Activation Of DNA Repair Pathways Research Articles

West Nile Virus Subgenomic RNAs Modulate Gene Expression in a Neuronal Cell Line.

Subgenomic flaviviral RNAs (sfRNAs) are small non-coding products of the incomplete degradation of viral genomic RNA. They accumulate during flaviviral infection and have been associated with many functional roles inside the host cell. Studies so far have demonstrated that sfRNA plays a crucial role in determining West Nile virus (WNV) pathogenicity. However, its modulatory role on neuronal homeostasis has not been studied in depth. In this study, we investigated the mechanism of sfRNA biosynthesis and its importance for WNV replication in neuronal cells. We found that sfRNA1 is functionally redundant for both replication and translation of WNV. However, the concurrent absence of sfRNA1 and sfRNA2 species is detrimental for the survival of the virus. Differential expression analysis on RNA-seq data from WT and ΔsfRNA replicon cell lines revealed transcriptional changes induced by sfRNA and identified a number of putative targets. Overall, it was shown that sfRNA contributes to the viral evasion by suppressing the interferon-mediated antiviral response. An additional differential expression analysis among replicon and control Neuro2A cells also clarified the transcriptional changes that support WNV replication in neuronal cells. Increased levels of translation and oxidative phosphorylation, post-translational modification processes, and activated DNA repair pathways were observed in replicon cell lines, while developmental processes such as axonal growth were deficient.

A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance.

A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.

DNA Damage by Radiopharmaceuticals and Mechanisms of Cellular Repair.

DNA is an organic molecule that is highly vulnerable to chemical alterations and breaks caused by both internal and external factors. Cells possess complex and advanced mechanisms, including DNA repair, damage tolerance, cell cycle checkpoints, and cell death pathways, which together minimize the potentially harmful effects of DNA damage. However, in cancer cells, the normal DNA damage tolerance and response processes are disrupted or deregulated. This results in increased mutagenesis and genomic instability within the cancer cells, a known driver of cancer progression and therapeutic resistance. On the other hand, the inherent instability of the genome in rapidly dividing cancer cells can be exploited as a tool to kill by imposing DNA damage with radiopharmaceuticals. As the field of targeted radiopharmaceutical therapy (RPT) is rapidly growing in oncology, it is crucial to have a deep understanding of the impact of systemic radiation delivery by radiopharmaceuticals on the DNA of tumors and healthy tissues. The distribution and activation of DNA damage and repair pathways caused by RPT can be different based on the characteristics of the radioisotope and molecular target. Here we provide a comprehensive discussion of the biological effects of RPTs, with the main focus on the role of varying radioisotopes in inducing direct and indirect DNA damage and activating DNA repair pathways.

H3.1K27me1 loss confers Arabidopsis resistance to Geminivirus by sequestering DNA repair proteins onto host genome

The H3 methyltransferases ATXR5 and ATXR6 deposit H3.1K27me1 to heterochromatin to prevent genomic instability and transposon re-activation. Here, we report that atxr5 atxr6 mutants display robust resistance to Geminivirus. The viral resistance is correlated with activation of DNA repair pathways, but not with transposon re-activation or heterochromatin amplification. We identify RAD51 and RPA1A as partners of virus-encoded Rep protein. The two DNA repair proteins show increased binding to heterochromatic regions and defense-related genes in atxr5 atxr6 vs wild-type plants. Consequently, the proteins have reduced binding to viral DNA in the mutant, thus hampering viral amplification. Additionally, RAD51 recruitment to the host genome arise via BRCA1, HOP2, and CYCB1;1, and this recruitment is essential for viral resistance in atxr5 atxr6. Thus, Geminiviruses adapt to healthy plants by hijacking DNA repair pathways, whereas the unstable genome, triggered by reduced H3.1K27me1, could retain DNA repairing proteins to suppress viral amplification in atxr5 atxr6.

Multifunctional Novel Nanoplatform for Effective Synergistic Chemo-Photodynamic Therapy of Breast Cancer by Enhancing DNA Damage and Disruptions of Its Reparation.

Photodynamic therapy (PDT) is an effective noninvasive therapeutic strategy that has been widely used for anti-tumor therapy by the generation of excessive highly cytotoxic ROS. However, the poor water solubility of the photosensitizer, reactive oxygen species (ROS) depleting by high concentrations of glutathione (GSH) in the tumor microenvironment and the activation of DNA repair pathways to combat the oxidative damage, will significantly limit the therapeutic effect of PDT. Herein, we developed a photosensitizer prodrug (CSP) by conjugating the photosensitizer pyropheophorbide a (PPa) and the DNA-damaging agent Chlorambucil (Cb) with a GSH-responsive disulfide linkage and demonstrated a multifunctional co-delivery nanoplatform (CSP/Ola nanoparticles (NPs)) together with DSPE-PEG2000 and PARP inhibitor Olaparib (Ola). The CSP/Ola NPs features excellent physiological stability, efficient loading capacity, much better cellular uptake behavior and photodynamic performance. Specifically, the nanoplatform could induce elevated intracellular ROS levels upon the in situ generation of ROS during PDT, and decrease ROS consumption by reducing intracellular GSH level. Moreover, the CSP/Ola NPs could amplify DNA damage by released Cb and inhibit the activation of Poly(ADP-ribose) polymerase (PARP), promote the upregulation of γ-H2AX, thereby blocking the DNA repair pathway to sensitize tumor cells for PDT. In vitro investigations revealed that CSP/Ola NPs showed excellent phototoxicity and the IC50 values of CSP/Ola NPs against MDA-MB-231 breast cancer cells were as low as 0.05-01 μM after PDT. As a consequence, the co-delivery nanoplatform greatly promotes the tumor cell apoptosis and shows a high antitumor performance with combinational chemotherapy and PDT. Overall, this work provides a potential alternative to improve the therapeutic efficiency of triple negative breast cancer cell (TNBC) treatment by synergistically enhancing DNA damage and disrupting DNA damage repair.

BLM helicase overexpressed in human gliomas contributes to diverse responses of human glioma cells to chemotherapy

Most of anti-tumour therapies eliminate neoplastic cells by introducing DNA damage which ultimately triggers cell death. These effects are counteracted by activated DNA repair pathways to sustain tumour proliferation capacity. RECQL helicases family, including BLM, participate in DNA damage and repair, and prevent the replication stress. Glioblastoma (GBM) is a common, malignant brain tumour that inevitably recurs despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Expression and functions of the BLM helicase in GBM therapy resistance have not been elucidated. We analysed expression and localisation of BLM in human gliomas and several glioma cell lines using TCGA datasets, immunostaining and Western blotting. BLM depleted human glioma cells were generated with CRISPR/Cas9 system. Effects of chemotherapeutics on cell proliferation, DNA damage and apoptosis were determined with flow cytometry, immunofluorescence, Western blotting and RNA sequencing. We found upregulated BLM mRNA levels in malignant gliomas, increased cytosolic localisation and poor survival of GBM patients with high BLM expression. BLM deficiency in LN18 and LN229 glioma cells resulted in profound transcriptomic alterations, reduced cell proliferation, and altered cell responses to chemotherapeutics. BLM-deficient glioma cells were resistant to the TMZ and PARP inhibitor treatment and underwent polyploidy or senescence depending on the TP53 activity. Our findings of high BLM expression in GBMs and its roles in responses to chemotherapeutics provide a rationale for targeting BLM helicase in brain tumours. BLM deficiency affects responses of glioma cells to chemotherapeutics targeting PARP1 dependent pathways.

The Complex Roles of DNA Repair Pathways, Inhibitors, Hyperthermia, and Contact Inhibition in Cell Cycle Halts.

The cell cycle has the capacity to safeguard the cell's DNA from damage. Thus, cell cycle arrest can allow tumor cells to investigate their own DNA repair processes. Cancer cells become extremely reliant on G1-phase cyclin-dependent kinases due to mutated oncogenes and deactivated tumor suppressors, producing replication stress and DNA damage during the S phase and destroying checkpoints that facilitate progression through the S/G2/M phase. DNA damage checkpoints activate DNA repair pathways to prevent cell proliferation, which occurs when the genome is damaged. However, research on how cells recommence division after a DNA lesion-induced arrest is insufficient which is merely the result of cancer cells' susceptibility to cell cycle arrest. For example, defects in the G1 arrest checkpoint may cause a cancer cell to proliferate more aggressively, and attempts to fix these complications may cause the cell to grow more slowly and eventually die. Defects in the G2-M arrest checkpoint may enable a damaged cell to enter mitosis and suffer apoptosis, and attempts to boost the effectiveness of chemotherapy may increase its cytotoxicity. Alternatively, attempts to promote G2-M arrest have also been linked to increased apoptosis in the laboratory. Furthermore, variables, such as hyperthermia, contact inhibition, nucleotide shortage, mitotic spindle damage, and resting phase effects, and DNA replication inhibitors add together to halt the cell cycle. In this review, we look at how nucleotide excision repair, MMR, and other variables, such as DNA replication inhibitors, hyperthermia, and contact inhibition, contribute to the outlined processes and functional capacities that cause cell cycle arrest.

PS-C26-12: SIGNIFICANCE OF PODOCYTE DNA DAMAGE AND GLOMERULAR DNA METHYLATION IN CKD WITH PROTEINURIA

Objective: Previously we have reported that altered DNA methylation is associated with DNA damage repair in podocytes in CKD, including diabetic nephropathy or IgA nephropathy (Cell Rep 2019, Sci Rep 2020). This study is aim to investigate the association of podocyte DNA damage and DNA methylation with clinical manifestations and renal prognosis in various types of kidney diseases, which has not been adequately investigated. Design and Method: Patients diagnosed with minor glomerular abnormalities (MGA, n = 33), membranous nephropathy (MN, n = 9) or diabetic nephropathy (DN, n = 10), following kidney biopsy at Keio University Hospital from 2015 to 2017 were included. We evaluated podocyte DNA double strand breaks (DSBs) using both long-distance PCR of nephrin gene and γH2AX immunostaining, and glomerular DNA methylation levels by immunostaining of 5mC, and examined the relationship between clinical parameters of patients, and DNA damage and methylation. Results: In MGA patients, both podocyte DNA damage and glomerular DNA methylation was associated with the amount of proteinuria. Moreover, glomerular DNA methylation was also associated with 3-year eGFR decline. We also examined in DN patients, in which podocyte DNA damage has been found to be involved in our previous study. It was demonstrated that podocyte DSBs were associated with a decline in eGFR over 6 months, whereas glomerular DNA methylation was associated with 2 year- and 3 year-eGFR decline. In addition, samples from MN patients were evaluated, in which activation of DNA repair pathways in the glomeruli has been suggested. Interestingly, the association of DNA DSBs or DNA methylation with proteinuria or kidney function was not indicated in MN. Conclusions: To our knowledge, this is the first study to demonstrate the possible association of podocyte DNA damage and glomerular DNA methylation with kidney function and its decline in MGA patients, whereas it was not observed in MN patients, despite a limitation of small sample size. Focusing on podocyte DNA damage and following DNA methylation changes may lead us to novel approaches to estimate renal prognosis in addition to traditional evaluation of renal biopsy as well as to develop novel therapeutic targets in specific types of CKD.

CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, understanding of DNA repair following Cas-mediated DNA cleavage remains incomplete. Using Cas12a ribonucleoprotein genome editing in the fungal pathogen, Magnaporthe oryzae, we detail non-canonical DNA repair outcomes from hundreds of transformants. Sanger and nanopore sequencing analysis reveals significant variation in DNA repair profiles, ranging from small INDELs to kilobase size deletions and insertions. Furthermore, we find the frequency of DNA repair outcomes varies between loci. The results are not specific to the Cas-nuclease or selection procedure. Through Ku80 deletion analysis, a key protein required for canonical non-homologous end joining, we demonstrate activity of an alternative end joining mechanism that creates larger DNA deletions, and uses longer microhomology compared to C-NHEJ. Together, our results suggest preferential DNA repair pathway activity in the genome that can create different mutation profiles following repair, which could create biased genome variation and impact genome engineering and genome evolution.

Complementary anti-cancer pathways triggered by inhibition of sideroflexin 4 in ovarian cancer

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy.

Effects of isolated and combined exposures of Boana curupi (Anura: Hylidae) tadpoles to environmental doses of trichlorfon and ultraviolet radiation

The biodiversity collapse strongly affects the amphibian group and many factors have been pointed out as catalytic agents. It is estimated that several events in the amphibian population decline worldwide may have been caused by the interaction of multiple drivers. Thus, this study aimed to evaluate the stressful effects of the exposure to environmental doses of trichlorfon (TCF) pesticide (0.5 μg/L; and an additional 100-fold concentration of 50 µg/L) and ultraviolet radiation (UV) (184.0 kJ/m² of UVA and 3.4 kJ/m² of UVB, which correspond to 5% of the daily dose) in tadpoles of the Boana curupi species (Anura: Hylidae). The isolated and combined exposures to TCF happened within 24 h of acute treatments under laboratory-controlled conditions. In the combined treatments, we adopted three different moments (M) of tadpole irradiation from the beginning of the exposures to TCF (0 h – M1; 12 h – M2; and 24 h – M3). Then, we evaluated tadpole survival, change in morphological characters, induction of apoptotic cells, lipid peroxidation (LPO), protein carbonyl content (PCC), glutathione S-transferase (GST), non-protein thiols (NPSH), and acetylcholinesterase (AChE), as well as the induction of genomic DNA (gDNA) damage. UVB treatment alone resulted in high mortality, along with a high level of apoptosis induction. Both UVA, UVB, and TCF increased LPO, PC, and AChE, while decreased GST activity. Regarding co-exposures, the most striking effect was observed in the interaction between UVB and TCF, which surprisingly decreased UVB-induced tadpole mortality, apoptosis, and gDNA damage. These results reinforce the B. curupi sensitivity to solar UVB radiation and indicate a complex response in face of UVB interaction with TCF, which may be related to activation of DNA repair pathways and/or inhibition of apoptosis, decreasing UVB-induced tadpole mortality.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

Abstract 2589: Effect of folfirinox with an ATR inhibitor on pancreatic tumor cells and its microenvironment

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is an extremely aggressive disease.There is a clear need of new strategies and new researches to treat and diagnose these patients. Regarding treatments, surgery is possible in only 20% of cases, and the chemotherapeutic molecule Gemcitabine is unfortunately lacking a good response rate. Recently, a new polychemotherapy oxaliplatin-based (FOLFIRINOX), which is a combination of 4 drugs: oxaliplatin, irinotecan, fluorouracil and leucovorin, has been approved. It has showed a significant increase of the overall survival inpatients compared to gemcitabine, but associated with more toxicity and still limited efficiency. Most of the drugs induce their toxicity by provoking DNA damages and replication stress, leading to the activation of DNA repair pathways. In this context,our research project proposes to find a synergistic association of FOLFIRINOX with specific inhibitors of DNA damage repair -Ataxia Telangiectasia and Rad3 related inhibitor (ATRi)- to increase the efficiency of the chemotherapy while reducing itstoxicity. The resistance to chemotherapy can come from the stroma that represents up to 80% of the tumor mass. The impact of the chemotherapies on the microenvironment can be a key to increase the treatments efficiency. That’s why in this project, we studied co-culture models to look at the effect of this new polychemotherapy on tumor cells and its microenvironment, in particular Cancer-Associated Fibroblasts (CAFs). Viability matrix in 2D & 3D in vitro co-culture of tumor cells with primary CAFs were carried out. DNA damage and proteins from the DNA damage repair pathways were analysed after treatments. Cell death and autophagy pathways were studied. In vivo, immunodeficient mice xenografted with ATCC and Patient Derived Xenograft models were treated with FOLFIRINOX and ATRi to evaluate the effect on tumor progression. A synergistic effect of the association was demonstrated in vitro independently of the KRAS, ATM, TP53, BRCA1/2 mutation statuts in several pancreatic models (ATCC and derived from PDX) and in co-culture with CAFs. A higher apoptosis and DNA damages were observed in tumor cells treated with the associated drugs. These results were associated with a decrease of DNA damage repair pathways leading to more apoptosis compared to the chemotherapy alone and an inhibition of the autophagy flux. Also, a phenotypic change in the cells was found after treating with ATRi and with an increase of this particular phenotype when the chemotherapy was added. A protective effect of the CAFs on tumor cells was observed and CAF secretome was analysed. In vivo, the association inhibits significantly the tumor growth compared to each treatment alone and no toxicity was observed. Now, validation of this polychemotherapy in vivo using co-culture models in immunodeficient and immunocompetent mice are crucial to confirm the therapeutic potential of this new treatment for PDAC. Citation Format: Marine Bruciamacchie, Nadia Vie, Véronique Garambois, Diego Tosi, Pierre-Emmanuel Colombo, Céline Gongora, Christel Larbouret. Effect of folfirinox with an ATR inhibitor on pancreatic tumor cells and its microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2589.

Abstract 227: Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure

Abstract Skin cancer is the most common form of cancer, with over 5 million cases reported in 2021. UVB exposure derived from sunlight is the major carcinogenic driver of skin cancer and poses a major public health risk. In response to UVB exposure, keratinocytes must activate DNA repair pathways to avoid mutagenesis. In addition to DNA repair pathways, cells initiate well-coordinated DNA damage response pathways to facilitate the repair process, including arresting global transcription. Here we report the use of kethoxal-assisted single-strand DNA-Sequencing (KAS-Seq) technology to profile the transcriptional dynamics that underlie the DNA damage response upon UVB exposure. Mechanistically, KAS-Seq involves the addition of an azide-tagged kethoxal group (N3-kethoxal) to DNA samples, which then reacts with guanines on single-stranded DNA (ssDNA). A “KAS signal” is therefore only obtained when DNA is unwound, allowing the N3-kethoxal reaction with accessible guanines to occur. Accordingly, in response to DNA damage, DNA can form ssDNA bubbles in order to facilitate DNA damage recognition and repair processes. KAS-Seq is therefore a suitable method to profile the transcriptional dynamics that occur in response to UVB-induced DNA damage. Here we show that UVB exposure in normal human keratinocytes (NHEK) cells induces a temporal and dynamic reprogramming in transcription using KAS-Seq. In terms of global transcription, our data show that the greatest loss of KAS signal occurs rapidly between 30mins and 3hrs post-UVB exposure, signifying transcriptional arrest. Interestingly, KAS signal is regained between 6-24hrs post-UVB, which is representative of a transition towards transcriptional recovery and induction of DNA damage processes. Furthermore, a more discrete and targeted analysis shows that the gain and loss of KAS signals primarily occur within the gene body, followed by promoter and intergenic regions. Overall, our data shows that transcriptional control in response to UVB-induced DNA damage is both dynamic and temporal. Further analysis is required to identify which transcriptional phenotype (transcriptional arrest or transcriptional recovery) is the most deleterious to the cell and DNA damage repair processes when disrupted. Additionally, we aim to further elucidate the molecular mechanisms that underlie these global changes in transcription, which will provide expanded insights into the wide-ranging oncogenic mechanisms that underlie UVB exposure. Citation Format: Emma A. Wilkinson, Tong Wu, Gayoung Park, Yan-Hong Cui, Chuan He, Yu-Ying He. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 227.

Heterochromatic repeat clustering imposes a physical barrier on homologous recombination to prevent chromosomal translocations

Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters. These clusters are refractory to DNA repair through homologous recombination (HR). The mechanisms by which pericentromeric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown. Here, we compare the spatial recruitment of HR factors upon double-stranded DNA breaks (DSBs) induced in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters. We show that while DSBs increase the accessibility of human pericentromeric heterochromatin by disrupting HP1α dimerization, mouse pericentromeric heterochromatin repeat clustering imposes a physical barrier that requires many layers of de-compaction to be accessed. Our results support a model in which the 3D organization of heterochromatin dictates the spatial activation of DNA repair pathways and is key to preventing the activation of HR within clustered repeats and the onset of chromosomal translocations.

Curcumin sensitizes carboplatin treatment in triple negative breast cancer through reactive oxygen species induced DNA repair pathway.

As patients with triple-negative breast cancer (TNBC) have a very weak response to hormone inhibition or anti-HER2 therapy, traditional chemotherapy is commonly used in these patients. Recently, carboplatin has been approved for the clinical treatment of TNBC. However, several patients exhibit resistance to carboplatin treatment. Therefore, strategies to enhance the antitumor effect of carboplatin need to be explored. In our study, we investigated the function of curcumin in increasing the response to carboplatin. MTT and colony formation assays were used to evaluate cell viability after carboplatin and curcumin treatment. In addition, we conducted flow cytometric and Western blot analyses to examine cellular apoptosis. Subsequently, molecular and biochemical experiments were used to explore the mechanism by which curcumin sensitized TNBC to carboplatin treatment. We demonstrated that different TNBC cells responded differently to carboplatin. Low-dose carboplatin killed CAL-51 cells but barely influenced CAL-51-R and MDA-MB-231 cells. To improve the sensitivity of resistant TNBC cells to carboplatin, combined treatment with curcumin was applied and was found to inhibit proliferation and induce apoptosis. Mechanistically, curcumin exerted its anticancer effect by increasing reactive oxygen species (ROS) production, which downregulated the DNA repair protein RAD51, leading to upregulation of γH2AX. As expected, ROS scavenger NAC reversed the inhibitory effect on growth and DNA repair pathway activity mediated by curcumin. Collectively, our data demonstrate that curcumin sensitizes TNBC to the anticancer effect of carboplatin by increasing ROS-induced DNA damage, thus providing an effective combination treatment strategy for TNBC.

The Activation оf DNА Repair Pathways after Ultra-Short Pulsed Electron Beam Irradiation in Human Cells

The aim of this work was to estimate the differences in activation of DNA repair pathways of DNA double-strand and single-strand breaks induced by ultrashort pulse electron beam irradiation in human K-562 cells. The activation of HR, NHEJ and BER DNA repair pathways was studied at non-lethal, sub-lethal and lethal doses of irradiation. Our results indicate that the activation of the specific repair pathways and repair kinetics depend on the irradiation dose.

Pharmacologic Ascorbate Enhances the Therapeutic Index of ATM-Inhibitor Based Chemoradiation for Colorectal Cancer

Ataxia telangiectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury.Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57bl/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors.Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated pATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT.P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

Host DNA repair response to oxidative damage is modulated by Trypanosoma cruzi in a strain-dependent manner

The conservation of genomic integrity and stability is essential for cell survival. DNA Damage Responses (DDRs) are considered of paramount importance for all living beings and involve mechanisms of cell cycle regulation and damage-specific DNA repair pathways. Hydrogen peroxide (H2O2) is a compound that, in supraphysiological concentrations, damages biomolecules including the DNA, causing base modifications and strand breaks. There is evidence that Trypanosoma cruzi, the protozoan that causes Chagas disease, interferes in the host cell's DNA metabolism. In order to investigate the influence of T. cruzi infection over the host cell capacity to withstand and repair DNA damage, we analyzed L6 cells infected with Berenice, and Colombiana T. cruzi strains according to their viability, proliferation, morphology, DNA degradation, expression of DNA repair, and cell cycle genes following H2O2 treatment. It was noted that T. cruzi infection might act as either a stressor or a protective element of host DNA, depending on the strain and H2O2 concentration. Cells infected with Berenice strain and treated with 0.8 mM H2O2 presented a reduced DNA damage response intensity (e.g., BER and HR). Infection with T. cruzi Colombiana prevented the activation of DNA repair pathways in response to 0.8mM and 1.6mM H2O2 (NER and MMR). Nevertheless, since cellular viability was not significantly compromised in Colombiana-infected cells following the oxidative insult, it is possible that the parasite directly influenced the host DNA repair machinery. Our results support the notion that T. cruzi is able to modulate the host cell DNA metabolism in a strain-dependent manner, an event which can be explored in future drug development strategies.

PARP1-modulated chromatin remodeling is a new target for cancer treatment.

Cancer progression requires certain tumorigenic mutations in genes encoding for different cellular and nuclear proteins. Altered expressions of these mutated genes are mediated by post-translational modifications and chromatin remodeling. Chromatin remodeling is mainly regulated by the chromatin remodeling enzyme complexes and histone modifications. Upon DNA damage, Poly-(ADP-ribose) Polymerase1 (PARP1) plays a very important role in the induction of chromatin modifications and activation of DNA repair pathways to repair the DNA lesion. It has been targeted to develop different anti-cancer therapeutic interventions and PARP inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for clinical use. But it has been found that the cancer cells often develop resistance to these PARP inhibitors and chromatin remodeling helps in enhancing this process. Hence, it may be beneficial to target PARP1-mediated chromatin remodeling, which may allow to reverse the drug resistance. In the current review, we have discussed the role of chromatin remodeling in DNA repair, how PARP1 regulates modifications of chromatin dynamics, and the role of chromatin modifications in cancer. It has also been discussed how the PARP1-mediated chromatin remodeling can be targeted by PARP inhibitors alone or in combination with other chemotherapeutic agents to establish novel anti-cancer therapeutics. We have also considered the use of PARG inhibitors that may enhance the action of PARP inhibitors to target different types of cancers.