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

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The Mechanism of Action of 6-Mercaptopurine1'2 I. Biochemical Effects 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 (3—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 ‘@C(AICA),from Southern Research Institute; glycine-1- osine monophosphate ; XMP, xanthosine monophosphate ; NAD, ‘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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. 540 Cancer Research Vol.25,May 1965 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. 37.5°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 (—25°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 4°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 12—15%. was removed by centnifugation and washed in the manner 6-MP incorporation in vivo.—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 4°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 8—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.—Cell-free extracts Dowex-1 (bromide form, XlO, 200—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 “45―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 4°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.
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