Some Metabolic Effects Exerted by Azaserine and Purine Analogs in Vivo@ @ ALAN C. SARTORELLI, HERBERT F. UPCHTJRCH,t ALLAN L. BIEBER, AND BARBARA A. BOOTH (Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut) SUMMARY The biochemical mechanisms involved in the synergistic anti-neoplastic activity of combinations of azaserine and certain purine analogs was studied in a subline of Sar coma 180 that neither was inhibited by purine analogs alone nor responded in a synergic manner to their combinations with azaserine. 6-Chloropurine, 6-thiogua nine, 6-mercaptopurine, and their ribonucleosides inhibited the incorporation of glycine-2-C4 into nucleic acid guanine of Sarcoma 180; in addition, the purinethiols decreased the incorporation of glycine into nucleic acid adenine of this neoplasm. In the purine analog-resistant neoplasm (Sarcoma 180/TG), the analogs did not decrease the utilization of isotopic glycine for the biosynthesis of guanine nucleotides; however, 6-thioguanine and 6-thioguanosine both inhibited the formation of adenine nucleotides de novo. Azaserine markedly depressed the synthesis of purine nucleotides de novo in both tumors. 6-Thioguanine and its ribonucleoside decreased the conversion of both hypoxanthine-8-C'4 and guanine-8-C'4 to nucleic acid purines in both cell lines. Thioguanine mononucleotide was formed from thioguanine by both tumors; however, considerably more thioguanine nucleotide was present in the sensitive cells than in the resistant ones. This difference appeared to be attributable to a greater capacity of Sarcoma 180 to maintain an intracellular concentration of analog nucleo tide. Pretreatment with azaserine caused an increase in the quantity of acid-soluble thioguanine nucleotide, as well as an increase in the amount of 6-thioguanine associated with the nucleic acids in both neoplasms. Combinations of azaserine (O-diazoacetyl-L-serine) and tribute to the carcinolytic activity of the drug combination certain purine analogs have been shown to produce a syner by : (a) incorporation into the nucleic acids, (b) interference gi8tic retardation of the growth of malignant cells; the use with the interconversion of purine nucleotides, or (c) in of these combinations on transplanted rodent neoplasms hibition of the de novo biosynthetic pathway for purine has been reviewed (31). The precise biochemical altera nucleotides ; none of these possibilities should be regarded tions responsible for the observed drug synergism have not as mutually exclusive. been elucidated, although metabolic effects induced by To gain information pertinent to the mode of action of these agents employed singly have received considerable combinations of azaserine and purine analogs, a subline of study and have been discussed in recent reviews (5, 14, 23, Sarcoma 180 was developed in which purine analogs neither 26, 28, 31, 43, 44). The metabolic alteration deemed re retarded growth nor enhanced the inhibitory activity of sponsible for the tumor-inhibitory effect of azaserine is the azaserine. Some biochemical effects of these agents on blockade of purine nucleotide biosynthesis de novo. Re purine metabolism and the biotransformations of thio cent evidence, however, has suggested that this site of guanine were compared in the parent neoplasm and in the action may be responsible only in part for the synergic in resistant variant. hibition demonstrated by combinations of azaserine and 6- chloropurine (38). 1\IATERIALS AND METHODS It has been suggested that various purine analogs con Tumor transplantation.—Experirnents were conducted * This research was supported in part by Grant CA-02817 from with Sarcoma 180 and a purine analog-resistant subline the National Cancer Institute, Public Health Service. (Sarcoma 180/TG) ; these neoplasms were maintained and t Present address : Biomedical Division, The Samuel Roberts employed throughout these experiments as ascitic growths Noble Foundation, Inc., Ardmore, Oklahoma. in female Ha/ICR Swiss mice 9—12weeks of age (A. R. @ Postdoctoral support provided by a Public Health Service Graduate Training Grant (CRTY-5012) ; present address, Depart Schmidt Co., Inc.). Transplantation of ascites cells was ment of Chemistry, Arizona State University, Tempe, Arizona. carried out by withdrawing peritoneal fluid from a donor Received for publication February 7, 1964. mouse bearing a 7-day tumor growth. The suspension was 1202 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1964 American Association for Cancer Research. SARTORELLI et al.—Effects of Azaserine and Purine Analogs 1203 centrifuged for 2 mm. (1600 X g), the supernatant pen C'4 (Tracerlab, Inc., 1.06 X 10@counts/min/@ig), 50 @tg.of toneal fluid was decanted, a tenfold dilution with isotonie adenine-8-C'4 (Calbiochem., 1.7 X 10@counth/min/@tg),50 saline was made, and 0.2 ml. of the cell suspension was in /Lg.of guanine-8-C'4 (Calbiochem., 1 X 10@counts/min/,@g), jected intrapenitoneally into each recipient animal. or 50 pig. of hypoxanthine-8-C'4 (Nuclear-Chicago Corp., Development of resislant subline (Sarcoma 180/TG).—The 9.4 X 10@counts/min/@ig), and 1 hour was allowed for in selection of a resistant population was achieved by the pro corporation of all isotopic substrates with the exception of cedures described earlier (9). Approximately 2 X 10@as guanine-8-C4, for which 20 minutes was allowed. The cites cells were transplanted into mice receiving three cells were then harvested, and the mixed nucleic acid and consecutive daily doses of a combination of 0.2 rng. of acid-soluble punines were isolated and their specific ac azaserine/kg and 40 rng of chloropurine/kg beginning 24 tivities determined (17, 32). hours after tumor implantation. After suitable time was Metabolism of 6-thioguanine.—Metabolites of thiogua allowed for the cell population to accumulate, tumor cells nine were isolated from acid-soluble extracts on columns from one such mouse were used for transplantation into a of Dowex-1-formate by a modification of the method of second group of animals. Resistance to purine analogs Moore and LePage (25). Thioguanine and thioguanosine when employed either alone or in combination with aza were eluted simultaneously, with 1 M formic acid ; then, senine was manifest by the 35th transplant generation. At with use of 4 M formic acid, 6-thiouric acid and thioguanine the 40th transfer, Sarcoma 180/TG was maintained on 0.2 nucleotide were separated as two distinct peaks. Concen lug. of azasenine/kg and 40 mg. chloropunine/kg for 2 con trations of metabolites were determined by measurement of secutive days beginning 24 hours after tumor implantation. the ultraviolet absorption in acid solution at 345 mz ac At generation 57, a subline of Sarcoma 180/TG was started cording to the following extinction coefficients : thioguanine which did not receive maintenance therapy with azaserine 17.0 X 10@;thiounic acid, 22.5 X 10@;and thioguanine and chionopunine ; during twelve consecutive transfers in nucleotide, 20.6 X 10g. Thioguanine associated with the the absence of the drug, the variant line retained its in nucleic acids was isolated as described in an earlier publi sensitivity to punine analogs; thus, the change was consid cation (41). ened heritable and relatively stable. All experiments were conducted with the subline of Drug therapy.—For any one experiment animals were Sarcoma 180/TG receiving maintenance doses of azaserine distributed into groups of five to ten mice of comparable and chioropurine and were carried out with transplant weight and maintained throughout the course of the cx generations 44 to 178. peniment on Purina Laboratory Chow pellets and water ad libitum. Each drug or drug combination was tested 2 or RESULTS more times, and the results obtained were averaged. The effects of several purine analogs and other inhibitors Drugs were administered by intrapenitoneal injection of nucleic acid biosynthesis on the survival time of mice beginning 24 hours after tumor implantation once or twice bearing either Sarcoma 180 or Sarcoma 180/TG ascites daily for 6 consecutive days, with combination treatments cells were measured ; the results are shown in Table 1. The administered simultaneously. Azaserine, 5-fluorouracil, glutarnine antagonist azaserine inhibited the growth of chioropunine, and 6-chioropurine nibonucleoside were ad both neoplasms to the same extent; chioropurine did not ministered in solution in isotonic saline, whereas amethop inhibit either tumor when given in daily doses up to 400 tern was dissolved in isotonic sodium bicarbonate. Other mg/kg (35), whereas thioguanine, thioguanosine, 6-mer purine analogs and their nibonucleosides were solubiized in captopurine, and 6-mercaptopunine ribonucleoside pro isotonic saline with sodium hydroxide and adjusted to pH longed survival to varying degrees only of those mice bear 7—8with hydrochloric acid. All drugs were administered ing the parent strain of Sarcoma 180. The combination of in volumes of 0.25—0.50ml. Controls given injections of azaserine with all the punine analogs tested produced syn comparable volumes of saline were included in each experi ergistic inhibition of Sarcoma 180; such treatment of Sar ment. Mice were weighed during the course of the expeni coma 180/TG resulted, however, in a survival time no ment, and the percentage change in body weight from on greater than that observed with azaserine alone. In addi set to termination of therapy was used as an indication of tion, 9-n-butylthioguanine administered in daily doses of drug toxicity. 2.8 or 11.2 mg/kg, as two equally divided doses injected at Evaluation of combinations of drugs as synergists was 12-hour intervals, enhanced the carcinostatic activity of based on two criteria : (a) the prolongation of the survival azasenine on Sarcoma 180; however, Sarcoma 180/TG time afforded by drug treatments, and (b) the number of proved to be resistant to this agent, and an anti-neoplastic animals surviving 50 days. All mice surviving over 50 effect attributable to azasenine alone was produced.