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PubMed1998

Effects of aphidicolin on Entamoeba histolytica growth and DNA synthesis.

We have previously demonstrated that DNA polymerase activity of Entamoeba histolytica is inhibited by aphidicolin, which is a specific inhibitor of eukaryotic nuclear replicative DNA polymerases. The present study was aimed to evaluate the effect of aphidicolin on growth and DNA synthesis by this parasite. Aphidicolin blocked the growth of axenic E. histolytica strain HM-1:IMSS. DNA synthesis was also inhibited by aphidicolin when assayed by incorporation of [3H]thymidine into the DNA. The inhibitory effect of aphidicolin on the growth of E. histolytica was abrogated by removal of the drug, and exposure to 3 microg/ml of the drug for at least 48 hr had little effect on the viability. Synchronous growth was observed in the recovery phase after removal of aphidicolin.

Animals↗
PubMed1998

Aphidicolin induces alterations in Golgi complex and disorganization of microtubules of HeLa cells upon long-term administration.

Treatment of HeLa cells with aphidicolin at 5 or 0.5 microg/ml induced cell cycle arrest at G1/S or G2/M phase, respectively, and was accompanied by unbalanced cell growth. Long-term administration of aphidicolin (more than 48 h) resulted in noticeable loss of reproductive capacity though cells were viable at the time of treatment. Immunofluorescence with anti-Golgi membrane protein monoclonal antibody (mAbG3A5) showed disfigurement of the characteristic mesh-like configuration when cells were treated for more than 48 h. Interestingly, we found that the fragmented Golgi complex formed a ring around the nucleus in more than 20% of the cells. Immunoelectron microscopy using mAbG3A5 antibody demonstrated that the stack structure of the fragmented Golgi complex in aphidicolin-arrested cells appeared partially broken up and seemed to have converted to a vesicle-like structure. Analysis using an antibody to tubulin and anticentrosome human autoimmune serum showed that alterations in the Golgi complex were induced even by the lower 0.5 microg/ml dose. These alterations were accompanied by both changes in the distribution of microtubules and an increase in the number of centrosomes. These cells lost their distinct perinuclear microtubule organizing center (MTOC). On the other hand, treatment with aphidicolin at 5 microg/ml did not induce multiplication of the centrosome although the loss of distinct MTOC was still evident. No changes took place in the Golgi complex, microtubule, or centrosome of cells treated with 0.5 microg/ml aphidicolin when cycloheximide was added simultaneously to the culture.

Aphidicolin↗
PubMed1993

Mass spectrometric identification and analysis of some aphidicolin metabolites in cancer patients.

We used gas chromatography/mass spectrometry to identify and quantitate some aphidicolin metabolites in plasma and urine of patients receiving aphidicolin-17-glycinate. The major metabolite found in plasma was 3-ketoaphidicolin, present at about one thent the concentrations of aphidicolin. 3-Ketoaphidicolin undergoes dehydroxymethylation to give 18-nor-3-ketoaphidicolin. This metabolite was also found in plasma and its concentration reached a maximum of 1% of aphidicolin. Small amounts of aphidicolin and 3-ketoaphidicolin were found free in urine but almost all of the drug was conjugated to glucuronic acid, as shown by mass spectrometric analysis of urine extracts and by enzymatic digestion with beta-glucoronidase followed by gas chromatographic/mass spectrometric analysis.

Animals↗
PubMed1997

Microtubule dependence of chromosome cycles in Xenopus laevis blastomeres under the influence of a DNA synthesis inhibitor, aphidicolin.

The spindle-assembly checkpoint of the cell cycle develops in Xenopus laevis embryos at the midblastula transition (MBT). Our previous experiments using animal-cap blastomeres indicate that the checkpoint is regulated by a mechanism that depends on age, but not on the nucleocytoplasmic (N/C) ratio (Clute and Masui, 1995). In the present study, the time of appearance of the spindle-assembly checkpoint is examined in animal-cap blastomeres whose N/C ratio is reduced by treatment with aphidicolin. Animal-cap blastomeres treated with aphidicolin from the 2-cell stage cleave more slowly after 4th cleavage, in a dose-dependent manner, but cleavage and chromosome cycles continue up to the 11th to 13th cleavage and then arrest. Blastomeres treated with aphidicolin have a reduced DNA content and N/C ratio compared to control blastomeres of the same age. Nevertheless, nocodazole-sensitive chromosome cycles appear at the same time as in control blastomeres, at 3 to 5 hr after 5th cleavage, regardless of the N/C ratio. The arrest in interphase caused by treating blastula stage animals caps with aphidicolin can be reversed by treatment with caffeine. The caffeine-induced mitosis becomes sensitive to nocodazole after the MBT, but not before. Therefore, the same mechanism which stabilizes maturation-promoting factor activity in the absence of a mitotic spindle also operates after the MBT in blastomeres that are treated with aphidicolin, if mitosis is induced by caffeine. This mechanism may involve the translation of a maternal mRNA at the time of the MBT, as suggested previously.

Animals↗
PubMed1999

Aphidicolin markedly increases the in vitro sensitivity to ara-C of blast cells from patients with AML.

Drug resistant cells often have an increased capacity to repair their DNA after damage by cytotoxic agents. Aphidicolin can inhibit this DNA repair. We describe a study of the effect of aphidicolin to modulate the sensitivity to cytotoxic drugs of blast cells from 13 patients with AML, 11 with de novo disease on presentation and 2 secondary to MDS. Three patients had relapsed following previous therapy and samples were received from 1 patient both on presentation and relapse. Blast cells were exposed to anthracyclines, antimetabolites or etoposide +/- aphidicolin (15 microM) for 48 hours. The MTT assay was used to measure cell survival and the LC50 (concentration of drug required for 50% cell kill) was calculated. Overall, there was a significant increase in sensitivity to ara-C on co-incubation with aphidicolin in 12/14 samples (p = 0.007). The median increase in sensitivity was 3.88-fold (range 1.26- to 80-fold). Interestingly, when patients were grouped according to in vitro sensitivity to ara-C, cells from resistant patients demonstrated the greatest increase in sensitivity (median 14-fold compared to 2-fold for the sensitive group, p = 0.02). Despite the documented evidence for altered DNA repair as a mechanism of resistance to the topoisomerase II inhibitors, we found no significant increase in sensitivity to daunorubicin, doxorubicin or etoposide on co-incubation with aphidicolin. Nevertheless, we believe the unparalleled modulation of ara-C warrants further investigation.

Antibiotics, Antineoplastic↗
PubMed1987

Aphidicolin-resistant mutator strains of mouse teratocarcinoma.

From among a series of stable, aphidicolin-resistant mutant strains of mouse teratocarcinoma, derived from a multipotent parental line (PSA-1-80), three were selected for further study on the basis of their comparatively high degrees of resistance and elevated frequencies of spontaneous forward mutation to 6-thioguanine and ouabain resistance. Fluctuation tests confirmed that they were mutator strains. Since each of the three mutants was isolated after multiple rounds of selection, and since a variety of biochemical abnormalities were observed, it is likely that a number of mechanisms, probably consisting of overlapping subsets, determine the phenotypes. Abnormalities in the metabolism of the nucleotide substrates for polymerization are likely to be of major importance in mutants designated Aph-2 and Aph-3, as there were marked alterations in the dCTP and dATP pool sizes. The specific activity of DNA polymerase alpha was also increased. For the case of Aph-3, which exhibited the greatest (400-fold) increase in resistance to aphidicolin, a mutation in the structural gene for DNA polymerase alpha may be an additional important component, since in vitro assays revealed that the isolated enzyme was resistant to aphidicolin. For the case of Aph-1 however, only minor alterations in dNTP pools were observed, and there was no increase in the specific activity of DNA polymerase alpha or in the aphidicolin resistance of the isolated DNA polymerase alpha, suggesting yet another mechanism(s) underlying the aphidicolin resistance/mutator phenotype. All three mutants formed subcutaneous tumors in syngeneic mice; both Aph-1 and Aph-2 were multipotent; whereas Aph-3 was nullipotent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Differential effects of aphidicolin on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells.

Aphidicolin clearly discriminated replicative DNA synthesis from unscheduled DNA synthesis. Aphidicolin inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells. The mode of inhibition of aphidicolin was a mixed type with respect to deoxycytidine triphosphate but was non-competitive with respect to the other three deoxynucleoside triphosphates. Aphidicolin did not affect the activity of unscheduled DNA synthesis in either bleomycin-treated permeable sarcoma cells or isolated rat liver nuclei. Considering the difference in sensitivity of DNA polymerase alpha and beta to aphidicolin, and other related information reported previously, the results are compatible with the idea that DNA polymerase alpha is involved in replicative DNA synthesis and DNA polymerase beta in unscheduled DNA synthesis in the present systems.

Animals↗
PubMed1983

Effect of aphidicolin on the elongation step of adenovirus DNA replication in vitro.

Adenovirus DNA synthesis carried out in vitro was inhibited by the aphidicolin. However, 30% of the DNA synthesis was resistant to aphidicolin even at a concentration of 200 micrograms/ml. When the distribution patterns of the radioactivity of the products synthesized in the presence of 50 micrograms/ml of the drug was examined after HindIII digestion of the product DNA, the radioactivity appeared preferentially in the fragments mapping nearest to the ends of the molecule. Pulse-chase experiment showed that the terminal fragments were synthesized with or without aphidicolin but that in the presence of aphidicolin the rate of elongation rapidly slowed down beyond this region, suggesting that a DNA polymerase sensitive to aphidicolin may participate in the synthesis of the internal region of adenovirus DNA.

Adenoviridae↗
PubMed1992

Potentiation of etoposide cytotoxicity against a human ovarian cancer cell line by pretreatment with non-toxic concentrations of methotrexate or aphidicolin.

Exposure of human ovarian cancer SW626 cell line to 0.08 mumol/l methotrexate or 25 mumol/l aphidicolin for 24 h caused no cytotoxicity but enhanced etoposide cytotoxicity. Methotrexate or aphidicolin treatment induced a reversible blockade at the beginning of S phase which was reversed upon drug removal with a consequent wave of synchronisation. The enhancement of etoposide cytotoxicity was not due to higher etoposide intracellular uptake in the methotrexate or aphidicolin-pretreated cells. The topoisomerase II content in methotrexate or aphidicolin pretreated SW626 cells was higher than in control cells assessed by western blotting or flow cytometry. The higher etoposide cytotoxicity observed after synchronization with methotrexate or aphidicolin was apparently unrelated to the number of drug-induced DNA-topoisomerase II complexes evaluated as DNA double strand breaks or DNA-protein crosslinks. These data support the view that etoposide-induced DNA-topoisomerase II complexes are more cytotoxic in cells which are in S-phase.

Aphidicolin↗
PubMed1993

DNA polymerase epsilon: aphidicolin inhibition and the relationship between polymerase and exonuclease activity.

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.

Animals↗
PubMed1985

Kinetics of DNA replication in C3H 10T1/2 cells synchronized by aphidicolin.

Aphidicolin is an inhibitor of DNA polymerase alpha and blocks nuclear DNA replication without interfering with mitochondrial DNA synthesis. The efficacy of this mycotoxin as a tool in cell synchronization was evaluated in C3H 10T1/2 clone 8 cells. At concentrations of 1-2 micrograms/mL, aphidicolin quickly reduced the [3H]thymidine uptake to less than 5% of control levels in the first 5 min of incubation. This inhibition was easily reversed by washing and refeeding cells with fresh medium. The synchronization protocol consisted of first blocking cells by confluence arrest, replating them at lower density, and then treating the cells with aphidicolin for 24 h. Once the inhibitor was removed, DNA replication started without any delay. The cell population traversed the S phase in about 8 h and synchronously doubled in cell number. Autoradiography studies revealed a labeling index of 89-93% during the S phase. However, it was also observed that 10T1/2 cells were able to enter S phase in the presence of aphidicolin. The extent of the ensuing replication in the nucleus was dependent on the time that cells remained arrested in early S phase. Analyses of the newly replicated DNA in alkaline sucrose gradients revealed a fairly homogeneous distribution of sizes of nascent DNA in synchronized cells pulse-labeled at the beginning of the S phase. Upon chase in nonradioactive medium, the average molecular weight of the nascent DNA increased linearly with time of DNA synthesis for 2 h. The apparent rate of DNA chain growth determined from pulse and chase experiments was 1.2 micron/min. This rate was strongly inhibited (93%) by aphidicolin at a concentration of 2 micrograms/mL.

Animals↗
PubMed1982

Effects of aphidicolin on cell proliferation, repair of potentially lethal damage and repair of DNA strand breaks in Ehrlich ascites tumour cells exposed to X-rays.

The effects of aphidicolin, a specific inhibitor of DNA alpha-polymerase, have been studied on various cellular end-points and on DNA strand break repair. In the concentration range 0.02-2 micrograms/ml DNA synthesis was strongly inhibited resulting in a concomittant loss of cell proliferation ability; RNA and protein synthesis were unaffected in this range. At these concentrations PLD repair in X-irradiated plateau-phase cells was unaffected even after 7 hours treatment with aphidicolin; however, at higher concentrations (greater than 2 micrograms/ml) PLD repair was inhibited. We show that in the low concentration range (less than 2 micrograms/ml) PLD repair can be seen in exponentially growing cells and from experiments with synchronized cells we establish that the PLD repair observed can be attributed to the S-phase population, the survival of G1-cells not being affected by aphidicolin. The 'promotion' of PLD repair in exponentially growing cells was in excess of that observed for the same cells in balanced salt solution in which PLD repair is usually observed. At high concentrations (greater than 2 micrograms/ml) of aphidicolin, both X-irradiated and control S-cells were killed increasingly as concentration increased. The repair of DNA strand breaks (single and double) was unaffected in the low concentration range, but a strong inhibition was observed at high concentration. It is concluded from these results that alpha-polymerase, which is strongly inhibited at low concentrations of aphidicolin as evidenced by the inhibition of DNA synthesis, plays no major part in the repair of DNA strand breaks or in the repair of PLD.

Animals↗
PubMed1982

Specificity and completeness of inhibition of DNA repair by novobiocin and aphidicolin.

Novobiocin and aphidicolin were both potent inhibitors of excision repair of u.v.-induced damage to DNA in human embryonic fibroblasts, and both also inhibited semiconservative DNA replication even more strongly. The mechanism of action of these two drugs is, however, different. Novobiocin inhibited repair replication without accumulating single-strand breaks, but aphidicolin inhibited repair replication with the accumulation of numerous single-strand breaks. Novobiocin appears to inhibit repair at an earlier stage than aphidicolin, which may indicate that DNA topoisomerases play a role in eukaryotic DNA repair. Digestion of DNA by exonuclease III indicated that repair patches in novobiocin-treated cells contained no excess 3'OH termini, whereas up to 40% of the repaired DNA in aphidicolin-treated cells had free 3'OH termini. Therefore, although aphidicolin resulted in the accumulation of single-strand breaks, many of the repair events escaped inhibition and the number of breaks is an underestimate of the true number of repair events.

Antibiotics, Antineoplastic↗
PubMed1991

Phase I and clinical pharmacological evaluation of aphidicolin glycinate.

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.

Adult↗
PubMed1980

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.

Adenoviruses, Human↗
PubMed1981

Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation.

Aphidicolin↗
PubMed1984

Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine.

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Animals↗
PubMed1984

Characterization of a mutant of Toxoplasma gondii resistant to aphidicolin.

Aphidicolin, a mycotoxin that inhibits eucaryotic DNA polymerase alpha, blocked the growth of Toxoplasma gondii in confluent cultured human fibroblasts. Aphidicolin immediately inhibited DNA synthesis by T. gondii while it had a delayed and less dramatic effect on RNA synthesis. A mutant of T. gondii resistant to aphidicolin was isolated with the aid of mutagenesis by ethylnitrosourea. Parasite growth measured three days after drug treatment and parasite DNA synthesis measured immediately after drug treatment were, respectively, five- and four-fold more resistant to aphidicolin in the mutant as compared with the wild type parasite. The mutant had a three-fold greater capacity than the wild type to incorporate uracil into its deoxycytidine triphosphate pool. This increased deoxycytidine triphosphate pool is the probable explanation for the mutant's resistance because this deoxynucleotide is known, in mammalian cells, to reverse the inhibition of DNA synthesis by aphidicolin in a competitive manner.

Animals↗