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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 -
Viruses May 2024Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological...
Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.
Topics: Animals; Antiviral Agents; Horses; Decitabine; Immunity, Innate; Herpesvirus 4, Equid; Fibroblasts; Herpesviridae Infections; Horse Diseases; Viral Load; Cell Line; Virus Replication; Drug Evaluation, Preclinical
PubMed: 38793627
DOI: 10.3390/v16050746 -
Journal of B.U.ON. : Official Journal... 2021Retraction of: 'Inhibitory effect of Aphidicolin - a tetracyclic diterpene - on the proliferation and apoptotic induction in human cervical cancer (HeLa) cells', by...
Retraction of: 'Inhibitory effect of Aphidicolin - a tetracyclic diterpene - on the proliferation and apoptotic induction in human cervical cancer (HeLa) cells', by En-Yan Yu, Rui-Yan Zhao, Dong-Sheng Wang, JBUON 2015;20(6):1480-1486; PMID:26854444. Following the publication of the above article, readers drew to our attention that part of the data was unreliable. The authors were requested to provide the raw data to prove the originality, but were unable to do so. After an investigation, the Editors of JBUON decided to retract this article. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.
PubMed: 33721476
DOI: No ID Found -
Emerging Topics in Life Sciences Dec 2023Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and...
Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.
Topics: Humans; DNA Replication; Chromosome Fragile Sites; DNA Replication Timing; Chromosomal Instability; DNA
PubMed: 37876349
DOI: 10.1042/ETLS20230023 -
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 -
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 -
International Journal of Molecular... Dec 2021The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable...
The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.
Topics: B7-H1 Antigen; Cell Cycle; Gene Expression Regulation, Neoplastic; Humans; Protein Transport; Squamous Cell Carcinoma of Head and Neck
PubMed: 34884892
DOI: 10.3390/ijms222313087 -
Molecules (Basel, Switzerland) Aug 2019Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2),...
Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC): 2.7 μM; SI: 114.8) compared with that of ACV (EC: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC): 0.7 μM; inhibition constant (): 0.3 μM) compared with reference drugs aphidicolin (IC: 0.8 μM; : 0.4 μM) and ACV triphosphate (ACV-TP) (IC: 0.9 μM; : 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.
Topics: Animals; Antiviral Agents; Benzoxepins; Carboxylic Acids; Chlorocebus aethiops; DNA-Directed DNA Polymerase; Herpesvirus 1, Human; Herpesvirus 2, Human; Humans; Lichens; Molecular Docking Simulation; Nucleic Acid Synthesis Inhibitors; Vero Cells; Viral Proteins; Virus Replication
PubMed: 31405197
DOI: 10.3390/molecules24162912 -
Journal of Radiation Research Mar 2024Telomere dysfunction induces chromosomal instability, which is a driving force in the development of cancers. To examine X-irradiation's effect on telomere integrity, we...
Telomere dysfunction induces chromosomal instability, which is a driving force in the development of cancers. To examine X-irradiation's effect on telomere integrity, we investigated X-ray-induced abnormalities in telomere signals detected by fluorescence in situ hybridization (telomere FISH) in mouse embryo fibroblast cells. The abnormalities were categorized as either extra telomere signals (ETSs) or loss of telomere signals (LTSs). The results indicated that low doses (0.3-0.5 Gy) of X-rays significantly induced ETS but not LTS and that ETS induction was saturated at doses above 0.5 Gy. In addition, treatment with hydrogen peroxide also induced ETS but not LTS. To clarify the involvement of radicals in inducing ETS, we examined the effect of ascorbic acid (AsA) on telomere FISH signals and found that pre-treatment with AsA (5 mM, 2 h), but not post-treatment, significantly suppressed the induction of ETS by X-irradiation. Importantly, neither pre- nor post-treatment with AsA affected X-ray-induced chromosome aberrations. These results suggest that oxidative DNA damage induced by radicals is involved in the induction of ETS. Furthermore, combined treatment with aphidicolin, a DNA replication inhibitor, elevated the induction of ETS by X-irradiation. This observation suggests that DNA replication stress, potentially triggered by oxidative DNA lesions within telomeres, may contribute to the induction of ETS resulting from X-irradiation. Based on these results, we propose that ETS is a sensitive biological marker of oxidative DNA damage in telomere structures.
Topics: Animals; Mice; In Situ Hybridization, Fluorescence; Telomere; Oxidative Stress; Chromosomal Instability; Chromosome Aberrations; DNA Damage
PubMed: 38171574
DOI: 10.1093/jrr/rrad102