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Current Opinion in Genetics &... Jun 2012Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. De novo CNVs are... (Review)
Review
Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. De novo CNVs are also a major cause of numerous genetic and developmental disorders. However, unlike many other types of mutations, little is known about the genetic and environmental risk factors for new and deleterious CNVs. DNA replication errors have been implicated in the generation of a major class of CNVs, the nonrecurrent CNVs. We have found that agents that perturb normal replication and create conditions of replication stress, including hydroxyurea and aphidicolin, are potent inducers of nonrecurrent CNVs in cultured human cells. These findings have broad implications for identifying CNV risk factors and for hydroxyurea-related therapies in humans.
Topics: Aphidicolin; Cells, Cultured; Chromosome Breakpoints; Chromosomes, Human; DNA Copy Number Variations; DNA Replication; Environment; Genes, cdc; Genetic Variation; Genome, Human; Humans; Hydroxyurea; Risk Factors; Stress, Physiological
PubMed: 22365495
DOI: 10.1016/j.gde.2012.01.009 -
Mutation Research Feb 2004The induction of chromosome damage in cultured human lymphocytes by in vitro treatments with aphidicolin (APC) and bleomycin (BLM) has been proposed as test of...
The induction of chromosome damage in cultured human lymphocytes by in vitro treatments with aphidicolin (APC) and bleomycin (BLM) has been proposed as test of sensitivity to mutagens. To assess their validity, we have investigated whether the individual expression of induced chromosome damage has a genetic rather than an environmental basis. Metaphase analysis for chromosomal aberrations (CA) and micronucleus (MN) assay in cytokinesis-blocked cells have been performed in peripheral blood lymphocytes from 19 healthy male twins (9 monozygotic and 10 dizygotic pairs), aged 70-78 years, after APC, BLM and APC+BLM treatments. Concordance between twins revealed a high genetic component in the sensitivity towards clastogenic action of APC both as percentages of CA and MN. The micronucleus assay demonstrated a genetic basis also in the expression of chromosome damage induced by BLM and APC+BLM treatments. Since twins were elderly people, to investigate the possible role of age, CA and MN frequencies were compared with those found in lymphocytes from 11 young male donors. Basal and APC-induced chromosome damage were clearly increased in the former. Following BLM and APC+BLM treatments, age significantly increased mitotic delay, as shown by the mitotic indexes (MI) and by the ratios between binucleated and mononucleated (B/M) cells.
Topics: Aged; Aphidicolin; Biomarkers; Bleomycin; Chromosome Aberrations; Humans; Male; Mutagens
PubMed: 14757193
DOI: 10.1016/j.mrfmmm.2003.10.006 -
Arzneimittel-Forschung 2002Twenty derivatives of aphidicolin were tested against HSV (herpes simplex virus), HCMV (human cytomegalovirus) and adenovirus in vitro. In addition, the antiviral...
Twenty derivatives of aphidicolin were tested against HSV (herpes simplex virus), HCMV (human cytomegalovirus) and adenovirus in vitro. In addition, the antiviral activity of aphidicolin (CAS 38966-21-1) in combination with aciclovir (CAS 59277-89-3) or cidofovir (CAS 113852-37-2) against HSV was determined. The antiviral effects were evaluated using plaque reduction assay in Vero cells or human Foreskin Fibroblasts (HFF) for HSV and HCMV, respectively. Combination indexes were calculated using the method of Chou and Talalay. Two derivatives (K14254 and K14266) that are considered to be prodrugs of aphidicolin were shown to inhibit HCMV and HSV replication comparably to aphidicolin. None of the tested substances inhibited adenovirus replication. Aphidicolin acted synergistically with aciclovir in a 1:1 molar ratio and with cidofovir in different ratios. Aphidicolin and its two antiviral active derivatives might represent useful additional tools for antiviral therapy of HSV and HCMV infections, especially in combination with clinically used drugs.
Topics: Adenoviridae; Antiviral Agents; Aphidicolin; Cidofovir; Cytomegalovirus; Cytosine; Dose-Response Relationship, Drug; Drug Synergism; Herpesvirus 1, Human; Humans; Indicators and Reagents; Organophosphonates; Organophosphorus Compounds; Virus Replication
PubMed: 12087926
DOI: 10.1055/s-0031-1299904 -
Journal of the National Cancer Institute Aug 1991The toxicity profile and the pharmacokinetics of aphidicolin glycinate, a water-soluble analogue of aphidicolin, have been evaluated in two consecutive phase I clinical... (Clinical Trial)
Clinical Trial
The toxicity profile and the pharmacokinetics of aphidicolin glycinate, a water-soluble analogue of aphidicolin, have been evaluated in two consecutive phase I clinical studies. In the first study, aphidicolin glycinate was given by 1-hour infusion for 5 consecutive days, every 3 weeks (daily x 5 study); in the second study, which was planned on the basis of the pharmacokinetic information obtained in the previous study, the drug was given by 24-hour continuous infusion. Treatment was repeated every 3 weeks. In the daily x 5 study, the daily dose was escalated from 12 mg/m2 to the maximum tolerated dose of 2250 mg/m2. Local toxicity was dose limiting. Elimination half-life was 2 +/- 0.2 hours (mean +/- SE) with aphidicolin being undetectable 6-8 hours after the end of the infusion. In the 24-hour continuous-infusion study, the dose was escalated from 435 mg/m2 to the maximum tolerated dose of 4500 mg/m2. Local toxicity was dose limiting, while other toxic effects were absent. The experimentally determined concentrations at the steady state were in agreement with those predicted on the basis of the available pharmacokinetic data. The targeted concentration at the steady state of 3 micrograms/mL was achieved at doses greater than or equal to 3000 mg/m2. Twenty-four-hour continuous infusion is the recommended schedule for clinical evaluations of aphidicolin glycinate as the synchronizing agent or in combination with cisplatin.
Topics: Adult; Aged; Antineoplastic Agents; Aphidicolin; DNA Polymerase II; Diterpenes; Drug Administration Schedule; Drug Evaluation; Female; Half-Life; Humans; Infusions, Intravenous; Male; Middle Aged
PubMed: 1886148
DOI: 10.1093/jnci/83.16.1160 -
Anti-cancer Drugs Jul 2000Disseminated neuroblastoma diseases are still indicated by a poor outcome despite treatment regimens including radiation therapy and high-dose chemotherapy with stem...
Disseminated neuroblastoma diseases are still indicated by a poor outcome despite treatment regimens including radiation therapy and high-dose chemotherapy with stem cell rescue. Therefore, new substances and treatment regimens are of interest. Aphidicolin (APH), a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, has a specific toxicity for neuroblastoma cells. Furthermore, it was shown to enhance the effects of X-ray radiation and chemotherapy on malignant cells. To find new substances, 20 APH derivatives were tested for their anti-neuroblastoma efficacy in vitro in UKF-NB-2 cells. Five derivatives had antitumoral activity in neuroblastoma cells. A relationship between the structure and the antitumoral efficacy showed that the hydroxyl groups at C-3 and C-18 are essential for the antitumoral effects. Furthermore, antitumoral effects of APH in combination with doxorubicin and vincristine, both part of commonly used treatment regimens for disseminated neuroblastoma diseases, were tested in the neuroblastoma cell line UKF-NB-2. APH was found to act synergistically with vincristine and synergistically to additive with doxorubicin depending on the molecular ratio of the substances in combination. This may offer the chance to use APH and its derivatives as additional tools in the treatment of neuroblastomas.
Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Aphidicolin; Cell Survival; Doxorubicin; Drug Synergism; Enzyme Inhibitors; Humans; Molecular Structure; Neuroblastoma; Nucleic Acid Synthesis Inhibitors; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vincristine
PubMed: 11001389
DOI: 10.1097/00001813-200007000-00009 -
Virology Jul 1986Aphidicolin-resistant mutants (Aphr) of Bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. Efficiency of plating (e.o.p.) of the...
Aphidicolin-resistant mutants (Aphr) of Bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. Efficiency of plating (e.o.p.) of the resistant mutants was not reduced at 500 microM aphidicolin, although e.o.p. of wild type phi 29 was less than 10(-5) at the same concentration of aphidicolin. By recombination and complementation analyses, both sites of the mutations, aph-71 and aph-101, of Aphr71 and Aphr101, respectively, were mapped in gene 2 which encodes phi 29 DNA polymerase. The activity of wild type phi 29 DNA polymerase, in a partially purified fraction, was inhibited by aphidicolin. DNA polymerases from Aphr71 and Aphr101, prepared in the same manner as that of wild type, were resistant to the drug. These results indicate that the acquisition of the aphidicolin resistance of Aphr71 and Aphr101 of bacteriophage phi 29 results from a structural alteration of phi 29 DNA polymerase which reduces sensitivity to aphidicolin.
Topics: Aphidicolin; Bacteriophages; Chromosome Mapping; DNA Replication; DNA-Directed DNA Polymerase; Diterpenes; Drug Resistance, Microbial; Genetic Complementation Test; Mutation; Nucleic Acid Synthesis Inhibitors; Recombination, Genetic
PubMed: 3087058
DOI: 10.1016/0042-6822(86)90368-5 -
International Journal of Radiation... Feb 1988Mouse L cells were synchronized in early S-phase by two 12 h incubations in medium containing aphidicolin (2 micrograms/ml), separated by 8 h in drug-free medium. The...
Mouse L cells were synchronized in early S-phase by two 12 h incubations in medium containing aphidicolin (2 micrograms/ml), separated by 8 h in drug-free medium. The relationship between X-ray-induced cell killing and DNA double-strand breakage was then examined for cells that had entered S-phase, G2-phase, mitosis, and G1-phase following release from aphidicolin and was compared to the response of asynchronous cultures. Aphidicolin-synchronized cells showed cycle phase-dependent changes in their dose-responses for both killing and DNA dsb. However, on the basis of the level of DNA dsb per unit length of DNA required to produce a lethal lesion, aphidicolin-synchronized cells were more sensitive to X-rays than were asynchronous cultures. This sensitivity peaked 2 h after release from aphidicolin treatment and then progressively declined towards the asynchronous culture value. It is argued that these results are due to deregulation of the temporal order of DNA replication following aphidicolin treatment, and can be incorporated into the critical DNA target size model (Radford, Hodgson, and Matthews, in preparation) by postulating that the targets for radiation action in mammalian cells are DNA-associated with potentially transcriptionally active proto-oncogenes or constitutive fragile sites.
Topics: Animals; Aphidicolin; Cell Cycle; Cell Division; Cell Survival; DNA; DNA Damage; Diterpenes; L Cells; Mice
PubMed: 3126159
DOI: 10.1080/09553008814550571 -
Virology Nov 1992African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by phosphonoacetic acid and cytosine arabinoside. In...
African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by phosphonoacetic acid and cytosine arabinoside. In contrast to all other alpha-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 microM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo, phosphonoacetic acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to phosphonoacetic acid showed the same resistance to aphidicolin as wild type virus.
Topics: African Swine Fever Virus; Aphidicolin; Cytarabine; DNA, Viral; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Kinetics; Phosphonoacetic Acid
PubMed: 1413523
DOI: 10.1016/0042-6822(92)90219-f -
DNA Repair May 2019Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the...
Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the comet assay, is widely used as biomarker to assess the antioxidant status in human studies. Here, the alkaline comet assay was now optimized for easy and time saving detection of repair capacity upon oxidative stress-induced DNA damage using the DNA polymerase inhibitor aphidicolin (APC) to block repair of hydrogen peroxide (HO) induced DNA damage. Addition of a DMSO-containing DNA damage stop solution was found suitable to replace washing steps for HO removal before APC block. Cell treatment with APC at 6 μM did not impact baseline DNA damage but could reliably block DNA repair after HO challenge in both fresh and cryopreserved samples thus omitting the use of a starting time point control. Under the conditions used, frozen cells, with or without an additional 4 h rest, showed the same repair capacity as their fresh counterpart. The intra assay coefficient of variation (CV) was 3.3%. To provide proof of principle, the modified assay was applied to cryopreserved PBMC from 19 participants of a short-term Brassica diet intervention study investigating potential health promoting effects of the food intervention. Then, a 33% increase in DRC (p ≤ 0.01) could be shown in samples after intervention (mean ± SD: 5.82 ± 1) as compared to baseline (mean ± SD: 4.38 ± 1.21). Individual samples from baseline and intervention showed an inter-individual CV of 27.65% (baseline) and 17.26% (intervention). Taken together this modified comet assay protocol allows the facilitated detection of DNA repair in fresh or cryopreserved human PBMC samples with a good sensitivity and reliability and could be useful in human studies addressing the antioxidant status and repair capacity of PBMC.
Topics: Adult; Aphidicolin; Comet Assay; Cryopreservation; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase; Dose-Response Relationship, Drug; Female; Healthy Volunteers; Humans; Hydrogen Peroxide; Leukocytes, Mononuclear; Male; Nucleic Acid Synthesis Inhibitors; Oxidative Stress; Time Factors; Young Adult
PubMed: 30889507
DOI: 10.1016/j.dnarep.2019.03.005 -
Journal of Medical Genetics May 2007Fragile sites are specific genomic loci that form gaps, constrictions and breaks on chromosomes exposed to replication stress conditions. In the father of a patient with...
Fragile sites are specific genomic loci that form gaps, constrictions and breaks on chromosomes exposed to replication stress conditions. In the father of a patient with Beckwith-Wiedemann syndrome and a pure truncation of 18q22-qter, a new aphidicolin-sensitive fragile site on chromosome 18q22.2 (FRA18C) is described. The region in 18q22 appears highly enriched in flexibility islands previously found to be the characteristic of common fragile site regions. The breakpoint was cloned in this patient. The break disrupts the DOK6 gene and was immediately followed by a repetitive telomere motif, (TTAGGG)(n). Using fluorescent in situ hybridisation, the breakpoint in the daughter was found to coincide with the fragile site in the father. The breakpoint region was highly enriched in AT-rich sequences. It is the first report of an aphidicolin-sensitive fragile site that coincides with an in vivo chromosome truncation in the progeny.
Topics: Aphidicolin; Base Sequence; Child; Chromosome Breakage; Chromosome Deletion; Chromosome Fragile Sites; Chromosomes, Human, Pair 18; Cloning, Molecular; DNA Mutational Analysis; Fathers; Female; Humans; Molecular Sequence Data; Pedigree; Repetitive Sequences, Nucleic Acid
PubMed: 17475918
DOI: 10.1136/jmg.2006.044628