[CANCER RESEARCH 41, 2235-2240, June 1981]0008-5472/81 /0041-OOOOS02.00

Template Specificity of DMA Binding by Nogalamycin and Its AnalogsUtilizing Competitive Fluorescence Polarization

Carol L. Richardson,1 Ava D. Grant, Sandra L. Schpok, William C. Krueger, and Li H. Li

Meloy Laboratories, Inc., Springfield, Virginia 22151 [C. L. R., A. D. GJ, and Research Laboratories. The Upjohn Company, Kalamazoo, Michigan 49001 [S L SW. C. K., L H. L]

ABSTRACT

Nogalamycin, an anthracycline antibiotic, interacts with DNA.This interaction is measured by a competitive fluorescencepolarization assay in which nogalamycin displaces acridineorange. The amount of acridine orange displaced is dependentupon the concentration and DNA-binding capability of the drug.The relative DNA-binding capacities of several nogalamycin

analogs are also determined by this method. A comparison ofthese results with circular dichroism and thermal denaturationyields a positive correlation. The base-pair specificities of these

compounds are also evaluated by competitive fluorescencepolarization using DNA's of differing base composition. These

results indicate that compounds containing the nogalose moiety generally prefer adenine and thymine. On the other hand,some of the 7-O-alkyl analogs appear to interact similarly withDNA's of differing base composition, and others show a preference for DNA's with high guanine and cytosine content.

Specificities obtained with this method are compared with DNAthermal denaturation and polymerase inhibition studies. Thepotential value of this relatively new competitive method for thestudy of DNA-reactive antitumor compounds is discussed.

INTRODUCTION

Nogalamycin, an anthracycline antibiotic, similar to manyother useful antitumor drugs, interacts with DNA (1, 5). Thenature of this interaction is intercalative (10, 12, 16, 17) andhas been studied by a variety of physicochemical and biologicalmethods (7, 9, 11). The renewed interest in this class ofcompounds is due, in part, to the recently synthesized structural analogs (19). The closely related dis and con isomers ofthe 7-O-alkyl derivatives presumably differ only in the configuration centered around a single carbon atom (Chart 1), buttheir biological and chemical activities differ significantly.

For this reason, these compounds are particularly suited forvalidation of a relatively new methodology. The AO2 displace

ment assay (13-15) measures the interaction of a compound

with DNA by competitive fluorescence polarization. Both thecompound and AO are mixed with DNA in a cuvet, and thefluorescence polarization value of the AO is measured. Thisvalue reflects the proportion of fluorochrome (AO) moleculesthat have been displaced by the drug. The method is rapid,requires very small quantities of DNA, and does not rely on thefluorescent properties of the test compound. The initial studies

1To whom requests for reprints should be addressed, at Meloy Laboratories,

Inc.. 6715 Electronic Drive, Springfield, Va. 22151.2 The abbreviations used are: AO, acridine orange; poly(dA-dT), alternating

deoxyadenylate and deoxythymidylate; poly(dG-dC), alternating deoxyguanylateand deoxycytidylate; Dso, drug concentration required for 50% displacement ofacridine orange from DNA as measured by fluorescence polarization.

Received December 29, 1980; accepted March 2, 1981.

contained herein are aimed at determining if this methodologycan detect differences in the DNA reactivity of nogalamycinanalogs. Circular dichroism and thermal denaturation are performed to provide comparative data.

The AO displacement assay has also been applied to thestudy of base-pair specificity for actinomycin D and an anthra-

cenedione analog (14). For the parent compound, nogalamycin, template specificity has been reported as a preference foradenine and thymine based on thermal denaturation of DNA(7"m)(2, 3) and flow dichroism studies (10); however, contradic

tory results were reported utilizing buoyant density techniques(7). Obviously, this situation required further investigation.

The determination of base-pair specificity of a series of

nogalamycin analogs provides information on the structuralfeature(s) involved. Base-pair specificities of several of these

analogs are also studied by Tmand DNA and RNA polymeraseinhibition using actinomycin D as a control.

The purpose of this work is 2-fold. Initially, nogalamycin and

its analogs are used to further validate the AO displacementassay and to determine if this method is sufficiently sensitive todifferentiate the structurally similar dis and con isomers. Thesecond objective is to elucidate the nucleotide base-pair spec

ificity of nogalamycin and its analogs.

MATERIALS AND METHODS

Drugs. Nogalamycin and its analogs were obtained from theUpjohn Company, Kalamazoo, Mich. Nogalamycin analogswere synthesized and purified by Wiley et al. (19). The preparation, chemistry, and structure of these compounds have beenreported (19, 20). The 7-O-alkyl derivatives exist in 2 isomerie

forms with respect to position 7. In the con isomer, the alkylgroup substitution is probably above the plane of the aromaticring; in the dis configuration, it is probably below the plane, asshown in Chart 1.

Materials. AO was purchased from the Aldrich ChemicalCompany, Milwaukee, Wis.; poly(dA-dT), actinomycin D, and

calf thymus DNA type I were from the Sigma Chemical Company, St. Louis, Mo.; Clostridium perfringens DNA and Micro-coccus luteus DNA poly(dG-dC) were obtained from MilesLaboratories, Elkhart, Ind.; and poly(dG-dC) was purchasedfrom P-L Biochemicals, Milwaukee, Wis.

AO Displacement Assay. This procedure has been described previously (14, 15). Dilutions of nogalamycin or analogwere prepared in 75% dimethyl sulfoxide. An aliquot (50 ftl)was added to 1.85 ml of 0.01 M sodium cacodylate buffer, pH6.7, in the cuvet. Following addition of AO (100 jitl of a 10 U.Msolution), the solution was mixed, and the initial fluorescencepolarization of the AO and test compound mixture was measured in a Model Fluoro I (distributed by Meloy Laboratories,Inc., Springfield, Va.), using excitation and emission filters at492 and 520 nm, respectively. Then, 50 fil of DNA were added

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C. L. Richardson et al.

,3 OH

CH,

CONFIGURATION

»3

OH

COMPOUND

NOGALAMYCIN

NOGALAMYCINIC ACID

NOGAMYCIN

7-0-METHYLNOGALAROL

7-0-METHYLNOGAROL

7-0-ETHYLNOGAROL

DIS-NOGALAROL

NOGALARENE '

7-DEOXYNOGALAROL

NOGALAMYCIN-N-OXIDE 2

CO2CH3

CO2H

H

CO2CH3

H

H

CO2CH3

C02CH3

COjCH3

CO2CH3

NOGALOSE

NOGALOSE

NOGALOSE

OCH3

OCH3

OCH2CH3

OH

THE OH ON CARBON 8 AND R-, ARE ABSENT; THE RING CONTAINING

THESE CARBONS BECOMES AROMATIC.

THE N HAS AN O ATTACHED IN ADDITION TO THE TWO CH3 GROUPS.

Chart 1. Structures of nogalamycin and its analogs based on present experimental evidence. The most probable structures of the dis and con isomers aregiven; however, it is possible that the correct structures are mirror images ofthose indicated.

in a concentration equivalent to 35% saturation [6 milliunits ofpoly(dG-dC), 1.9 ¡IMcalf thymus DNA, 1.6 /ÕMM. luteus DNA,and 1.3 /¿Mpoly(dA-dT) based on phosphate]. After addition of

the DNA, the sample in the cuvet was mixed, and the finalpolarization was measured.

The maximum binding was determined by addition of AO toDNA in the absence of test compound. Data were expressedas a percentage of maximum binding, and the concentration oftest compound that resulted in 50% displacement of the AOfrom DNA was defined as the D50.

Circular Dichroism. The interaction between calf thymusDNA and the nogalamycins was measured at 25°on a Gary 60

spectropolarimeter equipped with a Model 6003 circular di-

chroism attachment (VaríanInstruments, Monrovia, Calif.). Theinstrument was calibrated with 10-camphorsulfonic acid (8).

Thermal Denaturation. Drug-DNA interactions were evaluated by thermal melting of DNA (7m). The melting studies wereperformed on a Gilford 2400 spectrophotometer with a Model2527 thermoprogrammer in 0.01 M sodium phosphate buffer,pH 7.2.

Purification of DNA Polymerases. DNA polymerase a of

cultured L1210 cells was purified according to the procedureof Graham ef al. (6) with some modifications. L1210 cells (12g) were sonically dispersed and extracted twice with 0.8 M KCIbuffer. After ammonium sulfate fractionation (65% saturation),the aliquot was dialyzed against buffer composed of 50 mwTris-HCI (pH 7.8), 1 mM dithiothreitol, 1 mM EDTA, and 10%glycerol and was passed through a DEAE-cellulose columnthat was eluted with buffer containing 0.3 M KCI to removenucleic acids. Fractions containing DNA polymerase activitywere pooled, dialyzed, applied onto a phosphocellulose column, and eluted with buffer containing 0 to 0.05 M KCI. Afterthe removal of most of the nonenzymatic proteins, the columnwas further eluted with a buffered KCI gradient from 0.1 to 0.7M. DNA polymerase a was collected and used for subsequentstudies.

Assay of DNA Polymerase a Activity. The enzyme activitywas assayed in a 500-/il total reaction mixture containing 40mM Tris-HCI buffer (pH 7.8); 1.2 ITIMMgCI2; 20 mM KCI; 4 mM

dithiothreitol; 64 mM concentrations each of dATP, dCTP, anddGTP; 1 ¡UM[3H]dTTP or [3H]dGTP (7400 cpm/pmol); 10 ¿ig

calf thymus DNA, poly(dA-dT), or poly(dG-dC); and 50 ¡ulDNApolymerase. The reaction was incubated at 37°.At time points

0, 20, and 40 min, 50 ¡ulof the reaction mixture were added toeach of triplicate wells of a 96-well Falcon culture plate containing 100 /il of 20% trichloroacetic acid per well. The platewas placed on an ice bath, and 25 /il of salmon sperm DNA (4mg/ml) were then added to each well. The aliquot was collected on glass fiber paper by means of a Scatron Cell Harvester (Flow Laboratories, Rockville, Md.) and washed with10% trichloroacetic acid and absolute ethanol. Discs wereplaced in glass scintillation vials containing 0.5 ml of 0.5 Mperchloric acid and incubated for 1 hr at 70°.After incubation,

10 ml of BBS-3 scintillation counting solution were added. Thisscintillation fluid consisted of 100 ml BBS-3 Biosolvent (Beck-

man Instruments, Fullerton, Calif.) and 42 ml Liquifluor (NewEngland Nuclear, Boston, Mass.) per liter of toluene. Theradioactivity was determined in a Packard Tri-Carb liquid scin

tillation counter.Purification of RNA Polymerases. RNA polymerases I and

11were purified from isolated normal mouse nuclei (18) essentially according to the procedure of Blair (4). Briefly, nRNApolymerase was solubilized by sonic extraction of the nuclei inbuffer [50 mM Tris-HCI (pH 7.9), 0.1 mM EDTA, 0.5 mM

dithiothreitol, 5 mM MgCI2, and 25% glycerol] containing 0.3M (NH4)2SO4. The enzyme was precipitated with (NH4)2SO4(0.42 g/ml of extract), dissolved in buffer containing 0.05 M(NH4)2SO4, and dialyzed with buffer. After centrifugation at105,000 x g for 1 hr, the supernatant was applied onto aDEAE-Sephadex A-25 column and eluted with a linear gradient

of buffered (NH4)2SO4 solution (0.05 to 0.5 M). Although bothRNA polymerases I and II were isolated, Enzyme I was arelatively minor component in our preparation.

Assay of RNA Polymerase II Activity. The enzyme activitywas assayed in 200-¡ulvolumes containing 40 ¡ugof calf thymusDNA or 0.2 A260unit of poly(dA-dT) or poly(DG-dC); 62.5 mMTris-HCI (pH 7.9); 3 mM MnCI2; 85 mM (NH4)2SO4; 0.25 mMconcentrations each of ATP, CTP, and GTP; 2.5 /IM [5-3H]UTPor [3H]GTP (20 /iCi/pmol); and 10 to 20 /il of RNA polymeraseII. The reaction mixture was incubated at 37°for 20 min, the

acid-insoluble fraction was collected, and the radioactivity wasdetermined.

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Fluorescence Polarization Studies with Nogalamycin

RESULTS

Nogalamycin was analyzed for its ability to displace AO fromDNA by measuring the fluorescence polarization of the AO inthe presence of a constant concentration of DNA and increasing concentrations of the compound. Nogalamycin was foundto be effective at low concentrations, requiring less than 1 JUMto displace 0.5 ftM AO from the synthetic polynucleotide,poly(dA-dT). In contrast, the poly(dG-dC) required significantly

higher nogalamycin concentrations to obtain similar displacement (Chart 2). Dose-response effects were noted with all 4

polynucleotides; increasing concentrations of the compoundyielded decreasing AO binding.

Due to the competitive nature of this procedure, the dose-

response curves are often more sigmoidal than linear, especially as concentrations approach 0% of maximum binding.Hence, in order to obtain a reliable comparison value, the D50is chosen. In general, the more strongly a compound interactswith DNA, the lower is its D50.

The Dso's obtained through this procedure were compared

with values obtained with 2 classical methods, i.e., Tm andcircular dichroism, and these data are presented in Table 1.Both nogalamycin and d/s-nogamycin show the strongest in

teraction with DNA according to circular dichroism and Tmmeasurements. Results also indicate that compounds with thedis isomerie configuration bind better to calf thymus DNA thandoes the con isomer for each of the 7-O-alkyl pairs tested. The

dis ¡somersare also required in lower concentrations than arethe con isomers for displacement of AO from DNA, as shownby the D50's in Table 1 consistent with the circular dichroism

and Jm data. The lowest DSO,0.07 /IM, was obtained with d/s-nogamycin.

It was also of interest to examine the base compositionpreferences of these analogs with both classical methods andthe new methodology. Because the technique of circular dichroism yields difference spectra qualitatively different forpoly(dA-dT) than for poly(dG-dC), information on base se

quence preferences could not be obtained by this method.Thermal denaturation provided some useful information with

a variety of polynucleotides. As shown in Table 2, both nogalamycin and d/s-nogamycin cause the largest changes in melting temperatures with poly(dA-dT). As the G + C content of

the natural nucleotides was increased, the change in meltingtemperatures decreased. In contrast, the control compound,actinomycin D, did not alter the melting temperature of poly(dA-

dT) but affected the melting temperature of calf thymus DNAmore than of C. perfringens DNA. The latter has a higher A +T content (73%) than does calf thymus DNA (57%). Withactinomycin D, the M. luteus DNA did not melt. Similarly,poly(dG-dC) could not be tested with any of these compounds.

The AO displacement method does provide useful data withthe entire spectrum of polynucleotides. Values were determined with 0.5 /¿MAO at polynucleotide concentrations thatwere equivalent to 35% saturation. Thus, competitive conditions were optimized (13). The results clearly indicate betterdisplacement of AO from poly(dA-dT) than from other polynu

cleotides by nogalamycin (Chart 2). Table 3 summarizes theresults obtained with 12 nogalamycin analogs. Four of thecompounds show strikingly lower D50's (at least a 4-fold differ

ence) for poly(dA-dT) than for poly(dG-dC). The 4 compounds,nogalamycin, d/s-nogamycin, d/s-nogalamycinic acid, and nogalamycin A/-oxide, all contain the nogalose sugar moiety. In

Table 1Comparison of 3 methods lor measuring interaction between calf thymus DNA

and nogalamycin and its analogs

O

g 100

m

1 8°

¿

2 60

x<

OE111O.

40

20

POLY dG-dC

0.02 0.05 0.1 0.2 0.5 1.0

NOGALAMYCIN CONCENTRATION, u K

1.2

Chart 2. AO displacement assay for nogalamycin with 4 polynucleotides ofdiffering A + T content: poly(dAKlT); calf thymus DNA; M. luteus DNA; andpoly(dG-dC). The percentage of maximum binding of AO to DNA is plotted againstthe logarithmic drug concentration. The better the nucleotide-drug interaction,the lower is the nogalamycin concentration required to obtain 50% of maximumAO binding.

ACDa

(millidegrees ofCompound ellipticity)A7"mNogalamycind/s-Nogalamycinic

acidd/s-Nogamycin7-d/s-O-Methylnogalarol7-con-O-Methylnogalarol7-dis-O-Methylnogarol7-con-O-Methylnogarol7-d/s-O-Ethylnogarol7-con-O-Ethylnogarol1281186828215.0°4.411.75.03.14.003.9-2.5Dsoc

(MM)0.18

±0.02d0.21

±0.010.07±0.000.15±0.020.33±0.020.58±0.030.90±0.040.20±0.011.18±0.02

a Difference spectrum determined by circular dichroism at 465 nm. ¿CD,circular dichroism of DNA and compound mixture - circular dichroism of sepa

rate components.6 Determined by thermal melting of DNA with or without the compound in 0.01

M phosphate buffer, pH 7.2. The ratio of DNA (measured as phosphate) tocompound was 6.5. Drug concentration was 1.2 X 10~5 M. A7"m, 7~m(DNA and

compound mixture) - 7m (DNA alone). The Tm of calf thymus DNA is approximately 71°.

c Displacement of AO-DNA binding.d Mean ±S.D. of 4 separate determinations.

Table 2

Thermal denaturation of polynucleotides of differing A + T content withnogalamycin and its analogs

The thermal melting with or without the compound was determined in 0.01 Mphosphate buffer, pH 7.2. The ratio of polynucleotide (measured as phosphate)to compound was 6.5. Compound concentrations were 1.2 x 10~5 M.

CompoundNogalamycind/s-Nogamycind/s-Nogalamycinic

acid7-d/s-O-Ethylnogarol7-d/s-O-Methylnogarol7-con-O-MethylnogarolActinomycin

DPoly(dA-dT)(100%

A+T)24.6°18.33.29.78.26.90.0AT„C.

perfringensDNA(73%

A+T)15.4°11.34.04.15.21.97.6aCalf

thymusDNA(57%

A+T)15.0°11.74.43.95.00.08.6M.

luteusDNA(27%

A+T)6.2°7.42.51.82.90.6

A rm, rm (DNA and compound mixture) - T"m(DNA alone).

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C. L. Richardson et al.

contrast, none of the nogarol or nogalarol derivatives, whichlack this sugar moiety, show a markedly lower D50for poly(dA-dT) than for poly(dG-dC). Actinomycin D was chosen as acontrol compound because it does not interact with poly(dA-

dT) and shows a preference for polynucleotides with high G+ C content. Data obtained with the AO method indicate thatactinomycin D, even in a concentration as high as 50 ¡J.M,didnot displace the competing fluorochrome from poly(dA-dT).The relative D50's for poly(dG-dC), calf thymus DNA, and M.

luteus DNA were expected, based upon the known base composition preference of actinomycin D.

In an attempt to correlate this template specificity with biochemical effects, additional studies, such as inhibition of DNApolymerase using poly(dA-dT), calf thymus DNA, and poly(dG-

dC) as templates, were carried out (Table 4). Nogalamycinshowed less inhibition of this enzyme with poly(dG-dC) thanwith either calf thymus DNA or poly(dA-dT) as a template, in

agreement with a preference of this compound for poly(dA-dT).d/'s-Nogamycin behaved similarly. The results with actinomycin

D, however, were surprising in light of the well-known prefer

ence of this compound for guanine and cytosine and its poorinteraction with adenine and thymine. At a concentration ashigh as 5 UM, actinomycin D was not inhibitory when poly(dG-

dC) was the template, but a 30% inhibition was observed withpoly(dA-dT).

In order to determine whether this effect was enzyme related,a second enzyme, RNA polymerase II, was purified from mouseliver nuclei and tested with the same templates for inhibition bythese compounds. The results, given in Table 5, were predictable with actinomycin D; i.e., the greatest inhibition was obtained with poly(dG-dC) as the template. However, both noga-lamycin and oVs-nogamycin also showed the same trend, the

most inhibitory effect with the template of highest G + Ccontent.

Table 3

Interaction of nogalamycin and its analogs with polynucleotides of differing base composition as measuredby AO displacement assay

Displacement of AO from DNA by the compound was measured in 0.01 Mcacodylate buffer. pH 6.7. Theproportion of free AO molecules was determined by fluorescence polarization. The concentration of AO was0.5 JIM.Polynucleotide concentrations were adjusted to 35% of saturation with this AO concentration. TheAO and drug were added simultaneously, followed by addition of the polynucleotide.

CompoundNogalamycindis-Nogalamycinic

aciddis-NogamycinNogalamycin

W-oxide7-Deoxynogalaroldis-Nogalarol7-d/s-O-Methylnogalarol7-con-O-Methylnogalarol7-dis-O-Ethylnogarol7-con-O-Ethylnogarol7-dis-O-Methylnogarol7-con-O-MethylnogarolNogalareneActinomycin

DPoly(dA-dT)

(100% A +T)0.070.120.060.760.330.180.370.210.200.560.470.520.13>50.00DsotyCalfthymus DNA

(57% A -HT)0.180.210.075.500.220.250.150.330.201.180.580.900.271.60,M)M.luteus DNA

(27% A -HT)0.200.460.0710.000.240.250.180.250.270.680.180.640.350.61Poly(dG-dC)(0% A -HT)1.070.50O.2717.000.130.140.080.260.260.800.280.740.230.54

Table 4Inhibition of DNA polymerase a by nogalamycin and its analogs with different templates

The percentage of inhibition of purified DNA polymerase of L1210 ascites cells was calculated from therate of incorporation of [3H]dGTPor [3H)dTTP.The reaction mixture (0.5 ml) contained 20 fimol Tris-HCI (pH

7.8); 0.6 fimol MgCI2; 10 jimol KCI; 2 jimol dithiothreitol; 1.28 fimol each dATP. dCTP, and dTTP; 5 figtemplate polynucleotide, [3H)dGTP,or [3H)dTTP;and 50 /il enzyme (containing 6.25 fig protein). Incubationswere performed at 37°for 20 and 40 min.

CompoundNogalamycind/s-Nogamycind/s-Nogalamycinic

acid7-d/s-O-Methylnogarol7-con-O-MethylnogarolActinomycin

DDrug

concentration(;iM)11011010

50105010

501

510Poly(dA-dT)186443623838225753

541330

65Inhibition

(%)Calf

thymusDNA29

6778

8928

5844

5323

2559

7556poly(dG-dC)0

4282921

4532

39370

013

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Fluorescence Polarization Studies with Nogalamycin

Table 5

Inhibition of RNA polymerase II by nogalamycin and its analogs with differenttemplates

The conditions for assaying RNA polymerase II are described in the text.Reactions were carried out at 37°for 20 min.

Inhibition (%)Drug concen-

tration Calf thymusCompound (/ÃŒM) Poly(dA-dT) DMA Poly(dG-dC)

Nogalamycin

d/s-Nogamycin

1020

50

3185

72

4642

76

100100

92

Actinomycin D51004859 100100 100

DISCUSSION

The AO displacement assay has been applied to the study ofa few classical intercalators (e.g., actinomycin D, Adriamycin,and daunorubicin) and an anthraquinone derivative (14, 15);however, the ability of this method to differentiate closelyrelated chemical structures was untested. The availability of arange of nogalamycin derivatives with different activitiesprompted us to test the sensitivity of this method. Nogalamycinand all of the analogs showed dose-response effects charac

teristic of competitive binding with a variety of polynucleotides(Chart 2). However, the drug concentrations at which AOdisplacement became evident varied with the analog and thepolynucleotide template. In general, the better the interactionof the drug with DMA, the lower is the D60with the exception ofd/s-nogamycin. The results obtained with this analog indicated

better AO displacement than that for the parent compound(Table 1). This is not readily explained in relation to resultsobtained by other methods.

At the other end of the spectrum, the weakly DNA-reactive

compounds were more readily detected by this technique thanby Tmand circular dichroism. The interaction between 7-con-O-methylnogarol and DNA is not readily detectable by Tmmeasurements, but a dose-response effect is clearly obtained

in the AO assay, with a D50of 0.90 JIM.Both circular dichroism and thermal denaturation studies

indicate that the dis isomers interact more strongly with DNAthan do the con isomers (Table 1). The D50's obtained by this

method also show the same striking difference in activity withthis small change in molecular configuration. Thus, the AOdisplacement method is sufficiently sensitive to differentiateclosely related chemical structures.

The second facet of these studies involved base-pair speci

ficity of binding. The parent compound has been reported toprefer adenine and thymine (1, 2). However, analyses of buoyant density changes upon drug-polynucleotide binding suggested a better interaction between nogalamycin and GC ratherthan nogalamycin and AT (7). The AO displacement assay hasbeen applied to a base-pair specificity study of actinomycin D,

and the results were in agreement with the documented G +C preference of this drug. The base-pair specificity study was

carried out with nogalamycin and its analogs (Table 3), and theresults were compared with those of the Tmstudies (Table 2).Both methods confirmed the A + T preference reported fornogalamycin (1, 2). d/'s-Nogamycin behaved similarly. In con

trast, this A + T preference was not observed for most of the

analogs studied. In fact, a clear preference for poly(dA-dT)over poly(dG-dC) was obtained with only 4 of the 13 compounds: nogalamycin; d/s-nogamycin; d/s-nogalamycinic acid;and nogalamycin N-oxide. These are the 4 derivatives that

contain a nogalose moiety (Chart 1), indicating that the presence of this sugar influences the binding specificity of thesecompounds to DNA.

Additional template specificity studies were performed using3 DNA polymers to measure the inhibition of highly purifiedDNA polymerase of ascites L1210 cells by nogalamycin andits analogs. Upon initial examination of the data, the resultsappeared to confirm other studies that indicated greater enzyme inhibition with poly(dA-dT) as the template than withpoly(dG-dC) (Table 4). However, the control, actinomycin D,

did not behave as predicted. Enzyme inhibition with this drugalso suggested an A + T preference, although the enzymeactivity with poly(dA-dT) as template was completely inhibited

at higher drug concentrations (Table 4).Since actinomycin D and nogalamycin predominantly inhibit

RNA synthesis, highly purified RNA polymerase II of mouseliver nuclei was utilized in similar inhibition assays with actinomycin D, nogalamycin, and d/s-nogamycin. Results showed

that only 5 /IM actinomycin D gave 100 and 92% of the activityof this enzyme with the poly(dG-dC) template; significantly lessinhibition was obtained with poly(dA-dT). These data conflict

with the results of Tm, DNA polymerase inhibition, and AOdisplacement. It is tempting to speculate that the polymeraseitself is also responsible for the observed template specificities.Probably, the reactions among drug, DNA, and enzyme do notreflect merely the drug-DNA interaction but are significantlymore complex.

In summary, the AO displacement assay method is sufficiently sensitive to differentiate the structurally similar dis andcon isomers. In general, the method correlates well with classical techniques used to demonstrate the DNA reactivity of acompound. The base-pair specificity indicated by the Jm andAO displacement methods is a preference for A -l- T by noga

lamycin, as previously reported in the literature. A similarpreference for A + T was observed with nogamycin, nogalamycin acid, and nogalamycin N-oxide; these 3 compounds also

contain the nogalose moiety. These studies confirm the potential value of the AO displacement method for the determinationof relative DNA reactivities and elucidation of base-pair specificities of similar classes of compounds.

ACKNOWLEDGMENTS

We wish to thank Dr. Charles G. Smith of Revlon Health Care for helpfuldiscussions that initiated this study. We acknowledge the skillful technical assistance of L. M. Pschigoda and the assistance of R. Rasdorf in the preparationof this manuscript.

REFERENCES

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2. Bhuyan, B. K., and Smith, C. G. Differential interaction of nogalamycin withDNA of varying base composition. Proc. Nati. Acad. Sei. U. S. A., 54: 566-572, 1965.

3. Bhuyan. B. K., and Smith. C. G. Nogalamycin. In: A. C. Sartorelli and D. GJohns (eds.), Antineoplastic and immunosuppressive Agents, Part 2. pp.623-632. Berlin: Springer-Verlag, 1975.

4. Blair. D. G. R. DNA-dependent RNA polymerases of Ehrlich carcinoma,other murine ascites tumors, and murine normal tissues. J. Nati. Cancer

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2240 CANCER RESEARCH VOL. 41

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1981;41:2235-2240. Cancer Res   Carol L. Richardson, Ava D. Grant, Sandra L. Schpok, et al.   Analogs Utilizing Competitive Fluorescence PolarizationTemplate Specificity of DNA Binding by Nogalamycin and Its

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