The Mechanism of Action of 6-Mercaptopurine1'2 I. Biochemical Effects

Home , Adenylosuccinate, AICA ribonucleotide, Inosinic acid, Xanthosine monophosphate

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 Cornell University Medical College, New York, New York)

SUMMARY The tumor Sarcoma 180 (5-180) was compared to host liver in an effort to evaluate the inhibitory action of 6-MP.3 The concentration of endogenous adenine nucleotides in S-180 was about 40 % of that found in liver, that of guanine nucleotides was about the same in both tissues, while that of inosinate was much lower in tumor. The liver of normal mice and 5-180-bearing mice exhibited the same capacity to convert IMP to AMP. A slightly greater conversion of 6-MP to 6-thio IMP in 5-180 was noted but it was not by itself of sufficient magnitude to explain the anti-tumor specificity of the compound. An 5-180 sensitive to 6-MP and one resistant to it each showed about 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-thio IMP ; the inhibition, though low, was greaten in the tumor. The actions of 6-thio IMP, reported by others in mammalian systems, are analogous to those previously found in bacterial and avian systems; i.e., the conversion of IMP to AMP and XMP is inhibited. It is suggested that the basis for the anti-tumor specificity may lie in differences in the enzymatic capacity of tumors and host tissues to carry out the con versions blocked by 6-thio IMP.

Growth and isotope incorporation studies with bacterial tumor cells (1) have confirmed the observation that 6-thio systems (3â€”5,12, 13, 17, 18) have led to the hypothesis IMP is actually an effective competitive inhibitor of the that the active form of 6-MP is its ribonucleotide (thio conversion to xanthylate, the intermediate in the forma inosinate, 6-thio IMP), which inhibits the further conver tion of guanylate. sion by inosinate to other purine ribonucleotides (4, 5). Since the conversion of inosinate to other punine nibo This inhibition by thioinosinate of the formation of ade nucleotides occurs in both normal and tumor tissues, the nylate and guanylate has been demonstrated in cell-free selective susceptibility of tumors such as Sarcoma 180 preparations from Streptococcus faecalis and pigeon liver, (5-180) to the action of 6-MP must be considered in terms respectively (26). In L1210, studies on the incorporation of the total purine synthesis and interconversion mecha of labeled hypoxanthine into nucleic acid punines are con nisms. This paper presents some aspects of purine me sistent with the inhibition at the level of hypoxanthine, tabolism in 5-180 and host liver pertinent to nucleotide but only the conversion to adenylate seems to be seriously synthesis and conversion. The synthesis in vivo of 6-thio affected by 6-MP (10). More recent studies with partially IMP, the synthesis of purines de novo in in vitro systems, purified inosine-5'-phosphate-NAD-oxidoreductase (IMP and the inhibition of adenylate formation by 6-thio IMP dehydrogenase) from bacteria (15) and Ehrlich ascites in S-180 and liver have been studied.

1 This investigation was supported in part by funds from the National Cancer Institute, NIH, USPHS (CA-03190-08), and MATERIALS AND METHODS (5-K3-CA-16, 673-02). IMP@8@HC was obtained from Schwarz BioResearch, 2 A preliminary report has been presented (25). Inc. ; 6-MP-8-'4C and 4-amino-5-imidazole-carboxamide4- 3 The abbreviations used are : 6-MP, 6-mercaptopurine; 6-thio IMP, 6-thioinosinate; IMP, inosine monophosphate; AMP, aden â€˜@C(AICA),from Southern Research Institute; glycine-1- osine monophosphate ; XMP, xanthosine monophosphate ; NAD, â€˜4C,fromTracerlab. ATP (disodium salt) ; IMP (sodium nicotinamide adenine dinucleotide; AICA, 4-amino.5-imidazole salt); NAD (DPN); fructose-i ,6-diphosphate (sodium carboxamide; ATP, adenosine triphosphate; DPN, diphospho salt) ; a-ketoglutarate; L-glutamine ; nibose-5-phosphate pynidine nucleotide; TCA, trichloroacetic acid; GMP, guanosine monophosphate. (sodium salt); and tetrahydnofolic acid, were obtained Receivedfor publication September10,1964;revisedDecember from Sigma Chemical Company. 6-Thioinosinate (6-thio 23, 1964. IMP) was kindly supplied by Dr. A. Hampton. 539

HA/ICR Swiss mice (approximately 20 gm each) with With labeled AlGA (specific activity = 4.11 X 10@cpm/ a 5- or a 7-day-old subcutaneous implant of 5-180 were smole) as precursor, the incubation medium was that de kindly supplied by Dr. H. Schwartz and Dr. C. Reilly of scribed by Schulman and Buchanan (27). In all the incu Sloan-Kettering Institute. Mice with 11-day-old sub bations, there was 250 mg wet weight of tissue/mi. cutaneous implants of a resistant variant of 5-180 were After a 15-min equilibration period, the labeled pre supplied by Dr. D. Clarke. Necrotic tumors were dis cursor was added and the incubation carried out for 2 hr at carded in all instances. 37.5Â°Cin a Dubnoff metabolic shaker. The reaction was Acetone powders of the tissues were prepared by stopped by immersion of the vessels in an acetone-Dry Ice homogenizing the liver or tumor with 10 volumes of cold bath. Carrier IMP was added to give a final concentra acetone (â€”25Â°C)in a Waring Blendor, followed by filter tion of 5 @moIes/ml. All subsequent steps were carried ing and rehomogenizing the resuspended cake in fresh cold out at 4Â°Cunless otherwise specified. acetone. The last 2 steps were repeated twice. The yield Sufficient cold 60 % TCA was then added to give a final of acetone powder from different batches of liver was about TCA concentration of 10 %. The precipitated protein 25 % of the wet weight while that from 5-180 was 12â€”15%. was removed by centnifugation and washed in the manner 6-MP incorporation in vivo.â€”Mice with 5-day-old im described above. After ether extraction, the supernatant plants of S-l80 were given i.v. (caudal vein) injections of solutions were lyophilized to dryness. Since the incuba 6-MP-8-'4C (50 mg/kg body weight, specific activity = tions were carried out in duplicate, one set of samples was 2.3 X 10@cpm/,@mo1e). The animals were sacrificed at dissolved in a minimal volume of 0.01 N HC1 and the nu 2, 6, and 24 hr after a single injection and at 48 hr after 3 cleotides were isolated as barium salts (24). To minimize successive daily injections of the labeled compound. The the adsorption of traces of labeled precursor, unlabeled liver and tumor were removed and chilled in chopped ice. AICA or glycine was added during the precipitation and All subsequent steps, unless otherwise specified, were 2 reprecipitations. The radioactivity was determined carried out at 4Â°C. The chilled tissues were homogenized and was shown by paper chromatography (i-butyric briefly in a Potter-Elvehjem homogenizer with 1 volume acid-NH3) to be associated with inosinate. of cold distilled water. Sufficient cold 20 % TCA was The duplicate set of samples was dissolved in water, ad added to give a final concentration of 10 % TCA and ho justed to pH 11 with NH4OH and put on Dowex-1 (chlo mogenization was continued for another 3 min. The pre ride form, X10, 200-400 mesh) columns. These were cipitated protein was removed by centrifugation, resus washed with at least 100 volumes of water and 0.003 N HC1 pended and rehomogenized 3 more times with cold 5% until the eluates were free of radioactivity. Inosinate TCA. The combined supernatant solutions were adjusted was subsequently eiuted with 0.005 N HC1, lyophilized to to 0.01 N with respect to HBr and extracted 8â€”10times dryness, and finally dissolved in water for radioactivity de with ether to remove the TCA. termination. The identity was further confirmed spec The components of this acid-soluble fraction (adjusted trally and by paper chromatography. to PH 9 with concentrated NH4OH) were separated on Conversion of inosinate to adenylate.â€”Cell-free extracts Dowex-1 (bromide form, XlO, 200â€”400mesh) colunms were prepared by homogenizing acetone powder or frozen into 3 fractions : (a) feed and wash water, containing tissue in a medium containing 0.16 M KC1 and 0.05 M Tnis nucleosides, nicotinamide, amino acids, and flavins, (b) buffer, pH 7.4, (21), (4 gm wet weight or the equivalent 0.006 N HBr, containing free purines, kNIP, and IMP, and amount of acetone powder/b ml) in a Virtis â€œ45â€•ho (c) 0.18 N HBr, containing 6-thio I@1P with traces of GMP mogenizer for 3 min. To partially fractionate the active and ATP. These fractions were concentrated to dryness system(s) involved in this conversion, aliquots of the and taken up in a minimal measured volume of water. homogenate were centrifuged at 15,000 X g for 30 min and The radioactivities of aliquots of the fractions were de at 34,800 X g for 60 min5. All the operations were carried termined on infinitely thin films on aluminum planchets out at 4Â°C. in a Geiger-Muller internal flow counter. Paper chroma Each 10 ml of incubation mixture contained 2 gm wet tography of the 3rd fraction in various solvent systems weight or the equivalent amount of acetone powder; 2.5 (16) showed that all the radioactivity was associated with mmoles ATP ; 2.5 mmoles L-aspartate ; 5 mmoles fructose 6-thio IMP. The identity was further confirmed spec 1 ,6-diphosphate; 0.5 mmoies MgCl2; 5 mmoles sodium trally. phosphate buffer, pH 7.4; 2.5 mmoles a-ketoglutarate; 1 Purine synthesis de novo in cell-free preparations.â€” mmole NAD; and â€œxâ€•@tmolesIMP-8-'@C (specific ac Frozen 7-day-old implants of S-180 (sensitive) and 11-day tivity = 6.31 X 10@cpm/,@mole). old implants of S-180 (resistant) as well as their come In the comparative studies of acetone powder and sponding host livers were homogenized (3 min in a Virtis frozen tissue preparations of livers of tumor-bearing and â€œ45â€•)ina pH 7.4 phosphate medium (0.035 M sodium nontumor-bearing animals, the concentration of IMP was phosphate, 0.13 M KC1, 0.04 @iKHCO3, 0.01 M MgC12, and 1.25 j@moles/10 ml incubation. After a 4-hr incubation 0.015 M L-glutamme4). When glycine-1-'4C (specific ac at 20Â°C6in a Dubnoff metabolic shaker, the reaction was tivity = 1.24 X 10@ cpm/@@mole) was used as precursor, stopped and the incubation mixture deproteinized after the incubation medium was essentially that of Levenberg et at. (19) but with an additional 6 @molesATP/ml and a S The comparison of these fractions will be part of another slightly higher glycine concentration of 8.66 j@moles/ml. communication. 0 Incubations at this temperature have been shown to promote 4 The presence of glutamine stabilizes the purine-synthesizing accumulation of succinoadenylate (21), a compound of interest enzymes. (J. M. Buchanan, personal communication.) in studies of nucleotide interconversions.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. SALSER AND BALlSâ€”Action of 6-Mercaptopurine. I 541 ether extraction. The clear solution was neutralized and, TABLE 2 if necessary, stored at â€”20Â°C. The components of such INOSINATE SYNTHESIS BY LIVER AND solutions were separated on Dowex-1 (chloride form, X10, S-180 IN VITROâ€• 200-400 mesh) columns by a modification of the method of Deutsch and Nilsson (11). The identities of the various WEIGHTSensitiveResistantLiverS-180LiverS-ISOGlycine-1-'4CSYNTHESISM@OLE/GMWET fractions were checked spectrally and by paper chroma PaEculsoaIN0sINAm tography and the radioactivities were determined. This method of fractionation was used to determine the endoge nous nucleotide concentration of the 2 tissues. In the inhibition studies, the reaction mixtures contained 5.16 @molesIMP/lO ml. The levels of 6-thio IMP studied A.â€• were 1.94 and 4.85 @imoles/10ml. After a 2-hr incubation B.C 98 137 114 194 at 37.5Â°C, the reaction was stopped and deproteinization AIC-4-'4C carried out as described in the foregoing section. The A.b 28 35 26 34 solutions were lyophilized to dryness, taken up in freshly B.C95 29131 37104 28184 36 prepared 1 N HC1 and hydrolyzed for 21 hr at 100Â°C. The punines were isolated by chromatography on Dowex-50 a See â€œMaterials and Methods.â€• (hydrogen form, AGW, X8, 200-400 mesh) columns by b Precipitated as barium nucleotides and isolated by paper elution with 0.15 N HC1 for xanthine, 6-MP and hypox chromatography. anthine and 1.5 N HC1 for guanine and adenine. C Isolated by chromatography on Dowex-1 (chloride form).

RESULTS AND DISCUSSION TABLE 3 CONVERSION OF INOSINATE TO ADENYLATE The enzymatic conversion of 6-MP to its active form, IN LIVER PREPARATIONSâ€• 6-thio IMP, by 5-180 and host liver was studied. Table 1 summarizes the data obtained from a typical ex derivatives@' weight/hrLiver T issueAdenine rn@ixnole/gmwet periment in vivo. The 6-thio IMP levels of S-180 were consistently about 10â€”15%higher than that of host liver, from nontumor-bearing mice with the highest levels being found in animals sacrificed 2 Frozen tissue hr after injection. Catabolism of 6-thio IMP proceeded Acetone powder 39c at a steady rate, but a detectable amount was still present after 24 and 48 hr. Liver from S-180-bearing mice The synthesis de novo of purines by whole homogenates Frozentissue 42 of the 2 tissues under consideration is given in Table 2. Acetone powder41 40c A 6-MP-resistant variant of 5-180, and the corresponding host liver have also been included here. The liver of both a Whole homogenates were incubated as described in â€œMa terials and Methods.â€• groups of animals showed essentially the same degree of b See footnote 8. incorporation of the 2 precursors. The tumor, whether C Calculated on the basis that the acetone powder preparation sensitive or resistant, had greater capacity to synthesize is 25% of the wet weight of the liver (see â€œMaterials and purines than the respective liver, i.e., 1.4-fold (sensitive) Methodsâ€•). and 1.7-fold (resistant) with glycine as precursor, and 1.3- fold (sensitive) and 1.3-fold (resistant) with AICA as growing tumor. Consequently, it is not surprising that precursor. These differences do not seem great compared the concentration of free nibonucleotides is much lower in to the greater requirement for purines in the more rapidly the tumor than in the liver (see Table 4). Such differ ences, though small, may well be reflected in the relative TABLE 1 sensitivity of the 2 tissues to punine antagonists, such as 6-TmoINoSINAvsi SYNTHESIS IN LIVER AND 6-MP. 5-180 IN Vivoâ€• Preliminary studies with unlabeled IMP have shown

WaIGETLiverS-1801 6-THIOINOSINATZ/GM WaT that the net formation of AMP, GMP, or their intermedi Tna (hr)@'@LUOLZ ates (based on spectral data), was too low in cell-free ex tracts of both liver and tumor to be determined accurately. To facilitate the determination of small changes of this Injection nature, all subsequent studies were carried out with â€œC@ 2 X 10' X 10' labeled IMP. 6 0.69X 10' 0.82X 10' In all these comparative studies, the liver of the tumor 24 0.24 X 10' 0.28X 10@ bearing animals was used as the normal tissue since there is an ambiguity as to the exact tissue or cell of origin of 3 Injections 482.27 0.21X 10'2.47 0.24X 10' 5-180. Furthermore, the conversion to adenylate is the same in the liver of nontumor and 5180-bearing animals, a See â€œMaterials and Methods.â€• i.e., about 40 mMmoles/gm wet weight of tissue/hr. Table b Time interval between injection (or the last of 3 successive 3 summarizes the data obtained with frozen tissues and daily injections) of 6-mercaptopurine and sacrifice of the animal. acetone-powder preparations of these 2 tissues. The latter

TABLE 4 as a base line for such determinations, no great significance CONCENTRATION OF ENDOGENOUS NUCLEOTIDESâ€• should be attached to these small differences in the amount of synthesis between these 2 tissues in the presence of 6- NUCLEOTIDESbMHOLE/ WEIGHTAdenylateGuanylateInosinateLiver GM WET thio IMP. TISSUEPURINE CONCLUSIONS Biochemical studies on the mechanism of action of 6-MP in diverse systems in vitro have led to a number of sug @ Whole homogenate gested sites of inhibition by the active form, 6-thio IMP.9 Whole homogenate blankc 8.25 1.02 0.55 The common denominator is that they all involve facets Supernatant 15,000 X g blank' 7.82 0.85 0.52 of purine and punine nucleotide metabolism which are normally found in living systems. Yet, there is consider S-180 able variation in biologic response to 6-MP from species to Whole homogenate 2.90 0.76 0' species and even from tissue to tissue. It is conceivable Whole homogenate blankc 3.05 0.80 Od Supernatant 15,000 X g blankc7.63 2.740.90 0.680.48 0â€• that these variations, which are reflected by different de grees of sensitivity or resistance, may be attributed at a See â€œMaterials and Methods.â€• least in part to qualitative and/or quantitative differences b See footnote 8. in the enzymatic makeup of the systems. Consequently, C See footnote 7. Cell-free extracts from 4 gm wet weight of comparative studies of a sensitive tumor such as 5-180 tissue made up to 20 ml with buffer and adjusted with cold 60% and the inherently more resistant host tissues have to be trichloroacetic acid to give a final concentration of 7%. considered in terms of possible enzymatic differences of d The values were too low to be detected by the assay method this nature in punine synthesis and nucleotide intercon used. versions. TABLE 5 In certain bacteria (4, 7, 26) and some experimental neoplasms (8, 23), sensitivity to 6-MP can be attributed INHIBITION OF INOSINATE TO ADENYLATE CONVERSION IN CELL-FREE PREPARATIoNâ€• to a greater ability (compared to a resistant mutant) to convert this compound to 6-thio IMP. However, the DERWATIVES@' Mj@MOLE/GM @MOLE/1O ML INCUBATIONADENINE data presented here show that the capacity for this con WEIGHT/HRInosine, WET version in S-180 is not appreciably greater (only 10â€”15%)

monophosphate6-ThioinosinateLiverS-ISO5.160198 than that in host liver. In vitro experiments'0 show essen tially the same thing. These experimental values are in @ the same range as those observed in mice bearing a re 995.161.94188 201 @95 sistant variant of 5-180 (22). The higher values at 2 hr, @ the shortest experimental time period, followed by a steady 825.164.85 186 @78 decrease with time suggest that most of the synthesis prob @ @177 ably occurred within 2 hr. The 6-thio IMP persists for a 179 71@68 considerable period of time in both liver and 5-180. This rather small difference in synthesis of 6-thio IMP can a See â€œMaterials and Methods.â€• b Samples had been hydrolyzed to free purines prior to column hardly explain by itself the great difference observed in chromatography. the biologic response of the 2 tissues to 6-MP (9). An inhibition in the pathway de novo, probably at the values were adjusted to a wet weight basis (see â€œi\'iaterials phosphoribosylamidotransferase step, has recently been and Methodsâ€•). On the basis of these results, either suggested as the primary site of action of 6-MP in experi preparation can be used for studies of this nature. mental tumors in vivo (6). However, in biochemical Table 4 summarizes the endogenous purine nucleotide studies of this enzyme system, adenylate has been reported concentration of liver and S-180. These values were ob to be 5 % as effective as 6-thio IMP and the inhibitions by tamed from acid-soluble extracts of whole homogenates as the 2 compounds are additive (20). The endogenous well as the blanks for some of the incubations with Un adenylate concentration of 5-180 is over 1000 times greater dialyzed preparations.7 The concentration of adenine than the 6-thio IMP level at 2 hr after injection of an LDSO nucleotides8 in S-180 is about 40 % of that in the liver, dose of 6-MP. Thus, in this tumor, the contribution by while that of guanine nucleotides8 is but slightly less in the 6-thio IMP formed would be negligible and could 5-180. hardly by itself explain the observed anti-tumor specificity The inhibition by 6-thio II\1P of the conversion of found only with 6-MP. Although 6-thio IMP may exert inosinate to adenylate was examined in 15,000 X g super feed-back inhibition, it does not appear likely that the bio natants of liver and S-180. The results are given in Table 5 6-Thioinosinate has been reported to inhibit IMP dehydro 5. The inhibition in the liver at the 2 levels studied was genase (15, 26), succinoadenylic acid synthetase (2, 14,26), adenylo about half that observed in the tumor. Since the IMP succinase (2, 14), and glutamine PP-ribose-P amidotransferase concentrations used are in excess of those occurring in the (20). 10 Experiments carried out with cell-free homogenates of liver tissues, and because of errors inherent in using wet weight and S-180 essentially confirm the data obtained in vivo, i.e., the 7 These were tissue blanks used in assaying studies of the con incorporation by S-180 was consistently about 10â€”12%higher than version of inosinate to adenylate (to be published). by liver. (Salser, J. S., Wahrman, S., and Balis, M. E., unpub 5 This represents the sum of the mono-, di-, and triphosphates. lished data).

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. SALSER AND BALlsâ€”Action of 6-Mercaptopurine. I 543 logic result can be primarily attributed to that biochemical D. J. Purine Ribonucleotide Pyrophosphorylases and Resist action alone. Furthermore, the effects of 6-MP on in ance to Purine Analogues in Streptococcus faecalis. J. Biol. Chem.,256:1471â€”79,1961. corporation of formate and pre-formed purines (6) can be 8. Brockman, R. W., Kelley, G. G., Stutts, P., and Copeland, V. interpreted in terms of the â€œsparingâ€•action of 6-MP on Biochemical Aspects of Resistance to 6-Mercaptopurine in formate utilization (4) and the inhibition of xanthine oxi Human Epidermoid Carcinoma Cells in Culture. Nature, dase by 6-MP (28, 29). The latter would result in less 191: 46â€”71,1961. 9. Clarke, D. A., Philips, F. S., Sternberg, S. S., Hitchings, G. degradation of purines per se. Consequently, there would W., Stock, C. C., and Elion, G. B. 6-Mercaptopurine: Effects be less need for purine synthesis de novo and the utilization in Mouse Sarcoma 180 and in Normal Animals. Cancer Res., of tracer doses of purines would not be decreased. 13: 593â€”604,1953. Studies with bacteria have indicated that 6-MP exerts 10. Davidson, J. D. Studies on the Action of 6-Mercaptopurine in its inhibitory action as the ribonucleotide, 6-thio I1\1P, Sensitive and Resistant L1210 Leukemia in Vitro. Ibid., 20: 225â€”32,1960. which blocks the further conversion of IMP to AMP and 11. Deutsch, A., and Nilsson, R. Ion Exchange Chromatography GMP (4, 5). The inhibition of these nucleotide intercon of Inosine Phosphates. Acta Chem. Scand., 7: 1288â€”92, 1953. versions has subsequently been demonstrated to occur at 12. Elion, G. B., Hitchings, G. H., and Vanderwerif, H. Antago reasonable 6-thio IMP concentrations in bacterial (15, 26) nists of Nucleic Acid Derivatives. VI. Purines. J. Biol. Chem., 192: 508â€”18,1951. and avian (26) systems in vitro. The present study, as 13. Elion, G. B., Singer, S., Hitchings, G. H., Balis, M. E., and well as those of Atkinson et at. (1), indicate that these in Brown, G. B. Effects of Purine Antagonists on a Diamino hibitions do occur in mammalian systemsâ€• with 6-thio purine-Resistant strain of Lactobacillus casei. Ibid., 202: 647â€” IMi@ levels comparable to those which exist in tissues in 54, 1953. vivo. The lower endogenous level of IMP and the slightly 14. Hampton, A. Studies of the Action of Adenylosuccinase with 6-Thio Analogues of Adenylosuccinic Acid. Ibid., 237: 529â€”35, higher synthesis of 6-thio IMP in 5-180 (compared to host 1962. liver) could indicate some specificity. However, it does 15. . Reactions of Ribonucleotide Derivatives of Purine not seem likely that these differences alone can explain the Analogues at the Catalytic Site of Inosine 5'-Phosphate I)e degree of specificity observed in vivo with [email protected] The hydrogenase. Ibid., 258: 3068â€”74, 1963. 16. Hampton, A., and Maguire, H. Nucleotides. I. Synthesis of reported inhibitions in vitro by 6-thio Ii\'IP of the conver 6-Chloro-, 6-Mercapto-, and 2-Amino-6-mercapto-9-@-D- sion of IMP to succino-Ai\'IP and XMP suggest that the ribofuranosylpurine 5'-phosphate. J. Am. Chem. Soc., 83: basis of the selective susceptibility of tissues to 6-MP 150â€”57,1961. might be expected, in part, to lie in differences in the ability 17. Hutchison, D. J. Biological Activities of 6-Mercaptopurine: of tissues to carry out such interconversions. Attempts Effects on Streptococcus faecalis. Ann. N. Y. Acad. Sci., 60: 212â€”19,1954. to verify this suggestion have been carried out and will be 18. . Metabolism of Resistant Mutants of Streptococcus reported. faecalis. I. Isolation and Characterization of the Mutants. Cancer Res., 18: 214â€”19, 1958. ACKNOWLEDGMENTS 19. Levenberg, B., Hartman, S. C., and Buchanan, J. M. Biosyn thesis of the Purines, X. Further Studies in Vitro on the Met The authors wish to thank Dr. George B. Brown for his interest abolic Origin of Nitrogen Atoms 1 and 3 of the Purine Ring. in this work and his many helpful suggestions. J. Biol. Chem., 220: 379â€”90,1956. 20. McCollister, R. J., Gilbert, W. R., Ashton, D. M., and Wyn REFERENCES gaarden, J. B. Pseudofeedback Inhibition of Purine Synthesis 1. Atkinson, M. R., Morton, R. K., and Murray, A. W. Inhibition by 6-Mercaptopurine Ribonucleotide and Other Purine of Inosine 5'Phosphate Dehydrogenase from Ehrlich Ascites Analogues. Ibid., 239: 1560â€”63,1964. Tumour Cells by 6-Thioinosine 5'-Phosphate. Biochem. J., 21. Newton, A. A., and Perry, S. V. The Incorporation of N'@ 89: 167â€”72,1963. into Adenine Nucleotides and Their Formation from Inosine 2. . Inhibition of Adenylosuccinate Synthetase and Ade Monophosphate by Skeletal-Muscle Preparations. Biochem. nylosuccinate Lyase from Ehrlich Ascites-Tumour Cells by J.,74:127â€”36,1960. 6-Thioinosine 5'-Phosphate. Ibid., 92: 398â€”404,1964. 22. Paterson, A. R. P. The Formation of 6-Mercaptopurine Ribo 3. Balis, M. E., Hylin, V., Coultas, M. K., and Hutchison, D. J. side Phosphate in Ascites Tumor Cells. Can. J. Biochem. Metabolism of Resistant Mutants of Streptococcus faecalis. II. Physiol., 37: 1011â€”23,1959. Incorporation of Exogenous Purines. Cancer Res., 18: 220â€”25, 23. . The Development of Resistance to 6-Mercaptopurine 1958. in a Subline of the Ehrlich Ascites Carcinoma. Ibid., $8: 4. . Metabolism of Resistant Mutants of Streptococcus 1117â€”27,1960. faecalis. III. The Action of 6-Mercaptopurine. Ibid., 18: 440â€”44, 24. Saffram, M. Nucleotide Synthesis by Tissue Extracts. 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Metabolism of 4-amino-5-imidazolecarboxamide in Pigeon Liver. J. Biol. Chem., 196: 513â€”26, 1952. 11 Inhibition of succinoadenylate synthetase (noncompeti 28. Silberman, H. R., and Wyngaarden, J. B. 6-Mercaptopurine tively) and adenylosuccinase (competitively) from Ehrlich ascites as Substrate and Inhibitor of Xanthine Oxidase. Biochim. tumor cells by 6-thio IMP has recently been reported (2). Biophys. Acta, 47: 178-180, 1961. 12 It should be emphasized that the only normal tissue studied 29. Ultmann, J. E., and Feigelson, P. The Effects of 8-Azaguanine was the liver. It does not necessarily follow that all nonmalig and 6-Mercaptopurine on Purine Catabolism in the Rat. Can nant tissues behave like the liver. cer Res., 18: 1319â€”23,1958.

Josephine See Salser and M. Earl Balis

Cancer Res 1965;25:539-543.

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