Role of condensates in modulating DNA repair pathways and its implication for chemoresistance.

For cells, it is important to repair DNA damage, such as double-strand and single-strand DNA breaks, because unrepaired DNA can compromise genetic integrity, potentially leading to cell death or cancer. Cells have multiple DNA damage repair pathways that have been the subject of detailed genetic, biochemical, and structural studies. Recently, the scientific community has started to gain evidence that the repair of DNA double-strand breaks may occur within biomolecular condensates and that condensates may also contribute to DNA damage through concentrating genotoxic agents used to treat various cancers. Here, we summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double-strand breaks. We also describe evidence suggesting that condensates may be involved in the repair of other types of DNA damage, including single-strand DNA breaks, nucleotide modifications (e.g., mismatch and oxidized bases), and bulky lesions, among others. Finally, we discuss old and new mysteries that could now be addressed considering the properties of condensates, including chemoresistance mechanisms.

DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.

DNA ligase I (LIG1) joins DNA strand breaks during DNA replication and repair transactions and contributes to genome integrity. The mutations (P529L, E566K, R641L and R771W) in LIG1 gene are described in patients with LIG1-deficiency syndrome that exhibit immunodeficiency. LIG1 senses 3'-DNA ends with a mismatch or oxidative DNA base inserted by a repair DNA polymerase. However, the ligation efficiency of the LIG1 variants for DNA polymerase-promoted mutagenesis products with 3'-DNA mismatches or 8-oxo-2'-deoxyguanosine (8-oxodG) remains undefined. Here, we report that R641L and R771W fail in the ligation of nicked DNA with 3'-8-oxodG, leading to an accumulation of 5'-AMP-DNA intermediates in vitro. Moreover, we found that the presence of all possible 12 non-canonical base pairs variously impacts the ligation efficiency by P529L and R771W depending on the architecture at the DNA end, whereas E566K exhibits no activity against all substrates tested. Our results contribute to the understanding of the substrate specificity and mismatch discrimination of LIG1 for mutagenic repair intermediates and the effect of non-synonymous mutations on ligase fidelity.

Targeting the DNA Damage Response to Overcome Cancer Drug Resistance in Glioblastoma.

Glioblastoma multiforme (GBM) is a severe brain tumor whose ability to mutate and adapt to therapies is at the base for the extremely poor survival rate of patients. Despite multiple efforts to develop alternative forms of treatment, advances have been disappointing and GBM remains an arduous tumor to treat. One of the leading causes for its strong resistance is the innate upregulation of DNA repair mechanisms. Since standard therapy consists of a combinatory use of ionizing radiation and alkylating drugs, which both damage DNA, targeting the DNA damage response (DDR) is proving to be a beneficial strategy to sensitize tumor cells to treatment. In this review, we will discuss how recent progress in the availability of the DDR kinase inhibitors will be key for future therapy development. Further, we will examine the principal existing DDR inhibitors, with special focus on those currently in use for GBM clinical trials.

Strand breaks and chemical modification of intracellular DNA induced by cold atmospheric pressure plasma irradiation.

DNA damage in the A549 human lung cancer cell line treated with cold plasma irradiation was investigated. We confirmed that cold atmospheric plasma generated reactive oxygen and nitrogen species (RONS) in a liquid, and the intracellular RONS level was increased in plasma-irradiated cells. However, a notable decrease in cell viability was not observed 24 hours after plasma irradiation. Because RONS induce oxidative damage in cells, strand breaks and chemical modification of DNA in the cancer cells were investigated. We found that 8-oxoguanine (8-oxoG) formation as well as DNA strand breaks, which have been thoroughly investigated, were induced by plasma irradiation. In addition, up-regulation of 8-oxoG repair enzyme was observed after plasma irradiation.

Sensitive spectrofluorimetric and mass spectroscopic methods for the determination of nucleic acid damage induced by photosensitized anti-inflammatory drugs: Comparative study.

Anti-inflammatory drugs are reported to induce changes in nucleic-acids upon UV-irradiation. Such changes have the potential to cause apoptosis, carcinogenesis, and mutagenesis. In this work, the kinetics of the damage induced in DNA by some anti-inflammatory drugs were compared after UV-irradiation. Five commonly used anti-inflammatory drugs; diclofenac, ketoprofen, leflunomide, piroxicam and tolmetin, were studied. Simple, sensitive and eco-friendly methods for the analysis of DNA-damage were proposed including absorption spectroscopy, MALDI-TOF mass spectrometry and fluorescence using TbCl3. Results show that all drugs induced DNA-damage after UV-irradiation. Absorption spectroscopy results demonstrated hyperchromic shift in the absorption band characteristic to DNA, indicating distortion of the double-strand. Mass spectra showed a significant decrease of the molecular-ion-peak of DNA, together with peaks of smaller m/z that indicated the formation of DNA strand-breaks. TbCl3 fluorescence was observed to increase with incubation time of each drug with DNA, indicating the presence of more single-stranded regions in DNA due to damage. TbCl3 fluorescence was used to obtain the kinetics of the induced damage. Results show that DNA-damage occurred via photoinduced oxidative mechanism. Also, the potency of the studied drugs was examined on calf-thymus real DNA samples using TbCl3 fluorescence with ketoprofen and leflunomide being the most photogenotoxic anti-inflammatory drugs.

Pervasive Genomic Damage in Experimental Intracerebral Hemorrhage: Therapeutic Potential of a Mechanistic-Based Carbon Nanoparticle.

Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity in vitro and in vivo. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.

The role of single strand break repair pathways in cellular responses to camptothecin induced DNA damage.

Efficient DNA repair is critical for cell survival following exposure to DNA topoisomerase I (Top1) inhibitors camptothecin, a nature product from which the common chemotherapeutic drugs irinotecan and topotecan are derived. The camptothecin-derived agents exert their antitumor activities by specifically stabilizing the Top1-DNA covalent complexes (Top1cc) and blocking the DNA religation step. When exposed to these DNA damage agents, tumor cells quickly activate DNA damage response. This allows sufficient time to remove the Top1ccs and prevent tumor cells from apoptosis. Several repair pathways have been implicated in this process. One of the most relevant repair modes is DNA single strand break repair (SSBR) pathway. The expression level or mutagenesis of specific repair factors involved in SSBR pathway may play an indispensable role in individual's capacity of repairing camptothecin induced DNA damage. Therefore, understanding of the tolerance pathways counteracted to camptothecin cytotoxicity is crucial in alleviating chemotherapy resistance. This review focus on the SSBR pathway in repair camptothecin induced DNA damage, aiming to provide insights into the potential molecular determinants of camptothecin chemosensitivity.

Depurination of Colibactin-Derived Interstrand Cross-Links.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome.

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes-and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage-distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

XRCC1-mediated DNA repair is associated with progression-free survival of multiple myeloma patients after autologous stem cell transplant.

Autologous stem cell transplant (ASCT) with high-dose melphalan (HDM) is the standard treatment for fit multiple myeloma (MM) patients. It is generally believed that some DNA repair proteins impact the activity to repair melphalan-induced DNA damage, thus potentially contributing to the patient's clinical response. However, knowledge of these proteins is limited. In the current study, we investigated the roles of XRCC1, a protein involved in base excision repair and single-strand break repair, in melphalan response in MM cells. Small interfering RNA knockdown of XRCC1 significantly increased the accumulation of melphalan-induced DNA damage in MM cells and sensitized them to melphalan treatment, indicating that genetic variation in XRCC1 may impact response to melphalan treatment. We then evaluated the association between an XRCC1 variant with reduced activity, rs25487 (R399Q), and clinical outcomes of 108 MM patients with melphalan therapy. Our results showed that XRCC1 rs25487 was associated with prolonged progression-free survival (PFS) in MM patients. The adjusted hazard ratio for PFS between patients carrying rs25487 AA/AG and GG was 0.42 (95% confidence interval: 0.25, 0.84, P = .014). Taken together, these results indicate that XRCC1 is involved in the repair of melphalan-induced DNA damage and XRCC1 rs25487 variant with impaired DNA repair function influences the clinical responses of HDM in MM patients.

Electrochemical behaviour of anticancer drug lomustine and in situ evaluation of its interaction with DNA.

Electrochemical techniques were used to investigate the behavior of lomustine (CCNU) and its degradation in aqueous solution at a glassy carbon electrode (GCE). The in situ interaction of CCNU and chemically degraded CCNU (cdCCNU) with dsDNA was then investigated in dsDNA incubated solutions, using dsDNA electrochemical biosensors and comet assays. CCNU undergoes electrochemical reduction in two irreversible, diffusion-controlled, and pH-dependent redox processes, each with transfer of two electrons and one proton. At pH ≥ 10.1, the peak potential for the two processes was essentially pH-independent and involved only one electron. A mechanism was proposed for the reduction of CCNU in a neutral medium. In addition, it was found that CCNU underwent spontaneous degradation during incubation in aqueous solution, without the formation of electroactive degradation products. The degradation process was faster in basic media. Moreover, this pro-drug interacted with the DNA. Its metabolite(s) initially caused condensation of the double helix chains, followed by the unwinding of these chains. In addition, free guanine (Gua) was released from the dsDNA and oxidative damage to the DNA by the CCNU metabolite(s) was evidenced from the detection of 8-oxoGua and 2,8-oxoAde. These results were confirmed by the poly(dA)- and poly(dG)-polyhomonucleotide biosensors, which revealed the oxidative damage caused to both bases (guanine and adenine) of the dsDNA by the CCNU metabolite(s). The comet assay indicated breaks in the single strand DNA, complementing the results of the studies using differential pulse voltammetry. Conformational changes of dsDNA caused by CCNU and cdCCNU were confirmed using comet assays.

Synthetic lethality between BRCA1 deficiency and poly(ADP-ribose) polymerase inhibition is modulated by processing of endogenous oxidative DNA damage.

Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.

Roundup® confers cytotoxicity through DNA damage and Mitochondria-Associated apoptosis induction.

Glyphosate-based herbicides (GBH) are the most widely used pesticides in the world. The extensive use of them increases the potential human health risk, including the human inhalation toxicity risk. We studied the effect of the most famous GBH Roundup® (RDP) in the concentration range from 50 to 125 µg/mL on Mitochondria-Associated apoptosis and DNA damage in Human alveolar carcinoma cells (A549 cells). Alkaline comet assay, immunofluorescence assay and Flow Cytometric Analysis assay were employed to detect DNA damages and apoptosis of A549 cells. We found RDP caused concentration-dependent increases in DNA damages and proportion of apoptotic cells in A549 cells. RDP induced the DNA single-strand breaks and double-strand breaks; the collapse of mitochondrial membrane by increasing Bax/Bcl-2, resulting in the release of cytochrome c into cytosol and then activated caspase-9/-3, cleaved poly (ADP-ribose) polymerase (PARP) in human lung tissue cells. The results demonstrate that RDP can induce A549 cells cytotoxic effects in vitro at the concentration lower than the occupational exposures level of workers, which means RDP has a potential threat to human health.

Toward a better understanding of the mechanism of action for intra-arterial delivery of irinotecan from DC Bead(TM) (DEBIRI).

DC Bead is designed for the embolization of liver malignancies combined with local sustained chemotherapy delivery. It was first demonstrated around a decade ago that irinotecan could be loaded into DC Bead and used in a transarterially directed procedure to treat colorectal liver metastases, commonly referred to as drug-eluting bead with irinotecan (DEBIRI). Despite numerous reports of its safe and effective use in treating colorectal liver metastases patients, there remains a perceived fundamental paradox as to how this treatment works. This review of the mechanism of action of DEBIRI provides a rationale for why intra-arterial delivery of this prodrug from an embolic bead provides for enhanced tumor selectivity, sparing the normal liver while reducing adverse side effects associated with the irinotecan therapy.

Genotoxic effects of the cyanobacterial pentapeptide nodularin in HepG2 cells.

The cyanobacterial pentapeptide nodularin (NOD), mainly produced by genus Nodularia, is a potent inhibitor of protein phosphatases PP1 and PP2A, and causes animal mortality. The few studies available indicate that NOD is a potential non-genotoxic carcinogen. In the present study we evaluated NOD (0.01, 0.1 and 1 µg/ml) genotoxic activity in human hepatoma (HepG2) cells with the comet, γH2AX and cytokinesis block micronucleus cytome assays. In addition, induction of oxidative stress was studied. Moreover changes in the expression of selected genes from the P53 pathway, involved in the response to DNA damage (P53, GADD45α, CDKN1A, MDM2), apoptosis (BAX, BCL2) and oxidative stress (GPX1, GSR, GCLC, CAT, SOD1) were determined using qPCR. Non-cytotoxic concentrations induced time and dose dependant increase in reactive oxygen species (ROS) production and substantially increased the formation of oxidative DNA damage. In addition, elevated formation of micronuclei was detected. For the first time it has been shown that NOD deregulated the mRNA level of DNA damage (CDKN1A, GADD45α) and oxidative stress (GPX1, GSR, GCLC, CAT and SOD1) responsive genes and anti-apoptotic gene BCL2. Our results provide new evidence that NOD genotoxic effects are mediated through ROS production, already at low environmentally relevant concentrations.

DNA and chromosome damage induced by bleomycin in mammalian cells: An update.

Bleomycin (BLM) is an antibiotic isolated from Streptomyces verticillus. It has radiomimetic actions on DNA thus it has been widely used in clinical chemotherapy for the treatment of different types of cancer, including head and neck tumors, lymphomas, squamous-cell carcinomas and germ-cell tumors. Because of this, the study of BLM genotoxicity is of practical interest. This antibiotic is an S-independent clastogen and an agent that generates free radicals and induces single- and double-strand breaks in DNA. In the present review, we will summarize our current knowledge concerning the DNA and chromosome damage induced by BLM in mammalian cells, with emphasis on new developments published since 1991.

TDP1 is Critical for the Repair of DNA Breaks Induced by Sapacitabine, a Nucleoside also Targeting ATM- and BRCA-Deficient Tumors.

2'-C-cyano-2'-deoxy-1-ß-d-arabino-pentofuranosylcytosine (CNDAC) is the active metabolite of the anticancer drug, sapacitabine. CNDAC is incorporated into the genome during DNA replication and subsequently undergoes ß-elimination that generates single-strand breaks with abnormal 3'-ends. Because tyrosyl-DNA phosphodiesterase 1 (TDP1) selectively hydrolyzes nonphosphorylated 3'-blocking ends, we tested its role in the repair of CNDAC-induced DNA damage. We show that cells lacking TDP1 (avian TDP1-/- DT40 cells and human TDP1 KO TSCER2 and HCT116 cells) exhibit marked hypersensitivity to CNDAC. We also identified BRCA1, FANCD2, and PCNA in the DNA repair pathways to CNDAC. Comparing CNDAC with the chemically related arabinosyl nucleoside analog, cytosine arabinoside (cytarabine, AraC) and the topoisomerase I inhibitor camptothecin (CPT), which both generate 3'-end blocking DNA lesions that are also repaired by TDP1, we found that inactivation of BRCA2 renders cells hypersensitive to CNDAC and CPT but not to AraC. By contrast, cells lacking PARP1 were only hypersensitive to CPT but not to CNDAC or AraC. Examination of TDP1 expression in the cancer cell line databases (CCLE, GDSC, NCI-60) and human cancers (TCGA) revealed a broad range of expression of TDP1, which was correlated with PARP1 expression, TDP1 gene copy number and promoter methylation. Thus, this study identifies the importance of TDP1 as a novel determinant of response to CNDAC across various cancer types (especially non-small cell lung cancers), and demonstrates the differential involvement of BRCA2, PARP1, and TDP1 in the cellular responses to CNDAC, AraC, and CPT. Mol Cancer Ther; 16(11); 2543-51. ©2017 AACR.

The Use of PARP Inhibitors in Cancer Therapy: Use as Adjuvant with Chemotherapy or Radiotherapy, Use as a Single Agent in Susceptible Patients, and Techniques Used to Identify Susceptible Patients.

This chapter describes some of the techniques in use in our laboratories for the investigation of PARP inhibitors in clinical medicine. More specifically, we are involved in investigating the utility of PARP inhibitors in the treatment of hematopoietic malignancies. We are also actively investigating the properties of the PARP systems in cell biology. We begin the chapter with a very brief history of the invention and use of PARP inhibitors. We then explain the underlying logic of the use of PARP inhibitors either in combination with chemo- or radiotherapy or as single agents used alone. We then provide in full detail the protocols that we use to study PARP inhibitors in cell biology to identify patients that should be susceptible to PARP inhibitor treatment and to manage and investigate these patients throughout their treatment.

[EVALUATION OF GENOMIC PARAMETERS IN DUCTAL BREAST CANCER PATIENTS AND THE ABILITY OF IT'S CORRECTION].

The level of DNA single strand breaks, chromosomal abnormalities and sister chromatid exchanges and the possibility of its normalization with oligopeptide bioregulator Livagen and cobalt ions in the lymphocyte culture from patients with breast cancer have been studied. The results show that the genome of ductal breast cancer patients is characterized by the high density of DNA single strand breaks, high frequency of chromosomal abnormalities and increased levels of chromatin condensation. The usage of Livagen and cobalt in the form of modifying agents has a protective effect by all studied parameters. The obtained results allow us to conclude that research of lymphocytes of ductal breast cancer patients using the analysis conducted by us, can be useful in assessing the therapeutic effect in the treatment of breast cancer patients.