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Developmental Biology May 1997The spindle-assembly checkpoint of the cell cycle develops in Xenopus laevis embryos at the midblastula transition (MBT). Our previous experiments using animal-cap...
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.
Topics: Animals; Aphidicolin; Blastomeres; Cell Cycle; Chromosomes; DNA; Microtubules; Nocodazole; Nucleic Acid Synthesis Inhibitors; Xenopus laevis
PubMed: 9169045
DOI: 10.1006/dbio.1997.8540 -
Chemical & Pharmaceutical Bulletin Apr 1987
Topics: Aphidicolin; Chemical Phenomena; Chemistry; DNA Polymerase II; Diterpenes; In Vitro Techniques; Molecular Conformation
PubMed: 3115608
DOI: 10.1248/cpb.35.1641 -
In Vivo (Athens, Greece) 1994We have purified and characterized Pseudorabies virus (PRV) DNA polymerase from infected TK- mouse cells. PRV DNA polymerase has a 3'- > 5' exonuclease activity; it is...
We have purified and characterized Pseudorabies virus (PRV) DNA polymerase from infected TK- mouse cells. PRV DNA polymerase has a 3'- > 5' exonuclease activity; it is stimulated by ionic strength, requires magnesium for optimal activity and it is more sensitive to aphidicolin than eukaryotic and HSV-1 replicative DNA polymerases. Aphidicolin inhibits in vitro PRV DNA polymerase competitively with respect to dCTP with a Ki of 0.06 microM and completely blocks viral growth in vivo at 4.4 microM. The high sensitivity to aphidicolin of animal herpesvirus DNA polymerases might allow a topical use of this drug in the treatment of animal herpesvirus keratitis and stomatitis.
Topics: Animals; Aphidicolin; Cell Line; Herpesvirus 1, Suid; Mice; Nucleic Acid Synthesis Inhibitors; Virus Replication
PubMed: 7772734
DOI: No ID Found -
Cancer Chemotherapy and Pharmacology 1992Aphidicolin, a reversible inhibitor of DNA polymerase alpha and delta, has recently been reported to reverse the resistance to cisplatin (DDP) of an ovarian cancer cell...
Aphidicolin, a reversible inhibitor of DNA polymerase alpha and delta, has recently been reported to reverse the resistance to cisplatin (DDP) of an ovarian cancer cell line. We investigated the pharmacokinetics of aphidicolin in mice and examined its activity either alone or in combination with DDP in the DDP-sensitive M5076 (M5) murine reticular cell sarcoma as well as in a DDP-resistant subline (M5/DDP). The drug was cleared from plasma very rapidly (clearance, 41.6 ml min-1 kg-1), showing a half-life of 15 min. Aphidicolin concentrations in the tumor were approximately 50% of those found in plasma at steady state. Using several dose schedules and continuous infusions we failed to detect significant antitumor activity for aphidicolin glycinate. Potentiation of the activity of DDP by aphidicolin glycinate was moderate in mice bearing M5 tumor as well as in those bearing M5/DDP tumor. These data do not support the possible clinical use of aphidicolin in combination with DDP. However, further studies should be carried out in different tumor models before this possibility is conclusively ruled out.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aphidicolin; Cell Line; Cisplatin; Drug Administration Schedule; Drug Resistance; Drug Synergism; Female; Half-Life; Infusions, Intravenous; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Ovarian Neoplasms
PubMed: 1394802
DOI: 10.1007/BF00685597 -
Oncology Research 1993We have compared the effects of a number of inhibitors including aphidicolin, 2,4-dinitrophenol (DNP) and novobiocin on the in vitro cytotoxicity of several... (Comparative Study)
Comparative Study
We have compared the effects of a number of inhibitors including aphidicolin, 2,4-dinitrophenol (DNP) and novobiocin on the in vitro cytotoxicity of several topoisomerase II (topo II)-directed agents, using cultured murine Lewis lung carcinoma cells. These agents comprised amsacrine, CI-921 (9-[(2-methoxy-4-methylsulfonylamino)phenylamino]-N,5-dimethyl-4- acridinecarboxamide isethionate, isethionate, a derivative of amsacrine), DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide dihydrochloride, a new DNA intercalator with high solid tumor activity), daunorubicin, doxorubicin, epirubicin, etoposide, mitoxantrone, and teniposide. Novobiocin, an antibiotic that affects topo II action, reduced the cytotoxic effect of DACA as well as that of amsacrine and doxorubicin, and reduced the extent of G2-phase arrest by DACA. DNP, an uncoupler of mitochondrial respiration, inhibited drug action in a manner similar to that of novobiocin but to a smaller extent. Aphidicolin, a specific inhibitor of DNA polymerase-alpha, reduced the cytotoxic effect of amsacrine, CI-921, etoposide, and teniposide but not that of DACA, daunorubicin, doxorubicin, epirubicin, or mitoxantrone. The immediate effect of each topo II-directed agent on the incorporation of thymidine into DNA was also measured at a drug concentration (D10) that killed 90% of cells. Susceptibility to aphidicolin reversal was strongly correlated with inhibition of thymidine incorporation (r = 0.91; p < or = 0.001). The results suggest that the involvement of DNA replication in the cytotoxic action of topo II-directed agents differs according to the agent used.
Topics: Acridines; Amsacrine; Animals; Antineoplastic Agents; Aphidicolin; Cell Survival; DNA Replication; Doxorubicin; Mice; Thymidine; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Uridine
PubMed: 8260750
DOI: No ID Found -
Biochimica Et Biophysica Acta Oct 1994The mammalian DNA polymerase inhibitors aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair of DNA damage induced by... (Comparative Study)
Comparative Study
Effect of DNA polymerase inhibitors on repair of gamma ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-beta-D-arabinofuranosylcytosine.
The mammalian DNA polymerase inhibitors aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair of DNA damage induced by gamma rays or 4-nitroquinoline 1-oxide in normal human fibroblasts to an extent 2- to 4-fold greater than that seen with each inhibitor alone. Thus either aphidicolin modulates the rate of intracellular accumulation of araC 5'-triphosphate (araCTP), the presumed rate-limiting step in the genotoxic action of araC, or aphidicolin and araC inhibit repair by different mechanisms. To explore these possibilities, we compared the effects of aphidicolin, araC, araCTP, and 2',3'-dideoxythymidine triphosphate (ddTTP) on repair of DNA damage induced by 60Co gamma radiation in intact versus permeable human fibroblasts. Both aphidicolin and araC strongly inhibited repair in permeable cells, as indicated by the accumulation of DNA strand breaks in irradiated cultures that were subsequently treated with saponin (25 micrograms/ml; 10 min) and incubated for 2 h with either chemical. The extent of repair inhibition by each drug was comparable in intact and permeable cells, amounting to approximately 1.1 sites/10(8) daltons/2 h upon exposure to 150 Gy. The active metabolite of araC, araCTP, did not inhibit repair in intact cells, but did so in permeable cells to an extent within the range of that seen with araC or aphidicolin alone. The incidence of DNA strand breaks accumulating in gamma-irradiated permeable cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was approximately 2-fold greater than that arising in parallel cultures which had been incubated with optimal concentrations of each of the three drugs alone. Although the resolution of our assays compelled us to monitor repair events in moribund cell populations, we have reason to be confident that within the short post-irradiation period considered here, the observed drug-accumulated breaks truly represent functional repair inhibition and not merely abortive pathological responses. We thus conclude that (1) the accumulation of araCTP in intact cells is not limiting the ability of araC to inhibit DNA repair; and (2) the mode of the inhibitory action of araC/araCTP on gamma ray repair is different from that of aphidicolin. In contrast to the observations with these chemicals, ddTTP (20 microM), a potent inhibitor of DNA polymerase beta, did not produce any measurable effect on DNA repair in gamma-irradiated permeable fibroblasts, nor did it enhance the efficacy of araC, araCTP or aphidicolin to inhibit repair. These results strongly suggest that DNA polymerase beta plays no significant role in the repair of gamma radioproducts in human fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Aphidicolin; Arabinofuranosylcytosine Triphosphate; Cells, Cultured; Child; Cytarabine; DNA Repair; Drug Combinations; Fibroblasts; Humans; Nucleic Acid Synthesis Inhibitors; Saponins
PubMed: 7918688
DOI: 10.1016/0925-4439(94)90112-0 -
Proceedings of the National Academy of... Apr 1996To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline...
To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.
Topics: Aphidicolin; Apoptosis; Cell Cycle; Gene Amplification; Gene Expression; HeLa Cells; Humans; Models, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tetracycline; Tetrahydrofolate Dehydrogenase; Trimetrexate
PubMed: 8622946
DOI: 10.1073/pnas.93.8.3394 -
Methods in Molecular Biology (Clifton,... 2017Protozoans are single-celled eukaryotes and many of the best-studied protozoans are parasitic to humans (e.g., Plasmodium falciparum causing malaria and Trypanosoma...
Protozoans are single-celled eukaryotes and many of the best-studied protozoans are parasitic to humans (e.g., Plasmodium falciparum causing malaria and Trypanosoma brucei causing sleeping sickness). These organisms are distantly related to humans but with retained eukaryotic type of cellular processes, making them good model systems for studies of the evolution of basic processes like the cell cycle. Giardia intestinalis causes 250 million cases of diarrhea yearly and is one of the earliest diverging protozoans. It is possible to synchronize its cell cycle using compounds that inhibit different steps of the cell cycle and the detailed protocol is described here.
Topics: Aphidicolin; Cell Cycle; Flow Cytometry; Giardia lamblia; Plasmodium falciparum; Trypanosoma brucei brucei
PubMed: 27815907
DOI: 10.1007/978-1-4939-6603-5_15 -
BioEssays : News and Reviews in... Oct 1992It has been almost twenty-five years since Huberman and Riggs first showed that there are multiple bidirectional origins of replication scattered at approximately 100 kb... (Review)
Review
It has been almost twenty-five years since Huberman and Riggs first showed that there are multiple bidirectional origins of replication scattered at approximately 100 kb intervals along mammalian chromosomal fibers. Since that time, every conceivable physical property unique to replicating DNA has been taken advantage of to determine whether origins of replication are defined sequence elements, as they are in microorganisms. The most thoroughly studied mammalian locus to date is the dihydrofolate reductase domain of Chinese hamster cells, which will be used as a model to discuss the various methods of investigation. While several laboratories agree on the rough location of the 'initiation locus' in this large chromosomal domain, different experimental approaches paint different pictures of the mechanism by which initiation occurs. However, a variety of new techniques and synchronizing agents promises to clarify the picture for this particular locus, and to provide the means for identifying and isolating other origins of replication for comparison.
Topics: Animals; Aphidicolin; CHO Cells; Chromosomes; Cricetinae; Cricetulus; DNA Replication; DNA-Directed DNA Polymerase; Mammals; Mimosine; Models, Genetic; Nucleic Acid Synthesis Inhibitors; Replication Origin; Replicon; Tetrahydrofolate Dehydrogenase
PubMed: 1365877
DOI: 10.1002/bies.950141002 -
Journal of Bioscience and Bioengineering Mar 2018Chlorella viruses or chloroviruses contain a gene that encodes an enzyme that catalyzes chitin synthesis. This gene is expressed early in viral infections to produce...
Chlorella viruses or chloroviruses contain a gene that encodes an enzyme that catalyzes chitin synthesis. This gene is expressed early in viral infections to produce chitin on the outside of the Chlorella cell wall. Interestingly, chitin synthesis by microalgal Chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and CO into useful materials. However, during the final viral infection stage, the host cells are completely lysed, so chitin should be harvested before cells lyse. To increase chitin yields, slow-growing chlorovirus isolates were adopted and the viral replication process was modified with an inhibitor of DNA synthesis. The accumulation of chitin on the surface of Chlorella cells infected with one of nine chlorovirus isolates carrying the chitin synthase gene was compared with that of CVK2 (a standard virus)-infected cells. Chlorella cells infected with CVNF-1 (a slow-growing virus) accumulated chitin over the entire cell surface within 15 min post-infection (p.i.), and chitin continued to accumulate for up to 8 h p.i. before cells lysed. This was 2-fold longer than the chitin-accumulation period for cells infected with CVK2. The addition of aphidicolin delayed the progression of the virus replication cycle and extended the chitin-accumulation period of CVNF-1-infected cells to 12 h p.i. before cells lysed. Additionally, chitin production in the aphidicolin-treated CVNF-1-infected cells was approximately 6-fold higher than in CVK2-infected cells not treated with aphidicolin. Thus, chitin synthesis in a Chlorella-virus system may be prolonged by using slow-growing viral isolates treated with aphidicolin.
Topics: Aphidicolin; Cell Membrane; Cell Wall; Chitin; Chlorella; Phycodnaviridae; Virus Replication
PubMed: 29100685
DOI: 10.1016/j.jbiosc.2017.10.002