of RNA Cytosine and RNA : Differential Inhibition by Diazo-Oxo-Norleucine*


(Department of , 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 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 were about 10 times the concentrations required to in hibit completely the biosynthesis de novoof 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 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 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 biosynthetic the biosynthesis of purines and the amination of pathways. The objectives were to attempt to ex 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 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 (6, 12) and the conversion levels of DON, followed by measurement of the of phosphate to 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

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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 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

Glycine only 0

* 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 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-


SPECIFICACTIVITYOFRNA NUCLEOTIDES CONDITIONS (coCNTS/ifiN/VUOLE) (additions in Amóles) (per cent inhibition in parentheses)

Pre-incubationCells with acid-C14cubation:orotic not washed after pre-ir 1. No addition Glutamine,10No 0%)4,200 (0%)170 a. Glutamine, 10 DON, 0.14 further addition (-25%) (-66%) 3. DON,0.14Cells 10ation:Glutamine, 3,500(-38%)4,800 20(-96%)550 washed after pre-incub 4. No addition Glutamine, 10 ( 0%) ( 0%) 5. DON, 0.14Incubation Glutamine, 10Uracil5,600( 3,500 (-27%)Cytosine50050 (-91%)

The pre-incubation was for 15 minutes at 38°C.in the Krebs-Ringer phosphate medium. The final conditions for the 2-hour incubation were the same as in Table 2.

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incubated with the cells in the absence of gluta- glutamine for the amination of uridine nucleotides. mine. The inhibition was not reversed by the sub A recent report (17) that exposed to sequent addition of glutamine, even when the azaserine contain more glutamine than the con DON was removed from the medium. Glutamine trols lends support to this explanation. was, however, capable of delaying or partially The 10-40 per cent inhibition by DON of the preventing the inhibition when added simul incorporation of C14from orotic acid into RNA taneously with DON. It appears that in these may represent an actual decrease in RNA intact cells DON inhibits irreversibly the forma synthesis. This inhibition was minimal at levels of tion of RNA cytosine and that glutamine com DON which inhibited only purine nucleotide petes with DON for the site of the inhibition. formation and was maximal at levels which in These effects are similar to those observed for hibited both purine and cytidine nucleotide forma inhibition by DON of de novo purine biosynthesis tion. This observation may be related to the fact in intact cells (13), the formation of formylglycin- that the pre-existing intracellular pool of free amidine ribotide in a soluble enzyme system (12), cytidine nucleotides is small (11, 16) and much less- and the animation of uridine nucleotides to cyti- able to support continued RNA synthesis than the dine nucleotides in a soluble enzyme system (8). large pool of free nucleotides. It is of interest that these cells, despite complete inhibi DISCUSSION tion of de novo formation of purine and cytidine It is assumed that the observed specific activi nucleotides, were able to maintain synthesis (or ties of the RNA nucleotides are directly related to turnover) of RNA at 60% of their usual rate. It the isotope content of their corresponding free may be judged that the relative effectiveness of nucleotide precursors and therefore represent the DON in inhibition of growth of tissues and cells rates of biosynthesis de novo of the free cytidine may be diminished by the availability of pre nucleotides and purine nucleotides. This assump existing nucleotides or of nucleotides from sources tion is in accord with current concepts and with other than the de novo pathways. other analytical data obtained with these cell suspensions, part of which has been reported (11). ACKNOWLEDGMENTS It has also been previously shown that DON does The authors appreciate the valuable assistance of Mis» not affect the conversion of orotic acid to uridine Audrey Posey. nucleotides (11). REFERENCES From Table 2 it may be estimated that the 1. ABRAMS,R., and BENTLEV,M. Biosynthesis of Nucleic minimum amount of DON required for complete Acid Purines. III. Guanosine-5'-phosphate Formation inhibition of cytosine labeling in the absence of from Xanthosine-S'-phosphate and L-Glutamine. Arch. Biochem. & Biophys., 79:91-110, 1959. exogenous glutamine is probably of the order of 0.01 jumóle(10-%). Table 1A shows that under 2. ACKERMANN,W.W., and POTTEB,V. R. Enzyme Inhibi tion in Relation to . Proc. Soc. Exper. Biol. similar conditions purine labeling was inhibited 80 & Med., 7:21-9, 1949. per cent by 0.00075 Minole(6 X 10-8M). These re 8. BIRNS,M.; ESSNER,E.; and NOVIKOFF,A. B. The Cyto sults indicate that at least 10 times as much DON chemistry and Fine Structure of the Novikoff Hepatoma in Ascites Form. Proc. Am. Assoc. Cancer Research, 3:7, was required to inhibit the biosynthesis of cyti 1959. dine nucleotides as to inhibit the biosynthesis of 4. EHRLICH,J.; COFFEY,G. L.; FISHER,M. W.; HILLEOAS, purine nucleotides. A factor of 10 or more may be A. B.; KOHBERGER,D.L.; MACHAMER,H. E.; RIGHTSEL, similarly estimated for the inhibitions in the W. A.; and ROEGNER,F. R. 6-Diazo-5-oxo-L-norleucine, a New Tumor-inhibitory Substance. I. Biologie Studies. An- presence of glutamine. tibiot. & Chemotherapy, 6:487-97, 1956. As suspected previously (11), the low amounts 5. EIDINOFF,M. L.; KNOLL,J. E.; MARAÑO,B.;and CHE- of DON which cause the anomalous stimulation of ONG,L. Pyrimidine Studies. I. Effect of DON (6-Diazo-5- cytidine nucleotide formation do correspond to oxo-L-norleucine) on Incorporation of Precursors into those effective in inhibition of purine nucleotide Nucleic Acid Pyrimidines. Cancer Research, 18:105-9, formation. Our explanation of the anomaly then is 1958. that a progressive irreversible "titration" (2) of 6. GOLDTHWAIT,D.A. 5-Phosphoribosylamine, a Precursor of Glycinamide Ribotide. J. Biol. Chem., 222:1051-68, competing for glutamine occurs and that 1956. inactivation of the more sensitive2 enzyme per 7. GREENLEES,J., and LEPAGE, G. A. Purine Biosynthesis and Inhibitors in Ascites Cell Tumors. Cancer Research, mits an increased availability of intracellular 16:808-18, 1956. *Close comparison of Fig. 6 (in [12]) with Table 4 (8) sug 8. HURLBERT,R. B., and KÄMMEN,H. O. Formation of gests also that the formation of formylglycinamidine ribotide is Cytidine Nucleotides from Uridine Nucleotides by Soluble more sensitive to DON than the formation of cytidine nucleo Mammalian Enzymes: Requirements for Glutamine and tides. Guanosine Nucleotides. J. Biol. Chem., 236:448-49, 1960.

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9. HURLBERT,R. B.; SCHMITZ,H.; BRUMM,A. F.; and POT 11. MOORE,E. C., and LEPAGE,G. A. In Vivo Sensitivity of TER, V. R. Nucleotide Metabolism. II. Chromatographie Normal and Neoplastic Mouse Tissues to Azaserine. Can Separation of Acid-Soluble Nucleotides. J. Biol. Chetn., cer Research, 17:804-8, 1957. 209:-23-39, 1954. 15. NOVIKOFF,A. B. A Transplantable Rat Liver Tumor In 10. KÄMMEN,H.O., and HUHLBERT,R.B. Amination of TJri- duced by 4-Dimethylaminoazobenzene. Cancer Research, dine Nucleotides to Cytidine Nucleotides by Soluble Mam 17:1010-27, 1957. malian Enzymes: Role of Glutamine and Guanosine Nu 16. SCHMITZ,H.; POTTER, V. R.; HURLBERT,R. B.; and cleotides. Biochim. et biophys. acta, 30:195-96, 1958. WHITE,D. M. Alternative Pathways of Glucose Metabo lism. II. Nucleotides from the Acid-soluble Fraction of 11. . The Formation of Cytidine Nucleotides and RNA Normal and Tumor Tissues and Studies on Nucleic Acid Cytosine from Orotic Acid by the Novikoff Tumor in Vitro. Synthesis in Tumors. Cancer Research, 14:66-74, 1954. Cancer Research, 19:654-63, 1959. 17. TOMISEK,A. J.; REID, M. R.; and SKIPPER,H. E. Chro 12. LEVENBERO,B.;MELNICK,I.; and BUCHANAN,J.M. Bio matographie Studies of . IV. Reversal synthesis of the Purines: XV. The Effect of Aza-L- of Azaserine-induced Inhibition by Phenylalanine and and 6-Diazo-5-oxo-L-norleucine on Biosyn Tryptophan. Cancer Research, 19:489-93, 1959. thesis de novo.J. Biol. Chem., 225:163-76, 1957. 18. WYATT,G. R. Purines, Pyrimidines and Related Com 13. MOORE,E. C. Effects of Certain Furine Inhibitors in Nor pounds. In: E. LEDERERand M. LEDERER(eds.), Chro- mal and Xeoplastic Tissues. Dissertation, Univ. of Wis matography, p. 374. Amsterdam: Elsevier Publishing Co., consin, 1958. Dissertation Abstracts, XIX: 5, 1958. 1957.

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E. C. Moore and Robert B. Hurlbert

Cancer Res 1961;21:257-261.

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