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Topoisomerase II Binds to Ellipticine in the Absence or Presence of DNA.

CHARACTERIZATION OF ENZYME∙DRUG INTERACTIONS BY FLUORESCENCE SPECTROSCOPY (∗)
  • Stacie J. Froelich-Ammon
    Footnotes
    Affiliations
    Department of Biochemistry, Nashville, Tennessee 37232-0146
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  • Marcia W. Patchan
    Affiliations
    Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201-1503
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  • Neil Osheroff
    Correspondence
    To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, 654 Medical Research Bldg. I, Vanderbilt University School of Medicine, Nashville, TN 37232-0146 . Tel.: 615-322-4338; Fax: 615-343-1166
    Affiliations
    Department of Biochemistry, Nashville, Tennessee 37232-0146

    Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Richard B. Thompson
    Correspondence
    To whom correspondence may also be addressed: Dept. of Biological Chemistry, 108 N. Greene St., University of Maryland School of Medicine, Baltimore, MD 21201 . Tel.: 410-706-7142; Fax: 410-706-7122
    Affiliations
    Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201-1503
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  • Author Footnotes
    ∗ This work was supported by National Institutes of Health Grant GM33944, by Faculty Research Award FRA-370 from the American Cancer Society (to N. O.), and by Office of Naval Research Contract N00014-91-J-1572 (to R. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) L41827.
    § Trainee under National Institutes of Health Grant 5 T32 CA09582.
Open AccessPublished:June 23, 1995DOI:https://doi.org/10.1074/jbc.270.25.14998
      Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme∙DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD≈ 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme∙DNA complex (KD≈ 1.5 μM) or to the enzyme in the absence of nucleic acids (KD≈ 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II∙ellipticine∙DNA complex is formed via initial drug binding to either the enzyme or DNA.

      INTRODUCTION

      The ability to modulate the topological state of nucleic acids is critical to the survival of eukaryotic and prokaryotic cells(
      • Wang J.C.
      ,
      • Osheroff N.
      ,
      ,
      • Osheroff N.
      • Zechiedrich E.L.
      • Gale K.C.
      ,
      • Watt P.M.
      • Hickson I.D.
      ). Topoisomerases, the enzymes that modulate DNA topology in vivo, are involved in virtually every aspect of DNA metabolism (1-5). In addition to their critical physiological functions, topoisomerases are targets for a number of relevant chemotherapeutic agents. For example, ciprofloxacin, which is targeted to the prokaryotic type II topoisomerase, DNA gyrase, is the most active oral antibiotic currently in clinical use(
      • Hooper D.C.
      • Wolfson J.S.
      ,
      • Reece R.J.
      • Maxwell A.
      ,
      • Hooper D.C.
      • Wolfson J.S.
      ,
      • Maxwell A.
      ,
      • Hooper D.C.
      ). Furthermore, camptothecin-based drugs, which target eukaryotic topoisomerase I, and drugs such as etoposide, amsacrine, doxorubicin, mitoxantrone, and ellipticine, which target eukaryotic topoisomerase II, are effective agents for the treatment of several human cancers(
      • Liu L.F.
      ,
      • Liu L.F.
      ,
      • Pommier Y.
      ,
      • Corbett A.H.
      • Osheroff N.
      ). Although these drugs are derived from diverse structural classes and act through three different topoisomerases, they all exert their cytotoxic effects by enhancing enzyme-mediated DNA breakage within the genome(
      • Corbett A.H.
      • Osheroff N.
      ,
      • Kreuzer K.N.
      • Cozzarelli N.R.
      ,
      • Schneider E.
      • Lawson P.A.
      • Ralph R.K.
      ,
      • Zhang H.
      • D'Arpa P.
      • Liu L.F.
      ,
      • Giaccone G.
      ,
      • Chen A.Y.
      • Liu L.F.
      ).
      Despite the clinical importance of topoisomerase poisons, interactions between these agents and their enzyme targets are poorly understood. It is likely that formation of a ternary complex between topoisomerase, DNA, and drug is critical for nucleic acid breakage and subsequent cell death(
      • Reece R.J.
      • Maxwell A.
      ,
      • Maxwell A.
      ,
      • Corbett A.H.
      • Osheroff N.
      ,
      • Chen A.Y.
      • Liu L.F.
      ). However, the pathway by which this ternary complex is assembled has yet to be determined. Three possible mechanisms for complex formation exist. In the first, the drug binds only to the topoisomerase∙DNA complex and has minimal interactions with either the enzyme or nucleic acid independently. Support for this possibility is derived from studies that characterized the binding of camptothecin with topoisomerase I (
      • Hertzberg R.P.
      • Caranfa M.J.
      • Hecht S.M.
      ) and quinolones with DNA gyrase (21, 22). In both cases, drugs were found to interact almost exclusively with the enzyme∙DNA complex. In the second mechanism, the drug becomes part of the ternary complex primarily through interactions with DNA. Support for this possibility stems from the fact that many topoisomerase-targeted agents bind (in either an intercalative or nonintercalative fashion) to DNA in the absence of enzyme(
      • Waring M.J.
      ,
      • Chow K.-C.
      • MacDonald T.L.
      • Ross W.E.
      ). It should be noted, however, that no correlation has been observed between either the mode or strength of DNA binding and the cytotoxicity or antineoplastic activity of these drugs. In the third mechanism, the drug becomes part of the ternary complex primarily through direct interaction with the enzyme in the absence of DNA. Evidence for this last possibility comes from surface-enhanced Raman scattering studies that suggest an interaction between intoplicine and topoisomerase II (25).
      To further define the mechanism of action of antineoplastic drugs targeted to eukaryotic topoisomerases, interactions between yeast topoisomerase II and ellipticine were characterized. Ellipticine is an intercalative alkaloid (
      • Woodward R.B.
      • Iacobucci G.A.
      • Hochstein F.A.
      ,
      • Goodwin S.
      • Smith J.A.
      • Horning E.C.
      ) that stimulates topoisomerase II-mediated DNA breakage(
      • Ross W.E.
      • Bradley M.O.
      ,
      • Tewey K.M.
      • Chen G.L.
      • Nelson E.M.
      • Liu L.F.
      ,
      • Pommier Y.
      • Schwartz R.E.
      • Kohn K.W.
      • Zwelling L.A.
      ,
      • Pommier Y.
      • Schwartz R.E.
      • Zwelling L.A.
      • Kohn K.W.
      ), and along with several of its analogs, is currently in clinical trials(
      • Paoletti C
      • Auclair C.
      • Lesca P.
      • Tocanne J.F.
      • Malvy C.
      • Pinto M.
      ,
      • Auclair C.
      • Voisin E.
      • Banoun H.
      • Paoletti C.
      • Bernadou J.
      • Meunier B.
      ,
      • Charcosset J.Y.
      • Bendirdjian J.P.
      • Lantieri M.F.
      • Jacquemin-Sablon A.
      ,
      • Auclair C.
      ,
      • Buzdar A.U.
      • Hortobagyi G.N.
      • Esparza L.T.
      • Holmes F.A.
      • Ro J.S.
      • Froaschini G.
      • Lichtiger B.
      ,
      • Perez R.P.
      • Nash S.L.
      • Ozols R.F.
      • Comis R.L.
      • O'Dwyer P.J.
      ,
      • Ruckdeschel J.C.
      • Modi S.P.
      • el-Hamouly W.
      • Portuese E.
      • Archer S.
      ,
      • Rouesse J.
      • Spielmann M.
      • Turpin F.
      • Le Chevalier T.
      • Azab M.
      • Modesir J.M.
      ,
      • Jurayj J.
      • Haugwitz R.D.
      • Varma R.K.
      • Paull K.D.
      • Barrett J.F.
      • Cushman M.
      ,
      • Kattan J.
      • Durand M.
      • Droz J.P.
      • Mahjoubi M.
      • Marino J.P.
      • Azab M.
      ) for the treatment of human cancers. Results of the present study indicate that topoisomerase II is the primary cellular target of the drug. In addition, ellipticine shows little ability to inhibit the religation of cleaved DNA by topoisomerase II, suggesting that this agent enhances DNA breakage by increasing the forward rate of cleavage. Finally, as determined by steady state and frequency domain fluorescence spectroscopy, it appears that ellipticine forms a stable complex with topoisomerase II in the absence of DNA, and that the enzyme dictates the ionic state of the drug in the topoisomerase II∙ellipticine∙DNA complex. A model is proposed in which ellipticine enters the ternary complex through its prior association with either DNA or the enzyme and does not require the presence of a preformed topoisomerase II∙DNA complex.

      EXPERIMENTAL PROCEDURES

      Materials and Yeast Strains

      A 40-mer double-stranded oligonucleotide containing a characterized cleavage site for topoisomerase II (
      • Corbett A.H.
      • Zechiedrich E.L.
      • Osheroff N.
      ,
      • Sander M.
      • Hsieh T.
      ) was synthesized on an Applied Biosystems DNA Synthesizer. The sequence of one of the two complementary oligonucleotides is: 5′-TGAAATCTAACAATG↓CGCTCATCGTCATCCTCGGCACCGT-3′ where the arrow denotes the site of topoisomerase II-mediated DNA cleavage. Oligonucleotides were purified as described previously (
      • Froelich-Ammon S.J.
      • Gale K.C.
      • Osheroff N.
      ) and diluted to the appropriate concentration in 5 mM Tris-HCl, pH 7.4, 0.5 mM EDTA. Negatively supercoiled bacterial plasmid pBR322 DNA was prepared as described by Maniatis and co-workers(
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ). Ellipticine (Sigma) was solubilized as a 20 mM or 10 mM stock solution in dimethyl sulfoxide or ethanol, respectively, and stored at −20°C. The yeast strains employed for the present study were Saccharomyces cerevisiae JN394 with genotype ura3-52, leu2, trp1, his7, ade1-2, ISE2, rad52:LEU2, and JN394t2-1, whose genotype is isogenic to JN394 except for the replacement of the wild type topoisomerase II gene (TOP2+) with the top2-1 mutant allele. Dimethyl-POPOP1(
      The abbreviation used is: dimethyl-POPOP
      1,4-bis(4-methyl-5-phenyloxazolyl)benzene.
      ) and rose bengal were acquired from Eastman; ultrapure HEPES was from VWR; Tris-HCl and ethidium bromide were obtained from Sigma; SDS was purchased from E. Merck Biochemicals; proteinase K was from United States Biochemical Corp.; yeast nitrogen base, yeast extract, and Bacto-agar were from Difco. All other chemicals were analytical reagent grade.

      Yeast Topoisomerase II Overexpression and Purification

      Yeast topoisomerase II was overexpressed and purified by the procedure of Worland and Wang (
      • Worland S.T.
      • Wang J.C.
      ) as modified by Elsea et al.(
      • Elsea S.H.
      • Hsiung Y.
      • Nitiss J.L.
      • Osheroff N.
      ). Briefly, overexpression was achieved in yeast strain JEL 1 (transformed with YEpGAL1TOP2). Cells were induced by the addition of galactose in glucose-free media, grown for 16 h to an A600 of ~1.0, and harvested by centrifugation. Pellets were resuspended in buffer (50 mM Tris-HCl, pH 7.7, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 25 mM NaF, 1 mM Na2S2O5, 1 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride), quick-frozen, and stored at −80°C until use. Cells were lysed at 4°C, and all purification procedures were carried out at 4°C. Topoisomerase II was purified from cell extracts to apparent homogeneity (as determined by visualization on silver-stained polyacrylamide gels) by phosphocellulose column chromatography based on the protocol of Shelton et al.(
      • Shelton E.R.
      • Osheroff N.
      • Brutlag D.L.
      ). Approximately 20 mg of the type II enzyme was isolated from 25 g of frozen wet-packed yeast cells.

      Primary Cellular Target Assay

      The primary cellular target of ellipticine in yeast was determined by the protocol of Nitiss and co-workers(
      • Nitiss J.L.
      • Wang J.C.
      ,
      • Elsea S.H.
      • Osheroff N.
      • Nitiss J.L.
      ,
      • Nitiss J.L.
      • Liu Y.X.
      • Hsiung Y.
      ). Briefly, yeast strain JN394t2-1 was cultured in YPDA media at 25°C. Following the adjustment of logarithmically growing cells to a titer of 2 ´ 106 cells/ml, ellipticine (10-200 μM) was added to the medium. Cultures were incubated with the drug for 18 h at 25°C or 30°C. Cells were diluted into sterile water and plated in duplicate onto YPDA medium solidified with 1.5% Bacto-agar (Difco). Plates were incubated at 25°C or 30°C, and surviving colonies were counted.

      Topoisomerase II-mediated DNA Cleavage

      Assays were performed using a modification of the protocol described by Robinson and Osheroff(
      • Robinson M.J.
      • Osheroff N.
      ). Experiments were carried out in the absence of ATP, thus monitoring topoisomerase II-mediated cleavage that occurs prior to the strand passage event(
      • Osheroff N.
      ). DNA cleavage reactions contained 100 nM yeast topoisomerase II and 5 nM negatively supercoiled pBR322 in reaction buffer (20 mM HEPES, pH 7.9, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, and 2.5% glycerol) with a total volume of 20 μl. The DNA cleavage/religation equilibrium was established by incubating reaction mixtures at 28°C for 6 min. Cleavage products were trapped by the addition of 2 μl of 10% SDS, followed by the addition of 1.5 μl of 250 mM EDTA and 2 μl of a 0.8 mg/ml solution of proteinase K. Samples were incubated at 45°C for 20 min to digest the topoisomerase II, mixed with 2 μl of 10 mM Tris-HCl, pH 7.9, 0.05% bromphenol blue, 0.05% xylene cyanol, and 60% sucrose, and heated at 70°C for 2 min. Reaction products were resolved by electrophoresis in 1% agarose gels in 40 mM Tris acetate, pH 8.0, 2 mM EDTA, and gels were stained with 1 μg/ml ethidium bromide. DNA bands were visualized by transillumination with UV light (300 nm) and were photographed through Kodak 23A and 12 filters with Polaroid type 665 positive-negative film. The amount of DNA present was quantitated by scanning photographic negatives with an E-C Apparatus model EC910 scanning densitometer using Hoefer GS-370 Data System software. Alternatively, DNA cleavage bands were quantitated with an Alpha Innotech IS1000 Imaging System. In both cases, the densities of the bands were proportional to the amount of DNA present. The effects of ellipticine were studied over a range of 250 nM to 100 μM. All control samples contained an equal amount of drug diluent, either dimethyl sulfoxide or EtOH. No drug-induced DNA cleavage was seen in the absence of topoisomerase II.

      Topoisomerase II-mediated DNA Religation

      Assays were performed by a modification of the protocol of Robinson et al.(
      • Robinson M.J.
      • Martin B.A.
      • Gootz T.D.
      • McGuirk P.R.
      • Moynihan M.
      • Sutcliffe J.A.
      • Osheroff N.
      ). Reactions contained 100 nM yeast topoisomerase II and 5 nM negatively supercoiled pBR322. DNA cleavage/religation equilibria were established as described above. Topoisomerase II-mediated religation of cleaved DNA was induced by rapidly shifting samples from 28°C to 65°C. Religation was terminated by the addition of SDS (2 μl of 10%) at various time points. Following the cessation of religation, samples were treated with EDTA and proteinase K. Reaction products were analyzed by agarose gel electrophoresis and quantitated as described above.

      Steady-state and Frequency Domain Fluorescence Spectroscopy

      Steady-state fluorescence experiments were performed utilizing an SLM 8000C fluorometer with SLM software Version 4.0. Spectral analysis was carried out at 25°C. Fluorescence excited-state lifetimes were determined using an ISS K2 multifrequency phase-modulation fluorometer, and data were analyzed using the ISS software package Version 2.0. A Liconix 4214NB helium-cadmium laser emitting 3 milliwatts at 326 nm or 12 milliwatts at 442 nm was the excitation light source. For both steady-state and frequency domain experiments, emitted light was monitored while the emission polarizer was fixed at the magic angle (54.7°), and either a 420 nm or 520 nm interference band pass filter was employed to separate fluorescence from scattered light. Dimethyl-POPOP, with a lifetime value of 1.45 ns, or rose bengal, with a lifetime of 732 ps (in EtOH)(
      • Thompson R.B.
      • Gratton E.
      ), were used as references. Data were acquired at 25°C between 1 and 250 MHz. Samples contained the designated concentrations of yeast topoisomerase II and/or 40-mer oligonucleotide and ellipticine in a final volume of 500 μl of 20 mM HEPES, pH 7.9 (unless otherwise noted), 100 mM NaCl, 5 mM MgCl2, and 0.1 mM EDTA and were incubated for 6 min prior to fluorescence measurements. All chemicals were ultrapure grade to eliminate scatter and nonspecific fluorescence. The buffer background, shown as a reference in Fig. 6, was subtracted from intensities for binding calculations.
      Figure thumbnail gr6
      Figure 6:Ellipticine●topoisomerase II binding. The emission spectra for 1 μM ellipticine in the presence of topoisomerase II concentrations ranging from 0-150 nM are shown in the left panel. Samples were excited at 326 nm. A double reciprocal binding analysis is shown in the right panel. Data are plotted as 1/Δ fluorescence intensity at 420 nm versus 1/[topoisomerase II] and represent the averages of two independent experiments.

      RESULTS

      Primary Cellular Target of Ellipticine

      Ellipticine and several of its analogs are currently being evaluated for their clinical efficacy against human cancers(
      • Paoletti C
      • Auclair C.
      • Lesca P.
      • Tocanne J.F.
      • Malvy C.
      • Pinto M.
      ,
      • Auclair C.
      • Voisin E.
      • Banoun H.
      • Paoletti C.
      • Bernadou J.
      • Meunier B.
      ,
      • Charcosset J.Y.
      • Bendirdjian J.P.
      • Lantieri M.F.
      • Jacquemin-Sablon A.
      ,
      • Auclair C.
      ,
      • Buzdar A.U.
      • Hortobagyi G.N.
      • Esparza L.T.
      • Holmes F.A.
      • Ro J.S.
      • Froaschini G.
      • Lichtiger B.
      ,
      • Perez R.P.
      • Nash S.L.
      • Ozols R.F.
      • Comis R.L.
      • O'Dwyer P.J.
      ,
      • Ruckdeschel J.C.
      • Modi S.P.
      • el-Hamouly W.
      • Portuese E.
      • Archer S.
      ,
      • Rouesse J.
      • Spielmann M.
      • Turpin F.
      • Le Chevalier T.
      • Azab M.
      • Modesir J.M.
      ,
      • Jurayj J.
      • Haugwitz R.D.
      • Varma R.K.
      • Paull K.D.
      • Barrett J.F.
      • Cushman M.
      ,
      • Kattan J.
      • Durand M.
      • Droz J.P.
      • Mahjoubi M.
      • Marino J.P.
      • Azab M.
      ). Since these drugs stimulate topoisomerase II-mediated DNA breakage, and the stabilization of enzyme-DNA cleavage complexes induces cell death, it has been assumed that topoisomerase II is the primary cytotoxic target of this drug class. However, there is no direct evidence to support this assumption. Therefore, to determine the primary cellular target and cytotoxic mechanism of ellipticine, a yeast genetic system that exploits a temperature-sensitive chromosomal copy of the topoisomerase II allele (top2-1) was employed. At 25°C, enzyme activity in the top2-1 strain is ~100% (i.e. wild type), but at 30°C, activity is diminished to <10%(
      • DiNardo S.
      • Voekel K.
      • Sternglanz R.
      ). Since the enzyme is essential(
      • Uemura T.
      • Yanagida M.
      ,
      • Goto T.
      • Wang J.C.
      ,
      • Holm C.
      • Goto T.
      • Wang J.C.
      • Botstein D.
      ,
      • Uemura T.
      • Morikawa K.
      • Yanagida M.
      ), it is not possible to fully delete the activity.
      If topoisomerase II is the primary cellular target for ellipticine and if cytotoxicity is due to the stimulation of topoisomerase II-mediated DNA cleavage, a reduction in enzyme activity should greatly diminish drug-induced cell death. Conversely, if topoisomerase II is the primary target, but cell death results from the impairment of catalytic activity, cells with decreased levels of enzyme activity should be hypersensitive to ellipticine. Finally, if ellipticine targets other components in the cell, reduced levels of topoisomerase II should not dramatically affect drug toxicity.
      At 25°C, cell growth was abrogated by 20 μM ellipticine, and ~90% of the initial culture was killed by 200 μM drug (Fig. 1). However, at 30°C, no cell death was observed at any ellipticine concentration employed. Even at 200 μM ellipticine, ~30% cell growth occurred. These results identify topoisomerase II as the primary cellular target of ellipticine (at least in yeast) and demonstrate that the drug acts by converting the type II enzyme to a cellular poison.
      Figure thumbnail gr1
      Figure 1:Cytotoxicity of ellipticine toward yeast cells (JN394t2-1) that express a temperature-sensitive mutant topoisomerase II (top2-1). Ellipticine cytotoxicity at 25°C (●) and at 30°C (●) is shown for increasing concentrations of ellipticine at 18 h. Relative cell survival at time zero was set arbitrarily to 100%. Results are representative of four independent experiments.
      As a control, the cytotoxicity of ellipticine toward yeast cells that contain wild type topoisomerase II (JN394 cells) was determined at 25°C and 30°C (data not shown). Similar drug cytotoxicity was observed at either temperature. Furthermore, the relative survival curve resembled that of the top2-1 strain at 25°C. Thus, resistance of the JN394t2-1 cells to ellipticine at 30°C does not result from a difference in cellular efflux or metabolism of the drug at the elevated temperature.

      Effects of Ellipticine on Topoisomerase II-mediated DNA Religation

      The ability to cleave and religate DNA is not only central to the physiological functions of topoisomerase II, but also provides the basis for the action of antineoplastic agents targeted to the enzyme. As discussed above, these agents act by increasing the amount of topoisomerase-mediated DNA breakage(
      • Liu L.F.
      ,
      • Liu L.F.
      ,
      • Pommier Y.
      ,
      • Corbett A.H.
      • Osheroff N.
      ). In this regard, two drug mechanisms have been reported based on religation assays. Drugs that function by the first mechanism (including several quinolones and genistein) have little effect on religation and are presumed to act by enhancing the forward rate of cleavage(
      • Corbett A.H.
      • Osheroff N.
      ,
      • Robinson M.J.
      • Martin B.A.
      • Gootz T.D.
      • McGuirk P.R.
      • Moynihan M.
      • Sutcliffe J.A.
      • Osheroff N.
      ,
      • Jensen P.S.S.B.S.
      • Andersen A.H.
      • Christiansen K.
      • Alsner J.
      • Thomsen B.
      • Westergaard O.
      ,
      • Corbett A.H.
      • Guerry P.
      • Pflieger P.
      • Osheroff N.
      ). Agents that function by the second mechanism (including amsacrine and etoposide) strongly inhibit religation of the severed DNA and appear to act primarily at this step of the cleavage/religation event (52, 61, 63).
      To delineate the mechanism by which ellipticine enhances DNA breakage, the effects of this drug on topoisomerase II-mediated DNA religation were assessed. As determined by the conversion of supercoiled to linear DNA, 10 μM ellipticine produced maximal stimulation (~6-fold) of topoisomerase II-mediated DNA breakage (Fig. 2, inset). This concentration of ellipticine had virtually no effect on the apparent first order rate of religation (Fig. 2). In contrast, 25 μM etoposide (which produced equivalent cleavage levels to that of ellipticine) decreased the apparent first order rate of religation ~10-fold. This finding suggests that ellipticine stimulates enzyme-mediated DNA breakage primarily by increasing the forward rate of cleavage. This is the first strongly intercalative drug found to function by this mechanism(
      • Corbett A.H.
      • Osheroff N.
      ).
      Figure thumbnail gr2
      Figure 2:Effects of ellipticine on topoisomerase II-mediated DNA cleavage and religation. Results are shown for religation reactions performed in the absence of drug (○) or in the presence of 10 μM ellipticine (▲). Data for reactions performed in the presence of 25 μM etoposide (●) are shown for comparison. Data are plotted as the loss of linear DNA versus time. The proportion of linear DNA was set to 100% at time zero. The inset shows the effect of 0-10 μM ellipticine (x axis) on relative DNA cleavage (y axis). Levels of DNA cleavage in the presence of drug diluent were set to 1.0. Results of DNA cleavage and religation represent the averages of three independent experiments.

      Fluorescence Properties of Ellipticine

      Fluorescence spectroscopy is a sensitive technique that can be used to characterize ligand∙macromolecule binding and to describe the molecular environment of the bound ligand. However, the presence of 18 tryptophan residues per subunit of yeast topoisomerase II (
      • Giaever G.
      • Lynn R.
      • Goto T.
      • Wang J.C.
      ) makes it difficult to utilize the intrinsic fluorescence of the enzyme. Therefore, the interactions of ellipticine with DNA, topoisomerase II, and the enzyme∙DNA complex were elucidated by monitoring the fluorescence properties of ellipticine.
      Ellipticine can exist as a protonated or a deprotonated species (pKa = 7.4), and the fluorescence emission of the drug is highly pH-dependent (Fig. 3). At high pH, where the deprotonated form predominates, the drug fluoresces weakly with peak excitation at 360 nm (data not shown) and emission at 420 nm. In addition, deprotonated ellipticine has a short lifetime (~60 ps, Table I see Fig. 7) and a low quantum yield, suggesting that the fluorescence of this drug species is substantially quenched in water. This suggestion is supported by the fact that the fluorescence lifetime and apparent efficiency of ellipticine fluorescence increase in ethanol () or glycerol (not shown) at low temperatures.
      Figure thumbnail gr3
      Figure 3:pH-dependent emission of ellipticine. The fluorescence emission spectra for 10 μM ellipticine at pH values ranging from 2.0 to 10.3 are shown. Samples were excited at 326 nm.
      Table I:Ellipticine lifetime data
      Figure thumbnail gr7
      Figure 7:Frequency domain fluorescence lifetime measurements of ellipticine. Phase shifts, in degrees for free (●) and complexed (●) ellipticine, and demodulation ratios, in percent for free (□) and complexed (●) ellipticine in the ternary complex are shown. The residual values (top panel) represent the differences between the measured and best fit calculated values. Dimethyl-POPOP was utilized as a reference.
      At low pH, where the protonated form of ellipticine is most prevalent, the drug displays maximal absorption at 440 nm and emission at 520 nm. Both the lifetime (3.5 ns) and the apparent quantum yield of this species are considerably larger than that of the deprotonated drug. Since fluorescence assays were carried out at pH 7.9 (the optimal pH for topoisomerase II activity), both deprotonated and protonated ellipticine were present in an ~60:40 ratio. The dramatic differences in emission maxima and lifetimes of these two ionic species allow both forms to be monitored simultaneously.

      Ellipticine∙DNA Binding

      The binding of ellipticine to a double-stranded 40-mer oligonucleotide was characterized. This 40-mer contains a cleavage/recognition site for topoisomerase II(
      • Corbett A.H.
      • Zechiedrich E.L.
      • Osheroff N.
      ,
      • Sander M.
      • Hsieh T.
      ), and enzyme-mediated cleavage at this site is stimulated by the drug (not shown). In the presence of the oligonucleotide, the fluorescence intensity, anisotropy, and the lifetime of the protonated form of ellipticine increased (Fig. 4, left panel,). No changes in the fluorescence properties of the deprotonated species were observed in the presence of DNA. These findings are consistent with the anionic nature of the genetic material and suggest that protonated ellipticine binds to DNA. Similar results have been found for other intercalators such as ethidium bromide and acridine yellow(
      • Wahl P.
      • Paoletti J.
      • LePecq J.B.
      ,
      • Steiner R.F.
      • Kubota Y.
      ).
      Figure thumbnail gr4
      Figure 4:Ellipticine●DNA binding. The emission spectra for 1 μM ellipticine in the presence of oligonucleotide concentrations ranging from 25-200 nM are shown in the left panel. Samples were excited at 442 nm. A double reciprocal binding analysis is shown in the right panel. Data are plotted as 1/Δ fluorescence intensity at 520 nm versus 1/[oligonucleotide] and represent the averages of two independent experiments. The change in anisotropy of ellipticine with increasing concentrations of 40-mer is shown (inset).
      The affinity of ellipticine for DNA binding was monitored by quantitating the change in intensity at 520 nm. The apparent KD value was calculated (
      • Davenport D.
      ) from the slope of the line (KDI∞) in a plot of the inverse of the intensity increase (ΔI) versus the inverse of the DNA concentration (Fig. 4, right panel). The y-intercept (i.e. 1/ΔI∞) is defined as the inverse of the intensity enhancement expected when all the ellipticine is bound. The apparent KD (~65 nM) for DNA binding determined by this method is comparable to values reported previously for ellipticine (
      • Sorace R.A.
      • Sheid B.
      ,
      • Zegar I.
      • Gräslund A.
      • Bergman J.
      • Eriksson M.
      • Nordén B.
      ,
      • Larue L.
      • Rivalle C.
      • Muzard G.
      • Paoletti C.
      • Bisagni E.
      • Paoletti J.
      ,
      • Kohn K.W.
      • Waring M.J.
      • Glaubiger D.
      • Friedman C.A.
      ) and other cationic aromatic intercalators(
      • Wahl P.
      • Paoletti J.
      • LePecq J.B.
      ,
      • Steiner R.F.
      • Kubota Y.
      ). The ratio of the apparent maximal intensities (ΔI∞) of DNA-bound ellipticine and the free protonated drug is comparable to the ratio of the lifetimes of these two species. The increased steady-state anisotropy of the protonated drug in the presence of DNA (Fig. 4, inset in right panel), together with the concomitant increase in lifetime, provides strong evidence that the rotational diffusion of the drug is less rapid and/or its motion is restricted.

      Formation of a Ternary Topoisomerase II∙Ellipticine∙DNA Complex

      Formation of the topoisomerase II∙ellipticine∙DNA complex was monitored by adding increasing amounts of topoisomerase II to a solution of ellipticine and the oligonucleotide or by adding DNA to a solution containing enzyme and the drug. In either case, an increase in fluorescence intensity at 420 nm was observed, indicating that the deprotonated form of the drug is present in the ternary complex (Fig. 5, left panel). In addition, the lmax of the 420 nm peak was blue-shifted, suggesting that the drug in the ternary complex is located in a hydrophobic environment. Binding affinity was evaluated, as described above, by plotting the inverse of the apparent intensity change (ΔI) as a function of the inverse of the enzyme concentration at fixed drug and DNA concentrations (Fig. 5, right panel). The calculated apparent KD is ~1.5 μM, which is in the range of clinical efficacy for ellipticine(
      • Paoletti C
      • Auclair C.
      • Lesca P.
      • Tocanne J.F.
      • Malvy C.
      • Pinto M.
      ,
      • Auclair C.
      • Voisin E.
      • Banoun H.
      • Paoletti C.
      • Bernadou J.
      • Meunier B.
      ,
      • Charcosset J.Y.
      • Bendirdjian J.P.
      • Lantieri M.F.
      • Jacquemin-Sablon A.
      ,
      • Auclair C.
      ,
      • Buzdar A.U.
      • Hortobagyi G.N.
      • Esparza L.T.
      • Holmes F.A.
      • Ro J.S.
      • Froaschini G.
      • Lichtiger B.
      ,
      • Perez R.P.
      • Nash S.L.
      • Ozols R.F.
      • Comis R.L.
      • O'Dwyer P.J.
      ,
      • Ruckdeschel J.C.
      • Modi S.P.
      • el-Hamouly W.
      • Portuese E.
      • Archer S.
      ,
      • Rouesse J.
      • Spielmann M.
      • Turpin F.
      • Le Chevalier T.
      • Azab M.
      • Modesir J.M.
      ,
      • Jurayj J.
      • Haugwitz R.D.
      • Varma R.K.
      • Paull K.D.
      • Barrett J.F.
      • Cushman M.
      ,
      • Kattan J.
      • Durand M.
      • Droz J.P.
      • Mahjoubi M.
      • Marino J.P.
      • Azab M.
      ).
      Figure thumbnail gr5
      Figure 5:Topoisomerase II∙ellipticine┙DNA binding. The emission spectra for 1 μM ellipticine and 200 nM oligonucleotide in the presence of topoisomerase II concentrations ranging from 0-150 nM are shown in the left panel. Samples were excited at 326 nm. A double reciprocal binding analysis is shown in the right panel. Data are plotted as 1/Δ fluorescence intensity at 420 nm versus 1/[topoisomerase II] and represent the averages of two independent experiments.
      To further define the fluorescence properties of deprotonated ellipticine in the ternary complex, the frequency domain lifetimes were determined. A dramatic increase in lifetime from ~60 ps for free ellipticine to ~23 ns for the bound drug was observed (, see Fig. 7). This finding suggests that the quantum yield of bound ellipticine is much higher than the uncomplexed drug. The fact that ellipticine exhibits a stronger fluorescence (not shown) and a longer lifetime in a less polar solvent such as ethanol () supports the suggestion that the ellipticine binding site in the ternary complex is hydrophobic in nature. The relatively high XR2 value suggests that the emission may be more complex than the two-component model used to fit the data.
      In contrast to the emission increase at 420 nm, there was a decrease in intensity at 520 nm upon the addition of topoisomerase II, and the intensity of this peak was always less than that of free ellipticine. These data imply that deprotonated ellipticine is the major species present in the ternary complex. This suggestion is confirmed by the fact that the lifetimes observed for protonated ellipticine in the presence of enzyme and oligonucleotide were the same as those of the free or the DNA-bound drug.

      Formation of the Topoisomerase II∙Ellipticine Complex

      Although the protonated form of ellipticine binds to DNA, it is the deprotonated form that is present in the ternary complex. This finding implies that the enzyme rather than the DNA dictates the protonation state of the bound drug and suggests that topoisomerase II may play a role in recruiting the drug to the ternary complex. Therefore, the ability of ellipticine to bind directly to topoisomerase II in the absence of DNA was assessed.
      Addition of yeast topoisomerase II to ellipticine resulted in dramatic increases in the fluorescence intensity and lifetime of the deprotonated form of the drug (Fig. 6, left panel). These data are consistent with formation of a binary complex between deprotonated ellipticine and the enzyme. In contrast, no intensity increase at 520 nm was seen, and the lifetime and anisotropy of the protonated drug were that of free compound. Thus, it appears that there is no appreciable binding between protonated ellipticine and topoisomerase II.
      The dissociation constant for the binary complex was determined by varying enzyme at a fixed ellipticine concentration (Fig. 6, right panel). The apparent KD value (~160 nM) was ~10-fold lower than that calculated for the ternary complex. Under conditions approaching stoichiometric binding of the drug to topoisomerase II, a lifetime component of ~24 ns was observed (, Fig. 7). Thus, binding of the drug to the enzyme greatly increases the quantum yield of the deprotonated ellipticine. As expected, the large increase in lifetime was proportional to the increase in intensity (ΔI∞) of the bound over the free form.
      It was not possible to utilize lifetime analysis for the direct measurement of the fraction bound because of the large difference in the lifetimes of bound and free ellipticine and the difficulty in determining the lifetime of the free deprotonated form at the frequencies available. In addition, anisotropy of the topoisomerase∙ellipticine complex could not be measured because excitation near the peak of the absorption band at 360 nm, where the limiting anisotropy is maximal (r0 = 0.4, data not shown), resulted in overlap between the water Raman -OH stretching band emission and the sample emission.

      DISCUSSION

      Although topoisomerase II is the primary cellular target for a number of clinically relevant antineoplastic agents, virtually nothing is known about enzyme∙drug interactions. In general, binding studies have been hampered by the limited number of techniques available and the relatively large amount of enzyme required for these methods. However, by taking advantage of the optical properties of ellipticine and the ability to overexpress topoisomerase II in yeast, it was possible to characterize the interactions of this DNA cleavage enhancing drug with topoisomerase II and the enzyme∙DNA complex.
      A summary scheme depicting ellipticine binding to DNA, topoisomerase II, and the enzyme∙DNA complex is shown in Fig. 8. At neutrality, two species of ellipticine, a protonated and a deprotonated form, exist in equilibrium. While protonated ellipticine intercalates in free DNA, it is predominantly deprotonated ellipticine that binds to topoisomerase II in the absence of DNA and is present in the ternary complex. This finding implies an interaction between the drug and topoisomerase II in the ternary complex and further suggests that the enzyme dictates the ionic state of ellipticine. Furthermore, it appears that the binding of ellipticine to topoisomerase II is indicative of the enzyme∙drug interactions in the ternary complex, since a mutant type II topoisomerase that is hypersensitive to ellipticine has a higher intrinsic binding affinity for the drug.2(
      S. J. Froelich-Ammon, M. W. Patchan, S. H. Elsea, R. B. Thompson, and N. Osheroff, unpublished results.
      )
      Figure thumbnail gr8
      Figure 8:Model of ellipticine binding to DNA, topoisomerase II, and the topoisomerase II┙DNA complex. Protonated and deprotonated forms of ellipticine are shown. Topoisomerase II is depicted by the handlebar-shaped structure, and DNA is represented by the cylinders. The enzyme is shown binding to DNA at a point of helix-helix crossover as proposed by Zechiedrich and Osheroff (73) and Roca et al. (74). In this model, the topoisomerase II∙ellipticine∙DNA ternary complex (complex 3) is formed via the prior association of the drug with either the DNA (complex 1) or the enzyme (complex 2). Formation of the ternary complex results in increased levels of covalent topoisomerase II∙DNA cleavage complexes (complex 4).
      Based on previous studies that examined drug binding to topoisomerase I (20) or DNA gyrase(
      • Shen L.L.
      • Kohlbrenner W.E.
      • Weigl D.
      • Baranowski J.
      ,
      • Willmott C.J.R.
      • Maxwell T.
      ), it has been suggested that topoisomerase II-targeted agents may bind specifically to the enzyme∙DNA complex (14, 19). The generality of this theory is challenged by the finding that ellipticine and intoplicine (
      • Nabiev I.
      • Chourpa I.
      • Riou J.-F.
      • Nguyen C.H.
      • Lavelle F.
      • Manfait M.
      ) bind directly to the enzyme. Moreover, since the apparent dissociation constants for the topoisomerase II∙ellipticine and ellipticine∙DNA complexes are an order of magnitude lower than that of the ternary complex, it is likely that the formation of the topoisomerase II∙ellipticine∙DNA complex occurs via initial drug binding to either the enzyme or the DNA (Fig. 8). In the latter case, the ionic state of ellipticine bound to DNA must be converted by the enzyme to the deprotonated form during the formation of the ternary complex.
      It has been proposed that intercalative drugs alter the cleavage/religation equilibrium of topoisomerase II primarily through their effects on DNA structure(
      • Monnot M.
      • Mauffret O.
      • Simon V.
      • Lescot E.
      • Psaume B.
      • Saucier J.-M.
      • Charra M.
      • Belehradek Jr., J.
      • Fermandjian S.
      ). Two lines of evidence undercut this assertion. First, there appear to be dominant interactions between ellipticine and topoisomerase II in the ternary complex, indicating that there are points of contact between this drug and a hydrophobic portion of the enzyme. Second, the deprotonated form of ellipticine in the ternary complex is not the preferred species for DNA intercalation. While the deprotonation of ellipticine does not preclude intercalation, no evidence for the interaction between the deprotonated form of ellipticine and DNA was seen. This implies that interactions between the drug and DNA in the ternary complex differ from that in the ellipticine∙DNA complex. Because the enzyme and its nucleic acid substrates prefer different ionic states of the drug, the juxtaposition of ellipticine, the enzyme, and the DNA may produce an inherent stress in the ternary complex. If so, the resulting strain could contribute to the increased dissociation constant of the ternary complex and may be the underlying mechanistic basis for the enhancement of enzyme-mediated DNA cleavage.
      Before the design and clinical efficacy of novel topoisomerase II-targeted agents can be optimized, the mechanism of drug action must be understood. The current study shows that ellipticine interacts with the enzyme alone and raises the potential for multiple pathways for ternary complex formation. This finding presents a novel conceptual scaffold upon which to build new theoretical models for topoisomerase II∙drug∙DNA interactions.

      ACKNOWLEDGEMENTS

      We are grateful to the laboratory of Dr. Joseph Lakowicz and the Center for Fluorescence Spectroscopy (University of Maryland School of Medicine) for the use of the SLM 8000 steady state fluorescence spectrometer, to Dr. John Nitiss for expert advice and the yeast strains utilized, to Dr. Sarah H. Elsea and Michael Otto for helpful discussion, to Erin Hannah for assistance with yeast and preparation of pBR322 plasmid DNA, and to Paul Kingma, Dr. Kathy Latham, and Dr. Andrew Burden for critical reading of the manuscript.

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