The effect of adriamycin on Z-DNA formation and DNA synthesis.

The ability of adriamycin to inhibit Z-DNA formation induced by a high-salt environment was investigated. ADM inhibited this conversion, such that in poly (dG-dC) total inhibition was observed at 1 ADM: 9 base pairs and in eukaryotic DNA (calf thymus) at 1 ADM: 11,5 base pairs. Even at low ADM concentration, 1 ADM: 160 base pairs, some inhibition was observed. At similar ADM:DNA concentrations, an inhibition in DNA synthesis in cells in culture was observed, which showed some parallel with the inhibition of Z-DNA formation. A model is proposed where Z-DNA formation precedes DNA synthesis and where inhibition of the former could explain the antineoplastic nature of adriamycin.     

Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin.

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.     

Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates.

Transplasma membrane electron transport from HeLa cells, measured by reduction of ferricyanide or diferric transferrin in the presence of bathophenanthroline disulfonate, is inhibited by low concentrations of adriamycin and adriamycin conjugated to diferric transferrin. Inhibition with the conjugate is observed at one-tenth the concentration required for adriamycin inhibition. The inhibitory action of the conjugate appears to be at the plasma membrane since (a) the conjugate does not transfer adriamycin to the nucleus, (b) the inhibition is observed within three minutes of addition to cells, and (c) the inhibition is observed with NADH dehydrogenase and oxidase activities of isolated plasma membranes. Cytostatic effects of the compounds on HeLa cells show the same concentration dependence as for enzyme inhibition. The adriamycin-ferric transferrin conjugate provides a more effective tool for inhibition of the plasma membrane electron transport than is given by the free drug.     

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Is the Drug-Responsive NADH Oxidase of the Cancer Cell Plasma Membrane a Molecular Target for Adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC₅₀ for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.     

The SalGI restriction endonuclease. Enzyme specificity.

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.     

The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli.

The protein encoded by the gam gene of bacteriophage lambda ("gamma protein") is a specific inhibitor of the recBC enzyme of Escherichia coli. The lambda protein has been purified approximately 2,000-fold, and its structure and inhibitory activity have been characterized. It appears to be composed of two identical subunits of 16,500 daltons, inhibits all of the catalytic activities of the recBC enzyme with apparently equal efficiency, but has no effect upon any other E. coli or lambda-DNase tested. Inhibition does not occur unless recBC enzyme is exposed to gamma protein prior to reaction of the enzyme with DNA. The inhibitory activity is independent of temperature, and no catalytic activity has been detected that might fulfill the inhibitory function. It appears instead that the inhibition involves a stoichiometric, rather than a catalytic interaction between gamma protein and the enzyme. Reaction kinetics for the recBC enzyme inhibited by gamma protein show no anomalous protein--only a depressed rate. Inhibition is not competitive and does not appear to affect the enzyme's affinity for DNA. The enzyme remains inhibited after it is separated from "excess" gamma protein by gel filtration or sedimentation in a glycerol gradient, and inhibited enzyme has a reduced electrophoretic mobility compared to that of uninhibited enzyme. Gamma Protein inhibits recBC enzyme which has been reconstituted from cell-free extracts by complementation in vitro, but at least one of the complementing factors present in extracts from recB- cells does not by itself form a complex with gamma protein. The mechanism of inhibition and the implications of these results from gamma replication and recombination are discussed.     

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin

1. A mixture of NADPH and ferrodoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O₂ and superoxide radical is produced. 2. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine: xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. 3. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. 4. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Affinity labeling the DNA polymerase alpha complex. I. Pyridoxal 5'-phosphate inhibition of DNA polymerase and DNA primase activities of the DNA polymerase alpha complex from Drosophila melanogaster embryos

DNA polymerase alpha from Drosophila melanogaster embryos is a multisubunit enzyme complex which can exhibit DNA polymerase, 3'----5' exonuclease, and DNA primase activities. Pyridoxal 5'-phosphate (PLP) inhibition of DNA polymerase activity in this complex is time dependent and exhibits saturation kinetics. Inhibition can be reversed by incubation with an excess of a primary amine unless the PLP-enzyme conjugate is first reduced with NaBH4. These results indicate that PLP inhibition occurs via imine formation at a specific site(s) on the enzyme. Results from substrate protection experiments are most consistent with inhibition of DNA polymerase activity by PLP binding to either one of two sites. One site (PLP site 1) can be protected from PLP inhibition by any nucleoside triphosphate in the absence or presence of template-primer, suggesting that PLP site 1 defines a nucleotide-binding site which is important for DNA polymerase activity but which is distinct from the DNA polymerase active site. PLP also inhibits DNA primase activity of the DNA polymerase alpha complex, and primase activity can be protected from PLP inhibition by nucleotide alone, arguing that PLP site 1 lies within the DNA primase active site. The second inhibitory PLP-binding site (PLP site 2) is only protected from PLP inhibition when the enzyme is bound to both template-primer and correct dNTP in a stable ternary complex. Since binding of PLP at site 2 is mutually exclusive with template-directed dNTP binding at the DNA polymerase active site, PLP site 2 appears to define the dNTP binding domain of the active site. Results from initial velocity analysis of PLP inhibition argue that there is a rate-limiting step in the polymerization cycle during product release and/or translocation.     

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

DNA ligase activity in UV-irradiated monkey kidney cells.

The DNA ligase activity of monkey kidney CV-1 cells has been measured at different stages of culture growth and after different time intervals following ultraviolet irradiation. Results indicate that: - The level of enzyme activity is about twice higher in non synchronous, rapidly dividing cells than in confluent cultures. - UV-irradiation of cells induces a "de novo" synthesis of DNA ligase. - This induction is dose dependent in its extent and kinetics, and may lead to a DNA ligase level in UV-irradiated stationary cultures of the same order as observed in unirradiated exponentially growing cells. - This induction seems to be independent of semiconservative DNA synthesis since it is not affected by fluorodeoxyuridine.     

DNA synthesis in polyoma virus infection. V. Kinetic evidence for two requirements for protein synthesis during viral DNA replication.

Protein synthesis in polyoma virus-infected cells was inhibited by 99% within 4 min after exposure to 10 mug of cycloheximide per ml. Subsequent to the block in protein synthesis, the rate of viral DNA synthesis declined via inhibition of the rate of initiation of new rounds of genome replication (Yu and Cheevers, 1976). This process was inhibited with complex kinetics: within 15 min after the addition of cycloheximide, the rate of formation of closed-circular viral DNA was reduced by about one-half. Thereafter, DNA synthesis in cycloheximide-treated cells declined more slowly, reaching a level of 10% of untreated cells only after approximately 2 h. Protein synthesis was also required for normal closure of progeny form I DNA: in the presence of cycloheximide, DNA synthesis was diverted from the production of form I to form Ic, a monomeric closed-circular DNA component deficient in superhelical turns (Yu and Cheevers, 1976). Form I is replaced by Ic with first-order exponential kinetics. It is concluded that at least two proteins are involved in the control of polyoma DNA replication. One is apparently a stoichiometric requirement involved in the initiation step of viral DNA synthesis, since this process cannot be maintained at a normal rate for more than a few minutes in the absence of protein synthesis. The second protein requirement, governing the closure of newly synthesized progeny DNA, is considered distinct from the "initiation" protein on the basis of the kinetic data.     

Design of a nonuniformly distributed biocatalyst

The properties of a nonuniformly distributed biocatalyst, where the active enzymes are immobilized on the exterior or the interior portions o a solid support, are compared with those of a conventional biocatalyst which is uniformly distributed in a spherical geometry. To investigate the performance of nonuniformly distributed biocatalysts their effectiveness factors are computed and compared for six different enzyme distribution configurations: one-half core, one-half shell, one-third center space, one-third middle annulus, one-third outer shell, and the uniformly distributed. According to the results of numerical analysis, the biocatalyst performance of the exterior "shell" configuration is always far more effective for the immobilized enzymes with positive order reaction kinetics such as Michaelis-Menten and competitive product inhibition. However, in the case of negative order enzymatic reaction kinetics such as substrate inhibition, the interior "core" configuration of the biocatalyst can render far greater enzyme utilization efficiency.     

Kinetics of RNA polymerase-promoter complex formation: effects of nonspecific DNA-protein interactions.

The rates of formation of RNA polymerase-promoter open complexes at the galactose P2 and lactose UV5 promoters of E. coli were studied using polyacrylamide gels to separate the heparin-resistant complexes from unbound DNA. Both the apparent rate and extent of reaction at these promoters are inhibited at excess RNA polymerase. This inhibition, which can be relieved by the addition of non-promoter DNA, is interpreted to be the result of occlusion of the promoter site by nonspecifically bound polymerase. Additionally, biphasic kinetics are observed at both gal P2 and lac UV5, but not at the PR promoter of phage lambda. This behavior disappears when the concentration of RNA polymerase in the binding reaction is less than that of the promoter fragment. It is proposed that at excess enzyme nonspecifically bound polymerase molecules sliding along the DNA may "bump" closed complexes from the promoter site thereby reducing the rate of open complex formation. Kinetics mechanisms quantifying both the occlusion and bumping phenomena are presented.     

Quantification of formaldehyde-mediated covalent adducts of adriamycin with DNA.

Duplex DNA incubated with adriamycin, dithiothreitol (DTT), and Fe3+ under aerobic, aqueous conditions yields double-stranded (DS) DNA bands by denaturing polyacrylamide gel electrophoresis (DPAGE) analysis, characteristic of DNAs which are interstrand cross-linked. Another laboratory has provided evidence that formaldehyde produced under these conditions promotes the covalent linkage of adriamycin to one strand of DNA and suggested that this complex results in the anomalous DPAGE behavior. We provide herein strong support for this interpretation. We show: (a) that mixtures of DNA and adriamycin incubated with DTT/Fe3+, H2O2, or formaldehyde all show DS DNA bands on DPAGE, (b) that the DS DNA bands and the formaldehyde-mediated lesion (detected by an indirect, GC-MS analysis) form with similar time courses, and in similar amounts, and (c) that the DNA in the DS DNA bands contains approximately one such lesion per DNA, whereas the single-stranded DNA is devoid of it. These results further support the interpretation that adriamycin does not create interstrand cross-links in DNA, and that the DS DNA observed in DPAGE experiments derives from the formaldehyde-mediated monoadduct.     

Biochemical studies on the potentiation of antitumor activity of cisplatin by adriamycin

The RNA synthesis in vitro by E. coli RNA polymerase was found to be highly sensitive to cis-diamminedichloroplatinum (cisplatin) inhibition. The degree of inhibition was in proportion to the length of time of template preincubation with cisplatin, suggesting that cisplatin-template binding was involved in the inhibition of RNA polymerase. The effect of adriamycin on this inhibition was studied and it was found that adriamycin significantly enhanced the inhibitory effect of cisplatin and that the total effect was greater than the sum of the effects of each drug used individually. This synergistic effect was not observed when the effect of the combination of adriamycin and cisplatin on in vitro DNA synthesis was studied.     

Inhibition of plasma membrane NADH dehydrogenase by adriamycin and related anthracycline antibiotics

Doxorubicin (adriamycin) is cytotoxic to cells, but the biochemical basis for this effect is unknown, although intercalation with DNA has been proposed. This study suggests that the cytotoxicity of this drug may be due to inhibition of the plasma membrane redox system, which is involved in the control of cellular growth. Concentrations between 10^–6–10^–7 M adriamycin inhibit plasma membrane redox reactions >50%. AD32, a form of adriamycin which does not intercalate with DNA, but is cytotoxic, also inhibits the plasma membrane redox system. Thus, the cytotoxic effects of adriamycin, which limit its use as a drug, may be based on the inhibition of a transplasma membrane dehydrogenase involved in a plasma membrane redox system.