(CANCER RESEARCH 28, 1559-1565, Auguet 19681

SUMMARY

Hydroxyurea, a compound which inhibits the growth ofrapidly proliferating tissues in animals and man, is known toinhibit DNA synthesis, and studies in crude systems have mdicated that it does so by preventing the reduction of ribonucleotides to deoxyribonucleotides.

This study demonstrates hydroxyurea-induced inhibition ofthe reduction of ribonucleotides by a highly purified enzymesystem from Escherichia coli B. The inhibition (70% at 3 x10—s M under standard incubation conditions) is dependenton the concentration of hydroxyurea and on the duration ofexposure of the enzyme to hydroxyurea. The effect of hydroxyurea is specific for protein B2 of the ribonucleoside diphosphatereductase system. The inhibition of enzymatic activity is apparently irreversible, although there is negligible irreversiblebinding of ‘4C-labeledhydroxyurea to the enzyme protein.

INTRODUCTION

Hydroxyurea has been shown to inhibit the growth of rapidlyproliferating cells in vivo (18) and in vitro (15, 25) . It wasreported in 1963 to inhibit the growth of certain transplantedtumors in mice (26) and subsequently to be active againstchronic granulocytic leukemia and other malignant neoplasmsin man (1 1, 12, 14, 27). Although it does not appear to beuniquely active against any human tumor, it is of continuinginterest because it is an active chemotherapeutic agent of a newclass.

Recent studies have established that hydroxyurea and severalrelated hydroxamates cause inhibition of the synthesis of DNAbut not that of RNA or protein in cultured mammalian cells

1 This investigation was supported in part by Grant No. 67:52

from the Swedish Cancer Society, No. 470 from the DamonRunyon Fund, and CA 07897-04 from the NIH to Peter Reichard.

2 Leave of absence from the Sloan-Kettering Institute for Can

cer Research, New York, New York during the tenure of anAlfred P. Sloan Award in Cancer Research, August 1966 to July1967.

3 Address reprint requests to Dr. Irwin H. Krakoff, Sloan

Kettering Institute, 410 East 68th Street, New York, New York10021.

4 Special Postdoctoral Fellow of the National Institute of Gen

era! Medical Science, USPHS (Grant No. 1-F3-GM-32, 309-01).Received November 16, 1967; accepted April 13, 1968.

(7, 24, 30, 31), bacteria (8, 21), and regenerating rat liver (23).In all of these biologic systems the effect of hydroxyurea israpidly reversible.

An inhibition of the formation of deoxyribonucleotides fromribonucleotides has been suggested to be responsible for the biologic effect of hydroxyurea (1, 6, 17) . In an attempt to examinemore closely the effect of hydroxyurea on ribonucleotide reduction, the influence of this drug on the highly purified ribonucleoside diphosphate reductase of E. coli was investigated.

This enzyme system catalyzes the reduction of a ribonucleoside diphosphate (e.g., CDP)5 to the corresponding deoxyribonucleotide (e.g., dCDP) at the expense of TPNH (9, 13, 16),as depicted in the following reaction sequence:

[I] TPNH + 11+ + thioredoxin-S2 > thioredoxin(Sn)2 + TPN@.

[II] thioredoxin-(SH)2 + CDP ) thioredoxin-S2 +dCDP.

In Reaction I a specific disulfide bond of a small protein(thioredoxin-S@) is reduced in the presence of TPNH and theenzyme thioredoxin reductase. The reaction product, thioredoxin-(SH)2, serves as the reductant for the actual formationof deoxyribonucleotide as depicted in Reaction II. The latterreaction has an absolute requirement for two protein fractions(proteins B! and B2) and Mg2+ and a partial requirementfor ATP, which acts as a positive allosteric effector. Recentstudies in this laboratory (4) suggest that proteins B! andB2 are subunits of a catalytically active ribonucleoside diphosphate reductase complex.

Hydroxyurea was found to inhibit the overall sequence. Thiseffect was found to result from an apparently irreversible inactivation of protein B2 of the ribonucleoside diphosphatereductase complex by hydroxyurea.

a Abbreviations used : ATP, adenosine 5'-triphosphate ; dATP,2'-deoxyadenosine 5'-triphosphate ; CDP, cytidine 5'-diphosphate;dCDP, 2'-deoxycytidine 5'-diphosphate ; CMP, cytidine 5'-monophosphate; dCMP, 2'-deoxycytidine 5'-monophosphate ; GDP,guanosine 5'-diphosphate ; dGDP, 2'-deoxyguanosine 5'-diphosphate ; dTTP, 2'-deoxythymidine 5'-triphosphate ; TPNH, tnphosphopyridine nucleotide (nicotinamide adenine dinucleotidephosphate) reduced ; DTT, dithiothreitol; Tnis, (tnis)hydroxymethylaminomethane ; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid ; EDTA, ethylenediaminetetraacetic acid.

AUGUST 1968 1559

Inhibition of Ribonucleoside Diphosphate

Reductase by Hydroxyurea1

Irwin H. Krakoff,2@ Neal C. Brown,4 and Peter ReichardDepartment of Chemi.stry II, Karoliriska Institutet, Stockholm, Sweden

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CompoundConcentrations%activity

remainingI,Hydroxyurea5

x 103a25II,N-hydroxyurethan10—2M50III,Formamidoxime3

x 10@ M3X102M6810IV,Hydroxylamine3

x 10—4M11V,N-methylhydroxylamine12 x 102M20VI,Methoxyurea101

M85VII,N-hydroxylglycineamide5 x 10—2M

(maximumsolubility)94VIII,Urea10—1M100

Irwin H. Krakoff, Neal C. Brown, and Peter Rei chard

80

@60

C

°.40C-)

Hydroxyurea(Mx 10@)

Chart 1. Inhibition of CDP reduction by hydroxyurea under“standardassay conditions― (see text). Although the substrateis CDP-@H and the reduction is to dCDP-@H, the analytic procedure requires hydrolysis to dCMP. The reduction, therefore, isexpressed as m@moles dCMP synthesized/reaction vessel in thisand the other charts.

ALso shown in this chart is the effect of hydroxyurea on CDPreduction in the absence of ATP, a positive allosteric effector.Enzyme activity was measured at the designated concentrationsof ATP with 30 @gof protein Bi and 16 @gprotein B2. Activity inthe absence of hydroxyurea was assigned a value of 100%. In thepresence and absence of ATP this control value was respectively,13.4 and 6.5 m@moles of dCMP formed. CDP, cytidine 5'-diphosphate ; dCDP, 2'-deoxycytidine 5'-diphosphate ; dCMP, 2'-deoxycytidine 5'-monophosphate.

Inhibition of Ribonucleotide Reduction by Hydroxyurea.Under the conditions described above; the addition of HU tothe reaction mixture inhibited CDP reduction (Chart 1). Thedegree of inhibition was related to the concentration of bydroxyurea: 40% at 1.5 X 10@ M, 70% at 3 x 10@ M, and90% at 102 M. The reduction of GDP was found to be inhibited similarly by hydroxyurea.

Inhibition of Ribonucleotide Reduction by Other Hydroxamates. Ribonucleotide reduction was studied in the presenceof hydroxamates related to hydroxyurea, and the inhibitoryactivities of these compounds are compared with that of hydroxyunea in Table 1. Of the compounds tested, hydroxylaminecaused the most pronounced inhibition of the enzyme system.N-hydroxyurethan, formamidoxime, and N-methyl hydroxylamine were also found to be significantly inhibitory at concentrations •similar to those at which hydroxyurea was active.Methoxyurea, N-hydroxyglycine amide, and urea did not causesignificant inhibition even at higher concentrations. The possible significance of these observations will be discussed later.

Influence on Hydroxyurea Inhibition of Certain FactorsWhich Affect Ribonucleotide Reduction. As outlined in the

Table 1

Effect of some hydroxamates on nibonucleotide reduction. Eachof these compounds was evaluated for inhibitory activity under“standard assay conditions― (see text) at the concentrations listedabove.

introduction, the nibonucleotide reduction system is quite complex, and the following aspects of the system must be considered in connection with the inhibition by hydroxyurea : (a)

1560 CANCER RESEARCH VOL. 28

MATERIALS AND METHODS

Hydroxyurea was obtained from Miss Barbara Stearns,Squibb Institute for Medical Research, New Brunswick, NewJersey.

Hydroxyurea-14C, 327 microcuries/micromole, was obtainedfrom Dr. Jack Davidson, Department of Nuclear Medicine,National Institutes of Health, Bethesda, Maryland.

Proteins Bi and B2 (9, 20), thioredoxin (13), and thioredoxin reductase (16) were prepared in this laboratory bymethods described previously.

In most experiments, the activity of the ribonucleoside diphosphate reductase was assayed by measuring the formationof radioactive dCDP from 3H-labeled CDP (Schwarz Bioresearch) under standard conditions as described elsewhere (9,20) . Unless stated otherwise, the reaction was started by theaddition of protein B2 to the other components of the incubation mixture. Incubation was carried out at 23°C for 10 mmutes in a final volume of 0.130 ml and terminated by the addition of 1.0 ml of 1 @tHC1O4.

In experiments involving the kinetics of hydroxyurea inhibition of CDP and GDP reduction, the reaction was assayed bycoupling the formation of deoxyribonucleotides to the disappearance of TPNH (9).

With respect to proteins Bi and B2, one enzyme unit isdesignated as that amount of protein that catalyses the reduction of 1 millimicromole of ribonucleoside diphosphate per 10mm per 0.130 ml of standard incubation volume at 23°C. Thespecific activities of the proteins used in this study were asfollows: protein Bi, 600—1000units per mg, and protein B2,1000—5000units per mg. Unless stated otherwise, the enzymeactivity of B2 was always measured with an excess of proteinBi activity. Similarly, when a critical study of Bi activity wasnecessary, it was measured in the presence of an excess of B2activity.

RESULTS

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Ribonucleoside Reductase Inhibition

the hydrogen donor system, (b) substrate concentration, (c)allosteric effects, (d) protein Bi or B2.

Experiments were done to evaluate the effect of hydroxyureaon these aspects of the ribonucleotide reductase system:

When Reaction I (see Introduction) was studied separatelyin the absence of the complete CDP reductase system, addition of hydroxyurea at 102 M did not inhibit the thioredoxindependent oxidation of TPNH. In a second experiment, excessdithiol in the form of DTT was added to the complete CDPreductase system (Reactions I and II) and had no influenceon the inhibitory effect of hydroxyurea. Both of the aboveexperiments indicated that the action of hydroxyurea was dueneither to inactivation of dithiol nor to an effect on thioredoxinreductase.

The addition of a large excess of substrate (CDP) did notinfluence the inhibitory effect of hydroxyurea (Chart 2). Thisexperiment excludes the unlikely possibility that hydroxyureaexerts its effect by competition with the substrate.

The inhibition by hydroxyurea did not appear to depend onthe nature or the amount of allosteric effector present in thesystem. Chart 3 demonstrates that hydroxyurea inhibits CDPreduction both in the presence and in the absence of the positive allosteric effector, ATP. In addition, another positiveallosteric effector, deoxythymidine 5'-triphosphate, producedlittle or no influence on the inhibitory effect of hydroxyurea(Chart 3) . Although not shown in graphic form, addition ofthe negative allosteric effector dATP at a concentration of 1.5x 10@Malsohadnoeffectontheinhibitionbyhydroxyurea.

The inhibition of ribonucleotide reduction by hydroxyureawas found to be time dependent. A mixture of proteins Biand B2 was incubated with hydroxyurea at a concentrationof 102 M for varying periods of time in the absence of substrates. The reaction was started by addition of substrate andthe thioredoxin system. This addition diluted the hydroxyureato a noninhibitory concentration of less than 10@ M and al

lowed an assay of remaining activity. Under these conditions,the amount of inhibition of ribonucleotide reduction by hydroxyurea clearly was related to the duration of preincubation(Table 2).

Specificity of Hydroxyurea for Enzyme Protein B2. Twotypes of experiments were done to determine the specificityof hydroxyurea for protein Bi or B2. In the first (Chart 4),hydroxyurea was incubated with varying ratios of enzyme protein Bi and B2, i.e., a constant amount of protein Bi withvarying amounts of protein B2. Although hydroxyurea at 102M was markedly inhibitory at all ratios, a 5-fold lower concen

tration (2 x 10—sM) produced less inhibition in the presence

4

I@ I I

1 10 20 50 100

CDP- 3H(Mx 10@)

Chart 2. Effect of hydroxyurea on CDP reduction in the presence of varying concentrations of substrate CDP-@H. With theexception of the variations in substrate concentration, the assaywas performed under standard conditions with 17 @gof proteinBi and 17 i@gof protein B2. CDP, cytidine 5'-diphosphate ; HU,hydroxyurea.

HU,5xlO3M •

.@

.CC

I

3

2

1

0

.@

INo

1000.1 1.0

dTTP (Mx104)

Chart 3. Inhibition of CDP reduction by hydroxyurea in the presence of varying concentrations of a positive allosteric effector, dTTP.Except for the replacement of ATP with dTTP as the allosteric effector, the assay was performed under standard conditions with 19 @gof protein Bi and 9.0 j@gof protein B2. CDP, cytidine 5'-diphosphate ; dTTP, 2-deoxythymidine 5'-triphosphate; HU, hydroxyurea.

AUGUST 1968 1561

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Preincubation time,hydroxyurea,10-2 ii (mm)Enzyme

proteinin preincubation

mixture @Enzyme

activityremaüung

(mjimoleedCMPformed per. aaxnple)%inhibition0B1

andB22.701B1andB22.7202B1andB2223185B1andB21.394910B1andB20.438410B13.25010B2<0.01100

irwin H. Krakoff, Neal C. Brown, and Peter Reichard

Table 2

Effect of preincubation of hydroxyurea with the reaction mixture. 200 @gof protein B1 and80 @gof protein B2, either separately or in combination, were preincubated at 22°Cwith 102M hydroxyurea in 0.05 Tris-Cl (pH 7.0) in a volume of 0.090 ml. At various times after the

start of the preincubation, 0.010-mi fractions were removed from the mixture and rapidlydiluted to a final volume of 0.130 ml with substrate (CDP) and the other components of astandard assay mix to allow the measurement of enzyme activity. When Bi or B2 were preincubated separately with hydroxyurea, an excess of the complementary protein necessary forassay of enzyme activity was included in the standard assay mixture. dCMP, 2'-deoxycytidine5'-monophosphate ; CDP, cytidine 5'-diphosphate.

of increasing amounts of protein B2. This finding suggestedthat the relation of hydroxyurea to protein B2 was a determinant factor. Therefore, an experiment was done (Table 2)in which hydroxyurea (10—2 M) was incubated with proteinB1 and B2 separately before assay. In this study samples ofeither Bi or B2 were incubated with hydroxyurea and thenassayed after a 13-fold dilution with the other componentsof the standard incubation mix, including the complementaryenzyme. As shown in Table 2, preincubation of hydroxyureawith protein Bi did not impair its subsequent activity. However, preincubation of hydroxyurea with protein B2 produced

.@

F.,,

as.@

C>.,vs

I

@}ratio31i@2} units @/2.1 @I4.2 7/7 3.5/35

Chart 4. Effect of increasing concentrations of protein B2 on theinhibitory effect of hydroxyurea. The enzyme assays were carriedout under standard assay conditions at the two designated concentrations of hydroxyurea. The definition of an enzyme unit isgiven in the methods section. Protein Bi and protein B2 usedhad respective specific activities of 500 and 2000 units per mg.RU, hydroxyurea; dCMP, 2'-deoxycytidine 5'-monophosphate.

.@

N“Ia,

C

In

a,0E

E

4

3

2

1

HU,

lOmIn oomin180mln24@Incubation at 22°C@/2 3/3 1/is

Chart 5. Effect of prolonged incubation of hydroxyurea andprotein B2 on subsequent enzyme activity. Protein B2 at a concentration of 0.7 mg per ml was incubated at 22°C in 0.5 MHEPES-Na (pH 7.6) containing hydroxyurea at concentrationsof 10—2,10—s,and 1O—@M. At various times after the start of incubation, samples containing 1.4 jzg of protein B2 (0.002 ml) wereremoved for assay under standard conditions in the presence of15 j@gof protein B! and CDP-@H as substrate. HU, hydroxyurea;dCMP, 2'-deoxycytidine 5'-monophosphate ; HEPES-Na, N'-2 bydroxyethylpiperazine-N-2-ethanesulfonic acid sodium salt.

1562 CANCER RESEARCH VOL.28

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Table3Elapsed

timefollowing gelfiltration (hr)dCMP

synthesized(mj@mo1es/@gprotein)Controlhydroxyurea

treated22.71081.970241200

a nearly completely inactive enzyme. This finding clearly mdicated that the inhibitory effect of hydroxyurea was entirelyon protein B2 of the ribonucleoside diphosphate reductasecomplex.

The time-dependency of the effect of hydroxyurea on protein B2 was evaluated in an experiment shown in Chart 5.

When hydroxyurea was preincubated with protein B2, it wasobserved that with sufficient preincubation, i.e., up to 24 hours,100% inhibition could be obtained, even at a concentration ofhydroxyurea of 10@ M, which was not inhibitory under standard short-time incubation conditions.

Irreversibility of Inhibition. Since the biologic effects of hydroxyurea are rapidly reversed upon its removal from numerous biologic systems (7, 18, 24, 25, 30, 31), a series of studieswas done to evaluate the reversibility of hydroxyurea inhibition of ribonucleotide reduction. In the first experiment of thisseries, a complete reaction mixture was exposed to hydroxyureaat 5 X 10—sM, and ribonucleotide reduction was measured bythe TPNH assay. After inhibition was found to be established,the entire reaction mixture, containing proteins Bi, B2, andhydroxyurea, was passed through a column of Sephadex G-25to remove small molecules. The protein mixture was recovered,and upon assay no reductase activity was apparent. Controlsamples of the enzymes treated identically, except for exposure to hydroxyurea, exhibited satisfactory activity after recovery from the Sephadex column. These experiments demonstrated that the inhibition of B2 activity produced by hydroxyurea was not easily reversed even in the presence of thecomplementary enzyme protein.

The results of a second and more vigorous evaluation of theirreversibility of the effects of hydroxyurea are shown in Table3. In this experiment, protein B2 alone was exposed to by

droxyurea until loss of its activity was complete. The proteinwas then desalted by ifitration through Sephadex G-25 as described above and allowed to stand at room temperature.Aliquots were removed periodically for assay of ribonucleotidereductase activity. It was clear that hydroxyurea-inhibitedprotein B2 did not recover its activity even upon standing atroom temperature for prolonged periods (i.ip to 24 hours) aftergel filtration. Control samples, treated identically with theexception of exposure to hydroxyurea, deteriorated somewhatbut still retained more than 50% of their initial activity.

Lack of Binding of Hydroxyurea to Enzyme. To study anypossible binding of hydroxyurea to protein B2, the previousexperiment (Table 3) employing Sephadex was performed with14C-labeled hydroxyurea. The results illustrated in Chart 6show clear separation of radioactivity from the protein peakafter gel filtration of a mixture of hydroxyurea and protein B2.On the basis of the small amount of radioactivity found in association with the protein peak, the maximum binding whichcould have occurred was estimated to be less than 0.015m@moles of hydroxyurea per m@mole of enzyme protein.6

6 For this estimation we assume a molecular weight of 100,000

for protein B2 [S20@= 5.5 (3)]. The enzyme fraction used in thisstudy is believed to be 30% pure.

1,@ -

E

0.C-)

SEPHADEXCHROMATOGRAMENZYME PROTEIN82

HU- 14C

II I.@ , I . . .1

1.0@ 1.4@ 1@8 2.2@ 2@6 3.0 3.4 3.8 4.2

milliliters (0.lml fractions)

Chart 6. Gel filtration of a mixture of hydroxyurea-14C andprotein B2. Protein B2 (120 @g)was incubated for 3 hours at22°Cin 0.06 ml of 0.05 M HEPES-Na (pH 7.6) in the presenceof 10—3M hydroxyurea-14C (327 @curiesper @&mole). At this time,0.05 ml of the enzymatically inactive mixture was passed througha column (0.5 x 18 cm) of Sephadex G-25 previously equilibratedwith 0.05 M HEPES-Na (pH 7.6) and 0.001 M EDTA. Theeffluent was collected in fractions of 0.10 ml and examined spectrophotometrically at a wavelength of 280 m@ to localize protein.Subsequently 0.05 ml of each fraction was assayed for radioactivityby liquid scintillation counting. As a control an identical experiment was performed with a sample of B2 preincubated in theabsence of hydroxyurea. Only the chromatogram of the mixtureof hydroxyurea-14C and protein B2 is shown. Gel filtration andother procedures following the initial preincubation were performed at 4°C. HEPES-Na N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid sodium salt. EDTA, ethylenediaminetetraacetic acid.

100,000

10,000

i,cxm

100

n

A.

.-@ -@ - -

0.4

0.3

0.2.@E

0.1@0.

0

Failure of reactivation of enzyme B2 upon prolonged standingafter removal of hydroxyurea by gel filtration. The initial part ofthis experiment, involving incubation and gel filtration, is described in the legend of Chart 6. After gel filtration of the preincubation mixture and removal of samples for counting, the remainder of fractions containing the bulk of protein B2 waspooled, adjusted to a concentration of 0.35 mg/ml with 0.05 MHEPES-Na (pH 7.6) and allowed to stand at 22°C.At variousperiods 0.004 ml-samples (12 @gof B2) were removed for assayunder standard conditions with 30 @gof protein Bi. The activityof protein B2 before gel filtration was determined under standardconditions with 0.002 ml-fractions of the preincubation mix and30 @gof protein Bi. As mentioned in Chart 6, an identical experiment was performed simultaneously on a sample of protein B2preincubated in the absence of hydroxyurea-'4C. Before gel filtration, protein B2 in the control preincubation mixture had a specific activity of 2.0 m@imoles of dCMP formed per @gof protein.B2 in the hydroxyurea-treated mixture was completely inactive.dCMP, 2'-deoxycytidine 5'-diphosphate ; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfothc acid sodium salt.

AUGUST 1968 1563

Ribonucleoside Reductase Inhibition

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Irwin H. Krakoff, Neal C. Brown, and Peter Reichard

Nevertheless, assay of the protein peak (results in Table 3)showed complete inhibition of enzymatic activity. Controlsamples treated identically except for incubation with hydroxyurea showed satisfactory enzymatic activity. In a similar experiment in which hydroxyurea-14C was incubated with bothproteins Bi and B2 plus the other components of the reaction,the results were identical, i.e., there was clear separation of theradioactivity from the hydroxyurea-inhibited enzyme protein.These results clearly demonstrate that intact hydroxyurea wasnot bound irreversibly to protein B2. The possibility that hydroxyurea is transformed into a noncarbon-containing compound which remains bound to the protein is, of course, notexcluded by these experiments.

DISCUSSION

A number of reports have suggested that the effects of bydroxyurea on DNA synthesis are related to a dnIg-inducedinhibition of the cellular formation of deoxyribonucleotides.Recent work with E. coli ( 17) has correlated inhibition ofDNA synthesis with an hydroxyurea-dependent depletion ofcellular pools of deoxyribonucleoside triphosphates.

Hydroxyurea is also known to block the formation of deoxycytidylic acid in rabbit kidney cells ( 1) . Furthermore, solubleextracts of bone marrow obtained from humans or rats treatedwith hydroxyurea have a decreased ability to convert cytidylicto deoxycytidylic acid (6) . Hydroxyurea also inhibits the conversion of ribonucleotides to the corresponding deoxyribonucleotides in crude extracts of Ehrlich ascites and L cells (28).

Despite the increasing amount of evidence correlating thebiologic action of hydroxyurea with a specific effect on the formation of deoxyribonucleotides, alternative explanations existwhich presume different mechanisms of drug action.

The present study demonstrates clearly that hydroxyureainhibits the activity of ribonucleoside diphosphate reductase,a highly purified enzyme system catalyzing the reduction ofribonucleotides to deoxyribonucleotides in E. coli ; this inhibition occurs as a result of a specific interaction between bydroxyurea and a discrete subunit of the active enzyme cornplex. The specificity of this effect adds considerable evidencein support of the hypothesis that ribonucleotide reduction isa primary target for the biologic action of hydroxyurea andmakes certain alternative mechanisms less probable.

In general, support for alternative mechanisms has arisenfrom the observation that hydroxyurea is degraded in variousbiologic systems to hydroxylamine (2) . In the present studyit was found that the effect of hydroxylamine on protein B2appears to be identical to that of hydroxyurea. It was recentlydemonstrated that protein B2 is a nonheme iron protein witha characteristic spectrum at 350—420m@ (3) . In unpublishedexperiments (N. C. Brown and P. Reichard) it was found thaton treatment of protein B2 with either hydroxyurea or bydroxylamine, the loss of enzyme activity was paralleled byprofound spectral changes. From these results it appears possible that the inhibition of ribonucleotide reductase by bydroxyurea may be connected with a breakdown of hydroxyureato hydroxylamine, conceivably occurring at the enzyme surface.This possibility would, of course, not invalidate the conclusion

that ribonucleotide reductase is a main target for the biologicaction of hydroxyurea. Support for this concept comes fromthe study of several other hydroxamates illustrated in Table1, which shows a striking parallelism between the effects ofthese compounds on ribonucleotide reductase and specificeffects on DNA synthesis reported earlier by Young et at. (32).

Studies of the influence of exogenous deoxyribonucleosideson the inhibitory activity of hydroxyurea on DNA synthesishave shown varying results. In some in vitro systems, additionof deoxyribonucleosides resulted in protection from inhibitionby hydroxyurea (32, 33), while in other systems such an effectwas not found ( 10, 19, 29) . It should be noted, however, thatthe reversal of inhibition would require the added deoxyribonucleosides to be phosphorylated inside the cell. Many cellscontain catabolic enzymes which degrade deoxyribonucleosides.This may explain the varying results in different cell systems.

Several investigators have noted that the effects of hydroxyurea in bacteria (8, 21) , isolated mammalian cells (7, 15, 24,30) , and intact animals (1 1, 12, 18, 27) are rapidly reversible.This need not be inconsistent with the demonstration here ofthe apparent irreversibility of the inhibitory effect of hydroxyurea on the purified protein B2 of the ribonucleoside reductase.It might instead indicate that the proper conditions for reactivation of the enzyme in vitro have not yet been found orthat living cells have a marked and rapid capacity to replenishthe enzyme.

REFERENCES

1. Adams, R. L. P., Abrams, R., and Lieberman, I. Deoxycytidylate Synthesis and Entry into the Period of DeoxyribonucleicAcid Replication in Rabbit Kidney Cells. J. Biol. Chem., @41:903-905, 1966.

2. Borenfreund, E., Krim, M., and Bendich, A. ChromosomalAberrations Induced by Hyponitrite and HydroxylamineDerivatives. J. Natl. Cancer Inst., 3@: 667—669,1964.

3. Brown, N. C., Eliasson, R., Reichard, P., and Thelander, L.

Non-heme Iron as a Cofactor in Ribonucleotide Reductase

from E. coil. Biochem. Biophys. Res. Commun., 30: 522—527,1968.

4. Brown, N. C., Larsson, A., and Reichard, P. On the SubunitStructure of Ribonucleoside Diphosphate Reductase. J. Biol.

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Effect of Hydroxyurea on Pseudomonas aeruginosa. CancerRes., @4:1012—1019,1964.

9. Holmgren, A., Reichard, P., and Thelander, L. EnzymaticSynthesis of Deoxyribonucleotides VIII. The Effects of ATP(adenosine triphosphate) and dATP in the CDP ReductaseSystem from E. coli. Proc. Natl. Acad. Sci. U. S., 54: 830-836,1965.

1564 CANCER RESEARCH VOL. 28

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Ribonucleoside Reductase Inhibition

10. Kihlman, B. A., Erik@on, T., and Odmark, G. Effects ofHydroxyurea on Chromosomes, Cell Division and NucleicAcid Synthesis in Vicia Faba. Hereditas, 55: 386-397, 1966.

11. Krakoff, I. H., Murphy, M. L., and Savel, H. PreliminaryTrials of Hydroxyurea in Neoplastic Diseases in Man. Proc.Am. Assoc. Cancer Res., 4: 35, 1963.

12. Krakoff, I. H., Savel, H., and Murphy, M. L. Phase II Studiesof Hydroxyurea (NSC-32065) in Adults: Clinical Evaluation.Cancer Chemotherapy Rept., 40: 53—55,1964.

13. Laurent, T. C., Moore, E. C., and Reicbard, P. EnzymaticSynthesis of Deoxyribonucleotides IV. Isolation and Characterization of Thioredoxin, the Hydrogen Donor from Escherichiacoti B. J. Biol. Chem., @9:3436—3444,1964.

14. Lerner, H. J., and Beckloff, G. L. Hydroxyurea AdministeredIntermittently. J. Am. Med. Assoc., 19@: 1168—1170,1965.

15. Mohler, W. C. Cytotoxicity of Hydroxyurea (NSC-32065)Reversible by Pyrimidine Deoxyribosides in a MammalianCell Line Grown In Vitro. Cancer Chemotherapy Rept., $4:1-6,1964.

16. Moore, E. C., Reichard, P. and Thelander, L. Enzymatic Synthesis of Deoxyribonucleotides. V. Purification and Propertiesof Thioredoxin Reductase from Escherichia coli B. J. Biol.Chem., p39: 3445-3452, 1964.

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AUGUST 1968 1565

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1968;28:1559-1565. Cancer Res   Irwin H. Krakoff, Neal C. Brown and Peter Reichard  HydroxyureaInhibition of Ribonucleoside Diphosphate Reductase by

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