The effect of divalent nickel (Ni2+) on in vitro DNA replication by DNA polymerase alpha.

The effects of the carcinogenic metal nickel on DNA polymerase alpha (pol alpha) activity and fidelity have been analyzed. In the absence of Mg2+, the presence of Ni2+ ions at concentrations below 0.25 mM gave rise to a dose-dependent activation of pol alpha as monitored by [3H]dTMP incorporation into an activated DNA template. The apparent Km for Ni(2+)-dependent pol alpha incorporation of dTTP was estimated to be 25 microM, which was about 10 times higher than the Km for Mg2+ (2.3 microM). Above 0.25 mM, Ni2+ caused a dose-dependent inhibition of pol alpha activity and the Ki was calculated to be 1.5 mM. Scatchard analyses showed that Ni2+ binds to affinity-purified pol alpha and associated proteins at two tight binding sites with a Kd of approximately 50 microM and at eight weak binding sites with a Kd of approximately 4 mM. In the presence of 2 mM Mg2+, the addition of Ni2+ to the reactions caused an inhibition of polymerase activity. The inhibition patterns tended to switch from competitive to mixed-type to noncompetitive as a function of Ni2+ concentration. Lastly, Ni2+ increased the incorporation of the modified nucleotide dideoxy-CMP in reactions using varying ratios of dideoxy-CTP/dCTP.

New perspectives in the treatment of acute myeloid leukemia by hematopoietic growth factors.

The application of hematopoietic growth factors in the treatment on acute myeloid leukemia (AML) may principally aim at shortening the period of treatment associated neutropenia and reducing the rate of infectious complications by their post-therapeutic administration but may also be used to increase the sensitivity of leukemic blasts to antileukemic therapy by pretherapeutic growth stimulation. Both aspects were addressed in subsequent clinical phase II studies and preclinical investigations. In a first clinical trial, 36 patients with high-risk AML received granulocyte-macrophage colony-stimulating factor (GM-CSF) after successful cytoreductive chemotherapy and experienced a shortening of the period of post-therapeutic neutropenia by 6 to 9 days, leading to a significant reduction of treatment-associated deaths from 39% to 14%. In preclinical studies an enhancement of the cytotoxicity of cytosine arabinoside (AraC) on leukemic blasts could be shown by pretreatment with GM-CSF or IL-3. Investigations on the impact of hematopoietic growth factors on the intracellular metabolism of AraC indicated that this effect was primarily mediated by an increase in the activity of DNA-polymerase-alpha. The evaluation of different doses of AraC showed the most marked increase after the combination of GM-CSF with conventional rather than high doses of AraC. Based on these preclinical experiments, a prospective randomized trial was subsequently initiated investigating the effect of GM-CSF before and during induction, consolidation, and the first two cycles of maintenance chemotherapy in newly diagnosed AML. This ongoing trial has enrolled 67 patients at the current time. An early interim analysis showed no differences in remission rates but a tendency toward a longer remission duration in patients receiving GM-CSF. These data indicate that hematopoietic growth factors like GM-CSF in particular may provide a new perspective in the treatment of acute myeloid leukemia with the possibility of reducing treatment associated mortality and perhaps of increasing the efficacy of antileukemic treatment.

Murine cytomegalovirus DNA polymerase: purification, characterization and role in the antiviral activity of acyclovir.

Murine cytomegalovirus (MCMV) neither induces a viral thymidine kinase (TK) nor enhances the activity of a cellular TK. Nevertheless, MCMV is highly susceptible to 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV). The cellular TK is neither responsible for phosphorylation of ACV nor its anti-MCMV activity. This is clear from the findings that little ACV triphosphate is formed in MCMV-infected mouse embryo fibroblasts (MEF) and that the replication of MCMV is inhibited equally well by ACV in TK+ and TK- cells. Even if trace amounts of ACV triphosphate would be formed by enzymes other than TK, and ACV triphosphate would be responsible for the anti-MCMV activity of ACV, then the MCMV DNA polymerase ought to be highly sensitive to ACV triphosphate. To examine this possibility, the MCMV DNA polymerase was partially purified and characterized. The apparent Ki value of the MCMV DNA polymerase for ACV triphosphate indicates that the sensitivity of the MCMV DNA polymerase to ACV triphosphate is equivalent to that of the HSV DNA polymerase. Therefore, the trace amounts of ACV triphosphate that are formed in MCMV-infected MEF seem to be insufficient to inhibit MCMV DNA polymerase and may not play a key role in the anti-MCMV activity of ACV.

Chemical modifications of aminonaphthalenesulfonic acid derivatives increase effectivity and specificity of reverse transcriptase inhibition and change mode of action of reverse transcriptase and DNA polymerase alpha inhibition.

The reverse transcriptase (RT) inhibition and the specificity of 15 aminonaphthalenesulfonic acid derivatives were examined with RT of a simian immunodeficiency virus derived from an African green monkey (SIVagmTYO-7). The two compounds with the strongest RT inhibition (NF415) or the highest specificity (NF345), together with suramin, were evaluated against polymerase alpha-primase complex from calf thymus. We have also compared the kinetics of inhibition of the viral and the cellular polymerase by these three compounds. While RT inhibition followed a mixed competitive and non-competitive mechanism, inhibition of the DNA polymerase alpha was competitive for suramin and non-competitive for NF415 and NF345. Certain structural characteristics appeared to be common for specific RT inhibitors.

Mechanism of caffeine modulation of the antitumor activity of adriamycin.

We examined the effects of a combination of adriamycin (ADR) and caffeine on DNA and protein biosynthesis and on the activities of DNA polymerase alpha and beta in normal and tumor tissue. The decrease in DNA and protein biosynthesis in tumor produced by caffeine combined with ADR were 2.5 and 2.4 times greater, respectively, compared with ADR alone. The combination of caffeine and ADR enhanced the decrease in DNA polymerases activities in the tumor which was induced by ADR, the decreases being 1.8 and 1.6 times greater, respectively, than that of ADR alone. In contrast, these ADR-induced changes in normal tissues were not enhanced by the combination with caffeine. The combination with caffeine had no effect on ADR concentration in normal tissues, but in the tumor, it increased the ADR concentration to 2.1 times that of ADR alone. In vitro, ADR efflux from Ehrlich ascites carcinoma cells was significantly inhibited by exposure to caffeine. These findings indicate that the effect of caffeine on ADR concentration in the cell plays an important role in the mechanism by which caffeine enhances ADR antitumor activity.

Effects of Tokishakuyakusan, Keishibukuryogan and Unkeito on DNA polymerase alpha activity in PMS-treated immature rat uterus incubated in vitro.

We have recently found that Tokishakuyakusan (TS), Keishibukuryogan (KB) or Unkeito (UT) inhibits in vivo DNA polymerase alpha activity in the rat uterus stimulated by PMS. In this study, uteri resected 24 h after injection of PMS on day 27 of age were incubated in vitro with 20 micrograms/ml of extract of TS, KB or UT for 4 h. The DNA polymerase alpha activity in uteri tended to decrease after the addition of TS, KB or UT with significant difference (P < 0.05) compared with TS-, KB- and UT-untreated control groups. These results suggest that TS, KB or UT, especially KB, tends to inhibit directly the enzyme activity in rat uterus.

Catalytic properties of DNA polymerase alpha activity associated with the heart-stabilized nuclear matrix prepared from HeLa S3 cells.

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase alpha associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37 degrees C prior to subfractionation, as has been reported previously for DNA polymerase alpha bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase alpha activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase alpha activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase alpha activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2 M NaCl or 0.25 M (NH4)2SO4 and led us to consider that a 37 degree incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase alpha which is unlikely to be involved in DNA replication in vivo.

The anti-cancer drug camptothecin inhibits elongation but stimulates initiation of RNA polymerase II transcription.

Camptothecin is a widely used anti-tumor drug that specifically inhibits DNA topoisomerase I. It is believed that topoisomerase I participates in the process of transcription by relaxing torsional stress induced in the duplex DNA by the elongating RNA polymerase. We have assessed the effects of camptothecin on RNA polymerase II transcription from the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Using in vivo [3H]uridine pulse labeling and in vitro nuclear run-on techniques to estimate relative rates of transcription, it was found that camptothecin stimulated RNA synthesis from promoter-proximal sequences of the DHFR gene, while transcription from promoter-distal sequences was reduced. Furthermore, camptothecin caused a significant accumulation of RNA polymerases in the 5'-end of the DHFR gene. The effect of camptothecin on transcription was reversible, resulting in a wave of RNA synthesis recovery in a 5' to 3' direction through the DHFR gene following a chase with camptothecin-free medium. We conclude that camptothecin stimulates initiation but inhibits elongation of the RNA polymerase II transcribed DHFR gene.

Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid.

Mutations that conferred sensitivity to the pyrophosphate analog phosphonoacetic acid in bacteriophage T4 DNA polymerase were identified. The mutations were loosely clustered in four regions of the gene. As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to phosphonoacetic acid also altered sensitivity to nucleotide analogs. Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity. Kornberg (A. Kornberg, Science 163:1410-1418, 1969) envisioned a DNA polymerase active center which accommodates primer terminus and template DNAs and the incoming nucleotide. Some mutations identified on the basis of sensitivity to phosphonoacetic acid may be part of such an active center because single amino acid substitutions simultaneously alter several DNA polymerase functions.

Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1.

Introduction of a reactive 5-mercapto group into some of the cytosine and/or uracil bases of various oligo- and polynucleotides by partial thiolation resulted in several potent inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) in primary human lymphocytes. These compounds exhibited little if any toxicity against uninfected peripheral blood mononuclear cells and showed 15 to 75 times higher antitemplate activity against a p66/p51 HIV-1 recombinant reverse transcriptase (RT) than against the DNA polymerase alpha from human lymphocytes. In contrast, the unthiolated oligo- and polynucleotides are void of antitemplate activity, and their apparent inhibitory effect on HIV-1 closely paralleled their toxicity for the cells. Partially thiolated poly(dC) (MPdC) was the most potent of all the compounds tested against HIV-1 in peripheral blood mononuclear cells (50% effective concentration, 1.8 micrograms/ml or 0.019 microM), while showing low cytotoxicity (greater than 100 micrograms/ml). The corresponding unmodified poly(dC) showed no anti-HIV-1 activity at 50 micrograms/ml but had pronounced cytotoxicity. MPdC was also a potent inhibitor of HIV-1 RT (50% inhibitory concentration, 0.30 micrograms/ml). The inhibitory activities of thiolated homooligo(dCs) against both HIV-1 replication and HIV-1 RT increased with increasing chain length. The heterooligonucleotides included in this study were designed as structural analogs of portions of the natural primer of HIV-1 RT, i.e., tRNA(3Lys). An 18-mer analog of the 3' terminus, complementary (antisense) to the primer-binding site of the HIV-1 genome, was attached to an oligo(dC) tail and 5-thiolated; this increased its activity and decreased its toxicity. This compound will serve as a new lead in the development of more effective antitemplates against HIV-1.

Inhibition of DNA polymerase alpha expression and cell growth, a possible triple helix mechanism.

The antiproliferative effects mediated by a 14-mer homopyrimidine oligonucleotide (5' CTTTCT-CTTTTCTC3'), designed to form DNA triplex with a purine region of the DNA polymerase alpha promoter, were evaluated on the human breast cancer cell line MDA-MB 231. In order to stabilize the triple complex under physiologic conditions, replacement of cytosines by methylcytosines in the oligomer sequence was carried out. Band-shift analyses demonstrated a complete triplex formation between the radiolabeled target duplex DNA and the methylcytosine-modified oligomer at the concentration of 0.1 microM under physiologic pH and temperature. A single exposure of MDA-MB 231 cells to 0.5 microM methylcytosine-modified oligonucleotide was able to markedly reduce the cell number and the percentage of cells in DNA synthesis up to 58% and 66%, respectively, compared with controls. Furthermore, a 48% reduction in the amount of the DNA polymerase alpha mRNA was reported after treatment with the oligomer. In conclusion, data from the present study demonstrate that an oligonucleotide to DNA polymerase alpha promoter, designed to form a triple helix with target double-stranded DNA, inhibits the expression of the reporter gene at the biologic and molecular levels, suggesting a possible triplex-mediated mechanism of action.

High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants.

Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.

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.

Sequence-dependent termination of mammalian DNA polymerase reaction by a new platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-plati num(II) monohydrate).

We examined the mechanism of the inhibition of DNA synthesis by a new platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato+ ++)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis-diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase alpha were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase beta was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.

Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.