-
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 -
Environmental and Molecular Mutagenesis Oct 2020Genome instability is a hallmark of most human cancers and is exacerbated following replication stress. However, the effects that drugs/xenobiotics have in promoting...
Genome instability is a hallmark of most human cancers and is exacerbated following replication stress. However, the effects that drugs/xenobiotics have in promoting genome instability including chromosomal structural rearrangements in normal cells are not currently assessed in the genetic toxicology battery. Here, we show that drug-induced replication stress leads to increased genome instability in vitro using proliferating primary human cells as well as in vivo in rat bone marrow (BM) and duodenum (DD). p53-binding protein 1 (53BP1, biomarker of DNA damage repair) nuclear bodies were increased in a dose-dependent manner in normal proliferating human mammary epithelial fibroblasts following treatment with compounds traditionally classified as either genotoxic (hydralazine) and nongenotoxic (low-dose aphidicolin, duvelisib, idelalisib, and amiodarone). Comparatively, no increases in 53BP1 nuclear bodies were observed in nonproliferating cells. Negative control compounds (mannitol, alosteron, diclofenac, and zonisamide) not associated with cancer risk did not induce 53BP1 nuclear bodies in any cell type. Finally, we studied the in vivo genomic consequences of drug-induced replication stress in rats treated with 10 mg/kg of cyclophosphamide for up to 14 days followed by polymerase chain reaction-free whole genome sequencing (30X coverage) of BM and DD cells. Cyclophosphamide induced chromosomal structural rearrangements at an average of 90 genes, including 40 interchromosomal/intrachromosomal translocations, within 2 days of treatment. Collectively, these data demonstrate that this drug-induced genome instability test (DiGIT) can reveal potential adverse effects of drugs not otherwise informed by standard genetic toxicology testing batteries. These efforts are aligned with the food and drug administration's (FDA's) predictive toxicology roadmap initiative.
Topics: Animals; B-Lymphocytes; Biomarkers; Chromosome Aberrations; Cyclophosphamide; DNA Replication; Genome; Genomic Instability; Humans; Male; Rats; Rats, Sprague-Dawley; Whole Genome Sequencing
PubMed: 32078182
DOI: 10.1002/em.22364 -
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 -
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 -
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 -
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 -
Frontiers in Cell and Developmental... 2024The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome...
The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless-Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase-helicase uncoupling, we characterized the Timeless-Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless-Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless-Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.
PubMed: 38487270
DOI: 10.3389/fcell.2024.1346534 -
Current Computer-aided Drug Design 2020Urolithiasis is the process of forming stones in the kidney, bladder, and/or urinary tract. It has been reported that kidney stones are the third most common disorder...
BACKGROUND
Urolithiasis is the process of forming stones in the kidney, bladder, and/or urinary tract. It has been reported that kidney stones are the third most common disorder among urinary diseases. At present, surgical procedures and Extracorporeal Shock Wave Lithotripsy (ESWL) are commonly employed for the treatment of Urolithiasis. The major drawback of these procedures is the recurrence of stones.
METHODS
This study aimed to identify potential natural inhibitors against human Serum Albumin (SA) from the plant Scoparia Dulsis for Urolithiasis. As protein-ligand interactions play a key role in structure- based drug design, this study screened 26 compounds from Scoparia Dulsis and investigated their binding affinity against SA by using molecular docking. The three dimensional (3D) structure of SA was retrieved from Protein Data Bank (PDB) and docked with PubChem structures of 26 compounds using PyRX docking tool through Autodock Vina. Moreover, a 3D similarity search on the PubChem database was performed to find the analogs of best scored compound and docking studies were performed. Drug-likeness studies were made using Swiss ADME and Lipinski's rule of five was performed for the compounds to evaluate their anti-urolithiatic activity.
RESULTS
The results showed that citrusin c (Eugenyl beta-D-glucopyranoside) exhibited best binding energy of -8.1 kcal/mol with SA followed by aphidicolin, apigenin, luteolin and scutellarein. Two compounds (PubChem CID 46186820, PubChem CID 21579141) analogous to citrusin c were selected based on the lowest binding energy.
CONCLUSION
This study, therefore, reveals that these compounds could be promising candidates for further evaluation for Urolithiasis prevention or management.
Topics: Drug Design; Drug Discovery; Humans; Molecular Docking Simulation; Plant Extracts; Scoparia; Serum Albumin, Human; Urolithiasis
PubMed: 31393255
DOI: 10.2174/1573409915666190808125518 -
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 -
Methods in Molecular Biology (Clifton,... 2020Primary cultures of neurons of the peripheral nervous system have been successfully used for studying many aspects of neuronal development and survival, including...
Primary cultures of neurons of the peripheral nervous system have been successfully used for studying many aspects of neuronal development and survival, including investigations into the mechanisms of axon degeneration. In this chapter, we describe how to prepare and microinject dissociated cultures of sympathetic neurons of the superior cervical ganglion (SCG) specifically for use in highly controlled and targeted assays of axon survival and degeneration.
Topics: Animals; Aphidicolin; Axons; Axotomy; Fluorescent Dyes; Mice; Microinjections; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Nerve Growth Factor; Primary Cell Culture; Sensory Receptor Cells; Solutions; Superior Cervical Ganglion
PubMed: 32524470
DOI: 10.1007/978-1-0716-0585-1_3