Biochemical Aspects Ofmercaptopurine Inhibition and Resistance*

Home , Adenylosuccinate, Inosine, Inosinic acid, Nucleotide, Phosphoribosylamine, Purine

Biochemical Aspects of Mercaptopurine Inhibition and Resistance*

R. W. BROCKMAN

(Kettering-Meyer Laboratory, Southern Research institute, Birmingham, Alabama)

SUMMARY The observed effects of 6-mercaptopurine on purmne synthesis de novo can be at tributed to inhibition by 6-mercaptopurmne ribonucleotide of phosphoribosylamine synthesis. Azaserine-induced accumulation of@ formyiglycinamide ribonucleotide (FGAR) in human tumor cellsin culture was inhibitedby low concentrationsof 6- mercaptopurine and 6-thioguanine; such inhibition of FGAR accumulation did not occur in a mercaptopurmne-resistant subline that lacked inosinic and guanylic acid pyrophosphorylase activities, the enzyme system by which 6-mercaptopurmne ribo nucleotide and 6-thioguanylic acid are formed. Resistance to mercaptopurmne has been seen to be accompanied frequently by decrease or loss of these enzyme activities. Such loss of enzyme activity appears to be a genetically stable and heritable characteristic of resistant cells. Resistance to mercaptopurine by means other than loss of capacity for ribonucleotide formation is also evident.

INTRODUCTION of the metabolism of mercaptopurine, mechanisms Elion and Hitchings (28) described the syn by which it inhibits nucleic acid synthesis, and thesis of 6-mercaptopurmne over a decade ago. mechanisms by which biological systems may be Early reports of the inhibitory effects of mercapto come resistant to this analog. It is hoped that this purmne on microorganisms (29), experimental tu presentation may provide a framework for die mors (28), and mouse leukemias (48) were fol cussion, since aspects of the mechanisms of action lowed by extensive studies of the biological and of mercaptopurine and other purinethiols and of biochemical effects of this purine analog (56). resistance to these inhibitors are still under active investigation. Mercaptopurine is known to be metabolized by pathways of hypoxanthine metabolism and to METABOLISM OF MERCAPTOPURINE interfere with the synthesis and interconversion of Mercaptopurine closely follows the known purine nucleotides (see 1, 10, 11, 75). The analog pathways for the anabolism and catabolism of also suppresses immune responses (71). purine bases. The analog was oxidized to thiouric Considered in the present discussion are aspects acid by microorganisms and by mammalian cells

S The experimental work reported herein was supported (20, 30, 52, 56). The rate of oxidation of mer by the Cancer Chemotherapy National Service Center, Na captopurine by milk xanthine oxidase was relative tional Cancer Institute, under the National Institutes of ly slow, and the analog was a competitive inhibi Health Contract No. SA-43-ph-2483, and by grants from the tor of xanthine oxidation (72). Mercaptopurine Charles F. Kettering Foundation and the Alfred P. Sloan Foundation. was metabolized to hypoxanthine and to purine The abbreviations used are those accepted as standard by nucleotides in microorganisms (4, 20) ; desulfuriza the Journal of BiOlOgical Chemistry with certain additions or tion also occurred in miceasindicated by sulfate. exceptions. Su excretion and labeling of nutleic acid adenine AMP, GMP, IMP: adenosine-, guanosine-, and inOSine-5'- monophosphates. and guanine from mercaptopurine-8-C'4 (56). 6- ATP, GTP, UTP, CTP: adenosine-, guanosine-, uridine Methylmercaptopurmne was identified as an excre and cytidine-5'-triphosphates. tion product of the metabolism of mercaptopurine @ dUTP: deoxyuridine-5'-triphosphate. (68). Chart 1 summarizessome of the known dTTP: thymidine-5'-triphosphate. pathways of metabolism of 6-inerciaptopurine.' PRPP: 5-phosphoribosyl-1-pyrophosphate. FGAR: formylglycinamide ribonudeotide. 1 Elion et aL considered catabolism of tbiopuzine In their @@@ MP: 6-mercaptopurine. . contribution to this conference. :. 1191

Purine nucleoside phosphorylase was active for Carter (21) showed that mercaptopurine and the formation of mercaptopurine ribo- and deoxy hypoxanthine competed for a partially purified E. ribonucleosides (31, 81). Evidence that mercapto coli purine ribonucleotide pyrophosphorylase that purine inhibited inosine phosphorylase was oh was also active for guanine. Competition of mer tamed in E. coli (82) and in neoplasms grown in captopurmne and hypoxanthine for IMP pyrophos cell culture.2 Paterson (62) found that mercapto phorylase probably accounts for the observed in purine ribonucleoside was rapidly cleaved to the hibition of conversion of hypoxanthine to nucleo base by Ehrlich ascites cells, suggesting the action tides in microbial cells (13). The data summarized of purine nucleoside phosphorylase or hydrolase. in Table 1 show that in our studies the inhibition Kinase activity for inosine and mercaptopurine by mercaptopurine of nucleotide formation in S. ribonucleoside has not yet been demonstrated, al faecalis was specific for hypoxanthine and guanine though indirect evidence for its existence was oh under the experimental conditions used (see also tained in a study of the metabolism of inosine to [5]). Mercaptopurine was metabolized to the cor purine nucleotides in bacterial cells deficient in the responding ribonucleotide by mouse neoplasms capacity to metabolize hypoxanthine to IMP. and other tissues in vivo, but no evidence for the

Anobotism NP ribonucleoside @ phosphory,@@@@ pyrophosphorylas â€˜...(kinose)

@ 6-Mercaptopurins @.â€”.â€”& NP rsbonucleottde I'

1/ â€˜:ii Inosinic polyphosphotcc acid polynucleotides Co tabolism zanthins zanthine @ midase oxidase @ 6-Nercaptopurine 6-thioxonthins 6-thiouric acid L__,@6-methylmercaptopurine @ sulfate; hypoxanthin.;anabolic and catabolic products of purine metabolism Ca4ur 1.â€”Metabolism of 6-mercaptepurine in rim

SH Â®-OCH2 SH formation of the ribonucleoside di- and triphos + PPi phates was obtained in these studies (9, 60). How c:x + ever, Way et al. (85) obtained indirect spectro photometric evidence for further phosphorylation of mercaptopurine ribonucleoside-5'-phosphate by pork kidney enzyme preparations. OH OH Some radioactivity from mercaptopurine-S36 CHART 2.â€”Enzymatic synthesis of 6-mercaptopurine ribo and from mercaptopurine-8-C'4 was incorporated nucleotide by reaction of the base with 5-phosphoribosyl-1- into the nucleic acid fraction isolated from mouse pyrophosphate. tissues (7, 56), but unequivocal evidence of in corporation of the analog as the nucleotide, and From the results obtained it appeared that MP thus as an integral part of the nucleic acid chain, resistant Streptococcus faeca& (SF/MP) utilized has not yet been presented. No evidence was oh inosine for nucleotide formation equally as well as tamed for incorporation of mercaptopurmne into did the parent sensitive strain (SF/0).3 the nucleic acids of microorganisms (20) or of Mercaptopurine, like natural purine bases, was mouse leukemia cells in vivo (9) or in cell culture converted to the nucleotide by enzyme-catalyzed (16)@Hansenet at. (38, 39) observedthat radio reaction with 5-phosphoribosyl-1-pyrophosphate activity from mercaptopurmne-S35 associated with (PRPP) (53, 86) (Chart 2). ribonucleic acids was significantly decreased by

3 G. P. Wheeler and B. Bowden (personal communication, treatment of the nucleic acid fraction with hydro 1962). gen sulfide. This was so whether the tracer was ad 3B.W.Brockman,P.Chambers,C.S.Debavadi(unpub ministered in vivo and the nucleic acid subsequent lished observations). . ly isolated or whether ribonucleic acid was directly

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1963 American Association for Cancer Research. BROCKMANâ€”ASpecIS of Mercaptopurine Inhibition and Resistance 1193 exposed to mercaptopurine-S35 in vitro. Certain cursors such as formate, glycine, carbon dioxide, metal ions increased the radioactivity associated and ammonium salts (see [38]). Gots (83) showed with the nucleic acid fraction. Thus, some of the that exogenous purines, particularly adenine and mercaptopurine associated with the nucleic acid hypoxanthine, inhibited the accumulation of 5- fraction may be nonspecifically bound. amino-4-imidazolecarboxamide ribonucleoside in Anomalous nucleotides are known to be enzy a purine-requiring mutant, Escherichia coli B-96. matically incorporated into polynucleotides. For In a subsequent study with an adenine-requiring example, bromodeoxyuridine-5'-triphosphate and mutant it appeared that adenine, or adenine con dUTP can substitute for dTTP, and deoxyinosine geners, specifically prevented the formation of .5'-triphosphate can partially substitute for dGTP aminoimidazolecarboxamide derivatives (84) . Hen as substrates for DNA polymerase (6). Similarly, derson (41) observed that natural purine bases and .5-ribosyl-UTP, thymine ribonucleoside-5'-triphos nucleosides inhibited azaserine-induced formyl phate, 5-fiuorouridine-5'-triphosphate, and 5-bro TABLE 1 mouridine-.5'-triphosphate substitute completely or partially for UTP as substrates for microbial EFFECT OF MERCAPTOPURINE ON THE UTTL@ATION OF RNA polymerase; azaguanosine-5'-triphosphate EXOGENOUS PURINES FOR NUCLEOTIDE FORMA and inosine-5'-triphosphate also substitute partial TION BY Streptococcus faecaiis ly for GTP as substrates for this enzyme (44). Mercaptopurine nucleotides have not yet been re NUcLEOTTDESt IN TEE ported to be substrates for DNA or RNA poly FRACTIONCouut,/sec;SOLUBLE merase. Carbon (19) studied mercaptopurine ribo mercaptopurine-treated as % of control. nucleotide-5'-diphosphate as a substrate for the SUBSTRATERADIOACTIVE polynucleotide phosphorylase of Micrococcus lyso mercaptopurine550Level of deiktjcus. Although this enzyme is relatively non specific with respect to the base moiety of the sub lAg/miAdenine-8-C'4 @g/m1125 Mg/mI65.5 strate nucleotide and catalyzes polyribonucleotide formation from a number of purine or pyrimidine Hypoxanthine-8-C'4 8 17 38 ribonucleoside-5'-diphosphates (36), including hy Guanine-8-C'4 6 poxanthine and 8-azaguanine, mercaptopurine Inosine-8-C'4 93 ribonucleoside-5'-diphosphate was not active as a Xanthine-8-C'498 88103 8495 117 substrate. Instead, this analog inhibited the polymerization of ADP but had little effect on the S C. S. Debavadi and R. W. Brockman, unpublished obser. vations. polymerization of UDP or CDP by polynucleotide Exponentially growing cultures (65 ml.) of S. faccalis were phosphorylase (19). It is perhaps significant that treated with MP at the concentrations indicated; 15 mm. later the radioactive substrates indicated (5 Mc.) were added. 6-azauridine-5'-diphosphate, which has not been After incubation with substrate for 15 min. cells were sepa found to be incorporated into nucleic acids in vivo, rated from the medium by centrifugation, soluble extracts was likewise not a substrate for polynucleotide were prepared, and aliquots of the soluble fraction were analyzed by two-dimensional chromatography.radioautog phosphorylase (see [77]). This is not to say that raphy (see [171 for a detailed description of these procedures). polynucleotide phosphorylase is the enzyme in t Nucleotides separated include AMP, ADP, AlT, iMP, volved in incorporation of the analog into RNA GMP, GTP, and DPN. in vivo but rather to point out that this enzyme, which is less specific than RNA or DNA poly glycinamide ribonucleotide (FGAR) accumulation merases, apparently does not accept nucleotides of in Ehrlich ascites cells. It was not possible to de azauracil or mercaptopurine as a substrate. Clear fine precisely the active form of the purine in ly, the more direct approach to this question would hibitor or the inhibitory site in these studies with be an examination of appropriate mercaptopurine intact bacterial and mammalian cells. Wyngaar nucleotides as substrates for RNA and DNA den and Ashton (88), using a partially purified polymerases. enzyme system from pigeon liver, showed that adenine nucleotides, guanine nucleotides, and IMP INHIBITION OF BIOCHEMICAL inhibited phosphoribosylpyrophosphate amido PATHWAYS transferase, which catalyzes the following reaction; It is appropriate to consider first what is now PRPP + glutamineâ€”â€˜phosphoribosylamine termed feedback inhibition or end-product inhihi tion. A number of workers recognized that un + glutamate+ PP1. labeled purines prevented or depressed the incor Purine bases and ribonucleosides were inactive as poration into the purine structure of labeled pre inhibitors of the enzyme reaction, which is the

earliest irreversible reaction specifically concerned and other purinethiols that could be converted to with purine synthesis (Chart 3). This end-product nucleotides also inhibited FGAR accumulation in inhibition of phosphoribosylamine synthesis by azaserine-treated mouse neoplasms. Thus, certain purine nucleotides is analogous to the inhibition of purine analogs mimicked the natural purines in carbamylaspartate synthesis by pyrimidine nu inhibition of the de novo pathway of purine syn cleotides described by Pardee and his associates thesis in microorganisms and in mammalian cells. (32, 89).@ Simpson et (ii. (73) observed that mercaptopurine The observations that natural purines affected inhibited the incorporation of formate-C'4 into an early step in purine synthesis de novo was ex purine nucleotides and nucleic acids in Ehrlich tended to purine analogs by Gots and Gollub (35), ascites cells in vivo under conditions that did not affect the 4-amino-5-imidazolecarboxamide-C'4 PRPP + Glutamine labeling of these same fractions. These results, in agreement with those cited above, indicate that Purine nucleotides ______the de novo block occurs prior to the formation of and analogs Phosphoribosylamifle IMP. McCollister, Gilbert, and Wyngaarden (54) have now shown that MP ribonucleotide, 6-thio Glycinamide ribotide guanylic acid, and 8-azaguanylic acid inhibited phosphoribosylamine synthesis in enzyme prepa 1@ rations from avian liver; the corresponding free FG AR bases and nucleosides were inactive as inhibitors.

,__ Glutamine The enzyme-MP ribonucleotide dissociation con Axaserine stant (K1 = 5.3 x 1O@ M) was reported to be at least as low as that observed for any of the normal FGAmR ribonucleoside monophosphates.5 The inhibition of I phosphoribosylamine synthesis by mercaptopurine ribonucleotide appears to account for the often oh AIC ribotide served inhibition of purine synthesis de novo by mercaptopurine. 1@ Studies with microbial and mammalian sys I NP tems indicated that mercaptopurine also inter fered with purine interconversions known to take Nercapto purine place at the nucleotide level (see [1, 10, 11, 75]). @ ribonucleotide Salser et at. (67) demonstrated that mercapto purine ribonucleotide inhibited the conversion of SAMP XMP IMP to adenylosuccinicacidby enzymeprepara tions from S. faeca1is; levels of mercaptopurine / nucleotide equimolar with IMP gave approxi AMP GMP mately 50 per cent inhibition of this reaction. CHART 3.â€”Abbreviated pathway of purine synthesis de Hampton (see [37]) observed inhibition of the novo indicating known sites of action of mercaptopurine ribo cleavage of adenylosuccinic acid to AMP by mer nucleotide. Abbreviations: FRPP, 5-phosphoribosyl-i-pyro captopurine ribonucleotide.6 Similarly, conversion phosphate; FGAR, formyiglycinamide ribonucleotide; FGAmR, formylglycinamidineribonucleotide;AIC, 5(4)-ami 5 Caskey and Wyngaarden (22) ecently observed that ATP no-4(5).imidazolecarboxamide; SAMP, adenylosuccinic acid; was not inhibitory to phosphoribosylamine synthesis in XMP, xanthylic acid. partially purified avian liver preparations, whereas AMP and ADP were inhibitory. The method of isolation and purification who observed that mercaptopurine and 6-thio of the enzyme may be critical for the demonstration of feed guanine were particularly effective as inhibitors back inhibition, since Hartman (40) reported no inhibition of phosphoribosylamine synthesis by natural purine nucleotides of the accumulation of 5-amino-4-imidazolecar with a 1,000-fold purified enzyme from chicken liver. boxamide ribonucleoside by 1?. coli B-96. Sar C The chemically synthesized D-isomer of the analog of torelli and LePage (69) observed that thioguanine adenylosuccinic acid, in which a sulfur atom replaces the 6- inhibited the azaserine-induced accumulation of amino group, inhibited cleavage of adenylosuccinic acid to FGAR in Ehrlich ascitescells (see Chart 3). Le AMP (37) and of 5-N-succinylamino4-imidazolecarboxamide ribonucleotide to AIC ribonudeotide (55). This analog was Page and Jones (50) showed that mercaptopurine it.self cleaved to mercaptopurine rihonucleotide by adenylo 4For a retent discussionof such regulation of biosynthetic succinase, but enzymatic synthesis of this analog was not ob pathways see Moyed and Umbarger (58). served.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1963 American Association for Cancer Research. BRociu@&Nâ€”Aspects of Mercaptopurine Inhibition and Resistance 1195 of IMP to xanthylic acid was inhibited by mer loss of IMP pyrophosphorylase activity. This was captopurine ribonucleotide in a pigeon liver en observed in biological systems as diverse as micro zyme preparation (67). Mercaptopurine inhibition organisms and human neoplasms in cell culture of AMP formation was observed in L1Q1O leu (Table 3). GMP pyrophosphorylase activity was kemia cells in vitro (2.5). However, results of other frequently lost simultaneously with loss of IMP studies with tumor cells in ,,ivo indicated that mer pyrophosphorylase activity, whereas AMP pyro captopurine inhibition of AMP formation was in phosphorylase activity was retained by mercapto significant and that inhibition of GMP synthesis punine-resistant cells (9, 14, 15). Studies with men was slight under the experimental conditions used captopunine-resistant cells showed that loss of in these studies (73). Comparisons of the relative IMP pyrophosphorylase activity was accompanied effectiveness of mercaptopurine ribonucleotide as by loss of capacity to form mercaptopunine nibo an inhibitor of the enzymatic synthesis of AMP nucleotide. Table 4 presents specific data on and of xanthylic acid have not been reported for nucleotide formation from punines in mercapto mammalian cells but would be of interest in view purine-sensitive and -resistant human epidermoid of the apparently slight effect of mercaptopurine carcinoma cells in culture (15). Supernatant en on these pathways in some tumor cells in vivo. zyme fractions from sensitive cells actively cata Atkinson et al. (2, 3) observed that the enzy matic reaction of ATP with nicotinamide mono TABLE 2 nucleotide was inhibited by synthetic preparations ENZYMATIC REACTIONS INHIBiTED BY 6-MERCAPTO containing mercaptopurine ribonucleoside-5 â€˜-tn PIJRINE RIBONUCLEOTIDE* phosphate. Enzymatic formation of a mercapto punine-containing analog of DPN was also oh 1. PRPP+glutamine+H2Oâ€”@5-phosphoribosylamine+ served. It is possible that the formation of such a glutamate+PP@ DPN analog, as postulated by Kaplan et at. (46), 2. IMP+L-aspartate+GTP-@adenylosuccinate+GDP+P might be a factor in mechanisms of mercapto punine inhibition ; however, the formation of such S. AdenylosuccinateÂ±AMP+fumarate an analog in vivo has not been reported. The known 4. iMP+DPN@+H@O-*xanthylic acid+DPNH+H@ sites of inhibition by mercaptopurmne nucleotide are summarized in Table 2. 5. Nicotinamide mononucleotide+ATPDPN+PP1 Mercaptopunine and other analogs are known * 6-Mercaptopurine ribonucleoside-5'-monophosphate was to affect immune responses including antibody inhibitory in reactions 1-4; 6-mercaptopurine ribonucleoside. production, allergic reactions, and homograft reac 5'-triphospbate was reported to inhibit reaction 5 (see text). tions (see [71]). Mechanisms by which mercapto purine might alter these processes are not known, lyzed the formation of AMP, GMP, IMP, and but interference with protein synthesis is one mercaptopurine nibonucletotide. MP-resistant cells possibility. Mercaptopunine was reported to in had AMP pyrophosphorylase activity but lacked hibit induced enzyme synthesis (49) and to inhibit IMP-GMP pyrophosphorylase activity and failed leucine incorporation into protein (59) in mam to form mercaptopurine nibonucleotide. malian cells. However, Roy et al. (66) found no in Microorganisms both sensitive or resistant to hibition by MP nibonucleoside-5'-tniphosphate of mercaptopurine possess xanthylic acid pyrophos the enzymatic incorporation of leucine into micro phorylase activity (14) ; but such activity appears somal protein in vitro. Indirect effects on protein to be low in many mammalian cells (9, 15). From synthesis might be mediated through interference a study of purine metabolism and pyrophosphor with the formation of a specific RNA or, conceiv ylase activities of S. faeca1i@ mutants that were ably, by incorporation of the analog into an RNA resistant to purine analogs, it was concluded that fraction with subsequent interference with the at least three distinct purine ribonucleotide pyro formation or function of RNA template for pro phosphorylase activities exist: AMP, IMP-GMP, tein synthesis. Such effects have not yet been and xanthylic acid pyrophosphorylases (14). demonstrated, however, and mechanisms by Strains of S. faecalis resistant to mercaptopurine which punine analogs exert specific effects on im were found to be resistant to 8-azaguanine and mune response remain unknown. vice versa; sensitivity to analogs of adenine and xanthine was retained in mercaptopunine- and MECHANISMS OF RESISTANCE azaguanine-resistant mutants. Cells resistant to Resistance to mercaptopurmne is frequently ac 8-azaxanthine exhibited a specific loss of capacity companied by decrease or loss of capacity to form to metabolize xanthine and azaxanthine to nucleo mercaptopunine nibonucleotide and decrease or tides and were found to have lost xanthylic acid

TABLE 3 CoRRELATIONOF RESISTANCETO MERCAPTOPURINEANDLOSSOF CAPACITY OF CELLs To FoRM MERCAPTOPURINE RIBONUCLEOTIDE*

.. . or iou of mercapto Mercaptopurine-reiistant Decrease or loss of ribonucleotide ...... purine ribonucleotide formation cell imes. pyrophosphoryiaae activityDecrease .. ... vitroI.Microorganisms: in intact cells in vivo or in

(17)Saitnomelj,aStreptococcusfaecalisBrockman et al. (14, 18) et at. Salser et al. (67)Brockman

(45)Ii.Mouse typhimwiumKallÃ© and Gots

neoplasms in rico: L1210 leukemia a at. (9, 12, 18)Brockman el at. (9, 12) 62)m.MammalianEhrlich ascitesBrockman Paterson (61,

cells in culture: L cells and Aronow (84) Fibroblasts (AMK-Q) and Ove (51) P888(16)D98/AH@ leukemiatLieberman Brockman et al. (16),Davidson, Brockman et al. Ct al. (27)Tomizawa

(79)Iv.Human et al. (79) et a!. Adenocarcinoma 755Szybalski Brockman and KelleyllSZybalski

neoplasms in culture: H.Ep. #2* et at. (15) at al. (15) KB#Brockman Brockman and KeUy@@Brockman

a Reference is given here to complete papers except in those cases where the work has not been published in full aS @et(see [101). t P888 cellsresistant to azaguanine were cross-resistantto mercaptopurine (65). @ Human bone marrow cells resistant to 8-azahypoxanthine and cross-resistant to mercaptopurine. Â§Humanepidermoidcarcinomacells. #Humanneoplasmofepithelialorigin. II Unpublished observation.

TABLE 4 pyrophosphorylase activity; all other purine CoNvrniszoN OF B@s@sTORIBONucLE0TIDEsBYPYRO nucleotide pyrophosphorylase activities were re PHOSPRORYLASES FROM 6-MERCAPTOPURINE-SENSITIYE tamed. s. faecalis strains partially resistant to AND -RESISTANT HUMAN EPIDERMOID CARCINOMA 2-fluoroadenine exhibited a specific decrease in (H.Ep. #2) IN CELL CULTURE5 AMP pyrophosphorylase activity. These observa tions and similar findings in mammalian cells have RIBONVCLEOTIDK FORMED PER aided in understanding some of the observed HOURH Mo.PROTEININ1 inhibition and cross-resistance patterns of punine RIBONUCLEOTIDE analogs in microorganisms and in experimental roxumElAMOLES #5 H.Ep. @ .E p. @f neoplasms (43). MP-resistant @.y@js@nt (sensitive)H.Ep. Kalle and Gots (45) obtained mercaptopunine MPtAdenylic with MPtIi without resistant mutants of Salmonella typhimurium that acid exhibited loss of pyrophosphorylase activity for Guanylicacid 383 4 1 inosinic acid 889 < 1 <1 conversion of hypoxanthine and mercaptopurine Mercaptopurine ribo. to nucleotides with retention of GMP pyrophos nucleotide414 766429 <1359 <1 phorylase activity. Similarly, an azaguanine-re sistant mutant showed loss of GMP pyrophos (see [15]). phorylase activity with retention of IMP pyro t Resistant cell line was maintained on mercaptopurine. phosphorylase activity. Attempts to fractionate @ Resistant cell line was grown in the absence of MP for 60 generations. IMP and GMP pyrophosphorylaseactivities in

extracts from the parent organism were, however, resistant line; Aronowt made similar observations. unsuccessful. The possibility that these two en Mercaptopurine was discussed earlier as an in zyme activities might be attributable to isozymes7 hibitor of the conversion of IMP to adenylosuc was considered by these authors. cinic acid and to xanthylic acid and also as an The marked decreases or losses of IMP-GMP inhibitor of the enzymatic synthesis of phosphori pyrophosphorylase activities in mercaptopurine bosylamine. With azaserine used to block the resistant cells (Table 3) and the loss of capacity of formation of formylglycinamidine nibonucleotide, such cells to form mercaptopunine nibonucleotide thereby producing accumulations of FGAR (50, support the hypothesis that a nucleotide is the in 69; see Chart 2), we have made a study of the hibitory form of the analog. Efforts to inhibit men effect of puninethiols on FGAR accumulation in captopurine-resistant L1210 (57) or H.Ep. #2 mercaptopurine-sensitive and -resistant human

TABLE 5 EFFECTS OF PURINETHIOLS ON AZASERINE-INDUCED FORMYLGLYCINAMIDE R1B0- NUCLEOTIDE (FGAR) ACCUMULATION IN SENSITIVE AND RESISTANT HUMAN EPIDERMOID CnLLS IN CULTURE5

ACCUMULATION Di THE SOLUBLE (JLO/ML)FGAR-C'4 FRACTION (% or CONTROLWITHOUT INHIBITORCoNcxlira@tTIoN PURINETHIOLS)fAzaserinePurinethiolH.Ep.

#f/MPAzaserine #5/SH.Ep.

,605 counts/min counts/min

â€œ +6-Mercaptopurine 10 .05 87 10 0.1 36 10 0.5 8 119 10 5.0 <1 77

a' +6-Thioguanine 10 0.1 54 110 10 0.5 3 106 10 2.5 <1 10 5.0 118

â€œ +9-Cyclohexyl-6- mercaptopurine10 100 1 .085 10647,840 126

@ S Chumley, F. Hays, and R. W. Brockman (unpublished observations). t Rapidly growing cultures (approximately 3X107 cells/iÂ® ml) were treated with azaserine fol. lowed 30 min. later by purine analogs; 30 min. later the formate-C'4 (25 soc.) was added. After 2 hours' incubation with formats-C'4 cells were harvested by centrifugation; cell counts were made, and samples of the soluble extracts were prepared for analysis by two-dimensional chromatography radioautography as described previously (see [9, 16]). FGAR-C14 accumulation in 80-@zI. samples of the soluble extracts from azaserine-treated cells is expressed as counts/mm. Effects of purine analogs on FGAR accumulation are expressed as per cent of azaserine-treated control.

cells (47) with mercaptopunine nibonucleoside-5'- epidermoid carcinoma cells in culture. The mercap phosphate were unsuccessful. In the case of H.Ep. topurine-resistant H.Ep. #2 cells used, H.Ep. #2/ #2 cells this may be due to the presence of nucleo MP, lacked IMP-GMP pyrophosphorylase activ tidase activity in serum protein used in cell culture ity and thus failed to form mercaptopurine ribo medium as well as such activity by H.Ep. #2 cells nucleotide (see Table 4) (15) ; the resistant cells themselves.2 Tomizawa and Aronow (84) noted also failed to form 8-azaguanylic acid and 6-thio that IMP but not hypoxanthine or inosine sup guanylic acid. The results summarized in Table 5 ported growth of L cells in culture under condi show that both mercaptopurine and thioguanine tions such that an exogenous source of punines was were potent inhibitors of FGAR accumulation in required for growth. Tomizawa (83) reported that sensitive cells (H.Ep. p2/S). Low concentrations MP ribonucleotide inhibited this mercaptopurine (0.5 @@g/mi)of mercaptopurmne or thioguanine in hibited growth of H.Ep. #2/S cells. Concentrations 7 Enzymes that have the same origin and consist of similar but distinguishable proteins having the same biochemical of mercaptopunine and thioguanine lOX the levels action have been termed isozymes (see [87]). 8 L. Aronow (personal communication, 1962).

that were highly effective in H.Ep. @2/Scells were mented with thymine (42). Relative activities and without effect on FGAR accumulation in H.Ep. equilibria of inosine phosphorylase and of an mo #2/MP cellsand did not inhibitgrowthof the re sine kinase9 are factors to be considered. Thus, sistant cells. These observations are in agreement rapid breakdown of the ribonucleoside to the base with those of LePage and Jones (50), showing that and weak kinase activity might account for re mercaptopunine and thioguanine inhibited FGAR sistance of SF/MP to mercaptopunine ribonucleo accumulation in neoplastic cells. These results also side (Chart 4). provide indirect evidence that the inhibition of Alterations of pyrophosphorylase activities ac FGAR accumulation requires conversion of the companying resistance to mercaptopunine appear purine analog to the nucleotide, an observation in to be of genetic origin. H.Ep. #2/MI' cells grown in agreement with the more direct experimental find cell culture for many generations in the absence of ings of McCollister et al. (54) showing that nucleo mercaptopunine did not regain IMP-GMP pyro tides of mercaptopurine and thioguanine were the phosphorylase activity (15) ; similar results were active inhibitors of phosphonibosylamine syn obtained with L1210 mouse neoplasms in tiivo (9). thesis. It is of interest that 9-cyclohexyl-6-mercap Sirotnak et at. (74) have demonstrated transforma topurine, a potent inhibitor of growth of H.Ep. tion of Diplococc-us pneumoniae to azaguanine re #2/S and H.Ep. #2/MP cells in culture (47), was sistance by means of DNA from azaguanine without effect on FGAR accumulation at the con resistant cells. Recently, Szybalski et at. (80) iso centration used, again indicating that nucleotide lated a subline of D98 human bone marrow cells in formation was requisite for inhibition (see also culture that was resistant to 8-azahypoxanthine [501). These results also indicate that the 9-alkyl (D98/AH) and cross-resistant to mercaptopurine mercaptopurine inhibited growth by a mechanism and to other hypoxanthine and guanine analogs. different from that of mercaptopurine. D98/AH exhibited loss of IMP-GMP pyrosphos

Phosphorylase Kinase @ Base + ribose-l-phosphate ______Nucleoside Nucleotide AT P + + Phosphate ADP

CHART 4.â€”Metabolism of base to nucleoside and nucleotide

Observations that mercaptopunine-resistant mi phorylase activities (79). By means of DNA from croorganisms and neoplasms were cross-resistant the parent sensitive D98 cell line it was possible to to mercaptopurine nibonucleoside (62, 76; see also increase significantly the number of cells that [43])suggesteda lackofcapacityforphosphoryla could utilize hypoxanthine for growth under condi tion of the nucleoside. Paterson (62) found that tions of a requirement for an exogenous purine mercaptopurine nibonucleoside was rapidly broken (78). This result was produced specificallyby down to the free base and that mercaptopurmne DNA from D98 cells sensitive to azahypoxanthine ribonucleotide was not formed in significant and appears to be an example of transformation in amounts from the base or the nucleoside by intact mammalian cells. An interesting aspect of this ex mercaptopurine-resistant Ehrlich ascites cells. We periment, for the present discussion, is that the have observed, however, that mercaptopurine D98/AH cells treated with DNA from D98/S cells resistant S. faecalis lacking in IMP pyrophos and then exposed to medium containing ameth phorylase activity, and thus unable to utilize hy optenin, thymidine, and hypoxanthine (78) gave poxanthine-8-C'4 for purmne nucleotide formation, rise to a population of cells that possessed IMP did carry out conversion of inosine-8-C'4 to punine GMP pyrophosphorylase activity.'0 This re nucleotides.5 sult strengthens the hypothesis that the observed The lack of mercaptopurine inhibition of the loss of enzyme activity was a consequence of gene metabolism of inosine to punine nucleotides in mutation. SF/0 under conditions in which the metabolism Mechanisms of resistance to mercaptopurine of hypoxanthine was strongly inhibited (Table 1) and to other punine analogs by means other than also suggests direct conversion of inosine to IMP. loss of IMP-GMP pyrophosphorylase are evident Such a pathway would appear to be quantitatively 9 The existence of such a kinase can be inferred from studies limited, however, since a resistant S. faecalis line with intact growing cells. (SF/MP) was unable to grow on inosine as a sole Jo R. W. Brockman, P. Stutts, W. Szybalski, and E. H. source of purines in folic acid-free medium supple Szybalska (unpublished observation).

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1963 American Association for Cancer Research. BRocx@Nâ€”Aspects of Mercaptopurine Inhibition and Resistance 1199 from the observations of Sartorelli et at. (70), Non-sensitive Transplantable Mouse Tumors. Cancer Paterson (63, 64), and Bieber et al. (8). It was Res., 21:228â€”31,1961. 8. BIEBER,S.,and Poss@tLns,R.The Uptake of Purines and postulated that mercaptopurine was excluded from Formate into the DNA of 6-MP-sensitive and Resistant entry into resistant Ehrlich ascites cells on the Strains of Ca-7&5. Proc. Am. Assoc. Cancer Beg., 3:304, basis of observations that intact resistant cells 1962. failed to form mercaptopurine ribonucleotide even 9. BROCKMAN,R.W. A Mechanism of Resistance to 6-Mer though the enzymatic capacity for nucleotide captopurine: Metabolism of Hypoxanthine and 6-Mer captopurine by Sensitive and Resistant Neoplasms. Can formation could be demonstrated in the resistant cer lIes., 20:643â€”58, 1960. cells (63) . However, no differences in permeability 10. . Mechanisms of Resistance to Anticancer Agents. of sensitive and resistant L1210 cells to mercapto In: A. HADDOWand S. WELNHOUSE(eds.), Advances in purine were found (24). Davidson (26), in an Cancer Res., 7:129â€”284, 1963. 11. Baocxiu.tr@i,R. W., and ANDERSON,E.P. Biochemistry of examination of the IMP pyrophosphorylase activ Cancer. Ann. Rev. Biochem. (in press). ity of human leukemia cells from cases clinically 12. BROCK@&AN,R.W.; BsxNurr, L. L., JR.; SispsoN, M. S. resistant to MP, has so far found only one ex WaaON, A. R.; THOMSON, J. R.; and Sxxppna, H. E. A ample of a significant decrease in enzyme capacity Mechanism of Resistance to 8-Azaguanine. II. Studies for mercaptopurine ribonucleotide formation ; in with Experimental Neoplasms. Cancer Res., 19:856-69, 1959. the authors' laboratory no clear examples of de 13. BROCKMAN,R.W.; BnxNsrrr, L. L., Sn.; and SKIPPER, creased IMP pyrophosphorylase activity accom H. E. A Comparison of Purine Metabolism in Strains of panying clinical resistance to mercaptopurine have Streptococcusfaecaha Susceptible and Resistant to 6-Mer been observed. It is possible that the degree of re captopurine Inhibition. Proc. Am. Assoc. Cancer lIes., 2: 191â€”92,1957. sistance achieved clinically rarely approaches that 14. BROCKMAN,R. W.; DEBAVADI, C. S.; &rrrrrs, P.; and reached in experimental systems. Increased degra HUTCHISON, D. J. Purine Ribonucleotide Pyrophos dation of thioguanine by resistant neoplasms was phorylases and Resistance to Purine Analogues in Strepto observed (70) ; this mechanism of resistance is also coccusfaecali.. J. Biol. Chem., 236:1471â€”79, 1961. possible for mercaptopurine. Increased degrada 15. BROORMAN,R.W.; KELLEr,G. @.;S'i'irrrs,P.; and Cors LAND, V. BiochemicalAspects of Resistance to 6-Mercapto tion of azaguanine was not observed to occur in purine in Human Epidermoid Carcinoma Cells in Culture. resistance to this analog in Ll210 leukemia in vivo Nature, 191:469â€”71, 1961. (12).Decreasedsensitivityof certain reactionsto 16. BROCKMAN,R. W.; ROO6A,R. A.; LAW, L. W.; and Srurrs, MP ribonucleotide, such as interconversions of P. Purine Ribonucleotide Pyrophosphorylaae Activity and Resistance to Purine Analogs in P388 Murine Lymphocytic purine nucleotides (8), may be another mecha Leukemia. J. Cell. Comp. Physiol., 60:68-84, 1902. nism of resistance. 17. BROCEMAN,R. W.; SPARKS, M. C.; Hu'rcaieoN, D. J.; and SKIPPER, H. E. A Mechanism of Resistance to 8-Asa REFERENCES guanine. I. Microbiological Studies on the Metabolism of 1. ANDERSON,E. P., and LAW, L. W. Biochemistry of Can Purines and 8-Azapurines.Cancer Res., 19:177â€”88,1959. cer. Ann. Rev. Biochem., 29:577â€”608, 1960. 18. BROCKMAN, R. W.; SPARKS, C.; SIMpeoN, M. 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R. W. Brockman

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