[CANCER RESEARCH 37, 4202-4210, November 1977)

SUMMARY

The effects of antitumor intercalating agents on themethylation of RNA were studied by the use of solubletransfer RNA (tRNA) methylases from the Novikoft ascitestumor and nucleolar nibosomal RNA (rRNA) methybasesfrom the tumor and liver of the rat.

In the soluble tRNA methybase system, ellipticine at lowdrug levels preferentially inhibited methylation of tRNAadenine, whereas ethidium bromide and hycanthone preferentially inhibited methybation of tRNA guanine. At higherdrug levels, methylation of other tRNA bases was alsosignificantly inhibited by ethidium bromide and hycanthone; however, methylation of tRNA pyrimidine bases wasnot at all affected by eblipticine. Perturbation of methylacceptor sites by interaction of ellipticine with tRNA wasindicated by decreased inhibition with increased tRNA concentrations. With a partially purified adenine methylasepreparation, ellipticine was competitive with tRNA. Ethidium bromide and hycanthone appeared to interact with theenzymes rather than with the tRNA substrate, since theinhibition of a partially purified guanine methylase preparation was noncompetitive with respect to tRNA.

All three drugs also inhibited rRNA synthesis and methylation in isolated nucleoli. Methylation of RNA in isolatedtumor nucleoli was more susceptible to these drugs thanwas methybation of liver nucleolar RNA. The differentialwas the most marked in the case of ethidium bromide.Ethidium bromide administered to tumor-bearing ratscaused a marked reduction in the content of ANA and ofrRNA methylase activity in nucleoli prepared 16 hr later.Under these conditions ellipticine also caused a decreasein nucleolar RNA polymerase and rRNA methylase activities.The potential of these drugs in perturbation of RNA processing may make a significant contribution to their antitumon activity.

INTRODUCTION

Intercalating agents are a group of compounds, usuallyplanar hetero-polycyclic compounds, capable of intercabation between adjacent base pairs in DNA, as originallydescribed by Lerman (21). The structure and function of

DNA are perturbed as a result of intercalation. However, thebiological consequences are not uniform. Some intercalating agents exert a stronger effect on DNA replication,whereas others have a stronger effect on RNA transcription.These compounds are also capable of intercalation intodouble-stranded helical regions of RNA (5, 9, 19, 38),interfering with the processing of pre-nRNA (36), the terminab nucleotidyl transfer of tRNA (10), and the integrity andfunction of RNA in protein synthesis (2, 37, 39). Otherbiological effects unrelated to nucleic acid intercalationare also evident, for example, specific inhibition of certainenzymes by ethidium bromide (4) and hycanthone (35).

The antitumor effect of ellipticine (12, 18, 20) and hycanthone (13, 35, 40) has been well established. Althoughethidium bromide is not considered to be a practical antitumor agent, its efficacy against certain tumors has, nevertheless, been reported (11, 32). These drugs and theirderivatives affect preferentially the synthesis of rANA (3, 8,15, 40). Disruption of ribosome production in malignantcells certainly would cause malignant cells to cease growth(22). However, it is not obvious how these intercalatingagents achieve antitumor effects solely by intercalationinto nucleic acids, unless there are reasons to believe thatprincipal nucleic acid components accessible to intercalating agents may be different among malignant and normaltissues. We have demonstrated previously that rRNAmethylases appears to be favorable targets for the actionof chemotherapeutic agents, since the characteristics ofrRNA methylases and the sensitivity of these enzymes toinhibitors were distinctly different between rapidly growing hepatoma cells and resting liver (23). Thus, this studywas conducted to investigate effects of these intercalatingagents on nucleolar synthesis and methylation of RNA for abetter understanding of the mechanisms of their antitumoreffects. It is also anticipated that, by virtue of the ability ofintercalating agents to interact specifically with doublestranded helical regions of RNA (5, 9, 19, 38) and to alterthe tertiary structure of RNA (37), intercalating agents mayprove useful in the elucidation of structural requirementsfor RNA methybation. We report here the effects of ellipticine, ethidium bromide, and hycanthone on the methylationof tRNAand rRNAwith the use of enzymesystemsfrom ratNovikoff ascites hepatoma and liver.

I Supported by Grants CA-15086 from the National Cancer Institute and

G-635 from The Robert A. Welch Foundation (both awarded to M. C. L.),and Contract N01-CM-43786 (awarded to R. B. H.) from the Divison ofCancerTreatment,NationalCancerInstitute.

2 To whom requests for reprints should be addressed , at Department of

Biochemistry, The University of Texas System Cancer Center, Texas MedicalCenter, 6723 Bertner Ave., Houston, Texas 77030.

3 Summer college student trainee.

ReceivedJanuary17, 1977;acceptedAugust10, 1977.

MATERIALSAND METHODS

[3H]-S-Ado-Met4 (7.5 Ci/mmole) and [5-3HJUTP (12.6 Ci/

4 The abbreviations used are: [3H)-S-Ado-Met, S-adenosyl-L-(methyl

3H]methionine;TKMMG,0;05 M Tris-CI,pH 7.8:0.05M KCI:1mM MgCl2:5mM H5CH,CH,OH:20% glycerol.

4202 CANCERRESEARCHVOL. 37

Inhibition of RNA Methylation by Intercalating Agents1

Ming C. Liau,2 Grace W. Lln, Carol A. Knight,3 and Robert B. Hurlbert

Department of Biochemistry, The University of Texas System Cancer Center, M. 0. Anderson Hospital and Tumor Institute, Houston, Texas 77030

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Inhibitors of RNA Methy!ation

by chromatography on a column of Sephadex G-150. Concentrated guanine-nich methylases or adenine-nich methylases (0.5 ml) were applied to a column of SephadexG-150(1.3 x 57 cm), which had been previously equilibrated withTKMMG. The same solution was used to elute the enzymeat a flow rate of 2.3 mI/fraction/iS mm. The A2@profile wasrecorded by an automatic recorder. The assay of tRNAmethylase activity and the concentration of enzyme prepanations were as described above. The specific activities ofguanine methylases and adenine methylases were increased 3.8- and 2.5-fold, respectively, over DEAE-cellulose-purified enzyme preparations.

Assayof tRNA Methylases

For the assay of pH 5-precipitabbe enzymes, the standardreaction mixture in 0.25 ml contained 0.05 M Tnis-CI, pH7.8; 0.2 M NH4CI; 0.5 mM MgCI2; 5 mM dithiothreitol; 20 pgE. co/i B tRNA; 0.27 pM [3H]-S-Ado-Met (7.5 Ci/mmole);and 46 pg enzyme protein. For the assay of DEAE-cellubosepurified enzymes, 0.2 M NH4CI was replaced by 0.1 MNH4CI, since DEAE-cellubose-punified enzymes were inhibited at NH4CI concentrations above 0.1 M, and theamount of enzyme protein was 22 pg. The incubation wasallowed to proceed at 37°for 30 mm, unless otherwisestated. The methylated tRNA was precipitated with 5%tnichboroacetic acid, collected, and washed on Milbiporefilters (0.45 pm) for the determination of radioactivity. Ablank without E. coli B tRNA was routinely assayed andsubtracted.

Preparationand Incubationof Nucleoli

The preparation and incubation of nucleoli from Novikoffascites hepatoma and liver for the determination of RNAsynthesis and methylation were as previously described(23, 24).

AnalyticalProcedures

Determinations of RNA, DNA, protein, and radioactivitywere as previously described (23, 24). Analyses of methylated tRNA bases by alkaline hydrolysis of RNA followed byelectrophoretic separation of mononucleotides were aspreviously described (22).

RESULTS

Effectof IntercalatingAgentson tRNA Methylation

E. co!i B tRNA's were used as the methyl acceptors forthe assay of tRNA methybases from nat Novikoff tumor. E.co/i B tRNA's functioned effectively as methyl acceptors,as did methyl-deficient tRNA's, except for the detection ofenzyme activities forming 7-methylguanine and 5-methyburacil (7, 14, 27). The methylation of E. co!i B tRNA by the pH5-precipitabbe enzyme preparation from rat tumor was inhibited by ethidium bromide, ellipticine, and hycanthone,as shown in Chart 1. Ethidium bromide was the mosteffective inhibitor of the 3, and hycanthone was the least

mmole) were obtained from Amersham/Searle Corp., Arbington Heights, Ill. [a-32P]-CTP was kindly provided by Dr.E. C. Moore of this department. S-Adenosyl-L-methionineand nibonucleoside tniphosphate were obtained from P-LBiochemicals, Inc. , Milwaukee, Wis. ; Escherichia co!i BtRNA was obtained from General Biochemicals, Inc. , Chagrin Falls, Ohio; actinomycin D was obtained from MerckSharpe and Dohme, West Point, Pa.; and ethidium bromidewas obtained from Sigma Chemical Co. , St. Louis, Mo.Elbipticine/NSC 71795 and hycanthone/NSC 142982 wereprovided by the Drug Research and Development Divisionof the National Cancer Institute. Novikoff ascites hepatomacells were grown in 150- to 175-g Sprague-Dawley rats. Thecells were harvested for experiments on the 5th day aftertransplantation.

Preparationof tRNA Methylases

pH 5-precipitableEnzymePreparation.The preparationof pH 5-precipitabbe enzymes from high-speed cytosol extract of Novikoff ascites hepatoma cells was as previouslydescribed (25). The pH 5 precipitate was dissolved inTKMMG to make a protein concentration of approximately5 mg/mI.

DEAE-Cebbubose-PurlfledEnzyme Preparation. For thepurpose of studying modes of inhibition of tRNA methylation by intercalating agents, it was necessary to removetRNA from the pH 5-precipitable enzyme preparation.DEAE-cellubose was washed with 2 M NaCI solution toremove UV-absorbing materials and then with water untilthe eluate was free from chloride ion. The washed DEAEcellulose was equilibrated with TKMMG. Approximately 20mg pH 5-precipitable enzymes dissolved in TKMMG werepassed through a DEAE-cellulose column (1.3 x 10 cm).The column was eluted with TKMMG until unadsorbedproteins were washed off the column. tRNA methylaseswere then eluted with a 100-mb linear gradient of KCI from0.05 to 0.5 M in 0.05 M Tris-CI, pH 7.8:1 mM MgCI2:5 mMHSCH2CH2OH:20% glycerol, collecting 3 mI/fraction/i 5mm. The A2@profile was recorded by an automatic recorder. For the determination of enzyme profile, the reaction mixture in 0.5 ml contained: 0.05 M Tnis-CI, pH 7.8; 0.1M KCI: 5 mM dithiothreitob; 25 @gE. co/i B tRNA; 0.13 pM[3H1-S-Ado-Met; and a 5O-@LIaliquot from each fraction. Theincubation and assay of labeled tRNA were as previouslydescribed (25). The active fractions were pooled and concentrated to contain approximately 1 mg protein per ml bydialysis under reduced pressure against 0.05 M Tnis-CI, pH7.8:0.05 M KCI:1, mM MgCl2:5 mM HSCH2CH@OHwith aSelectron Colbodion Bag Apparatus (Schleicher andSchuell, Inc., Keene, N. H.). The specific activity of thisenzyme preparation was increased 6.5-fold over the pH 5-precipitabbe enzyme preparation.

FurtherPurificationof GuanineMethylasesandAdenineMethylase. tRNA methylaseswere resolvedinto 2 majorfractions by chromatography on a column of DEAE-cebbulose (Chart 2). One fraction was enriched in guanine methylases (64%) eluted between 0.16 and 0.21 M KCI, whereasthe other fraction was enriched in adenine methylase (63%)eluted between 0.21 and 0.27 M KCI. Further purification ofguanine methylases and adenine methybase was achieved

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Effect of intercalating agentson the methylation ofindividualtRNAbasesby tumormethylasesThe

incubation was conducted as described in Chart 1 witha5-foldincrease of the reaction mixture. The methylated tRNAwasprecipitated

and washed with 0.4 N perchboric acid in thepresenceof0.5 mg carrier yeastRNAand wasthen hydrolyzedin 0.3 NKOHat37°for 18 hr. The methylated mononucleotides liberatedbyalkaline

hydrolysis were separated by paper electrophoresisandassayedas previously described (26). The distribution of labelinthe

methylated mononucleotides in the control wascytosine,18.5±0.7% (S.E.); adenine,27.7 ±1.0%; guanine, 52.5 ±0.8%;and

uracib, 1.3 ±0.5%, representing 42,000 cpmtotal.Total

meth- Individual basemethylationylation% (% control activity)

Molar ratio control(drug:tRNA)° activity Cytosine Adenine GuanineUracilEllipticine32

83 107 24 1041046474 133 14 8410612854 82 10 6510319243 103 5 40105Ethidiumbromide13

79 133 96 501242655 132 56 2415881

- 23 62 20 88210415 55 15 128Hycanthone69

88 166 93 6210410375 127 81 5310713861 122 71 36863456 12 7 4 10

M. C. Liau et a!.

effective. Complete inhibition of tRNA methylation couldbe obtained at a drug:protein (w/w) ratio of about 1 forethidium bromide and about 3 for hycanthone, whereasthe maximal inhibition by ellipticine was limited to 60 to70% at a drug:protein ratio above 1. As will be describedlater, the modes of inhibition by these intercalating agentsare apparently different, and methylation of some bases,particularly pyrimidine bases, is insensitive to eblipticine.The methylation of individual tRNA bases was affectedby these intercalating agents to different degrees, as shownin Table 1. The methylation of adenine was selectivelyinhibited by ellipticine at lower drug levels, and the methylation of guanine was also modestly inhibited at higher druglevels. The methylation of cytosine and uracil was notaffected by ellipticine, even when the extent of inhibitionreached 60%. In contrast, ethidium bromide and hycanthone preferentially inhibited the methylation of guanineat lower drug levels, although the methylation of adeninewas also inhibited significantly, especially at higher levelsof ethidium bromide and hycanthone. The methybationof pynimidines was stimulated by ethidium bromide andhycanthone at lower drug levels and was inhibited onlyat relatively high drug bevels. It appears that the methylationof purine bases is preferentially affected by these intercalating agents. Our previous results indicated that 1-methybadenine was the only methylated adenine detectable in thisheterologous system, whereas guanine methylation yielded4 methybated products (25). The effect of these intercalatingagents on individual guanine methylation was then studied,and the results are shown in Table 2. The formation of 1-methylguaninewas affectedby eblipticineto thegreatestextent, and N2-methybguanine, the major methylated guanine, was affected to the least extent. Although both ethidium bromide and hycanthone preferentially inhibited guanine methylation, the sensitivity of individual guanine methylation to these 2 drugs was not in the same order. Thedifferential effect of these intercalating agents on individualbase methylation suggests strongly that mechanisms of

C-)

Amounts of Drug (nmobes)Chart 1. Inhibition by intercalating agents of tRNA methylation by pH 5-

precipitabbe enzyme preparation from tumor cells. The preparation andassay of tRNA methylases were as described in ‘Materialsand Methods.―Ellipticine was dissolved in dimethyl sulfoxide and was added in a volumenot to exceed 10@ 10 @ldimethyl sulfoxide inhibited 15 ±4% (SE.) of theenzyme activity. Ethidium bromide and hycanthone were dissolved in water.The drugs in the indicated amounts were allowed to interact with tRNA inthe reactionmixture at O@for 10 mm before the addition of enzymesand[3H)-S-Ado-Met. The control activity was 69 ±6 pmobesof methyl-3H groupsincorporated into tRNA per mg protein.

inhibition or modes of interaction between tRNA and theseintercalating agents may be different. To test these possibilities, we further purified tRNA methylases, since the pH5-precipitable enzyme preparation contained substantialamounts of cellular tANA, which might interfere with thepurpose of the study.

The removal of tRNA from the pH 5 precipitate enzymepreparation was achieved by chromatography on DEAEcellulose. tRNA methybases were resolved into 2 majorfractions by a KCI gradient, as shown in Chart 2. Onefraction was ebuted at 0.16 to 0.21 M KCI, which wasenriched in guanine methybases (the distribution of methylated bases by this fraction was 2% cytosine, 33% adenine,64% guanine, and 1% uracil), and the other, eluted at 0.21to 0.27 M KCI, was enriched in adenine methylase (thedistribution of methylated bases by this fraction was 2%cytosine, 63% adenine, 34% guanine, and 1% uracil). TheUV-absorbing peak at 0.4 M KCI was lANA.

With the combined guanine- and adenine-nich methylases, the inhibition of tANA methylation by ellipticine wasdiminished by increasing amounts of tRNA substrate, butwas relatively independent of the amount of protein (asshown in Tables 3 and 4), suggesting that the mechanismof inhibition is due mainly to the blocking of the tRNAacceptor sites. On the other hand, the inhibition of tANAmethylation by ethidium bromide and hycanthone was diminished by increasing amounts of enzymes,but was relalively independent of the amount of tANA. Thus, inhibition

Table 1

a Based on

26,000.an average molecular weight of E. coli tRNA of

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Effect of intercalating agents on the methylationoftANA-guanineby tumormethylasesThe

incubation was conducted as described in Table 1.ThemethylatedtRNAwasprecipitatedand washedwith 0.4 Nperchboricacid

in the presence of 0.5 mg carrier yeast RNA and wasthenhydrolyzedin I N HCI at 100°for 1 hr. Methybatedguanineswereseparated

from other methylatedcomponents bychromatographyofthe HCIhydrolysateon a columnof Dowex50WX12as previ

ously described (25). The separation of individualmethybatedguanineswas carried out by paper chromatography aspreviouslydescribed(25).The distribution of label in the methylatedguaninesin

the control was 1-methylguanine,3.5 ±1.5%:7-methylguanine,1.9 ±0.2%: N2-dimethylguanine, 19.6 ±2.8%: and N2-methyl

guanine,75.0±4.6%,representing24,000cpmtotal.Individual

guaninemethylation(%control activity)

Total methylation of N2-Di-N2-Molar

ratio guanine 1-Methyl- 7-Methyl- methyl- Methyl(drug:tRNA) % control guanine guanine guanineguanineEblipticine32

99 69 90 1001006486 65 75 8388Ethidiumbromide13

41 24 32 52403913 3 13 2210Hycanthone41

79 100 93 638212345 64 49 40 45

S 10 15 20 25 30 35

Inhibitors of RNA Methy!ation

separated from, adenine methylase, which was found predominantly in the 1st active peak (Chart 3b). Base analysisof the methylated tANA by guanine methylase fractions, asindicated by the arrows in Chart 3a, Showed a distributionof 89, 10.5, and 0.5% in N2-methybguanine, N2-dimethylguanine, and 1-methyladenine, respectively. The comesponding analysis of adenine methylase fractions as mdicated by the arrows in Chart 3b showed a distribution of84, 12, and 4% in 1-methyladenine, N2-methybguanine, andN2-dimethybguanine, respectively. Further purification ofadenine methylase was impractical, as the enzyme preparation became very unstable. These enzyme preparationswere used for the study of inhibitory mechanisms by theseintercalating agents.

The K,@,(tANA) values are 8.4 and 1.5 pM for guaninemethylase and adenine methylase preparation , respectively.The inhibition of guanine methylases by ethidium bromideand hycanthone is noncompetitive with respect to tRNA (asshown in Chart 4), whereas the inhibition of adenine methylase by elbipticine is competitive with respect to tANA (asshown in Chart 5). Thus, different modes of drug actionare clearly indicated.

Effectsof IntercalatingAgentson rRNA Methylation

The methylation of rRNA in isolated nucleoli preparedfrom tumor and liver was also inhibited by these intencalating agents, as shown in Chart 6. The relative effectivenessof these 3 intercalating agents for inhibition of tumor rRNAmethylation was of the same order as the inhibition oftRNA methylation. There was a noticeable difference in thesensitivity to these drugs of tumor and liver nucleolam rRNAmethylation. Whereas significant inhibition of tumor rRNAmethylation could be demonstrated at a drug:DNA ratio of0.2, 5- to 10-fold such ratios were required to achieve a

: comparabledegreeof inhibitionin livernucleoli.Wehavea no satisfactory explanation for this differential effect of

intercalating agents on the tumor and liver nucleolar rRNAmethylation. We have noticed that tumor nucleoli weremuch larger than liver nucleoli, and the RNA:DNA ratio ofisolated tumor nucleoli was 2-fold greater than that ofisolated liver nucleoli (23). It is possible that the nibosomeproducing apparatus in isolated liver nucleoli is bettershielded from interaction with intercalating agents.

Table 5 illustrates that some polynucleotides interferewith the inhibitory activity of these intercalating agents onthe methylation of tumor nucleolar rANA. The inhibitoryactivity of ellipticine was significantly reduced by DNA,rRNA, polyinosinate, pobyguanybate, and polyadenylate, butwas unaffected by poly(Cl), polycytidylate, polyuridylate,and ApA. DNA and nRNA were the only polynucleotideswith even a slight effect to reverse the inhibitory activity ofethidium bromide and hycanthone. These results demonstrate that the inhibition of rRNA methylation by thesedrugs may be analogous to the inhibition of tRNA methylation. Namely, the inhibition by ellipticine is done primarilyby blocking the acceptor sites, and the binding of elbipticineto the acceptor sites can be competed with effectively bypolynucleotides, as shown in Table 5. One characteristiccommon to those polynucleotides that compete effectivelywith rRNA methyl-acceptor sites for ellipticine is the punine

Table 2

C

@ -

a

Fractions

Chart 2. Chromatography on DEAE-cellubose of the pH 5-precipitableenzyme preparation from tumor cells. Chromatography was carried out asdetailed in “Materialsand Methods.―The active fractions indicated by thearrowswerepooledandconcentrated.ThetRNAmethybatedbythispurifiedenzyme preparation showed a distribution of methylated mononucleotidesasfollows:cytosine,0.2%;adenine,30.7%;guanine,69.0%;anduracil,0.1%.

by ethidium bromide and hycanthone may be due more tointeraction with the enzymes than to interaction with tRNA.The extent of inhibition of tRNA methylation by theseintercalating agents remained approximately the samewhether [3H]-S-Ado-Met was used at subsaturation level,0.27 pM, or saturation level, 10.7 pM, indicating littleinterference with the use of [3H]-S-Ado-Met.

For the study of inhibitory mechanisms, guanine- oradenine-nich methylases obtained from DEAE-cellubosechromatography were further purified by chromatographyon a column of Sephadex G-150. Guanine methylases(Chart 3a) were eluted after, and were almost completely

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The tRNA methylases were prepared as described in Chart 2. The assay was conductedasdescribedin “Materialsand Methods.― The amount of enzyme protein was 22 @g,andtheamounts

of tRNA varied from 10 to 100 @tg.Intercalating agents were allowed to interact withtRNAinthe reaction mixture at 0°for 10 mm before the addition of enzyme preparation and [3H]-S-Ado

Met.Ellipticine

Ethidium bromide HycanthoneAmounts Control activity

of (pmoles methyltRNA:tRNAgroups incorpo- drug % inhibi- tRNA: % inhibi- tRNA: % inhibi

(/Lg) rated/mg protein) (w/w) tion drug tion drugtion10

350 ±22― 1 81 ±12 1 72 ±2 0.3 35 ±720450±14 2 66± 4 2 71±6 0.7 44±340571 ±14 4 53 ± 1 4 77 ±4 1.3 48 ±375610±55 8 42± 6 8 75±4 2.547±131

00 540 ±42 10 47 ± 4 10 73 ±3 5.0@ 54 ± 6

The preparation and assay of partially purified tumor tRNA methylases were as describedinTable3, except that the amount of tRNA was fixed at 20 j.@gand the amounts of enzymeproteinvariedfrom 11 to 88 @g.Intercalating agents were allowed to interact with the enzymes inthereaction

mixture at 0°for 10 mm before the addition of tRNA and[3H)-S-Ado-Met.Control

activity Ellipticine Ethidium bromide HycanthoneAmounts (pmobes methylof protein groups incorpo- Protein: % inhibi- Protein: % inhibi- Protein: % inhibi

(ILg) rated/mg tRNA) drug tion drug tion drugtion11

188± 23° 2.2 34±11 4.3 79±3 1.874±622424 ± 41 4.3 41 ± 6 9.1 67 ±8 3.7 68 ±344822 ± 79 9.1 44 ± 3 16.7 51 ±8 7.1 51 ±666859 ±102 12.5 47 ± 2 25.0 45 ±2 11.1 34 ±288911 ±112 25.0 49 ± 5 33.3 23 ±1a

Mean ± SE.

M. C. Liau et a!.

Table 3

Inhibition of partially purified tumor tRNA methylase activity by intercalating agents relative to theamounts of tRNA

a The data are averages of 3 determinations, expressed as mean ± SE.

Table 4inhibition of tRNA methylase activity by intercalating agents relative to the amounts of enzyme

protein

base content, and some less obvious aspects of secondarystructure are probably involved. It is possible that theellipticine-sensitive acceptor sites of rRNA have a certainstructure, which can be deduced from more systematicstructure-activity comparison.

Comparisonof Effects on Synthesisand MethylationofNucleolarRNA

We have demonstrated previously that RNA synthesisand RNA methybationin isolatednucleoliare separateprocesses (24). It is of interest to see how these intercalating agents may affect these 2 processes. As shown inChart 7, ellipticine inhibited methylation more than synthesis of RNA, and ethidium bromide inhibited RNA synthesismore than methylation (especially in the case of liver),while hycanthone affected the 2 processes to similar degrees.As expected,actinomycinD inhibitedRNA synthesismore than RNA methylation; when 90% of RNA synthesiswas inhibited , RNA methybation still took place to a substantial level, 65 and 79% of the control values for tumor andliver nucleoli, respectively. In the case of ethidium bromide,there is an obvious difference with respect to the sensitivityof tumor and liver nucleolar RNA methylation. These resultssuggest that the inhibition of rRNA synthesis and rRNAmethylation by ethidium bromide is mediated by indepen

dent mechanisms and that ethidium bromide affects preferentialby the methybation of tumor nucleolar rRNA. Previously we demonstrated that homopolymers like polyinosinate, polyguanylate, and polyadenylate and oligonucleotides inhibited exclusively the methylation of tumor nucleobar rRNA (22), and we attributed the differential sensitivityof rANA methylation between tumor and liver to differentcharacteristics of rRNA methylases (23).

The nearest neighbor frequency analysis of the ANAtranscribed in the presence of these drugs revealed thatthese drugs did not significantly change the nearest neighbor frequency, whereas actinomycin D showed a tendencyslightly to increase A + U content of the ANA (Table 6). Ifthe inhibition of RNA synthesis by these drugs is mediatedby the blocking of the DNA templates as a result of intercalation, there is probably no preference for binding to particubar base pairs.

Effects of IntercalatingAgents AdministeredIn Vivo onthe NucleolarRNAContenUandEnzymeActivities

If the mechanisms of inhibition of rANA methybation byellipticine and ethidium bromide are as proposed, theadministration of ellipticine or ethidium bromide would beexpected to produce different effects on nucleolar RNAcontent and the activities of RNA polymerase and rANA

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Inhibitors of RNA Methy!ation

ase and rANA methylase activities. These experiments weredesigned to examine the bong-term effects triggered by theinitial drug actions; therefore, the results obtained do notnecessarily reflect the initial effects of drug actions. Theseresults show that the nucleobar RNA produced in the presence of ellipticine is not degraded as readily as the nucleobarRNA produced in the presence of ethidium bromide.

DISCUSSION

The requirement of secondary and tertiary structure forthe methylation of certain tANA bases has been clearlyindicated by Shenshneva et a!. (34) in the formation of 5-methylcytosine and 1-methybadenine, which required thepresence of both parts of the enzymatically split molecule,whereas either part alone failed to function as methylacceptor. Since most methylated bases in tRNA are locatedin the same positions of different molecules (1, 6, 28, 29),the participation of secondary and tertiary structure tospecify the recognition of tANA by tANA methylases isstrongly implicated. According to Munns and Sims (28),methylation of tRNA occurred successively at differentstages of tRNA maturation, implying different structuralrequirements for individual base methybation. Methylatedadenine is located in the single-stranded TOjSCloop, whichis involved in the tertiary interaction with the D loop (31).Our data shows methylation of tANA adenine to be mostsensitive to elbipticine. Thus, the tertiary structure betweenthe TOIC loop and the D loop appears to be sensitivelyperturbed by interaction with ellipticine. The next substructune to be affected by ellipticine is the D stem, where N2-methylguanine and N2-dimethylguanine are located (16).These 2 methybated guanines are major methylated guaninederivatives detectable under present conditions, since 1-methylguanine and 7-methylguanine were formed inefticiently with E. co!i B tANA as substrate (25) and were labileand destroyed during alkaline hydrolysis (22).

The structural requirements for the methylation of rRNAare virtually unknown. Because the methyl acceptor sitesare very close to ANA polymerases engaged in the tran

S

Chart 4. Inhibition of guanine methylases by ethidium bromide and hycanthone: Lineweaver-Burk plots of the reaction rates as a function of tRNAconcentrations.The reactionmixture in 0.1 ml contained0.05N Tris-CI,pH7.8; 0.1 M NH@CI;5 mM MgCI2; 5 m@.idithiothreitol; 10.7 pM [31-I]-S-Mo-Met(1.9 Ci/mmole); 8 @Lgpurified guanine methylases; and E. coli B tRNA (MW.26,000)varyIngfrom 3.8 to 19.2 @M.Ethidiumbromideor hycanthonewasusedat 6.4or 110nmoles/tube,respectively.Theincubationwasconductedat 30°for 30 mm. @,control.

methylases. The administration of ethidium bromide willprobably produce effects similar to those of poly(l)(C) (23,26), since both compounds show a similar inhibitory mechanism against tumor rRNA methylases. Indeed, the in vivoeffects of ethidium bromide, as presented in Table 7, werevery similar to those produced by poby(l)(C); there was asubstantial loss of nucleolar ANA, and the loss of rRNAmethylase activity far exceeded the loss of ANA pobymeraseactivity. In contrast, ellipticine caused a slight increase innucleolar RNA content and similar losses of ANA polymer

Elutisu Volume (ml)

Chart 3. Further purification of guanine methylases and adenine methylassby SephadexG-150chromatography.Theguanine-richmethylasepreparation eluted at 0.16 to 0.21 PAKCI (a) or the adenine-rich methybasepreparation ebuted at 0.21 to 0.27 N KCI (b) obtained from DEAE-cellulosechromatography of the pH 5-precipitable enzyme preparation as shown inChart 2 were separately chromatographed on a column of Sephadex G-150as described in “Materialsand Methods.―Guanine methybase(a) andadenine methylase (b) fractions (arrows) were pooled and concentrated.

@dinmBrnmide

— 0.1 0.1 0.2

1/tRNA , jiM1

(lIiptici@,@―

/

—0.6 —0.4 —0.2 0.1 0.2 0.3

1/tRNA , JlM@1Chart 5. InhibItion of adenine methylase by elbipticine: Lineweaver-Burk

plots of the reaction rates as a function of tRNA concentrations. Thereaction mixture in 0.1 ml contained 0.05 PATris-CI, pH 7.8; 0.25 PANH@Cl;0.5 m@.iMgCI5; 5 mM dithiothreitob; 10.7 @.tM[3H]-S-Ado-Met (1.9 Ci/mmole);12 @gpurified adenine methylase; and E. coii B tRNA varying from 3.8 to19.2 @M.Eblipticine was used at a concentration of 41 nmobes/tube. Theincubation was conducted at 30°for 30 mm. @,control.

NOVEMBER1977 4207

.=@

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1 2 3

agentsTheassay was as described in Chart 6. The drugs were allowed to interact withpolynucleotidesin

the reaction mixture before mixing with the nucleolar pellet and[3H)-S-Ado-Met.%

control activity@ control activity@ control activity

(togetherwith@ ethidi WI (together withellipticine) bromide)hycanthone)%con

trol ac- Expec- Expec- ExAdditions tivity Found teda Found teda FoundpectedEllipticine,

35 ;L948Ethidiumbromide, 20 @g33Hycanthone,

25 @g73DNA,20 @.Lg 95 72 46 45 31 7969tRNA,30 @Lg 112 73 54 41 37 9182Polyinosinate,

25 /Lg 77 61 37 31 25 6156Polyguanylate,25 @xg 74 57 36 3024Polyadenylate,25 @.tg 66 69 32 23 22 5048Poly

(Cl), 50 @Lg 103 49 47 3534Pobycytidylate,50@ 98 38 46 3332Polyuridylate,

50 lLg 95 51 50 2631ApA,150 @g 52 23 25 18 17

M. C. Liau et a!.

...... . . Tn.@r

......‘........

aC-)

a

ill

SI

SI

45

25

a

C-)

a

aC-)

5.2 5.4 5.5 5.5 1.0[thidium Iromids/ONAEblipticius,@.5N A Iycant@sus,

I DNA

Chart 6. Inhibition of tumor and liver nucleolar rRNA methylation by intercalating agents. The reaction mixture in 0.25 ml contained 0.25 N sucrose; 0.05NTris-CI,pH7.8;0.04MNH4F;1mMMgCl2;0.2mMEDTA:0.9to 1.2 @M[‘Hj-S-Ado-Met;andanamountofnucleolicontainingapproximately50 @gDNA.Elbipticinewasdissolvedin dimethylsulfoxideandwasaddedin a volumenot to exceed10 @I;10@ dimethylsulfoxideinhibited 10 ±5%of the nucbeola.rRNA methylation. The drugs were allowed to interact with nucleobar preparation in the reaction mixture at 0°for 10 mm before the addition of [21IJ-S-AdoMet. The control activity for tumor nucleoli was 77 ±9 and for liver nucleoli it was 24 ±3 pmoles of methyl-3H groups incorporated into nucleolar RNA permg DNA.

Table5Reversalby polynucleotidesof the inhibition of tumor nucleolar RNAmethylationby intercalating

a These are the data expected if the intercalating agents and polynucleotides or ApA affect

nucleolar RNA methylation independently.

scniption of rDNA (24), DNA may play a certain robe indetermining the conformation of methyl acceptor sites,which are evidently susceptible to binding and perturbationby ellipticine.

The inhibition of RNA methylation by ethidium bromideand hycanthone via inactivation of enzymes rather thanRNA substrates is an unexpected finding. Ethidium bromidedefinitely interacts with tANA, preferentially at the D stem(33). The binding of ethidium bromide to the D stem maynot perturb the structure of tRNA sufficiently to affect itsfunction as methyl acceptor, since polyamines that sharethe same binding sites of ethidium bromide (33) promotetRNA methylation (17, 30). The enzymes specific for themethylation of guanine at the binding sites of ethidium

bromide are preferentially inactivated by ethidium bromide.Thus, interactions between methylating enzymes and ethidium bromide may also involve a high degree of specificity.

The demonstration that these intercalating agents affecttumor nucleolar ANAsynthesis and methylation more thanliver systems provides a good basis for considering themuseful in the development of cancer chemotherapeuticagents. We have elaborated previously on reasons for theuse of rRNA methylase inhibitors as selective antitumoragents, based on findings that the regulatory status ofrRNA methylases was demonstrably different between tumon and liver (22) and that many rANA methylase inhibitorsshown to be active against tumor enzymes were devoid ofeffect on normal liver enzymes (26). Ethidium bromide

CANCERRESEARCHVOL. 374208

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Nucleoli were prepared from Novikoff hepatomacells and incubated with [a-32PJ-CTPin the reaction mixture permittingRNAsynthesis.

The hydrolysisof labeled RNAby alkali and the separation of labeled mononucleotides by paper electrophoresis wereasdescribed

in Table I.Frequency

of nearestneighbor toCRNAsyn- (fraction of total CTP incorporated)

Molar ratio thesis % of(drug:DNA) control CpC ApC GpCUpCControl0

100 0.39 0.10 0.290.21±0.01―±0.01 ±0.02±0.01Actinomycin

D0.00355 0.37 0.12 0.290.230.00530 0.37 0.13 0.280.22Ellipticine0.53

75 0.38 0.11 0.290.220.8167 0.39 0.11 0.300.20Ethidiumbromide0.12

55 0.40 0.10 0.300.200.2138 0.38 0.11 0.310.21Hycanthone1.22

50 0.39 0.10 0.280.231.9836 0.40 0.12 0.270.22a

Mean ± S.E.

Effects of intercalating agents administered in vivo onthenucleolarRNAcontent and enzymeactivitiesRats

bearing Novikoff ascites hepatoma on the 4th dayweregiven1 i.p.-injected dose of eblipticine (5 mg dissolved in 1mldimethyl

sulfoxide) or ethidium bromide (5 mg dissolved in 1ml0.9%NaCI solution). Control rats received I ml 0.9% NaCIsolutionor

dimethyl sulfoxide. Ratsweresacrificed 16hr later, andnucleoliwerepreparedfrom control or drug-treatedtumor cells forthedeterminations

of nucleic acid and enzyme activities. [3HJ-CTP(0.2mM;specific activity, 0.124 Ci/mmole) or 0.7 @M[methyl-3H]-S-Ado-Met

(specific activity, 7.5 Ci/mmole) were used for theassaysofRNA polymerase or RNA methylase activities. The control activi

ties (0.9% NaCI solution) were 10.1 ±1.2 or 0.12 ±0.01 nmolesoflabeledCMP or methyl groups incorporated into RNA per mgDNAfor

RNA polymerase or RNA methylase activities,respectively.%

controlactivityRNA

po- RNA methDrugs RNA:DNA bymeraseylasesControl

1.46 ±0.05@ 100100Dimethylsulfoxide 1.48 ±0.03 73 ±12 72 ±2Elbipticine

1.55 ±0.05 57 ±9 50 ±11Ethidiumbromide 1.08 ±0.29 66 ±9 28 ±4a

Mean ± SE.

Inhibitors of RNA Methy!ation

C-)

ill

@ SI

@ SI

@ 40

@ 20

@L J@L@ : j@@ @L

DNA0.10 0.25 155 1.31 1.15 0.52 .10 1.33 0.72 1.20 2.50

Table 6Inhibition of tumor nucleolar RNAsynthesisby intercalating agent:

Effect on nearest neighbor frequency

Table 7

appears to be a selective agent to disrupt the ribosomeproducing processes of the Novikoff tumor nucleoli andmay provide a good model for further structural modification to enhance its specificity as an inhibitor of rRNAmethylases.

NOVEMBER1977 4209

Drugs: !@c.L_I .IJIiL

@j@: 5J3 5.25 151

Chart 7. Effects of intercalating agents on the synthesis and methylation of tumor and liver nucleolar RNA. The reaction mixture, in addition to thosedescribed in Chart 6, contained 0.4 m@.iATP and 0.2 m@.iGTP, CTP. and UTP. The synthesis of RNA was measured with 0.2 m@[3H)UTP (specific activity,0.2 Ci/mmole), and the methylation of RNA was measured with 1.24 @M[3H1-S-Ado-Met. The control activities for RNA synthesis were 7.11 ±1.07 and0.83 ±0.12 nmoles UMP incorporated per mg DNA for tumor and liver nucleoli, respectively, and those of RNA methybation were 132 ±14 and 35 ±4pmoles methyl groups incorporated per mg DNA for tumor and liver nucleoli, respectively.

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4210 CANCER RESEARCH VOL. 37

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1977;37:4202-4210. Cancer Res   Ming C. Liau, Grace W. Lin, Carol A. Knight, et al.   Inhibition of RNA Methylation by Intercalating Agents

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