Biosynthesis of RNA Cytosine and RNA Purines: Differential Inhibition by Diazo-Oxo-Norleucine* E. C. MOOREANDROBERTB.HURLBERT (Department of Biochemistry, The University of Texas 31. D. Anderson Hospital and Tumor Institute, Houston, Texas) SUMMARY The effects of 6-diazo-5-oxo-L-norleucine (DON) on the conversions of orotic acid- 6-C14to RNA pyrimidines and of glycine-2-C14 to RNA purines were studied in sus pensions of Novikoff tumor cells in vitro. The concentrations of DON required to inhibit the animation step in the biosyn thesis of cytidine nucleotides were about 10 times the concentrations required to in hibit completely the biosynthesis de novoof purine nucleotides. The degree of inhibition in both cases was diminished by the presence of glutamine in the medium. The inhibition by DON of the animation step in cytidine nucleotide formation was shown to be irreversible. The effect of glutamine was to delay or partially prevent the inhibition. The formation of cytidine nucleotides was observed to be stimulated by the minimal concentration of DON effective in the inhibition of purine nucleotide formation ; an ex planation of this anomal}- is offered. 6-Diazo-5-oxo-L-norleucine (DON), an analog Page (13, 14). An anomalous effect was also noted : of glutamine, is a tumor-inhibitory antibiotic (4) whereas certain concentrations of DON inhibited and a potent and useful inhibitor of several reac completely the conversion of orotic acid to cyto tions in the biosynthesis of nucleotides. In soluble sine compounds, lower concentrations in the enzyme systems, DON completely and irreversi presence of glutamine stimulated the conversion. bly inhibits the conversion of formylglycinamid We have compared more closely the effects of ribotide to formylglycinamidine ribotide (12) in DON on purine and pyrimidine biosynthetic the biosynthesis of purines and the amination of pathways. The objectives were to attempt to ex uridine nucleotides to form cytidine nucleotides (8, plain the anomalous effect mentioned above and 10) in the biosynthesis of pyrimidines. Both of to provide data bearing on the mechanism of the these reactions utilize adenosine triphosphate antibiotic and antitumor activities of the com (ATP) and the amide group of glutamine. Two pound. This comparison was made directly and other reactions in the biosynthesis of purines also simultaneously by incubation of suspensions of inhibited in a similar way by DON (or by the less Novikoff tumor cells with C'Mabeled orotic acid potent analog azaserine) are the formation of and C14-labeled glycine in the presence of various phosphoribosyl amine (6, 12) and the conversion levels of DON, followed by measurement of the of xanthosine phosphate to guanosine phosphate isotope content of the RNA uridylic, cytidylic, (D- In a study of the utilization of orotic acid-C14 adenylic, and guanylic acids. In additional experi ments the stimulatory effect and the irreversibility in rat tumor cells in vitro (11), it was noted that the formation of cytosine compounds was less of the inhibitory effect of DON on the formation of sensitive to inhibition by DON than was the RNA cytosine from orotic acid were studied in formation of purine nucleotides from glycine-C14 more detail. in mouse tissues, as reported by Moore and Le- MATERIALS AND METHODS *This investigation was supported by a grant from the Orotic acid-6-C14 and glycine-2-C14 were pur American Cancer Society (P-146A). chased from Tracerlab, Inc., and from Volk Received for publication August 29, I960. Radiochemical Company, respectively. 257 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. 258 Cancer Research Vol. 21, February 1961 The Novikoff tumor, in both the solid (15) and ployed for determination of nucleotide concentra ascites (3) forms, was maintained in rats by intra- tions were e28o= 13,000 for cytidylic acid and peritoneal transplantation. Preparation and incu «ano=9,900, 14,200, and 11,800 for uridylic, bation of the tissues followed in general the pro adenylic, and guanylic acids, respectively. cedures used by Kämmenand Hurlbert (11) for the solid tumor and the procedures used by Moore RESULTS (13) and Greenlees and LePage (7) for the ascites The effects of DON on the formation of RNA form. The exact incubation conditions are de pyrimidines from orotic acid-6-C14 and the forma scribed with the tables. At the end of each incuba tion of RNA purines from glycine-2-C14 were de tion the cells were centrifuged and washed once termined simultaneously in the experiment re with fresh glucose-salts medium, then extracted ported in Table 1. This comparison was made and washed twice with cold perchloric acid. The possible by the knowledge that labeled orotic acid nucleic acids were extracted with hot 10 per cent is incorporated almost exclusively into pyrimidine sodium chloride. After two alcohol precipitations nucleotides and that the portion of the labeled TABLE1 EFFECTSOFDONo\ INCORPORATIONOFGLYCINE-C"ANDOROTICAcio-C14INTORNA SPECIFICACTIVITYOF RNTANCCLEOTIDES CONDITIONS (COUNTB/UIN/VMOLE) (p*r cent inhibition or stimulation in parentheses) C'«-Precur«orA.* /¿moles)0.0 Glycine only 0<n Oy.440f Glycine+orotic 0.0 l,100/( U/°; U/oJ ( 0%) ( 0%) Glycine+orotic 0.00075 210 (-82%) 180 (- 75%) 5,600 (- 3%) 600 (+ 58%) Glycine+oroticB.f 4.fi0.0 380(-67%)11,200\, 170 (-67%)6,400\, 3,300(-43%)120 60 (-84%)230 Glycine only 0<n 0(n5.100J1 Glycine+orotic 0.0 9,600/( 0%) u/°; 5,400 ( 0%) 510 ( 0%) Glycine+orotic 0.00075 8,400 (-19%) 5,300 (- 8%) 4,800 (-11%) 470 (- 8%) Glycine+orotic 0.45 980 (-91%) 390 (- 93%) 3,700 (-31%) 1,100 (+115%) Glycine+oroticDON( 4.5Adenine1,2001,550 (-95%)Guanine600\,0 (-100%)Uracil405,8003,300 (-39%)Cytosine20380260 (- 49%) * Each flask contained 0.4 ml. (packed) of Novikoff ascites tumor cells and 10.0 ml. of Robinson's medium in a total volume of 12.3 ml. with Nj-6% COSas «asphase.As noted, 1.7 /¿molesoforotic acid-6-C14(1.15X10* counts, min) and/or 2.0 /¿molesof glycine-2-C14(3.35X10«counts/min) were added. t Each flask contained 0.4 ml. (packed) of Novikoff solid tumor mince, 2.8 ml. of Krebs-Ringer phosphate medium with glu cose and bicarbonate, and 30 timólesof L-glutamine in a total volume of 4.1 ml., with O2-5% CO»asgas phase. As noted, 0.57 limole of orotic acid (0.38X10* counts/min) and/or 2.0 jumólesofglycine (3.85X106 counts/min) was added. The flasks were incubated for 2 hours at 38°C.The RNA nucleotides were prepared as described in the text. of the sodium nucleates, the RNA was hydrolyzed glycine used for nucleic acid biosynthesis is in with sodium hydroxide at 37°C.Details of these corporated primarily into the purine nucleotides. procedures have been previously published (11). The experiment consisted of two parts. In part A, The resulting RNA nucleotides were separated by suspensions of Novikoff ascites tumor cells were chromatography on Dowex-1 (formate) columns incubated anaerobically in Robinson's medium by gradient elution with formic acid (9). In under essentially the conditions used in experi Experiment I, the cytidylic acid, which was con ments on purine nucleotide biosynthesis in mouse taminated with other radioactive materials de ascites tumor cells (7, 13). In part B, minces of the rived from the glycine-2-C14, was purified by Novikoff solid tumor were incubated aerobically chromatography on paper with the isopropanol- in a modified Krebs-Ringer medium with added HC1 solvent system of Wyatt (18). The other glutamine as described in the experiments on nucleotides did not require further purification. pyrimidine nucleotide formation (11). In both The specific activity of each nucleotide was de parts the DON level was varied and the amount of termined from the amount of radioactivity and radioactivity incorporated from glycine alone into the amount of nucleotide in the center tubes of the the RNA cytosine was determined as a control. ion-exchange peak or in the central portion of the The data in Table 1 confinn the previous indi paper spot. The spectropho tome trie constants em- cations of a difference in sensitivity to DON of the Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. MOOREANDHuRLBERT—InhilñtionbyDON of Cytosine and Furine Formation 259 systems forming RXA cytosine and RNA purines. (flasks 3 and 5). Then glutamine was added to The incorporation of glycine into the RNA purines flasks 1, 3, 4, and 5, radioactive orotic acid was of 0.4 ml. (packed volume)1 of tumor was almost added to all, and the incubation was continued completely inhibited by 0.00075 p moles of DON (6 under the conditions of Table 2 for 2 hours. In X 10-8M) in the absence of exogenous glutamine flasks 4 and 5, the cells were washed free of the pre- (part A), or by 0.45 jumólesofDON (1 X 10-<M) incubation medium before the incubation in fresh in the presence of 30 /¿molesofglutamine (part B), medium with orotic acid and glutamine. This while the incorporation of orotic acid into RNA amount of DON, selected from Table 2 to cause cytosine Was stimulated by these lower levels of only partial inhibition in the presence of gluta- inhibitor. A high level, 4.5 Amóles,of DON in mine, caused a complete inhibition when pre hibited the formation of both cytosine and purines under both sets of conditions. TABLE 2 A closer estimation of the amounts of DON re STIMULATIONANDINHIBITIONOFRNA quired to stimulate and inhibit cytidine nucleotide CYTOSINEBIOSYNTHESISBYDON biosynthesis alone was provided by the experi ment reported in Table 2.