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.     

Mechanism of inhibition of herpes simplex virus and vaccinia virus DNA polymerases by aphidicolin, a highly specific inhibitor of DNA replication in eucaryotes.

The inhibition in vitro of herpes simplex virus 1 and vaccinia virus DNA polymerases by aphidicolin is primarily noncompetitive with dGTP, dATP, dTTP, DNA, and Mg2+ and competitive with dCTP in analogy with the mode of inhibition of cellular alpha-polymerase. The degree of inhibition of viral or cellular growth in vivo can be quantitatively predicted by the degree of inhibition of the isolated replicative DNA polymerases at the same concentration of aphidicolin in suitable conditions (limiting dCTP concentration). Thus, the only in vivo target for aphidicolin is probably the replicative DNA polymerase, and aphidicolin is a highly specific inhibitor of replicative nuclear DNA synthesis in eucaryotes. This, coupled with the lack of mutagenic effect, represents a valuable property for an anticancer drug. The specificity of inhibition (contrary to the aspecific effect on almost all DNA polymerases by a true competitive inhibitor, such as 1-beta-D-arabinofuranosylcytidine 5'-triphosphate) and the structure of the drug, which does not resemble that of the triphosphates, suggest that aphidicolin must recognize a site common only to the replicative DNA polymerases of eucaryotes and different from the binding site for deoxyribonucleic triphosphates and DNA, which should be similar in reparative and procaryote-type DNA polymerase; the aphidicolin binding site is probably very near to, or even overlaping with, the binding site for dCTP so that the drug mimics a competitive effect with this nucleotide.     

Effect of aphidicolin on vaccinia virus: isolation of an aphidicolin-resistant mutant.

Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 microM or more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100-fold by 80 microM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 microM aphidicolin. In infected cells, aphidicolin concentrations over 10 microM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 microM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 microM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 microM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 microM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 microM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.     

In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis

The aerial parts of the plant are generated by groups of rapidly dividing cells called shoot apical meristems. To analyze cell behavior in these structures, we developed a technique to visualize living shoot apical meristems using the confocal microscope. This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerizing microtubules) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems. Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation were inhibited, suggesting an important role for DNA synthesis in growth and patterning.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.     

Arrest of chain growth of replicon-sized intermediates by aphidicolin during rat fibroblast cell chromosome replication

The effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 micrograms/ml) causing 90-95% inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2-5 x 10(-7) Da); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high-molecular-weight DNA. This merging of the intermediates was associated with DNA synthesis in gaps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine-DNA leader with long-wave ultraviolet light; when the cells had been pulse-labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin-arrested intermediates was cut into fragments of the original size by long-wave ultraviolet light irradiation. The arrest of chain elongation at the replicon-size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNAs.     

Characterization of a Mutant of Toxoplasma gondii Resistant to Aphidicolin1

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.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos.

We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polyermase alpha. These cells fail to proliferate in the presence of aphidicolin. In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo. In continuous exposure to aphidicolin (2 micrograms/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.     

Mutations in the herpes simplex virus DNA polymerase gene conferring hypersensitivity to aphidicolin.

Fourteen mutants known or likely to contain mutations in the herpes simplex virus DNA polymerase gene were examined for their sensitivity to aphidicolin in plaque reduction assays. Eleven of these exhibited some degree of hypersensitivity to the drug; altered aphidicolin-sensitivity correlated with altered sensitivity to the pyrophosphate analog, phosphonoacetic acid. The DNA polymerase specified by one of these mutants, PAAr5, required roughly seven-fold less aphidicolin to inhibit its activity by 50% than did polymerase specified by its parental strain. Mutations responsible for the aphidicolin-hypersensitivity phenotype of PAAr5 were mapped to an 0.8 kbp region in the herpes simplex virus DNA polymerase locus. These data taken together indicate that 1) mutations in the herpes simplex virus DNA polymerase gene can confer altered sensitivity to aphidicolin, 2) that the HSV polymerase is sensitive to aphidicolin in vivo, and 3) that amino acid alterations which affect aphidicolin binding may affect the pyrophosphate exchange-release site as well, suggesting that aphidicolin binds in close proximity to this site.     

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.     

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.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

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.     

Structural basis for inhibition of DNA replication by aphidicolin

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.     

The mode of inhibitory action by aphidicolin on eukaryotic DNA polymerase alpha.

The mode of action by aphidicolin on DNA polymerase alpha from the nuclear fraction of sea-urchin blastulae was studied. The inhibition of DNA polymerase alpha by aphidicolin was uncompetive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template-primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase alpha by competing with dCTP with a Ki value of 0.5 microgram/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC). (dG)12-18 or poly(dA) . (dT)12-18. Similar results were obtained for DNA polymerase alpha (B form) from mouse myeloma MOPC 104E.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors

Summary Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase ase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent K _m for dCTP and the apparent K _i for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.Abbreviations ara-A 9--d-arabinofuranosyl adenine - ara-C 1--d-arabinofuranosyl cytosine - aph aphidicolin     

Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.     

Endoreduplication is not inhibited but induced by aphidicolin in cultured cells of tobacco

Endoreduplication is a common process in plants that allows cells to increase their DNA content. In the tobacco cell cultures studied in this work it can be induced by simple hormone deprivation. Mesophyll protoplast-derived cells cultured in the presence of NAA (auxin) and BAP (cytokinin) keep on dividing, while elongation and concomitant DNA endoreduplication are induced and maintained in a medium containing only NAA. If aphidicolin is given to the two types of culture, no effect is observed on elongating, endoreduplicating cells. However, the cells programmed for division switch to elongation and DNA endoreduplication. Thus aphidicolin, an inhibitor of the replicative DNA polymerases, alpha and delta, does not inhibit endoreduplication, and furthermore actually induces it when the mitotic cell cycle is blocked. DNA duplication and cell growth can only be completely blocked if ddTTP, an inhibitor of DNA polymerase-beta, is given together with aphidicolin. This result implies that an aphidicolin-resistant DNA polymerase, such as the repair-associated DNA polymerase-beta, can mediate DNA synthesis during endoreduplication and can substitute for polymerases-alpha and -delta when the latter are inhibited. Similar results are obtained in cultures of the BY-2 cell line by withdrawing auxins from the culture medium. In this cell line endoreduplication is induced only in a small proportion of the cells. A greater proportion of the cells are blocked in the G(2) phase of the cell cycle.