A Mechanism of Resistance to 6-Mercaptopurine: Metabo Lism of Hypoxanthine and 6-Mercaptopurine by Sensitive and Resistant Neoplasms*

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A Mechanism of Resistance to 6-Mercaptopurine: Metabo lism of Hypoxanthine and 6-Mercaptopurine by Sensitive and Resistant Neoplasms*

R. W. BROCKMAN WITHTHETECHNICALASSISTANCEOFMARGUERITES.SIMPSON,JOANM. DAVIS,ANDPATRICIASTUTTS (Kettering-Meyer Laboratory,^ Southern Research Institute, Birmingham, Alabama)

SUMMARY Leukemia L1210 ascites tumor cells sensitive to inhibition by 6-mercaptopurine were observed to metabolize hypoxanthine-8-C14 and 6-mercaptopurine-S36 to ribonucleotide derivatives in vivo. Hypoxanthine-8-CH extensively labeled adenylic and guanylic acids of L1210 nucleic acids. Soluble enzyme preparations from L1210 ascites cells catalyzed the reactions of adenine, guanine, 8-azaguanine, hypoxanthine, and 6- mercaptopurine with 5-phosphoribosyl-l-pyrophosphate (PRPP) to yield 5'-ribonucleotide derivatives. Lines of L1210 resistant to 6-mercaptopurine failed to form significant amounts of inosinic acid or 6-mercaptopurine ribonucleotide in vivo.The nucleic acids of the drug- resistant leukemic cells exposed in vivo to hypoxanthine-8-C14 were not extensively la beled. Soluble enzyme preparations from the L1210 ascites cells resistant to purine antagonists did catalyze the reaction of adenine with PRPP to yield adenylic acid but did not yield significant amounts of the 5'-ribonucleotides of guanine, 8-azaguanine, hypoxanthine, or 6-mercaptopurine. These findings support the interrelated hypotheses that (a) the metabolism of 6- mercaptopurine to a ribonucleotide derivative constitutes a lethal synthesis and that (b) an effective mechanism of resistance to 6-mercaptopurine in L1210 mouse leukemia is a decrease in the enzymic capacity of the drug-resistant leukemic cells to form 6-mercap topurine ribonucleotide.

Neoplasms sensitive to inhibition by 8-azagua- revealed a significant decrease in the capacity nine were observed to metabolize guanine and 8- of the 8-azaguanine-resistant neoplasms to form azaguanine to the corresponding ribonucleotide ribonucleotide derivatives of guanine, 8-azagua- derivatives in vivo; resistance to 8-azaguanine was nine, hypoxanthine and 6-mercaptopurine (14). Re- found to be accompanied by decreased capacity suits of studies with bacteria that are sensitive to form 8-azaguanine ribonucleotides (8, 13). A or resistant to 8-azaguanine and 6-mercaptopurine study of the capacity of enzyme preparations from support the hypothesis that resistance to these 8-azaguanine-sensitive and -resistant neoplasms purine analogs is a consequence of the decreased to catalyze the reaction of purines and purine capacity of the resistant cells to synthesize the analogs with 5-phosphoribosyl-l-pyrophosphate fraudulent ribonucleotide derivatives (11, 12, 14). * This study was supported by the Cancer Chemotherapy Thej present â€¢. report . extends / \ these. â€¢investigations . â€¢ , ., National Service Center, National Cancer Institute, under OD drug-resistance to (a) an investigation of the National Institutes of Health Contract Number SA-43-ph- m mm metabolism of hypoxanthine and 6-mercap- 1741, and by grants from the Charles F. Kettering Foundation topurine in leukemia L1210 ascites tumor cells and the Alfred P. Sloan Foundation. and in 6-mercaptopurine, 6-thioguanine, and 8- t Affiliated with the Sloan-Kettering Institute for Cancer azaguanine-resistant lines of L1210;1 (b) & study Research, New York. 1Lines of Dr. L. W. Law, National Cancer Institute. Received for publication December 18, 1959. L1210/MP = 6-mercaptopurine-resistant; L1210/TG = 6- 643

of the in vivo conversion of 6-mercaptopurine-S36 and normal tissues was obtained by eluting the to the ribonucleotide derivative in L5178-Y ascites 6-mercaptopurine-S35 ribonucleotide from the pa cells,1 in Sarcoma 180 ascites cells, and in L4946 per with water and evaporating the eluates on Lymphoma, Sarcoma 180, and Adenocarcinoma planchÃ©isfor counting in gas-flow proportional 755 grown as subcutaneously implanted tumors; counters. The activity of S35was corrected for the and (c) a comparison of the capacity of enzyme radioactive decay which took place over the period preparations from sensitive and resistant L1210 of time required for accomplishing electrophoretic, ascites cells to catalyze the reaction of purines radioautographic, and quantitative analyses. and purine analogs with 5-phosphoribosyl-l-pyro- Soluble enzyme preparations were prepared phosphate. from L1210 ascites tumor cells as described pre viously (8). These preparations were assayed for MATERIALS AND METHODS their capacity to catalyze the reactions of purines Radioactive substrates used in this investiga and purine analogs with 5-phosphoribosyl-l-pyro- tion were the same as those described previously phosphate. (8). The magnesium salt of 5-phosphoribosyl-l- pyrophosphate (PRPP) was obtained from Pabst RESULTS Metabolism of hypoxanthine-8-Clt in vivo : anal Laboratories, Milwaukee, Wisconsin. The in vivo tracer experiments with hypoxan- ysis of alcohol-soluble fraction.â€”Hypoxanthine is thine-8-C14, 6-mercaptopurine-8-C14, and 6-mer extensively anabolized in vivo by the parent drug- captopurine-S35 were carried out in the manner sensitive L1210 ascites cells to yield nucleotide described in previous experiments (8). The pro derivatives of adenine, guanine and hypoxanthine cedure is briefly outlined here. including AMP, ADP, ATP, DPN, IMP, and Hypoxanthine-8-C14 or 6-mercaptopurine-8-C14 GMP (Table 1). Radioactive nucleoside-5'-di- and was injected intraperitoneally into mice bearing triphosphate derivatives of hypoxanthine and gua sensitive or resistant L1210 ascites cells. The radio nine were not conclusively identified although active substrate (10 /tc/mouse) was given in two they may have been present in small amounts. equally divided doses 1 hour apart. One hour after This result is in marked contrast with the anabo- the second injection the neoplasms and normal lism of hypoxanthine by L1210/MP, L1210/TG, tissues were removed and extracted with hot 80 and L1210/8-Aza (Table 1). These differences are per cent ethanol-water. The extracts were con clearly revealed in a comparison of the radio- centrated and lyophilized for Chromatographie, autogram patterns (Fig. 1). In the drug-resistant electrophoretic, and radioautographic analyses as lines of L1210 the only radioactive ribonucleotide previously described (8). The solid residue remain present in any detectable quantity was adenylic ing after ethanol extraction served as the starting acid, and it was present only in trace amounts. point for the isolation of nucleic acids, which An examination of the results of a similar analysis of the fate of hypoxanthine-8-C14 in non- were analyzed for radioactivity by chemical oxida tion of organic carbon to C14O2 for gas phase neoplastic tissues from animals bearing L1210 counting by a modification of the Bernstein- revealed that significantly more catabolic than Ballentine technic (57). anabolic products were present in soluble extracts The 6-mercaptopurine-S35 was similarly admin from liver and spleen (Table 2). For example, istered at a level of 40 ^c/mouse in two equally allantoin accounted for considerably more than divided doses 1 hour apart. One hour after the half of the total radioactivity found in liver and second injection the neoplasm and normal tissues spleen. A similar result was obtained in analyses were removed for analysis. The concentrated ex of the soluble extracts of liver and spleen from tracts were subjected to high-voltage paper elec- mice bearing drug-resistant L1210 ascites lines. trophoresis (20 volts/cm) in ammonium formate Pronounced predominance of anabolism over ca- buffer (pH 3.5) to isolate 6-mercaptopurine-S35 tabolism in drug-sensitive neoplasms and the in ribonucleotide (see Table 3). The radioactive ribo verse relationship in non-neoplastic tissues could nucleotide was located by exposing the electro- account for the degree of selective toxicity that phoresis strips to x-ray film. Quantitative data certain of the purine analogs have on neoplasms. on the amount of 6-mercaptopurine-S35 ribonucleo Allantoin was the major product of catabolism of hypoxanthine-8-C14 found in extracts of the tide present in sensitive or resistant neoplasms sensitive and resistant lines of L1210. There do thioguanine-resistant; L1210/8-Aza = 8-azaguanine-resistant. not appear to be significant differences in the L1210/TG and L1210/8-Aza are also resistant to 6-mercapto- amounts of radioactive uric acid and allantoin purine (89). L5178-Y is the L5178 ascites tumor cell line of Law which was passed through tissue culture by Dr. Glenn remaining in sensitive and resistant cells under Fischer, Yale University. the experimental conditions used. In in vivo ex-

periments such as these it is not possible to L1210 may well be derived from the large pools specify how much of the degradation products of radioactive nucleotides present in the sensitive were formed in the ascites cells and how much cells. might have been formed in cells of the host. Metabolism of hypoxanthine-8-Cu in vivo : label Results of the enzyme studies discussed below ing of nucleic acid purines.â€” Hypoxanthine-8-C14 suggest that soluble enzyme preparations from extensively labels the nucleic acid purines of L1210

TABLE 1 METABOLISMOFHYPOXANTHINE-S-C"BYL1210ASCITESCELLSintito

(COUNTS/SEC)CatabolicUETABOUCPRODUCTSDETECTEDINSOLUBLEEXTBACTst

ABCITESCELLS*L1210L1210/MPproductsXÂ«3.8<2.0 products^Ad2.40 NUCLEICt(MC/MOLE)764.310.3 ACID

DP31.20

L1210/TG 0 <1.0 15.1 0 0 <1.0 0 0 0 0 0 6.2 L1210/8-AzaRADIOACTIVE0U.A.3.12.53.5All.24.428.642.1Anabolic0AdR21.600AMP192.9<2.0<2.0A 0ATP8.200HxR5.100IMP44 0GMP21.200INCORPORATIONINTO5.5

* Ascites tumor cells were removed for analysis 2 hours after the initial injection of radioactive substrate. Total amount of hypoxanthine-8-C14 injected intraperitoneally was 10 /ic/mouse in two doses 1 hour apart. t Aqueous alcohol extracts were analyzed by two-dimensional chromatography-radioautography. Radioactive compounds were eluted, and activity was determined in internal gas flow proportional counters to a probable error of Â±1per cent except when radioactivity was quite low. A zero indicates that no radioactive compound could be detected by radioautography. ÃŽNucleicacid samples were oxidized to carbon dioxide, and the activity was determined in the gas phase(57);activity expressed as /ic/mole of carbon. RNA and DNA were not separated. Â§Radioactive DPN was detected in L1210 ascites cells; counts per second = 17.1. Abbreviations: Ad = adenine; AdR = adenosine; AMP, ADP, ATP = adenosine-5'-mono-t di-, and triphosphates; HxR = inosine; IMP = inosine-5'-phosphate; GMP = guanosine-5'-phosphate; Xa = xanthine; U.A. = uric acid; All. = allantoin.

these ascites cells do not possess significant capaci TABLE2 ty to degrade hypoxanthine-8-C14 (Table 3) since COMPARISONOFTHEMETABOLISMOFHYPOXAN- very little radioactive xanthine and no radioactive THINE-8-C14 in tito BY NEOPLASTIC allantoin could be detected in the enzyme incuba ANDNON-NEOPLASTICCELLS tion medium. However, degradative enzymes may have been lost or inactivated in these preparations. HADIO-ACTIVITTor It is also possible that the neoplasms have less PORATIONINTONUCLEICACIDÂ§(Â«c/mole)7645379 degradative capacity than non-neoplastic tissue TOTAL)Hypo(PER CENT OF TISSUEL1210 TIVITY*(counts/sec)399118142DISTRIBUTION (6, 50) and that much of the radioactive allantoin present in the soluble extracts of the ascites cells may have arisen by degradation of hypoxanthine- xanthine624Catabolicproductsf87461Anabolicproducisi862435INCOR 8-C14in non-neoplastic tissues, particularly in the ascitesLiver#Spleen#TOTALRADIOAC liver. Significantly more radioactive hypoxanthine was detected in drug-sensitive than in drug-resist * From an aliquot portion of soluble extract of cells (see ant cells (Fig. 1). The experiments of Davidson footnotes * and f, Table 1). (21) suggest that decreased permeability of re t Catabolic products are xanthine, uric acid, and allantoin; sistant L1210 ascites cells is probably not a the latter compound accounted for most of the radioactivity in the products of degradation. factor in such a difference. The observations of Ã•Anabolicproducts include adenine, adenosine, adenosine Sartorelli and LePage (55, 56) raise the question mono-, di- and triphosphate, inosine, inosinic acid, guanylic of whether increased degradation of the radioac acid, and, in the case of LlalO, diphosphopyridine dinucleotide. The adenine nucleotides account for 50 per cent or more tive substrate by resistant cells could account of the radioactive compounds isolated. for the absence of hypoxanthine in resistant cells. Â§Seefootnote ÃŽ,Table1. The results of the enzyme studies (Table 3) do # Extracts of liver and spleen from mice bearing drug-resist ant ascites cells (see Table 1) exhibited patterns of metabolism not suggest increased degradation by resistant generally similar to non-neoplastic tissues from mice bearing cells, but this possibility is not entirely eliminated sensitive L1210. Catabolic products accounted for an average of 80 per cent of the total radioactivity, hypoxanthine for for the reasons mentioned above. Some of the approximately 8 per cent, and anabolic products, primarily radioactive hypoxanthine present in drug-sensitive adenine nucleotides, for the remainder.

but serves as a poor precursor of the nucleic acid captopurine-8-Cli in vivo.â€”Investigations of the purines of L1210/MP, L1210/TG, and L1210/8- metabolism of 6-mercaptopurine-S35 in mamma Aza (Table 1). As would be anticipated, the extent lian cells have shown that sulfur-35 is found in of labeling of nucleic acid purines parallels the 6-methylmercaptopurine (54), 6-thiouric acid, capacity of the cell lines to form purine ribonu- and sulfate (24, 27, 41). The intermediate oxida cleotides from hypoxanthine-8-C14. tion product 6-thioxanthine, as well as 6-thiouric Samples of the nucleic acid fraction isolated acid, has been isolated from bacterial cells (16). from L1210 were further analyzed by alkaline Hypoxanthine and anabolites of hypoxanthine are hydrolysis of RNA to yield ribonucleotides. These formed from 6-mercaptopurine in bacterial (1, were then separated by high-voltage paper electro- 12, 16) and mammalian cells (24). The use of phoresis using pH 3.5 ammonium formate buffer 6-mercaptopurine-S35 simplified the quantitative (45). Radioactive ribonucleotides were detected analysis of the capacity of sensitive and resistant by exposing the electrophoresis strips to x-ray L1210 cells to form 6-mercaptopurine ribonucleo- film; approximately equal amounts of radioactive tide in vivo in that the formation of radioactive adenylic and guanylic acids were present in the hypoxanthine derivatives was eliminated. Alcohol- hydrolysate. The nucleic acids isolated from the soluble extracts of ascites cells and of normal drug-resistant ascites cells were not sufficiently tissues of the host were subjected to paper electro radioactive to detect any significant amount of phoresis in ammonium formate buffer, pH 3.5. the ribonucleotides by this technic. From a comparison of the migration of 6-mercap Metabolism of 6-mercaptopurine-S3& and 6-mer- topurine, 6-mercaptopurine ribonucleotide, and

TABLE 3 COMPARISONOFTHECAPACITYOFENZYMEPREPARATIONSFROMDRUG-SENSITIVEAND-RESISTANT ASCITESTUMORCELLSTo CATALYZETHEREACTIONOFPURINESANDPURINEANALOGSWITH 5-PHOSPHORIBOSYL-l-PYROPHOSPHATE TO YIELD RIBONUCLEOTIDES

RADIOACTIVITYASOF TOTAL*LlÂ«10t27235022432412PER CENT OF RADIOACTIVESUBSTRATEAdenine-8-C1*Guanine-8-C148-Azaguanine-2-C14Hypoxanthine-8-C146-Mercaptopurine-S35PRODUCTS REACTIONAdenineCatabolicOF ENZYME 1210/8-AÂ»3945761180324

xanthine)Anabolic(hypoxanthine, acids)GuanineUnidentifiedCatabolic(inosine, inosinic and adenylic

acid)Anabolic(xanthine, uric acid)8-Azaguanine(guanosine, guanylic

Catabolic(8-azaxanthine)Anabolic 465236lot53393526L1Z10/MP4944759IS181080200942487121L1Ã•10/TG171964349431446513903787121L76096139271 acid)HypoxanthineCatabolic(8-azaguanosine, 8-azaguanylic

acid)Anabolic(xanthine, uric acid)6-Mercaptopurine,(inosine, inosinic ribonucleosideÂ§Catabolic6-mercaptopurine acid)Anabolic(6-thiouric (6-mercaptopurine ribonucleotide)DISTRIBUTION

* Enzyme reaction mixtures were analyzed by two-dimensional chromatography (phenol-water- n-butanol-propionic acid- water), with the exception of the 6-mercaptopurine reaction mixture, which was analyzed by one-dimensional chromatography (isopropanol-water: ammonia atmosphere). Chromatograms were exposed to x-ray film, and the radioactive areas of the chromatograms were eluted with water. The eluates were evaporated on planchets and radioactivity determined quantitatively in gas-flow proportional counters. t Traces of radioactive uric acid and allanoin could be detected when adenine was the substrate for the enzyme preparation from L1210. t Small amounts of allantoin could be detected. Â§6-Mercaptopurine and 6-thioxanthine were not separated by Chromatographie analysis or by electrophoresis at pH 9.0 (see Table 4). Exposure of 6-mercaptopurine to alkaline medium (Tris buffer, chromatography solvents) resulted in artifacts, one of which was tentatively identified as a disulfide of 6-mercaptopurine.

known metabolic derivatives of 6-mercaptopurine tabolism of 6-mercaptopurine-S36 by other mouse upon electrophoresis in formate buffer (Table 4), neoplasms are summarized in Table 5. Di- and it is evident that 6-mercaptopurine-S35 ribonucleo- triphosphate derivatives of 6-mercaptopurine ribo tide can be separated from other metabolites. nucleoside were not detected by the technics used; A quantitative comparison of the formation of the formation of such derivatives is probably 6-mercaptopurine-S35 ribonucleotide by sensitive a prerequisite to any incorporation of 6-mercapto and resistant lines of L1210 ascites cells and by purine into nucleic acids. representative host tissues is presented in Chart The products of metabolism of 6-mercaptopu- 1. Results of a similar comparison of the me- rine-8-C14 in vivoby L1210 and L1210/MP ascites tumor cells, by liver, and by spleen from these TABLE 4 same animals were analyzed by descending chro CHROMATOGRAPHICANDELECTROPHORETIC matography in 70 per cent isopropanol-water (am ANALYSISOFMETABOLICDERIVATIVES monia atmosphere) on Whatman- 3MM paper OF6-MERCAPTOPURINE and by paper electrophoresis (44). The basis for

migration value*0.560.740.520.430.620.100.130.130.140.550.340.430.540.13Relative Compound6-Mercaptopurine6-Methylmercaptopurine6-Thioxanthine6-Thiouricdistance! 600 pH9.0-6-9064-6929089-7-4-2-710040064SO(Â¡a88t78102IOS9810243635561100180 S.S pH o 400

acid6-Mercaptopurine ribonu-cleoside6-Mercapto-9-$-D-ribofuran-osylpurine-5'-phosphate(6-MPRP)Â§6-MPRP 15 -USE -i en-zyme)#6-MPRP(S. faecalis enzyme)#6-MPRP(L1210 riro)||HypoxanthineXanthineUric(L1210, in LI2IO LI2IO/MP LI2IO/MP LI2IO/TG U2IO/B-AIO (LAW) (HUTCHISON) CHART1.â€”Conversionof 6-mercaptopurine-S35 to the ribo acidInosineInosinic nucleotide in vivo by ascites tumor cells that are sensitive acidSodium (L1210) or resistant (L1210/MP) to inhibition by 6-mercapto sulfateRF purine. The 6-thioguanine-(L1210/TG)- and 8-azaguanine- (L1210/8-Aza)-resistant neoplasms are also resistant to 6- mercaptopurine. The 6-mercaptopurine-S36 ribonucleotide was * Whatman SMM papers were developed overnight by descending chromatography with 70 per cent isopropanol- separated from other metabolites present in soluble extracts of water in an ammonia atmosphere (45). ascites cells or normal tissues by paper electrophoresis in am monium formate buffer. See Table 4 and footnotes | and Â§of t Compounds were subjected to electrophoresis on What man 3MM paper in 0.05 M ammonium formate buffer (pH Table 5 for details of preparation and analysis of samples. 3.5) at a potential gradient of approximately 20 volts/cm for 2 hours or in 0.05 M sodium tetraborate (pH 9) at a potential the analysis by these technics of the 6-mercapto gradient of approximately 15 volts/cm for 1.5 hours (45). purine derivatives obtained in aqueous alcohol- The distance in centimeters that IMP migrated from the origin is defined as 100 units; migration of other compounds is ex soluble extracts of cells is given in Table 4 (45). pressed relative to IMI' migration. A negative sign indicates In the present experiments the radioactive areas migration toward the cathode. ÃŽSyntheticsample obtained from Dr. T. L. Loo, National were eluted from the chromatograms with water; Cancer Institute, Bethesda, Md. Electrophoretic mobility was the eluates were then lyophilized and subjected determined on a freshly prepared solution of 6-thiouric acid to further analysis by electrophoretic, Chromato dissolved in dilute NaOH. An aged solution yielded a product with a relative migration value of 120. graphie, and radioautographic technics. The fol Â§Synthesized by Dr. Howard J. Schaeffer and Miss Jean- lowing metabolic derivatives of 6-mercaptopurine- ette Thomas, Southern Research Institute. 8-C14were separated and identified by these tech # Biosynthesized by reaction of 6-mercaptopurine-S35 with nics: hypoxanthine, 6-mercaptopurine (not sepa 5-phosphoribosyl-l-pyrophosphate in the presence of enzyme preparations from StreptococcusJaecalis orLl210 ascites tumor rated from 6-thioxanthine or inosine that might cells. be present), 6-thiouric acid, 6-mercaptopurine || Alcohol-soluble extract of L1210 ascites tumor cells from mice injected with 6-mercaptopurine-S35. The action of bovine ribonucleoside, 6-mercaptopurine ribonucleotide, intestinal phosphatase (Armour and Co.) on 6-MPRP yielded and lesser amounts of as yet unidentified radio 6-mercaptopurine-S35 ribonucleoside, which was identified by co-chromatography with a synthetic sample of 6-MP ribo active derivatives. Some of these derivatives ap nucleoside. pear to be artifacts formed from 6-mercaptopurine

and derivatives in the alkaline media used for itself might be incorporated into nucleic acid. chromalography and electrophoresis. Instability The results recorded in Table 6 show that there of 6-mercaptopurine and derivatives prevented was relatively little incorporation of the purine use of the two-dimensional solvent system (phenol- nucleus of 6-mercaptopurine-8-C14 into nucleic water: n-butanol-propionic acid-water) used suc acids as such or incorporation as adenylic and cessfully in analyzing metabolites of hypoxanthine. guanylic acids under the conditions of these ex It was noted that radioactive 6-mercaptopurine periments. No positive evidence for incorporation and derivatives tended to streak upon chromatog- of 6-mercaptopurine as such into nucleic acid was obtained in these experiments. TABLE 5 Purine ribonudeotide pyrophosphorylase activity METABOLISMOF6-MERCApropuniNE-S36TO of soluble enzyme preparations from sensitive and THE RlBONUCLEOTIDE DERIVATIVE BY resistant leukemia L1210 ascites tumor cells?â€”In MOUSENEOPLASMSinVivo* a previous study the capacity of enzyme prepara-

TABLE 6 to purine-S36 LABELINGOFTHENUCLEICACIDS NeoplumttSarcoma therapy with ribonu- OF i.i-.'1'iASCITESCELLSAND 6-mercaptopurineSensitivecleotideÂ§ (counta/sec)1019 OFNORMALMOUSETISSUESBY CAHBON-14FROM6-MERCAPTO- 180 (ascites) PURINE-8-C14* L5178-Y(ascites)Adenocarcinoma SensitiveSensitive 306268786824

755 Activity of (tumor)L4946Sarcoma 180 Borderline Asrites cells nucleic acid t sensitivityResistant or normal tissue (MC/mole of (lymphoma) carbon) Ll210/A-Meth (ascites)#ResponseResistant6-Mercapto- L1210 12.9 Liver 2.20 * Chart 1 summarizes the metabolism of 6-mercaptopurine- Spleen 5.01 SÂ»by leukemia L1210. L1210/MP 2.15 t Neoplasms were removed for analysis 2 hours after the Liver 1.95 initial injection of radioactive substrate. The total amount of 6-mercaptopurine-S36 injected intraperitoneally was 40 Spleen 4.44 /ic/mouse in two doses 1 hour apart. * Ascites cells and tissues were t Lyophilized aqueous alcohol extracts of ascites cells were redissolved in 1 ml. of water per 4 X 10" cells extracted; 0.1-ml. removed for analysis 2 hours after samples were analyzed electrophoretically in ammonium for the initial injection of radioactive substrate. The total amount of 6- mate buffer (pH 3.5). Lyophilized aqueous alcohol extracts mercaptopurine-8-C14 injected in of solid tumors were redissolved in 1 ml. water/gm tissue extracted; 0.1-ml. samples were similarly analyzed electro traperitoneally was 10 Â¿ic/mouse phoretically (see Table 4). in two equally divided doses 1 hour Â§Paper electrophoresis strips were exposed to x-ray film apart. to locate radioactive compounds; water eluates from papers t See footnote t, Table 1. were evaporated on planchÃ©isforcounting in gas-flow propor tional counters. tions from L1210 and Ll210/8-Aza lymphomatous #The amethopterin-resistant L1210 ascites line carried tumors to catalyze the formation of ribonudeotide in this laboratory does not respond to 6-mercaptopurine ther derivatives of purines and purine analogs was apy (61.) compared (14). Enzyme preparations from L1210 raphy in most of the one-dimensional solvents tumor were found to be significantly more active examined. The systems used in this study, though than those of Ll210/8-Aza tumor in catalyzing not ideal, permitted qualitative identification of the reactions of guanine, 8-azaguanine, hypoxan most of the radioactive derivatives. thine, and 6-mercaptopurine with 5-phosphoribo- The labeling of nucleic acid purities by C14from 1Similarly prepared enzymes from human leukemic cells 6-mercaptopurine-8-Cu.â€”The extent of labeling catalyzed the formation of ribonudeotide derivatives of natural of nucleic acids of L1210, L1210/MP, or nor purine bases and of 6-mercaptopurine, 6-thioguanine, and mal mouse tissues by 6-mercaptopurine-8-C14 was 8-azaguanine. Human leukemic cells were obtained from Dr. R. R. Ellison and Dr. J. H. Burchenal, Sloan-Kettering Insti shown to be low (Table 6) in comparison with tute for Cancer Research, New York, New York, and from Dr. the radioactivity of the nucleic acids of these S. D. Palmer, University Hospital, University of Alabama same cells and tissues from animals receiving School of Medicine, Birmingham, Alabama. Dr. J. D. David hypoxanthine-8-C14 (Table 1). Elion, Bieber, and son, National Cancer Institute, has made similar observations in independently conceived experiments (personal communica Hitchings (24) suggested that 6-mercaptopurine tion).

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1960 American Association for Cancer Research. BROCKMANâ€”ResistancetoMercaptopurine 649 syl-1-pyrophosphate (PRPP). The present study by the enzyme preparations. It can generally has now been extended to include enzyme prepara be said that the extent of degradation of substrates tions from the other resistant lines of L1210 ascites by the soluble enzyme preparations from resistant tumor cells. The data summarized in Table 3 cells was no greater than that by similar enzyme present a comparison of the capacities of enzymes preparations from sensitive cells. Exceptions to from L1210, L1210/MP, L1210/TG, and L1210/8- this generalization are that enzyme preparations Aza to catalyze reactions of adenine-8-C14, gua- from Ll210/8-Aza and L1210/TG deaminated nine-8-C14, 8-azaguanine-2-C14, hypoxanthine-8- guanine to xanthine more extensively than did C14 and 6-mercaptopurine-S35 with PRPP.3 The those from L1210; 8-azaguanine was more exten products of the enzyme reaction were separated sively deaminated by Ll210/8-Aza than by L1210. and detected by two-dimensional chromatograph- In every case unreacted purine or purine analog ic-radioautographic analysis (8), with the excep was still available in the reaction mixture when tion of the 6-mercaptopurine-S35 reaction mixture, enzyme action was terminated. which was analyzed by one-dimensional chroma- The activities of enzyme preparations from tography in 70 per cent isopropanol-water (am the spleens of animals bearing the ascites cell monia atmosphere). The results of this comparison lines were also assayed for capacity to form ribo show that enzymes from L1210 catalyze the con nucleotide derivatives. It was observed that these version of adenine to adenylic and inosinic acids. enzyme preparations catalyzed ribonucleotide for Inosine and hypoxanthine were also formed along mation; data on the activity of enzyme prepara with small amounts of xanthine. If it is assumed tions from spleen under these experimental condi that the inosine present was largely derived from tions have been presented (8). inosinic acid, then the over-all conversion of ade nine to nucleotide derivatives by the L1210 en DISCUSSION zyme preparation is seen to proceed in 50 per Results of studies on the mechanism of action cent yield under the experimental conditions used of 6-mercaptopurine in experimental neoplasms (Table 3). It is interesting that the similarly (43) led to the conclusion (58) that this drug prepared enzymes from the resistant L1210 lines (a) inhibited the labeling of nucleic acid purines formed adenylic acid in approximately 50 per by radioactive glycine and formate (33, 40) ; (b) in cent yield under the same experimental conditions hibited the incorporation of hypoxanthine into without forming appreciable amounts of inosinic nucleic acid purines; and (c) did not inhibit adenine acid or inosine. Very little hypoxanthine was incorporation into nucleic acid purines. Studies in formed from adenine by enzyme preparations from microbiological systems have shown that resistance resistant cells. In striking contrast to this result to 6-mercaptopurine is accompanied by altered with adenine as substrate, it was found that the purine metabolism (1-3, 9, 11-15, 26, 34, 35). L1210 enzyme preparation was significantly more The hypothesis that the conversion of 6-mercap active in catalyzing the reactions of guanine, 8- topurine and 8-azaguanine to ribonucleotide de azaguanine, hypoxanthine, and 6-mercaptopurine rivatives constitutes a "lethal synthesis" is sup with PRPP than were similar enzyme preparations ported by the results of studies comparing the from L1210/MP, L1210/TG, and Ll210/8-Aza. intermediary metabolism of these purine analogs Thus, only very small amounts of 6-mercapto in drug-sensitive and in drug-resistant bacterial purine ribonucleotide and 8-azaguanylic acid were and neoplastic cells (8, 9, 11-15). Evidence that produced in the incubation mixtures with en 8-azaguanylic acid could inhibit an 8-azaguanine- zymes from the drug-resistant L1210 cells. It resistant organism, thus circumventing resistance was observed that the reaction of 8-azaguanine to the base, stems from the observation that with PRPP in the presence of enzymes from 8-azaxanthine was metabolized to 8-azaguanylic L1210 yielded 8-azaguanylic acid and rather large acid by an 8-azaguanine-resistant strain of Strep amounts of 8-azaguanosine. This result is in agree tococcusfaecalis and that 8-azaxanthine inhibited ment with the results of the previous studies of growth of this organism. A strain of S. faecalis the metabolism of 8-azaguanine in vivoin bacterial resistant to inhibition by 8-azaxanthine failed and neoplastic cells (8, 12, 44). to form 8-azaguanylic acid from this analog (12). By means of the chromatographic-radioauto- From the work of Way and Parks (65), Lukens graphic technic used, it was also possible to ex and Herrington (42), and Carter (17), it is evident amine the extent of degradation of the substrates that the enzymes which catalyze the formation 3There does not appear to be an enzyme possessing catalyt of the natural purine ribonucleotides also catalyze ic activity for the reaction of xanthine with PRPP in these the formation of fraudulent purine ribonucleotides. preparations. Paterson (49) has demonstrated the formation

of 6-mercaptopurine ribonucleotide in Ehrlich as- Thus specific sites of inhibition by ribonucleotide cites tumor cells sensitive to 6-mercaptopurine derivatives of purine analogs are known. and has reported that drug-resistant cells fail In the area of pyrimidine metabolism Hand- to form the fraudulent ribonucleotide.4 Tomizawa schumacher and Pasternak (30) and Skoda and and Aronow (64), in a study of 6-mercaptopurine Sorm (63) have shown that 6-azauracil is metabo metabolism in Earle's "L" strain of mouse fibro- lized to 6-azauridylic acid in drug-sensitive bacteria blasts in tissue culture, observed the presence and in neoplasms (48); it was demonstrated that of 6-mercaptopurine ribonucleotide in the medium 6-azauridylic acid blocks the synthesis of uridylic in which sensitive cells were grown but not in acid from orotic acid. The first demonstration medium in which 6-mercaptopurine-resistant cells that resistance to a pyrimidine analog could arise were grown. Sartorelli, LePage, and Moore (55, 56) from a metabolic failure was the observation by found that Ehrlich ascites cells sensitive to inhibi Handschumacher (29) that microorganisms resist tion by 6-thioguanine formed more 6-thioguanylic ant to 6-azauracil lacked the capacity to form acid than did a drug-resistant line and that the the ribonucleoside derivative. Skoda et al. (62) sensitive cells incorporated more 6-thioguanylic have also recently demonstrated an effect of 6- acid than did the 6-thioguanine-resistant cells. azauridine-5'-diphosphate on pply nucleo tide syn In this case increased degradation of 6-thioguanine thesis in studies using bacterial enzyme systems. by the resistant cells was considered to be a Comprehensive studies of the metabolism of 5- significant factor. Remy and Smith (52) found fluorouracil in neoplasms by Heidelberger et al. that 2,6-diaminopurine ribonucleotide was formed (7, 18, 20, 31, 32) and in bacteria by Cohen et by sensitive but not by 2,6-diaminopurine-resist- al. (19) have demonstrated conclusively that 5- ant Escherichia coli. fluoro-2'-deoxyuridylic acid is an inhibitor of thy- Indirect evidence that a ribonucleotide deriva midylic acid synthesis and hence of DNA syn tive of 6-mercaptopurine is an active inhibitor thesis. Reichard, SkÃ¶ld,and Klein (51) have shown has recently been presented by Davidson (21, 22), that the development of resistance to 5-fluorouracil who showed that 6-mercaptopurine inhibited the in Ehrlich ascites tumor cells resulted in loss of conversion of inosinic acid to adenylic acid in capacity of resistant cells to metabolize uracil L1210 ascites tumor cells in vitro. Altered cell to uridine and uridylic acid. It has also been ob permeability as a factor in resistance to 6-mercap served that bacteria resistant to 5-fluorouracil topurine in L1210 has been excluded experimen lack the capacity to metabolize uracil or 5-fluoro tally (20). Salser and Balis (53) found that 6- uracil to the corresponding ribonucleotide deriva mercaptopurine ribonucleotide inhibited the con tives (10). version of inosinic acid to adenylosuccinic acid The accumulated evidence from these studies by an enzyme preparation from S. faecalisf con thus provides strong support for the following version of inosinic acid to xanthylic acid by an interelated hypotheses : enzyme preparation from pigeon liver was al 1. Purine and pyrimidine analogs can be metab so inhibited by 6-mercaptopurine ribonucleotide. olized to ribonucleotide derivatives by drug-sensi Hampton et al. (28) observed that the 6-mercapto tive cells. The formation of such fraudulent ribo- analog of adenylosuccinic acid inhibited the action nucleotides can be viewed as a lethal synthesis. of adenylosuccinase in cleavage of adenylosuccinic 2. In certain cases cells that are resistant to acid; Miller et al. (47) observed similar inhibition purine and pyrimidine analogs show decreased by this analog of the enzymic cleavage of succino- activity of enzymes catalyzing the lethal synthesis 5-amino-4-imidazolecarboxamide ribonucleotide. of fraudulent nucleotides. *Paterson has recently observed that a 6-mereaptopurine- When considered in conjunction with theories resistant Ehrlich ascites cell line was also resistant to 6-mercap on gene-enzyme relationships (4, 5, 36) and in topurine ribonueleoside, in agreement with the findings of the light of current concepts of the chemical basis Skipper et al. (59). In in vitro experiments the drug-resistant of heredity (46), these hypotheses lead to the view Ehrlich ascites cells formed approximately one-tenth as much 6-mercaptopurine ribonucleotide from 6-mercaptopurine ribo that resistance to purine analogs could arise by nueleoside as did the drug-sensitive cells; much of the ribo- gene mutations which result in loss of ribonucleo nucleoside appears to have been hydrolyzed to 6-mercapto tide pyrophosphorylase activity (Chart 2). purine (personal communication from Dr. A. R. P. Paterson, Results of studies using enzyme preparations The University of British Columbia, Vancouver). from drug-resistant microorganisms (11, 15), and 6Inhibition of the conversion of inosinic acid to adenylo of studies using bacterial and mammalian enzyme succinic acid by 6-mercaptopurine ribonucleotide in enzyme preparations (8, 17, 38, 42, 65) have shown that preparations from Escherichia coli has been observed (personal communication from Dr. Alexander Hampton, Sloan-Kettering there are several distinct ribonucleotide pyrophos- Institute for Cancer Research, New York). phorylases. From the results of studies on the

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1960 American Association for Cancer Research. BROCKMANâ€”Resistance to Mercaptopurine 651 capacity of 6-mercaptopurine- and 8-azaguanine- We are indebted to Dr. Elizabeth P. Anderson and Dr. resistant cells to form purine ribonucleotides, it J. D. Davidson, National Cancer Institute, and to Dr. Howard E. Skipper, Dr. L. L. Bennett, Jr., Dr. Glynn P. appears that a single enzyme may catalyze the Wheeler, and Dr. Frank M. Schabel, Jr., Southern Research formation of inosinic and guanylic acids or that Institute, for their valuable advice and interest in this study. We the enzymes responsible for the formation of these are also indebted to Miss Linda Simpson and Mrs. Jane Hazel- nucleotides are closely linked genetically. Detailed rig for the quantitative determinations of radioactivity, and to studies with purified enzyme preparations are re Mr. J. R. Thomson and Miss Gail Yerby for assistance in the biological aspects of these experiments. quired in order that these possibilities may be explored more fully. REFERENCES Resistance to 6-mercaptopurine, to 6-thiogua- 1. BALIS,M. E.; HYLIN,V.; COULTAS,M.K.; and HUTCHI nine, and to 8-azaguanine in L1210 did not result SON,D. J. Metabolism of Resistant Mutants of Streptococ in decreased activity of adenylic acid pyrophos- cus faeealis. II. Incorporation of Exogenous Purines. Can cer Research, 18:220-25, 1958. phorylase. It is of considerable interest in this 2. . Metabolism of Resistant Mutants of Streptococcus light that an adenine analog, 4-aminopyrazolo faeealis. III. The Action of 6-Mercaptopurine. Ibid., pp. [3,4-d] pyrimidine (APP) is inhibitory to neo- 440-44. 3. BALIS,M. E.; LEVIN,D. H.; BROWN,G. B.; EUON, G. B.; HYPOXANTHINE INOSINIC ACID NATHAN,H. C.; and HITCHINGS,G. H. The Effects of GUANINE GUANYLIC ACID 6-Mercaptopurine on Lactobacillus casei. Arch. Biochem. & Biophys., 71:358-66, 1957.

GENE- -ENZYME FORMING 4. BEADLE,G. W. Biochemical Genetics. Chem. Rev., 37:15- SYSTEM 96, 1945. 5. . The Role of the Nucleus in Heredity. In: The 8-AZAGUANINE 8-AZAGUANYLIC ACID Chemical Basis of Heredity, pp. 3-22. Baltimore: The 6-MERCAPTOPURINE 6-MERCAPTOPURINE RIBONUCLEOTIDE Johns Hopkins Press, 1957. 6. BENNETT,L.L., JR.; SKIPPER,H. E.; SIMPSON,L.;WHEEL CHART2.â€”A biochemical basis for inhibition by purine ER,G. P.; and WILCOX,W.S. Searches for Exploitable Bio analogs and for resistance to such analogs. The enzyme- chemical Differences between Normal and Cancer Cells. catalyzed formation of 8-azaguanylic acid and 6-mercapto V. Cellular Conservation of Purines. Cancer Research, purine ribonucleotide can be viewed as lethal syntheses. Ac 20:62-81, 1960. cording to this view a genetically controlled reduction of en 7. BOSCH,L.;HARBERS,E.;and HEIDELBERGER,C.Studies on zyme activity in resistant cells would result in decreased syn Fluorinated Pyrimidines. V. Effects on Nucleic Acid thesis of fraudulent ribonucleotides. Metabolism in Vitro. Cancer Research, 18:335-43, 1958. 8. BROCKMAN,R.W.; BENNETT,L. L., JR.; SIMPSON,M. S.; plasms resistant to 6-mercaptopurine (59, 60), WILSON,A. R.; THOMSON,J. R.; and SKIPPER,H. E. A and it would be interesting to know in this case Mechanism of Resistance to 8-Azaguanine. II. Studies with whether APP is converted to a ribonucleotide Experimental Neoplasms. Cancer Research, 19:856-69, 1959. by adenylic acid pyrophosphorylase. Resistance 9. BROCKMAN,R.W.; BENNETT,L. L., JR.; and SKIPPER, to 6-mercaptopurine in L1210 can now be seen H. E. A Comparison of Purine Metabolism in Strains of to result in: (a) decreased capacity of resistant Streptococcusfaeealis Susceptible and Resistant to 6-Mer cells in vivo to convert hypoxanthine, 6-mercapto captopurine Inhibition. Proc. Am. Assoc. Cancer Re purine, guanine, and 8-azaguanine to ribonucleo search, 2:191, 1957. 10. BROCKMAN,R.W.; DAVIS,J. M.; and STUTTS,P. Metabo tides; (o) decreased capacity of cell-free enzyme lism of Uracil and 5-Fluorouracil by Drug-sensitive and preparations from resistant cells to catalyze the Drug-resistant Bacteria. Biochim. et Biophys. Acta, 40: conversion of these same purines and analogs to 22-32, 1960. ribonucleotides; (c) biological cross-resistance to 11. BROCKMAN,R.W., and HUTCHISON,D.J. Decreased Ribo 8-azaguanine of 6-mercaptopurine-resistant L1210 nucleotide Pyrophosphorylase Activity in S. faeealis Mu tants Resistant to Purine Analogs. Fed. Proc., 18:197, used in these experiments (39) ; and (d) no decrease 1959. in adenylic acid pyrophosphorylase activity. 12. BROCKMAN,R.W.; SPARKS,M. C.; HUTCHISON,D.J.; and Kornberg (37) has termed the reactions by SKIPPER,H. E. A Mechanism of Resistance to 8-Azagua- which purine bases and also uracil yield ribonucleo nine. I. Microbiological Studies on the Metabolism of tides as "salvage pathways." It seems likely that Purines and 8-Azapurines. Cancer Research, 19:177-88, 1959. the extent of operation of these salvage mech 13. BROCKMAN,R.W.; SPARKS,M. C.; and SIMPSON,M. S. A anisms is one of the factors in determining the Comparison of the Metabolism of Purines and Purine sensitivity or resistance of some experimental Analogs by Susceptible and Drug-resistant Bacterial and neoplasms to agents such as 6-mercaptopurine, Neoplastic Cells. Biochim. et Biophys. Acta, 26:671-72, 1957. 6-thioguanine, and 8-azaguanine. 14. BROCKMAN,R.W.; SPARKS,M. C.; SIMPSON,M. S.; and SKIPPER,H. E. Decreased Ribonucleotide Pyrophosphory ACKNOWLEDGMENTS lase Activity in Streptococcusfaeealis and L1210 Leukae The authors are grateful to Dr. L. W. Law, National Cancer mia Resistant to Purine Antagonists. Biochem. Pharm., Institute, for the experimental leukemias used in this work. 2:77-79, 1959.

15. BBOCKMAN,R. W.; SPARKS,M. C.; STUTTS,P.; and chemical and Metabolic Investigations. J. Biol. Chem., SCHABEL,F. M., JR. Biochemical Basis for Resistance to 234:1255-62, 1959. Furine Antagonists in Streptococcusfaecalis. Bact. Proc., 32. HEIDELBERGER,C.; CHAUDHURI,N. K.; DANNEBERG, p. 52, 1959. P.; MOOREN, D.; GRIESBACH,L.; DUSCHINSKT,R.; 16. CAREY,N. H., and MANDEL,H. G. Relation between the SCHNITZER,R.J.; PLEVEN,E.; and SCHEINER,J.Fluorinat Inhibition of Growth of Bacillus cereus and Metabolism of ed Pyrimidines, a New Class of Tumor-Inhibitory Com 6-Mercaptopurine. Abstr., Am. Soc. Pharmaeol. Exper. pounds. Nature, 179:663-66, 1957. Therap., 1958. 33. HEIDELBERGER,C., and KELLER, R. A. The Effects of 17. CARTER,C.E. Reaction of 6-Mercaptopurine with Inosine- Twenty-nine Compounds on Nucleic Acid and Protein and Guanosine-5'-phosphate Pyrophosphorylase Purified Biosynthesis in Slices of Flexner-Jobling Carcinoma and from E. coli. Biochem. Pharm., 2:105-11, 1959. Rat Spleen. 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FIG. 1.â€”Comparison of the intermediary metabolism of L1210/8-Aza = 8-azaguanine-resistant \ also resistant to hypoxanthine-8-C14 in vivo in ascites tumor cells by means of L1210/TG = 6-thioguanine-resistant / 6-mercaptopurine. two-dimensional chromatographic-radioautographic analysis Radioactive compounds identified are: of extracts of cells. Dotted circles locate faintly radioactive 52 Adenine All. Allantoin 29A AMP from ATP areas. 25 Adenosine UA Uric acid 11 IMP 3 Hypoxanthine 50 DPN 16 GMP L1210 = parent, drug-sensitive leukemia. 5 Inosine 51 Unidentified 17 ADP L1210/MP = 6-mercaptopurine-resistant. 44 Xanthine 29 AMP 34 ATP

All All UA UA

29 29A 29 5l II 16 50

L I2IO/MP Ascites L I2IO/TG Ascites Hypoxanthine-8-C" Hypoxanthine-8-C14

All UA All UA 29 29

FIG.l

Biosynthesis of the Purines. XXV. The Enzymatic Cleav Counting of C14-Labeled Carbon Dioxide. Int. J. App. age of N-(5-Amino-l-Ribosyl-4-Imidazolylcarbonyl)-L- Radiation & Isotopes, 3:172-5, 1958. Aspartic Acid S'-Phosphate. J. Biol. Chem., 234:1806-11, 58. SKIPPER,H. E. On the Mechanism of Action of 6-Mercap 1959. topurine. Ann. New York Acad. Sc., 60:315-21, 1954. 48. PASTERNAK,C. A., and HANDSCHUMACHER,R.E. The 59. SKIPPER,H. E.; MONTGOMERY,J.A.; THOMSON,J.R.; and Biochemical Activity of 6-Azauridine: Interference with SCHABEL,F.M., JR. Structure-Activity Relationships and Pyrmidine Metabolism in Transplantable Mouse Tumors. Cross-Resistance Observed on Evaluation of a Series of J. Biol. Chem., 234:2992-97, 1959. Furine Analogs against Experimental Neoplasms. Cancer 49. PATERSON,A.R. P. The Formation of 6-Mercaptopurine Research, 19:425-37, 1959. Riboside Phosphate in Ascites Tumor Cells. CaÃ±ad.J. 60. SKIPPER,H. E.; ROBINS,R. K.; THOMSON,J.R.; CHENG, Biochem. & Physiol., 37:1011-23, 1959. C. C.; BROCKMAN,R.W.; and SCHABEL,F.M., JR. Struc 50. POTTER,V. R. The Biochemical Approach to the Cancer ture-Activity Relationships Observed on Screening a Problem. Fed. Proc., 17:691-97, 1958. Series of Pyrazolopyrimidines against Experimental Neo 51. REICHARD,P.;SKÃ–LD,O.;and KLEIN,G. Possible Enzymic plasms. Cancer Research, 17:579-96, 1957. Mechanism for the Development of Resistance against 61. SKIPPER,H. E., and THOMSON,J.R. Effects of a Series of Fluorouracil in Ascites Tumors. Nature, 183:939^11, 1959. Tumor-inhibiting Agents and Related Compounds on 52. REMT,C. N., and SMITH,M. S. Metabolism of 2,6-Diami- L1210 Leukemia and Drug-resistant Lines Thereof. In: nopurine: Conversion to 5'-Phosphoribosyl-2-methylami- Investigations of Diverse Systems for Cancer Chemo no-6-aminopurines by Enzymes of Escherichia coli. J. Biol. therapy Screening. Cancer Research, Suppl. No. 3, pp. Chem., 228:325-38, 1957. 44-46, 1955. 53. SALSER,J. S., and BALIS,M. E. Studies on the Mechanism 62. SKODA,J.; KARA,J.; SORMOV,Z.;and SORM,F. Inhibition of Action of 6-Mercaptopurine in Cell-free.Preparations. of Escherichia coli Polynucleotide Phosphorylase by 6-Aza- Fed. Proc., 18:315, 1959. uridine Diphosphate. Biochim. et Biophys. Acta, 33:579- 54. SARCIONE,E. J., and STUTZMAN,L.6-Methylmercapto- 80, 1959. purine: Identification as Metabolite of 6-Mercaptopurine 63. SKODA,J.,and Â§ORM,F.The Accumulation of Orotic Acid, in Vivo and Its Activity in Vitro. Proc. Soc. Exper. Biol. Uracil and Hypoxanthine by Escherichia coli in the Pres & Med., 101:766-73, 1959. ence of 6-Azauracil and the Biosynthesis of 6-Azauridylic 55. SARTORELLI,A.C., and LEPAGE,G. A. Metabolic Effects Acid. Coll. Czech. Comm., 24:1331-37, 1959. of 6-Thioguanine. II. Biosynthesis of Nucleic Acid Purines 64. TOMIZAWA,S.,and ARONOW,L. Studies on Drug Resist in Vivo and in Vitro. Cancer Research, 18:1329-35, 1958. ance in Mammalian Cells. II. 6-Mercaptopurine Resist 56. SARTORELLI,A.C.; LEPAGE, G. A.; and MOORE,E. C. ance in Mouse Fibroblasta. J. Pharm. & Exper. Therap., Metabolic Effects of 6-Thioguanine. I. Studies on Thio- 128:107-14, 1960. guanine-resistant and -sensitive Ehrlich Ascites Cells. 65. WAY,J. L., and PARKS,R. E., JR. Enzymatic Synthesis of Cancer Research, 18:1232:39, 1958. 5'-Phosphate Nucleotides of Furine Analogues. J. Biol. 57. SIMPSON,L. A Simplified Procedure for Proportional Chem., 231:467-80, 1958.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1960 American Association for Cancer Research. A Mechanism of Resistance to 6-Mercaptopurine: Metabolism of Hypoxanthine and 6-Mercaptopurine by Sensitive and Resistant Neoplasms

R. W. Brockman, Marguerite S. Simpson, Joan M. Davis, et al.

Cancer Res 1960;20:643-653.

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