The earlier observations of the inhibition in vitro by6-thio IMP of the conversion of inosinate to adenylate andto guanylate in Streptococcus faecalis and pigeon liver, nespectively (24), have subsequently been confirmed bystudies of other bacterial species (11, 12) and certain mammalian systems (2, 23). Inasmuch as such conversions(Chart 5) constitute an integral part of purine metabolismin tissues in general, these results suggest that the selective

susceptibility of tumors such as Sarcoma 180 (5-180) tothe action of 6-MP could be partly attributed to qualita

1 This investigation was supported in part by funds from theNational Cancer Institute, National Institutes of Health, USPHS(CA-03190-08 and 5-K3-CA-16,673-02). A preliminary reporthas been presented (20—22).

2 Abbreviations used are: IMP, inosine monophosphate; AMP,

adenosine monophosphate; XMP, xanthosine monophosphate;6-MP, 6-mercaptopurine; ATP, adenosine triphosphate; 6-thioIMP, 6-thioinosinate; GTP, guanosine triphosphate; NAD,nicotinamide adenine dinucleotide; DPN-diphosphopyridinenucleotide ; SAICAR, succinoaminoimidazolecarboxamide ribonucleotide; CMC-carboxymethyl cellulose; Tris, tris(hydroxymethyl) aminomethane; TCA, trichioroacetic acid.

Received for publication September 10, 1964; revised December23, 1964.

tive and/or quantitative differences in these nucleotideconversions in tumor tissues compared to host tissues. Totest the tenability of such a proposal, more detailed studiesof the enzymatic reactions involved in purine ribonucleotide interconversions have been undertaken with S-180 andhost liver as the experimental system. .

Studies with bacterial (10) and ascites tumor cell (5)systems indicate that some purine and pyrimidine analogsmight owe their inhibitory effects to their ability to function as nepressors of enzyme synthesis. This possibility

has also been investigated by assaying for the enzymes ofnucleotide interconversion (Chart 5) in tumors and liversof animals which have been treated with 6-MP.

The results of these studies indicate that differences inenzymatic capacity between 5-180 and host liver do existand suggest that these differences may be of sufficientmagnitude to play an important part in the anti-tumorspecificity of 6-MP in 5-180.

MATERIALS AND METHODS

IMP-8-'4C was obtained from Schwanz BioResearch,Inc.; ATP (disodium salt), IMP (sodium salt), GTP (tn

544

The Mechanism of Action of 6@Mercaptopurine1

II. Basis for Specificity

JOSEPHINE SEE SALSER AND M. EARL BALlS

(Division of Nucleoprotein Chemistry, Sloan-Kettering Institute for Cancer Research, Sloan-Kettering Division of CornellUniversity Medical College, New York, New York)

SUMMARY

The conversion of IMPS to AMP and XMP by tumors and liver was examined aspart of the study of the basis for the anti-tumor specificity of 6-MP. Liven preparations convert IMP to AMP more efficiently than do those from tumors. In incubations with tumor preparations, but not with liver, an accumulation of succinoadenylate was observed. A 6-MP resistant 5-180 was examined and found to resembleliven in its capacity to form adenylate. The succinoadenylate formed was less thanhalf that found with sensitive 5-180. These findings, which indicate that a lack ofadenylosuccinase may be a major cause of deficiency in adenine synthesis, were confirmed by direct assays for this activity. In line with the observations that the sameenzyme catalyzes the cleavage of both succinoadenylate and succinoaminoimidazolecarboxamide nibonucleotide (SAICAR) in other species, the ability of 5-180 to cleavethese 2 substrates, compared to liver, was reduced to the same extent. Similar studiesof the conversion of IMP to XMP indicate that the tumor also had less capacity tosynthesize XMP than did the host liver.

Tissues from mice which had received prior treatment with 6-MP were somewhatless active in these conversions than those from controls. The reduction in the capacity to synthesize AMP appears to be greaten in both tissues. The relative effectappears to be greater in the tumors than in the livers.

These data suggest that the basis for the tissue specificity of 6-MP may be attnibutable in part to reduced enzymatic capacity of certain tissues to synthesize AMPand XMP from IMP. Since this capacity seems to be less in tumors, inhibition by6-thio IMP of these metabolic steps might be more critical in tumors.

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SALSER AND BALlS—ACtiOn of 6-Mercaptopurine. II 545

sodium salt), NAD ($-DPN), L-glutamme, fructose-i ,6-diphosphate (sodium salt), a-ketoglutarate, reduced glutathione (crystalline), and pynidoxyl phosphate wereobtained from Sigma Chemical Company.

Xanthine oxidase was purchased from Worthington Biochemical Corporation. Yeast adenylosuccinase was prepared from Fleischmann's active dry yeast (StandardBrands, Inc.) according to the method of Carter and Cohen(6).

SAICAR was prepared from a Biogen culture of Escherichia coti strain B973accordingto the method of Cots andGollub (9). Succinoadenylate was prepared by themethod of Carter and Cohen (6).

HA/ICR Swiss mice (approximately 20 gm each) with a3- or 7-day-old s.c. implant of 5-180 were kindly suppliedby Dr. H. Schwartz and Dr. C. Reilly of this institute.Mice with 11-day-old s.c. implants of a resistant variantof 5-180 were supplied by Dr. C. Clarke. In all instances,tumors with perceptible necrosis were discarded. C57black mice (approximately 20 gm each) with 9- and 18-day-old s.c. implants of Adenocarcinoma E 0771 weresupplied by Dr. K. Suguira of this institute.

Mice with 3-day-old implants of 5-180 were given i.p.injections of 6-MP (50 mg/kg body weight) for 4 successivedays. Control animals were given CMC-saline injections.The animals were sacrificed on the 7th day and the tumorand liver removed.

The preparation and use of acetone powders of the 2tissues have been discussed elsewhere (23).

PREPARATION OF CELL-FREE EXTRACTS

A. Extracts converting inosinate to adenylate.—Acetonepowder of frozen tissue was homogenized in a medium containing 0.16 M KC1 and 0.05 i@iTnis HC1 buffer, pH 7.4(17) (4 gm wet weight/ml) in a Virtis “45―homogenizerfor 3 min. To partially fractionate the active system(s)involved in this conversion, aliquots of the homogenatewere centrifuged at 15,000 X g for 30 min, and at 34,800 Xg for 60 min. All the operations were carried out at 4°C.

B. Extract,@ of adenylosuccina.se assay.—Fresh or frozentissue was homogenized in a Virtis “45―homogenizer for 2min in 0.025 Mphosphate buffer, pH 7.2 (4 gui wet weight!10 ml). The preparation was cleared of cellular debris bycentrifuging at 10,000 X g for 30 mm. All preparationswere carried out at 4°C.

C. Extractsconvertinginosinateto guanylate.—Acetonepowder or frozen tissue was homogenized in a Virtis “45―homogenizer with 0.05 M phosphate buffer, pH 7.4 (2 guiwet weight/b ml). Dialyzed 20,000 X g supematantswere prepared according to the method used by Lagerkvistfor pigeon liver extracts (14).

ASSAY M@iriioDs

A. Conversion of inosinate to adenylate.—Unless otherwise designated, each 10 ml of incubation mixture contained the extract from 2 gui wet weight of the tissue (orthe equivalent amount of acetone powder@); 2.5 mmolesATP; 2.5 mmoles L-aspartate; 5 mmoles fructose-i ,6-di

S We are indebted to Dr. S. Gots of the University of Pennsyl

vania for the stock culture of B. cell B97 and to Dr. L. Kaplanof this institute for the Biogen run.

4 The yield of acetone powder from liver was about 25% of the

wet weight while that from S-iSO was 12-15%.

phosphate; 0.5 mmole MgCl2; 5 mmoles sodium phosphatebuffer, pH 7.4; 2.5 mmoles a-ketoglutarate; 1 mmoleNAD ; and “x―@smolesIMP-8-'@C (specific activity, 6.31 X10@cpm/pmole). In studies comparing the ener@r sourcein this conversion, 2.5 mmoles of GTP were used in placeof ATP, NAD, fructose-i ,6-diphosphate, and a-ketoglutarate. The tissue extracts were added to the substratesolution after a 30-min equilibration period, and the incubations were carried out either at 37.5°Cfor 2 hr (unlessotherwise specified) or at 20°Cfor 4 hr in a Dubnoff metabolic shaker. The lower temperature was chosen for someof the experiments since the incorporation studies ofNewton and Perry (17) have shown that succinoadenylateaccumulated in skeletal muscle preparations incubated atthis temperature.

At the end of the incubation, the reaction was stoppedby immersion of the vessels in an acetone Dry Ice bath.Sufficient cold 60 % TCA was added to the reaction mixtureto give a final TCA concentration of 7 %. The precipitated protein was removed by centrifugation in the coldand resuspended and washed twice with cold 5 % TCA.The combined supernatant solutions were extracted 10times with ether to remove the TCA. The clear solutionwas either lyophiized to dryness or neutralized and storedat —20°C.

The dry samples were taken up in freshly prepared 1 NHC1 and hydrolyzed for 24 hr at 100°C. The free purineswere isolated by chromatography on Dowex-50 (hydrogenform, AGW, X8, 200-400 mesh) columns with HC1 (1).

The components of neutralized and deproteinized mixtunes, on the other hand, were separated on Dowex-1(chloride form, X10, 200-400 mesh) columns by a modification of the method of Deutsch and Nilsson (7). This consisted of stepwise elution with (a) 0.003 N HO!, (b) 0.005 NHO!, (c) 0.01 N HC1-0.02 M NaCl, (d) 0.01 N HC1-0.05 MNaC1, (e) 0.01 N HC1-0.2 M NaC1, (J) 0.01 N HC1, and (g)1.0 N HC1. The identities of the various fractions werechecked spectrally and by paper chromatography. Theidentity of succinoadenylate was further confirmed by incubating it with yeast adenylosuccinase (6).

The radioactivities of the various compounds were determined on infinitely thin films on aluminum planchets ina Geiger-Muller internal flow counter.

B. Adenylosuccinase activity.—The cleavage of succinoadenylate was followed spectrally, i.e., the decrease inOD@ (6), and by paper chromatography. Each mixturecontaining 449 @imolesof substrate in 0.4 ml phosphatebuffer (pH 7.2) and 2.0 ml tissue extract was incubated at37.5°C. The reaction was stopped by heating for 3 minat 100°C,cooling rapidly, and centrifuging. For spectraldeterminations, the precipitate was resuspended andwashed with buffer. The combined supennatant solutionswene made up to a final volume of 5 ml. To isolate theadenylate formed, replicate samples were similarly depnoteinized, the precipitate washed with water, and the supernatant solution concentrated to a minimal volume forpaper chromatography (i-AmOH—K2HPO4) (6).

The cleavage of SAICAR was followed by using a modifled Bratton-Marshall procedure in which coupling was delayed 8 miii after diazotization (9). Each incubation mixtune consisted of 2.0 ml tissue extract and 0.25 ml buffer(pH 7.2) containing 640 @molesof substrate.

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TISSUE PREPARATIONLWER

(m@imoIe/gm wet wt./hr)S-iSO (m,@mo1e/gm wetwt./hr)@&@p,b

ADP,ATPSuccino AMPAMP,

ADP,ATP,SuccionAMPWhole

homogenateSupernatant

15,000Xg15,000Xg'34,800Xg74

115203800

00021

41772612

2027

7

546 Cancer Research Vol.25,May 1965

C. Conversion of inosinate to guanylate.—The initialincubations were carried out in the complex medium descnibed by Lagerkvist (14). Each incubation mixturecontained the dialyzed extract from 2 gm wet weight oftissue or the equivalent amount of acetone powder@; 1.41j@moles IMP-8-'4C (specific activity, 5.26 X 10@cpm/j@mole); 4 j@moles ATP ; 100 j.smoles fructose-i ,6-diphosphate; 20 j@moles NAD ; 120 j@moles nicotinamide; 120j@moles L-aspartate; 120 @imolesL-glutamate; 120 j@molesL-glutamine; 120 @&molesMgSO4; 150 @igpynidoxal phosphate; and 1000 @molessodium phosphate buffer, pH 7.4(20 ml total volume). This will be referred to as “complexmedium― in the text and tables.

Since the conversion to xanthylate is the step inhibitedby 6-thio IMP (Chart 5), subsequent incubations (20 ml)consisted of dialyzed extracts from 2 gm wet weight, 20

@@molesNAD, 120 Mmoles of phosphate buffer, pH 7.4, 40j@molesof glutathione, and “x―Mmoles of IMP-8-'4C (referred to as “simplemedium―).

The tissue extracts were added to the substrate after a20-mm equilibration period and the incubations werecarried out for 90 mm (unless otherwise specified) at 37.5°Cin a Dubnoff metabolic shaker. The reaction was stoppedby immersing each vessel for 3 mm in a boiling water bath,cooled to 37.5°C, and further incubated for 30 min with2500 units of xanthine oxidase to remove any xanthinethat might have arisen from degradation of IMP. At theend of this incubation, the reaction was stopped byfreezing, and the acid-soluble fraction was prepared in themanner previously described in the assay of the conversionof inosinate to adenylate.

The components of the acid-soluble fraction (adjustedto pH 9 with concentrated NH4OH) were separated onDowex-1 (bromide form, X10, 200—400mesh) columns into3 fractions, namely, (a) feed water wash, containing nicotinamide and nucleosides, (b) 0.0005 N HBr, containingfree purines, and (c) 0.18—1.0N HBr, containing combinednucleotides.

All 3 fractions were concentrated by lyophilization forsubsequent column chromatography. The nucleotidefraction was adjusted to 1 N with respect to HC1 and hydrolyzed for 21 hr at 100°C. The punines were isolatedfrom these fractions by chromatography on Dowex-50(hydrogen form, AGW X8, 200-400 mesh) by either stepwise (1) or gradient elution with HC1. With a 16 X 1 cmresin-bed column, a suitable linear gradient (18) was obtamed by feeding 1920 ml of 5 N HC1 into a mixer chambercontaining400 mlof 1 N HC1; in turn, thiswasfedintoa second mixer chamber containing 400 ml of 0.1 N HC1. Therate of flow of the eluent was regulated to about 0.5 ml/min.

The nicotinamide-nucleoside fraction was chromatographed on paper after an initial fractionation with HC1gradient. The identities of the various components werechecked spectrally and by paper chromatography and theradioactivities were determined.

RESULTS AND DISCUSSION

The conversion of inosinate to adenine nibonucleotidesby various preparations of liver and 5-180 is summarizedin Table 1. In the liver, the activity of the adenine

TABLE 1

CONVERSION OF INoSINA@m TO ADENINE RIBONUCLEOTIDESBY VARIOUS TISSUE PREPARATIONS―

a See “Assay Methods. “ All these incubations were carried

out at 20°Cfor 4 hr with an IMP concentration of 2.58 pmoles/incubation.

b Abbreviations used are : AMP, adenosine monophosphate;

ADP, adenosine diphosphate; ATP, adenosine triphosphate.C This incubation was carried out under the same conditions

but with 5.16 @.imolesof IMP/incubation.

nucleotide fraction5 was consistently about 3 times thatfound in 5-180. The latter, however, accumulated onethird of its total adenine derivatives in the form of the intermediate, succinoadenylate, which was not found in thevarious liver preparations under these experimental conditions. The total adenine derivatives formed by 5-180were about one-half that of the liver. Doubling of thesubstrate concentration essentially doubled the conversionwithout any effect on the relative ability of the two tissues.

The most active of the preparations was the 15,000 X gsupernatant solution ; this has been used for all subsequentstudies. The 34,800 X g supernatant solution was aboutas active as the whole homogenate. These results suggestthat at 15,000 X @isome of the degradative enzymesystem(s) were removed.6 At 34,800 X g some of the enzyme systems involved in the regeneration of ATP (andperhaps, subsequently GTP) were probably also removed.

A number of workers (16, 17, 25, 26) have shown that inmammalian tissues ATP and an ATP-regenerating systemcould substitute for the GTP required (15) in the conversion of inosinate to adenylate. Studies with liven and 5-180 are in agreement with these observations (Table 2).The endogenous guanine nucleotides, 0.92 and 0.75 j@moles/gm wet weight of liver and S-180, respectively (23), supplemented by added ATP and substrates for ATP negeneration, seem to be as efficient as added GTP in theserelatively crude extracts. Consequently ATP was usedin the studies presented here. A 3-fold increase in theATP and substrates required for its regeneration did notaffect the conversion.

The results of varying substrate concentrations (Table3) show that the formation of adenylate was proportionalto substrate concentration at and below 5.16 pmoles/10 mlincubation. Higher concentrations had no effect on 5-180and caused only a 20 % increase in liver. At the higherconcentrations, the difference between the two tissues be

6 For the purpose of this study, this represents the sum of the

mono-, di-, and triphosphates.0 There was less inosine in the incubations with either super

natant fraction than in those with whole homogenates.

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ATP@'GTP@TOTAL

ADENINE DERIVATIVESd(m@ano1e/gmwetwt./hr)LiverS-iSO—

—

+

+++—

+—

-<2

185@ 198

@ 2@

201<2

9195

10499

\VETWEIGId' (gin)TOTAL

ADENINE DERIVATWES@ (mg@mole/2 hrincubation)LiverS-iSO1

24i95

40279695

200404

IMP@(@mo1es/1O mlincubation)TOTAL

ADENI@E DERIVATWESC (m@imole/gm wetwt./hr)LiverS-1800.645

1.292.58

5.16

7.7412.9028

6298

108ilSd1982012O8d24323715

25515661d

9599108d10498

AMP@, ADP, ATP(inpmoles/gm wet wt./hr)Succino-AMP(m@imoles/gm wetwt./hr)Sensitive'@

LiverS-180

Resistant'

LiverS-i80208

78

2021500

32

013

SALSER AND BALls—Action of 6-Mercaptopurine. II 547

TABLE 2

COMPARISON OF ENERGY SOURCES IN THE CONVERSION

OF INOSINATE TO ADENYLATE―

TABLE 4

EFFECT OF VARYING ENZYME CONCENTRATION ON THE

CONVERSION OF INO5INATE TO ADENYLATE―

a See “Assay Methods.― These were incubations 5.16 @smoles

inosine monophosphate/lO ml incubation and 15,000 X g supernatant, carried out at 37.5°C. Aliquots of tissue extract (5 gm

wet weight/b ml) were used in this study.b Wet weight of tissue in each incubation.

C See Table 2.

CHART 1.—Conversion of inosinate to adenylate. Supernatant(15,000 X g) from 2 gm wet weight tissue incubated 30-240 mmwith 5.16 @zmolesinosine monophosphate at 37.5°C. The productwas isolated after various times of incubation by column chromatography (see “Assay Methods―).

TABLE 5CoNvERSION OF INOSINATE TO ADENINE RIBONUCLEOTIDES

BY SENSITIVE AND RESISTANT S-180@

a See “Assay Method.― These incubations were carried out

at 37.5°Cfor 2 hr with 5.16 @moleIMP/lO ml incubation. The15,000 X g supernatant of the tissue preparation was used.

b Adenosine triphosphate. The energy source consisted of 2.5

mmoles ATP, 5 mmoles fructose-i ,6-diphosphate, 2.5 mmolesa-ketoglutarate, and 1 mmole nicotinamide adenine dinucleotide/10 ml incubation, designated as (+). Three times this amountwas used in the experiment indicated as +++.

C Guanosine triphosphate. 2.5 mmoles GTP were substituted

for the aforementioned ATP system (footnote b).d All these samples were hydrolyzed to free purines before

chromatography. Neither succinoadenine nor its riboside wasdetectable in these samples. The conversion was measured aslabeled adenine isolated.

TABLE 3

EFFECT OF VARYING SUBSTRATE CONCENTRATION ON

THE CONVERSION OF INOSINATE TO ADENYLATE―

a,

E

0E

E

a,

C

Time (minutes)

a See “AssayMethods.― Incubations were carried out at37.5°Cfor 2 hr with 15,000 X g supernatant.

b IMP, inosine monophosphate.

C See Table 2.

d These are average values from 4 hr incubations at 20°C;

succinoadenosine monophosphate is included in the 5-180 values.

comes magnified, i.e., the capacity in S-iSO is only about40 % that in the liver. The results obtained with the twodifferent incubation conditions were essentially the same.No succinoadenine derivatives, however, were found onfractionation of the mixtures incubated at 37.5°C.

Under the experimental conditions used in these studies,the conversion to adenylate was proportional to theamount of tissue added, (Table 4) and proceeded linearlyfor more than 3 hr (Chart 1).

In the resistant variant of S-180 studied (Table 5), theconversion to adenine nibonucleotides was about 80 % that

a See “AssayMethods.― Incubations were carried out at20°Cfor 4 hr with 5.16 @molesinosine monophosphate and 15,000x g supernatant.

b Abbreviations used are: AMP, adenosine monophosphate;

ADP, adenosine diphosphate; ATP, adenosine triphosphate;6MP, 6-mercaptopurine.

C The terms “sensitive― and “resistant― refer to response of

S-180 to 6MP therapy.

of the corresponding host liver; less than 10 % (in contrastto the 30 % observed in sensitive 5-180) accumulated inthe form of succinoadenylate. The enzymatic activity ofa preparation of sensitive tumor studied at the same timewas about half that of the resistant variant. The liver of

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AMPS',,ai@p,ATP (mpinoles/ginwet wt./hr)Succino

AMP(rnpmoles/

gin wetwt./hr)Control

(uninjected)LiverS-180

Control (CMC-saline)Liver5-180

Treated (6-MP, 50 mg/kg body wt.)cLiver5-180203

77

21274

173520

27

032

018

0

Tissue preparationInosinemonopho,-

phate (j*inoles/incubation)Xanthylate

(mpmolesjgm wetwt./hr)HA/ICR

Swiss micebearing7-day-old5-180implantLiver1.41315-1801.4111Liver2.82805-1802.8220C57

black micebearingE0771implant9-day

implantLiver2.82232E07712.823318-day

implantLiver1.41170

548 Cancer Research Vol.25,May 1965

the host, however, exhibited the same enzymatic capacityregardless of which tumor was carried.

Treatment of the host animals with a therapeutic dose of6-MP resulted in a slight decrease in the enzymatic activity under consideration. The ability of the liver andtumor preparations of these animals to form adenylate wasfound to be 80 % and 65 %, respectively, of the controlvalues (Table 6). The total over-all conversion by 5-180was about 35 % that of the corresponding liver. Just asin the control animals, succinoadenylate was found onlyin the tumor; about 30 % of the total adenine nucleotideswereinthisform.

TABLE 6EFFECT OF 6-MERCAPTOPURINE (6-MP) THERAPY ON THE

CONVERSION OF INOSINATE TO ADENINE

RIBONUCLEOTIDES―

The accumulation of succinoadenylate in extracts of5-180 suggests that there may be a lower level of adenylosuccinase activity in this tissue. This activity has beenassayed with both succinoadenylate (Chart 2) andSAICAR (Chart 3) as substrate. The activity was determined by following the spectral change. In the caseof succinoadenylate, the product in replicate samples wasisolated by paper chromatography; the two values obtained corresponded closely. In both instances, the enzymatic activity in 5-180 was 35-40 % that in the liverpreparations.

Table 7 summarizes the data on the conversion of inosinate to xanthylate. This study has been extended to indude the tissues of C57 black mice bearing the tumor E0771. The conversion to xanthylate in extracts of these

In

I.@

.cC.)

CHART 3.—Cleavage of succinoaminoimidazolecarboxamideribonucleotide by liver and 5-180 extracts. Each 2.25 ml incubation (pH 7.2) containing 640 mpmoles of substrate and 2 mltissue extract (see Chart 2) was carried out at 37.5°C.The reaction was followed by measuring the aminoimidazolecarboxamideribonucleotide (AICAR) formed using a modified Bratton-Marshall procedure (9).

TABLE 7

CONVERSION OF INOSINATE TO XANTHYLATE BY

VARIOUS TISSUE PREPARATIONS―

Time (minutes)

a See “Materials and Methods,― and “AssayMethods.― Incubations were carried out at 20°Cfor 4 hr with 5.16 @smolesmosine monophosphate and 15,000 X g supernatant.

b Abbreviations used are : AMP , adenosine monophosphate;

ADP, adenosine diphosphate; ATP, adenosine triphosphate;CMC, carboxymethyl cellulose.

C Mice with 3-day-old s.c. implants of S-iSO were injected i.p.

-or 4 successive days.

a,

E

a,C.)

E

0

CHART 2.—Cleavage of succinoadenylate by liver and 5-180extracts. Each incubation mixture (2.4 ml) contained 449 mgimoles succinoadenylate and 2 ml tissue extract (10,000 X g supernatant of 4 gui wet weight/b ml, pH 7.2). Incubations were carned out for various times at 37.5°C.The reaction was followedby determining decrease in OD,,o (@, A) and by paper chromatographic isolation of the adenosine monophosphate (AMP)formed (0, @).

150

100

Time(minutes)

a See “Assay Methods.― Incubations were carried out with

the complex incubation medium. In all instances, the amountof guanylate formed was negligible.

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0 90 180 240

XANTBYLATE(m@mo1e/gmwetwt./hr)LiverS-iSOControl

UninjectedCarboxymethyl cellulose saline

Treated (50 mg/kg body wt.)b80847020

1914

INOSINI:(j@mo1e/incubation)XANTRYLATE

(mpmole/gm wetwt./hr)LiverS-iSO0.713

1.4262.852

7.130

12.83425

551171201841881936

153436414648

SALSER AND BALlS—ActiOn of El-Mercaptopurine. II 549

tumors is much lower than that in the corresponding livenpreparations, i.e., about 25—30% in 5-180 and 12 % and30 % in E 0771. In the relatively slow growing tumor,E 0771, two different implants have been examined todetermine if age of the tumor can be correlated with enzymatic capacity. In spite of the lower substrate concentration used in the particular assay, the extracts of 18-dayold implants were actually found to be more efficient inthis conversion than those from 9-day-old implants. Withthe same set of assay conditions, the liver preparationsfrom the animals with the older implants seemed to be lessefficient. These observations may bear some relationshipto the lesser response of older, more established tumors totherapy.

The conversion of inosinate to xanthylate by extracts ofliver and 5-180 was studied at several different concentrations of IMP (Table 8). The relative conversion of the 2tissues was the same at all the concentrations studied;furthermore, the saturation concentration seemed to bebe the same for both tissues, i.e., 7.13 j@moles. The highervalues observed here compared to Table 7 could probablybe attributed to the protective effect of the glutatbione(12) in the “simplemedium― (see “AssayMethods―) usedin these studies.

The ability of extracts to catalyze the formation ofxanthylate with time was studied. It can be seen thatwith both tissues, the reaction was linear for about 3 hr

TABLE 8EFFECT OF VARYING SUBSTRATE CONCENTRATION ON THE

CONVERSION OF INOSINATE TO XANTHYLATE―

TABLE 9EFFECT OF 6-MERCAPTOPURINE THERAPY ON THE CONVERSION

OF INOSINATE TO XANTHYLATE―

a See “AssayMethods.― Incubations were in “complexmedium― with 2.82 @moleinosine monophosphate.

b Mice with 3-day-old s.c. implants of 5-180 were injected i.p.

for 4 successive days.

1-aspartateGTp(orATP)@@

IMP ‘ @5uccino-AMP-.-. AMP

.1@ NAD “>6-THIOINOSINATE/

d@ XMP ‘ GMP

CHART 5.—Inhibition of nucleotide interconversions by 6-thioinosinate. Dotted hne8 indicate sites of inhibition. GTP,guanosine triphosphate; ATP, adenosine triphosphate; AMP,adenosine monophosphate; IMP, inosine monophosphate; XMP,xanthosine monophosphate ; GMP, guanosine monophosphate;NAD, nicotinamide adenine dinucleotide.

(Chart 4). The relative activity of the 2 preparations wasthe same at all the time periods studied, i.e., extracts ofS-180 had only about one-fourth of the enzymaticcapacity exhibited by those of host liven.

Preparations from tissues of animals which had receivedtherapeutic doses of 6-MP showed only a slight reductionin the capacity to convert inosinate to xanthylate (Table9).

CoNcLusIoNs

In studying the mechanism of action of a drug, it isevident that even though the site of action may be common to many living systems, there is considerable variationin biologic response. Qualitatively different responsescould result from quantitative reduction in the enzymaticcapacities of a system. Numerous examples can be citedfrom studies with bacteria, e.g., (a) a 50-fold increase in amutant's resistance to 6-MP is accompanied by a 30%reduction in its ability (compared to the parent) to convent this compound in its active form, 6-thio IMP (13, 24);(b) a total loss of ability of a mutant to grow on 2 ,6-diaminopunine as a purine source occurs concomitantly witha 30 % reduction in its ability to incorporate this substrate(3). It is conceivable that the selective susceptibility oftumors to various chemotherapeutic agents is due, in part,to such a quantitative reduction in certain enzymaticcapacities.

The lower endogenous inosinate concentration in 5-180,in spite of its greaten capacity for purine synthesis de novoand its slightly greaten capacity to synthesize the activecompound, 6-thio IMP (23), cannot explain the degree ofspecificity seen in vivo. It seems more likely to lie, at

a See “AssayMethods.― Incubations carried out in “simplemedium.―

a,

E0,

E

0.

Time (minutes)

CHART 4.—Formation of xanthylate from inosinate by tissueextracts. Incubations were carried out for 45—270mm with 7.13pmoles inosine monophosphate in the “simplemedium― describedin “Assay Methods.― The product was isolated after varioustimes of incubation by column chromatography. (XMP, xanthosine monophosphate.)

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550 Cancer Research

least in part, in differences in the nucleotide conversions(Chart 5) shown to be inhibited by 6-thio IMP (24).

From the data presented, the over-all conversions ofinosinate to adenine and xanthine derivatives in 5-180 areabout 50 % and 25—30%, respectively, of those found in theliver. Consequently, inhibition by 6-thio IMP of the further conversion of inosinate could result in reduction of thetumor's inherently lower enzymatic capacity below thethreshold required for growth. A similar reduction of theprocess in liver might not cause a serious deficiency. Therelative reduction would be less in the liver, which has a“reserve―capacity, as reflected by its higher endogenousnucleotide concentrations (23).

The lower adenylosuccinase activity (about 35—45% ofthat in the liver) in 5-180 is consistent with the observedaccumulation of this intermediate (about 30 % of the totaladenine derivatives) in the incubation mixtures of thetumor. This lowered enzymatic capacity has been furthen substantiated by the accumulation of SAICAR in5-180 (compared to liver) in purine synthesis de novo withaspartate-14C as precursor.7 The similarity in the enzymaticcleavages of the 2 substrates (succinoadenylate andSAICAR) suggests that in these 2 tissues, as in microbialsystems (9), the same enzyme is involved. Although allthe parameters measured indicate qualitative identity ofthe enzyme in liver and in 5-180, the possibility of an“altered―enzyme8 in 5-180 has to be considered.

The usefulness of any anti-cancer agent stems from thefact that host tissues are inherently more resistant to theaction of the agent than the tumor. Such host resistancemay be because the enzymatic makeup of the host cellsis qualitatively and/or quantitatively different from thetumor cell. The more efficient the agent is, the greaterthe difference between the two. Consequently, resistancein tumors can, in some cases, be considered as a shift incertain enzymatic capacities to those found in the host.The foregoing data indicate that the host tissue studied isrelatively resistant by virtue of an excess of enzymeswhich are involved in the sequence of metabolic steps inhibited by 6-thio IMP (Chart 5). In the resistant variantstudied, the tumor seems to have undergone a change,making it resemble the host liver. Its resistance could beattributed partially to this shift in enzymatic capacity.

It is possible that anti-purines of chemotherapeuticvalue (e.g., 6-MP, by virtue of its structural similarity),could affect purine interconversions through enzyme repression. The results obtained from treated animals showonly a small decrease in enzymatic activity of both liverand 5-180. Nevertheless, this reduction, however small,could increase the differential between the 2 tissues, i.e.,enhancing the inherently lower enzymatic capacity of thetumor. It is possible, however, that these observed reductions are not specific for the particular enzyme systemsstudied but is merely a secondary manifestation of thetoxicity of the drug. Similar lowering of enzymatic conversions have been observed with other purine analogs(4, 22).

It is too early to extrapolate to tumors in general even

7 J. S. Salser and M. E. Balis, unpublished observations.

8 Partridge and co-workers (8, 19) have shown altered proper

ties (thermolability, inhibition by metal and urea) of adenylosuccinase in a number of Neurospora mutants.

though in the conversion to xanthylate, E 0771 parallelsthe results obtained with 5-180. The available data havebeen obtained from cell-free preparations of liver (the onlyhost tissue studied) and tumor tissues, and do not necessarily reflect the over-all picture of in vivo systems. Theydo lend support to the concept that quantitative and/orqualitative biochemical differences between tissues couldserve as a basis for effective anti-tumor chemotherapy.Ultimately, other host tissues will have to be studied inorder to obtain a more complete understanding of the response in the intact animal.

ACKNOWLEDGMENTSThe authors wish to thank Dr. George B. Brown for his interest

in this work and his many helpful suggestions, as well as Mrs.Elizabeth Carroll and Miss Anne Manz for their competent technical assistance.

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2. Atkinson, M. R., Morton, R. K., and Murray, A. W. Inhibitionof Inosine 5'-Phosphate Dehydrogenase from Ehrlich AscitesTumour Cells by 6-Thioinosine 5'-Phosphate. Biochem. J.,89: 167—72,1963.

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6. Carter, C. E., and Cohen, L. H. The Preparation and Properties of Adenylosuccinase and Adenylosuccinic Acid. J. Biol.Chem., @@5:17—30,1956.

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8. Giles, N. H., Partridge, C. W. H., and Nelson, N. J. TheGenetic Control of Adenylosuccinase in Neuro8pora crassa.Proc.Natl.Acad. Sci.U. S.,48:305—17,1957.

9. Gots, J. S., and Gollub, E. G. Sequential Blockade in AdenineBiosynthesis by Genetic Loss of an Apparent BifunctionalDeacylase. Thid., 4$: 826—34,1957.

10. Gots, J. S., and Gollub, E. G. Purine Analogues as FeedbackInhibitors. Proc. Soc. Exp. Biol. Med., 101: 641—43,1959.

11. Hampton, A. Studies of the Action of Adenylosuccinase with6-Thio Analogues of Adenylosuccinic Acid. J. Biol. Chem.,@S7:529—35,1962.

12. Hampton, A. Reactions of Ribonucleotide Derivatives ofPurine Analogues at the Catalytic Site of Inosine 5'-PhosphateDehydrogenase. Ibid., p58: 3068—74,1963.

13. Hutchison, D. J. Metabolism of Resistant Mutants of Streptococcus faecali8. I. Isolation and Characterization of the Mutants. Cancer Res., 18: 214-19, 1958.

14. Lagerkvist, U. Biosynthesis of Guanosine-5'-Phosphate. I.Xanthosine-5'-Phosphate as an Intermediate. J. Biol. Chem.,235: 138-42, 1958.

15. Lieberman, I. Enzymatic Synthesis of Adenosine-5'-Phosphatefrom Inosine-5'-Phosphate. Ibid., 525: 327—39,1956.

16. Newton, A. A., and Perry, S. V. Incorporation of Nitrogen15 in the 6-Amino Group of Adenosine Triphosphate by Muscle Extracts. Nature, 179: 49—SO,1957.

17. Newton, A. A., and Perry, S. V. The Incorporation of N15intoAdenine Nucleotides and Their Formation from Inosine Monophosphate by Skeletal-Muscle Preparations. Biochem. J.,74: 127—36,1960.

18. Parr, C. W. The Separation of Sugars and of Sugar Phosphatesby Gradient Elution from Ion-Exchange Columns. Ibid.,66: xxvii—xxviii, 1954.

19. Patridge, C. W. H. Altered Properties of the Enzyme Adenylo

Vol.25,May 1965

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SALSER AND B.@u@Is—Action of 6-Mercaptopurine. II 551

succinase Produced by Interallelic Complementation at theAD-4 Locus in Neurospora cras@a. Biochem. Biophys. Res.Commun., 8: 613—19, 1960.

20. Salser, J. S., and Balis, M. E. Conversion of Inosmic Acid(IMP) in Normal and Tumor Tissues. Proc. Am. Assoc. Cancer Res., 5: 265, 1961.

21. Salser, J. S., and Balis, M. E. Action of Antipurines on Nucleotide Interconversion. Ibid., 3: 357, 1962.

22. Salser, J. S., and Balis, M. E. Further Studies on NucleotideInterconversions in Liver and Sarcoma 180. Federation Proc.,21: 164°,1962.

23. Salser, J. S., and Balls, M. E. The Mechanism of Action of6-Mercaptopurine I. Biochemical Effects. Cancer Res., @5:539—43,1965.

24. Salser, J. S., Hutchison, D. J., and Balis, M. E. Studies onthe Mechanism of Action of 6-Mercaptopunine in Cell-FreePreparations. J. Biol. Chem., SS6: 429—32,1960.

25. Yefimochkina, E. F. The Conversion of Inosinate to Adenylatein Muscle Extracts. Biokhimiya, @5:607—16,1960.

26. Yefimochkina, E. F., and Braunstein, A. E. The Amination ofInosinic Acid to Adenylic Acid in Muscle Extracts. Arch.Biochem. Biophys., 85: 350—52,1959.

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1965;25:544-551. Cancer Res   Josephine See Salser and M. Earl Balis  SpecificityThe Mechanism of Action of 6-Mercaptopurine: II. Basis for

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