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Metabolism of Resistant Mutants of Streptococcusfaecalis III. The Action of 6-Mercaptopurine*t M. EARLBALIS,VALIAHYLIN, M. KATHARINE COULTAS,ANDDORRISJ. HUTCHISON (Laboratories of the Sloan-Kettering Institute for Cancer Research, New York, N.Y.) The compound 6-mercaptopurine (6-MP) has been shown to possess definite antitumor (13) properties and to inhibit the growth of several microorganisms (14-16). This inhibition has been reversed by several purines (14-16), and it is possible that the antitumor action is also con cerned with purine metabolism. The present exper iments were undertaken in an attempt to elucidate the action of 6-MP by studying its effects on bacteria which are resistant to it. Several such strains have been isolated and characterized (17), and certain aspects of their metabolism have been described (4, 17). In the present study the followbig mutants have been used: SF/O Parent (ATCC No. 8043) SF/MP Resistant to 6-MP SF/MPcc Resistant to 6-MP, isolated inde pendently of SF/MP SF/MP/A Double mutant, resistant to 6-MP and A-methopterin SF/DAP Resistant to 2,6-diammopurine SF/A Resistant to A-methopterin SF/A/O Partial revert of SF/A SF/A/MP Double mutant, resistant to Amethopterin and 6-MP MATERIALS AND METHODS Reagents.—The6-MP-8-C" was obtained from the Southern Research Institute, Birmingham, Alabama, and the sodium fonnate-C14 from Isotopes Specialties Co., Burbank, Califor nia. Bacteria and media.—Theisolation, maintenance, and char acterization of the strains of Streptococcusfaecalia have been described elsewhere (16, 17). The standard inocula used in these experiments were grown and prepared according to the following regimen: Two successive transfers were made into 5 ml. of liquid medium identical with that on which the culture is carried; the third transfer with the use of saline-washed inocu lum was then made into 5 ml. of an unlabeled equivalent of the medium which was to be used in the labeling experiment. The * A preliminary report of this work has been presented (3). t This investigation was supported in part by funds from the National Cancer Institute, National Institutes of Health, Public Health Service (Grant # CY3190, C2699), and from the U.S. Atomic Energy Commission—Contract # AT(301)-910. Received for publication November 27, 1957. inoculum used was one which gave approximately 8 X 10' cells/ml. In each experiment, 400 ml. of a folie acid-(l m¿tg/ml) or thymine-(l jug/ml) supplemented purine and pyrimidinefree medium (F-PP) was further supplemented with the labeled purine at a concentration of 3.8 X 10~~* M. This medium was sterilized at 121° C. for 15 minutes; if the labeled compound was heat-labile, it was omitted from the medium, sterilized by filtration through an ultrafine sintered glass filter, and added aseptically to the cooled medium. Just prior to inoculation, a K'-ml. aliquot was removed aseptically from each flask to serve as a titration blank. The flasks were then inoculated so that there were approximately 8 X IO6cells/ml and incubated at 35°C. for 18 hours. At the termination of the incubation period, 19 ml. were removed from each flask, and the rest of the cells were harvested by centrifugation. A 5-ml. aliquot was handled aseptically, since it was to be used as the inoculum for a series of controls to determine the resistance and purine requirement for the mutants after the growth under the above conditions. A 2-ml. sample was used to determine the amount of growth by turbidity measurement on a Coleman Junior Spectrophotometer, and a 12-ml. sample was used for titer determination. Controls on A-methopterin and 6-MP resistance as well as purine requirements were made on each culture for each experi ment; the saline-washed suspension prepared from the 5-ml. aliquot removed from the experimental flasks was used as such. The basal medium for the resistance controls was the one sup plemented with PGA at 1 mjug/ml, while the response to adenine, guanine, hypoxanthine, and xanthine was determined in this medium and a medium supplemented with thymine at 1 Mg/ml rather than the PGA. The controls were set up in 13 X 100-mm. tubes which con tained 2 ml. of medium. These tubes were sterilized at 121° C. for 8 minutes, cooled, and inoculated with a saline-washed sus pension diluted so that each inoculated tube contained 8 X 10s cells/ml. Turbidity measurements were made after incubation for 18 hours at 35°C. Isolation of nucleic add purines.—Theharvested celb were washed successively with cold trichloroacetic acid, alcohol, and ether. The washed cells were treated at 37°C. for 24 hours with 1 ml. of l N sodium hydroxide per 180 mg. of bacteria (20). The degraded pentosenucleic acid (PNA) was separated from the deoxypentosenucleic acid (DNA) by acidification with HC1 and trichloroacetic acid, followed by the addition of 1.5 volumes of etbanol. The DNA was collected by centrifugation. The individual purines were isolated (8) by hydrolysis of the nucleic acids, precipitation of silver purines, regeneration of the purines as hydrochlorides, and separation of the individual purines by paper chromatography. The radioactivities were determined as has been previously described (7). Infinitely thin Sims on aluminum planchéiswere measured in a Geiger-Müller flow counter with helium-isobutane gas. All the plancheta con tained sufficient radioactivity to result in measurements of at least twice those of the background, and the activities were determined to within standard errors of less than 5 per cent (18), except where noted. 440 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1958 American Association for Cancer Research. BALISet al.—Streptococcus faecalis and 6-Mercaptopurine RESULTS Table 1 shows the effect of 6-MP on the ability of the wild strain and seven mutants to synthesize nucleic acid adenine and guanine from exogenous xanthine and hypoxanthine. Xanthine is the only purine which all the strains are known to utilize for this purpose (4,17). 6-MP had some inhibitory effect on the conversion of xanthine into nucleic acid guanine, but greatly depressed its incorpora tion into nucleic acid adenine in all the strains except the two single mutants which are resistant to its growth inhibitory effect, SF/MP and SF/ MPcc. If, however, hypoxanthine was used as the labeled precursor instead of xanthine, 6-MP caused a clearly marked depression of the incorporation of the exogenous purine into both the nucleic acid adenine and guanine. This depression was very much greater in the two 6-MP-resistant single mutants than in any of the other strains. In both these experiments, effects produced by 6-MP on the incorporation of the exogenous hypoxanthine or xanthine into nucleic acid purines were of the same order of magnitude in both the DNA and UNA. The effect of 6-MP on the possible utilization 441 of inosinic acid by the organisms is shown in Table 2. Labeled inosinic acid was not available, so the method of reciprocal labeling was employed. The fraction of the nucleic acid purines which was synthesized de novo is the ratio of the relative specific activity (RSA) of the nucleic acid purines obtained from bacteria grown in the presence of the potential precursor to the RSA obtained from the control bacteria where both the media con tained sodium formate-C14 (7). In Experiment 3, Table 2, are shown the control values for a series of determinations. In Experiment 4, inosinic acid was present, and, since there was no reduction in the amount of formate used for the synthesis of the nucleic acid purines, it is clear that exoge nous inosinic acid did not serve as a purine source for these bacteria. In Experiment 5, both inosinic acid and 6-MP were added, and certain of the mutants showed a very large drop in the amount of formate incorporated into the nucleic acid pu rines. This might be due to the utilization of either 6-MP or inosinic acid as a purine precursor. ¡How ever, the addition of 6-MP caused a marked reduc tion in the incorporation of formate-C14 by five of the seven strains examined. This indicated that TABLE1 EFFECTOFO-MERCAPTOPUHINE ONTHEUTILIZATION OFEXOGENOUS PURINES FOBNUCLEIC ACIDSYNTHESIS HIP.t EXP. 1 AdeninePNA AdeninePNA.77.19.59.11.64.83.52.54DNA.74.21.81.14.951.20.67.74GuaninePNA.68.16.40.09.68.89.60.57 STRAIN DNA.03* .03*1.40 SF/O 1.10.03* SF/MP .03*.77 SF/MP/A SF/MPcc .58.10 SF/DAP .11.05* .07*.03* SF/A .03*.01* SF/A/0 .02*GuaninePNA.81.82.561.001.001.22.58.60DNA1.30.75.881.30.97.63 SF/A/MP The bacteria were grown in media which contained 1 X 10~3moles/liter of 6-mercaptopurine and 3.8 X 10~* moles/liter of the labeled purine. In Exp. 1 the labeled precursor was xanthine; in Exp. 2 it was hypoxanthine. The activities of the isolated purines have been divided by the activities of the corresponding purines from con trol bacteria grown identically, except for the omission of 6-mercaptopurine. * Error of radioactivity assay < 15 per cent (18). TABLE 2 UTILIZATIONOFLABELEDFORMATEBY S. faecalis EXP. 8 EXP. 4 EXP.«le «AdeninePNA8*104*0.1*DNA8*104*0.1*GuaninePNA8*104*0.1* Adenine Guanine Adenine GuanineAdenine>NA1918ID•1tttt18PNA6188*18103*0.1*DNA9178*U148*0.1*GuaninePNA7178*U103*0.1*DNA7178*18 PNA DNA PNA DNA PNA DNA PNA DNA STRAIN 21 22 Õ2 18 «2 23 28 SF/O 20 18 19 18 18 18 SF/MP 17 SF/MP/A 17 17 17 17 17 U •i1« SI 18 SF/MPcc to tt 88 22 SF/DAP tt tt tt tt tt tt 2Õ tt SF/A/O tt tt M 10 U 18 1* SF/A/MP 1» 1» Values reported are relative specific activities. (RSA) =- c0110^/"""/1110^ °*isolated baae IQQ counta/ min/ mole of precursor Exp. 3, medium contained 20 ¿ig/ml«odiumformate-C'14. Èxp.. 4,, medium contained 20 /ig/ ml sodium formate-C14 plus 3.8 X 10~*moles/liter of inosmic acid.. Ezp. 5, medium contained 20 //ir/ml sodium formate-C14 plus 3.8. X 10~*moles/liter ~ of inoainic acid and 1 X 10~*molea/Iiter of 6-mercaptopurine. Exp. 6, medium contained 20 tai/nú sodium formate-C14 plus 1 X 10~* moles/liter of 6-mercaptopurine. * Error in radioactivity assay < IS per cent (18). Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1958 American Association for Cancer Research. 442 Cancer Research these strains, instead of synthesizing purines de novo, were utilizing either the 6-MP or the inosinic acid. Experiment 6 showed that, with at least four of the strains, 6-MP produced a similar depression of formate incorporation in the absence of inosinic acid. In these strains, therefore, the 6-MP and not the inosinic acid was being utilized as a source of purines for nucleic acid synthesis. It is thus probable that in the experiments in Table 2 none of the strains used inosinic acid. Two strains in which incorporation of the formate-C14 into nucleic acid purines was not de pressed in the presence of 6-MP were the two resistant single mutants—SF/MP and SF/MPcc (Table 2, Experiments 3 and 5). Confirmation of Vol. 18, May, 1958 it did so to a lesser extent than did any of the other strains except SF/MP. At the other extreme, SF/A/MP utilized 6-MP almost exclusively as a source of nucleic acid purines unless provided with an alternative purine in the form of xanthine or hypoxanthine. Comparison of the results in Table 3 with those in Table 2 shows considerable con firmation. For example, in the case of SF/A/MP, Exps. 3 and 4 show that, in the presence of 6-MP, incorporation of formate into nucleic acid, adenine, and guanine was reduced to 0.1/19 or 0.5 per cent of its control level, suggesting that about 99.5 per cent of nucleic acid adenine and guanine were derived from the 6-MP.1 Table 3, Exp. 8, shows by direct measurement that about TABLE 3 INCORPORATION OFEXOGENOUS O-MERCAPTOPURINE INTONUCLEICACID PURINESIN MEDIUMCONTAINING FOLICACID mjjg/ml)EXP. 9AdeninePNA625*5024560474DNA685*6024880281GuaninePNA416*2920434048Ex».AdeninePNA453*S3172435344410GuaninePNA02»0IS10779Values 8Guanine 7Adenine(1 EXP. AdenineSTBAIN PNA54 DNA017971 PNA PNASF/0SF/MPSF/MP/ASF/MPccSF/DAPSF/ASF/A/OSF/A/MP6190£0976486DNA PNA DNA 7085GuanineDNA 7083 86Exp. as:Relativespecificactivity(RSA)counts/min/molemiint.s/min/inoie reported purinexoffenous Exp. Exp. Exp. Exp. 7, contained 8, contained 9, contained 10, contained 100 X 100 X 100 X 3.8 X > U I I II L3/ 111I1J/ 1LHJ1C CAUgCUL/1» eisolated Dunne100. JJU11LIC lO-'MO-MP-S-C" and 3.8 X 10"* Mhypoxanthine. lO^MO-MP-S-C". lO^MO-MP-S-C" and 3.8 X 10~6 Mxanthine. 10-*M6-MP-8-C» and S.8 X IQ-" Mxanthine. * Error of radioactivity assay < IS per cent (18). the ability of the other strains to use 6-MP as a source of nucleic acid purines is afforded by experiments in which 6-MP itself was the labeled precursor, as may be seen in Table 3, experiments 7 and 8. 6-MP was extensively utilized by strains SF/A/O and SF/A/MP as a precursor for nucleic acid purines, and the extent of this utilization was not greatly modified by the presence of hypo xanthine. Furthermore, even in the presence of xanthine seven of the eight strains studied incor porated labeled 6-MP extensively into their nucleic acid purines, and this incorporation was quite substantial even if the unlabeled xanthine and labeled 6-MP were present in the medium in equal concentrations (Exps. 9 and 10 in Table 3). The only strain which did not incorporate 6-MP ex tensively was SF/MP, one of the single mutants which is resistant to 6-MP. In this connection it will be noted that, although the other 6-MPresistant single mutant SF/MPcc utilized 6-MP, 79-86 per cent of the nucleic acid purines were derived from this source. It is interesting to note that, throughout these experiments, irrespective of the precursor used, the RNA and DNA had, within the limits of experi mental error, the same specific activities (5). Fur thermore, the effects of 6-MP were the same on both nucleic acids. DISCUSSION In a previous paper the effects of 6-MP on the synthesis of purines de novo and the conversion of one purine derivative into another in Lactobacitttis casei were described (6). It was suggested that, in this organism, the enzyme which normally converts hypoxanthine and other purines to their nucleotides also converts 6-MP to 6-MP nucleo1The basis for this calculation has been discussed in more detail elsewhere (7). Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1958 American Association for Cancer Research. BALIS et al.—Streptococcus tide2 and that the latter competes with inosinic acid and prevents its conversion to adenine and guanine derivatives. If it is assumed that 6-MP acts on S. faecalis by the same mechanism, the simplest type of mutant to be expected is one which cannot convert 6-MP to its nucleotide.3 It would further be expected that such a mutant would also be unable to convert other purines to their nucleotides. SF/MP seems to be a mutant of this type. It has already been shown that it requires xanthine for optimal growth (17). It can convert hypoxanthine, adenine, and guanine to their nucleotides, but to a slight extent. This latter process is inhibited by 6-MP, but it makes such a small contribution to the economy of the cell that its inhibition has no effect on growth. The cells can still utilize exogenous xanthine exten sively (4, 17), and the amount of 6-MP nucleotide formed is presumably too small to inhibit utiliza tion of the large amount of xanthine nucleotide formed. The two single mutants which are resistant to 6-MP, SF/MP and SF/MPcc, do have certain common characteristics which distinguish them from the other strains: 6-MP depresses their ca pacity to utilize hypoxanthine, but not xanthine or formate, as precursors of nucleic acid purines (Tables 1 and 2). They differ, however, in that SF/MPcc uses hypoxanthine and 6-MP as sources of nucleic acid purines, but SF/MP does not. Moreover, SF/MPcc is distinguished from the wild strain not only by its resistance to 6-MP but also by the fact that it requires less folie acid for optimal growth (17). It is quite possible, therefore, that this mutant's resistance to 6-MP is due to the high rate at which it can synthesize purines de novo. In this connection it must be remembered that 6-MP is not inhibitory to the wild strain when sufficient exogenous purine is available, i.e., 6-MP is inhibitory only if the supply of purines is limited. Presumably in SF/MPcc the rate of purine syn thesis de novo is so high that, under the conditions of our experiments, the supply of purines was always abundant. If the 6-MP resistance of SF/ MPcc is indeed due to the rapidity with which it can synthesize purines de novo, it might be anticipated that it should also be somewhat re sistant to folie acid antagonists such as A-methopterin. This has been shown to be the case (17). 1 It has been shown that an enzyme from pigeon liver which converts hypoxanthine and guanine to their nucleotides can also synthesize a phosphoribosyl derivative of 6-MP from 6-MP (19). 3Work by Dr. R. W. Brockman (private communication) has shown that SF/MP cannot convert these purines into their free nucleotides, in confirmation of this hypothesis. faecalis and 6-Mercaptopurine 443 The present experiments have disclosed no dif ferences between the double mutants, SF/MP/A and SF/A/MP, on the one hand, and the sensitive mutants on the other. The varying ability of the strains to use 6-MP as a purine source is almost certainly a factor in 6-MP resistance. This is particularly striking in the case of the double mutant SF/A/MP and may provide an explana tion for the resistance of this strain. There is a precedent for the observation that a quantitative change in the ability of bacteria to use a substrate should result in qualitative changes in growth response (1). In accordance with the mechanism suggested above for the action of 6-MP (6), it is possible that some of the postulated 6-MP nucleo tide might be converted to inosinic acid and that this might be sufficient to reverse the action of the remaining 6-MP nucleotide. It should perhaps be emphasized that, although for convenience the present studies have been limited to nucleic acids, it is not suggested that 6-MP exerts a specific effect on nucleic acid me tabolism per se. On the contrary, it seems more probable that it may inhibit conversion of inosinic acid to adenine and guanine derivatives. If this is so, its effects on tumors and microorganisms may be due to its interfering with syntheses of nucleic acids or purine-containing coenzymes or both (7, 11, 12). It seems not unlikely that the mechanisms of the effects on tumors and microorganisms may have much in common. It has been shown that varying tissues including tumors depend upon de novo synthesis to greater or lesser degrees relative to their ability to use preformed purines (4, 9, 10). Furthermore, two leukemias, Line I and Line I/A, which differ in their sensitivity to A-methopterin, differ also in the extent to which they utilize exogenous formate for the synthesis of RNA pu rines (2). If the view advanced above, that the 6-MP resistance of SF/MPcc is due to its increased ability to synthesize purines de novo, is correct, it is at least conceivable that the resistance of certain tumors to antagonists of purine synthesis or utilization may be explicable on similar lines. SUMMARY The effect of 6-mercaptopurine on S. faecalis (ATCC No. 8043) and several of its mutants has been investigated. Some mutants are able to use the inhibitor as a source of nucleic acid purines; to some extent, resistance may be due to that fact. Two single mutants are resistant to 6-MP for other reasons. The first appears to lack the enzyme system which converts certain purines, including 6-MP, to nucleotides, and it is thought that the Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1958 American Association for Cancer Research. 444 Cancer Research nucleotide of 6-MP is the true inhibitor. The second has a greater ability to synthesize purines de novo and thus can better withstand the purine antagonist. These facts emphasize the multiple mechanisms which can lead to resistance. ACKNOWLEDGMENTS The authors wish to thank Dr. George Bosworth Brown for his continued interest and very helpful discussions of this project. REFERENCES 1. BALIS,M. E.; BHOOKE,M. S.; BROWN,G. B.; and MAOASAMK, B. The Utilization of Purines by Purineless Mu tants of Aerobacter acrogenes. 3. Biol. Chem., 219:917-26, 1956. 2. BALIS,M. E., and DANOIS,J. Effects of A-methopterin on Nucleic Acid Synthesis in Leukemic Spleen Breis, Cancer Research, 15:607-8, 1955. 3. BALIS,M. E., and HUTCHISON,D. J. The Utilization of 6Mercaptopurine by Streptococcusfaecalis. Fed. Proc., 16 : 149, 1957. 4. BALIS,M. E.; HTLIN, V.; COULTAS,M. K.; and HUTCHI SON,D. J. Metabolism of Resistant Mutants of Streptococcu>faecalis. II. Incorporation of Exogenous Purines. Can cer Research, 18:220-25, 1958. 5. BALIS,M. E., and LAKK,C. T. Synthesis of PNA and DNA in Escherichia coli. Fed. Proc., 13:178, 1954. 6. BALIS,M. E.; LEVIN,D. H.; BROWN,G. B.; ELION,G. B.; NATHAN,H. C.; and HITCHINOS,G. H. The Effects of 6Mercaptopurine on Lactobaclllus casei. Arch. Biochem. & Biophys., 71:858-66, 1957. 7. BALIS,M. E.; LEVIN,D. H.; BROWN,G. B.; ELION,G. B.; VANDERWERFF, H.; and HITCHINQS,G. H. The Incorpora tion of Exogenous Purines into Pentose Nucleic Acid by Lactobacillus casei. J. Biol. Chem., 196:729-47, 1952. 8. 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SCHMIDT,G., and THANNHAUSER, S. J. A Method for the Determination of Deoxyribonucleic Acid, Ribonucleic Acid and Phosphoproteins in Animal Tissues. J. Biol. Chem., 161:83-89, 1945. Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1958 American Association for Cancer Research. Metabolism of Resistant Mutants of Streptococcus faecalis: III. The Action of 6-Mercaptopurine M. Earl Balis, Valia Hylin, M. Katharine Coultas, et al. Cancer Res 1958;18:440-444. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/18/4/440 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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