Cyclin D1) plays an important role in tumorigenesis and metastasis in multiple cancers including HCC. Herein, this study was designed to explore the role of CCND1 in regulating LCSCs differentiation and 5-Fu resistance in HCC cells. Methods: The CCND1 mRNA level was examined by qRT-PCR. The protein levels of γ-H2AX (a DNA damage marker) and RAD51 (a DNA repair protein) were examined by Western blot. CD133 was used as a LCSC marker and CD133+ cell percentage in HCC cells was detected by flow cytometry. Results: CCND1 silencing decreased CD133+ cell percentage in HepG2 and SMMC-7721 cells. Furthermore, CCND1 silencing significantly increased protein level of γ-H2AX and decreased that of RAD51 under 5-Fu exposure. Moreover, CCND1 silencing enhanced the sensitivity of HepG2 and SMMC-7721 cells to 5-Fu, which was effectively abrogated by RAD51 upregulation. Conclusion: Collectively, CCND1 silencing suppresses LCSCs differentiation and overcomes 5-Fu resistance in HCC.

There are seven homologs in eukaryotic RAD51 gene family which are conserved among animals and plants, and those are RAD51, DMC1, RAD51c, XRCC3, RAD51b, RAD51d and XRCC2. The first four of them are important in the process of homologous recombination, but also the DNA repair mechanism, while the other three show normal meiosis. RAD51, DMC1, RAD51c and XRCC3 have lineages that are divergent from the other three paralogs, showing potential functional redundancy. The repair mechanism also includes single- or double-strand break rejoining during replication, recombination and DNA damage, which is made by the DNA ligase enzymes. There are many DNA ligase enzymes, and the sequenced genome of Arabidopsis thaliana showed a homologue XRCC4 of the human DNA ligase IV binding protein. Arabidopsis thaliana encoded also homologues for the other six vertebrate Rad51 proteins. Our Results showed the XRCC2 and XRCC3 are interacting with Rad51c. Tow complexes will be formed; BCDX2 (RAD51B-RAD51C-RAD51D-XRCC2) and CX3 (RAD51C-XRCC3). The two complexes have a function at two distinct stages of homologous recombinational DNA repair.

Songwei Huan,1,* Tao Gui,1,* Qiutong Xu,1,* Songkuan Zhuang,2 Zhenyan Li,1 Yuling Shi,3 Jiebin Lin,3 Bin Gong,1 Guiqiang Miao,1 Manseng Tam,4 Huan-Tian Zhang,1 Zhengang Zha,1 Chunfei Wu3 1Institute of Orthopedic Diseases and Department of Bone and Joint Surgery, The First Affiliated Hospital, Jinan University, Guangzhou 510630, Guangdong, People’s Republic of China; 2School of Life Science, Xiamen University, Xiamen, Fujian 361005, People’s Republic of China; 3Department of Orthopedics, The Third Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510405, People’s Republic of China; 4IAN WO Medical Center, Macao Special Administrative Region, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhengang Zha; Chunfei Wu Tel +86-20-38688617; Tel +86-13610075186Fax +86-20-38688000; Email tbri@jnu.edu.cn; wuchunfei666@163.comBackground: Chondrosarcoma is the second-most common type of bone tumor and has inherent resistance to conventional chemotherapy. Present study aimed to explore the therapeutic effect and specific mechanism(s) of combination BET family protein and HDAC inhibition in chondrosarcoma.Methods: Two chondrosarcoma cells were treated with BET family protein inhibitor (JQ1) and histone deacetylase inhibitors (HDACIs) (vorinostat/SAHA or panobinostat/PANO) separately or in combination; then, the cell viability was determined by Cell Counting Kit-8 (CCK-8) assay, and the combination index (CI) was calculated by the Chou method; cell proliferation was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation and colony formation assay; cell apoptosis and reactive oxygen species (ROS) level were determined by flow cytometry; protein expressions of caspase-3, Bcl-XL, Bcl-2, γ-H2AX, and RAD51 were examined by Immunoblotting; DNA damage was determined by comet assay; RAD51 and γ-H2AX foci were observed by immunofluorescence.Results: Combined treatment with JQ1 and SAHA or PANO synergistically suppressed the growth and colony formation ability of the chondrosarcoma cells. Combined BET and HDAC inhibition also significantly elevated the ROS level, followed by the activation of cleaved-caspase-3, and the downregulation of Bcl-2 and Bcl-XL. Mechanistically, combination treatment with JQ1 and SAHA caused numerous DNA double-strand breaks (DSBs), as evidenced by the comet assay. The increase in γ-H2AX expression and foci formation also consistently indicated the accumulation of DNA damage upon cotreatment with JQ1 and SAHA. Furthermore, RAD51, a key protein of homologous recombination (HR) DNA repair, was found to be profoundly suppressed. In contrast, ectopic expression of RAD51 partially rescued SW 1353 cell apoptosis by inhibiting the expression of cleaved-caspase-3.Conclusion: Taken together, our results disclose that BET and HDAC inhibition synergistically inhibit cell growth and induce cell apoptosis through a mechanism that involves the suppression of RAD51-related HR DNA repair in chondrosarcoma cells.Keywords: chondrosarcoma, JQ1, HDAC, apoptosis, RAD51, DNA repair

Targeting DNA damage response (DDR) pathway has been proposed as an approach for amplifying tumor-specific replicative lesions. RAD51 plays a central role in the DDR process, and thus represents a promising anti-tumor target. We here report the discovery of a series of next generation RAD51 inhibitors that can prevent RAD51 foci formation. The lead compounds dramatically impaired human cancer cell growth, induced cell cycle arrest in S-phase, and resulted in elevated γH2AX. Furthermore, cancer cells became sensitized to chemotherapy and other DDR inhibitors. Dosed either as a single agent or in combination with cisplatin, the compounds significantly inhibited tumor growth in vivo. By upregulating ATR-CHK1 signaling, the RAD51 inhibitors increased surface PD-L1 levels in various tumor cells, suggesting a potential combination of RAD51 inhibitors with PD-1/PD-L1 blockade. Overall, our findings provide the preclinical rationale to explore RAD51 inhibitors as monotherapy or in combination with chemotherapy, immunotherapy or DDR-targeting therapy in cancer treatment.

Methyltransferase-like 3 (METTL3) and N6-methyladenosine (m6A) are involved in many types of biological and pathological processes, including DNA repair. However, the function and mechanism of METTL3 in DNA repair and chemotherapeutic response remain largely unknown. In present study, we identified that METTL3 participates in the regulation of homologous recombination repair (HR), which further influences chemotherapeutic response in both MCF-7 and MDA-MB-231 breast cancer (BC) cells. Knockdown of METTL3 sensitized these BC cells to Adriamycin (ADR; also named as doxorubicin) treatment and increased accumulation of DNA damage. Mechanically, we demonstrated that inhibition of METTL3 impaired HR efficiency and increased ADR-induced DNA damage by regulating m6A modification of EGF/RAD51 axis. METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. We further demonstrated that the m6A ‘reader,’ YTHDC1, bound to the m6A modified EGF transcript and promoted EGF synthesis, which enhanced HR and cell survival during ADR treatment in BC. Our findings reveal a pivotal mechanism of METTL3-mediated HR and chemotherapeutic drug response, which may contribute to cancer therapy.

Background: There is increasing interest in enhancing the response of the PARP inhibitor olaparib, which is currently approved for pancreatic ductal adenocarcinoma (PDAC) patients with defects in DNA damage repair associated with germline BRCA1/2 mutations. Moreover, agents that can mimic these defects in the absence of germline BRCA1/2 mutations are an area of active research in hopes of increasing the number of patients eligible for treatment with PARP inhibitors. The extent to which regorafenib, an FDA-approved tyrosine kinase inhibitor, can be used to enhance the efficacy of PARP inhibitors in PDAC cells without known BRCA1/2 mutations remains to be investigated., Methods: Comet assay, cell cycle analysis, western blotting, and immunofluorescent detection of H2AX S139 were used to evaluate the extent to which regorafenib induces DNA damage in PDAC cell lines. The effects of regorafenib, either alone or in combination with PARPi inhibitors, on PDAC cell death were assessed by Annexin V/PI co-staining assay in cell lines and by immunohistochemistry staining for cleaved caspase-3 in mouse tumors and in ex vivo slice cultures of human PDAC tumors. Flow cytometry-based analysis was used to evaluate the ability of regorafenib to reprogram PDAC tumor microenvironment., Results: We now show that regorafenib, a tyrosine-kinase inhibitor with efficacy in several gastrointestinal malignancies, can enhance the response of olaparib in pancreatic cancer. While regorafenib induces DNA damage and limits the ability of PDAC cells to resolve the damage, regorafenib by itself does not induce apoptosis. However, regorafenib in combination with olaparib further induces DNA damage in vitro, in tumor-bearing mice, and in ex vivo slice cultures of human PDAC tumors, resulting in increased apoptosis compared to olaparib alone. Notably, we show that the efficacy of the combination treatment is not dependent on cytolytic T cells., Conclusions: Together, these findings demonstrate that regorafenib can attenuate DNA damage response and potentiate the efficacy of PARP inhibitors in PDAC tumors., Competing Interests: Declarations. Ethics approval and consent to participate: For human studies, pancreatic tissue was obtained from patients with PDAC undergoing resection on a protocol approved by the Institutional Review Board of Northwestern University. Informed consent was obtained from patients before resection. All resected specimens were deidentified. All experiments on human specimens were performed in accordance with relevant guidelines and regulations. All mouse studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) Office at Northwestern University. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Objective: This study investigates the mechanism underlying sorafenib resistance in hepatocellular carcinoma cells (HCC), focusing on DNA damage repair (DDR) pathways to develop targeted therapeutic strategies., Methods: Bioinformatics analysis was used to screen genes associated with sorafenib resistance, which was further demonstrated by western blotting. Cell proliferation was determined using the EdU assay. The presence of binding sites between valproic acid (VPA) and NOTCH1 was analyzed by molecular docking. Comet and flow cytometry assays evaluated DNA damage and cell cycle arrest induced by VPA in sorafenib-resistant cells, with further mechanistic insights gained via western blotting and co-immunoprecipitation (Co-IP)., Results: We found that NOTCH1/ATM axis plays a vital role in the prognosis of patients with liver cancer and in the behavior of sorafenib-resistant cells. HCC resistant to sorafenib exhibited enhanced cell proliferation ability. Moreover, overexpression of NOTCH1 in sorafenib-sensitive HCC cells significantly increased liver cancer cell proliferation. Conversely, silencing NOTCH1 expression in sorafenib-resistant HCC cell lines reduced their proliferative activity. Additionally, VPA enhanced the therapeutic efficacy against sorafenib-resistance cells by modulating NOTCH1/ATM/p-BRCA1/p-CHK2/γ-H2AX signaling axis and homologous recombination (HR) activity., Conclusion: Targeting NOTCH1 and ATM is a promising strategy to overcome sorafenib resistance in HCC, particularly through the combined use of VPA and sorafenib., Competing Interests: None., (AJTR Copyright © 2024.)

RAD51 is related to the bacterial RecA protein and is best known for its role in homologous recombination-mediated repair of DNA damage. Here, we report an unexpected function of RAD51 in the maintenance methylation of genomic DNA, a function that is separable from its role in homologous recombination. First, it acts as an inhibitor of the E3 ubiquitin ligase UHRF1. Deficiency in RAD51 causes excessive ubiquitination and degradation of the DNA methyltransferase DNMT1, leading to the loss of global DNA methylation. Second, RAD51 helps UHRF1 to monoubiquitinate histone H3 to generate DNMT1 recruiting signal. It binds H3 directly, enabling UHRF1 to bind and ubiquitinate H3 more readily. Disrupting the interaction between RAD51 and H3 diminishes DNMT1 recruitment and the failure of maintenance methylation of genomic DNA. Thus, RAD51 dually regulates UHRF1. These results establish RAD51 as a guardian of the integrity of both the genome and the epigenome., Competing Interests: Competing interests statement:The authors declare no competing interest.

Our previous study demonstrated that the linear quadratic model appeared to be not well-suited for high dose per fraction due to an observed increase in α/β ratio as the dose per fraction increased. To further validate this conclusion, we draw the cell survival curve to calculate the α/β ratio by the clone formation experiment and then convert the fractionated radiation dose into an equivalent single hypofractionated radiation dose comparing with that on the survival curve. Western Blot and laser confocal immunofluorescence were used to detect the expression of γ-H2AX and RAD51 after different fractionated modes of radiation. We constructed a murine xenograft model, and changes in transplanted tumor volume were used to evaluate the biological effects after different fractionated radiation. The results demonstrated that when fractionated radiation dose was converted into equivalent single hypofractionated radiation dose, the effectiveness of hypofractionated radiation was overestimated. If a larger α/β ratio was used, the discrepancy tended to become smaller. γ-H2AX was higher in 24 h after a single high dose radiation than the continuous expression of the DNA repair marker RAD51. This implies more irreparable damage in a single high dose radiation compared with fractionated radiation. In the murine xenograft model, the effectiveness of hypofractionated radiation was also overestimated, and additional fractions of irradiation may be required. The conclusion is that after single hypofractionated radiation, the irreparable damage in cells increased (α value increased) and some repairable sublethal damage (β value) was converted into irreparable damage (α value). When α value increased and β value decreased, the ratio increased., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Introduction: Several biomarkers are currently available to address targeted treatments in cancer patients, with lung malignancies representing one of the best examples., Case Description: We report the case of a patient affected by advanced non-small cell lung cancer with an uncommon histology and a complex biology. The use of a large next-generation sequencing (NGS) NGS panel allowed us to identify an extremely rare BRAF mutation (V600Q), a MET amplification, a high tumor mutational burden, a germline pathogenetic BRCA1 mutation and a homologous recombination deficiency through RAD51 assay. The treatment decision was driven by the abundance of molecular information., Conclusions: This case highlights that an attentive and critical evaluation of molecular reports is key for the tailoring of treatment algorithms at the patient-level scale., Competing Interests: Declaration of conflicting interestThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: FF attended advisory board for BeiGene. MT received speakers’ and consultants’ fees from Astra-Zeneca, Pfizer, Eli-Lilly, BMS, Novartis, Roche, MSD, Boehringer Ingelheim, Otsuka, Takeda, Pierre Fabre, Amgen, Merck, Sanofi. MT received institutional research grants from Astra-Zeneca, Boehringer Ingelheim.All remaining authors have no conflict of interest to declare.

Two recombinases, RAD51 and DMC1, catalyze meiotic break repair to ensure crossovers (COs) between homologous chromosomes (interhomolog) rather than between sisters (intersister). FIDGETIN-LIKE-1 (FIGL1) downregulates both recombinases. However, the understanding of how FIGL1 functions in meiotic repair remains limited. Here, we discover new genetic interactions of Arabidopsis thaliana FIGL1 that are important in vivo determinants of meiotic repair outcome. In figl1 mutants, compromising RAD51-dependent repair, either through the loss of RAD51 paralogs (RAD51B or XRCC2) or RAD54 or by inhibiting RAD51 catalytic activity, results in either unrepaired breaks or meiotic CO defects. Further, XRCC2 physically interacts with FIGL1 and partially counteracts FIGL1 activity for RAD51 focus formation. Our data indicate that RAD51-mediated repair mechanisms compensate FIGL1 dysfunction. FIGL1 is not necessary for intersister repair in dmc1 but is essential for the completion of meiotic repair in mutants such as asy1 that have impaired DMC1 functions and interhomolog bias. We show that FIGL1 attenuates interhomolog repair, and ASY1 counteracts FIGL1 to promote interhomolog recombination. Altogether, this study underlines that multiple factors can counteract FIGL1 activity to promote accurate meiotic repair., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing in vitro fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo EDC treatment of rats and immunostaining of treated oocytes Basic Protocol 2: In vitro fertilization and immunostaining of early-stage embryos., (© 2024 Wiley Periodicals LLC.)

Hexavalent chromium [Cr(VI)] is a known lung carcinogen and a driving mechanism in human lung cells for Cr(VI)-induced lung cancer is chromosome instability, caused by prolonged Cr(VI) exposure inducing DNA double-strand breaks, while simultaneously inhibiting the repair of these breaks. In North Atlantic right whales, Cr(VI) induces breaks but does not inhibit repair. It is unclear if this repair inhibition is specific to human lung cells or occurs in other species, as it has only been considered in humans and North Atlantic right whales. We evaluated these outcomes in rodent cells, as rodents are an experimental model for metal-induced lung carcinogenesis. We used a guinea pig lung fibroblast cell line, JH4 Clone 1, and rat lung fibroblasts. Cells were exposed to two different particulate Cr(VI) compounds, ranging from 0 to 0.5 ug/cm 2 , for 24 or 120 h and assessed for cytotoxicity, DNA double-strand breaks, and DNA double-strand break repair. Both particulate Cr(VI) compounds induced a concentration-dependent increase in cytotoxicity and DNA double-strand breaks after acute and prolonged exposures. Notably, while the repair of Cr(VI)-induced DNA double-strand breaks increased after acute exposure, the repair of these breaks was inhibited after prolonged exposure. These results are consistent with outcomes in human lung cells indicating rodent cells respond like human cells, while whale cells have a markedly different response., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nitro-fatty acids (NO 2 -FAs) are endogenous pleiotropic lipid mediators regarded as promising drug candidates for treating inflammatory and fibrotic diseases. Over the past two decades, the anti-inflammatory and cytoprotective actions of NO 2 -FAs and several molecular targets have been identified. More recently, preclinical studies have demonstrated their potential as prospective cancer therapeutics with favorable safety and tumor-selective profiles. In this review, we describe the mechanisms of action, with a focus on NO 2 -FA antineoplastic and chemosensitizing effects. We also address the potential therapeutic applications of endogenous and structurally modified NO 2 -FAs species in cancer treatment., Competing Interests: Declaration of interests F.J.S. and C.A.N. acknowledge financial interests in Creegh Pharmaceuticals Inc. F.J.S. has a financial interest in Furanica, Inc. The authors have no other conflicts of interest to declare. All descriptions, interpretations, and opinions expressed in the manuscript are solely those of the contributing authors and do not necessarily represent the views (s) of the affiliated institutions, organizations, or funding bodies., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ovarian cancer (OC) is the deadliest malignancy of the female reproductive system. The standard first-line therapy for OC involves cytoreductive surgical debulking followed by chemotherapy based on platinum and paclitaxel. Despite these treatments, there remains a high rate of tumor recurrence and resistance to platinum. Recent studies have highlighted the potential anti-tumor properties of metformin (met), a traditional diabetes drug. In our study, we investigated the impact of met on the anticancer activities of cisplatin (cDDP) both in vitro and in vivo. Our findings revealed that combining met with cisplatin significantly reduced apoptosis in OC cells, decreased DNA damage, and induced resistance to cDDP. Furthermore, our mechanistic study indicated that the resistance induced by met is primarily driven by the inhibition of the ATM/CHK2 pathway and the upregulation of the Rad51 protein. Using an ATM inhibitor, KU55933, effectively reversed the cisplatin resistance phenotype. In conclusion, our results suggest that met can antagonize the effects of cDDP in specific types of OC cells, leading to a reduction in the chemotherapeutic efficacy of cDDP., Competing Interests: Declaration of competing interest We declare the manuscript is an original work which has not been previously published in whole or in part and is not under consideration for publication in any other journals. Its publication has been approved by all co-authors. All authors have contributed to this work and state no existing competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Rationale: Both bulk RNA-sequencing and GEO database upon chemotherapy to non-small cell lung cancer (NSCLC) cells reveal that ZNF131 (Zinc Finger Protein 131) maybe a crucial transcriptional factor involved. However, it is a recently discovered protein with largely unexplored expression patterns and biological functions. Methods: Bioinformatics analyses and immunohistochemistry staining were assessed to detect both mRNA and protein levels of ZNF131 in NSCLC specimens and cell lines. Next, colony formation assay, MTT assay, EdU assay, transwell assay, flow cytometric analysis, sphere formation assay, western blotting analysis, mouse xenograft model analysis, immunofluorescence assay, and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of ZNF131 interaction on proliferation, invasion, stemness, chemotherapy sensitivity. RNA-sequencing assay, RNA-microarray, and ChIP-sequencing assay were used to identify candidate downstream target genes. Further, liquid chromatography-tandem mass spectrometry analysis, GST pull-down assay, and immunoprecipitation assays were performed to evaluate the interactions between ZNF131, BACH1, and CUL3. Results: ZNF131 was elevated in NSCLC specimens and cell lines, which significantly correlates with advanced TNM stage and poor prognosis in NSCLC patients. ZNF131 overexpression promotes NSCLC cell proliferation, invasion, and stemness both in vitro and in vivo . ZNF131 appears to target the RAD51 gene within a well-defined region (-668bp to -403bp) of the RAD51 promoter. ZNF131 contributes to RAD51-dependent homologous recombination (HR), primarily through its Zinc Finger and BTB domains. ZNF131-BACH1 interaction, mediated by their respective BTB domains, enhances the stability of both proteins, effectively preventing their ubiquitin-mediated degradation by CUL3. The ZNF131-BACH1 partnership significantly amplifies RAD51-dependent HR, resulting in expedited resistance to both radiotherapy and chemotherapy in NSCLC patients. Desoxyrhaponticin was shown to halt NSCLC progression and orchestrate a synergistic effect together with chemotherapy at least partially by targeting ZNF131. Conclusions: Our findings indicate that ZNF131 exhibits heightened expression in NSCLC, driving essential processes such as proliferation, invasion, and stemness by transcriptionally activating RAD51. The ZNF131-BACH1 interaction serves as a crucial enhancer, further boosting RAD51 transcription and ultimately accelerating therapy resistance in NSCLC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background/Objectives : Irinotecan, a camptothecin (CPT) derivative, is commonly used as a first-line therapy for colorectal cancer (CRC), but resistance remains a significant challenge. This study aims to explore the therapeutic potential of FL118, another CPT derivative, with a focus on overcoming resistance to irinotecan. Methods: The effects of FL118 on CRC cells were evaluated, and bioinformatics analysis was performed on RNA-seq data. Transfection was conducted to observe the knockdown effect of survivin, and the in vivo efficacy of FL118 was assessed using a xenograft model. Results : FL118 induces apoptosis, G2/M arrest, and DNA damage. A notable mechanism of action of FL118 is a reduction in survivin levels, which downregulates the expression of RAD51, a key marker of homologous recombination, and attenuates DNA repair processes. Given that SN38 is the active metabolite of irinotecan, FL118 reduces cell viability and RAD51 in SN38-resistant LOVO cells. Conclusions : Our findings provide effective insights into the antitumor activity of FL118 and its potential as a therapeutic agent for overcoming irinotecan resistance in CRC.

Meiosis is the developmental program that generates gametes. To produce healthy gametes, meiotic recombination creates reciprocal exchanges between each pair of homologous chromosomes that facilitate faithful chromosome segregation. Using fission yeast and biochemical, genetic, and cytological approaches, we have studied the role of CDK (cyclin-dependent kinase) in the control of Swi5-Sfr1, a Rad51-recombinase auxiliary factor involved in homolog invasion during recombination. We show that Sfr1 is a CDK target, and its phosphorylation downregulates Swi5-Sfr1 function in the meiotic prophase. Expression of a phospho-mimetic sfr1-7D mutant inhibits Rad51 binding, its robust chromosome loading, and subsequently decreases interhomolog recombination. On the other hand, the non-phosphorylatable sfr1-7A mutant alters Rad51 dynamics at late prophase, and exacerbates chromatin segregation defects and Rad51 retention observed in dbl2 deletion mutants when combined with them. We propose Sfr1 phospho-inhibition as a novel cell-cycle-dependent mechanism, which ensures timely resolution of recombination intermediates and successful chromosome distribution into the gametes. Furthermore, the N-terminal disordered part of Sfr1, an evolutionarily conserved feature, serves as a regulatory platform coordinating this phospho-regulation, protein localization and stability, with several CDK sites and regulatory sequences being conserved., (© 2024. The Author(s).)

Introduction: RAD51 is a pivotal DNA repair gene managing double-stranded DNA break recognition and repair. RAD51 high expression was associated with adverse outcomes in other cancer types. This study aims to investigate the tumor microenvironment and immune landscape in the RAD51 high-expressed Hepatocellular Carcinoma (HCCs)., Methods: A total of 467 patients from two large independent cohorts with clinical and transcriptomic data were obtained. The cohort was dichotomized based on the median RAD51 gene expression. xCell and Gene Set Enrichment Analysis (GSEA) were used., Results: RAD51 high-expressed HCCs were associated with worse recurrence-free, progression-free, disease-specific, and overall survival (all P < 0.05). While RAD51 high-expressed HCCs were associated with intratumoral heterogeneity, homologous recombination deficiency, and fraction altered scores, mutation or neoantigens were not increased in this group. xCell analysis demonstrated inconsistent immune cell infiltration between two cohorts. Cytolytic activity as well as GSEA with immune-related gene sets also demonstrated inconsistent results between two cohorts as well. On the other hand, RAD51 expression was significantly increased in higher-grade tumors, larger tumors, and higher clinical stages. RAD51 high-expressed HCCs were found to have elevated proliferation score. Furthermore, GSEA exhibited significant enrichment of all the cell proliferation-related gene sets in the Hallmark collection, including E2F targets, G2M checkpoint, Mitotic spindle, MYC targets, and MTORC1 signaling consistently in both cohorts (all false discovery rate < 0.25)., Conclusions: RAD51 high-expressed HCCs were associated with worse survival and with increased cell proliferation and were not necessarily associated with immune infiltration or inflammation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro . These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.