Growth and isotope incorporation studies with bacterialsystems (3—5,12, 13, 17, 18) have led to the hypothesisthat the active form of 6-MP is its ribonucleotide (thioinosinate, 6-thio IMP), which inhibits the further conversion by inosinate to other purine ribonucleotides (4, 5).This inhibition by thioinosinate of the formation of adenylate and guanylate has been demonstrated in cell-freepreparations from Streptococcus faecalis and pigeon liver,respectively (26). In L1210, studies on the incorporationof labeled hypoxanthine into nucleic acid punines are consistent with the inhibition at the level of hypoxanthine,but only the conversion to adenylate seems to be seriouslyaffected by 6-MP (10). More recent studies with partiallypurified inosine-5'-phosphate-NAD-oxidoreductase (IMPdehydrogenase) from bacteria (15) and Ehrlich ascites

1 This investigation was supported in part by funds from the

National Cancer Institute, NIH, USPHS (CA-03190-08), and(5-K3-CA-16, 673-02).

2 A preliminary report has been presented (25).

3 The abbreviations used are : 6-MP, 6-mercaptopurine; 6-thio

IMP, 6-thioinosinate; IMP, inosine monophosphate; AMP, adenosine monophosphate ; XMP, xanthosine monophosphate ; NAD,nicotinamide adenine dinucleotide; AICA, 4-amino.5-imidazolecarboxamide; ATP, adenosine triphosphate; DPN, diphosphopynidine nucleotide; TCA, trichloroacetic acid; GMP, guanosinemonophosphate.

Receivedfor publication September 10,1964;revisedDecember23, 1964.

tumor cells (1) have confirmed the observation that 6-thioIMP is actually an effective competitive inhibitor of theconversion to xanthylate, the intermediate in the formation of guanylate.

Since the conversion of inosinate to other punine nibonucleotides occurs in both normal and tumor tissues, theselective susceptibility of tumors such as Sarcoma 180(5-180) to the action of 6-MP must be considered in termsof the total purine synthesis and interconversion mechanisms. This paper presents some aspects of purine metabolism in 5-180 and host liver pertinent to nucleotidesynthesis and conversion. The synthesis in vivo of 6-thioIMP, the synthesis of purines de novo in in vitro systems,and the inhibition of adenylate formation by 6-thio IMPin S-180 and liver have been studied.

MATERIALS AND METHODS

IMP@8@HC was obtained from Schwarz BioResearch,Inc. ; 6-MP-8-'4C and 4-amino-5-imidazole-carboxamide4-‘@C(AICA), from Southern Research Institute; glycine-1-‘4C,from Tracerlab. ATP (disodium salt) ; IMP (sodiumsalt); NAD (DPN); fructose-i ,6-diphosphate (sodiumsalt) ; a-ketoglutarate; L-glutamine ; nibose-5-phosphate(sodium salt); and tetrahydnofolic acid, were obtainedfrom Sigma Chemical Company. 6-Thioinosinate (6-thioIMP) was kindly supplied by Dr. A. Hampton.

539

The Mechanism of Action of 6-Mercaptopurine1'2

I. Biochemical Effects

JOSEPHINE SEE SALSER AND M. EARL BALlS

(Divi8ion of Nucleoprolein Chemistry, Sloan-Kettering Institute for Cancer Research, Sloan-Kettering Divi8ion of CornellUniversity Medical College, New York, New York)

SUMMARY

The tumor Sarcoma 180 (5-180) was compared to host liver in an effort to evaluatethe inhibitory action of 6-MP.3 The concentration of endogenous adenine nucleotidesin S-180 was about 40 % of that found in liver, that of guanine nucleotides was aboutthe same in both tissues, while that of inosinate was much lower in tumor. The liverof normal mice and 5-180-bearing mice exhibited the same capacity to convert IMPto AMP. A slightly greater conversion of 6-MP to 6-thio IMP in 5-180 was notedbut it was not by itself of sufficient magnitude to explain the anti-tumor specificity ofthe compound. An 5-180 sensitive to 6-MP and one resistant to it each showedabout one and a half times as much capacity to synthesize purines in vitro as host liver.

The conversion of IMP to AMP by either 5-180 or liver was inhibited by 6-thioIMP ; the inhibition, though low, was greaten in the tumor. The actions of 6-thioIMP, reported by others in mammalian systems, are analogous to those previouslyfound in bacterial and avian systems; i.e., the conversion of IMP to AMP and XMPis inhibited. It is suggested that the basis for the anti-tumor specificity may lie indifferences in the enzymatic capacity of tumors and host tissues to carry out the conversions blocked by 6-thio IMP.

on April 8, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

540 Cancer Research Vol.25,May 1965

HA/ICR Swiss mice (approximately 20 gm each) witha 5- or a 7-day-old subcutaneous implant of 5-180 werekindly supplied by Dr. H. Schwartz and Dr. C. Reilly ofSloan-Kettering Institute. Mice with 11-day-old subcutaneous implants of a resistant variant of 5-180 weresupplied by Dr. D. Clarke. Necrotic tumors were discarded in all instances.

Acetone powders of the tissues were prepared byhomogenizing the liver or tumor with 10 volumes of coldacetone (—25°C)in a Waring Blendor, followed by filtering and rehomogenizing the resuspended cake in fresh coldacetone. The last 2 steps were repeated twice. The yieldof acetone powder from different batches of liver was about25 % of the wet weight while that from 5-180 was 12—15%.

6-MP incorporation in vivo.—Mice with 5-day-old implants of S-l80 were given i.v. (caudal vein) injections of6-MP-8-'4C (50 mg/kg body weight, specific activity =2.3 X 10@cpm/,@mo1e). The animals were sacrificed at2, 6, and 24 hr after a single injection and at 48 hr after 3successive daily injections of the labeled compound. Theliver and tumor were removed and chilled in chopped ice.All subsequent steps, unless otherwise specified, werecarried out at 4°C. The chilled tissues were homogenizedbriefly in a Potter-Elvehjem homogenizer with 1 volumeof cold distilled water. Sufficient cold 20 % TCA wasadded to give a final concentration of 10 % TCA and homogenization was continued for another 3 min. The precipitated protein was removed by centrifugation, resuspended and rehomogenized 3 more times with cold 5%TCA. The combined supernatant solutions were adjustedto 0.01 N with respect to HBr and extracted 8—10timeswith ether to remove the TCA.

The components of this acid-soluble fraction (adjustedto PH 9 with concentrated NH4OH) were separated onDowex-1 (bromide form, XlO, 200—400mesh) colunmsinto 3 fractions : (a) feed and wash water, containingnucleosides, nicotinamide, amino acids, and flavins, (b)0.006 N HBr, containing free purines, kNIP, and IMP, and(c) 0.18 N HBr, containing 6-thio I@1P with traces of GMPand ATP. These fractions were concentrated to drynessand taken up in a minimal measured volume of water.The radioactivities of aliquots of the fractions were determined on infinitely thin films on aluminum planchetsin a Geiger-Muller internal flow counter. Paper chromatography of the 3rd fraction in various solvent systems(16) showed that all the radioactivity was associated with6-thio IMP. The identity was further confirmed spectrally.

Purine synthesis de novo in cell-free preparations.—Frozen 7-day-old implants of S-180 (sensitive) and 11-dayold implants of S-180 (resistant) as well as their comesponding host livers were homogenized (3 min in a Virtis“45―)in a pH 7.4 phosphate medium (0.035 M sodiumphosphate, 0.13 M KC1, 0.04 @iKHCO3, 0.01 M MgC12, and0.015 M L-glutamme4). When glycine-1-'4C (specific activity = 1.24 X 10@ cpm/@@mole) was used as precursor,the incubation medium was essentially that of Levenberget at. (19) but with an additional 6 @molesATP/ml and aslightly higher glycine concentration of 8.66 j@moles/ml.

With labeled AlGA (specific activity = 4.11 X 10@cpm/smole) as precursor, the incubation medium was that described by Schulman and Buchanan (27). In all the incubations, there was 250 mg wet weight of tissue/mi.

After a 15-min equilibration period, the labeled precursor was added and the incubation carried out for 2 hr at37.5°Cin a Dubnoff metabolic shaker. The reaction wasstopped by immersion of the vessels in an acetone-Dry Icebath. Carrier IMP was added to give a final concentration of 5 @moIes/ml. All subsequent steps were carriedout at 4°Cunless otherwise specified.

Sufficient cold 60 % TCA was then added to give a finalTCA concentration of 10 %. The precipitated proteinwas removed by centnifugation and washed in the mannerdescribed above. After ether extraction, the supernatantsolutions were lyophilized to dryness. Since the incubations were carried out in duplicate, one set of samples wasdissolved in a minimal volume of 0.01 N HC1 and the nucleotides were isolated as barium salts (24). To minimizethe adsorption of traces of labeled precursor, unlabeledAICA or glycine was added during the precipitation and2 reprecipitations. The radioactivity was determinedand was shown by paper chromatography (i-butyricacid-NH3) to be associated with inosinate.

The duplicate set of samples was dissolved in water, adjusted to pH 11 with NH4OH and put on Dowex-1 (chloride form, X10, 200-400 mesh) columns. These werewashed with at least 100 volumes of water and 0.003 N HC1until the eluates were free of radioactivity. Inosinatewas subsequently eiuted with 0.005 N HC1, lyophilized todryness, and finally dissolved in water for radioactivity determination. The identity was further confirmed spectrally and by paper chromatography.

Conversion of inosinate to adenylate.—Cell-free extractswere prepared by homogenizing acetone powder or frozentissue in a medium containing 0.16 M KC1 and 0.05 M Tnisbuffer, pH 7.4, (21), (4 gm wet weight or the equivalentamount of acetone powder/b ml) in a Virtis “45―homogenizer for 3 min. To partially fractionate the activesystem(s) involved in this conversion, aliquots of thehomogenate were centrifuged at 15,000 X g for 30 min andat 34,800 X g for 60 min5. All the operations were carriedout at 4°C.

Each 10 ml of incubation mixture contained 2 gm wetweight or the equivalent amount of acetone powder; 2.5mmoles ATP ; 2.5 mmoles L-aspartate ; 5 mmoles fructose1 ,6-diphosphate; 0.5 mmoies MgCl2; 5 mmoles sodiumphosphate buffer, pH 7.4; 2.5 mmoles a-ketoglutarate; 1mmole NAD; and “x―@tmoles IMP-8-'@C (specific activity = 6.31 X 10@cpm/,@mole).

In the comparative studies of acetone powder andfrozen tissue preparations of livers of tumor-bearing andnontumor-bearing animals, the concentration of IMP was1.25 j@moles/10 ml incubation. After a 4-hr incubationat 20°C6in a Dubnoff metabolic shaker, the reaction wasstopped and the incubation mixture deproteinized after

S The comparison of these fractions will be part of another

communication.0 Incubations at this temperature have been shown to promote

accumulation of succinoadenylate (21), a compound of interestin studies of nucleotide interconversions.

4 The presence of glutamine stabilizes the purine-synthesizing

enzymes. (J. M. Buchanan, personal communication.)

on April 8, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

PaEculsoaIN0sINAm

SYNTHESISM@OLE/GMWETWEIGHTSensitiveResistantLiverS-180LiverS-ISOGlycine-1-'4C

A.―B.C

AIC-4-'4CA.b

B.C95

98

2829131

137

3537104

114

2628184

194

3436

T issueAdenine derivatives@'rn@ixnole/gmwetweight/hrLiver

from nontumor-bearing miceFrozen tissueAcetone powder

Liver from S-180-bearing miceFrozentissueAcetone powder41

39c

4240c

Tna (hr)@'@LUOLZ6-THIOINOSINATZ/GM WaTWaIGETLiverS-1801

Injection2624

3 Injections482.27

X 10'0.69X 10'0.24 X 10'

0.21X 10'2.47

X 10'0.82X 10'0.28X 10@

0.24X 10'

SALSER AND BALlS—Action of 6-Mercaptopurine. I 541

ether extraction. The clear solution was neutralized and,if necessary, stored at —20°C. The components of suchsolutions were separated on Dowex-1 (chloride form, X10,200-400 mesh) columns by a modification of the methodof Deutsch and Nilsson (11). The identities of the variousfractions were checked spectrally and by paper chromatography and the radioactivities were determined. Thismethod of fractionation was used to determine the endogenous nucleotide concentration of the 2 tissues.

In the inhibition studies, the reaction mixtures contained5.16 @molesIMP/lO ml. The levels of 6-thio IMP studiedwere 1.94 and 4.85 @imoles/10ml. After a 2-hr incubationat 37.5°C, the reaction was stopped and deproteinizationcarried out as described in the foregoing section. Thesolutions were lyophilized to dryness, taken up in freshlyprepared 1 N HC1 and hydrolyzed for 21 hr at 100°C. Thepunines were isolated by chromatography on Dowex-50(hydrogen form, AGW, X8, 200-400 mesh) columns byelution with 0.15 N HC1 for xanthine, 6-MP and hypoxanthine and 1.5 N HC1 for guanine and adenine.

RESULTS AND DISCUSSION

The enzymatic conversion of 6-MP to its active form,6-thio IMP, by 5-180 and host liver was studied.Table 1 summarizes the data obtained from a typical experiment in vivo. The 6-thio IMP levels of S-180 wereconsistently about 10—15% higher than that of host liver,with the highest levels being found in animals sacrificed 2hr after injection. Catabolism of 6-thio IMP proceededat a steady rate, but a detectable amount was still presentafter 24 and 48 hr.

The synthesis de novo of purines by whole homogenatesof the 2 tissues under consideration is given in Table 2.A 6-MP-resistant variant of 5-180, and the correspondinghost liver have also been included here. The liver of bothgroups of animals showed essentially the same degree ofincorporation of the 2 precursors. The tumor, whethersensitive or resistant, had greater capacity to synthesizepurines than the respective liver, i.e., 1.4-fold (sensitive)and 1.7-fold (resistant) with glycine as precursor, and 1.3-fold (sensitive) and 1.3-fold (resistant) with AICA asprecursor. These differences do not seem great comparedto the greater requirement for purines in the more rapidly

TABLE 1

TABLE 2INOSINATE SYNTHESIS BY LIVER AND

S-180 IN VITRO―

a See “Materials and Methods.―b Precipitated as barium nucleotides and isolated by paper

chromatography.C Isolated by chromatography on Dowex-1 (chloride form).

TABLE 3

CONVERSION OF INOSINATE TO ADENYLATE

IN LIVER PREPARATIONS―

a Whole homogenates were incubated as described in “Materials and Methods.―

b See footnote 8.

C Calculated on the basis that the acetone powder preparation

is 25% of the wet weight of the liver (see “Materials andMethods―).

growing tumor. Consequently, it is not surprising thatthe concentration of free nibonucleotides is much lower inthe tumor than in the liver (see Table 4). Such differences, though small, may well be reflected in the relativesensitivity of the 2 tissues to punine antagonists, such as6-MP.

Preliminary studies with unlabeled IMP have shownthat the net formation of AMP, GMP, or their intermediates (based on spectral data), was too low in cell-free extracts of both liver and tumor to be determined accurately.To facilitate the determination of small changes of thisnature, all subsequent studies were carried out with “C@labeled IMP.

In all these comparative studies, the liver of the tumorbearing animals was used as the normal tissue since thereis an ambiguity as to the exact tissue or cell of origin of5-180. Furthermore, the conversion to adenylate is thesame in the liver of nontumor and 5180-bearing animals,i.e., about 40 mMmoles/gm wet weight of tissue/hr. Table3 summarizes the data obtained with frozen tissues andacetone-powder preparations of these 2 tissues. The latter

6-TmoINoSINAvsi SYNTHESIS IN LIVER AND5-180 IN Vivo―

a See “Materials and Methods.―b Time interval between injection (or the last of 3 successive

daily injections) of 6-mercaptopurine and sacrifice of the animal.

on April 8, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TISSUEPURINE

NUCLEOTIDESbMHOLE/GM WETWEIGHTAdenylateGuanylateInosinateLiver

@ Whole homogenateWhole homogenate blankcSupernatant 15,000 X g blank'

S-180Whole homogenateWhole homogenate blankcSupernatant 15,000 X g blankc7.63

8.257.82

2.903.052.740.90

1.020.85

0.760.800.680.48

0.550.52

0'Od0―

@MOLE/1O ML INCUBATIONADENINEDERWATIVES@' Mj@MOLE/GMWETWEIGHT/HRInosine,

monophosphate6-ThioinosinateLiverS-ISO5.160198

@201 @95995.161.94188

@186 @78825.164.85@177@179 @6871

542 Cancer Research Vol.25,May 1965

TABLE 4

CONCENTRATION OF ENDOGENOUS NUCLEOTIDES―

as a base line for such determinations, no great significanceshould be attached to these small differences in the amountof synthesis between these 2 tissues in the presence of 6-thio IMP.

CONCLUSIONSBiochemical studies on the mechanism of action of 6-MP

in diverse systems in vitro have led to a number of suggested sites of inhibition by the active form, 6-thio IMP.9The common denominator is that they all involve facetsof purine and punine nucleotide metabolism which arenormally found in living systems. Yet, there is considerable variation in biologic response to 6-MP from species tospecies and even from tissue to tissue. It is conceivablethat these variations, which are reflected by different degrees of sensitivity or resistance, may be attributed atleast in part to qualitative and/or quantitative differencesin the enzymatic makeup of the systems. Consequently,comparative studies of a sensitive tumor such as 5-180and the inherently more resistant host tissues have to beconsidered in terms of possible enzymatic differences ofthis nature in punine synthesis and nucleotide interconversions.

In certain bacteria (4, 7, 26) and some experimentalneoplasms (8, 23), sensitivity to 6-MP can be attributedto a greater ability (compared to a resistant mutant) toconvert this compound to 6-thio IMP. However, thedata presented here show that the capacity for this conversion in S-180 is not appreciably greater (only 10—15%)than that in host liver. In vitro experiments'0 show essentially the same thing. These experimental values are inthe same range as those observed in mice bearing a resistant variant of 5-180 (22). The higher values at 2 hr,the shortest experimental time period, followed by a steadydecrease with time suggest that most of the synthesis probably occurred within 2 hr. The 6-thio IMP persists for aconsiderable period of time in both liver and 5-180. Thisrather small difference in synthesis of 6-thio IMP canhardly explain by itself the great difference observed inthe biologic response of the 2 tissues to 6-MP (9).

An inhibition in the pathway de novo, probably at thephosphoribosylamidotransferase step, has recently beensuggested as the primary site of action of 6-MP in experimental tumors in vivo (6). However, in biochemicalstudies of this enzyme system, adenylate has been reportedto be 5 % as effective as 6-thio IMP and the inhibitions bythe 2 compounds are additive (20). The endogenousadenylate concentration of 5-180 is over 1000 times greaterthan the 6-thio IMP level at 2 hr after injection of an LDSOdose of 6-MP. Thus, in this tumor, the contribution bythe 6-thio IMP formed would be negligible and couldhardly by itself explain the observed anti-tumor specificityfound only with 6-MP. Although 6-thio IMP may exertfeed-back inhibition, it does not appear likely that the bio

5 6-Thioinosinate has been reported to inhibit IMP dehydro

genase (15, 26), succinoadenylic acid synthetase (2, 14,26), adenylosuccinase (2, 14), and glutamine PP-ribose-P amidotransferase(20).

10 Experiments carried out with cell-free homogenates of liver

and S-180 essentially confirm the data obtained in vivo, i.e., theincorporation by S-180 was consistently about 10—12%higher thanby liver. (Salser, J. S., Wahrman, S., and Balis, M. E., unpublished data).

a See “Materials and Methods.―b See footnote 8.

C See footnote 7. Cell-free extracts from 4 gm wet weight of

tissue made up to 20 ml with buffer and adjusted with cold 60%

trichloroacetic acid to give a final concentration of 7%.d The values were too low to be detected by the assay method

used.

TABLE 5

INHIBITION OF INOSINATE TO ADENYLATE CONVERSION

IN CELL-FREE PREPARATIoN―

a See “Materials and Methods.―

b Samples had been hydrolyzed to free purines prior to column

chromatography.

values were adjusted to a wet weight basis (see “i\'iaterialsand Methods―). On the basis of these results, eitherpreparation can be used for studies of this nature.

Table 4 summarizes the endogenous purine nucleotideconcentration of liver and S-180. These values were obtamed from acid-soluble extracts of whole homogenates aswell as the blanks for some of the incubations with Undialyzed preparations.7 The concentration of adeninenucleotides8 in S-180 is about 40 % of that in the liver,while that of guanine nucleotides8 is but slightly less in5-180.

The inhibition by 6-thio II\1P of the conversion ofinosinate to adenylate was examined in 15,000 X g supernatants of liver and S-180. The results are given in Table5. The inhibition in the liver at the 2 levels studied wasabout half that observed in the tumor. Since the IMPconcentrations used are in excess of those occurring in thetissues, and because of errors inherent in using wet weight

7 These were tissue blanks used in assaying studies of the con

version of inosinate to adenylate (to be published).5 This represents the sum of the mono-, di-, and triphosphates.

on April 8, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

SALSER AND BALls—Action of 6-Mercaptopurine. I 543

logic result can be primarily attributed to that biochemicalaction alone. Furthermore, the effects of 6-MP on incorporation of formate and pre-formed purines (6) can beinterpreted in terms of the “sparing―action of 6-MP onformate utilization (4) and the inhibition of xanthine oxidase by 6-MP (28, 29). The latter would result in lessdegradation of purines per se. Consequently, there wouldbe less need for purine synthesis de novo and the utilizationof tracer doses of purines would not be decreased.

Studies with bacteria have indicated that 6-MP exertsits inhibitory action as the ribonucleotide, 6-thio I1\1P,which blocks the further conversion of IMP to AMP andGMP (4, 5). The inhibition of these nucleotide interconversions has subsequently been demonstrated to occur atreasonable 6-thio IMP concentrations in bacterial (15, 26)and avian (26) systems in vitro. The present study, aswell as those of Atkinson et at. (1), indicate that these inhibitions do occur in mammalian systems― with 6-thioIMi@ levels comparable to those which exist in tissues invivo. The lower endogenous level of IMP and the slightlyhigher synthesis of 6-thio IMP in 5-180 (compared to hostliver) could indicate some specificity. However, it doesnot seem likely that these differences alone can explain thedegree of specificity observed in vivo with S@180.12 Thereported inhibitions in vitro by 6-thio Ii\'IP of the conversion of IMP to succino-Ai\'IP and XMP suggest that thebasis of the selective susceptibility of tissues to 6-MPmight be expected, in part, to lie in differences in the abilityof tissues to carry out such interconversions. Attemptsto verify this suggestion have been carried out and will bereported.

ACKNOWLEDGMENTS

The authors wish to thank Dr. George B. Brown for his interestin this work and his many helpful suggestions.

REFERENCES

1. Atkinson, M. R., Morton, R. K., and Murray, A. W. Inhibitionof Inosine 5'Phosphate Dehydrogenase from Ehrlich Ascites

Tumour Cells by 6-Thioinosine 5'-Phosphate. Biochem. J.,89: 167—72,1963.

2. . Inhibition of Adenylosuccinate Synthetase and Adenylosuccinate Lyase from Ehrlich Ascites-Tumour Cells by6-Thioinosine 5'-Phosphate. Ibid., 92: 398—404,1964.

3. Balis, M. E., Hylin, V., Coultas, M. K., and Hutchison, D. J.Metabolism of Resistant Mutants of Streptococcus faecalis. II.Incorporation of Exogenous Purines. Cancer Res., 18: 220—25,1958.

4. . Metabolism of Resistant Mutants of Streptococcusfaecalis. III. The Action of 6-Mercaptopurine. Ibid., 18: 440—44,1958.

5. Balis, M. E., Levin, D. H., Brown, G. B., Elion, G. B., Nathan,H. C., and Hitchings, G. W. The Effects of 6-Mercaptopurineon Lactobacillus casei. Arch. Biochem. Biophys., 71: 358—66,1957.

6. Bennett, L. L., Jr., Simpson, L., Golden, J., and Barker, T. L.The Primary Site of Inhibition by 6-Mercaptopurine on thePurine Biosynthetic Pathway in Some Tumors in Vivo. CancerRes., 28: 1574—80,1963.

7. Brockman, R. W., Debavadi, C. S., Stutts, P., and Hutchison,

11 Inhibition of succinoadenylate synthetase (noncompetitively) and adenylosuccinase (competitively) from Ehrlich ascitestumor cells by 6-thio IMP has recently been reported (2).

12 It should be emphasized that the only normal tissue studiedwas the liver. It does not necessarily follow that all nonmalignant tissues behave like the liver.

D. J. Purine Ribonucleotide Pyrophosphorylases and Resistance to Purine Analogues in Streptococcus faecalis. J. Biol.Chem.,256:1471—79,1961.

8. Brockman, R. W., Kelley, G. G., Stutts, P., and Copeland, V.Biochemical Aspects of Resistance to 6-Mercaptopurine inHuman Epidermoid Carcinoma Cells in Culture. Nature,191: 46—71,1961.

9. Clarke, D. A., Philips, F. S., Sternberg, S. S., Hitchings, G.W., Stock, C. C., and Elion, G. B. 6-Mercaptopurine: Effectsin Mouse Sarcoma 180 and in Normal Animals. Cancer Res.,

13: 593—604,1953.10. Davidson, J. D. Studies on the Action of 6-Mercaptopurine in

Sensitive and Resistant L1210 Leukemia in Vitro. Ibid., 20:225—32,1960.

11. Deutsch, A., and Nilsson, R. Ion Exchange Chromatographyof Inosine Phosphates. Acta Chem. Scand., 7: 1288—92, 1953.

12. Elion, G. B., Hitchings, G. H., and Vanderwerif, H. Antagonists of Nucleic Acid Derivatives. VI. Purines. J. Biol. Chem.,192: 508—18,1951.

13. Elion, G. B., Singer, S., Hitchings, G. H., Balis, M. E., andBrown, G. B. Effects of Purine Antagonists on a Diaminopurine-Resistant strain of Lactobacillus casei. Ibid., 202: 647—

54, 1953.14. Hampton, A. Studies of the Action of Adenylosuccinase with

6-Thio Analogues of Adenylosuccinic Acid. Ibid., 237: 529—35,1962.

15. . Reactions of Ribonucleotide Derivatives of PurineAnalogues at the Catalytic Site of Inosine 5'-Phosphate I)ehydrogenase. Ibid., 258: 3068—74, 1963.

16. Hampton, A., and Maguire, H. Nucleotides. I. Synthesis of6-Chloro-, 6-Mercapto-, and 2-Amino-6-mercapto-9-@-D-ribofuranosylpurine 5'-phosphate. J. Am. Chem. Soc., 83:150—57,1961.

17. Hutchison, D. J. Biological Activities of 6-Mercaptopurine:Effects on Streptococcus faecalis. Ann. N. Y. Acad. Sci., 60:212—19,1954.

18. . Metabolism of Resistant Mutants of Streptococcusfaecalis. I. Isolation and Characterization of the Mutants.Cancer Res., 18: 214—19, 1958.

19. Levenberg, B., Hartman, S. C., and Buchanan, J. M. Biosynthesis of the Purines, X. Further Studies in Vitro on the Metabolic Origin of Nitrogen Atoms 1 and 3 of the Purine Ring.J. Biol. Chem., 220: 379—90,1956.

20. McCollister, R. J., Gilbert, W. R., Ashton, D. M., and Wyngaarden, J. B. Pseudofeedback Inhibition of Purine Synthesisby 6-Mercaptopurine Ribonucleotide and Other PurineAnalogues. Ibid., 239: 1560—63,1964.

21. Newton, A. A., and Perry, S. V. The Incorporation of N'@into Adenine Nucleotides and Their Formation from InosineMonophosphate by Skeletal-Muscle Preparations. Biochem.J.,74:127—36,1960.

22. Paterson, A. R. P. The Formation of 6-Mercaptopurine Riboside Phosphate in Ascites Tumor Cells. Can. J. Biochem.Physiol., 37: 1011—23,1959.

23. . The Development of Resistance to 6-Mercaptopurinein a Subline of the Ehrlich Ascites Carcinoma. Ibid., $8:1117—27,1960.

24. Saffram, M. Nucleotide Synthesis by Tissue Extracts. In: S.P. Colowick and N. 0. Kaplan (eds.), Methods in Enzymology,Vol. II, pp. 501—4,New York: Academic Press, Inc., 1955.

25. Salser, J. S., and Balis, M. E. Conversion of Inosinic Acid(IMP) in Normal and Tumor Tissues. Proc. Am. Assoc. Cancer Res., 3: 256, 1961.

26. Salser, J. S., Hutchison, D. J., and Balis, M. E. Studies onthe Mechanism of Action of 6-Mercaptopurine in Cell-freePreparations. J. Biol. Chem., 235: 429—32, 1960.

27. Schulman, M. P., and Buchanan, J. M. Biosynthesis of thePurines. II. Metabolism of 4-amino-5-imidazolecarboxamidein Pigeon Liver. J. Biol. Chem., 196: 513—26, 1952.

28. Silberman, H. R., and Wyngaarden, J. B. 6-Mercaptopurineas Substrate and Inhibitor of Xanthine Oxidase. Biochim.Biophys. Acta, 47: 178-180, 1961.

29. Ultmann, J. E., and Feigelson, P. The Effects of 8-Azaguanineand 6-Mercaptopurine on Purine Catabolism in the Rat. Cancer Res., 18: 1319—23,1958.

on April 8, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1965;25:539-543. Cancer Res   Josephine See Salser and M. Earl Balis  EffectsThe Mechanism of Action of 6-Mercaptopurine: I. Biochemical

  Updated version

  http://cancerres.aacrjournals.org/content/25/4_Part_1/539

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/25/4_Part_1/539To request permission to re-use all or part of this article, use this link

on April 8, 2021. © 1965 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from