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GeroScience Feb 2022Accumulation of senescent cells may drive age-associated alterations and pathologies. Senolytics are promising therapeutics that can preferentially eliminate senescent...
Accumulation of senescent cells may drive age-associated alterations and pathologies. Senolytics are promising therapeutics that can preferentially eliminate senescent cells. Here, we performed a high-throughput automatized screening (HTS) of the commercial LOPAC®Pfizer library on aphidicolin-induced senescent human fibroblasts, to identify novel senolytics. We discovered the nociceptin receptor FQ opioid receptor (NOP) selective ligand 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole (MCOPPB, a compound previously studied as potential anxiolytic) as the best scoring hit. The ability of MCOPPB to eliminate senescent cells in in vitro models was further tested in mice and in C. elegans. MCOPPB reduced the senescence cell burden in peripheral tissues but not in the central nervous system. Mice and worms exposed to MCOPPB also exhibited locomotion and lipid storage changes. Mechanistically, MCOPPB treatment activated transcriptional networks involved in the immune responses to external stressors, implicating Toll-like receptors (TLRs). Our study uncovers MCOPPB as a NOP ligand that, apart from anxiolytic effects, also shows tissue-specific senolytic effects.
Topics: Analgesics, Opioid; Animals; Anti-Anxiety Agents; Caenorhabditis elegans; Cellular Senescence; High-Throughput Screening Assays; Humans; Ligands; Mice; Narcotic Antagonists; Opioid Peptides; Piperidines; Receptors, Opioid; Senotherapeutics; Nociceptin
PubMed: 34820764
DOI: 10.1007/s11357-021-00487-y -
Frontiers in Genetics 2021The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile... (Review)
Review
The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.
PubMed: 34567068
DOI: 10.3389/fgene.2021.708860 -
PLoS Biology Sep 2021Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On the one hand, the de novo induction of cell fate identities requires the...
Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On the one hand, the de novo induction of cell fate identities requires the establishment and subsequent maintenance of epigenetic information to harness developmental gene expression. On the other hand, the embryo depends on cell proliferation, and every round of DNA replication dilutes preexisting histone modifications by incorporation of new unmodified histones into chromatin. Here, we investigated the possible relationship between the propagation of epigenetic information and the developmental cell proliferation during Xenopus embryogenesis. We systemically inhibited cell proliferation during the G1/S transition in gastrula embryos and followed their development until the tadpole stage. Comparing wild-type and cell cycle-arrested embryos, we show that the inhibition of cell proliferation is principally compatible with embryo survival and cellular differentiation. In parallel, we quantified by mass spectrometry the abundance of a large set of histone modification states, which reflects the developmental maturation of the embryonic epigenome. The arrested embryos developed abnormal stage-specific histone modification profiles (HMPs), in which transcriptionally repressive histone marks were overrepresented. Embryos released from the cell cycle block during neurulation reverted toward normality on morphological, molecular, and epigenetic levels. These results suggest that the cell cycle block by HUA alters stage-specific HMPs. We propose that this influence is strong enough to control developmental decisions, specifically in cell populations that switch between resting and proliferating states such as stem cells.
Topics: Animals; Aphidicolin; Cell Cycle; Cell Proliferation; Embryo, Nonmammalian; Enzyme Inhibitors; Epigenesis, Genetic; Histone Code; Hydroxyurea; Xenopus laevis
PubMed: 34491983
DOI: 10.1371/journal.pbio.3001377 -
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 -
The Journal of Cell Biology Aug 2021Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost...
Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress-response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle-TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle-TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.
Topics: Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Carrier Proteins; Cell Nucleolus; DNA Damage; DNA Replication; DNA, Ribosomal; DNA-Binding Proteins; Genomic Instability; HCT116 Cells; HeLa Cells; Humans; Hydroxyurea; Microscopy, Fluorescence; Nuclear Proteins; Phosphoproteins; Protein Binding; Protein Transport; Signal Transduction
PubMed: 34100862
DOI: 10.1083/jcb.202008085 -
International Journal of Molecular... May 2021DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication...
DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.
Topics: Aphidicolin; Cell Line, Tumor; Chromatin; DNA Damage; DNA Replication Timing; Epigenesis, Genetic; Genetic Loci; Histone Code; Humans; Models, Biological; Stress, Physiological
PubMed: 34066960
DOI: 10.3390/ijms22094959 -
Nature Communications Apr 2021The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around...
The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.
Topics: Aphidicolin; Calreticulin; Clonal Hematopoiesis; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Humans; Leukemia, Myeloid; Myeloid Progenitor Cells; Poly (ADP-Ribose) Polymerase-1; Repressor Proteins; Sequence Deletion; Serine-Arginine Splicing Factors; DNA Polymerase theta
PubMed: 33911081
DOI: 10.1038/s41467-021-22803-y -
Genetics Jun 2021Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and...
Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
Topics: Animals; Aphidicolin; Cells, Cultured; DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Deoxyadenine Nucleotides; Dideoxynucleotides; Female; G2 Phase; Indoles; Mice; Nucleic Acid Synthesis Inhibitors; Oocytes; Primary Cell Culture; Rad51 Recombinase; Tetrahydroisoquinolines
PubMed: 33792683
DOI: 10.1093/genetics/iyab054 -
Molecular Cancer Research : MCR Jul 2021Activating protein 2 alpha (AP-2α; encoded by ) functions as a tumor suppressor and influences response to therapy in several cancer types. We aimed to characterize...
Activating protein 2 alpha (AP-2α; encoded by ) functions as a tumor suppressor and influences response to therapy in several cancer types. We aimed to characterize regulation of the transcriptome by AP-2α in colon cancer. CRISPR-Cas9 and short hairpin RNA were used to eliminate expression in HCT116 and a panel of colon cancer cell lines. AP-2α target genes were identified with RNA sequencing and chromatin immunoprecipitation sequencing. Effects on cell cycle were characterized in cells synchronized with aphidicolin and analyzed by FACS and Premo FUCCI. Effects on invasion and tumorigenesis were determined by invasion assay, growth of xenografts, and phosphorylated histone H3 (PHH3). Knockout of induced significant alterations in the transcriptome including repression of , identified as a primary gene target of AP-2α. Loss of AP-2α delayed progression through S-phase into G-M and decreased phosphorylation of AKT, effects that were mediated through regulation of . Buparlisib (BKM120) repressed invasiveness of HCT116 and a panel of colon cancer cell lines; however, loss of AP-2α induced resistance to buparlisib. Similarly, buparlisib repressed PHH3 and growth of tumor xenografts and increased overall survival of tumor-bearing mice, whereas, loss of AP-2α induced resistance to the effect of PI3K inhibition. Loss of AP-2α in colon cancer leads to prolonged S-phase through altered activation of AKT leading to resistance to the PI3K inhibitor, Buparlisib. The findings demonstrate an important role for AP-2α in regulating progression through the cell cycle and indicates that AP-2α is a marker for response to PI3K inhibitors. IMPLICATIONS: AP-2α regulated cell cycle through the PI3K cascade and activation of AKT mediated through TGM2. AP-2α induced sensitivity to Buparlisib/BKM120, indicating that AP-2α is a biomarker predictive of response to PI3K inhibitors.
Topics: Aminopyridines; Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; HCT116 Cells; Humans; Mice; Morpholines; Phosphoinositide-3 Kinase Inhibitors; RNA Interference; RNA-Seq; S Phase; Transcription Factor AP-2; Xenograft Model Antitumor Assays
PubMed: 33753551
DOI: 10.1158/1541-7786.MCR-20-0867 -
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