Evidence of a common site of action for the antitumor agents, hydroxyurea and guanazole

Hydroxyurea and guanazole were used as selective agents in tissue culture to obtain independent Chinese hamster ovary cell lines resistant to the cytotoxic effects of hydroxyurea or guanazole. In all cases tested a cell line selected for resistance to one of the antitumor agents exhibited resistance to both drugs. This result supports the view that these two drugs act at a common site.     

Bacterial type II topoisomerases as targets for antibacterial drugs

Bacterial type II DNA topoisomerases are essential enzymes for correct genome functioning and cell growth. Gyrase is responsible for maintaining negative supercoiling of bacterial chromosome, whereas topoisomerase IV acts in disentangling daughter chromosomes following replication. Type II DNA topoisomerases possess an ATP binding site, which can be treated as a target for antibacterial drugs. Resolving crystal structures of protein fragments consisting of an ATP binding site complexed with ADPNP/antibiotics have proven to be valuable for the understanding of the mode of action of existing antibacterial agents and presented new possibilities for novel drug design. Coumarins, quinolones and cyclothialidines are diverse group of antibiotics that interfere with type II DNA topoisomerases, however their mode of action is different. Recently a new class of antibiotics, simociclinones, was characterized. Their mechanism of action towards gyrase is entirely distinct from already known modes of action, therefore demonstrating the potential for development of novel anti-bacterial agents.     

Computational analysis of the chiral action of type II DNA topoisomerases

It was found recently that bacterial type II DNA topoisomerase, topo IV, is much more efficient in relaxing (+) DNA supercoiling than (-) supercoiling. This means that the DNA-enzyme complex is chiral. This chirality can appear upon binding the first segment that participates in the strand passing reaction (G segment) or only after the second segment (T segment) joins the complex. The former possibility is analyzed here. We assume that upon binding the enzyme, the G segment forms a part of left-handed helical turn. This model is an extension of the hairpin model introduced earlier to explain simplification of DNA topology by these enzymes. Using statistical-mechanical simulation of DNA properties, we estimated different consequences of the model: (1) relative rates of relaxation of (+) and (-) supercoiling by the enzyme; (2) the distribution of positions of the G segment in supercoiled molecules; (3) steady-state distribution of knots in circular molecules created by the topoisomerase; (4) the variance of topoisomer distribution created by the enzyme; (5) the effect of (+) and (-) supercoiling on the binding topo II with G segment. The simulation results are capable of explaining nearly all available experimental data, at least semiquantitatively. A few predictions obtained in the model analysis can be tested experimentally.     

Phylogenomics of type II DNA topoisomerases

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.     

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.     

Binding of two DNA molecules by type II topoisomerases for decatenation

Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.     

A unique type II topoisomerase mutant that is hypersensitive to a broad range of cleavage-inducing antitumor agents

Bacteriophage T4 provides a useful model system for dissecting the mechanism of action of antitumor agents that target type II DNA topoisomerases. Many of these inhibitors act by trapping the cleavage complex, a covalent complex of enzyme and broken DNA. Previous analysis showed that a drug-resistant T4 mutant harbored two amino acid substitutions (S79F, G269V) in topoisomerase subunit gp52. Surprisingly, the single amino acid substitution, G269V, was shown to confer hypersensitivity in vivo to m-AMSA and oxolinic acid [Freudenreich, C. H., et al. (1998) Cancer Res. 58, 1260-1267]. We purified this G269V mutant enzyme and found it to be hypersensitive to a number of cleavage-inducing inhibitors including m-AMSA, VP-16, mitoxantrone, ellipticine, and oxolinic acid. While the mutant enzyme did not exhibit altered DNA cleavage site specificity compared to the wild-type enzyme, it did display an apparent 10-fold increase in drug-independent DNA cleavage. This suggests a novel mechanism of altered drug sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex, resulting in an increase in the concentration of cleavage intermediates available to inhibitors. Mutations that alter drug sensitivities tend to cluster within two specific regions of all type II topoisomerases. Residue G269 of gp52 lies outside of these regions, and it is therefore not surprising that G269V leads to a unique mechanism of drug hypersensitivity. We believe that this mutant defines a new category of type II topoisomerase mutants, namely, those that are hypersensitive to all inhibitors that stabilize the cleavage complex.     

Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases

Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase–quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions −2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme–drug complex.     

Anaphase chromatid motion: involvement of type II DNA topoisomerases.

Sister chromatids are topologically intertwined at the onset of anaphase: their segregation during anaphase is known to require strand-passing activity by type II DNA topoisomerase. We propose that the removal of the intertwinings involves at the same time the traction of the mitotic spindle and the activity of topoisomerases. This implies that the velocity of the chromatids is compatible with the kinetic constraints imposed by the enzymatic reaction. We show that the greatest observed velocities (about 0.1 microns s-1) are close to the theoretical upper bound compatible with both the diffusion rate (calculated here within a probabilistic model) and the measured reaction rate of the enzyme.     

Multimodal action of antitumor agents on DNA: the ellipticine series

Most cytotoxic anticancer agents interact directly or indirectly with nuclear DNA, the ultimate target for this class of compounds. For a given type of drug both direct and indirect action at the DNA level usually causes various types of interference or damage. This multimodal mechanism of action is well illustrated by antitumor drugs in the ellipticine series which may bind to DNA through intercalation, may undergo covalent binding, may generate oxidizing species, and may interfere with the catalytic activity of topoisomerase II. The antitumor activity of these compounds may, therefore, result from alternative cytotoxic events. The present review summarizes information obtained with ellipticine compounds on the relation between the nature of the drugs' action on DNA and their cytotoxic and/or antitumor activity. The occurrence of topoisomerase-mediated DNA cleavage appears to be responsible for antitumor activity. The capability of the drugs to interfere with the action of topoisomerase II requires the presence of an oxidizable phenolic group on their structure. This feature (or a related one) is shared by all antitumor drugs acting on this enzyme.     

Evidence that the antitumor agent hydroxyurea enters mammalian cells by a diffusion mechanism

Very little information is available about the process responsible for the uptake of the antitumor agent hydroxyurea by mammalian cells. Therefore we have investigated the transport of hydroxy[14C]-urea into Chinese hamster ovary cells. Using a convenient and reproducible 2 min. assay we found that hydroxyurea was taken up in a linear nonsaturable fashion between 0.01 mM and 100 mM drug. The Km for hydroxyurea uptake was essentially zero and the Vmax appeared to be infinite, suggesting a diffusion mechanism. The observation that intracellular drug concentrations were consistently less than medium concentrations indicated that uptake was not an active process. Experiments performed with the metabolic inhibitor sodium azide, and investigations at different assay temperatures also gave results consistent with a mechanism of drug diffusion. In total, the results obtained in this study are in agreement with the proposal that hydroxyurea enters the cell primarily by a process of diffusion. These observations provide a more complete understanding of the mode of action of this widely used drug.     

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.     

Epitope distribution of randomly established monoclonal antibodies against human type II DNA topoisomerases

The epitopes of about 100 monoclonal antibodies against human type II DNA topoisomerase were mapped along the enzyme molecules. Although they were randomly and independently established, epitope sites were unevenly distributed the toward N-terminal or C-terminal region. We suggest that the central catalytic domain is hidden inside the molecule and inaccessible to the antigen recognition sites. Using antibodies, we demonstrate the distinct localization of isoforms of Topo II in cultured cells. Some particularly useful antibodies are listed.     

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.     

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.     

Evidence for 5-HT2 involvement in the mechanism of action of hallucinogenic agents

The affinities (Ki values) of twenty two psycho-active agents, including LSD, 5-OMe DMT and a series of phenalkylamine derivatives, for cortical 5-HT1 and 5-HT2 binding sites were compared with two measures of behavioral activity. It was found that a significant correlation (r = 0.938) exists between the 5-HT2 binding affinities of these agents and their ED50 values as determined in tests of stimulus generalization using 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) as the training drug. Furthermore, for fifteen of these agents where human data were available, a significant correlation (r = 0.924) also exists between 5-HT2 binding affinities and their human hallucinogenic potencies. The results of this study suggest that the mechanism of action of these agents involves 5-HT2-related events.     

The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases.

The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.