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Antiviral Research Nov 2020Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection...
Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.
Topics: Animals; Antiviral Agents; Cell Line; Decitabine; Drug Combinations; Drug Discovery; Drug Synergism; Ganciclovir; Herpesviridae Infections; Herpesvirus 1, Equid; High-Throughput Screening Assays; Horses; Rabbits; Valganciclovir
PubMed: 32926887
DOI: 10.1016/j.antiviral.2020.104931 -
Nature Communications Jul 2020Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to...
Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.
Topics: Aphidicolin; Cell Line; Chromatin Immunoprecipitation Sequencing; Chromosome Fragile Sites; Chromosome Mapping; DNA; DNA Replication Timing; Fibroblasts; Gene Regulatory Networks; Genome, Human; Genomic Instability; High-Throughput Nucleotide Sequencing; Humans; Neoplasms; Nucleic Acid Conformation; Sensitivity and Specificity; Transcription, Genetic
PubMed: 32680994
DOI: 10.1038/s41467-020-17448-2 -
Cell Research Nov 2020DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA...
DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.
Topics: Cell Line, Tumor; Chromosome Breakage; Chromosome Fragile Sites; DNA; DNA Replication Timing; Genome, Human; Genomic Instability; Humans; Mitosis; Molecular Sequence Annotation; Neoplasms; Replication Origin; Sequence Analysis, DNA
PubMed: 32561860
DOI: 10.1038/s41422-020-0358-x -
Genes Jun 2020Components of the nuclear pore complex (NPC) have been shown to play a crucial role in protecting against replication stress, and recovery from some types of stalled or... (Review)
Review
Components of the nuclear pore complex (NPC) have been shown to play a crucial role in protecting against replication stress, and recovery from some types of stalled or collapsed replication forks requires movement of the DNA to the NPC in order to maintain genome stability. The role that nuclear positioning has on DNA repair has been investigated in several systems that inhibit normal replication. These include structure forming sequences (expanded CAG repeats), protein mediated stalls (replication fork barriers (RFBs)), stalls within the telomere sequence, and the use of drugs known to stall or collapse replication forks (HU + MMS or aphidicolin). Recently, the mechanism of relocation for collapsed replication forks to the NPC has been elucidated. Here, we will review the types of replication stress that relocate to the NPC, the current models for the mechanism of relocation, and the currently known protective effects of this movement.
Topics: DNA; DNA Damage; DNA Repair; DNA Replication; Genomic Instability; Humans; Nuclear Pore; Telomere
PubMed: 32526925
DOI: 10.3390/genes11060635 -
Biomolecules & Therapeutics Nov 2020Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated...
Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominantnegative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser, p-CREB-Ser, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser, p-Akt-Ser, p-CREB-Ser, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.
PubMed: 32394671
DOI: 10.4062/biomolther.2020.007 -
Plant Cell Reports Aug 2020Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments...
Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (HO), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.
Topics: Apoptosis; Cell Nucleus; DNA Damage; DNA Fragmentation; DNA Replication; Gene Expression Regulation, Plant; Glutathione; Hydrogen Peroxide; Interphase; Meristem; Mitosis; Onions; Protein Biosynthesis; Reactive Oxygen Species; Seedlings; Transcription, Genetic; Uracil
PubMed: 32328702
DOI: 10.1007/s00299-020-02545-9 -
Genes Mar 2020Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells...
Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council strain (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene () in a lymphocytes' CFS, and Duchenne muscular dystrophy gene ( in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.
Topics: Cell Line; Cells, Cultured; Chromosome Fragile Sites; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 3; DNA Replication; Dystrophin; Female; Humans; Male; Membrane Proteins; Nerve Tissue Proteins; Organ Specificity
PubMed: 32204553
DOI: 10.3390/genes11030326 -
Cancer Research Apr 2020Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule...
Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (, and ) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.
Topics: Aphidicolin; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Colorectal Neoplasms; DNA Damage; DNA Polymerase I; DNA Polymerase II; DNA Polymerase beta; Drugs, Investigational; Enzyme Inhibitors; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Lung Neoplasms; Neoplasm Proteins; Poly-ADP-Ribose Binding Proteins; RNA, Small Interfering; Retinoids
PubMed: 32161100
DOI: 10.1158/0008-5472.CAN-19-1372 -
Nature Microbiology May 2020Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular...
Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.
Topics: Cell Line; DNA Ligase ATP; DNA Replication; DNA, Circular; DNA, Viral; Flap Endonucleases; Genome, Viral; Hepatitis B virus; Hepatocytes; Humans; Proliferating Cell Nuclear Antigen; Replication Protein C; Virion; Virus Replication
PubMed: 32152586
DOI: 10.1038/s41564-020-0678-0 -
Mutagenesis Apr 2021In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal...
In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.
Topics: 2-Acetylaminofluorene; Carcinogens; DNA Adducts; DNA Damage; Fluorenes; Humans; Hydroxyacetylaminofluorene; Micronucleus Tests; Mutagens; Skin
PubMed: 31816077
DOI: 10.1093/mutage/gez044