Studies on the Mechanism of Action of 6-Mercaptopurine in Sensitive and Resistant L1210 Leukemia in Vitro*

JACKD. DAVIDSON

(Clinical Pharmacology and Experimental Tlierapeutics Service, National Cancer Institute, Betliesda, Md.)

SUMMARY A study was made of the effects of 6-mercaptopurine (6-MP) upon nucleic acid metabolism in L1210 leukemia cells in vitro. Pharmacological concentrations of 6-MP inhibited the incorporation of both hypoxanthine and glycine into adenine nucleotides. Higher concentrations of 6-MP inhibited the incorporation of hypoxanthine into both adenine and guanine nucleotides. In a subline of L1210 which is resistant to 6-MP there was much less utilization of hypoxanthine than in the sensitive line, and incorporation into both adenine and guanine moieties was strongly inhibited by 6-MP. On the contrary, utilization of glycine for nucleotide purine synthesis was unaffected by 6-MP. These findings support the hypothesis that in L1210 leukemia 6-MP is metabolized to its ribotide, and this produces a metabolic block in the conversion of inosinic acid to adenylic acid. They further indicate that, in the L1210 cells resistant to 6-MP, there is a very limited capacity to convert 6-MP and hypoxanthine to ribotides. This leads to competition between 6-MP and hypoxanthine and to formation of insufficient 6-MP ribotide to cause the critical block.

Although 6-mercaptopurine (6-MP) has been purines isolated from L1210 ascites leukemia cells studied with respect to its antitumor activity since have been studied. The results obtained suggest 1952, there is still relatively little information re that the primary antimetabolic activity of 6-MP garding the mechanism of its action. Skipper re in sensitive cells is the inhibition of the conversion viewed the available data in 1954 (18) and ad of inosinic acid to adenylic acid. duced appreciable evidence for the involvement of hypoxanthine metabolism in the action of 6-MP,1 MATERIALS AND METHODS but he felt it was impossible to be specific as to the General.â€”Aliquots of L1210 cell suspensions biochemical reactions concerned. In particular were incubated for 1 or 2 hours in the presence of 6-MP seemed to inhibit the de novobiosynthesis of various radioactive nucleic acid precursors with and purines as measured by the inhibition of the in without 6-MP. The purines of the nucleic acids corporation of labeled formate into the combined and/or the acid-soluble fractions were then isolat DNA and RNA of several mouse tumors. ed and their specific activities determined. In the present work the effects of 6-MP upon L1210 cells.â€”Lymphoid leukemia L12102 was the incorporation of hypoxanthine and glycine into grown in female DBA/2 mice in the ascites form. This parent line of leukemia is sensitive to 6-MP * Presented in part before the 50th annual meeting of the American Association for Cancer Research, April, 1059, At and will be referred to as L1210S. It was carried lantic City, N.J., and published in abstract form in the scien by weekly transplantation by the intraperitoneal tific Proceedings of that meeting. injection of 0.1 ml. of a 1:4000 dilution of 1-week- 1Abbreviations used: 6-MP = 6-mercaptopurine; 6-MP- old donor ascitic fluid. For experimental studies, R-P = 6-mercaptopurine nucleotide; L1210S = leukemia cells groups of ten to 30 mice were implanted with 0.1 sensitive to 6-MP; L1210R = leukemia cells resistant to 6-MP; RNA = ribonucleic acid; DNA = deoxyribonucleic acid; ml. of a 1:10 dilution of ascitic fluid, and the PI = inorganic phosphorus; TCA = trichloroacetic acid; L1210S cells were harvested on the 5th day of IMP = inosine-5'-phosphate; AMP = adenosine-5'-phos- 2The L1210 ascites leukemia strains were obtained from phate; TRIS = trishydroxymethyl aminomethane. Dr. Lloyd Law, National Cancer Institute, Bethesda, Mary Received for publication August 3, 1959. land.

225

growth. All dilutions were made with Locke's phosphate was counted at infinite thinness with an solution. end-window Geiger-MÃ¼ller tube through a 24 The resistant leukemia, L1210R, was a subline mg/sq cm aluminum absorber to removed the C14 in which resistance to 8-azaguanine has been de beta radiation. C14samples from experiments in veloped. This leukemia showed complete cross- cluding P32were monitored for P32contamination resistance to 6-MP (11). These cells were grown in by measurement of radioactivity on the liquid the same manner as the L1210S but owing to scintillation counter under conditions of low am slightly slower growth were found optimal for use plification which gave maximal sensitivity for P32 on the 7th day of their growth. Under the above and complete insensitivity for C14.All the P32em conditions the ascitic fluid from both leukemias ployed was fresh, so that its content of P33, with was free of gross blood. During the period of these energies similar to C14,was less than 4 per cent of studies the two lines of L1210 were checked at its total radioactivity. intervals for their respective sensitivity and re Chemical isolations.â€”At the conclusion of the sistance to 6-MP. aerobic incubations the contents of flasks were For experiments, tumor-bearing mice were sac poured into one-third their volume of 20 per cent rificed by cervical dislocation. The ascitic fluid was TCA at 0Â°C.to yield a final concentration of 5 collected with heparin and immediately packed in per cent. The resultant precipitates were stirred ice. The pooled cells were washed twice by centrif- for 2 minutes and allowed to stand at least 15 min ugation and resuspension in 10-20 volumes of utes at 0Â°C.before being centrifuged. The super buffer3 at 2Â°C.and finally suspended in a volume natant solutions were used for the isolation of the of buffer sufficient to yield the equivalent of 0.5- PÂ¡and acid-soluble fractions. The protein precipi 1.0 nil. of packed cells in 5 ml. Such 5-ml. aliquota tates were reextracted successively with 10 vol of the cell suspension were placed in 25-ml. Erlen- umes each of 5 per cent TCA and twice with 95 per meyer flasks along with 5 ml. of additional buffer cent ethanol in the cold. The precipitates were then containing the necessary quantity of 6-MP4 to extracted twice with 10-ml. portions of 95 per cent provide the desired final concentration. The con ethanol at boiling. This was followed by extraction centrations used ranged about the concentration of the nucleic acids with 5 ml. of 10 per cent sodi calculated to prevail in the total body water of um chloride first for 1 hour and with a second 5 ml. mice given 50-75 mg/kg (8). for J hour, both with constant stirring at 100Â°C. The radioisotopic precursors were added in Foaming was suppressed with General Electric 0.2-1.0 ml. isotonic saline solution after the flasks Antifoam 60. During these extractions the pH was had been warmed in the Dubnoff shaker incubator maintained at close to 7, initially by the addition bath for 2 minutes. of 0.1 N sodium hydroxide and later by back titrat Radioactivity.â€”The hypoxanthine-8-C14, spe ing with 0.1 N hydrochloric acid. The further steps cific activity 5.4 mc/mmole, and glycine-2-C14, in the precipitation of the combined RNA and specific activity 2.08 me/inmole, were obtained DNA, deproteinization, separation of RNA from from Isotopes Specialty Co., California. It was DNA by alkaline hydrolysis, and determination of felt necessary to use hypoxanthine of very high specific activities of nucleic acid P32were done as specific activity to avoid excessive concentrations previously described (7). and minimize the possibility of the "tracer" com The tissue PÂ¡was isolated from the initial TCA peting with 6-MP. Even in these experiments 5 (ic. supernatant fraction by the methods of earlier of hypoxanthine resulted in 1 X 10~4 M as the work (7) but with initial ether extraction of the tracer concentration in flasks with 4 X 10~4 M TCA. 6-MP. The C14was assayed by liquid scintillation The acid-soluble purines were isolated from the counting (6). initial 5 per cent TCA supernatant solution by ion In some experiments 5-10 Â¡jc.ofP32orthophos- exchange chromatography on Dowex-50 X 8 resin, phate was added to each flask as an internal stand 200-400 mesh, with either stepwise or gradient ard for comparison of the rates of nucleic acid syn elution with hydrochloric acid. In the stepwise thesis. P32 was obtained as high specific activity elution of Dowex-50 columns the hydrochloric orthophosphate from Oak Ridge, Tenn. The radio- acid concentrations employed for elution of the various purines were: xanthine, 0.5 N; hypoxan 3 The "Tris-salts buffer" used for cell incubations contained : thine, l N; 6-MP, l N (follows hypoxanthine); NaCl, 0.125 M; KC1, 0.005 M; MgCl2, 0.001 M; Tris, 0.05 si; guanine, 2.5 N; and adenine, 4 N. Unhydrolyzed and glucose, 0.0056 M, at pH 7.6. acid-soluble fractions contained significant xan 4 Tlie 6-mercaptopurine was kindly provided by Dr. George thine, hypoxanthine, and the 6-MP. They yielded Hrtchings of Burroughs Wellcome & Company, Tuekahoe, New York. only traces of adenine and guanine. The nucleo-

lu. Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. DAVIDSONâ€”Mechanism of Action of 6-Mercaptopurine 227 tides of the acid-soluble fractions which failed to glycine equal to 1000 times the amount of glycine- bind to Dowex-50 resin from 0.2 N hydrochloric C14employed in the incubations. The purines were acid were hydrolyzed in l N hydrochloric acid at recovered from the washed silver precipitates by 100Â°C.The hydrolysates were chromatographed dissociation in l N hydrochloric acid and were on Dowex-50 and yielded traces of xanthine, no then subjected to Chromatographie separation on hypoxanthine or 6-MP, and appreciable amounts Dowex-50. of adenine and guanine. For separation of purines RESULTS in acid hydrolysates of total acid-soluble fractions, In the first experiments with hypoxanthine-8- linear gradient elution (14) on Dowex-50 was C14 and L1210 cells with 6-MP L1210S and found superior to the stepwise system. With L1210R were employed in a 2-hour incubation 12 X 1 cm. resin bed columns a suitable gradient (Table 1). In this there were six flasks incubated was obtained with 400 ml. of 4 N hydrochloric acid per strain of cells. The values cited are the aver being fed into a mixer chamber containing 400 ml. ages of duplicate determinations for the controls TABLE 1 EFFECTOFG-MERCAPTOPUHINEONTHEINCORPORATIONOFHYPOXANTHINE-S-CÂ» INTONUCLEICACIDSOFSENSITIVEANDRESISTANTL1210 LEUKEMIACELLSinVitro

HYPOXANTHINE(C11or /MIN/MMOLEX10-Â«)RNAAdenine45.526.4(58%)*20.4(45%)5.17.63(12%).40(8%)Guanine28.626.6(93%)24.4(85%)5.78.72(12%).45(8%)DNAAdenine9.4COUNTS

STRAINL1210-sensitiveControl6-MPLEUKEMIA

76.0(64%) M6-MP8X10-

Each flask contained 540 cu. mm. of cells incubated 2 hours in 10.4 ml. buffer. WithLl210S there was 5.0 /tc. hypoxanthine-8-C14 per flask to give 1 X 10~4Mhypoxanthine. Because of the high incorporation in the L1210S study, only 2.5 Â¿tc.hypoxanthinewas used in the L1210R ex periment. Each figure represents the average of results obtained from separate analyses of duplicate flasks. *Figures in parentheses represent per cent of control value.

0.1 N hydrochloric acid. RNA and DNA purines and the same for each of two 6-MP concentrations. were isolated by hydrolysis of the RNA nucleo- The duplicates agreed within 5 per cent. In the tides for 1 hour and the DNA for 20 minutes in study with L1210S, 6-MP inhibited the incorpora l N hydrochloric acid at 100Â°C.After dilution to tion of hypoxanthine into adenine moieties of both 0.25 N these hydrolysates were loaded onto Dow RNA and DNA by about 50 per cent and did not ex-50 columns with appropriate hypoxanthine affect the incorporation into guanine moieties. In (200 /Â¿g.)and/or nonradioactive orthophosphate the case of L1210R the depression of utilization (2 mg. P) as carrier scavengers of possible con was more profound and involved both guanine and taminating radioactivity from the tracer com adenine moieties equally. Owing to the high order pounds employed in the incubations. The purines of utilization of hypoxanthine observed in the were eluted as described for the chromatography L1210S study, the amount of hypoxanthine used of the acid-soluble fraction. The various purine in the subsequent L1210R experiment was re fractions were quantitated by ultraviolet spectro- duced by one-half. This, plus the fact that the photometry, and appropriate aliquota were count studies involved crops of different cells and were ed for C14as described. performed separately, makes any direct compari In the glycine-2-C14 experiments, all purine son of the degree of hypoxanthine utilization by fractions were processed through silver purine pre the two cell lines only suggestive. cipitation in the presence of added nonradioactive To evaluate the apparently greater utilization

of hypoxanthine by L1210S, a critical study of the label into the DNA adenine as into DNA gua two cell lines without 6-MP was done (Table 2). nine, while L1210R showed a 2:1 ratio in the In this experiment identical packed volumes of opposite direction. The RNA data were not as 5-day growth of L1210S and 7-day growth of dissimilar for the two lines of cells as the DNA L1210R were incubated and analyzed, each in data but again indicated significantly poorer duplicate and simultaneously. Orthophosphate-P32 utilization of hypoxanthine by L1210R. was included with the hypoxanthine-C14 tracer as The experiments with hypoxanthine in L1210S an internal standard to measure the rates of nu- showed that 6-MP caused inhibition of utilization of the hypoxanthine that appeared to be specific TABLE2 for adenine moieties. Since this might reasonably COMPARISONOFTHE RELATIVEUTILIZATIONOFHYPO- be owing to a blocking of the conversion of ino- XANTHINE-8-C"BYL1210SANDL1210RFORNU- sinic acid to adenylic acid (Chart 1), this was fur CLEicACIDFURINEFORMATIONinVitro ther investigated with glycine-2-C14 used as a tracer of the de novobiosynthesis of purines (Table L1210R 3). In the case of L1210S, 6-MP produced signifi (counts/min/*tmo]e72.5 LH10S:L1210R0.70.7cant inhibition of incorporation of glycine into the adenine of both RNA and DNA with no effect L1210 inorganic P85 upon utilization for guanine moieties. These ob RNA PÂ» .55 .74 DNAPÂ»Acid-soluble servations are qualitatively analogous to those .143706.432.481.05.174401.70 0.80.83.8 found with hypoxanthine as the tracer. With hypoxan L1210R the 6-MP was entirely without effect upon thineRNA glycine utilization, in direct contrast to its pro adenine-C14 found inhibition of all incorporation of hypoxan RNAguanine-C14DNA .89.06 2.817.5 thine in these cells. These results are consistent adenine-C14 with the 6-MP block in L1210R being located on DNA guanine-C14L1210S .5598.0 .11Ratio 5.0 the pathway from free hypoxanthine to inosinic acid and with this pathway being incapable of pro Each figure represents the average of separate analyses of ducing sufficient 6-mercaptopurine ribotide to duplicate flasks. Each flask of both cell lines contained the cause inhibition of the conversion of inosinic acid equivalent of 900 cu. mm. packed cells in a total volume of 10.2 ml., Tris-salts buffer,3 6.5 inc.P31,and 2.32 nc. hypoxan- to adenvlic acid. thine-8-C14(final concentration 4.55 X 10~6M).

cleic acid synthesis without regard to the sources of purine moieties. The specific activities of the tissue Pi fractions isolated at the end of the 1-hour XANTHYLIC CUANYLIC incubation serve as a measure of the comparability AGIO * "*"XACIO of the P32available to the two cell lines for nucleic acid synthesis. The two were comparable, and XANTHOSINE GIUNCHINE specific activities of the RNA and DNA phos phorus were grossly similar, with L1210R having incorporated slightly more P32 than L1210S. To assure that no considerable difference existed in endogenous hypoxanthine pools in the two cell preparations, the acid-soluble hypoxanthine frac CHART1.â€”Nucleicacid purine interrelationships tions were recovered and shown to conform to about the same order of equivalence exhibited by In these same glycine experiments P32 was in the PÂ¡and nucleic acid radiophosphates. In the cluded in the incubations, as in the experiments of face of the similarity of the rates of nucleic acid Table 2, to enable more valid comparison of the synthesis in these preparations of the two lines of relative extent of glycine utilization by the two cells, the incorporation of hypoxanthine into the lines of leukemic cells (Table 4). In these studies DNA adenine of L1210S was 20 times as great the L1210S experiment was done at a different as in L1210R. Incorporation into DNA guanine time than the experiment with L1210R, and the showed a 5:1 differential in the same direction. amounts of cells were unfortunately not equal The pattern of utilization of hypoxanthine in (Table 3, footnotes). The specific activity of the the two cell lines was also qualitatively different inorganic phosphorus from L1210R was about in that the L1210S incorporated twice as much twice that from L1210S. This difference is not

strictly a function of the smaller volume of fects of 6-MP upon them as did the nucleic acid L1210S cells employed, and the disparity may re purine data. A further advantage to the use of flect differences in the sizes of the cells, degree of these acid-soluble purines was that they provided packing, the relative content of PÂ¡,or differences about 10 times the counting rates given by the in equilibria of the PÂ¡poolswith other labile phos analogous RNA purines. For these studies, ali- phate pools. The DNA-P32 specific activities were quots of a large crop of each line of cells were unequal in the opposite direction, with DNA from pipetted into eight incubation flasks. Two of these L1210 50 per cent higher than that of L1210R. flasks were processed as controls without any This more rapid DNA synthesis in L1210S should 6-MP. The remaining six had 6-MP concentra perhaps be said to be 3 times the L1210R rate tions which increased by a factor of 3.3 for each after allowance for the respective PÂ¡specific ac successive flask. These were the only experiments tivities. In this framework there is no evidence in which duplicate flasks were not employed for that the resistant leukemia, which used hypoxan- each determination. The results of separate ex-

TABLE 3 EFFECTOFO-MERCAPTOPURINEONTHE INCORPORATIONOFGLYciNE-2-C14INTOTHE NUCLEICACIDPURINESOFSENSITIVEANDRESISTANTL1210 LEUKEMIACELLSinVitro

INCORPORATIONopOLTCiNB-2-CÂ» (CÂ» COCÃ•T8/MIN//JMOLEX10-Â»)

STRAINLl210-sensitiveLECKEMU

Control 6-MP3.2X10~4M 10.5(63%)9.4(57%)11.212.2(109%)8.2(106%) MLl210-resistant6-MP 6. 4X 10-Â« 70(59%)191192(100%)39.1(92%)675664(99%) 7.8(101%)69

Control 6-MP3.2X10~4M 70(100%) 6-MP6.4X10~4MRNAAdenine11879(67%)*194(101%)Guanine42.339.1(92%)620(93%)DNAAdenine16.611.4(102%)Guanine7.770(100%)

Each flask contained 12.3 ml. of cell suspension with 5 pc. glycine-2-C14in Tris-salts buffer. The packed-cell volumes represented per flask were 1150 cu. mm. for L1210S and 775 cu. mm. forLl210R. Each figure represents the average of results obtained from separate analyses of duplicate flasks. * Figures in parentheses represent per cent of control value.

thine so much less than the sensitive leukemia, is TABLE4 compensating for this by greater dependence on COMPARISONOFTHERELATIVEUTILIZATION the de novo production of adenine moieties. In the OF GLYCINE-2-C" BY L1210S AND L1210R case of guanine moieties, however, there is a very FOR NUCLEICACID FURINE FORMATION in Vitro much greater utilization of glycine in L1210R than in L1210S. To explore the relationship between 6-MP con L1210S L1Z10R (countÂ»/mln,Vmole centration and the utilization of hypoxanthine by X10-Â») L1210 cells, experiments were performed in which Inorganic phosphorus UO 307 each line of cells was incubated for 1 hour with a RNA P32 1.19 1.52 series of concentrations of 6-MP. To facilitate this DNAPÂ» .31 .20 work the measurements of specific activity were RNA adenine C" 118 191 made on purines derived from the nucleotides of RNA guanine C14 42.3 675 the acid-soluble fractions of the cells rather than DNA adenine CÂ» 16.6 11.2 from isolated RNA and DNA. Such isolations had DNA guanine C14 7.7 69.0 been done in experiments of Table 1, and the re sultant data showed the same interrelationships These data are from the same experiments represented in Table 3. Each flask had 7.3 /Â¿c.P32 of adenine and guanine specific activities and ef- in it, and only the control flasks are considered.

periments with L1210S and with L1210R are extract increased with increasing 6-MP concentra plotted in Chart 2. In these experiments the tion to an amount which at the highest 6-MP con "tracer" hypoxanthine amounted to 4.2 X 10~5M centration was 2j times that of the control flask in all flasks, and no significant effect of the 6-MP content. The specific activity of the hypoxanthine, was apparent until its concentration approached however, remained quite constant and did not fall or exceeded this value. In L1210S the first two ef below the control flask values. The increase in the fective levels, 1.08 X 10~4 and 3.6 X 10~4, de amount of hypoxanthine may have been due to pressed incorporation into the adenine without two factors: (a) desulfuration of 6-MP itself to affecting the guanine significantly. At higher con yield hypoxanthine (3) and (6) inhibition by centrations the guanine was also affected. In the 6-MP of enzymic conversion of hypoxanthine to case of L1210R even the lowest concentration of other compounds. These two factors could operate simultaneously to preserve the specific activity of the hypoxanthine, since increased dilution with LI2IOS hypoxanthine arising from 6-MP could be offset by a reduction of endogenous dilution due to in o X 2 hibition of enzymes. X N.a. DISCUSSION s r The results of these studies can best be inter preted by referring to the diagram of metabolic O IO"5 IO"4 IO"5 pathways given in Chart 1 and by considering 6-MERCAPTOPURINE(molanty) them in relation to data from a number of recently published papers from other laboratories. The literature on 6-MP has very recently been exten sively reviewed by Mandel (13) and by Hand- O schumacher and Welch (10). X 2 Recent studies have indicated that in tumors sensitive to 6-MP, the 6-MP is metabolized to its ribotide and that in several sublines of tumors and microorganisms the development of resistance to O 10"Â° IO'* IO"3 6-MP has correlated with loss of the ability to 6- MERCAPTOPURINE(molanty) produce 6-MP-nucleotide (1, 15). It was further shown that this deficiency of L1210R is not due to CBAKT2.â€”Effectof 6-mercaptopurine on the incorporation of hypoxanthine-8-C14 into acid-soluble nucleotide purines of failure of 6-MP to enter the cells (5) and is ap sensitive and resistant L1210 leukemia cells in vitro. parently due to loss of active enzymic path The control figures represent the averages of results ob way (2). tained from separate analyses of duplicate flasks. Only single The present studies indicate that in L1210S, flasks were used for each concentration of 6-MP. where 6-MP-R-P is produced, both hypoxanthine With both lines of cells each flask contained 800 cu. mm. of cells incubated 1 hour in 11.0 ml. Tris-salts buffer. There was (Table 1) and glycine (Table 3) are inhibited in 2.8 tie. hypoxanthine-8-C14 per flask to give 4.2 X 10~6 M their incorporation into adenine nucleotides. The hypoxanthine. apparent common pathway for these two precur sors lies between IMP and AMP. Adenylosuccinic 6-MP depressed incorporation of hypoxanthine- acid appears to be an intermediary in this reaction 8-C14into both adenine and guanine, and the de sequence (12), and in the experiments of Chart 2 pression ran fairly parallel and linear with increas the purine base of this intermediate, 6-succinami- ing 6-MP concentration. These data are all in the nopurine, was sought directly and with carrier form of unadjusted specific activities of the isolat 6-succinaminopurine. The native compound could ed purines. Incidental to the isolation of the ade not be isolated, and synthetic material added as a nine and guanine it was expedient to isolate such carrier to the acid hydrolysate of the acid-soluble other purines as were moderately abundant in the nucleotides was recovered devoid of radioactivity. acid-soluble extracts. These included xanthine and Carter (4) has indicated that 6-succinaminopurine hypoxanthine. Some of the hypoxanthine repre might be partially destroyed by the acid hydroly sented recovered tracer compound, and the xan sis, so that this, or perhaps an even greater acid thine was radioactive because of its partial deriva lability of adenylosuccinic acid, may account for tion from labeled hypoxanthine. The amount of the failure to demonstrate the passage of hypoxan- hypoxanthine recovered from the acid-soluble cell thine-C14 through adenylosuccinic acid in this sys-

tern. In any event, no evidence was obtained Streptococcus faecalis. They found 50 per cent in whether 6-MP caused its inhibition before or after hibition of the conversion of IMP to "succino- adenylosuccinic acid. adenylic acid" (adenylosuccinic acid) by an equi- In L1210R Brockman et ai.5 found no formation molar concentration of 6-MP-R-P. This appears of 6-MP-R-P and no measurable conversion of analogous to the situation observed in the present hypoxanthine to inosinic acid. It was anticipated work with L1210S and further defines the locus of that the present studies might similarly show no the inhibition which our work could only indicate utilization of hypoxanthine on which to measure to lie between IMP and AMP. a 6-MP effect. Table 1 shows that hypoxanthine This paper has shown that one of the actions of was used but that use of it for both adenine and 6-MP in cells sensitive to the antitumor activity guanine nucleotide synthesis was very profoundly of 6-MP is to inhibit the production of adenine inhibited by 6-MP. A hypothesis to explain this is nucleotides. The mechanism by which this may that there is a very limited amount of inosinic acid inhibit growth has not been defined. A shortage of pyrophosphorylase enzyme in L1210R. This adenine nucleotides could not only inhibit nucleic would perhaps lead to competition between 6-MP acid synthesis but could impair numerous reac and hypoxanthine for the available enzyme with tions that require adenine-containing co-factors. consequent inhibition of the incorporation of hy The results relative to 6-MP resistance in L1210 poxanthine. The same paucity of enzyme could be leukemia are more explicit. From the data given responsible for insufficient conversion of 6-MP to here and those published by others, resistance cor 6-MP-R-P to induce the internucleotide block be relates with the loss by the cell of the capacity to tween IMP and AMP. The absence of this latter convert free 6-MP to its nucleotide derivative, block would explain why 6-MP had no effect on which appears to be the form responsible for anti- glycine utilization in L1210R (Table 3). The defi tumor activity. This mechanism of resistance ciency of inosinic acid pyrophosphorylase in seems to be a rather general one, since it has been L1210R was indicated by Brockman et al. (2) and demonstrated in several tumor and bacterial cell seems apparent in the very poor utilization of systems with respect to both purine and pyrimi- hypoxanthine by L1210R as compared with dine analogs (1, 9, 16). This raises a serious ques L1210S in Table 2. tion whether all purine and pyrimidine anti- The dose-response experiments with 6-MP in metabolites may not be destined to frequent fail L1210 with the use of hypoxanthine-C14 (Chart 2) ure as antitumor agents owing to this mechanism. were undertaken to determine whether the types Since nucleotides do not appear to be able to enter of inhibition found in L1210S and in L1210R, re mammalian cells, it may be desirable to direct spectively, could be demonstrated at different efforts toward preparing derivatives of analog drug concentrations in the converse leukemias. It nucleotides in the hope of obtaining compounds does appear that extremely high concentrations of that will enter cells and still not require metabolic 6-MP can inhibit the incorporation of hypoxan conversion to nucleotides before becoming active. thine into guanine nucleotides in L1210S just as ACKNOAVLEDGMENTS lower concentrations do in L1210R. This can be The author wishes to express his appreciation to Mrs. visualized as an overwhelming amount of 6-MP Elizabeth Marsden for her able technical assistance. successfully competing with hypoxanthine for a normally abundant but finite amount of inosinic REFERENCES acid pyrophosphorylase. In the case of L1210R, 1. BROCKMAN,R.AV.;SPARKS,M. C.; and SIMPSON,M. S. however, even the lowest concentration of 6-MP, A Comparison of the Metabolism of Purines and Purine Analogs by Susceptible and Drug-resistant Bacterial and which was ineffective in L1210S, produced inhibi Xeoplastic Cells. Biochim et. Biophys. acta, 26:671-72, tion of incorporation of hypoxanthine into both 1957. guanine and adenine moieties. This effect on gua Ã.BROCKMAN,R. AV.; SPARKS,M. C.; SIMPSON,M. S.; and nine moieties at low 6-MP concentration would SKIPPER,H. E. Decreased Ribonucleoside Pyrophosphor- seem to favor the hypothesis that all effects in ylase Activity of Streptococcusfaecalis and L1210 Leukemia Resistant to Purine Antagonists. Biochem. Pharm., 2:77- L1210R are being mediated through a blocking of 79, 1959. its limited content of inosinic acid pyrophosphor 3. CARET,N. H., and MANDEL,H. G. Relation between the ylase. Inhibition of Growth of Bacillus cereus and Metabolism of Salser and Balis (17) have recently reported 6-Mercaptopurine. Fed. Proc., 18:200, 1959. 4. CARTER,C. E. Synthesis of 6-Succinaminopurine. J. Biol. studies of the effects of synthetic 6-MP-R-P on Chem., 223:139-46, 1956. nucleotide interconversions in cell-free extracts of 5. DAVIDSON,J.D. Permeability of Resistant L1210 Leuke mia Cells to 8-Azaguanine and 6-Mercaptopurine. Proc. 1Personal communication from Dr. R. AA".Brockman. Am. Assoc. Cancer Research, 2:290, 1958.

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Jack D. Davidson

Cancer Res 1960;20:225-232.

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