Inhibitor Of DNA Polymerase Alpha Research Articles

The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

A High-Throughput Assay for DNA Replication Inhibitors Based upon Multivariate Analysis of Yeast Growth Kinetics

Mcm2–7 is the molecular motor of eukaryotic replicative helicase, and the regulation of this complex is a major focus of cellular S-phase regulation. Despite its cellular importance, few small-molecule inhibitors of this complex are known. Based upon our genetic analysis of synthetic growth defects between mcm alleles and a range of other alleles, we have developed a high-throughput screening (HTS) assay using a well-characterized mcm mutant (containing the mcm2DENQ allele) to identify small molecules that replicate such synthetic growth defects. During assay development, we found that aphidicolin (inhibitor of DNA polymerase alpha) and XL413 (inhibitor of the DNA replication-dependent kinase CDC7) preferentially inhibited growth of the mcm2DENQ strain relative to the wild-type parental strain. However, as both strains demonstrated some degree of growth inhibition with these compounds, small and variable assay windows can result. To increase assay sensitivity and reproducibility, we developed a strategy combining the analysis of cell growth kinetics with linear discriminant analysis (LDA). We found that LDA greatly improved assay performance and captured a greater range of synthetic growth inhibition phenotypes, yielding a versatile analysis platform conforming to HTS requirements.

Single Arm, Multi-Center, Phase II Study Of Clofarabine In Chinese Pediatric Patients With Refractory Or Relapsed Acute Lymphoblastic Leukemia

BackgroundClofarabine is a second generation purine nucleoside which inhibits DNA polymerase alpha and ribonucleotide reductase, leading to the depletion of intracellular deoxynucleotide triphosphate pools, disruption of mitochondrial function and apoptosis. A phase II study was conducted to evaluate the safety and efficacy of clofarabine in pediatric patients with refractory or relapsed acute lymphoblastic leukemia (ALL) in China. MethodPatients aged 1-21 years with histologically confirmed, relapsed and/or refractory ALL were eligible. Clofarabine was administered intravenously for 5 days at 52 mg/m2/d during induction and consolidation. The primary end point was overall remission rate (ORR; complete remission [CR] plus CR with incomplete counts [CRi]). Blood samples were obtained from patients who agreed to blood drawing for pharmacokinetic determinations. ResultsA total of 44 patients were treated in this study, in which 43 were evaluable for response. The median age was 13 years (range: 5 to 22 years), and the majority were B cell-ALL (75%, 33/44). The overall remission rate was 44.2% (19/43), including two complete remissions (CR), and 17 CRis. 39 patients had progressive disease at cut-off time, the overall median remission duration was 2.9 months, and the median remission duration was 3.4 months for patients achieved CR or CRi. Seven CR/CRi patients had chance to got HSCT after clofarabine treatment. Subgroup analysis showed that patients younger than 14 years and with B cell-ALL had a higher ORR than patients older than 14 years and with T cell-ALL, and the difference were statistically significant (age < 14 years vs. age ≥14 years: 60.9% vs. 25%, p =0.0308; B cell-ALL vs. T cell-ALL: 57.6% vs. 0, p=0.002). The most common (≥10%) adverse reactions (ADR) were hematological toxicity, gastrointestinal reaction, and abnormal liver function, and the most common grade 3/4 non-hematologic ADRs was ALT elevation (13.6%). PK analysis demonstrated that after multiple administrations the mean elimination half-life of clofarabine was 6.43 h, and the Tmax was around 2 h. The mean Cmax and exposure to clofarabine over the dosing period (AUC 0-24) achieved 581 ug/L and 2602.56 ug/L, respectively. It seems that there is no significant difference of PK profiles between Chinese and Caucasian patients. ConclusionSingle clofarabine treatment is effective in Chinese pediatric patients with multiple relapsed or refractory ALL with acceptable safety profiles. Disclosures:No relevant conflicts of interest to declare.

Abstract 4069: The p47 Phox mediated redox-signal promotes myogenesis via p38 MAPK regulated microRNA-206

Abstract miR-206 is reported that one of the skeletal muscle specific myogenic factors which are halt the cell cycle via inhibition of DNA polymerase-alpha. So far, MyoD and AP1 are as well as known for miR-206 transcription factors. In this study, we take the evidence of another transcription factor MEF-2 which have a capacity for miR-206 expression in myogenesis. In addition, a novel miR-206 transcription factor is belonging to p38 MAPK activities that can be regulated by p47 Phox mediated redox-signal. It is hypothesis for a novel role of myogenic miR-206 regulation that p47 Phox generated redox-signal contributes to skeletal muscle differentiation via p38 MAPK activated miR-206. Followed the hypothesis, we were analysis the miR-206 promoter region (&amp;gt;-2k) by MatInspector (Promotor analysis tool, www.genomatix.de) for found out another plausible transcription factors and compare the miR-206 expression levels in SB203580 (p38 MAPK inhibitor), DPI (p47 Phox inhibitor) treated and differentiation condition by real-time PCR. Then, measuring the differentiation induced ROS during myogenesis by FACS. Moreover, we analyzed the expressions of myogenic factors and cell cycle involved molecules, including DNA polymerase-alpha, with immunoblotting. As a result, we hunt out a specious myogenic transcription factor MEF-2 that was not reported as a miR-206 expression factor. Both MEF-2 upstream activator p38 MAPK and p38 MAPK stimulating redox generator p47 Phox inhibition were accompanied with miR-206 down regulation. Besides, hindering of p38 MAPK and p47 Phox observed restoration of DNA polymerase-alpha transcription/translation levels in myogenic condition. Accordingly, we suggest that ROS activated p38 MAPK down-stream transcription factor MEF-2 may showed to a novel regulation factor of miR-206 in mouse skeletal muscle myogenesis. Key words: microRNA-206(miR-206), p47 Phox, p38 MAPK, MEF-2 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4069.

Penta-O-galloyl-beta-D-glucose induces S- and G1-cell cycle arrests in prostate cancer cells targeting DNA replication and cyclin D1

We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring hydrolyzable gallotannin, inhibited the in vivo growth of human androgen-independent p53-mutant DU145 prostate cancer (PCa) xenograft in athymic nude mice without adverse effect on their body weight. We have also shown that PGG induced caspase-mediated apoptosis in the DU145 cells and the androgen-dependent human p53-wild-type LNCaP cells. Here, we investigated the cell cycle effects of PGG in these and other PCa cells. Our data show that treatment with subapoptotic doses of PGG induced S-arrest, whereas higher doses of PGG induced not only S-arrest but also G(1) arrest. We show, for the first time, that irrespective of the p53 functional status of the PCa cell lines, PGG exerted a rapid (within 2 h) and potent inhibition (inhibitory concentration by 50% approximately 6 microM) of 5-bromo-2'-deoxyuridine incorporation into S phase cells. In isolated nuclei, PGG inhibited DNA replicative synthesis with superior efficacy than a known DNA polymerase alpha inhibitor, aphidocolin. In addition to the S-arrest action, we have found a close association of downregulation of cyclin D1 with G(1) arrest induced by PGG. Overexpressing this G(1) cyclin abolished G(1) arrest, but hastened the S-arrest induction by PGG. Together, our data indicate that PGG induced PCa S-arrest probably through DNA replicative blockage and induced G(1) arrest via cyclin D1 downregulation to contribute to anticancer activity. Our data raise the hypothesis that PGG may be a novel inhibitor of DNA polymerases.

DNA polymerase alpha inhibition by aphidicolin and fragile site expression in prometaphase chromosomes of the Italian Mediterranean River Buffalo (Bubalus bubalis, 2n=50)

The present study reports on the expression and localization of “fragile sites” (FS) on prometaphase chromosomes of two groups of river buffaloes (Bubalus bubalis, 2n=50; Mediterranean Italian breed), reared in two different farms, with the aim to characterize chromosome fragility in this species. Totally, 400 aphidicolin induced breakages were identified and localized on the standardized ideogram of the river buffalo karyotype. Preliminary results can be synthesized as follows: (a) aphidicolin showed a remarkable decondensing effect on chromosome structure, enabling further studies at high resolution level; (b) the chromosomal expression of the breakages was not different in the two groups of animals; (c) the most fragile chromosomes were the inactive-X, chromosomes 9, 8 and active-X, showing 42, 32, 31 and 30 breakages, respectively; (d) the breaks were localized in the RBG-negative bands (corresponding to eterochromatic regions) or at the band-interband regions; (e) the chromosomal distribution of the break sites was not random and only partially related to chromosome length. The study is in progress to determine the relative incidence of the fragile sites at chromosomal band level, in order to construct a ‘fragile-site map’ of river buffalo, which could be utilized for genetic improvement programs of the species.

Clofarabine in Children with Refractory/Relapsed Acute Leukemia: Results of 2 Phase II Open Label Studies.

Background: Clofarabine is a second generation purine nucleoside analogue synthesized to improve the properties of its congeners, fludarabine and cladribine. Clofarabine inhibits DNA polymerase alpha and ribonucleotide reductase, leading to the depletion of intracellular deoxynucleotide triphosphate pools, disruption of mitochondrial function and apoptosis.Methods: Clofarabine was administered by IV infusion over 2 hrs for 5 days at 52 mg/m2/day and repeated every 2–6 weeks based on bone marrow response and recovery of normal hematopoiesis. An independent review panel confirmed all results.Results: The median age of the 103 children (61 with ALL and 42 with AML) was 12 years (range 1–22 yrs).ALL: The overall remission rate (CR+CRp) was 20%; 30% (18/61) of patients showed a response (7CR, 5CRp, 6PR). Responses were noted in 15 of 50 (30%) patients with B- lineage ALL, 2 of 6 (33%) with T-cell ALL. Responders received a median of 3 prior induction regimens; 50% (9/18) had prior HSCT and 50% (9/18) were refractory to the preceding induction regimen. Response rate in refractory patients was 26% (9/35). After clofarabine treatment, 10 patients proceeded to transplant (including 8 responders). 6 of 10 patients who received a transplant were alive at last follow up (survival range: 30.1+ – 145.1+ wks). Response duration in 6 patients with CR or CRp who did not receive a transplant ranged from 4.3 to 58.6 weeks; 2 patients maintained CR for 47.9 and 58.6 weeks after clofarabine therapy. Median overall survival for the patients who achieved at least a PR was 66.6 weeks compared to 12.9 weeks for all patients.AML: The response rate for AML patients was 26% (1 CRp, 10 PR). Responders had received a median of 2 prior induction regimens, 36% (4/11) had prior HSCT and 55% (6/11) patients were refractory to the preceding induction regimen. Response rate in refractory patients was 21% (6/28). One patient who had received 5 prior induction regimens achieved CRp. 13 (31%) patients (1CRp, 6PR, 3NE, 3TF) underwent HSCT after completing clofarabine therapy, 5 of whom were alive at last follow-up (survival range: 62.7+ – 160.1+ wks). Many patients proceeded to HSCT as soon as a donor was identified without waiting for the patient to go into remission, making remission difficult to assess. Median overall survival for patients who achieved at least a PR was 32.1 wks compared to 23.4 wks for all patients.Safety: The side effect profile was not unexpected in this heavily pretreated population. The ≥ gr 3 drug related AEs occurring in ≥10% patients included febrile neutropenia for ALL patients; febrile neutropenia, pyrexia, nausea, neutropenia, and hypotension for AML patients.Conclusion: Clofarabine shows significant activity in heavily pretreated children with multiply relapsed acute leukemia, allowing otherwise refractory patients to proceed to HSCT. Durable responses were seen in ALL patients with no donor options who continued therapy with single agent clofarabine. Combination studies incorporating clofarabine in pediatric patients with relapsed ALL and AML are ongoing.

Sulfo-Quinovosyl-Acyl-Glycerol (SQAG), a Eukaryotic DNA Polymerase Inhibitor and Anti-Cancer Agent

It was found that a class of sulfolipids known as sulfo-quinovosyl-acyl-glycerols (SQAGs) from ferns and algae are potent inhibitors of eukaryotic DNA polymerase alpha and beta and effective anti-neoplastic agents. In developing a procedure for the chemical synthesis of sulfolipids, many derivatives and stereoisomers of SQAGs have been obtained including sulfo-quinovosyl-monoacyl-glycerols (SQMGs) and sulfo-quinovosyl-diacyl-glycerols (SQDGs). This review describes studies on the structure-function relationship between synthetic SQAGs and DNA polymerase alpha and beta, and the relationship to cytotoxic activity. The major action was probably dependent on the fatty acid effect, which was reported previously, although each of the SQAGs was a much stronger inhibitor than just the fatty acid present in the SQAGs. The inhibitory effect could be influenced by the chain size of fatty acids in the SQAGs. The sulfonyl group in quinovose was also needed to inhibit the enzymes. Lineweaver-Burk plots of SQAGs indicated that DNA polymerase alpha was non-competitively inhibited, but the SQAGs were effective as antagonists of both the template-primer DNA-binding and the nucleotide substrate-binding of DNA polymerase beta. Based on these results, the molecular actions of SQAGs and drug design strategies for developing new anti-neoplastic agents were discussed.

Structural analysis of epolactaene derivatives as DNA polymerase inhibitors and anti-inflammatory compounds

Epolactaene (compound 1), a neuritogenic compound found in human neuroblastoma cells, was found to show anti-inflammatory activity in vivo in this study. DNA polymerases and DNA topoisomerase II (topo II) were some of the major molecular targets of compound 1. Since the agent seems to be a potential pharmaceutical medicine, we synthesized derivatives chemically and obtained seven compounds, 1 to 7 to screen clinically more efficient epolactaene derivatives. A comparison of its structural derivatives revealed that the long alkyl side chain seemed to have an important role in the inhibitory effect. Notably, C18-alkyl chain conjugated epolactaene (compound 5) was the strongest inhibitor of DNA polymerase alpha, beta, lambda (pol alpha, beta, lambda) and topo II, with IC50 values of 13, 135, 4.4 and 5 microM, respectively, and 500 microg of compound 5 caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 65.0%). Compound 5 did not influence the activities of plant or prokaryotic DNA polymerases, or of other DNA metabolic enzymes such as telomerase, RNA polymerase and deoxyribonuclease I. Based on these results, the relationship among the three-dimensional structure of epolactaene derivatives and the inhibition of polymerases and topo II, and anti-inflammation is discussed.

Chromosomal Instability at Common Fragile Sites in Seckel Syndrome

Seckel syndrome (SCKL) is a rare, genetically heterogeneous disorder, with dysmorphic facial appearance, growth retardation, microcephaly, mental retardation, variable chromosomal instability, and hematological disorders. To date, three loci have been linked to this syndrome, and recently, the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) was identified as the gene mutated at the SCKL1 locus. The ATR mutation affects splicing efficiency, resulting in low levels of ATR in affected individuals. Elsewhere, we reported increased instability at common chromosomal fragile sites in cells lacking the replication checkpoint gene ATR. Here, we tested whether cells from patients carrying the SCKL1 mutation would show increased chromosome breakage following replication stress. We found that, compared with controls, there is greater chromosomal instability, particularly at fragile sites, in SCKL1-affected patient cells after treatment with aphidicolin, an inhibitor of DNA polymerase alpha and other polymerases. The difference in chromosomal instability between control and patient cells increases at higher levels of aphidicolin treatment, suggesting that the low level of ATR present in these patients is not sufficient to respond appropriately to replication stress. This is the first human genetic syndrome associated with increased chromosome instability at fragile sites following replication stress, and these findings may be related to the phenotypic findings in patients with SCKL1.

The effects of dehydroaltenusin, a novel mammalian DNA polymerase alpha inhibitor, on cell proliferation and cell cycle progression.

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase alpha in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 microM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.

Fibroblast growth factor-10 attenuates H2O2-induced alveolar epithelial cell DNA damage: role of MAPK activation and DNA repair.

Fibroblast growth factor-10 (FGF-10), an alveolar epithelial cell (AEC) mitogen that is critical for lung development, may promote AEC repair. We determined whether FGF-10 attenuates H2O2-induced, A549 and rat alveolar type II cell DNA damage. We show that FGF-10 prevents H2O2-induced DNA damage assessed by an alkaline elution, ethidium bromide fluorescence as well as by a comet assay. Mitogen-activated protein kinase inhibitors abolished the protective effect of FGF-10 against H2O2-induced DNA damage yet had no effect on H2O2-induced DNA damage. A Grb2-SOS inhibitor (SH3 binding peptide), an Ras inhibitor (farnesyl transferase inhibitor 277), and an Raf-1 inhibitor (forskolin) each prevented FGF-10- and H2O2-induced A549 cell ERK1/2 phosphorylation. Also, FGF-10 and H2O2 each induced negligible ERK1/2 phosphorylation in Ras dominant-negative (N17) cells. Inhibitors of Ras and Raf-1 blocked the protective effect of FGF-10 against H2O2-induced DNA damage but had no effect on H2O2-induced DNA damage. Furthermore, cold conditions and aphidicolin, an inhibitor of DNA polymerase-alpha, -delta, and -epsilon, each blocked the protective effects of FGF-10, suggesting a role for DNA repair. We conclude that FGF-10 attenuates H2O2-induced AEC DNA damage by mechanisms that involve activation of Grb2-SOS/Ras/RAF-1/ERK1/2 pathway and DNA repair.

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.

Inhibition of replication induces non-apoptotic cell death in fibroblast cell lines derived from LEC rats.

Hydroxyurea (HU), an anticancer drug, inhibits ribonucleoside diphosphate reductase and reduces pool sizes of deoxyribonucleoside triphosphate (dNTP). The reduction of dNTP results in inhibition of DNA replication. The cytotoxic effect of HU was investigated using fibroblast cell lines from LEC rats. LEC rat cells showed significantly higher sensitivity to HU than did cell lines from control WKAH rats. No significant differences were observed between the percentages of apoptotic cells in either LEC or WKAH rat cells that had been treated with HU and those that had not been treated with HU. LEC rat cells also showed significantly higher sensitivity to aphidicolin, which blocks DNA synthesis by inhibiting DNA polymerase alpha, than did WKAH rat cells. In both LEC and WKAH rat cells, intensified bands of p53 protein were observed immediately after treatment with HU. Although the high level of p53 protein persisted in WKAH rat cells until 6 hr post-incubation time after treatment with HU, the level of p53 protein had decreased at 6 hr post-incubation time in LEC rat cells. When the cells were X-irradiated in the absence or presence of HU, the ratio of the surviving fraction without HU to that with HU only slightly increased after X-irradiation in WKAH rat cells. In contrast, the ratio in LEC rat cells significantly increased after X-irradiation in a dose-dependent manner.

Selective inhibition of mammalian DNA polymerase alpha by vitamin D2 and D3.

As described previously (H. Togashi et al. Biochem Pharmacol. 1998;56:583-590), the irradiated products of provitamin D(2) (ergosterol) inhibit the activities of eukaryotic DNA polymerases. In this report, therefore, we investigated whether vitamin D and its related compounds inhibited the activities of DNA polymerases. As expected, vitamin D(2) and vitamin D(3) were found to be selective inhibitors of mammalian DNA polymerase alpha (pol alpha) with IC(50) values of 123 and 96 micro M, respectively. On the other hand, provitamin D(2), provitamin D(3), and the active form of vitamin D(3) such as 1alpha,25-dihydroxyvitamin D(3) could not influence any of the DNA polymerase activities. Interestingly, vitamin D(3)-3beta-sulfate was a much stronger pol alpha inhibitor with an IC(50) value of 7.1 micro M. Vitamin D(2), vitamin D(3), and vitamin D(3)-3beta-sulfate could prevent the growth of NUGC-3 human gastric cancer cells with LD(50) values of 133, 77, and 44 micro M, respectively, but provitamin D(2) and provitamin D(3) could not. The cells were halted at the G1 phase in the cell cycle by these compounds.

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs.

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.

Proposed mechanism for the reaction catalyzed by a diterpene cyclase, aphidicolan-16beta-ol synthase: experimental results on biomimetic cyclization and examination of the cyclization pathway by ab initio calculations.

To examine the mechanism of the cyclization reaction catalyzed by aphidicolan-16beta-ol synthase (ACS), which is a key enzyme in the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha, skeletal rearrangement of 2a and biomimetic cyclization of 4b were employed. The structures of the reaction products, which reflect penultimate cation intermediates, allowed us to propose a detailed reaction pathway for the Lewis acid-catalyzed cyclizations and rearrangements. Isolation of these products in an aphidicolin-producing fungus led us to speculate that the mechanism of the ACS-catalyzed cyclization reaction is the same as that of a nonenzymatic reaction. Ab initio calculations of the acid-catalyzed reaction intermediates and the transition states indicate that the overall reaction catalyzed by ACS is an exothermic process though the reaction proceeds via an energetically disfavored secondary cation-like transition state. In conjunction with the solvent effect in the acid-catalyzed reactions, this indicates that the actual role of ACS is to provide a template which enforces conformations of the intermediate cations leading to the productive cyclization although it has been believed that the cation-pi interaction between cation intermediates and aromatic amino acid residues in the active site is important for the enzymatic catalysis. This study provided important information on the role of various cationic species, especially secondary cation-like structures, in both nonenzymatic and enzymatic reactions.

Dehydroaltenusin, a Mammalian DNA Polymerase α Inhibitor

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol alpha) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol alpha. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as delta and epsilon; it also showed no effect even on the pol alpha activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 microm. Dehydroaltenusin-induced inhibition of mammalian pol alpha activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol alpha, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol alpha. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol alpha, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.

Auxin Induction of Cell Cycle Regulated Activity of Tobacco Telomerase

Telomerase activity was measured at each phase of the cell cycle in synchronized tobacco (Nicotiana tabacum) BY-2 cells in suspension culture with the use of the telomeric repeat amplification protocol assay. The activity was low or undetectable at most phases of the cell cycle but showed a marked increase at early S phase. The induction of telomerase activity was not affected by the S phase blockers aphidicolin (which inhibits DNA polymerase alpha) or hydroxyurea (which inhibits ribonucleotide reductase), but it was prevented by olomoucine, an inhibitor of Cdc2/Cdk2 kinases that blocks G(1)-S cell cycle transition. These results suggest that the induction of telomerase activity is not directly coupled to DNA replication by conventional DNA polymerases, but rather is triggered by the entry of cells into S phase. Various analogs of the plant hormone auxin, including indole-3-acetic acid, alpha-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, potentiated the increase in telomerase activity at early S phase; the growth-inactive analog 2,3-dichlorophenoxyacetic acid, however, had no such effect. Potentiation by indole-3-acetic acid of the induction of telomerase activity was dose dependent. Together, these data indicate that telomerase activity in tobacco cells is regulated in a cell cycle-dependent manner, and that the increase in activity at S phase is specifically inducible by auxin.

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity.

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase α at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase α activity. © 1999 Cancer Research Campaign