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

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Studies on the Mechanism of Action of 6-Mercaptopurine in Sensitive and Resistant L1210 Leukemia in Vitro* 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 nucleo- tides. 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.—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.—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 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1960 American Association for Cancer Research. 226 Cancer Research Vol. 20, February 1960 growth. All dilutions were made with Locke's phosphate was counted at infinite thinness with an solution. end-window Geiger-Müller 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.—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 0°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 0°C.before being centrifuged. The super buffer3 at 2°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- P¡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 100°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.—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 P¡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 ¡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. DAVIDSON—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.
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