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Journal of Virology Nov 1980The inhibition in vitro of herpes simplex virus 1 and vaccinia virus DNA polymerases by aphidicolin is primarily noncompetitive with dGTP, dATP, dTTP, DNA, and Mg2+ and...
The inhibition in vitro of herpes simplex virus 1 and vaccinia virus DNA polymerases by aphidicolin is primarily noncompetitive with dGTP, dATP, dTTP, DNA, and Mg2+ and competitive with dCTP in analogy with the mode of inhibition of cellular alpha-polymerase. The degree of inhibition of viral or cellular growth in vivo can be quantitatively predicted by the degree of inhibition of the isolated replicative DNA polymerases at the same concentration of aphidicolin in suitable conditions (limiting dCTP concentration). Thus, the only in vivo target for aphidicolin is probably the replicative DNA polymerase, and aphidicolin is a highly specific inhibitor of replicative nuclear DNA synthesis in eucaryotes. This, coupled with the lack of mutagenic effect, represents a valuable property for an anticancer drug. The specificity of inhibition (contrary to the aspecific effect on almost all DNA polymerases by a true competitive inhibitor, such as 1-beta-D-arabinofuranosylcytidine 5'-triphosphate) and the structure of the drug, which does not resemble that of the triphosphates, suggest that aphidicolin must recognize a site common only to the replicative DNA polymerases of eucaryotes and different from the binding site for deoxyribonucleic triphosphates and DNA, which should be similar in reparative and procaryote-type DNA polymerase; the aphidicolin binding site is probably very near to, or even overlaping with, the binding site for dCTP so that the drug mimics a competitive effect with this nucleotide.
Topics: Aphidicolin; Binding Sites; Binding, Competitive; Cell Division; DNA Replication; Deoxycytosine Nucleotides; Diterpenes; Humans; Nucleic Acid Synthesis Inhibitors; Simplexvirus; Vaccinia virus
PubMed: 6253671
DOI: 10.1128/JVI.36.2.457-464.1980 -
Investigative Ophthalmology & Visual... Feb 2014To create an in vivo model of vector-mediated trabecular meshwork (TM) ablation and replacement.
PURPOSE
To create an in vivo model of vector-mediated trabecular meshwork (TM) ablation and replacement.
METHODS
We generated a conditionally cytotoxic, trackable vector, HSVtkiG, that expressed herpes simplex virus 1 thymidine kinase (HSVtk) and enhanced green fluorescent protein (eGFP). We optimized HSVtkiG ablation in vitro with ganciclovir (GCV) in comparison to eGFP control vector GINSIN and investigated the mechanism. Right eyes of 24 rats were then injected intracamerally with either HSVtkiG or GINSIN, before intraperitoneal GCV was administered 1 week later. Intraocular pressure, central corneal thickness (CCT), and slit-lamp exams were assessed for 8 weeks. Transduction and ablation were followed by gonioscopic visualization of eGFP. Histology was obtained with TM cell counts and immunohistochemistry markers of inflammation.
RESULTS
Transduction and ablation parameters were established in vitro. Apoptosis was the cause of cell death. In vivo, transduction was seen gonioscopically to be targeted to the TM, followed by disappearance of eGFP marker fluorescence in HSVtkiG-transduced cells after injection of GCV. Ablation resulted in an IOP decrease of 25% in HSVtkiG-injected eyes 2 days after GCV but not in GINSIN or noninjected control eyes (P < 0.05). Trabecular meshwork cellularity was decreased at the time of lowest IOP and recovered thereafter, while CCT remained unchanged. Inflammation was absent.
CONCLUSIONS
A vector-based system for inducible ablation of cells of the outflow tract was developed. Trabecular meshwork ablation lowered IOP, and recovery of cellularity and IOP followed. This model may be useful to study pressure regulation by the TM, its stem cells, and migration patterns.
Topics: Animals; Anterior Chamber; Antiviral Agents; Aphidicolin; Aqueous Humor; Cell Line; Female; Ganciclovir; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; Humans; Immunodeficiency Virus, Feline; Injections, Intraocular; Intraocular Pressure; Rats; Rats, Sprague-Dawley; Trabecular Meshwork; Transduction, Genetic
PubMed: 24448264
DOI: 10.1167/iovs.13-12890 -
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 -
Oncotarget Jun 2016Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit...
Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit their clinical use. Here, we report that the DNA polymerase inhibitor aphidicolin, which displayed negligible cytotoxicity as a single agent in primary CLL cells, markedly synergizes with fludarabine and cladribine via enhanced apoptosis. Importantly, synergy was recorded regardless of CLL prognostic markers. At the molecular level, aphidicolin enhanced purine analog-induced phosphorylation of p53 and accumulation of γH2AX, consistent with increase in DNA damage. In addition, aphidicolin delayed γH2AX disappearance that arises after removal of purine analogs, suggesting that aphidicolin causes an increase in DNA damage by impeding DNA damage repair. Similarly, aphidicolin inhibited UV-induced DNA repair known to occur primarily through the nucleotide excision repair (NER) pathway. Finally, we showed that fludarabine induced nuclear import of XPA, an indispensable factor for NER, and that XPA silencing sensitized cell lines to undergo apoptosis in response to fludarabine. Together, our data indicate that aphidicolin potentiates the cytotoxicity of purine analogs by inhibiting a DNA repair pathway that involves DNA polymerases, most likely NER, and provide a rationale for manipulating it to therapeutic advantage.
Topics: Antineoplastic Agents; Aphidicolin; Apoptosis; Cladribine; DNA Damage; DNA Repair; Drug Synergism; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Vidarabine
PubMed: 27223263
DOI: 10.18632/oncotarget.9525 -
Endocrine Journal Apr 1993We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase...
We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase genes and repress the expression of the c-myc oncogene in BeWo choriocarcinoma cells. In order to determine whether c-myc downregulation played a role in the induction of these placental genes, we treated BeWo choriocarcinoma cells with aphidicolin (APH), and 6-diazo-5-oxo-L-norleucine (DON), and compared the effects of these drugs with that of MTX. All of these drugs downregulate c-myc gene expression. At the doses used, both APH and DON repress c-myc expression to approximately the same extent, as well as inducing morphologic changes in BeWo similar to that of MTX, although neither stimulated hCG alpha expression. Both DON and APH stimulate placental alkaline phosphatase gene expression, but only MTX and DON stimulate cholesterol side chain cleavage enzyme gene expression. This indicates that the downregulation of c-myc gene expression is insufficient to stimulate the expression of all the placental genes stimulated by MTX.
Topics: Alkaline Phosphatase; Aphidicolin; Cholesterol; Choriocarcinoma; Chorionic Gonadotropin; DNA, Neoplasm; Diazooxonorleucine; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Methotrexate; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Uterine Neoplasms
PubMed: 7951513
DOI: 10.1507/endocrj.40.263 -
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 Plant Cell May 2004Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle...
Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle progression. In animals, ATR is an essential gene. Here, we find that Arabidopsis (Arabidopsis thaliana) atr-/- mutants were viable, fertile, and phenotypically wild-type in the absence of exogenous DNA damaging agents but exhibit altered expression of AtRNR1 (ribonucleotide reductase large subunit) and alteration of some damage-induced cell-cycle checkpoints. atr mutants were hypersensitive to hydroxyurea (HU), aphidicolin, and UV-B light but only mildly sensitive to gamma-radiation. G2 arrest was observed in response to gamma-irradiation in both wild-type and atr plants, albeit with slightly different kinetics, suggesting that ATR plays a secondary role in response to double-strand breaks. G2 arrest also was observed in wild-type plants in response to aphidicolin but was defective in atr mutants, resulting in compaction of nuclei and subsequent cell death. By contrast, HU-treated wild-type and atr plants arrested in G1 and showed no obvious signs of cell death. We propose that, in plants, HU invokes a novel checkpoint responsive to low levels of deoxynucleotide triphosphates. These results demonstrate the important role of cell-cycle checkpoints in the ability of plant cells to sense and cope with problems associated with DNA replication.
Topics: Aphidicolin; Arabidopsis; Arabidopsis Proteins; Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Cycle Proteins; DNA Replication; Gene Deletion; Hydroxyurea; Molecular Sequence Data; Phenotype; Protein Serine-Threonine Kinases; Ultraviolet Rays
PubMed: 15075397
DOI: 10.1105/tpc.018903 -
Journal of Virology Jul 1993Small DNA viruses have been historically used as probes of cellular control mechanisms of DNA replication, gene expression, and differentiation. Polyomavirus (Py) DNA...
Small DNA viruses have been historically used as probes of cellular control mechanisms of DNA replication, gene expression, and differentiation. Polyomavirus (Py) DNA replication is known to be linked to differentiation of may cells, including myoblasts. In this report, we use this linkage in myoblasts to simultaneously examine (i) cellular differentiation control of Py DNA replication and (ii) an unusual type of cellular and Py DNA synthesis during differentiation. Early proposals that DNA synthesis was involved in the induced differentiation of myoblasts to myotubes were apparently disproved by reliance on inhibitors of DNA synthesis (cytosine arabinoside and aphidicolin), which indicated that mitosis and DNA replication are not necessary for differentiation. Theoretical problems with the accessibility of inactive chromatin to trans-acting factors led us to reexamine possible involvement of DNA replication in myoblast differentiation. We show here that Py undergoes novel aphidicolin-resistant net DNA synthesis under specific conditions early in induced differentiation of myoblasts (following delayed aphidicolin addition). Under similar conditions, we also examined uninfected myoblast DNA synthesis, and we show that soon after differentiation induction, a period of aphidicolin-resistant cellular DNA synthesis can also be observed. This drug-resistant DNA synthesis appears to be subgenomic, not contributing to mitosis, and more representative of polyadenylated than of nonpolyadenylated RNA. These results renew the possibility that DNA synthesis plays a role in myoblast differentiation and suggest that the linkage of Py DNA synthesis to differentiation may involve a qualitative cellular alteration in Py DNA replication.
Topics: Animals; Aphidicolin; Cell Differentiation; Cell Line; DNA; DNA, Viral; Gene Expression Regulation, Viral; In Situ Hybridization; In Vitro Techniques; Mice; Muscles; Nucleic Acid Synthesis Inhibitors; Polyomavirus; Virus Replication
PubMed: 8389922
DOI: 10.1128/JVI.67.7.4169-4181.1993 -
Drug Discoveries & Therapeutics 2016Human Embryonic Lung fibroblasts (HEL cells) are widely used as a normal cell in studies of cell biology and can be easily maintained in the resting phase. Here we aimed...
Human Embryonic Lung fibroblasts (HEL cells) are widely used as a normal cell in studies of cell biology and can be easily maintained in the resting phase. Here we aimed to discover compounds that exhibit cytotoxicity against HEL cells in the dividing phase, but not in the resting phase. The cytotoxicity of each compound against HEL cells either in the resting phase or in the dividing phase was determined by MTT assay. Ratios of the IC50 of cells in the resting phase and that of cells in the dividing phase (RRD) for these compounds were compared. We selected 44 compounds that exhibited toxic effects on HEL cells in the dividing phase from a chemical library containing 325 anticancer drugs and enzyme inhibitors. The RRD values of those compounds were widely distributed. Paclitaxel and docetaxel, which are clinically used as anticancer drugs, had RRD values larger than 2000. On the other hand, the RRD value of dimethyl sulfoxide, an organic solvent, was 1. The cytotoxic effect of paclitaxel on HEL cells in the dividing phase was attenuated by aphidicolin, hydroxyurea, and nocodazole, confirming that the cytotoxic effects of paclitaxel are dependent on cells being in the dividing phase. Thapsigargin, whose RRD value was 800, the third highest RRD value in the library, exhibited therapeutic effects in a mouse model of FM3A ascites carcinoma. We suggest that compounds with high RRD values for HEL cells are candidate anticancer chemotherapy seeds.
Topics: Animals; Antineoplastic Agents; Aphidicolin; Carcinoma; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; Dimethyl Sulfoxide; Disease Models, Animal; Docetaxel; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Fibroblasts; Hydroxyurea; Lung; Mice; Nocodazole; Paclitaxel; Peritoneal Neoplasms; Resting Phase, Cell Cycle; Solvents; Taxoids; Thapsigargin; Xenograft Model Antitumor Assays
PubMed: 27594296
DOI: 10.5582/ddt.2016.01049 -
Cytometry. Part a : the Journal of the... Apr 2006DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through...
Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.
BACKGROUND
DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM.
MATERIALS AND METHODS
HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content.
RESULTS
While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation.
CONCLUSIONS
The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.
Topics: Aphidicolin; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Cycle Proteins; DNA Damage; DNA Replication; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; HL-60 Cells; Histones; Humans; Hydroxyurea; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Point Mutation; Protein Serine-Threonine Kinases; Serine; Tumor Suppressor Proteins
PubMed: 16528719
DOI: 10.1002/cyto.a.20241