Excision repair of DNA in the presence of aphidicolin

During excision repair of UV light or dimethyl sulphate (DMS)-induced damage to DNA the patch size for actively replicating KB or T98G cells is around 20 nucleotides. When confluent T98G cells or 'quiescent' KB cells are used the patch size is around 10 nucleotides. This value can be increased to around 20 nucleotides in T98G cells if a large excess of BrdUrd is included in the repair incubation medium. With 'quiescent' KB cells the patch size is not increased by excess BrdUrd. For all of these experimental conditions, when excision repair of UV or DMS damage takes place in the presence of aphidicolin, the patch size is found to be several times that found in its absence. Given the inhibitory specificity of aphidicolin for DNA polymerase alpha these results provide additional evidence that DNA polymerase alpha plays a role in the excision repair of DNA damaged by UV light or DMS. It is postulated that aphidicolin interrupts the processivity of the DNA polymerase alpha holoenzyme and allows an exonuclease to enlarge the repair site.     

The inhibition of DNA repair in primary rat hepatocyte cultures by aphidicolin: Evidence for the involvement of alpha polymerase in the repair process

The effect of aphidicolin on the repair of chemically induced DNA damage in rat hepatocytes was examined. Alkaline elution analysis of DNA damage and autoradiographic examination of unscheduled DNA synthesis both indicate that the repair of DNA damage was inhibited by aphidicolin. Because aphidicolin has been shown to be a specific inhibitor of alpha polymerase, these results suggest that the alpha polymerase plays an active role in the repair of rat hepatocyte DNA.     

Inhibition of repair of X-ray-induced DNA damage by heat: the role of hyperthermic inhibition of DNA polymerase alpha activity

HeLa S3 cells growing in suspension have been used to investigate possible mechanisms underlying the inhibitory action of hyperthermia (44 degrees C) on the repair of DNA strand breaks as caused by a 6-Gy X-irradiation treatment. The role of hyperthermic inactivation of DNA polymerase alpha was investigated using the specific DNA polymerase alpha inhibitor, aphidicolin. It was found that both heat and aphidicolin (greater than or equal to 2 micrograms ml-1) could decrease DNA repair rates in a dose-dependent way. When the applications of heat and aphidicolin were combined, each at nonmaximal doses, no full additivity in effects was observed on DNA repair rates. When the heat and radiation treatment were separated in time by postheat incubation at 37 degrees C, restoration to normal repair kinetics was observed within 8 h after hyperthermia. When heat was combined with aphidicolin addition, restoration of the aphidicolin effect to control level was also observed about 8 h after hyperthermia. It is suggested that although DNA polymerase alpha seems to be involved in the repair of X-ray-induced DNA damage, and although this enzyme is partially inactivated by heat, other forms of heat damage have to be taken into account to explain the observed repair inhibition.     

Effects of aphidicolin and/or 2',3'-dideoxythymidine on DNA repair induced in HeLa cells by four types of DNA-damaging agents

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.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

Reduced repair of X-ray-induced DNA lesions in cells without functioning DNA polymerase alpha

X irradiation of cells induces damage in the DNA, which can be detected as fragmentation of the DNA in alkali. To examine whether DNA polymerase alpha plays a role in the X-ray-induced fragmentation of the DNA, cells with and without functioning DNA polymerase alpha have been compared. We have used the drug aphidicolin, which is a specific inhibitor of polymerase alpha. The results show that DNA of aphidicolin-treated cells is more easily fragmented in alkali than DNA of untreated cells. This is paralleled by a lower repair replication in cells without functioning DNA polymerase alpha. Hence polymerase alpha is involved in the repair process of lesions induced by X irradiation.     

Effect of nalidixic acid on DNA repair in rat hepatocytes

Nalidixic acid, a DNA topoisomerase inhibitor, has been reported to inhibit DNA repair in some mammalian systems. To investigate the effect of nalidixic acid on DNA repair in cultured rat hepatocytes, DNA damage was induced by ultraviolet light or N-methyl-N-nitro-N-nitrosoguanidine. The presence of aphidicolin, a DNA polymerase alpha inhibitor resulted in a decrease in DNA repair. Nalidixic acid had no inhibitory effect. Neither aphidicolin nor nalidixic acid induced DNA repair. These results indicate that nalidixic acid does not damage DNA or inhibit DNA repair processes in hepatocytes.     

Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.     

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.     

Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha

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.     

Aphidicolin inhibits repair of DNA in UV-irradiated human fibroblasts

Aphidicolin, a specific inhibitor of DNA polymerase α, is shown to inhibit DNA repair in human diploid fibroblasts. Although aphidicolin has no apparent effect on the DNA of unirradiated cells, it causes a large number of strand breaks to accumulate in UV-irradiated cellular DNA. The number of breaks is the same as the number observed following a similar dose of ultraviolet light when cells are treated with arabinofuranosyl cytosine (araC) and hydroxyurea (HU), known inhibitors of repair. Moreover, two-dimensional paper chromatography shows that aphidicolin completely blocks removal of pyrimidine dimers. These observations are discussed in light of the proposed roles of DNA polymerases α β in DNA replication and repair and the action of aphidicolin on polymerase α.     

DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation

In confluent, stationary phase cells, an aphidicolin-sensitive DNA polymerase mediates UV-induced excision repair, but the situation in growing cells is still controversial. The sensitivity of repair synthesis to aphidicolin, an inhibitor of DNA polymerases alpha and delta, was determined in growth phase and confluent normal human fibroblasts (AG1518) using several techniques. Repair synthesis in confluent cells was always inhibited by aphidicolin, no matter which measurement technique was used. However, the inhibition of repair synthesis in growth-phase cells by aphidicolin was only detectable when techniques unaffected by changes in nucleotide metabolism were used. We conclude that UV-induced repair synthesis in growing cells is actually aphidicolin sensitive, but that this inhibition can be obscured by changes in nucleotide metabolism. Employing butylphenyl-deoxyguanosine triphosphate, a potent inhibitor of polymerase alpha and a weak inhibitor of delta, we have obtained evidence that polymerase delta is responsible for repair synthesis in growth-phase cells following UV irradiation.     

The regulation of the DNA repair process in mammalian cells. IV. The role of DNA polymerases in the epidermal growth factor regulation of the repair of single-stranded DNA breaks induced by ionizing radiation in Swiss 3T6 mouse cells]

A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase alpha and delta (aphidicolin) and DNA polymerase beta (2', 3'-dideoxythymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gr) or by gamma-ray irradiation (150 Gr) is more sensitive to aphidicolin independently of cell proliferating status. Aphidicolin inhibits the recovery of single-strand DNA in quiescent and mitogen-stimulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase alpha. It is suggested that the regulation action of mitogens on the DNA SSB repair may be determined by qualitative changes of this enzyme or of some conditions in which it functions. The involvement of DNA polymerase delta in this process is not excluded.     

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells.

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.     

Effect of aphidicolin on de novo DNA synthesis, DNA repair and cytotoxicity in gamma-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells. We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes

In view of the possible utilization of aphidicolin, a specific inhibitor of DNA polymerase α, in the treatment of neoplastic diseases, it seemed important to assess the mutagenic effect of the drug and the possible modification induced by metabolic activation in the liver. This paper shows that aphidicolin lacks mutagenicity in the Ames' $\text{Salmonella}$-microsome test in agreement with our previous observation that it does not induce DNA repair synthesis in HeLa cells. During the studies of mutagenicity we have observed that aphidicolin is converted to inactive derivative(s) by rat liver microsomal oxidases. The reaction is dependent on time and temperature and requires NADP⁺ and glucose-6-P. The metabolites are not mutagenic and they do not induce DNA repair synthesis in HeLa cells. Therefore the possible anti-cancer use of aphidicolin is not hampered by its partial metabolic inactivation in liver. Our results suggest however that aphidicolin will possibly be clinically useful at concentrations higher than those expected from our studies with human DNA polymerase $\text{α}\text{in vitro}$ and human neoplastic cell lines $\text{in vivo}$. The metabolic derivative(s) of aphidicolin is inactive both against cellular DNA polymerase α and Herpes simplex viral DNA polymerase.     

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells.     

Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study.

A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.