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Archives of Toxicology May 2021The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human...
The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.
Topics: Biological Monitoring; Comet Assay; Cryopreservation; DNA Damage; DNA Repair; Humans; Hydrogen Peroxide; Leukocytes, Mononuclear
PubMed: 33666708
DOI: 10.1007/s00204-021-03012-4 -
Nitric Oxide : Biology and Chemistry May 2021Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by...
Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 μM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.
Topics: Acyltransferases; Animals; Aorta; Aphidicolin; Cattle; Cell Death; Cell Nucleus; Cell Survival; Endothelial Cells; Nitric Oxide; Nitric Oxide Synthase Type III
PubMed: 33592314
DOI: 10.1016/j.niox.2021.02.001 -
PloS One 2021Homotypic or heterotypic internalization of another, either living or necrotic cell is currently in the center of research interest. The active invasion of a living cell...
Homotypic or heterotypic internalization of another, either living or necrotic cell is currently in the center of research interest. The active invasion of a living cell called entosis and cannibalism of cells by rapidly proliferating cancers are prominent examples. Additionally, normal healthy tissue cells are capable of non-professional phagocytosis. This project studied the relationship between non-professional phagocytosis, individual proliferation and cell cycle progression. Three mesenchymal and two epithelial normal tissue cell lines were studied for homotypic non-professional phagocytosis. Homotypic dead cells were co-incubated with adherent growing living cell layers. Living cells were synchronized by mitotic shake-off as well as Aphidicolin-treatment and phagocytotic activity was analyzed by immunostaining. Cell cycle phases were evaluated by flow cytometry. Mesenchymal and epithelial normal tissue cells were capable of internalizing dead cells. Epithelial cells had much higher non-professional phagocytotic rates than mesenchymal cells. Cells throughout the entire cell cycle were able to phagocytose. The phagocytotic rate significantly increased with progressing cell cycle phases. Mitotic cells regularly phagocytosed dead cells, this was verified by Nocodazole and Colcemid treatment. Taken together, our findings indicate the ability of human tissue cells to phagocytose necrotic neighboring cells in confluent cell layers. The origin of the cell line influences the rate of cell-in-cell structure formation. The higher cell-in-cell structure rates during cell cycle progression might be influenced by cytoskeletal reorganization during this period or indicate an evolutionary anchorage of the process. Recycling of nutrients during cell growth might also be an explanation.
Topics: Cell Cycle; Cell Division; Cell Line; Entosis; Epithelial Cells; Humans; Mesenchymal Stem Cells; Neoplasms; Phagocytosis
PubMed: 33544774
DOI: 10.1371/journal.pone.0246402 -
JCI Insight Mar 2021Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from...
Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.
Topics: Animals; Aphidicolin; Cell Cycle; Cell Differentiation; Cell Proliferation; DNA Replication; Diabetes Mellitus; Humans; Induced Pluripotent Stem Cells; Insulin; Insulin-Secreting Cells; Islets of Langerhans; Mice; Pancreas; Pluripotent Stem Cells; Stem Cell Transplantation; Transplants
PubMed: 33529174
DOI: 10.1172/jci.insight.141553 -
Cell Death Discovery Jan 2021Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB)...
Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-βgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.
PubMed: 33462217
DOI: 10.1038/s41420-020-00393-w -
FEBS Letters Mar 2021We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous...
We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between the FA protein FANCD2 and the non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in the FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediate stalled replication fork restart, and prevent nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability.
Topics: Aphidicolin; Cell Survival; DNA; DNA Repair; DNA Replication; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Fibroblasts; Genome, Human; Genomic Instability; Humans; Hydroxyurea; Mitomycin; Primary Cell Culture; Protein Binding; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases
PubMed: 33423298
DOI: 10.1002/1873-3468.14035 -
Nucleic Acids Research Jan 2021The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic...
The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.
Topics: Aphidicolin; Breast; Breast Neoplasms; Cells, Cultured; Chromosomal Instability; Chromosome Fragile Sites; DNA Breaks; DNA Copy Number Variations; DNA Repair; DNA, Neoplasm; Epithelial Cells; Female; Gene Amplification; Gene Duplication; Genes, erbB-2; Genetic Variation; Genomic Instability; Humans; Keratins, Hair-Specific; MRE11 Homologue Protein; Neoplasm Proteins; Whole Genome Sequencing
PubMed: 33290559
DOI: 10.1093/nar/gkaa1136 -
Analytical Biochemistry Feb 2021The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is...
The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.
Topics: Bromodeoxyuridine; Carbocyanines; Cell Cycle; DNA; DNA Replication; Flow Cytometry; Fluorescent Dyes; HL-60 Cells; HeLa Cells; Humans; Jurkat Cells; Reproducibility of Results; Staining and Labeling
PubMed: 33159846
DOI: 10.1016/j.ab.2020.114002 -
The Journal of Biological Chemistry 2021DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types...
DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5'-triphosphate-induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.
Topics: Animals; Cell-Free System; DNA Replication; Mitosis; Nuclear Envelope; Xenopus laevis
PubMed: 33148697
DOI: 10.1074/jbc.RA120.015142 -
Viruses Oct 2020Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal...
Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C/H Strain in French Horses Showing no Major Impact on the Strain Behaviour.
Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C (H) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as U clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C mutation. When cultured in vitro, the C EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A and G EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C strain and two other A/G field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C and A strains were more sensitive to aciclovir than the G strain, based on viral load measurement.
Topics: Animals; DNA-Directed DNA Polymerase; Disease Outbreaks; France; Genotype; Herpesviridae Infections; Herpesvirus 1, Equid; Horse Diseases; Horses; Male; Mutation; Open Reading Frames; Retrospective Studies; Viral Load; Viral Proteins
PubMed: 33066315
DOI: 10.3390/v12101160