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Cell Biology International Reports Aug 1983In order to clarify in vivo target(s) of aphidicolin, the cell cycle phase at which normal human diploid cells were accumulated with this drug was examined by flow...
In order to clarify in vivo target(s) of aphidicolin, the cell cycle phase at which normal human diploid cells were accumulated with this drug was examined by flow cytofluorometry. More than 80% of the cells moved through S phase and were accumulated at G2 phase at the drug dose under which growth of the cells was completely inhibited but there was no apparent cell death. On the other hand, at the higher dose, cell detachment occurred through the first entry of the cells to early S phase. Apparently, in vivo effects of aphidicolin are much more complicated than simple inhibition of DNA synthesis.
Topics: Aphidicolin; Cell Line; Cell Survival; DNA; Diterpenes; Dose-Response Relationship, Drug; Fibroblasts; Humans; Interphase; Lung
PubMed: 6413078
DOI: 10.1016/0309-1651(83)90111-x -
Carcinogenesis Dec 1993Both aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC) inactivate DNA polymerases alpha, delta and epsilon, and according block long-patch excision repair in...
Both aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC) inactivate DNA polymerases alpha, delta and epsilon, and according block long-patch excision repair in mammalian cells. We report here that in normal human fibroblasts both compounds strongly inhibit the repair of damage induced by UV or 4-nitroquinoline-1-oxide in the transcriptionally active c-myc gene, as indicated by the appearance of DNA strand breaks in carcinogen-treated cultures that were subsequently incubated in the presence of either polymerase inhibitor. We further demonstrate that the repair of UV photoproducts in the c-myc gene can be monitored by photolysis (313 nm) of DNA repaired in the presence of bromodeoxyuridine (BrdUrd). In UV-irradiated cultures, the incidence of aphidicolin- or araC-accumulated strand breaks was approximately 70% of that detected by the BrdUrd photolysis assay. Our data therefore implicate a critical role for DNA polymerases alpha, delta and/or epsilon in gene-specific repair in human cells. The techniques described here may prove useful in the study of DNA repair in defined sequences of the human genome following exposure to a diverse array of physical and chemical genotoxic agents.
Topics: 4-Nitroquinoline-1-oxide; Aphidicolin; Cell Line; Cytarabine; DNA Repair; Fibroblasts; Genes, myc; Humans; Mutagens; Transcription, Genetic; Ultraviolet Rays
PubMed: 8269635
DOI: 10.1093/carcin/14.12.2621 -
Bioorganic & Medicinal Chemistry Letters Feb 2016Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current...
Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current therapies. Thus, the exploration of innovative scaffolds, ideally with distinct mechanisms of action, is urgently needed. The natural product aphidicolin and its effects on cell cycle division have been widely studied; it is a potent inhibitor of parasitic cells. In the present study, we report for the first time the semisynthesis of a series of aphidicolin derivatives, their unique structural features, and demonstration of their activity against Trypanosoma cruzi cells. Two demonstrated high potency and selectivity against parasitic amastigote cells, and thus show promise as new leads for Chagas disease treatment.
Topics: Aphidicolin; Chagas Disease; Humans; Parasitic Sensitivity Tests; Structure-Activity Relationship; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 26810263
DOI: 10.1016/j.bmcl.2016.01.033 -
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 -
Journal of Natural Products Aug 2011Six new secondary metabolites including two aphidicolin analogues, inflatins A (1) and B (2), and four chlamydosporol derivatives, inflatins C-F (3-6), have been...
Six new secondary metabolites including two aphidicolin analogues, inflatins A (1) and B (2), and four chlamydosporol derivatives, inflatins C-F (3-6), have been isolated from the crude extract of Tolypocladium inflatum. The structures of 1-6 were determined mainly by NMR experiments, and 4 and 5 were further confirmed by X-ray crystallography. The absolute configurations of C-16 in 1 and C-5 in 3 were deduced via the circular dichroism data of the in situ formed [Rh₂(OCOCF₃)₄] complexes, whereas that of 4 was assigned by X-ray crystallography using Cu Kα radiation. Compounds 1 and 2 showed modest cytotoxicity against a panel of eight human tumor cell lines.
Topics: Aphidicolin; Ascomycota; Crystallography, X-Ray; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Pyrones
PubMed: 21812410
DOI: 10.1021/np200431k -
International Journal of Radiation... Feb 1992The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, was studied on DNA synthesis, PLD-recovery and DNA double-strand break rejoining in...
The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, was studied on DNA synthesis, PLD-recovery and DNA double-strand break rejoining in X-irradiated human fibroblasts. In unirradiated, exponentially growing cells, aphidicolin (0.5-5 micrograms ml) inhibited DNA synthesis almost completely. This effect depended not only on aphidicolin concentration but also on the duration of pre-incubation. The action of aphidicolin was found to be reversible. When aphidicolin had been removed, colony forming ability was not affected in aphidicolin pretreated cells. Aphidicolin pretreated and irradiated cells showed a reduction in PLD-recovery, dependent on aphidicolin concentration and duration of pretreatment. The initial number of DNA double-strand breaks (calibrated by 125I decay) was not affected by aphidicolin. However, after incubation for 90 min in the presence of aphidicolin there was a large reduction in double-strand break rejoining. With long incubation periods in aphidicolin rejoining was almost completely inhibited.
Topics: Adult; Aphidicolin; Cell Survival; DNA; DNA Damage; DNA Repair; Female; Fibroblasts; Humans
PubMed: 1351906
DOI: 10.1080/09553009214550811 -
Biochemistry Aug 1993Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and exonuclease activity. These substrates were used...
Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and exonuclease activity. These substrates were used to examine the mechanism of inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol epsilon.DNA complex. Importantly, aphidicolin binds equally well regardless of the identity of the next template base to be replicated (Ki approximately 0.6 microM). Hydrolysis of synthetic templates of defined sequence by the 3'-->5' exonuclease was examined. pol epsilon preferred to hydrolyze single-stranded DNA 3-fold better than double-stranded DNA (Vmax/KM), while under Vmax conditions single-stranded DNA was hydrolyzed 100-fold faster than double-stranded DNA. Aphidicolin did not inhibit exonuclease activity on single-stranded DNA; however, activity on double-stranded DNA was partially inhibited. Formation of an E.[template.primer].aphidicolin ternary complex inhibits exonuclease activity. However, even under conditions where the polymerase site is completely blocked by a template-primer, the exonuclease retains significant activity.
Topics: Animals; Aphidicolin; Base Sequence; Cattle; DNA; DNA Polymerase II; DNA-Directed DNA Polymerase; Exonucleases; Kinetics; Molecular Sequence Data; Nucleic Acid Synthesis Inhibitors; Oligodeoxyribonucleotides; Poly dA-dT; Substrate Specificity; Thymus Gland
PubMed: 8395209
DOI: 10.1021/bi00084a025 -
Blood Dec 1991The commitment process of a human megakaryoblastic cell line (MEG-O1) induced with phorbol ester, TPA, was investigated with special reference to glycoprotein (GP)...
The commitment process of a human megakaryoblastic cell line (MEG-O1) induced with phorbol ester, TPA, was investigated with special reference to glycoprotein (GP) IIb/IIIa expression, multinuclear formation, and DNA replication. TPA (10(-7) mol/L) completely inhibited cellular division in MEG-O1, but did not suppress de novo DNA synthesis. Two days' culture with 10(-7) mol/L TPA was sufficient for MEG-O1 cells to initiate an irreversible commitment process. These cells could not resume cell growth and expressed GP IIb/IIIa antigen; some of them showed multinuclear form and DNA polyploidy even after removal of TPA from the culture medium. DNA histogram analysis showed that, upon treatment with TPA, the percentage of cells whose DNA ploidy was more than 8N was 5 to 10 times higher than that of control cells. Precise analysis using cell size fractionation by centrifugal elutriation method showed that there was strong correlation between the percentage of multinuclear cells and DNA polyploidy in TPA-treated cells. The percentage and staining intensity of GP IIb/IIIa and other megakaryocytic phenotypes such as von Willebrand factor and PAS staining were highest in large multinuclear cell populations, suggesting that these cells are the most differentiated population in this system. In TPA-treated cells, the activity of DNA polymerase alpha, a marker for cell growth, remained at the same level as in control cells. Aphidicolin, a specific inhibitor of DNA polymerase alpha, completely inhibited the differentiation induction of MEG-O1 cells with TPA measured by either GP IIb/IIIa expression or multinuclear cell formation. Therefore, DNA replication appears to be involved in the process of phenotypic expression as well as endomitosis in megakaryocyte differentiation of MEG-O1 cells. Aphidicolin was also effective in inhibiting megakaryocytic differentiation of other leukemia cell lines such as human erythroleukemia (HEL) and K562 cell lines induced with TPA, suggesting the close interplay of DNA replication and phenotypic expression in megakaryopoiesis.
Topics: Aphidicolin; Cell Differentiation; Cell Division; DNA; DNA Replication; DNA-Directed DNA Polymerase; Flow Cytometry; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Megakaryocytes; Nucleic Acid Synthesis Inhibitors; Platelet Membrane Glycoproteins; Ploidies; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured
PubMed: 1742484
DOI: No ID Found -
American Journal of Human Genetics Jan 1992Common chromosomal fragile sites appear to be ubiquitous in humans and other mammals, and, although the molecular basis and function of these sites remain an enigma, it...
Common chromosomal fragile sites appear to be ubiquitous in humans and other mammals, and, although the molecular basis and function of these sites remain an enigma, it has been speculated that they may be a cytogenetic expression of gene activity. A population survey of 28 twin pairs was conducted to assess the heritability of common fragile-site expression. Our data yielded a heritability estimate of .88 for total site expression, suggesting that these sites may result from some common process that is under relatively stringent genetic control. An analysis of the expression of individual autosomal sites revealed that expression on both homologues in the same cell occurred more frequently than expected.
Topics: Adolescent; Aphidicolin; Child; Chromosome Aberrations; Chromosome Fragile Sites; Chromosome Fragility; Genetics, Population; Heterozygote; Homozygote; Humans; Metaphase; Twins
PubMed: 1729897
DOI: No ID Found -
Biochimie Feb 1992We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene. We confirmed that the purified...
We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene. We confirmed that the purified protein, of molecular weight 90,000, possesses a 3'----5' exonuclease activity in addition to DNA polymerizing activity in a single polypeptide. Its DNA polymerizing activity was sensitive to the drug aphidicoline, which is a specific and direct inhibitor of the alpha-like DNA polymerases including eukaryotic replicative DNA polymerases. Aphidicolin had no detectable effect on the 3'----5' exonuclease activity. The inhibition by aphidicolin on the polymerizing activity of polymerase II was competitive with respect to dNTP and uncompetitive with respect to template DNA. This mode of action is the same as that on eukaryotic DNA polymerase alpha. The apparent Ki value calculated from Lineweaver-Burk plots was 55.6 microM.
Topics: Aphidicolin; DNA; DNA Polymerase II; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Kinetics; Nucleotides; Recombinant Proteins
PubMed: 1581388
DOI: 10.1016/0300-9084(92)90036-e