Specific Inhibitor Of DNA Polymerase Research Articles

Discovery of a novel DNA polymerase inhibitor and characterization of its antiproliferative properties

ABSTRACTChromosomal duplication is targeted by various chemotherapeutic agents for the treatment of cancer. However, there is no specific inhibitor of DNA polymerases that is viable for cancer management. Through structure-based in silico screening of the ZINC database, we identified a specific inhibitor of DNA polymerase δ. The discovered inhibitor, Zelpolib, is projected to bind to the active site of Pol δ when it is actively engaged in DNA replication through interactions with DNA template and primer. Zelpolib shows robust inhibition of Pol δ activity in reconstituted DNA replication assays. Under cellular conditions, Zelpolib is taken up readily by cancer cells and inhibits DNA replication in assays to assess global DNA synthesis or single-molecule bases by DNA fiber fluorography. In addition, we show that Zelpolib displays superior antiproliferative properties to methotrexate, 5-flourouracil, and cisplatin in triple-negative breast cancer cell line, pancreatic cancer cell line and platinum-resistant pancreatic cancer cell line. Pol δ is not only involved in DNA replication, it is also a key component in many DNA repair pathways. Pol δ is the key enzyme responsible for D-loop extension during homologous recombination. Indeed, Zelpolib shows robust inhibition of homologous recombination repair of DNA double-strand breaks and induces “BRCAness” in HR-proficient cancer cells and enhances their sensitivity to PARP inhibitors.

First synthesis of (S)-(+)-hymenoic acid, a DNA polymerase λ inhibitor isolated from Hymenochaetaceae sp.

Hymenoic acid, isolated from cultures of the fungus, Hymenochaetaceae sp., is a specific inhibitor of DNA polymerase λ. The first synthesis of (S)-(+)-hymenoic acid was achieved by starting from trans-1,4-cyclohexanedimethanol and methyl (R)-(-)-3-hydroxyisobutyrate, and Julia-Kocienski olefination was employed as the key step.

Influence of particle size on the in vitro and in vivo anti-inflammatory and anti-allergic activities of a curcumin lipid nanoemulsion.

The polyphenolic compound, curcumin, is a natural yellow pigment component of turmeric. It exerts various biological effects, such as anti-inflammatory effects, and we have previously demonstrated that curcumin is a specific inhibitor of DNA polymerase λ. Curcumin is characterized by poor bioavailability as it is water-insoluble, is poorly absorbed and is systemically eliminated. In order to increase the bioavailability of curcumin, in this study, we produced a curcumin-loaded lipid nanoemulsion of various particle sizes (50, 100 and 200 nm). The curcumin lipid nanoemulsion was prepared by a modified thin-film hydration method followed by sonication. To identify the optimal particle size which exhibits the strongest physiological activity, we investigated the inhibitory effects of the obtained nanoemulsions against inflammatory and allergic activities. In in vitro cell culture experiments, the 100-nm curcumin lipid nanoemulsion showed the most prominent inhibitory effect on the production of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in RAW264.7 murine macrophages, and on the release of β-hexosaminidase induced by the calcium ionophore, A23187, in rat basophilic leukemia RBL-2H3 cells. In an in vivo experiment, in which mice were administered the curcumin-loaded lipid nanoemulsion of various particle sizes, the 100-nm curcumin lipid nanoemulsion showed the most prominent anti-inflammatory and anti-allergic effects, inhibiting 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory ear edema and immunoglobulin E (IgE)-induced passive cutaneous anaphylactic (PCA) reaction. The effects of particle size on serum curcumin absorption were also assessed in mice, and the 100-nm lipid nanoemulsion showed the greatest absorption. The results from our study suggest that the physiological activities of curcumin lipid nanoemulsions differ depending on particle size. Our data indicate that the curcumin lipid nanoemulsion with a particle size of 100 nm has potential for use in enhancing the bioavailability and medical value of curcumin.

Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-cotaining molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase β, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase α decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase β. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase β. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.

Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha.

The fungal diterpene, aphidicolin, is a well-known specific inhibitor of DNA polymerase alpha. Terpenoids are an important class of natural products. However, identification of the biosynthetic gene cluster in terpenoids is relatively rare compared with another important class of natural products, polyketides. To explore a reliable identification method for the biosynthetic gene cluster in fungal diterpenoids, cloning of the biosynthetic gene cluster of aphidicolin was employed. The application of a simple PCR method for genome walking based on the sequence of cDNA encoding aphidicolan-16beta-ol synthase (ACS) allowed us to analyze a 15.6-kb region of the Phoma betae genomic DNA. Six ORFs, PbGGS, ACS, PbP450-1, PbP450-2, PbTP, and PbTF were found in this region, and respectively expected to encode geranylgeranyl diphosphate synthase, diterpene synthase, two cytochrome P-450s, the transporter and transcription factor. Their amino acid sequences and introns were deduced by a corresponding cDNA analysis. This study shows that simple PCR-based genome walking without constructing a genomic DNA library is useful for identification of a small gene cluster. We propose a general strategy for the cloning the biosynthetic genes of fungal diterpenoids by using fungal GGS.

Control mechanisms of in vitro lens transdifferentiation of Xenopus laevis outer cornea

The isolated outer cornea cultured in presence of acidic Fibrob‐last Growth Factor (aFGF) can reprogramme its differentiation into lens fibres. The capacity of aFGF to promote lens differentiation is not linked to its mitogenic activity. In previous studies (Bosco et al., 1997) it was observed that the lens transdifferentiation of corneal cells occurs also when DNA replication and cell proliferation are prevented by addition of aphidicolin (a specific inhibitor of DNA polymerase in eukaryotes), to the culture medium. Experiments in which either transcription or translation have been inhibited by actinomycin D and cycloheximide, respectively, have now shown that the corneal cells are unable to transdiffer‐entiate into lens fibres. These results indicate that the multi‐step process lens‐transdifferentiation of corneal cells can be controlled also at these levels.

Checkpoint control on mitochondrial division inCyanidioschyzon merolae

It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red algaCyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase α, using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin onC. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3′-dihexyloxacarbocyanine iodide (DiOC6; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division inC. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA.

In vitro lens transdifferentiation of Xenopus laevis outer cornea induced by Fibroblast Growth Factor (FGF).

It has been shown that lens regeneration from outer cornea of larval Xenopus laevis is dependent on neural retina both in vivo and in tissue culture. The isolated outer cornea cultured in the presence of bovine brain-derived acidic Fibroblast Growth Factor (aFGF) is able to reprogram the differentiation into lens fibers, although this transdifferentiative process is not coupled with the formation of a normally organized lens. The capacity of aFGF to promote lens differentiation from cornea is not linked to its mitogenic activity. The cultured corneal cells can transdifferentiate into lens fibers in the presence of aFGF when DNA replication and cell proliferation are prevented by addition of aphidicolin, a specific inhibitor of DNA polymerase in eukaryotes, to the culture medium.

Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.

The effects of aphidicolin, a specific inhibitor of DNA polymerase α, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 × 10-6 M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube-like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin-positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube-like elements may develop simply by cell fusion without cell division and DNA synthesis.

Phage P4 DNA replication in vitro.

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.

Synthesis of 1-O-acylglycerol 2,3-cyclic phosphate: Determination of the absolute structure of PHYLPA, a specific inhibitor of DNA polymerase α

Four possible stereoisomers of PHYLPA, a specific inhibitor of DNA polymerase α, were synthesized in enantioselective manners. These isomers were examined for inhibition activity for DNA polymerase α, and the structure of PHYLPA was established as sodium 1-0-[(9′S,1O′R)-9′,10′-methanohexadecanoyl]-sn-glycerol-2,3-cyclic phosphate.

Biological properties of some benzopsoralen derivatives.

The biological activity of some benzopsoralen derivatives, prepared with the aim of obtaining new drugs for photochemotherapy, has been studied. The more interesting compounds are 4-hydroxymethyl-4',5'-benzopsoralen and 4-hydroxymethyl-4',5'-tetrahydro-benzopsoralen, which were found to be active in the dark also: DNA and RNA synthesis were both inhibited in Ehrlich cells, even if in a partially reversible fashion, while protein synthesis remained unaffected. In Chinese hamster ovary cells cultured in vitro, the clonal growth was strongly inhibited by incubation in the dark with both drugs, while a number of chromosomal aberrations was observed in the fraction of growing cells. Using alkaline elution, DNA strand breaks were detected. In addition, in the presence of aphidicolin, a specific inhibitor of DNA polymerase, the clonal growing capacity was completely restored; in contrast, the number of DNA strand breaks remained unchanged. All these results suggest that DNA topoisomerases are probably the target of these two benzopsoralens. These compounds are also good sensitizers; by UV-A irradiation they have a good capacity to produce singlet oxygen, but they appeared to be unable to induce erythemas on guinea-pig skin. Under UV-A light, they induced a strong inhibition of DNA synthesis in Ehrlich cells. Thus, benzopsoralens appear to be capable of inducing strong antiproliferative effects by two different mechanisms, by UV-A irradiation and in the dark.

Two different mechanisms are involved for the bleomycin-induced DNA repair synthesis in permeabilized HeLa cells

Bleomycin-induced DNA repair synthesis in the permeabilized HeLa cells was sensitive to aphidicolin, an inhibitor of DNA polymerase α and δ, and to dideoxythymidine triphosphate (ddTTP), a specific inhibitor of DNA polymerase β. Upon combined treatment with these inhibitors, the DNA repair synthesis was inhibited to an even higher degree. This indicated that the aphidicolin- and ddTTP- sensitive DNA repair syntheses may occur by independent mechanisms. The structure of incomplete repair patches being accumulated in the presence of these inhibitors was investigated by digestion of DNA with exonuclease III after incubation with Klenow fragment and T4 DNA ligase. The results have suggested that the patch accumulating in the presence of aphidicolin is a single-stranded gap made by excision enzyme(s), whereas that accumulating in the presence of ddTTP may be generated by strand displacement.

Selective abrogation of alloreactivity via priming in the presence of aphidicolin, a specific inhibitor of DNA polymerase.

Aphidicolin, a specific and direct inhibitor of eukaryotic DNA polymerase alpha, was used to investigate its impact on immunologic reactions in vitro. Dose response curve of the inhibitory effect was studied in murine and human primary allogeneic responses, as well as the proliferative responses to both PHA and Con A mitogens. The presence of aphidicolin during the allosensitization phase in secondary MLR of mice splenocytes resulted in complete abolishment of the subsequent response directed against the priming alloantigens, whereas alloreactivity to unrelated alloantigen-bearing cells was inhibited to a much lesser degree. The allosensitized aphidicolin-treated cells lost the ability to respond to subsequent PHA stimulation, but were capable of exerting a high responsiveness to Con A. The presence of aphidicolin during the allosensitization phase in secondary MLR of human mononuclear cells resulted in markedly decreased alloreactivity directed against the priming cells, but spared the subsequent response to unrelated alloantigens and to both PHA and Con A mitogenic stimuli. It is suggested that aphidicolin may be used for selective inactivation of proliferating cells without interfering with immunologic functions of other quiescent subsets. Aphidicolin may thus be a useful agent for induction of specific unresponsiveness in experimental models of allogeneic transplantation.

Involvement of DNA polymerases in the repair of DNA damage by benzo[ a]pyrene in cultured Chinese hamster cells

Mechanisms for induction of single-strand scissions in DNA by S9-activated benzo[ a]pyrene (B[a]P) and their repair in cultured Chinese hamster V79 cells were investigated with inhibitors of DNA-repair synthesis using alkaline sucrose gradient sedimentation analysis. The marked induction of single-strand scissions in DNA was observed following 3 h treatment of V79 cells with 5 μg/ml of B[a]P. These DNA lesions were repaired to the control level within 4 h after removal of B[a]P. The simultaneous addition of inhibitors of DNA-repair synthesis, 1-β- D-arabinofuranosylcytosine (araC) plus hydroxyurea with B[a]P did not increase the formation of DNA single-strand scissions. When these inhibitors were added after removal of B[a]P, however, they significantly blocked the rejoining of DNA-strand scissions. On the other hand, when aphidicolin, a specific inhibitor of DNA polymerase α, was used instead of araC, a partial inhibition of the rejoining was observed, and further addition of 2′,3′-dideoxythymidine, an inhibitor of DNA polymerase β, augmented the inhibitory effect. These results indicate that B[a]P-induced single-strand scissions of DNA in V79 cells could be repaired mostly by excision repair which involved DNA polymerase α and a non-α polymerase, presumably polymerase β.

Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments.

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.

Involvement of DNA-polymerase activities in mouse-blastocyst differentiation in vitro

Full-grown blastocysts were cultured for 707–92 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 α, (2) dideoxythymidine (d 2Thd), the precursor of dideoxythymidine triphosphate (d 2TTP), a specific inhibitor of DNA polymerase β and (3) d 2TTP 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 α during the first part of gestation instead of DNA polymerase β, as has previously been shown to be the case for mid-gestation rat trophoblast cells.

Effects of aphidicolin on the repair and fixation of potentially lethal damage sensitive to β-araA

The effects of aphidicolin, a specific inhibitor of DNA polymerase a, on the repair and fixation of potentially lethal damage (PLD) sensitive to treatment with β-araA—a drug acting via inhibition of DNA polymerases a and β-have been studied. Three μg/ml of aphidicolin given after irradiation did not produce any significant modification in cell survival. More damage was nevertheless fixed by a given concentration of β-araA in the presence of aphidicolin to a degree that 10 μM β-araA given together with 3 μg/ml aphidicolin produced an effect similar to that of 70 μM β-araA given alone. Although aphidicolin significantly enhanced the effectiveness of β-araA in fixing PLD, it did not significantly increase the sector of lesions affected. Combined treatment with β-araA and aphidicolin reduced or eliminated the shoulder from the survival curve without affecting the slope, an effect similar to that observed after treatment of cells with β-araA alone. The presence of aphidicolin during the time interval trey allowed for repair of the β-araA sensitive PLD resulted in a significant reduction in the repair rate. Based on these results, the necessity of distinction between inhibition of a repair process and fixation of the damage undergoing repair is discussed, as well as the possible involvement of DNA polymerase α and β in the repair and fixation of the DNA damage that underlies repair of PLD.

Effects of aphidicolin on the recovery of ribosomal RNA synthesis and on the repair of potentially lethal damage in UV irradiated simian and human cells

Transcription analysis have been employed as probe for monitoring DNA damage and repair in order to elucidate whether or not aphidicolin, a specific inhibitor of DNA polymerase α, affects the repair of UV damage. Results show that aphidicolin inhibits in a concentration-dependent way the recovery of ribosomal RNA transcription in both human and simian cells, indicating a direct involvement of α polymerase in DNA repair. This conclusion was further supported by the inhibitory action of aphidicolin on the repair of potentially lethal damage in UV-irradiated monkey cells.

Excision repair in Cockayne syndrome

Cockayne syndrome (CS) is a genetic disorder showing cellular sensitivity to the lethal effects of UV-irradiation. No defects in unscheduled DNA synthesis or in daughter-strand repair have been detected after UV-irradiation of CS cells. We have studied several aspects of excision repair, particularly at early times after UV-irradiation, and with one exception, we have not been able to detect any difference in the response of normal and CS cells to UV-irradiation, by measuring: (1) the rate of formation of incision breaks in the presence of 1-β- d - abinofuranosylcytosine (araC); (2) the amount of repair replication as measured by equilibrium centrifugation; (3) the ligation of repaired DNA to pre-existing DNA; (4) the digestibility of repaired DNA after treatment of nuclei with micrococcal nuclease. The single exception was a pair of CS strains from sibling donors in which the rate of uncoupled incision due to the presence of either araC or the specific inhibitor of DNA polymerase α, aphidicolin, was slightly faster than in other cells studied. This effect was absent in the heterozygous parents. However, since this was not seen in two other CS strains in the same genetic complementation group, we can not attribute this increased rate of incision to the defective CS gene. We conclude that, within the limits of resolution of these techniques, CS cells do not have a detectable defect in excision repair.