Effect of aphidicolin on DNA synthesis in HSV-1 infected and uninfected Vero cells

The effect of aphidicolin on DNA synthesis in herpes simplex virus type 1 (HSV-1) infected and uninfected Vero cells was determined by isodensity banding of [³²P]-labelled DNA. A 50% inhibition of HSV-1 DNA synthesis was observed at 0.07 μM aphidicolin while 2.1 and 1.3 μM were required to inhibit the cellular DNA synthesis to 50% in infected and uninfected Vero cells, respectively. When the viral DNA synthesis was totally inhibited by 10 μM aphidicolin, the cellular DNA synthesis was inhibited to about 90% in both infected and uninfected cells. Aphidicolin inhibited the cellular DNA synthesis in HSV-1 infected and uninfected Vero cells remaining in the presence of 250 μM foscarnet to the same extent as the DNA synthesis in the absence of foscarnet.     

In vitro DNA strand scission and inhibition of nucleic acid synthesis in L1210 leukemia cells by a new class of DNA complexers, the anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles)

CI-937 and CI-942 belong to a new class of DNA complexers, the anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles), and are being further developed as antitumor drugs based on their curative properties against murine solid tumour models. The biochemical effects of these agents were studied in L1210 leukemia in relation to other clinically used intercalators. After a 1-hr exposure, CI-937 and CI-942 reduced the cloning efficiency of L1210 cells by 50% at 3.0 X 10(-8) and 1.5 X 10(-7) M respectively. Based on an ethidium displacement assay, these drugs bound strongly to DNA, reducing the fluorescence of an ethidium-DNA complex by 50% at concentrations of 23 and 33 nM for CI-937 and CI-942 respectively. This was comparable to mitoxantrone at 15 nM, but much more potent than Amsacrine which required over 1.3 microM. A distinct property of the anthrapyrazoles was a much more potent inhibitory effect on whole cell DNA synthesis than on RNA synthesis. After L1210 cells were exposed to drug for 2 hr the concentration needed to inhibit DNA synthesis by 50% was 0.33 and 0.57 microM for CI-937 and CI-942, respectively, whereas 2.0 and 11.3 microM were required to inhibit RNA synthesis by the same extent. This was in contrast to Adriamycin and mitoxantrone which inhibited both activities equally at similar concentrations. It was apparent that the inhibition of these processes was not due to substrate depletion since intracellular ribonucleoside and deoxyribonucleoside triphosphates either remained constant or were elevated after a 2-hr exposure to 1 or 10 microM drug. A similar discriminatory effect was observed on DNA and RNA polymerase in permeabilized cells, and the inhibition of nucleic acid synthesis in this system could be reversed by exogenously added DNA. Since the high incidence of cardiotoxicity associated with the administration of anthracyclines has been related to the formation of reactive oxygen species, the ability of the anthrapyrazoles to augment superoxide dismutase sensitive oxygen consumption was observed in a rat liver microsomal system. CI-937 and CI-942 induced 5- and 10-fold less oxygen consumption than Adriamycin, producing rates of 12.4, 24.2 and 138.9 nmoles/min/mg microsomal protein, respectively, at a drug concentration of 0.5 mM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of arabinosyladenine in herpes simplex virus-infected and uninfected cells: Correlation with inhibition of DNA synthesis and role in antiviral selectivity

The metabolism of 9-β-D-arabinofuranosyladenine (ara-A, vidarabine) and its effects on DNA synthesis were compared in uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells. In the absence of an inhibitor of adenosine deaminase, ara-A was deaminated to 9-β-D-arabinofuranosylhypoxanthine and phosphorylated to ara-A-5′-mono-, di- and triphosphates in both types of cells. When an inhibitor of adenosine deaminase (coformycin) was added to cell cultures, nucleotides were the only metabolites detected—primarily the 5′-triphosphate of ara-A (aATP). Detailed studies performed in the presence of coformycin established that the net rate and extent of aATP formation were the same in uninfected and HSV-1-infected cells. After a 12-hr exposure to 50 μM ara-A, intracellular concentrations of aATP were approximately 40 μM. Levels of aATP correlated directly with inhibition of total DNA synthesis. Approximately 0.7 μM aATP was required for 50% inhibition of total DNA synthesis in both uninfected and HSV-1-infected cells. Following removal of ara-A-containing culture medium, aATP levels in uninfected cells declined with a half-life of 3.2 hr. In marked contrast, the half-life in HSV-1-infected cells was 9.3 hr; this may explain why as little as a 3-hr exposure to ara-A resulted in a significant HSV-1 titer reduction. Taken together, the data show that when ara-A was removed from culture medium, levels of aATP persisted longer in HSV-1-infected cells thereby prolonging antiviral activity. This effect could be important in vivo where levels of ara-A oscillate with dosing schedule.     

Reversal of the cytotoxicity of 3'-amino-3'-deoxythymidine by pyrimidine deoxyribonucleosides.

Mammalian cell replication is strongly inhibited by 3′-amino-3′deoxythymidine (3′-aminothymidine). This cytotoxieity can be specifically prevented or reversed by pyrimidine 2′-deoxyribonucleosides. The addition of 50 μM 2′-deoxycytidine to L1210 cells treated with 10 μM 3′ the population doubling time from about 38 hr to 17 hr. The control cells doubled every 13 hr. Another cytotoxic effect produced by 3′-aminothymidine is a dose- and time-dependent increase in cell volume. 2′-Deoxycytidine can effectively prevent and reverse this increase. 3′-Aminothymidme appears to be a potent selective inhibitor of DNA synthesis in L1210 cells. The incorporation of [³H]thymidine into DNA was inhibited by 50 per cent at 1 μM 3′-aminothymidine, a concentration which reduced L1210 replication by about 65 per cent. The rate of incorporation of [³H] adenine into DNA, another measure of DNA synthesis, was reduced similarly by 3′-aminothymidine. and 2′-deoxycytidine eliminated this inhibition as well. An effect on RNA or protein synthesis was not detected. The incorporation of [³H] uridine or [³H] adenine into RNA, or of tritiated amino acids into protein, was not reduced by 25 μM 3′-aminothymidine. These results suggest that selective disruption of DNA metabolism may account for the cytotoxicity of 3′-aminothymidine.     

Relationship between the oxidation potential of benzene metabolites and their inhibitory effect on DNA synthesis in L5178YS cells

The effects of benzene and its metabolites on the rate of DNA synthesis were measured in the mouse lymphoma cell line, L5178YS. The direct toxicity of benzene could be distinguished from that of its metabolites since bioactivation of benzene in L5178YS cells was not observed. Cells were exposed to benzene, phenol, catechol, hydroquinone, p-benzoquinone, or 1,2,4-benzenetriol over the range of 1.0 X 10(-7) to 1.0 X 10(-2) M for 30 min, and the rate of DNA synthesis was measured at various times after chemical washout. Cell viability and protein synthesis were determined by trypan blue dye exclusion and [3H]leucine incorporation, respectively. Effects were designated as "DNA specific" when DNA synthesis was inhibited in the absence of discernible effects on cell membrane integrity and protein synthesis. Concentrations of benzene as high as 1 mM had no effect on DNA synthesis. Comparison of the effects at the maximum nontoxic dose for each compound showed that catechol and hydroquinone were the most effective, inhibiting DNA synthesis by 65%. Phenol, benzoquinone, and benzenetriol inhibited DNA synthesis by approximately 40%. Maximum inhibition was observed 60 min after metabolite washout in each case. Benzoquinone was the most potent inhibitor of DNA synthesis, followed by hydroquinone, benzenetriol, catechol, and phenol with ED50 values of 5 X 10(-6), 1 X 10(-5), 1.8 X 10(-4), 2.5 X 10(-4), and 8.0 X 10(-4), respectively. Cyclic voltammetric experiments were performed on the hydroxylated metabolites of benzene to assess the possible involvement of a redox-type mechanism in their inhibition of DNA synthesis. The ease of oxidation of these metabolites correlated with their ED50 values for inhibition of DNA synthesis (r = 0.997). This suggests that oxidation of phenol or one of its metabolites may be necessary for production of the species involved in inhibition of DNA synthesis.     

Limited ability of thymidine to relieve mitotic inhibition by pyrimethamine in human fibroblasts.

Pyrimethamine (PY), an antimalarial drug with antileukaemic properties, is a folate analogue which inhibits the formation of tetrahydrofolic acid (THFA). As a result it blocks the biosynthesis of glycine, methionine, purines, pantothenate and thymidine. It also prevents the formylation of methionyl-tRNA and hence precludes mitochondrial protein synthesis. In human fibroblast cultures treated with PY at concentrations of 30, 100 or 200 μg/ml in the presence of the essential metabolites listed above, except thymidine, DNA synthesis and mitosis were arrested. The ability of thymidine to prevent this depended on the PY concentration and the duration of exposure to the drug. Thus, in the presence of thymidine. cells cultured in 30 μg/ml PY exhibited normal growth even after 2 days in contact with the drug; at 100 μg/ml PY. DNA synthesis continued but there was a reduction in the mitotic rate. which became more marked as the exposure time to the drug increased; at 200 μg/ml PY, there was no DNA synthesis or mitosis. These results indicate that the antimitotic effect of PY cannot be wholly accounted for by thymidine starvation. Oxygen uptake measurements showed a markedly decreased respiratory activity in fibroblasts which had been in contact with 100 μg/ml PY for 24 hr in the presence of the above metabolites, including thymidine. It is proposed that a major factor in the antimitotic activity of PY is the inhibition of mitochondrial protein synthesis.     

异靛甲体外抑制DNA和RNA生物合成的抗癌作用机理

以同位素参入法及光谱法观察异靛甲对核酸合成的抑制作用和药物与DNA相互作用。结果表明,异靛甲对DNA,RNA合成有较强的抑制作用,其IC_(50)分别为9和15μmol/L,药物作用非常迅速。30μmol/L异靛甲作用15μmin后DNA,RNA合成被抑制95％以上。实验还表明,异靛甲不能损伤DNA模板,也不抑制DNA拓扑异构酶及DNA多聚酶Ⅰ,但能明显抑制T7 RNA多聚酶,在’100μmol/L浓度下对mRNA的合成抑制达70％以上。     

Incorporation into nucleic acids of the antiherpes guanosine analog buciclovir, and effects on DNA and protein synthesis

Using cells expressing herpes simplex virus (HSV) thymidine kinase, we investigated the metabolism of the acyclic antiherpes guanosine analog buciclovir, in relation to the effects of the drug on viral DNA and protein synthesis. In these cells the predominant metabolite of buciclovir was its triphosphate, as in the HSV-1 infected Vero cells investigated in parallel. Further metabolism of buciclovir led to incorporation into RNA and DNA. Buciclovir inhibited DNA synthesis, not RNA synthesis, and prevented an increase in the size of newly synthesized DNA. To study the relative effects of BCV on cellular and viral DNA synthesis, human TK-cells transformed to a TK+ phenotype with HSV-2 DNA, were infected with HSV-1. In these HSV-1 infected cells buciclovir-triphosphate caused a preferential inhibition of viral DNA synthesis. Despite incorporation of buciclovir into RNA, and the presence of buciclovir-triphosphate from the time of infection onwards, no effect was observed on the synthesis of the beta proteins ICP-6 and ICP-8. Presumably as a consequence of inhibition of viral DNA synthesis, the synthesis of a beta gamma protein (gD) and a gamma protein (gC) were inhibited, and synthesis of the beta proteins (ICP-6 and ICP-8) was not shut-off. Glycosylation of gC that was still synthesized, was not inhibited. Thus, the biological effects of buciclovir can be explained by its inhibition of DNA synthesis.     

Inhibition of DNA, RNA and protein synthesis and chromatin alteration by N-hydroxyphenacetin

The effects of N-hydroxyphenacetin on DNA function and structure were investigated to elucidate the involvement of phenacetin in analgesic nephropathy and transitional cell carcinoma. N-Hydroxyphenacetin or a metabolite inhibited synthesis of DNA, RNA and protein; DNA inhibition was greater at higher pH. No single-strand breaks were detectable in DNA after N-hydroxyphenacetin treatment and no appreciable effect on cell viability was observed at concentrations up to 5 mM. N-Hydroxyphenacetin-induced alteration to chromatin structure was detected using nucleoid sedimentation analysis. Direct binding to plasmid DNA was not observed. These observations are consistent with a role for phenacetin metabolites in renal disease.     

Bleomycin inhibition of DNA synthesis in isolated enzyme systems and in intact cell systems.

Blcomycin (BLM) inhibits DNA and RNA synthesis in different isolated enzyme systems. The inhibition effect can be reduced by adcling RNA to the reaction mixture. The activity of the RNA dependent DNA polymerase and of a cell-free protein synthesizing system is not affected by BLM. The antibiotic reduces cell proliferation (L5178y mouse lymphoma cells) in vitro at low concentrations by cytostatis and at higher concentrations by cytotoxicity. In BLM-treated L5178y cells DNA synthesis is strongly reduced, while RNA and protein synthesis are not affected. In vivo, using growing quail oviducts, cell proliferation and cytodifferentiation are markedly inhibited after BLM treatment. This is attributed to the observed inhibition of DNA synthesis. RNA and protein synthesis as well as gene expression are not influenced by BLM under the conditions used.     

Non-selective decrease of collagen synthesis by cultured fetal lung fibroblasts after non-lethal doses of ethanol

Addition of ethanol to cultured fetal lung fibroblasts resulted in decreases of both collagen and noncollagen protein syntheses. The inhibitory effect of ethanol on protein synthesis was dependent on the concentration of ethanol and the number of treatments with ethanol. Significant inhibition of collagen and noncollagen protein synthesis was observed 3 hr after a single treatment in 0.25% (v/v) ethanol. The maximum inhibitory effect of ethanol on protein synthesis was observed at 6 hr after drug addition. Inhibition of protein synthesis was observed when either proline or glycine was used as the precursor amino acid. An inhibition of alcohol dehydrogenase did not block the ethanol-mediated inhibition of protein synthesis. Ethanol, added to cell cultures throughout the log phase, inhibited cell growth during the late log and stationary phases. Ethanol inhibition of collagen and noncollagen protein synthesis was reversed when the cell cultures were washed and suspended in fresh media for 24 hr. These inhibitory effects of ethanol on macromolecular syntheses were not engendered by killing of cells. The viability of the cells, as indicated by trypan blue exclusion, was not affected significantly at the concentrations of ethanol used. The inhibitory effect of ethanol on protein synthesis also did not originate from drug-mediated inhibition of precursor amino acid uptake. Polysomes isolated from ethanol-treated fibroblasts incorporated proline into protein at a rate which was reduced commensurate with cellular protein synthesis. The resultant inhibition by ethanol of protein synthesis was not attributable to a direct effect of drug on polysomes. Treatment of fetal lung fibroblasts with ethanol also caused a marked inhibition of radioactive thymidine and uridine incorporation, indicating a reduction of both total cellular DNA and RNA synthesis. Accordingly, the decrease of protein synthesis resulted from inhibition of RNA synthesis. Furthermore, messenger RNA synthesis may have decreased since polysomes isolated from ethanol-treated fibroblasts synthesized less protein in the wheat germ cell-free system. Unlike other biochemical variables that were inhibited by ethanol treatment, the level of prolyl hydroxylase activity was elevated significantly. The elevated level of prolyl hydroxylase activity, however, was related neither to the rate of collagen polypeptide synthesis nor to the degree of proline hydroxylation of cellular collagen. The data suggest that the growth-retarding effects of nonlethal doses of ethanol on fetal development may result from inhibition of macromolecular synthesis in fetal fibroblasts.     

Biological properties of benzodifuran derivatives

The antiviral activity and the effect on DNA synthesis of two benzodifuran compounds were studied. DNA and some RNA viruses were significantly inhibited by concentrations ranging from 15 to 30 nM/ml. The inhibition of DNA synthesis in host cells was obtained with concentrations higher than those inhibiting virus replication. A favourable ratio between antiviral activity and inhibition of DNA synthesis of the host cells is present in these compounds. This activity is substantially due to the ability of the compounds to complex with DNA.     

Antiviral activity of 2,3-dihydro-1H-imidazo[1,2-b]pyrazole in herpes simplex virus type 1-infected mammalian cells.

2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a known inhibitor of DNA synthesis, has been shown to be a useful drug for the synchronization of mammalian cells in culture. Recent studies in our laboratory indicate that IMPY may possess significant antiviral activity against herpes simplex virus (HSV) type 1. IMPY. at a concentration of 80 μg/ml or 0.73 mM, reduced syncytia formation approximately 80 per cent. A 50 per cent inhibitory dose was calculated for each drug in order to compare potency in syncytia reduction of IMPY with that of arabinosyladenine (ara-A) and arabinosyl-hypoxanthine (ara-H). Our results indicated that the antiviral potencies of the three drugs were ranked in the order ara-A > ara-H > IMPY, the 50 per cent inhibitory doses being 22, 195 and 309 μM respectively. Utilizing the microplate procedure of Sidwell and Huffman [Appl. Microbiol.22, 797 (1971)], inhibition of viral cytopathic effect was rated against drug cytotoxicity and a virus rating (VR) established. A virus rating of 0.68 was calculated for IMPY. In comparison, VR values of 0.84 and 0.66 were obtained for ara-A and ara-H respectively. In contrast to the syncytia reduction studies, IMPY appeared to possess antiviral activity equivalent to that of ara-H according to the criteria of the virus rating assay. A technique was developed for evaluating the degree of selectivity (°S) of a drug with respect to its differential effect on viral and cellular DNA synthesis. IMPY was found to possess a negative selectivity at all concentrations studied, reflecting the fact that it inhibited cellular DNA synthesis more than viral DNA synthesis. In contrast, ara-A and ara-H both expressed positive degrees of selectivity in that they inhibited viral DNA synthesis more extensively than cellular DNA synthesis.     

Selective inhibition of herpesvirus DAN synthesis by foscarnet

The inhibition of cellular and herpesvirus DNA synthesis by phosphonoformate (INN; foscarnet sodium) has been determined after isopycnic separation of cellular and viral DNA in CsCl gradients. The DNA synthesis was determined as the incorporation of ortho[³²P]phosphate and [³H]thymidine into DNA. A 50% inhibition of herpes simplex virus DNA synthesis was observed at 50 μM phosphonoformate. At this concentration cellular DNA synthesis was not inhibited. At 500 μM phosphonoformate more than 95% of the viral DNA synthesis was inhibited, while the cellular DNA synthesis in infected and uninfected cells were inhibited to about 10%. The same results were obtained in both Vero and GMK cells and using either ortho[³²P]phosphate or [³H]thymidine to label the newly synthesized DNA. The 50% inhibitory concentration of phosphonoformate was similar for inhibition of herpes DNA synthesis and plaque reduction.     

Effect of methylmercury (II) on the synthesis of deoxyribonucleic acid, ribonucleic acid and protein in HeLa S3 cells.

Exposing HeLa S3 cells to increasing concentrations of methylmercury CH₃Hg(II) for varying periods of time results in an abrupt cessation of intracellular events, such as DNA, RNA and protein syntheses, once the organomercurial concentration in the growth medium has exceeded a certain threshold, the value of which lies between 1 and 10 μM. The synthetic activities, expressed as the ability of the cells to incorporate ³H-labeled thymidine, uridine and leucine into the acid-insoluble fraction of the cells, decrease exponentially with time at a given methylmercury concentration and, at a given time, they cease in an abrupt, highly cooperative manner when plotted against the logarithm of the CH₃Hg(II) concentration, yielding sigmoidally shaped dose-response curves. The inhibition of intracellular macromolecular synthesis is reversible up to a certain CH₃Hg(II) level. For instance, cells that had been kept for 6 hr at CH³Hg(II) concentrations ranging from 2.5 to 10 μM did recover, albeit with lag periods that increased in length with increasing organomercurial concentration, as evidenced by their ability to continue RNA synthesis once the intoxicating medium had been removed. However, cells that had been exposed to 20 μM CH₃Hg(II) and above did not recover, at least not at incubation periods up to 91 hr (4.3 generation times), after termination of the 6 hr methylmercury treatment. In general, macromolecular synthesis decreases in the sequence DNA ~ RNA > protein, followed by cell death as soon as protein synthesis has become inhibited to a major degree. The acute effects of CH₃Hg(II) on intracellular DNA, RNA and protein syntheses were marginally different when HeLa S3 cells were exposed to methylmercury in three modes: as monolayers, as suspensions prepared from freshly trypsinized monolayer cells, and as suspension-cultures, demonstrating that, for instance, trypsin treatment of the cells increases their sensitivity toward methylmercury intoxication only slightly.     

Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, isolated nuclei and purified polymerases

The effects of lead acetate on DNA and RNA synthesis have been investigated with intact HeLa cells, isolated nuclei, and purified DNA and RNA polymerases. No inhibition of DNA or RNA synthesis in intact cells was found even after exposure to 0.5 mM lead acetate for 18 hr. In contrast, both DNA and RNA synthesis in isolated nuclei were inhibited by lead (with 50% inhibition at approximately 150 and 80 microM respectively). Similarly, both HeLa DNA polymerase alpha and RNA polymerase II were inhibited, with 50% inhibition obtained at approximately 150 and 20 microM lead acetate respectively. The inhibition of nucleic acid synthesis in isolated nuclei can thus be accounted for by inhibition of the polymerases. The sensitivity of Escherichia coli DNA polymerase I to lead acetate was found to be significantly greater than the HeLa DNA polymerase alpha (50% inhibition at only 10 microM), but the sensitivity of the E. coli RNA polymerase was the same as that of the HeLa enzyme.