aphidicolin and 2--3--dideoxythymidine

Ultraviolet radiation is known to induce skin cancer. The induction of DNA damage caused by UV-B and UV-C was investigated using cultured L-132 cells. DNA strand breaks assayed by the alkaline elution procedure occurred in a dose-dependent manner, the extent of the strand breaks were inversely well correlated with the number of viable L-132 cells after 24 h incubation. About a 10-fold dose of UV-B irradiation was required to induce a similar degree of strand breaking to that induced by UV-C. Similarly about a 10-fold dose of UV-B was required to produce a similar amount of pyrimidine dimers, such as cyclobutane-type dimers and pyrimidine-(6-4)-pyrimidone photoproducts, which were determined by ELISA using the specific monoclonal antibody, to that produced by UV-C. Strand breaks induced by UV-B, however, were not fully repaired in viable cells remaining after incubation of cells for a longer period of time, although UV-C-induced strand breaks were repaired in a time-dependent manner. Furthermore, an experiment with a cell-free system, where the induction of strand breaks by repair enzymes did not take place, indicated that UV-B caused significantly more direct DNA strand breaks than that caused by one-tenth the dose of UV-C. The data shown here suggest that UV-B-induced DNA damage is mediated, at least in part, via a different mechanism from the UV-C induced one.

Topics: Antibodies, Monoclonal; Aphidicolin; Cell Line; Dideoxynucleosides; DNA; DNA Damage; DNA Repair; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Lung; Photolysis; Pyrimidine Dimers; Ultraviolet Rays

To test whether DNA injury contributes to TNF-induced cytotoxicity, we attempted to enhance DNA injury by inhibiting its repair and then assessing effects on cytotoxicity. DNA repair, assayed as unscheduled DNA synthesis, was first detected in TNF-sensitive targets by 2-3 h of incubation with TNF. Targets resistant to TNF cytotoxicity did not demonstrate significant repair replication. Repair preceded the detection of TNF-induced DNA injury, which was subsequently demonstrated by a double-stranded DNA fragmentation assay, sedimentation of DNA in neutral and alkaline sucrose gradients, and gel electrophoresis of extracted DNA. This suggested that early during exposure to TNF, DNA repair proceeds more rapidly than strand breakage. To inhibit repair, nontoxic concentrations of aphidicolin (inhibitor of DNA polymerase-alpha) and dideoxythymidine (inhibitor of DNA polymerase-beta and gamma) were used. Aphidicolin inhibited repair and consistently sensitized to TNF cytotoxicity, decreasing the ID50 for TNF at least 10- to 50-fold. In contrast, dideoxythymidine had no effect on repair or cytotoxicity. Deoxycytidine, which competitively inhibits binding of aphidicolin to DNA polymerase, blocked the sensitization in a concentration-dependent fashion. In targets sensitized with aphidicolin, TNF-induced strand breakage was accelerated, being detected by 4 h of culture in the sucrose gradient assay. Sensitization to TNF was not due to a heightened activation of poly (ADP-ribose) polymerase. These results indicate that TNF-induced strand breakage participates in TNF-induced cytotoxicity and that the level of DNA repair plays a role in determining relative sensitivity of targets.

Topics: Aphidicolin; Cell Survival; Dideoxynucleosides; DNA; DNA Damage; DNA Repair; DNA Replication; Female; Humans; Poly(ADP-ribose) Polymerases; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

DNA synthesis in epimastigote forms of Trypanosoma cruzi was studied autoradiographically by incorporation of [3H]thymidine in the nuclear and kinetoplast DNA. Both DNA were heavily labelled. Three eukaryotic DNA polymerase inhibitors (aphidicolin, aracytidine, and dideoxythimidine) were chosen to study the nuclear and kinetoplast DNA synthesis in vivo. Inhibition was mainly observed with the nuclear DNA.

Topics: Animals; Aphidicolin; Autoradiography; Cytarabine; Dideoxynucleosides; DNA, Circular; DNA, Kinetoplast; DNA, Protozoan; Nucleic Acid Synthesis Inhibitors; Trypanosoma cruzi

DNA repair synthesis induced by methyl methanesulfonate in preconditioned HeLa cells in which DNA replicative synthesis had been highly suppressed was inhibited by aphidicolin (an inhibitor of DNA polymerases alpha and delta) and dideoxythymidine (ddThR, an inhibitor of DNA polymerase beta). Incomplete repair patches sensitive to exonuclease III were accumulated in the presence of aphidicolin while not in the presence of ddThR. These patches were comopleted by the combined action of Klenow fragment and T4 DNA ligase, indicating that the single-stranded gaps were formed during the repair synthesis. Moreover, ddThR had little effect on the repair synthesis in the presence of aphidicolin. Thus, the results suggest that the single-stranded gaps may be sealed first by aphidicolin-sensitive polymerase followed by ddThR-sensitive DNA polymerase on the same site of the repair patch.

Topics: Aphidicolin; Cells, Cultured; Dideoxynucleosides; Diterpenes; DNA Repair; DNA-Directed DNA Polymerase; DNA, Single-Stranded; HeLa Cells; Humans; Methyl Methanesulfonate; Thymidine

We examined the effect of specific inhibitors of DNA polymerases alpha and delta, and beta, on cisplatin (DDP) cytotoxicity in DDP-sensitive and -resistant human 2008 ovarian carcinoma cells. Under conditions of continuous exposure to drug combinations, neither aphidicolin glycinate (AG) nor dideoxythymidine enhanced the cytotoxicity of DDP in either cell line as determined by clonogenic survival assays. However, when clonogenic survival was determined following short-term drug exposure, AG exhibited strong synergism with DDP in the DDP-resistant, but not the DDP-sensitive cells, as indicated by median effect analysis of the data. DNA polymerase alpha mRNA levels were the same in both cell lines under basal conditions. DDP-sensitive cells, but not DDP-resistant cells, were able to increase their expression of DNA polymerase alpha in response to DDP exposure. Levels of mRNA for DNA polymerase beta and for the human DNA repair gene ERCC-1 were not elevated in resistant cells, either under basal conditions or 18 hr after a 1 hr exposure to IC20 concentrations of DDP. In another human ovarian carcinoma cell line, A2780, AG and DDP were synergistic in both DDP-sensitive and -resistant variants in short-term exposure. We conclude that DNA polymerases alpha and/or delta play a role in the DDP sensitivity of human ovarian carcinoma cells.

Topics: Antibiotics, Antineoplastic; Aphidicolin; Blotting, Northern; Cell Line; Cell Survival; Cisplatin; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; Female; Humans; Ovarian Neoplasms; Plasmids; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

HeLa cells treated with 10 J/m2 of ultraviolet (UV) radiation were examined for inhibition of DNA excision by inhibitors of DNA polymerase-alpha and -beta. DNA repair synthesis and excision were inhibited by aphidicolin, a specific inhibitor of DNA polymerase-alpha. Decreased release of radioactive nucleotides from UV-damaged DNA by the inhibitor indicates that the action of DNA polymerase-alpha activity is closely associated with DNA excision. Dideoxythymidine also inhibited DNA repair synthesis but failed to block the excision, suggesting that polymerization by DNA polymerase-beta might precede removal of damaged DNA.

Topics: Antibiotics, Antineoplastic; Aphidicolin; Cells, Cultured; Dideoxynucleosides; Diterpenes; DNA Polymerase II; DNA Repair; HeLa Cells; Humans; Ultraviolet Rays

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.

Topics: 2-Acetylaminofluorene; Animals; Aphidicolin; Cells, Cultured; Cytarabine; Dideoxynucleosides; Diterpenes; DNA Damage; DNA Repair; Hydroxyurea; In Vitro Techniques; Liver; Male; Nalidixic Acid; Nucleic Acid Synthesis Inhibitors; Rats; Topoisomerase I Inhibitors

When chick pineal glands were cultured in the dark with aphidicolin from midphotoperiod, the increase of serotonin N-acetyltransferase (NAT) activity was stimulated and the time of peak NAT activity was advanced. The peak level of NAT activity was also reached sooner on the 2nd day of culture. The increase of NAT activity was also stimulated in glands cultured under diurnal illumination, but the time of peak activity was not advanced. Effects with glands explanted into culture in the dark at other times were smaller and the time of peak NAT activity was not changed. Cytosine arabinoside and dideoxythymidine also stimulated the increase of NAT activity and advanced the time of peak activity with glands cultured in the dark from midphotoperiod. 3-Aminobenzamide markedly stimulated the increase of NAT activity both in the dark and under diurnal lighting when pineal glands were explanted into culture at mid- or late photoperiod. In contrast, with glands in culture from earlier in the photoperiod, aminobenzamide had no effect upon the increase of NAT activity up to the peak level found with control glands. Thereafter results were variable. Effects of cordycepin upon development of NAT activity were similar to those of 3-aminobenzamide but less marked. Incorporation of thymidine into acid-insoluble material in the dark was very markedly inhibited by aphidicolin, cytosine arabinoside, and dideoxythymidine, but only slightly by cordycepin. Aminobenzamide strongly inhibited incorporation by glands cultured from midphotoperiod, but had little effect with glands in culture from near the end of the photoperiod. We adopt the working hypothesis that excision repair of DNA may be a major component in the mechanism of the chick pineal clock.

Topics: Acetyltransferases; Animals; Aphidicolin; Arylamine N-Acetyltransferase; Benzamides; Cells, Cultured; Chickens; Circadian Rhythm; Cytarabine; Darkness; Deoxyadenosines; Dideoxynucleosides; Diterpenes; DNA Repair; DNA Replication; Kinetics; Light; Pineal Gland; Thymidine

The alkaline sucrose density gradient centrifugation method was modified to permit detection of 1 lesion/10(9) daltons of DNA. With this technique, the involvements of DNA polymerases in DNA repair of damage by dimethyl sulfate, UV irradiation, neocarzinostatin, and bleomycin were studied in HeLa cells with the aid of the DNA polymerase inhibitors aphidicolin and 2',3'-dideoxythymidine. DNA repair after UV-induced damage seemed to involve only polymerase alpha, while repair of damage by the other three agents involved both polymerase alpha and a non-alpha polymerase, probably polymerase beta. But repair after damage by dimethyl sulfate differed from that after damage by neocarzinostatin or bleomycin with respect to the co-operations of polymerase alpha and polymerase beta: in repair of dimethyl sulfate-induced damage, both polymerases operated on the same lesions, whereas after damage by neocarzinostatin or bleomycin, polymerase alpha and polymerase beta functioned independently on different lesions.

Topics: Aphidicolin; Bleomycin; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Repair; HeLa Cells; Humans; Hydroxyurea; Kinetics; Sulfuric Acid Esters; Thymidine; Ultraviolet Rays; Zinostatin

Full-grown blastocysts were cultured for 70-92 h in Dulbecco's modified Eagle's medium supplemented with fetal calf serum. They were treated with (1) aphidicolin, a specific inhibitor of eucaryotic DNA polymerase alpha, (2) dideoxythymidine (d2Thd), the precursor of dideoxythymidine triphosphate (d2TTP), a specific inhibitor of DNA polymerase beta and (3) d2TTP itself. Cytophotometric measurements of both the DNA content and the nuclear areas were performed. The results show firstly that trophectoderm differentiation into primary giant trophoblast cells is already irreversibly programmed at the blastocyst stage and does not depend on any DNA replication cycle from that stage onwards. Secondly, the typical enlargement of the trophoblastic nuclei, which occurs at the peri-implantation period, is not related to a proportional increase in DNA content. Thirdly, the onset and progress of DNA endoreduplication in trophoblastic cells is carried out by DNA polymerase alpha during the first part of gestation instead of DNA polymerase beta, as has previously been shown to be the case for mid-gestation rat trophoblast cells.

Topics: Animals; Aphidicolin; Blastocyst; Cell Differentiation; Cells, Cultured; Dideoxynucleosides; Dideoxynucleotides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Replication; Female; Gonadotropins; Male; Mice; Thymidine; Thymine Nucleotides; Trophoblasts

We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.

Topics: Aphidicolin; Dideoxynucleosides; Diterpenes; DNA; DNA Polymerase II; DNA Repair; Fibroblasts; Humans; Skin; Thymidine; Ultraviolet Rays

The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain.

Topics: Animals; Aphidicolin; Cells, Cultured; Dideoxynucleosides; Diterpenes; DNA; DNA Polymerase I; DNA Polymerase II; DNA Repair; Hydroxyurea; Mice; Mice, Inbred C57BL; Neurons; Thymidine; Ultraviolet Rays

Excision repair of u.v. damage in human fibroblasts is more sensitive to inhibitors of DNA polymerase alpha (cytosine arabinoside, aphidicolin) than to an inhibitor of polymerase beta (dideoxythymidine), which indicates a greater role in repair for polymerase alpha than for polymerase beta. These inhibitors all generate shortened patches with free 3' termini; the detailed structure of these patches was investigated in permeable cells or isolated nuclei by degradation of DNA with exonuclease III and by resynthesis with DNA polymerase I (Klenow fragment) and T4 DNA ligase. The structure of the shortened patches appears to be a short stretch of DNA synthesized in the 5'----3' direction within a longer single-strand gap. The single-strand gap ahead of the 3' terminus can be bridged only by the combined action of polymerase and ligase. This structure implies that excision must involve removal of an oligonucleotide or widening of a gap by 5'----3' exonuclease action to produce a single-strand region wide enough to be a substrate for polymerase alpha. There is no evidence for structures generated by nick translation or strand displacement.

Topics: Aphidicolin; Cell Line; Cell Nucleus; Cytarabine; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Repair; DNA Replication; Fibroblasts; Humans; Thymidine; Ultraviolet Rays

Excision repair of ultraviolet damage in human fibroblasts was partially inhibited by drugs that block DNA polymerases alpha or beta (cytosine arabinoside, aphidicolin and dideoxythymidine) causing a reduction in unscheduled synthesis and an accumulation of single-strand breaks. The strand breaks accumulated in the presence of aphidicolin could be resealed within 30 min after removal of the drug, but those accumulated by cytosine arabinoside took many hours. Digestion of repaired DNA with exonuclease III or S1 nuclease revealed that even the highest concentration of polymerase inhibitors, singly or in combination, that produced maximal accumulation of single-strand breaks only blocked 37-86% of repair sites. Use of single-strand break frequencies to measure the number of repair events can therefore be in error by as much as a factor of 3. The blocked patches with free 3'OH termini were, on average, 22% of normal length, corresponding to between 6 and 17 bases (assuming a normal patch of 25-75 bases in length). Patches that remained unsealed in vivo were also resistant to sealing by T4 ligase in vitro. The data are more consistent with a mechanism of repair in which long single-strand gaps are first made by excision enzymes and subsequently filled in by DNA polymerase alpha. Strand displacement or nick translation mechanisms seem unlikely.

Topics: Aphidicolin; Cells, Cultured; Cytarabine; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Repair; DNA Replication; Fibroblasts; Humans; Kinetics; Nucleic Acid Synthesis Inhibitors; Skin; Thymidine

Topics: Animals; Aphidicolin; Arabinose; Dideoxynucleosides; Diterpenes; DNA Repair; Humans; Hydroxyurea; Thymidine

Topics: Aphidicolin; Benzamides; Cell Line; Cytarabine; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Repair; DNA Replication; DNA, Single-Stranded; Embryology; Fibroblasts; Humans; Hydroxyurea; Molecular Weight; Nucleic Acid Synthesis Inhibitors; Skin; Thymidine; Ultraviolet Rays

Topics: Animals; Aphidicolin; Cell Line; Cell Nucleus; Cricetinae; Cricetulus; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; DNA Replication; DNA, Mitochondrial; Kinetics; Lung; Mitochondria; Thymidine