Lead exposure in the general population of the Metropolitan Area of Barcelona: blood levels and related factors

Bacterial and archeal type I topoisomerases, including topoisomerase I, topoisomerase III and reverse gyrase, have different potential roles in the control of DNA topology including regulation of supercoiling and maintenance of genetic stability. Analysis of their coding sequences in different organisms shows that they belong to the type IA family of DNA topoisomerases, but there is variability in organization of various enzymatic domains necessary for topoisomerase activity. The torus-like structure of the conserved transesterification domain with the active site tyrosine for DNA cleavage/rejoining suggests steps of enzyme conformational change driven by DNA substrate and Mg(II) cofactor binding, that are required for catalysis of change in DNA linking number.     

Effects of DNA methylation on topoisomerase I and II cleavage activities

DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.     

Holmium-YAG laser in outlet impingement of the shoulder. Mid-term results

Type II DNA topoisomerases are enzymes that are capable of transporting one duplex DNA through another. Recent experimental results, including the structure of a fragment of yeast topoisomerase II, have provided new insights into the mechanism of the strand passage reaction. Other results have begun to define the role of ATP in the catalytic cycle and illuminate how DNA breaks mediated by topoisomerase II can occur.     

Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs

The budding yeast Saccharomyces cerevisiae has been exploited to investigate the cytotoxic mechanisms of drugs that target DNA topoisomerases. This model organism has been used to establish eukaryotic DNA topoisomerase I or II as the cellular target of specific antineoplastic agents, to define mutations in these enzymes that confer drug resistance and to elucidate the cellular factors that modulate cell sensitivity to DNA topoisomerase-targeted drugs. These findings have provided valuable insights into the critical activities of these enzymes and how perturbing their functions produces DNA damage and cell death.     

Evaluation of bleached kraft mill process water using Microtox(R), Ceriodaphnia dubia, and Menidia beryllina toxicity tests

We show herein that human DNA topoisomerase II beta is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants. A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta.     

Involvement of topoisomerases in the initiation of simian virus 40 minichromosome replication

Topoisomerases provide the unlinking activity necessary for replication fork movement during DNA replication. It is uncertain, however, whether topoisomerases are also required for the initiation of replication. To investigate this point, we have performed pulse-chase experiments with SV40 minichromosomes as template to distinguish between the initiation and the elongation of replication. Using an unfractionated cytosolic extract as a source of replication functions, we found that the addition of topoisomerases at the initiation step significantly increased the number of active chromatin templates, whereas addition of topoisomerases at the elongation step had only minor effects. Minichromosomes with an extended chromatin structure as well as protein-free DNA required less topoisomerase for effective replication initiation. We could exclude the possibility that topoisomerases enhance the origin binding of T antigen, the SV40 replication initiator, and propose instead that the arrangement of nucleosomes influences the diffusion of supercoils during initial DNA unwinding. Efficient initiation therefore requires a high local concentration of topoisomerases to relax the torsional stress.     

Effects of nucleosomes and anti-tumor drugs on the catalytic activity of type II DNA topoisomerase from rat testis

To gain insight into the relative catalytic efficiencies of mammalian type I and type II DNA topoisomerases, in the cellular context, we have used naked DNA and DNA incorporated into nucleosomes as substrates. We observed that the relaxation activity of both the enzymes declined with DNA containing increasing densities of nucleosomes; however, kinetic analysis revealed that topoisomerase I seemed less affected than topoisomerase II. The addition of histone H1, in stoichiometric amounts, to naked DNA or minichromosomes lessened the activity of topoisomerase II, and required 7-fold less for complete inhibition when the latter was used as the substrate. To ascertain if the observed differences are specific to topoisomerase II from testis, we examined the effect of nucleosomes on the catalytic efficiency of its isoform from liver. Interestingly, the suppression of relaxation activity of liver topoisomerase II required substrates containing higher mass ratios of histone octamer/DNA. Studies on the effect of nucleosomes on the action of teniposide displayed significant differences in the kinetics of the reaction, in its IC50 values, and have provided biochemical evidence for the first time that nucleosomes increased inhibition caused by teniposide. Further, this feature appears to be specific for topoisomerase II-directed drugs and is not shared by the generic class of either DNA-intercalating or non-DNA-intercalating ligands.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.     

Coralyne and related compounds as mammalian topoisomerase I and topoisomerase II poisons

DNA topoisomerases are nuclear enzymes responsible for modifying the topological state of DNA. The development of agents capable of poisoning topoisomerases has proved to be an attractive approach in the search for novel cancer chemotherapeutics. Coralyne, an antileukemic alkaloid, has appreciable structural similarity to the potent topoisomerase I and II poison, nitidine. Analogues of coralyne were synthesized and evaluated for their activity as topoisomerase I and topoisomerase II poisons. These analogues were also evaluated for cytotoxicity in the human lymphoblast cell line, RPMI 8402, and its camptothecin-resistant variant, CPT-K5. The pharmacological activity of these analogues exhibited a strong dependence on the substitution pattern and the nature of substituents. Several 1-benzylisoquinolines and 3-phenylisoquinolines were also synthesized. These compounds, which incorporate only a portion of the ring structure of coralyne, were evaluated as topoisomerase poisons and for cytotoxicity. These structure-activity studies indicate that the structural rigidity associated with the coralyne ring system may be critical for pharmacological activity. The presence of a 3,4-methylenedioxy substituent on these coralyne analogues was generally associated with enhanced activity as a topoisomerase poison. 5,6-Dihydro-3,4-methylenedioxy-10,11-dimethoxydibenzo[a,g]quinoliz inium chloride was the most potent topoisomerase I poison among the coralyne analogues evaluated, having similar activity to camptothecin. This analogue also possessed exceptional potency as a topoisomerase II poison. Despite the pronounced activity of several of these coralyne derivatives as topoisomerase I poisons, none of these compounds had cytotoxic activity similar to camptothecin. Possible differences in cellular absorption between these coralyne analogs, which possess a quaternary ammonium group, and camptothecin may be responsible for the differences observed in their relative cytotoxicity.     

DNA topoisomerase targeting drugs: mechanisms of action and perspectives

The nuclear enzymes DNA topoisomerases I and II appeared as cellular targets for several antitumor drugs: campthotecin derivatives interacting with topoisomerase I, and actinomycin D, anthracycline derivatives, elliptinium acetate, mitoxantrone, epipodophyllotoxine derivatives, amsacrine and a new olivacine derivative, NSC-6596871 (S 16020-2), which interact with topoisomerase II. The functions of these enzymes are numerous and important since they are critical for DNA functions and cell survival. Despite the fact that they share the same target, topoisomerase II inhibitors have different mechanisms of action. Two principle types of induced alterations are involved in cellular resistance to topoisomerase II drugs: qualitative or quantitative alteration of the enzyme and/or increased drug efflux due to overexpression of P-glycoprotein. S 16020-2, a new olivacine derivative with a high antitumor activity against solid tumors, shows a potent cytotoxic effect against tumor cells expressing P-glycoprotein. This observation suggests that the comprehension of the respective effects of topoisomerase inhibitors and the precise knowledge of their mechanisms of resistance would improve the use of this therapeutic class in the clinic within rational chemotherapeutic combinations.     

DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities

DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria. Though clearly related, based on amino acid sequence similarity, they each play crucial, but distinct, roles in the cell. Gyrase is involved primarily in supporting nascent chain elongation during replication of the chromosome, whereas topoisomerase IV separates the topologically linked daughter chromosomes during the terminal stage of DNA replication. These different roles can be attributed to differences in the biochemical properties of the two enzymes. The biochemical activities, physiological roles, and drug sensitivities of the enzymes are reviewed.     

The response of eukaryotic topoisomerases to DNA damage

Beyond the known mutagenic properties of DNA lesions, recent evidence indicates that several forms of genomic damage dramatically influence the catalytic activities of DNA topoisomerases. Apurinic sites, apyrimidinic sites, base mismatches, and ultraviolet photoproducts all enhance topoisomerase I-mediated DNA cleavage when they are located in close proximity to the point of scission. Furthermore, when located between the points of scission of a topoisomerase II cleavage site, these same lesions (with the exception of ultraviolet photoproducts) greatly stimulate the cleavage activity of the type II enzyme. Thus, as found for anticancer drugs, lesions have the capacity to convert topoisomerases from essential cellular enzymes to potent DNA toxins. These findings raise exciting new questions regarding the mechanism of anticancer drugs, the physiological functions of topoisomerases, and the processing of DNA damage in the cell.     

Inhibitors of DNA topoisomerases]

DNA topoisomerases are enzymes regulating the conformational state of DNA in every aspect of genetic processes by catalyzing transient cleavage and religation of DNA strands. The enzymes are targets of some of the important anticancer drugs. Many candidates of anticancer drugs are being screened via inhibition of the enzymes. In the present review, I discuss the role of DNA topoisomerases in genetic processes in mammalian cells, characteristics and mode of action of topoisomerase inhibitors, and resistance of tumor cells to the anticancer drugs.     

Transforming growth factor-beta, endothelin-1, and c-fos expression in necrotizing/crescentic IgA glomerulonephritis

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.