- Email: [email protected]
Studies on the mode of action of azaserine
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
64, 423-436
(1956)
Studies on the Mode of Action of Azaserine’ L. L. Bennett, Jr., F. M. Schabel, Jr. and Howard E. Skipper From the Kettering-Meyer Laboratory (dsliafed with Sloan-Kettering Southern Research Institute, Birmingham, Alabama Received
March
Institute),
19, 1956
INTRODUCTIOK
The isolation and proof of structure of azaserine (O-diazoacetyl-nserine) has been described by Fusari et al. (1, 2). The marked inhibitory effect of this compound on growth of Sarcoma 180 was discovered by stock et al. (3). In studies with Sarcoma 180, Clarke and co-workers (4) observed the potentiation of the tumor-inhibiting action of 6mercaptopurine by azaserine. This observation, when considered in terms of current concepts of sequential blocking and combination chemot’herapy (s-7), suggested purine biosynthesis as a site of action of azaserine in the mammal. Preliminary results from t’his laboratory (8) showed that azaserine markedly inhibited de novo synthesis of purines in the mouse; confirmation of this observat.ion in another syst.em was obta.ined by Hartman, Levenberg, and Buchanan (9), who found that azaserine inhibited synthesis of inosinic acid in a pigeon liver enzyme system. The present study is a confirmat’ion and extension of the earlier report (8) on the prevention of azaserine-induced inhibition in bacteria by purines and related compounds and the effects of azaserine on polynucleotide synthesis by normal and neoplastic cells in the intact mouse. EXPERIMENTAL
Bacterial Xtudies A solid medium technique previously described (10) was used in initial studies of the prevention of inhibition by nznserine in Eschcrichia coli (A.T.C.C. 9637)2 r This investigation was supported by grants from the American Cancer Society, the Alfred I’. Sloan Foundation, and the C. F. Kettering Foundation. 2 Obtained from Dr. Bernard 1). Davis and carried in this laboratory since 1952. 423
424
BENNETT,
JR.,
SCHABEL,
JR.
AND
SKIPPER
TABLE I of Various Compounds in Reversing Azaserine-Induced Inhibition oj E. coli (Solid Jfedill??l) l’aper disks were saturated with solutions containing 1 mg.jml. of the various reversal agents except for the methionine-adenine combination which contained 0.5 mg./ml. of each. The concentrations of azaserine inhibited visible growth of E. coli on control plates. Effectiveness
Growth (cm. from paper disk) concentration, wg./&. Azaserine 0.10 0.12 0.14 0.16 -
Compound
I. 2. 3. 4. 5.
I’henvlalanine Tryptophan Methionine Adeninc Methionine + adenine
2.3 2.3 1.6 2.3 2.0
TABLE li;$ectiveness
of Various Inhibition
2.3 2.0 0.6 2.0 2.0
2.0 2.0 0 1.2 2.0
II
Compounds in Reversing ~2raserine-Il,(~k~~c(~ of lC. coli (Liquid Mediunr) Additives
Additives
Guanine
Adenine
Hypoxanthine
Xanthine
Thymine
4-Amino-5-imidazolecarboxamide
Tryptophan
2.0 2.0 0 0 1.3
M./ml.
0 1 10 50 1 10 100 1 10 100 500 1 10 100 500 1 10 100 500 10 100 1000 1 10
0
100 100 100 100 95 96 92 100 100 100 100 100 100 100 86 100 100 100 100 96 96 74 100 100
Per cent growth Azaserine. eg./ml. O.di
14 71 77 92 70 80 65 71 80 80 86 75 76 76 76 0 0 16 2 40 69 28 71 86
0.095 5
51 74 90 62 70 65 71 76 78 54 70 71 74 84 0 0 0 0 5 28 21 70 84
ACTION
TABLE E$ectiveness
425
OF AZASERINE
III
of 4-Amino-5-imidazolecarboxamide in Reversing Induced Inhibition of E. cob (Liquid Medium) Per cent
4-Amino-Simidazolecarboxnmide
Azaserine 0.05
0.06
0.07
81 80 81 78 83 85 81 85 78 77 67
57 60 56 67 67 69 78 81 71 73 51
growth
concentration, '
Azaserine-
pg./&
0.08
0.09
14 24 29 26 27 44 58 71 70 66 29
1 6 0 4 11 17 24 36 44 43 17
0.11
0.12
0.13
0 0 0 0 0 0 0 0 14 13 0
0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
pg./ml. 0
100
88
1 5 10 25 50 100 200 400 500 1000
100
91 90 91 91 91 86 90 87 81 80
104 103 104 104 106 10G 96 96 87
0
0 0 0 7 10 17 24 36 36 23
(Table I). This procedure entailed the application of filter-paper disks saturated with known concentrations of candidate reversal agents to the surface of minimal agar cultures of E. coli containing inhibitory levels of azaserine. Further investigation of the effectiveness of certain candidate reversal agents was then carried out by means of a liquid medium technique (Table II). In all liquid medium experiments, the basal medium of Davis and Mingioli (11) was used, and all turbidimetric measurements were made at 24 hr. In view of the importance of the observation that 4.amino-5-imidazolecarboxamide (AIC), as well as a variety of purines, will prevent inhibition of growth by azaserine, a more extensive study of this compound was made (Table III). Similar studies Ivere carried out to determine the effectiveness of nL-met,hionine in preventing inhibition of growth of E. coli by azaserine (Table IV). Since n-glutamine has been reported to reverse azaserine inhibition of inosinic acid synthesis in pigeon liver (9, 12), a study was also made of the effectiveness of this compound in preventing azaserine inhibition in E. coli (Table V).
In Kvo Tracer Studies CWLabeZed Compounds. Adenine-8-C’” (1 mc./mmole), sodium formate-C*” (1 or 2 mc./mmole), and serine-3-C’” (1.4 mc./mmole) were purchased from Isotopes Specialties Company, and gly-tine-l-C4 (1.3 mc./mmole) from Tracerlab, Inc. Hypoxanthine-B-Cl4 (5.2 mc./mmolc) was synthesized by reaction of 4,5-diamino6-hydroxypyrimidine with C1b-labcled formic acid. 4.Amino-5.imidazolecarboxamide-4-W (1 mc./mmole) was prepared from ethyl cyanoacetate-3-W by the method of Shaw and Woolley- (13).
426
BENNETT,
JR.,
SCHABEL,
JR.
TABLE Effectiveness Induced
AND
SKIPPER
IV
of DL-Methionine in Reversing AzaserineInhibition of E. coli (Liquid Medium) Per cent
growth
DL-Methionine
Azaserine
-
pg./ml.
0
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
100 101 101 100 100 104 103 99 100 99 100 97
88 88 82 83 86 82 83 88 89 83 84 84
82 82 79 84 85 82 80 85 87 80 85 89
67 63 68 70 80 71 82 85 85 80 83 85
38 42 36 63 65 74 78 78 80 83 82 84
5 22 29 20 34 60 82 79 80 78 82 83
11 9 20 11 42 34 68 78 76 75 82 80
0 5 0 9 0 12 47 60 68 64 79 79
0 0 0 0 0 3 18 54 70 73 76 71
0 0 0 0 0 0 24 43 62 66 66 74
0.12
0.13
.P&Z./ml. 0 1 5 10 25 50 100 200 300 400 500 1000
concentration,
TABLE Effectiveness
V
of L-Glutamine in Reversing Azaserine-Induced Inhibition of E. coli (Liquid Medium) Per cent
I L-Glutamine
Azaserine 0
0.05
0.06
0.07
86 82 83 85 78 96 99 100 107 105 112 112
82 81 83 86 74 88 97 99 105 105 109 114
60 68 67 77 68 90 95 100 102 109 110 114
growth
concentration,
pg./ml.
0.08
0.09
14 18 45 50 32 45 49 56 65 61 79 84
5 8 10 8 13 13 26 19 24 26 27 36
0.10 ___~
0.11
M./ml.
0 1 5 10 25 50 100 200 300 400 500 1000
100
100 100 100 100 100 100 107 107 113 112 114
-
-
-
4 5 5 9 13 14 18 32 19 17 15 29
0 0 0 0 8 9 13 12 14 19 15 20
0 0 0 0 9 8 9 6 15 15 14 24
-
0 0 0 0 0 6 6 12 5 13 17 24
ACTION
OF AZASERINE
427
Isolation Procedures. Isolation and counting of adenine and guanine from both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were carried out as described earlier (14). Tissue samples of about 5 g. wet weight were used. In those experiments in which thymine was also isolated, the following procedure was used. The DNA and RNA purines were isolated as usual (14). The ammoniacal solution, from which the DNA purines had been precipitated as the silver salts, was then adjusted to pH 4 with hydrochloric acid, after which the resulting silver chloride was removed by centrifugation and washed with water. The combined supernatant and washings were passed through a column (1 cm. x 1 sq. cm.) of Norit A. The column was then washed with 15 ml. of water, after which the nucleotides were eluted from the column with a 50:50:1 mixture of ethanol-water-ammonium hydroxide. This procedure for separating nucleotides on charcoal is essentially that of Graham.3 The eluate containing the pyrimidine nucleotides was evaporated to dryness in a stream of air, and t,he dry residue was digested for 2 hr. at 90-95” with 0.3 ml. 60% perchloric acid (15). This solution was adjusted to pH 5 with 5Oyopotassium hydroxide, and the resulting potassium perchlorate was removed by centrifugation and washed with water. The combined supernatant and washings were then applied as a band to a large sheet of Whatman No. 1 filter paper, and the chromatogram was developed ascending in the n-but,anol-water-formic acid solvent, of Markham and Smith (16) : R, values: thymine, 0.56; cytosine, 0.22. The thymine band was eluted in duplicate or t.riplicate as described for purines (14), and the purity and concentration of each eluted sample was determined with a Beckman model DU spectrophotometer. The criteria of purity were the position of the ultraviolet absorption maximum and the ratios of optical density readings: 250/260 and 280/260. Infinitely thin samples on stainless steel planchets were used for assay of the radioactivities (14). Evaluation of Procedures. Prior to more ext.ensive studies on the effects of azaserine on nucleic acid metabolism in vivo, a series of individual experiments was carried out over a period of several months to test t,he over-all procedural variation (biologic, isolation, and activity assay) which is inherent in such tracer studies in the int,act animal. Xine groups of five to ten mice bearing Sarcoma 180 were administered formate-Cl4 (10 pc./25 g. mouse) by intraperitoneal injection. After 6 hr. the mice were sacrificed, the tumors and intestines of a given group were pooled for isolation of DNA and RNA purines, and activities were assayed in a gas-flow counter (14). The results of these experiments are presented in Tnblc VI. Prelinrinary Experiments. In pilot experiments, groups of six mice each bearing Sarcoma 160 or Adenocarcinoma Eo 771 were administered azaserine intraperitoneally immediately before injection of formate-Cr4, glycine-1-CY4, serine-3-Cr4, or adcnine-S-CY4. The specific activities of combined nucleic acids (CKA) isolated from tumors, livers, and intestines were compared with those from concurrently run groups of control mice that received no azaserine. The procedures employed in these isolations and assays for radioactivity have been described previously (14). The results of these experiments are presented in Table VII.
3 A. F. Graham, personal communication.
428
BENiVETT,
JR.,
SCHABEL,
JR,
AND
SKIPPER
Studies with Sarcoma 180. The data presented in Table VI suggest that, under the conditions employed, any results of treatment which are reflected by a change in specific activities of greater than 40y0 are probably significant. In fact, these experiments indicate the most extreme variations to be expected, since they were carried out over a period of several months with different lots of labeled formate and with animals of varying weights from different shipment,s. The experiments reported below are probably more reproducible than those presented in Table VI since, in these, control and treated groups of animals xvere from the same shipment lot, were carefully matched for weight, and were administered labeled compounds from the same solution. In an extension of the experiment,s reported in Table VII, a series of experiments was carried out in which individual DNA and RXA purincs were isolated from azaserine-treated and control groups of mice which had been administered formate-C14, glycine-1-C lb, 4-amino-5-imidasolecarbosamide-4C’4, hypoxanthineTABLE Reproducibility
VI
of Incorporation
=
Specific activities,
i
Sa 180 Expt. No.
Ad
-Avg. Avg. dev. Max. dev. mean Avg. error,a Max. error,a
_-
‘% ‘%
Gil
-
I-
cozmts/sec./pg. purine Intestine
-IRNA
DKA
--
Ad
Gu
Ad
-~ GU
RNA
Ad
I-
Gu
0.37 0.43 0.30 0.38 0.34 0.31 0.26 0.24 0.23
0.29 0.33 0.25 0.27 0.25 0.24 0.20 0.19 0.25 i- ---
0.70 0.66 0.56 0.79 0.6s 0.59 0.49 0.51 0.63 __-
0.46 0.41 0.36 0.42 0.38 0.38 0.29 0.27 0.55 ~.
0.46 0.44 0.45 0.57 0.40 0.37 0.45 0.40 0.37 0.38 0.41 0.41 0.32 0.33 0.34 0.36 0.44 -__ -- ---
0.32
0.25 --
0.62 I_-
0.39 I_
0.39 ----
0.42 I_-
0.75 --~
0.69 --
0.03 0.08
0.08 0.17
0.06 0.16
0.04 0.07
0.06 0.15
0.07 0.15
0.08 0.14
I .I-
0.06 0.11
from
Studies
(Formate-U4)
19 33
12 32
I’
-
13 27
15 41
10 18
13 36
0.81 0.77 0.66 0.83 0.90 0.77 0.63 0.70 0.69 ~--_-~
-
./
9 20
terminated a hlaximum and average error from mean. ,411 experiments hr. Pooled tissues from five to ten mice were used in each experiment.
0.76 0.77 0.68 0.80 0.76 0.70 0.55 0.60 0.59 ---
12
i :20 at 6
ACTION
429
OF SZASERINE
8XY4, or adenine-8.CY4. The labeled precursors were chosen so that data on inhibition of carbon-14 incorporation int,o the polynucleotide purines might be useful in indicating the area of the sit,e of blockade by azaserine in the int,act animal. The results of these experiments are presented in Table VIII. Studies with Human Tumors. Studies of the effects of azascrine on utilization of formate-Cl4 and glycine l-CL” for purinc s),nthesis were also carried out with heterologous implants of a human tumor. For these experiments, implants of a human sarcoma (H&l) wre grown subcutnneowly in ~~ist:tr rats (Charles River strain) which were treated with cortisone (IT, 18). The isolations and assays were carried out as described above. The results are presented in Table IS. In these experiments and in thr other experiments described above, azaserine was given at high levels (relative to the maximum tolerated multiple injection dose) in order that maximum inhibitor?- effects might be obtained. RESULTS
ASD
DISCUSSION
The data in Table I indicate that phenylalanine, t,rypt.ophan, methionine, or sdenine is effective in prevent,ing inhibit,ion by azaserine of E. coli grown in solid medium. Many other compounds were tested as reversal agents by use of the solid medium technique; it is noteworthy that, of these, tyrosine and phenylpyruvic acid were effective reversal agents whereas serine, p-aminobeneoic acid, and thymine were ineffective. Kaplan and Stock (19) have observed earlier that azaserine-induced inhibition of E. coli could be prevented by phenylalanine, tyrosine, or tryptophan, and, on the basis of work with E. coli mutant,s, suggested TABLE E$ects of Azaserine Expt. no. 1
2 3 4 5
on Incorporation
Labeled compound
Forma&C” Formate-Cl? Formate-Cl4 Formate-Cl4 Glycine-1-C’” Glycine-1-C’” Serine-3-C14 Serine-3-Cla Adenine-8-C’” Adenine-8-C”
VII of Cl4 into Tissue Nucleic Treatment
Sone Azaserine Xone Azaserine Sone dzaserine None
Azaserine Xone Aznserine
Acids
CSA specific activities Tumor Intestine Liver uc./mole carbon
37a 85 63 13 26 7 27 9 45 40
75 9 90 14 20 8 44 11 120 132
13 3 5 3 4 4 5 3 71 62
a Adenoearcinoma Eo 771; all other experiments were carried out wit.h mice bearing Sa 180. Azsserine was administered when indicated in a single dose of 30 mg./kg. immediately prior to injection of a labeled precursor. All experiments were terminated 6 hr. after injection of the labeled compounds.
430
BENNETT,
JR.,
SCHABEL,
TABLE
JR.
AND
SKIPPER
VIII
Effects of Azaserine on Synthesis of Nucleic Acid Purines Sarcoma 180 and Host Tissues’ Specific activities
Labeled precursor and azaserine level
-Formate-Cl4 (azasei-ine: 20 mg./ kg.)
Glycine-1-C” (asaserine: 20 mg./ kg.1 AIC-4-Cl4 (azaserine : 20 mg./kg.)
AIC-4-Cl4 (azaserinc : 10 mg./kg.)
Hypoxanthine-8 Cl4 (azaserine mdkg.)
: 20
Hypoxanthine-8 Cl4 (azaserine mg./kg.)
: 20
Adenine-8-C’” (azaserine: mdk.)
20
Compound
DNA:
Ad Gil Th RNA: Ad Gu DNA: Ad Gu RNA: Ad Gu DNA: Ad Gu RNA: Ad GU DNA: Ad Gu RSA: Ad Gu DNA: Ad Gu WA: Ad Gu D?JA: Ad Gu RNA: Ad Gu DNA: Ad Gu RNA: Ad Gu
(treated groups as percentages of controlsjb
Sa 180
35 33 95 41 35 <8 <9 <7 9 83
(0.26) (0.20) (0.20) (0.49) (0.29) (0.25) (0.23) (0.45) (0.33) (0.58)
100 (0.44)
100 108 136 146 133 128 74 85 73 87 120 100 135 119 135
by
(1.1) (0.61) (0.55) (0.41) (1.2) (0.70) (0.076, (0.067, (0.15) (0.097, (0.053, (0.048 (0.092, (0.053: (1.0) (0.17) (1.6) (0.26)
Intestine
23 28 145 35 36 :19 ~25 13 16 51 93 96 135 64 116 84 122 35 59 69 87 61 96 96 122 70 146 104 195
Liver
(0.32) (0.33) (0.11) (0.63) (0.55) (0.16) (0.16) (0.30) (0.25) (0.19) (0.15) (0.69) 43 (0.35) (0.32) 58 (0.15) (0.28) (0.24) (0.99) 92 (0.26) (0.50) 92 (0.13) (0.37) 69 (0.16) (0.29) 100 (0.11) (0.78) 110 (0.30) (0.48) 133 (0.12) (0.28) 63 (0.16) (0.21) 66 (0.13) (0.44) 90 (0.20) (0.27) 83 (0.17) (1.1) 78 (0.31) (0.11) - (<0.07) (2.4) 82 (0.77) (0.18) 67 (0.057:
I-
Spleen
46 (1.9) 39 (1.3) 48 (0.97) 48 (0.50)
30 23 65 47 17 48 83 03 91 21 20 43 31 46 00 24
(1.0) (0.62) (0.75) (0.49) (0.35) (0.21) (0.65) (0.34) (1.1) (0.63) (0.42) (0.51) (1.7)
(1.1) (1.3) (0.72) -
a Five to ten mice were used in each control and each treated group. Each mouse was administered azaserine intraperitoneally at the indicated level immediately before injection of the labeled compound. All labeled compounds except AIC were given intraperitoneally at a level of 10 pc.,/25 g.; the AIC level was 5 +./25 g. Animals were sacrificed 6 hr. after administration of the labeled compounds. b The values in parentheses are the specific activities (counts/sec./pg. purine or thymine) of the control groups.
ACTION
431
OF AZASERINE
TABLE
IX
Effects of Azaserine on Synthesis of Nucleic Acid Purines by a Human Tumor (HS-i) and by Intestine of the Host Rat Specific activity,
cornls/sec./gg. #urine Rat intestine
Human tumor Treatment DNA
Formate-Cl4
RNA
RNA
DNA
0.66
0.61
1.2
0.97
Glycine-lC’4 Note: Azsserine was administered intraperitoneally at a level of 20 mg./kg. immediately prior to the injection of the labeled compound at a level of 10 PC./ 25 g. Animals were sacrificed 6 hr. after administration of the labeled compounds.
that azaserine is inhibitory for at least two sites of synthesis: (a) the condensation of indole and serine to form tryptophan, and (6) the synthesis of tyrosine and phenylalanine from their precursors. The data in Table I and the more extended data in Tables II and III confirm the prevention of inhibition by aromatic amino acids and show, in addition, that when minimal inhibiting levels of azaserine are used, purines (adenine, guanine, hypoxanthine, and xanthine), MC, and methionine are also effective reversal agents. Glutamine (Table V) also prevented inhibition of E. coli by azaserine-a result that might be expected from the observations t,hat glutamine reverses the azaserine-induced inhibition of inosinic acid synthesis in a pigeon liver system (9, 12). From these results, it appears that in E. coli azaserine blocks in more than one metabolic area, and that purine synthesis is one of the sites of action. Preliminary studies of the effect of azaserine on utilization of labeled precursors for synthesis of nucleic acid purines in tumor-bearing mice indicated that azaserine profoundly inhibited incorporation of de nouo precursors, formate, glycine, and the P-carbon of serine, into nucleic acids wit,hout inhibiting utilization of preformed purines (Table VII). The more extended study in which individual purincs were isolated from both RNA and DNA following administration of various labeled precursors (Table VIII) confirms these observations and indicates a marked inhibitory effect of azaserine on de nouo synbhesis of both adenine and
432
BENNETT,
JR.,
SCHABEL,
JR.
AND
SKIPPER
guanine. It is interesting that similar effects on de novo purine synthesis were observed in a heterologous implant of a human tumor (Table IX). Inhibition of de novo purine synthesis by azaserine has also been noted by LePage and Greenlees (20) in studies with the Ehrlich and RC3HED ascites tumors and by Heidelberger and Keller (21) in studies lvith slices of Flexner-Jobling carcinoma and rat spleen; no very marked inhibition of protein synthesis was observed in these experiments. Fernandes et al. (22) have found azaserine to be effective in viva in inhibit,ing glycine incorporation into acid-soluble purines and nucleic acid purincs of several tumors and spleens of the host mice; under the same conditions, utilization of adenine was stimulated. Since formate is a precursor of the methyl carbon atom of thymine (23)) an indication of the specificity of aznserine as an inhibitor of purine synthesis can be obtained by comparing its effects on formate incorporation into purines and into thymine. The fact that, in the same experiment in which formate incorporation into purines was markedly inhibited, formate incorporation into thymine was unaffected or increased (Table VIII) suggests that the observed inhibition of de novo purine synthesis by azaserine is not the result of a general interference with one-carbon metabolism. Large inequalit,ies in effecm on formate incorporation into purines and thymine have also been observed in studies with N-methylformamide (24) and in some (25, 26), but not all (27, 28), studies with the antifolics, aminopterin and A-methopterin. The data in Table VIII clearly indicate that in all tissues azaserine inhibits purine synthesis from either formate or glycine and fails to inhibit incorporation of adenine. In the experiments with AIC and hypoxanthine, some variations in effect were noted either between duplicate runs or from tissue to tissue; two complete experiments with each of these precursors are present’ed in order to illustrate the variations observed. In the two AIC experiments, different levels of azaserine were used; whereas, in the two hypoxanthine experiment’s, the levels of azaserine were the same. Azaserine inhibited incorporation of both AIC and hypoxanthine into intestine DNA adenine, and inhibited slightly incorporation of hypoxanthine into intestine DlSA guanine in one experiment but not in the other. These inhibitions were, however, much smaller than those observed with format’e and glycine. At the higher level employed (20 mg./kg.), azaserine inhibited utilization of AIC by spleen and liver, but no inhibition was observed at the lower level (10 mg./kg.) . In both DNA and RNA of Sarcoma 180 and RlYA of intestine, azaserine consistently failed to inhibit utilization of either RIC or hypoxanthine.
ACTION
433
OF AZASERINE
For the purpose of discussing the inhibition data with azaserine, a diagram of the biosynthetic pathway to nucleic acid purines is desirable. The de novo synthesis of purines has been worked out most thoroughly in pigeon liver, notably by Buchanan and by Greenberg (9, 12, 29-32). Pigeon liver utilizes formate, glycine, and AIC for synthesis of inosinic acid. These same precursors are utilized for the synthesis of nucleic acid purines by the mouse and rat (29). In both t’he mammal and microorganisms, administration of labeled formate and glycine results in the labeling of the same posit,ions in the nucleic acid purines as is observed in uric acid isolated after administration of these same precursors to pigeons (29, 33). Following administration of hypoxanthine-Cl4 to mice and rats, nucleic acid adenine and guanine are labeled almost equally; approximately equal labeling of adenine and guanine is also obtained by administration of labeled formate or glycine (34). Furthermore, an extract of rabbit bone marrow has been shown to convert inosine or inosinic acid to acid-soluble adenine and guanine compounds (35). On the basis of these observations, it is not unreasonable to assume the existence of a general biosynthetic pathway to purines. illthough it is realized that the complet,e de novo pathway to nucleic acid purines has not been worked out in any one biological system, a generalized scheme, based on the literature cited above, is presented below as a basis for discussion and correlation of the data with azaserine obt,ained in several diverse systems. Glycine
+ glycintlmide
ribotide
formylglycinnmidine
+ formylglycinamide ribotide
ribotide
--) 5-aminoimidazole
--) ribotide
+
Ad-R 5-aminohimidazolecarboxamide
ribotide
7
L
I
7
+ Hx-R
polynucleotides Gu-R
If AIC, hypoxanthine, and adenine enter this biosynthetic pathway by conversion to the corresponding ribotides, then the failure of azaserine to inhibit incorporation of these precursors into nucleic acid purines while inhibiting utilization of formate and glycine for purine synthesis would indicat’e that the site of blockade lies before the formation of AIC ribotide. These results obtained in the intact animal are consistent with those of Hartman et al. (9) who have shown that, in a pigeon liver system, azaserine inhibited synthesis of inosinic acid and caused an accumulation of formylglycinamide ribotide. These authors concluded that azaserine exerted an inhibitory action on de novo synthesis of purines at
434
BENNETT,
JR., SCHABEL,
JR. AND SKIPPER
a metabolic site subsequent to the formation of formylglycinamide ribotide. Additional evidence, obtained by this same group of workers, indicated that azaserine behaved as a glutamine antagonist in inhibiting reaction of glutamine with formylglycinamide ribotide to yield formylglycinamidine ribotide (12,32). Working in still another system, Tomisek et al. (36) have observed the accumulation of radioactive formylglycinamide ribotide and formylglycinamide riboside by azaserine-treated E. coli grown in a medium containing formate-Cl4 or glycine-CL4. In these experiments, azaserine inhibited incorporation of formate and glycine into purines, purine nucleosides, and purine nucleotides. Thus, data obtained in three different biological systems-pigeon liver, E. coli, and the intact tumor-bearing mouse-show that azaserine inhibits at an early stage of de lzozlopurine synthesis, and these data are consistent with the concept that the site of blockade lies somewhere between formylglycinamide ribotide and 5-amino-4-imidazolecarboxamide ribotide. ACKNOWLEDGMENTS
The authors wish to thank Dr.
1. Azaserine-induced inhibition of E. coli was significantly prevented by (a) 4-amino-5-imidazolecarboxamide (AIC), adenine, guanine, hypoxanthine, or xanthine; (b) methionine; and (c) glutamine; and, as has already been observed by others, by phenylalanine, tyrosine, or tryptophan. 2. In studies carried out on the effect of azaserine on purine synthesis in the tumors, intestines, livers, and spleens of mice bearing Sarcoma 180, it was found that azaserine inhibited utilization of formate-Cl4 and glytine-l-G4 for purine synthesis, failed to affect utilization of CY4-labeled adenine, AIC, and hypoxanthine, and failed to affect utilization of formate for thymine biosynthesis. Azaserine also inhibited utilization of formate-Cl4 and glycine-l-C?4 for purine synthesis by heterologous implants of a human sarcoma growing in the rat. 3. The results indicate that azaserine inhibits purine synthesis in the
ACTION
OF AZASERINE
435
intact animal at a stage prior to the formation of 5-amino-kimidaeolecarboxamide ribotide. REFERENCES
1. FUURI, S. A., FROHBRDT, R. P., RYDER, A., HASKELL, T. H., JOHANNESSEN, D. W., ELDER, C. C., AND BARTZ, Q. R., J. Am. Chem. Sot. 76, 2878 (1954). 2. FUSARI, S. A., HASKELL, T. I-I., FROIIARDT, R. I'., AND BARTZ, Q. R.,J. Am. Chem. Sot. 76, 2881 (1954). 3. STOCK, C. C., REILLY, H. C., BUCBLEY, S. M., CLARKE, D..4., ASD RHOADS, c. P., iVat7tre 173, 71 (1954). 4. CLBRKE, D. -4., PHILIPS, F. S., STOCK, C. C., ELION, G. B., ASD HITCHINGS, G. H., Abstracts of Papers, p. 12-M. 125th?rlceting, Am. Chem. Sot., Kansas City, 1954. 5. POTTER, V. R., Proc. Sot. Erptl. Bid. Med. 76, 41 (1951). 6. Law, L. W., Cancer Research 12, 871 (1952). 7. SKII'PER, H. I?., THOMSOX,J. R., ASD BELL, M., Cancer Research 14, 503 (1954). 8. SKIPPER, I-I. E., BESNETT, L. L., JR., AXD SCHABEL, F. M., JR., Federation hoc. 13, 29s (1954). 9. HARTM\IAK,S.C.,LEVENRERG, B., .~NDBUCH.AS~N,J.M., J. Am. Chem.Soc.77, 501 (1955). 10. SKIPPER, H. E., SCHAREL, F. hf., JR., BIXNS, V., THOMSOX, J. R., and WHEELER, G. I'., Cancer Research 16, 143 (1955). 11. DAVIS, B. D., ASD ?VfINGIOLI,E. S., J. Bacterial. 60, 17 (1950). 12. BUCIIAN.~~, J. nl., LEVESBERG, B., .~ND LUKE%, L. N., Abstracts of Papers, p. 12-C. 12Sth Meeting, Am. Chem. Sot., hkmenpolis, 1955. 13. SHAW, E., AXD WOOLLEY, 1). W., .J. Biol. Chem. 181, 89 (1949). 14. BENNETT, L. L.,JR.,S~PI%R,H.E.,STOCIC,C.C.,.~ND R~o~~s.,C.I'.,Cancer Rescarch 15, 4% (1955). 15. MARSHAK, A., ;\SD VOGEL, H. J., J. Biol. Chem. 189, 597 (1951). 16. MARKMAN, R., AXD Smm, J. I)., Biochem. J. 45, 294 (1949). 17. TOOL~X, H. W., Cuncer Xcseard~ 13, 3S9 (1953). 18. TOOLAN, H. W., Cancer Research 14, 660 (1954). 19. K.~PLAX, L., .4xD STOCK, C. C., Federation Proc. 13, 239 (1954). 20. LEPAGE, G. A. APED GREESLEES, J., Cancer Research, Suppl. No. 3, p. 102 (1955). 21. HEIDELBERGER, C., AXD KELLER, R. A., Cancer Research, Suppl. No. 3, p. 106 (1965). 22. FERSASDEF, J. F., LEPAGE, G. A.,.~xD LINDNER, A., Cancer Research 16, 154 (1956). 23. TOTTER, J. R., VOLKIN, E., AND CARTER, C. E., J. Am. Chem. Sot. 73, 1521 (1951). 24. BARCLAY, R. K., GARFINEEL, E., AND STOCK, C. C., J. Biol. Chem. 208, 875 (1954). 25. TOTTER, J. R., AND BEST, 9. N., Arch. Biochem. and Biophys. 64, 318 (1955). 26. BALIS, hf. E., AXD ~.~NCIS, J., Cancer Research 16, 603 (1955). 27. GOLDTHWAIT, D. A., AXD BENDICH, A., J. Biol. Chem. 196, 841 (1952).
436
BENNETT,
JR.,
SCHABEL,
JR.
.4ND
SKIPPER
28. SKIPPER, H. E., BENNETT, L. L., JR., AND LAW, L. W., Cancer Research 12, 677 (1952). 29. REICHARD, P., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 2, Chap. 23. Academic Press, New York, 1955. 30. LEVENBERQ, B., HARTMAN, S. C., AND BUCHANAN, J. M., Federation Proc. 14, 243 (1955). 31. GOLDTHWAIT, D. A., GREENBERG, G. R., AND PEABODY, R. A., Biochim. et Biophys. Acta 18, 148 (1955). 32. LEVENBERG, B., AND BUCHANAN, J. M., J. Am. Chem. Sot. 78, 504 (1956). 33. SUTTON, W. B., AND WERKMAN, C. H., Arch. Biochem. and Biophys. 47, 1 (1953). 34. BENNETT, L. L., JR., AND SKIPPER, H. E., Arch. Biochem. and Biophys. 64, 566 (1955). 35. ABRAM, R., AND BENTLEY, M., Arch. Biochem. and Biophys. 58, 109 (1955). 36. TOMISEK, A. J., KELLY, H. J., AND SKIPPER, H. E., Arch. Biochem. and Biophys. 64, 437 (1956).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
64, 423-436
(1956)
Studies on the Mode of Action of Azaserine’ L. L. Bennett, Jr., F. M. Schabel, Jr. and Howard E. Skipper From the Kettering-Meyer Laboratory (dsliafed with Sloan-Kettering Southern Research Institute, Birmingham, Alabama Received
March
Institute),
19, 1956
INTRODUCTIOK
The isolation and proof of structure of azaserine (O-diazoacetyl-nserine) has been described by Fusari et al. (1, 2). The marked inhibitory effect of this compound on growth of Sarcoma 180 was discovered by stock et al. (3). In studies with Sarcoma 180, Clarke and co-workers (4) observed the potentiation of the tumor-inhibiting action of 6mercaptopurine by azaserine. This observation, when considered in terms of current concepts of sequential blocking and combination chemot’herapy (s-7), suggested purine biosynthesis as a site of action of azaserine in the mammal. Preliminary results from t’his laboratory (8) showed that azaserine markedly inhibited de novo synthesis of purines in the mouse; confirmation of this observat.ion in another syst.em was obta.ined by Hartman, Levenberg, and Buchanan (9), who found that azaserine inhibited synthesis of inosinic acid in a pigeon liver enzyme system. The present study is a confirmat’ion and extension of the earlier report (8) on the prevention of azaserine-induced inhibition in bacteria by purines and related compounds and the effects of azaserine on polynucleotide synthesis by normal and neoplastic cells in the intact mouse. EXPERIMENTAL
Bacterial Xtudies A solid medium technique previously described (10) was used in initial studies of the prevention of inhibition by nznserine in Eschcrichia coli (A.T.C.C. 9637)2 r This investigation was supported by grants from the American Cancer Society, the Alfred I’. Sloan Foundation, and the C. F. Kettering Foundation. 2 Obtained from Dr. Bernard 1). Davis and carried in this laboratory since 1952. 423
424
BENNETT,
JR.,
SCHABEL,
JR.
AND
SKIPPER
TABLE I of Various Compounds in Reversing Azaserine-Induced Inhibition oj E. coli (Solid Jfedill??l) l’aper disks were saturated with solutions containing 1 mg.jml. of the various reversal agents except for the methionine-adenine combination which contained 0.5 mg./ml. of each. The concentrations of azaserine inhibited visible growth of E. coli on control plates. Effectiveness
Growth (cm. from paper disk) concentration, wg./&. Azaserine 0.10 0.12 0.14 0.16 -
Compound
I. 2. 3. 4. 5.
I’henvlalanine Tryptophan Methionine Adeninc Methionine + adenine
2.3 2.3 1.6 2.3 2.0
TABLE li;$ectiveness
of Various Inhibition
2.3 2.0 0.6 2.0 2.0
2.0 2.0 0 1.2 2.0
II
Compounds in Reversing ~2raserine-Il,(~k~~c(~ of lC. coli (Liquid Mediunr) Additives
Additives
Guanine
Adenine
Hypoxanthine
Xanthine
Thymine
4-Amino-5-imidazolecarboxamide
Tryptophan
2.0 2.0 0 0 1.3
M./ml.
0 1 10 50 1 10 100 1 10 100 500 1 10 100 500 1 10 100 500 10 100 1000 1 10
0
100 100 100 100 95 96 92 100 100 100 100 100 100 100 86 100 100 100 100 96 96 74 100 100
Per cent growth Azaserine. eg./ml. O.di
14 71 77 92 70 80 65 71 80 80 86 75 76 76 76 0 0 16 2 40 69 28 71 86
0.095 5
51 74 90 62 70 65 71 76 78 54 70 71 74 84 0 0 0 0 5 28 21 70 84
ACTION
TABLE E$ectiveness
425
OF AZASERINE
III
of 4-Amino-5-imidazolecarboxamide in Reversing Induced Inhibition of E. cob (Liquid Medium) Per cent
4-Amino-Simidazolecarboxnmide
Azaserine 0.05
0.06
0.07
81 80 81 78 83 85 81 85 78 77 67
57 60 56 67 67 69 78 81 71 73 51
growth
concentration, '
Azaserine-
pg./&
0.08
0.09
14 24 29 26 27 44 58 71 70 66 29
1 6 0 4 11 17 24 36 44 43 17
0.11
0.12
0.13
0 0 0 0 0 0 0 0 14 13 0
0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
pg./ml. 0
100
88
1 5 10 25 50 100 200 400 500 1000
100
91 90 91 91 91 86 90 87 81 80
104 103 104 104 106 10G 96 96 87
0
0 0 0 7 10 17 24 36 36 23
(Table I). This procedure entailed the application of filter-paper disks saturated with known concentrations of candidate reversal agents to the surface of minimal agar cultures of E. coli containing inhibitory levels of azaserine. Further investigation of the effectiveness of certain candidate reversal agents was then carried out by means of a liquid medium technique (Table II). In all liquid medium experiments, the basal medium of Davis and Mingioli (11) was used, and all turbidimetric measurements were made at 24 hr. In view of the importance of the observation that 4.amino-5-imidazolecarboxamide (AIC), as well as a variety of purines, will prevent inhibition of growth by azaserine, a more extensive study of this compound was made (Table III). Similar studies Ivere carried out to determine the effectiveness of nL-met,hionine in preventing inhibition of growth of E. coli by azaserine (Table IV). Since n-glutamine has been reported to reverse azaserine inhibition of inosinic acid synthesis in pigeon liver (9, 12), a study was also made of the effectiveness of this compound in preventing azaserine inhibition in E. coli (Table V).
In Kvo Tracer Studies CWLabeZed Compounds. Adenine-8-C’” (1 mc./mmole), sodium formate-C*” (1 or 2 mc./mmole), and serine-3-C’” (1.4 mc./mmole) were purchased from Isotopes Specialties Company, and gly-tine-l-C4 (1.3 mc./mmole) from Tracerlab, Inc. Hypoxanthine-B-Cl4 (5.2 mc./mmolc) was synthesized by reaction of 4,5-diamino6-hydroxypyrimidine with C1b-labcled formic acid. 4.Amino-5.imidazolecarboxamide-4-W (1 mc./mmole) was prepared from ethyl cyanoacetate-3-W by the method of Shaw and Woolley- (13).
426
BENNETT,
JR.,
SCHABEL,
JR.
TABLE Effectiveness Induced
AND
SKIPPER
IV
of DL-Methionine in Reversing AzaserineInhibition of E. coli (Liquid Medium) Per cent
growth
DL-Methionine
Azaserine
-
pg./ml.
0
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
100 101 101 100 100 104 103 99 100 99 100 97
88 88 82 83 86 82 83 88 89 83 84 84
82 82 79 84 85 82 80 85 87 80 85 89
67 63 68 70 80 71 82 85 85 80 83 85
38 42 36 63 65 74 78 78 80 83 82 84
5 22 29 20 34 60 82 79 80 78 82 83
11 9 20 11 42 34 68 78 76 75 82 80
0 5 0 9 0 12 47 60 68 64 79 79
0 0 0 0 0 3 18 54 70 73 76 71
0 0 0 0 0 0 24 43 62 66 66 74
0.12
0.13
.P&Z./ml. 0 1 5 10 25 50 100 200 300 400 500 1000
concentration,
TABLE Effectiveness
V
of L-Glutamine in Reversing Azaserine-Induced Inhibition of E. coli (Liquid Medium) Per cent
I L-Glutamine
Azaserine 0
0.05
0.06
0.07
86 82 83 85 78 96 99 100 107 105 112 112
82 81 83 86 74 88 97 99 105 105 109 114
60 68 67 77 68 90 95 100 102 109 110 114
growth
concentration,
pg./ml.
0.08
0.09
14 18 45 50 32 45 49 56 65 61 79 84
5 8 10 8 13 13 26 19 24 26 27 36
0.10 ___~
0.11
M./ml.
0 1 5 10 25 50 100 200 300 400 500 1000
100
100 100 100 100 100 100 107 107 113 112 114
-
-
-
4 5 5 9 13 14 18 32 19 17 15 29
0 0 0 0 8 9 13 12 14 19 15 20
0 0 0 0 9 8 9 6 15 15 14 24
-
0 0 0 0 0 6 6 12 5 13 17 24
ACTION
OF AZASERINE
427
Isolation Procedures. Isolation and counting of adenine and guanine from both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were carried out as described earlier (14). Tissue samples of about 5 g. wet weight were used. In those experiments in which thymine was also isolated, the following procedure was used. The DNA and RNA purines were isolated as usual (14). The ammoniacal solution, from which the DNA purines had been precipitated as the silver salts, was then adjusted to pH 4 with hydrochloric acid, after which the resulting silver chloride was removed by centrifugation and washed with water. The combined supernatant and washings were passed through a column (1 cm. x 1 sq. cm.) of Norit A. The column was then washed with 15 ml. of water, after which the nucleotides were eluted from the column with a 50:50:1 mixture of ethanol-water-ammonium hydroxide. This procedure for separating nucleotides on charcoal is essentially that of Graham.3 The eluate containing the pyrimidine nucleotides was evaporated to dryness in a stream of air, and t,he dry residue was digested for 2 hr. at 90-95” with 0.3 ml. 60% perchloric acid (15). This solution was adjusted to pH 5 with 5Oyopotassium hydroxide, and the resulting potassium perchlorate was removed by centrifugation and washed with water. The combined supernatant and washings were then applied as a band to a large sheet of Whatman No. 1 filter paper, and the chromatogram was developed ascending in the n-but,anol-water-formic acid solvent, of Markham and Smith (16) : R, values: thymine, 0.56; cytosine, 0.22. The thymine band was eluted in duplicate or t.riplicate as described for purines (14), and the purity and concentration of each eluted sample was determined with a Beckman model DU spectrophotometer. The criteria of purity were the position of the ultraviolet absorption maximum and the ratios of optical density readings: 250/260 and 280/260. Infinitely thin samples on stainless steel planchets were used for assay of the radioactivities (14). Evaluation of Procedures. Prior to more ext.ensive studies on the effects of azaserine on nucleic acid metabolism in vivo, a series of individual experiments was carried out over a period of several months to test t,he over-all procedural variation (biologic, isolation, and activity assay) which is inherent in such tracer studies in the int,act animal. Xine groups of five to ten mice bearing Sarcoma 180 were administered formate-Cl4 (10 pc./25 g. mouse) by intraperitoneal injection. After 6 hr. the mice were sacrificed, the tumors and intestines of a given group were pooled for isolation of DNA and RNA purines, and activities were assayed in a gas-flow counter (14). The results of these experiments are presented in Tnblc VI. Prelinrinary Experiments. In pilot experiments, groups of six mice each bearing Sarcoma 160 or Adenocarcinoma Eo 771 were administered azaserine intraperitoneally immediately before injection of formate-Cr4, glycine-1-CY4, serine-3-Cr4, or adcnine-S-CY4. The specific activities of combined nucleic acids (CKA) isolated from tumors, livers, and intestines were compared with those from concurrently run groups of control mice that received no azaserine. The procedures employed in these isolations and assays for radioactivity have been described previously (14). The results of these experiments are presented in Table VII.
3 A. F. Graham, personal communication.
428
BENiVETT,
JR.,
SCHABEL,
JR,
AND
SKIPPER
Studies with Sarcoma 180. The data presented in Table VI suggest that, under the conditions employed, any results of treatment which are reflected by a change in specific activities of greater than 40y0 are probably significant. In fact, these experiments indicate the most extreme variations to be expected, since they were carried out over a period of several months with different lots of labeled formate and with animals of varying weights from different shipment,s. The experiments reported below are probably more reproducible than those presented in Table VI since, in these, control and treated groups of animals xvere from the same shipment lot, were carefully matched for weight, and were administered labeled compounds from the same solution. In an extension of the experiment,s reported in Table VII, a series of experiments was carried out in which individual DNA and RXA purincs were isolated from azaserine-treated and control groups of mice which had been administered formate-C14, glycine-1-C lb, 4-amino-5-imidasolecarbosamide-4C’4, hypoxanthineTABLE Reproducibility
VI
of Incorporation
=
Specific activities,
i
Sa 180 Expt. No.
Ad
-Avg. Avg. dev. Max. dev. mean Avg. error,a Max. error,a
_-
‘% ‘%
Gil
-
I-
cozmts/sec./pg. purine Intestine
-IRNA
DKA
--
Ad
Gu
Ad
-~ GU
RNA
Ad
I-
Gu
0.37 0.43 0.30 0.38 0.34 0.31 0.26 0.24 0.23
0.29 0.33 0.25 0.27 0.25 0.24 0.20 0.19 0.25 i- ---
0.70 0.66 0.56 0.79 0.6s 0.59 0.49 0.51 0.63 __-
0.46 0.41 0.36 0.42 0.38 0.38 0.29 0.27 0.55 ~.
0.46 0.44 0.45 0.57 0.40 0.37 0.45 0.40 0.37 0.38 0.41 0.41 0.32 0.33 0.34 0.36 0.44 -__ -- ---
0.32
0.25 --
0.62 I_-
0.39 I_
0.39 ----
0.42 I_-
0.75 --~
0.69 --
0.03 0.08
0.08 0.17
0.06 0.16
0.04 0.07
0.06 0.15
0.07 0.15
0.08 0.14
I .I-
0.06 0.11
from
Studies
(Formate-U4)
19 33
12 32
I’
-
13 27
15 41
10 18
13 36
0.81 0.77 0.66 0.83 0.90 0.77 0.63 0.70 0.69 ~--_-~
-
./
9 20
terminated a hlaximum and average error from mean. ,411 experiments hr. Pooled tissues from five to ten mice were used in each experiment.
0.76 0.77 0.68 0.80 0.76 0.70 0.55 0.60 0.59 ---
12
i :20 at 6
ACTION
429
OF SZASERINE
8XY4, or adenine-8.CY4. The labeled precursors were chosen so that data on inhibition of carbon-14 incorporation int,o the polynucleotide purines might be useful in indicating the area of the sit,e of blockade by azaserine in the int,act animal. The results of these experiments are presented in Table VIII. Studies with Human Tumors. Studies of the effects of azascrine on utilization of formate-Cl4 and glycine l-CL” for purinc s),nthesis were also carried out with heterologous implants of a human tumor. For these experiments, implants of a human sarcoma (H&l) wre grown subcutnneowly in ~~ist:tr rats (Charles River strain) which were treated with cortisone (IT, 18). The isolations and assays were carried out as described above. The results are presented in Table IS. In these experiments and in thr other experiments described above, azaserine was given at high levels (relative to the maximum tolerated multiple injection dose) in order that maximum inhibitor?- effects might be obtained. RESULTS
ASD
DISCUSSION
The data in Table I indicate that phenylalanine, t,rypt.ophan, methionine, or sdenine is effective in prevent,ing inhibit,ion by azaserine of E. coli grown in solid medium. Many other compounds were tested as reversal agents by use of the solid medium technique; it is noteworthy that, of these, tyrosine and phenylpyruvic acid were effective reversal agents whereas serine, p-aminobeneoic acid, and thymine were ineffective. Kaplan and Stock (19) have observed earlier that azaserine-induced inhibition of E. coli could be prevented by phenylalanine, tyrosine, or tryptophan, and, on the basis of work with E. coli mutant,s, suggested TABLE E$ects of Azaserine Expt. no. 1
2 3 4 5
on Incorporation
Labeled compound
Forma&C” Formate-Cl? Formate-Cl4 Formate-Cl4 Glycine-1-C’” Glycine-1-C’” Serine-3-C14 Serine-3-Cla Adenine-8-C’” Adenine-8-C”
VII of Cl4 into Tissue Nucleic Treatment
Sone Azaserine Xone Azaserine Sone dzaserine None
Azaserine Xone Aznserine
Acids
CSA specific activities Tumor Intestine Liver uc./mole carbon
37a 85 63 13 26 7 27 9 45 40
75 9 90 14 20 8 44 11 120 132
13 3 5 3 4 4 5 3 71 62
a Adenoearcinoma Eo 771; all other experiments were carried out wit.h mice bearing Sa 180. Azsserine was administered when indicated in a single dose of 30 mg./kg. immediately prior to injection of a labeled precursor. All experiments were terminated 6 hr. after injection of the labeled compounds.
430
BENNETT,
JR.,
SCHABEL,
TABLE
JR.
AND
SKIPPER
VIII
Effects of Azaserine on Synthesis of Nucleic Acid Purines Sarcoma 180 and Host Tissues’ Specific activities
Labeled precursor and azaserine level
-Formate-Cl4 (azasei-ine: 20 mg./ kg.)
Glycine-1-C” (asaserine: 20 mg./ kg.1 AIC-4-Cl4 (azaserine : 20 mg./kg.)
AIC-4-Cl4 (azaserinc : 10 mg./kg.)
Hypoxanthine-8 Cl4 (azaserine mdkg.)
: 20
Hypoxanthine-8 Cl4 (azaserine mg./kg.)
: 20
Adenine-8-C’” (azaserine: mdk.)
20
Compound
DNA:
Ad Gil Th RNA: Ad Gu DNA: Ad Gu RNA: Ad Gu DNA: Ad Gu RNA: Ad GU DNA: Ad Gu RSA: Ad Gu DNA: Ad Gu WA: Ad Gu D?JA: Ad Gu RNA: Ad Gu DNA: Ad Gu RNA: Ad Gu
(treated groups as percentages of controlsjb
Sa 180
35 33 95 41 35 <8 <9 <7 9 83
(0.26) (0.20) (0.20) (0.49) (0.29) (0.25) (0.23) (0.45) (0.33) (0.58)
100 (0.44)
100 108 136 146 133 128 74 85 73 87 120 100 135 119 135
by
(1.1) (0.61) (0.55) (0.41) (1.2) (0.70) (0.076, (0.067, (0.15) (0.097, (0.053, (0.048 (0.092, (0.053: (1.0) (0.17) (1.6) (0.26)
Intestine
23 28 145 35 36 :19 ~25 13 16 51 93 96 135 64 116 84 122 35 59 69 87 61 96 96 122 70 146 104 195
Liver
(0.32) (0.33) (0.11) (0.63) (0.55) (0.16) (0.16) (0.30) (0.25) (0.19) (0.15) (0.69) 43 (0.35) (0.32) 58 (0.15) (0.28) (0.24) (0.99) 92 (0.26) (0.50) 92 (0.13) (0.37) 69 (0.16) (0.29) 100 (0.11) (0.78) 110 (0.30) (0.48) 133 (0.12) (0.28) 63 (0.16) (0.21) 66 (0.13) (0.44) 90 (0.20) (0.27) 83 (0.17) (1.1) 78 (0.31) (0.11) - (<0.07) (2.4) 82 (0.77) (0.18) 67 (0.057:
I-
Spleen
46 (1.9) 39 (1.3) 48 (0.97) 48 (0.50)
30 23 65 47 17 48 83 03 91 21 20 43 31 46 00 24
(1.0) (0.62) (0.75) (0.49) (0.35) (0.21) (0.65) (0.34) (1.1) (0.63) (0.42) (0.51) (1.7)
(1.1) (1.3) (0.72) -
a Five to ten mice were used in each control and each treated group. Each mouse was administered azaserine intraperitoneally at the indicated level immediately before injection of the labeled compound. All labeled compounds except AIC were given intraperitoneally at a level of 10 pc.,/25 g.; the AIC level was 5 +./25 g. Animals were sacrificed 6 hr. after administration of the labeled compounds. b The values in parentheses are the specific activities (counts/sec./pg. purine or thymine) of the control groups.
ACTION
431
OF AZASERINE
TABLE
IX
Effects of Azaserine on Synthesis of Nucleic Acid Purines by a Human Tumor (HS-i) and by Intestine of the Host Rat Specific activity,
cornls/sec./gg. #urine Rat intestine
Human tumor Treatment DNA
Formate-Cl4
RNA
RNA
DNA
0.66
0.61
1.2
0.97
Glycine-lC’4 Note: Azsserine was administered intraperitoneally at a level of 20 mg./kg. immediately prior to the injection of the labeled compound at a level of 10 PC./ 25 g. Animals were sacrificed 6 hr. after administration of the labeled compounds.
that azaserine is inhibitory for at least two sites of synthesis: (a) the condensation of indole and serine to form tryptophan, and (6) the synthesis of tyrosine and phenylalanine from their precursors. The data in Table I and the more extended data in Tables II and III confirm the prevention of inhibition by aromatic amino acids and show, in addition, that when minimal inhibiting levels of azaserine are used, purines (adenine, guanine, hypoxanthine, and xanthine), MC, and methionine are also effective reversal agents. Glutamine (Table V) also prevented inhibition of E. coli by azaserine-a result that might be expected from the observations t,hat glutamine reverses the azaserine-induced inhibition of inosinic acid synthesis in a pigeon liver system (9, 12). From these results, it appears that in E. coli azaserine blocks in more than one metabolic area, and that purine synthesis is one of the sites of action. Preliminary studies of the effect of azaserine on utilization of labeled precursors for synthesis of nucleic acid purines in tumor-bearing mice indicated that azaserine profoundly inhibited incorporation of de nouo precursors, formate, glycine, and the P-carbon of serine, into nucleic acids wit,hout inhibiting utilization of preformed purines (Table VII). The more extended study in which individual purincs were isolated from both RNA and DNA following administration of various labeled precursors (Table VIII) confirms these observations and indicates a marked inhibitory effect of azaserine on de nouo synbhesis of both adenine and
432
BENNETT,
JR.,
SCHABEL,
JR.
AND
SKIPPER
guanine. It is interesting that similar effects on de novo purine synthesis were observed in a heterologous implant of a human tumor (Table IX). Inhibition of de novo purine synthesis by azaserine has also been noted by LePage and Greenlees (20) in studies with the Ehrlich and RC3HED ascites tumors and by Heidelberger and Keller (21) in studies lvith slices of Flexner-Jobling carcinoma and rat spleen; no very marked inhibition of protein synthesis was observed in these experiments. Fernandes et al. (22) have found azaserine to be effective in viva in inhibit,ing glycine incorporation into acid-soluble purines and nucleic acid purincs of several tumors and spleens of the host mice; under the same conditions, utilization of adenine was stimulated. Since formate is a precursor of the methyl carbon atom of thymine (23)) an indication of the specificity of aznserine as an inhibitor of purine synthesis can be obtained by comparing its effects on formate incorporation into purines and into thymine. The fact that, in the same experiment in which formate incorporation into purines was markedly inhibited, formate incorporation into thymine was unaffected or increased (Table VIII) suggests that the observed inhibition of de novo purine synthesis by azaserine is not the result of a general interference with one-carbon metabolism. Large inequalit,ies in effecm on formate incorporation into purines and thymine have also been observed in studies with N-methylformamide (24) and in some (25, 26), but not all (27, 28), studies with the antifolics, aminopterin and A-methopterin. The data in Table VIII clearly indicate that in all tissues azaserine inhibits purine synthesis from either formate or glycine and fails to inhibit incorporation of adenine. In the experiments with AIC and hypoxanthine, some variations in effect were noted either between duplicate runs or from tissue to tissue; two complete experiments with each of these precursors are present’ed in order to illustrate the variations observed. In the two AIC experiments, different levels of azaserine were used; whereas, in the two hypoxanthine experiment’s, the levels of azaserine were the same. Azaserine inhibited incorporation of both AIC and hypoxanthine into intestine DNA adenine, and inhibited slightly incorporation of hypoxanthine into intestine DlSA guanine in one experiment but not in the other. These inhibitions were, however, much smaller than those observed with format’e and glycine. At the higher level employed (20 mg./kg.), azaserine inhibited utilization of AIC by spleen and liver, but no inhibition was observed at the lower level (10 mg./kg.) . In both DNA and RNA of Sarcoma 180 and RlYA of intestine, azaserine consistently failed to inhibit utilization of either RIC or hypoxanthine.
ACTION
433
OF AZASERINE
For the purpose of discussing the inhibition data with azaserine, a diagram of the biosynthetic pathway to nucleic acid purines is desirable. The de novo synthesis of purines has been worked out most thoroughly in pigeon liver, notably by Buchanan and by Greenberg (9, 12, 29-32). Pigeon liver utilizes formate, glycine, and AIC for synthesis of inosinic acid. These same precursors are utilized for the synthesis of nucleic acid purines by the mouse and rat (29). In both t’he mammal and microorganisms, administration of labeled formate and glycine results in the labeling of the same posit,ions in the nucleic acid purines as is observed in uric acid isolated after administration of these same precursors to pigeons (29, 33). Following administration of hypoxanthine-Cl4 to mice and rats, nucleic acid adenine and guanine are labeled almost equally; approximately equal labeling of adenine and guanine is also obtained by administration of labeled formate or glycine (34). Furthermore, an extract of rabbit bone marrow has been shown to convert inosine or inosinic acid to acid-soluble adenine and guanine compounds (35). On the basis of these observations, it is not unreasonable to assume the existence of a general biosynthetic pathway to purines. illthough it is realized that the complet,e de novo pathway to nucleic acid purines has not been worked out in any one biological system, a generalized scheme, based on the literature cited above, is presented below as a basis for discussion and correlation of the data with azaserine obt,ained in several diverse systems. Glycine
+ glycintlmide
ribotide
formylglycinnmidine
+ formylglycinamide ribotide
ribotide
--) 5-aminoimidazole
--) ribotide
+
Ad-R 5-aminohimidazolecarboxamide
ribotide
7
L
I
7
+ Hx-R
polynucleotides Gu-R
If AIC, hypoxanthine, and adenine enter this biosynthetic pathway by conversion to the corresponding ribotides, then the failure of azaserine to inhibit incorporation of these precursors into nucleic acid purines while inhibiting utilization of formate and glycine for purine synthesis would indicat’e that the site of blockade lies before the formation of AIC ribotide. These results obtained in the intact animal are consistent with those of Hartman et al. (9) who have shown that, in a pigeon liver system, azaserine inhibited synthesis of inosinic acid and caused an accumulation of formylglycinamide ribotide. These authors concluded that azaserine exerted an inhibitory action on de novo synthesis of purines at
434
BENNETT,
JR., SCHABEL,
JR. AND SKIPPER
a metabolic site subsequent to the formation of formylglycinamide ribotide. Additional evidence, obtained by this same group of workers, indicated that azaserine behaved as a glutamine antagonist in inhibiting reaction of glutamine with formylglycinamide ribotide to yield formylglycinamidine ribotide (12,32). Working in still another system, Tomisek et al. (36) have observed the accumulation of radioactive formylglycinamide ribotide and formylglycinamide riboside by azaserine-treated E. coli grown in a medium containing formate-Cl4 or glycine-CL4. In these experiments, azaserine inhibited incorporation of formate and glycine into purines, purine nucleosides, and purine nucleotides. Thus, data obtained in three different biological systems-pigeon liver, E. coli, and the intact tumor-bearing mouse-show that azaserine inhibits at an early stage of de lzozlopurine synthesis, and these data are consistent with the concept that the site of blockade lies somewhere between formylglycinamide ribotide and 5-amino-4-imidazolecarboxamide ribotide. ACKNOWLEDGMENTS
The authors wish to thank Dr.
1. Azaserine-induced inhibition of E. coli was significantly prevented by (a) 4-amino-5-imidazolecarboxamide (AIC), adenine, guanine, hypoxanthine, or xanthine; (b) methionine; and (c) glutamine; and, as has already been observed by others, by phenylalanine, tyrosine, or tryptophan. 2. In studies carried out on the effect of azaserine on purine synthesis in the tumors, intestines, livers, and spleens of mice bearing Sarcoma 180, it was found that azaserine inhibited utilization of formate-Cl4 and glytine-l-G4 for purine synthesis, failed to affect utilization of CY4-labeled adenine, AIC, and hypoxanthine, and failed to affect utilization of formate for thymine biosynthesis. Azaserine also inhibited utilization of formate-Cl4 and glycine-l-C?4 for purine synthesis by heterologous implants of a human sarcoma growing in the rat. 3. The results indicate that azaserine inhibits purine synthesis in the
ACTION
OF AZASERINE
435
intact animal at a stage prior to the formation of 5-amino-kimidaeolecarboxamide ribotide. REFERENCES
1. FUURI, S. A., FROHBRDT, R. P., RYDER, A., HASKELL, T. H., JOHANNESSEN, D. W., ELDER, C. C., AND BARTZ, Q. R., J. Am. Chem. Sot. 76, 2878 (1954). 2. FUSARI, S. A., HASKELL, T. I-I., FROIIARDT, R. I'., AND BARTZ, Q. R.,J. Am. Chem. Sot. 76, 2881 (1954). 3. STOCK, C. C., REILLY, H. C., BUCBLEY, S. M., CLARKE, D..4., ASD RHOADS, c. P., iVat7tre 173, 71 (1954). 4. CLBRKE, D. -4., PHILIPS, F. S., STOCK, C. C., ELION, G. B., ASD HITCHINGS, G. H., Abstracts of Papers, p. 12-M. 125th?rlceting, Am. Chem. Sot., Kansas City, 1954. 5. POTTER, V. R., Proc. Sot. Erptl. Bid. Med. 76, 41 (1951). 6. Law, L. W., Cancer Research 12, 871 (1952). 7. SKII'PER, H. I?., THOMSOX,J. R., ASD BELL, M., Cancer Research 14, 503 (1954). 8. SKIPPER, I-I. E., BESNETT, L. L., JR., AXD SCHABEL, F. M., JR., Federation hoc. 13, 29s (1954). 9. HARTM\IAK,S.C.,LEVENRERG, B., .~NDBUCH.AS~N,J.M., J. Am. Chem.Soc.77, 501 (1955). 10. SKIPPER, H. E., SCHAREL, F. hf., JR., BIXNS, V., THOMSOX, J. R., and WHEELER, G. I'., Cancer Research 16, 143 (1955). 11. DAVIS, B. D., ASD ?VfINGIOLI,E. S., J. Bacterial. 60, 17 (1950). 12. BUCIIAN.~~, J. nl., LEVESBERG, B., .~ND LUKE%, L. N., Abstracts of Papers, p. 12-C. 12Sth Meeting, Am. Chem. Sot., hkmenpolis, 1955. 13. SHAW, E., AXD WOOLLEY, 1). W., .J. Biol. Chem. 181, 89 (1949). 14. BENNETT, L. L.,JR.,S~PI%R,H.E.,STOCIC,C.C.,.~ND R~o~~s.,C.I'.,Cancer Rescarch 15, 4% (1955). 15. MARSHAK, A., ;\SD VOGEL, H. J., J. Biol. Chem. 189, 597 (1951). 16. MARKMAN, R., AXD Smm, J. I)., Biochem. J. 45, 294 (1949). 17. TOOL~X, H. W., Cuncer Xcseard~ 13, 3S9 (1953). 18. TOOLAN, H. W., Cancer Research 14, 660 (1954). 19. K.~PLAX, L., .4xD STOCK, C. C., Federation Proc. 13, 239 (1954). 20. LEPAGE, G. A. APED GREESLEES, J., Cancer Research, Suppl. No. 3, p. 102 (1955). 21. HEIDELBERGER, C., AXD KELLER, R. A., Cancer Research, Suppl. No. 3, p. 106 (1965). 22. FERSASDEF, J. F., LEPAGE, G. A.,.~xD LINDNER, A., Cancer Research 16, 154 (1956). 23. TOTTER, J. R., VOLKIN, E., AND CARTER, C. E., J. Am. Chem. Sot. 73, 1521 (1951). 24. BARCLAY, R. K., GARFINEEL, E., AND STOCK, C. C., J. Biol. Chem. 208, 875 (1954). 25. TOTTER, J. R., AND BEST, 9. N., Arch. Biochem. and Biophys. 64, 318 (1955). 26. BALIS, hf. E., AXD ~.~NCIS, J., Cancer Research 16, 603 (1955). 27. GOLDTHWAIT, D. A., AXD BENDICH, A., J. Biol. Chem. 196, 841 (1952).
436
BENNETT,
JR.,
SCHABEL,
JR.
.4ND
SKIPPER
28. SKIPPER, H. E., BENNETT, L. L., JR., AND LAW, L. W., Cancer Research 12, 677 (1952). 29. REICHARD, P., in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 2, Chap. 23. Academic Press, New York, 1955. 30. LEVENBERQ, B., HARTMAN, S. C., AND BUCHANAN, J. M., Federation Proc. 14, 243 (1955). 31. GOLDTHWAIT, D. A., GREENBERG, G. R., AND PEABODY, R. A., Biochim. et Biophys. Acta 18, 148 (1955). 32. LEVENBERG, B., AND BUCHANAN, J. M., J. Am. Chem. Sot. 78, 504 (1956). 33. SUTTON, W. B., AND WERKMAN, C. H., Arch. Biochem. and Biophys. 47, 1 (1953). 34. BENNETT, L. L., JR., AND SKIPPER, H. E., Arch. Biochem. and Biophys. 64, 566 (1955). 35. ABRAM, R., AND BENTLEY, M., Arch. Biochem. and Biophys. 58, 109 (1955). 36. TOMISEK, A. J., KELLY, H. J., AND SKIPPER, H. E., Arch. Biochem. and Biophys. 64, 437 (1956).