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Pinnipeds use a variety of acoustic information underwater for social interactions, hunting, and predator avoidance. Thus, the ability to accurately localize a sound source in the environment can have a clear survival value. Nonetheless, the sound localization mechanisms for seals underwater still have to be clarified, especially for sounds received in the median plane. In this study, the sound localization abilities of five harbor seals for high-frequency noise band stimuli were measured underwater in the median plane. The seals' minimum audible angles (MAAs) were determined for two different high-frequency noise band stimuli using a two-alternative forced-choice procedure. Noise 1 had a frequency range between 8 and 16 kHz. Noise 2 contained frequencies between 14 and 16 kHz. Psychoacoustic results for the tested harbor seals show that the seals were able to localize these stimuli in the median plane underwater with MAAs between 5.1° and 17.5°. The results suggest that spectral cues improve the seals' ability to localize high-frequency sound signals in the median plane.

DNA nano-structures present appealing new means for monitoring different molecules. Here, we demonstrate the assembly and utilization of a surface-attached double-stranded DNA catenane composed of two intact interlinked DNA nano-circles for specific and sensitive measurements of the life essential topoisomerase II (Topo II) enzyme activity. Topo II activity was detected via the numeric release of DNA nano-circles, which were visualized at the single-molecule level in a fluorescence microscope upon isothermal amplification and fluorescence labeling. The transition of each enzymatic reaction to a micrometer sized labeled product enabled quantitative detection of Topo II activity at the single decatenation event level rendering activity measurements in extracts from as few as five cells possible. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In an underwater environment the physical characteristics of sound propagation differ considerably from those in air. For this reason, sound localization underwater is associated with difficulties, especially in the median plane. It was the approach of the present study to investigate whether harbor seals (Phoca vitulina) are able to determine the direction of a tonal signal form above or below in the underwater environment. Minimum audible angles (MAAs) or the angular range in which the animals could localize a pure tone stimulus in the vertical plane were obtained for frequencies from 0.35 up to 16 kHz. Testing was conducted with four male harbor seals in a semi-circle area of 6 m in diameter in about 2.5 m depth, by using a two alternative forced choice method. The results show that harbor seals are able to localize a pure tone in the median plane under water with a high performance for low frequency stimuli between 350 Hz and 2 kHz with MAAs ranging from below 2.5° up to about 25°. For higher frequencies the animals show strong individual differences.

Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2-3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIβ in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIβ have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIβ. The quinone induced ~4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ~2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ~2-4 times faster with the β isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIβ when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase IIβ.

(-)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea (Camellia sinensis) leaves, and many of its cellular effects are consistent with its actions as a topoisomerase II poison. In contrast to genistein and several related bioflavonoids that act as interfacial poisons, EGCG was the first bioflavonoid shown to act as a covalent topoisomerase II poison. Although studies routinely examine the effects of dietary phytochemicals on enzyme and cellular systems, they often fail to consider that many compounds are altered during cooking or cellular metabolism. To this point, the majority of EGCG and related catechins in green tea leaves are epimerized during the brewing process. Epimerization inverts the stereochemistry of the bond that bridges the B- and C-rings and converts EGCG to (-)-gallocatechin gallate (GCG). Consequently, a significant proportion of EGCG that is ingested during the consumption of green tea is actually GCG. Therefore, the effects of GCG and related epimerized green tea catechins on human topoisomerase IIα and IIβ were characterized. GCG increased levels of DNA cleavage mediated by both enzyme isoforms with an activity that was similar to that of EGCG. GCG acted primarily by inhibiting the ability of topoisomerase IIα and IIβ to ligate cleaved DNA. Several lines of evidence indicate that GCG functions as a covalent topoisomerase II poison that adducts the enzyme. Finally, epimerization did not affect the reactivity of the chemical substituents (the three hydroxyl groups on the B-ring) that were required for enzyme poisoning. Thus, the activity of covalent topoisomerase II poisons appears to be less sensitive to stereochemical changes than interfacial poisons.

By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully., Competing Interests: The authors have declared that no competing interests exist.

Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg(2+) ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N-gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.

An organometallic ruthenium complex of quinolone antibacterial agent ofloxacin, [(η(6)-p-cymene)RuCl(O,O-oflo)]·2.8H(2)O (1·2.8H(2)O), was isolated, and its crystal structure was determined. In this "piano-stool" complex, quinolone is bidentately coordinated to the metal through the ring carbonyl and one of the carboxylic oxygen atoms. Interactions of the title complex with DNA were studied by spectroscopic methods [electronic, fluorescence, and circular dichroism (CD)] and atomic force microscopy (AFM). It was established that the electrostatic attraction between the ruthenium complex and DNA in a solution is important for binding because interactions were observed only in a solution with low ionic strengths. An induced-CD (ICD) signal was observed in a solution of DNA and the title complex, which proves interaction between ruthenium and macromolecules. Competitive binding between cisplatin and 1 to DNA revealed that cisplatin prevents binding of 1. Our experiments revealed that binding of the title complex to DNA occurs also if guanine N7 is protonated. AFM has shown that the title complex provokes DNA shrinkage. Preliminary biological tests have also been performed.

Background: Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research., Methods/principal Findings: Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more potent., Conclusions/significance: As a first-in-class anticancer quinolone derivative, voreloxin is a toposiomerase II-targeting agent with a unique mechanistic signature. A detailed understanding of voreloxin's molecular mechanism, in combination with its evolving clinical profile, may advance our understanding of structure-activity relationships to develop safer and more effective topoisomerase II-targeted therapies for the treatment of cancer.

Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17)(q22;q21) involving the PML and RARA genes is associated with exposure to agents targeting topoisomerase II (topoII), particularly mitoxantrone and epirubicin. We previously have shown that mitoxantrone preferentially induces topoII-mediated DNA damage in a "hotspot region" within PML intron 6. To investigate mechanisms underlying epirubicin-associated t-APL, t(15;17) genomic breakpoints were characterized in 6 cases with prior breast cancer. Significant breakpoint clustering was observed in PML and RARA loci (P = .009 and P = .017, respectively), with PML breakpoints lying outside the mitoxantrone-associated hotspot region. Recurrent breakpoints identified in the PML and RARA loci in epirubicin-related t-APL were shown to be preferential sites of topoII-induced DNA damage, enhanced by epirubicin. Although site preferences for DNA damage differed between mitoxantrone and epirubicin, the observation that particular regions of the PML and RARA loci are susceptible to these agents may underlie their respective propensities to induce t-APL.

Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17) translocation is a well-recognized complication of cancer treatment with agents targeting topoisomerase II. However, cases are emerging after mitoxantrone therapy for multiple sclerosis (MS). Analysis of 12 cases of mitoxantrone-related t-APL in MS patients revealed an altered distribution of chromosome 15 breakpoints versus de novo APL, biased toward disruption within PML intron 6 (11 of 12, 92% vs 622 of 1022, 61%: P = .035). Despite this intron spanning approximately 1 kb, breakpoints in 5 mitoxantrone-treated patients fell within an 8-bp region (1482-9) corresponding to the "hotspot" previously reported in t-APL, complicating mitoxantrone-containing breast cancer therapy. Another shared breakpoint was identified within the approximately 17-kb RARA intron 2 involving 2 t-APL cases arising after mitoxantrone treatment for MS and breast cancer, respectively. Analysis of PML and RARA genomic breakpoints in functional assays in 4 cases, including the shared RARA intron 2 breakpoint at 14 446-49, confirmed each to be preferential sites of topoisomerase IIalpha-mediated DNA cleavage in the presence of mitoxantrone. This study further supports the presence of preferential sites of DNA damage induced by mitoxantrone in PML and RARA genes that may underlie the propensity to develop this subtype of leukemia after exposure to this agent.

Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations. Treatment of cells with cobalt generates DNA strand breaks and covalent protein-DNA complexes. However, the basis for these effects is not well understood. Since the toxic events induced by cobalt resemble those of topoisomerase II poisons, the effect of the metal on human topoisomerase IIalpha was examined. The level of enzyme-mediated DNA scission increased 6-13-fold when cobalt(II) replaced magnesium(II) in cleavage reactions. Cobalt(II) stimulated cleavage at all DNA sites observed in the presence of magnesium(II), and the enzyme cut DNA at several "cobalt-specific" sites. The increased level of DNA cleavage in the presence of cobalt(II) was partially due to a decrease in the rate of enzyme-mediated religation. Topoisomerase IIalpha retained many of its catalytic properties in reactions that included cobalt(II), including sensitivity to the anticancer drug etoposide and the ability to relax and decatenate DNA. Finally, cobalt(II) stimulated topoisomerase IIalpha-mediated DNA cleavage in the presence of magnesium(II) in purified systems and in human MCF-7 cells. These findings demonstrate that cobalt(II) is a topoisomerase II poison in vitro and in cultured cells and suggest that at least some of the genotoxic effects of the metal are mediated through topoisomerase IIalpha.

TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996) Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues. Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore, this study characterized the basis for drug action. Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide. Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme. TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide. This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions.

TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells. However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known. Therefore, the primary cellular target of the drug and its mode of action were determined. TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro. The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha. A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity. Results indicate that topoisomerase II is the primary cellular target of TAS-103. In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex. TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide. Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme. Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.

To further characterize the mechanistic basis for cellular resistance/hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and ellipticine. The mutant enzyme contained a single point mutation that converted Gly437 --> Ser (top2G437S). Purified top2G437S displayed wild-type enzymatic activity in the absence of drugs but exhibited two properties that were not predicted by the cellular resistance phenotype. First, in the absence of ATP, it was hypersensitive to all of the drugs examined and hypersensitivity correlated with increased drug affinity. Second, in the presence of ATP, top2G437S lost its hypersensitivity and displayed wild-type drug sensitivity. Since the resistance of yeast harboring top2G437S could not be explained by alterations in enzyme-drug interactions, physiological levels of topoisomerase II were determined. The Gly437 --> Ser mutation reduced the stability of topoisomerase II and decreased the cellular concentration of the enzyme. These findings suggest that the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability. This study highlights the need to analyze both the biochemistry and the physiology of topoisomerase II mutants with altered drug sensitivity in order to define the mechanistic bridge that links enzyme function to cellular phenotype.

The effects of extracellular sodium on platelet aggregation and calcium mobilization in platelets stimulated with either rattlesnake (Crotalus atrox) lectin (RSL) or alpha-thrombin were compared. The absence of extracellular sodium had no effect on platelet aggregation or calcium mobilization in response to all levels of RSL tested. In contrast platelet aggregation was sodium-dependent in response to less than or equal to .2 units/ml alpha-thrombin. Surprisingly, calcium mobilization occurred in platelets treated with a threshold level of alpha-thrombin in the absence of external sodium. Thus sodium-dependent platelet aggregation in response to a low dose of thrombin apparently is not the result of sodium-dependent calcium mobilization.