-
Scientific Reports Aug 2022Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal...
Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called "R-loops" in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break "hotspots". We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Repair; Neural Stem Cells; R-Loop Structures
PubMed: 35927309
DOI: 10.1038/s41598-022-17452-0 -
Cell Reports Sep 2020Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific...
Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.
Topics: Adenosine Triphosphatases; Aphidicolin; Chromatin; Chromosome Fragile Sites; DNA; DNA Replication; DNA-Binding Proteins; Epithelial Cells; Female; G2 Phase; HCT116 Cells; Humans; Male; Mitosis; Models, Biological; Multiprotein Complexes; Stress, Physiological
PubMed: 32966795
DOI: 10.1016/j.celrep.2020.108177 -
Biology Oct 2022In recent years, amniotic fluids have gained attention in cancer research. They have an influential role in protecting embryos against several anomalies. Chick early...
In recent years, amniotic fluids have gained attention in cancer research. They have an influential role in protecting embryos against several anomalies. Chick early amniotic fluid (ceAF)-amniotic fluid isolated from growing chicken-has been used in many other studies, including myocardial infarctions and skin regeneration. In this study, we employed ceAF's promising therapeutic applications against tumorigenesis in both in vitro and in vivo studies. We selected three robust proliferating tumor cell lines: BCaP37, MCF7, and RKO. We found that selective dosage is required to obtain maximum impact to deter tumorigenesis. ceAF not only disrupted the uniform colonies of tumor cell lines via disturbing mitochondrial transmembrane potential, but also arrested many cells at growing G1 state via working agonistically with aphidicolin. The significant inhibition of tumor metastasis by ceAF was indicated by in vivo models. This leads to apoptosis analysis as verified by annexin-V staining stays and immunoblotting of critical proteins as cell cycle meditators and apoptosis regulators. Not only on the protein level, but we also tested ceAF's therapeutic potentials on mRNA levels as indicated by quantitative real-time PCR summarizing the promising role of ceAF in deterring tumor progression. In conclusion, our study reveals the potent role of ceAF against tumorigenesis in breast cancer and colon carcinoma. Further studies will be required to determine the critical components present in ceAF and its purification to narrow down this study.
PubMed: 36358278
DOI: 10.3390/biology11111577 -
SLAS Discovery : Advancing Life... Jul 2019Mcm2-7 is the molecular motor of eukaryotic replicative helicase, and the regulation of this complex is a major focus of cellular S-phase regulation. Despite its...
Mcm2-7 is the molecular motor of eukaryotic replicative helicase, and the regulation of this complex is a major focus of cellular S-phase regulation. Despite its cellular importance, few small-molecule inhibitors of this complex are known. Based upon our genetic analysis of synthetic growth defects between alleles and a range of other alleles, we have developed a high-throughput screening (HTS) assay using a well-characterized mutant (containing the allele) to identify small molecules that replicate such synthetic growth defects. During assay development, we found that aphidicolin (inhibitor of DNA polymerase alpha) and XL413 (inhibitor of the DNA replication-dependent kinase CDC7) preferentially inhibited growth of the strain relative to the wild-type parental strain. However, as both strains demonstrated some degree of growth inhibition with these compounds, small and variable assay windows can result. To increase assay sensitivity and reproducibility, we developed a strategy combining the analysis of cell growth kinetics with linear discriminant analysis (LDA). We found that LDA greatly improved assay performance and captured a greater range of synthetic growth inhibition phenotypes, yielding a versatile analysis platform conforming to HTS requirements.
Topics: Alleles; DNA Replication; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Reproducibility of Results; Synthetic Lethal Mutations; Yeasts
PubMed: 30802412
DOI: 10.1177/2472555219829740 -
Scientific Reports Nov 2019Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an...
Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.
Topics: Aphidicolin; Biophysical Phenomena; Cell Cycle; Cell Size; Cytoplasm; Flow Cytometry; HeLa Cells; Humans; Viscosity
PubMed: 31712575
DOI: 10.1038/s41598-019-52758-6 -
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 -
Journal of Physiology and Pharmacology... Apr 2023Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental...
Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental neurotoxicity test (DNT) was established with the human neural progenitor cell line. ReNcell CX cells were exposed to neurotoxic chemicals (aphidicolin, hydroxyurea, cytosine arabinoside, 5-fluorouracil, and ochratoxin A) or non-neurotoxic chemicals (sodium gluconate, sodium bicarbonate, penicillin G, and saccharin). Propidium iodide (PI) was used to evaluate cell viability. BrdU and Ki-76 were employed to determine cell proliferation. Based on the cell viability and proliferation, mathematical models were built by linear discriminant analysis. Furthermore, the neurotoxic-considered chemicals inhibited cell cycle progression at the protein level, supporting the biomolecular rationale for the predictive model. Overall, these results show that the new test method can be used to determine the potential developmental neurotoxicants or new drug candidates.
Topics: Humans; Ki-67 Antigen; Neural Stem Cells; Neurotoxicity Syndromes; Cell Line
PubMed: 37453095
DOI: 10.26402/jpp.2023.2.07 -
Cell Reports Sep 2020Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a...
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.
Topics: Aphidicolin; Cell Line; Chromosome Breakage; DNA; DNA Damage; DNA Repair; DNA Replication; Fibroblasts; Fragile X Mental Retardation Protein; Fragile X Syndrome; Genome, Human; Humans; Models, Biological; Mutation; R-Loop Structures; RNA; Stress, Physiological
PubMed: 32966779
DOI: 10.1016/j.celrep.2020.108179 -
Viruses Apr 2024The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including...
The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.
Topics: HIV-1; Humans; mRNA Cleavage and Polyadenylation Factors; T-Lymphocytes; HeLa Cells; Macrophages; Virus Integration; Cell Nucleus; Capsid Proteins; HIV Infections; Capsid
PubMed: 38793552
DOI: 10.3390/v16050670 -
Nucleic Acids Research Jul 2021Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome...
Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.
Topics: Acid Anhydride Hydrolases; Animals; Aphidicolin; Cell Line; Chromosome Fragile Sites; DNA Copy Number Variations; DNA Replication; Genetic Loci; Humans; Mice; Mutation; Neoplasm Proteins; Promoter Regions, Genetic; R-Loop Structures; Ribonuclease H; Stress, Physiological; Transcription, Genetic
PubMed: 34181717
DOI: 10.1093/nar/gkab559