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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry

and Molecular

Biology, Inc.

Vol. 265, No. 19, Issue of July 5, pp. 11377-11381, 1990 Printed in U.S. A.

Cytotoxic Mechanisms of Glutamine in Mouse L 12 10 Leukemia*

Antagonists
(Received for publication, January 29, 1990)

Stephen
From

D. Lyons,

Melissa
of Biochemistry,

E. Sant,

and Richard
of Sydney,

I. ChristophersonS
Sydney, New South Wales 2006, Australia

the Department

University

The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent amidotransferases in vitro. Experiments performed with mouse L1210 leukemia growing in culture show that each antagonist has different sites of inhibition in nucleotide biosynthesis. Acivicin is a potent inhibitor of CTP and GMP synthetases and partially inhibits N-formylglycineamidine ribotide (FGAM) synthetase of purine biosynthesis. DON inhibits FGAM synthetase, CTP synthetase, and glucosamine-6-phosphate isomerase. Azaserine inhibits FGAM synthetase and glucosamine-6-phosphate isomerase. Large accumulations of FGAR and its di- and triphosphate derivatives were observed for all three antagonists which could interfere with the biosynthesis of nucleic acids, providing another mechanism of cytotoxicity. Acivicin, azaserine, and DON are not potent inhibitors of carbamyl phosphate synthetase II (glutamine-hydrolyzing) and amidophosphoribosyltransferase in leukemia cells growing in culture although there are reports of such inhibitions in vitro. Blockade of de nouo purine biosynthesis by these three antagonists results in a “complementary stimulation” of de nouo pyrimidine biosynthesis.

L-Glutamine provides the amino group for biosynthesis of a variety of metabolites including the purine intermediates and derivatives, 5-phosphoribosylamine, N-formylglycineamidine (FGAM),l GMP, NAD, and NADP, and the pyrimidine intermediates carbamyl phosphate and CTP, and glucosamine 6-phosphate (GlcN-6-P). The L-glutamine antagonists, acivicin, azaserine, and DON, have anti-cancer activity attributed
*This work was supported by Project Grant 880119 from the National Health and Medical Research Council, Grant A08415281 from the Australian Research Council, and Grants AI183 and AK123 from the Utah Foundation for HPLC detectors. The costs of publication of this article were defraved in _ part bv_ the pavment of -page __ charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelv to indicate this fact. $ To whom correspondence should be addressed. ’ The abbreviations used are: FGAM. N-formvlplvcineamidine ribotide; P-Rib-PP, 5-phosphoribosyl ‘l-pyropgo&hate; lo-CHOH,folate. A”‘-formvl tetrahvdrofolate: FGAR. N-formvlglvcineamide ribotide; AIR, 5-aminoimidazole ribotide; SAICAR, N-succino-5-aminoimidazole-4-carboxamide ribotide; AICAR, 5-aminoimidazole-4carboxamide ribotide; sAMP, adenylosuccinate; Ord, orotidine; Oro, orotate; OMP, orotidine 5’-monophosphate; amido PRTase, amidophosphoribosvltransferase; CPSase, carbamvl phosphate svnthetase i1 (gcutaminehydrolyzing); DON, 6-diazo-5-dxolr,-norleuciie; FGARDP/TP, di- and triphosphate derivatives of FGAR: Henes. 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid; HPLC; high pies&e liquid chromatography; X,.,, wavelength of maximal ultraviolet absorbance.
” -”

to potent irreversible inhibition of amidotransferases involved in nucleotide biosynthesis (Livingston et al., 1970; Tso et al., 1980). Most of these amidotransferases are inhibited by all three glutamine antagonists in uitro. For example, Jayaram et al. (1975) found that of the seven amidotransferases involved in nucleotide metabolism, amidophosphoribosyltransferase, GMP synthetase, carbamyl phosphate synthetase II (glutamine hydrolyzing) were not significantly affected by azaserine while FGAM synthetase, CTP synthetase, GlcN-6P isomerase,2 and NAD synthetase were inhibited. DON inhibited all seven amidotransferases in uitro; acivicin inhibited six of the seven amidotransferases by more than 90% while amidophosphoribosyltransferase was inhibited by only 10%. Carbamyl phosphate synthetase II, CTP synthetase, amidophosphoribosyltransferase, FGAM synthetase, and GMP synthetase from rat hepatoma are potently inhibited by acivicin in uiuo when enzymic activities are subsequently assayed in cell extracts (Aoki et al., 1982; Weber et al., 1984; Elliott and Weber, 1985). Inhibition of purified target enzymes in vitro and inactivation of amidotransferases in uiuo with subsequent assay of enzymic activities in cell extracts determine all susceptible reactions but do not discriminate between possible mechanisms of cytotoxicity in uiuo. An antagonist may inhibit several amidotransferases in a pathway, but blockade of the most proximal reaction provides the primary cytotoxic effect; flux through the pathway to subsequent reactions is blocked and inhibition at distal amidotransferases would then be of minor significance. Data presented in this paper show that acivicin, DON, and azaserine each have unique modes of action in growing leukemia cells. Susceptible amidotransferases for each antagonist in uiuo were identified as metabolic crossouerpoints, where cellular metabolites prior to the blockade accumulate and subsequent metabolites deplete relative to those in untreated cells (Rolleston, 1972; Rognstad, 1979; Christopherson and Duggleby, 1983). The methodology described here can be generally applied to the determination of cytotoxic mechanisms of inhibitors of de nouo nucleotide biosynthesis in growing cells.
EXPERIMENTAL PROCEDURES DISCUSSION AND RESULTS3

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Metabolic crossover points have been used to define reactions of nucleotide metabolism in growing leukemia cells
* GlcN-6-P isomerase catalyzes the conversion of Fru-6-P + GlcN6-P which is a precursor for the svntheses of UDP-GlcNAc and UDPGalNAc: GlcG-6-P + GlcNAc-6-P ++ GlcNAc-1-P + UDP-GlcNAc ++ UDP-GalNAc. 3 Portions of this paper (including “Experimental Procedures,” “Results,” and Figs. 1 and 4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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“Elution leukemia surfaces” cells.org by guest. 4). At least two significant metabolic crossover points or sites of inhibition are induced by each glutamine antagonist investigated (Table II). No [Ylbicarbonate is incorporated into GTP in the presence of acivicin indicating that inhibition of GMP synthetase is complete and the primary site of inhibition in purine biosynthesis (data not shown).#A ACIVICIN of Amidotransferases which become rate-limiting in the presence of a glutamine antagonist leading to cytotoxicity..5 mM (Fig. >0. magenta. 1978). Significant incorporation of [Ylbicarbonate into 4-carboxy&aminoimidazole ribotide and subsequently into ATP. 1964. Purified GMP synthetase is totally inhibited by DON (Jayaram et al. These procedures enable determination in growing cells of an inhibited reaction which has substrates and products with significant ultraviolet absorption (Figs. blue. We have identified and quantified the di.OlO.006. 3 and 4) may lead to the greater host toxicity displayed by this antagonist (Catane et al. Accumulation of N-succino-5-aminoimidazole-4-carboxamide ribotide. Incorporation of FGAR triphosphate into nucleic acids or direct inhibition of RNA or DNA polymerases by FGAR derivatives may be additional mechanisms of cytotoxicity for all three glutamine antagonists. 1970). 1966. 1975). but the site of inhibition most on NAD I AMP UMP AICAFI IMP j Downloaded from www. >0.3 which is nearly 3-fold higher than the normal concentration of ATP in mammalian cells (-3.Inhibition 25 . >0. Rowe et al. The representation of data as elution surfaces is described in the text.. Four hours after exposure to 25 pM azaserine. However. 3 and 4). 2. it is known that azaserine induces morphological irregularities in the genetic material of malignant and normal cells with associated increases in cellular size and DNA content (Hansen and Vandevoorde. 2013 UDphugars NADP CDP UDP ADP sAMP SAICAR FIG.and triphosphate derivatives of FGAR. The full-scale deflection is 0. FGAR triphosphate reaches a cellular concentration of 9. The metabolic consequences of these higher phosphorylated forms of a natural purine intermediate are not known. >O. l-3).004. green.5 mM). 1985). >O. yellow. red. . Two cultures in the presence of [“Clformate (50 PM). and the more highly charged “FGAR polyphosphates” remained unresolved at or near the origin of chromatograms after development with solvent.jbc.005 absorbance unit. Accumulation of FGAR and depletion of adenine and guanine nucleotides are consistent with inhibition of FGAM synthetase by acivicin.OOO. Livingston et al. The levels of FGAR derivatives accumulated may be a result of the relative proportions of FGAM synthetase and amidophosphoribosyltransferase inactivated by each antagonist. and one was exposed to acivicin (25 pM) for 4 h.002. and XMP and depletion of guanine nucleotides defines a metabolic crossover point at GMP synthetase in acivicintreated leukemia cells (Figs. >0. 1979.012. The greater accumulation of FGAR derivatives induced by azaserine (Figs. on February 21. Earhart and Neil. thin layer chromatography was used for separating purine precursors.. cyan. Rosenbloom et al. and DON. azaserine. after addition of acivicin (data not shown).008. >0. Metabolites from 50-ml samples were extracted and analyzed by showing effects (55 ml) were grown of acivicin HPLC. 1 and 2) and/or which incorporate 14C from a radiolabeled precursor (Figs. possibly by interference of these metabolites with RNA synthesis in nondividing cells. and the colored contours have the following absorbance ranges: block.. 1968. IMP. 5-aminoimidazole-4-carboxamide ribotide. Because DON and azaserine are potent inhibitors of FGAM synthetase.. indicates that this antagonist is not a potent inhibitor of FGAM synthetase in growing cells although Elliott and Weber (1985) demonstrated potent inhibition of FGAM synthetase by acivicin in vitro in assays of extracts from drug-treated rat hepatoma. purine intermediates distal to the blockade disappear and inhibition of GMP synthetase could not be observed. Accumulation of higher phosphorylated derivatives of FGAR in response to azaserine treatment has been reported (Bennett and Smithers.

1987). and maintenance of ATP levels required for metabolic functions of normal resting (Go phase) cells. on metabolites B. -.Inhibition of Amidotransferases . potent inhibition. DON and acivicin both induce accumulation of UTP and depletion of ATP. azaserine. and CTP (Fig. amidophosphoribosyltransferase. DON. Glutamine Amidotransferase Acivicin DON Azaserine antagonist Inhibition AmidoPRTase FGAM synthetase GMP synthetase NAD synthetase CPSase CTP synthetase GlcN-6-P isomerase 0 -0 0 0 0 -- 0 0 0 0 0 0 0 0 --- - 30 Time (min) FIG. CPSase. c. Acivicin and DON also inhibit CTP synthetase (Table II) which would enhance accumulation of UTP. and azaserine exert their cytotoxic effects via inhibition of different groups of glutamine amidotransferases (Table II). Id). 1980). and levels profiles of Fig. consistent with inhibition of DNA synthesis. Glutamine Nucleotide Acivicin DON ATP ADP GTP GDP UTP UDP CTP CDP NAD NADP UDP-Glc/Gal UDP-GlcNAc/GalNAc 86 78 52 58 180 140 3. a. carbamyl phosphate synthetase II (glutamine hydrolyzing). of Cultures (55 ml) were grown in the presence of [‘%]formate from 24 h after inoculation at a density of 4 X lo4 cells/ml. A. but different amidotransferases are affected. . methotrexate (NSC 740) induces incorporation of dUTP into DNA (Goulian et al. Effects of glutamine cells which incorporate antagonists [Wlformate. 1) although the depletion of ATP in acivicin-treated cells is minor compared with the corresponding depletion induced by DON (Table I). Acivicin and DON block both de nouo purine and pyrimidine biosynthesis in growing cells. amidophosphoribosyltransferase. unbalanced nucleotide pools may lead to genetic miscoding. 1986) or pyrazofurin (Sant et al. were are expressed antagonist Azaserine relative to untreated cells.. and o.9 67 94 95 160 170 % of control/cell 39 48 35 61 290 190 4. Cells were exposed to drug (25 pM) for 4 h. and metabolites from 50-ml samples were extracted and analyzed by HPLC. only 14C profiles are shown..org by guest. . and NAD synthetase are not major sites of inhibition in growing cells. 1980). These antagonists are still being evaluated as potential anti-cancer agents long after their discovery.jbc.. Cellular metabolites were extracted from a 50-ml culture sample and analyzed by HPLC as described under “Experimental Procedures. augmenting the accumulation of UTP induced by acivicin or DON and causing the relative increases in UTP and CTP induced by azaserine (Fig. which does not inhibit CTP synthetase (Table II). 3. 1) may be misincorporated into RNA. Acivicin. which may be attributed to sparing of &phosphoribosyl I-pyrophosphate from the orotate phosphoribosyl- transferase reaction and of glutamine from the CTP synthetase reaction. partial inhibition.” Peaks representing nucleotides of interest in absorbance profiles. and inhibition of amidophosphoribosyltransferase would spare both glutamine and 5-phosphoribosyl lpyrophosphate. Table I).. AmidoPRTase. acivicin. which may be generally toxic. 1. All three glutamine antagonists block the purine pathway (Table II) and induce accumulation of UTP relative to the level of ATP after drug treatment (Fig. DON induces S phase arrest (Huber et al.. Kemp et aL. Acivicin blocks progression of the cell cycle in G. potent inhibition of either the de novo pyrimidine or purine pathway is accompanied by “complementary stimulation” of flux through the unaffected pathway (Lyons. 1975. absorbance which incorpob. Accumulation of these metabolites could stimulate pyrimidine biosynthesis. The eluate was monitored concurrently by ultraleukemia violet absorbance and “C detectors. and C are metabolites with ultraviolet rate [“Clformate d. Thornwaite and Allen. For example. 1975). 3.5 14 97 99 350 50 29 46 21 49 67 66 50 80 68 66 100 34 proximal in the pathway induces the cellular deficiencies of nucleotides. A general phenomenon has been observed for many inhibitors of nucleotide biosynthesis. in contrast to results obtained in uitro (Jayaram et al. 60 90 u Partial inhibition of amidophosphoribosyltransferase by DON is consistent with the reduced accumulation of FGAR derivatives relative to azaserine although inhibition of FGAM synthetase is complete for both antagonists. which suggests that RNA and/or DNA synthesis is affected. Blockade of the purine pathway at FGAM synthetase or GMP synthetase would spare cellular glutamine from utilization in purine biosynthesis. no inhibition.-. 2013 . A detailed knowledge of their mechanisms of Downloaded from www. and are unidentified. particularly guanine nucleotides. carhamyl phosphate synthetase II. GTP. on February 21. DON. Acivicin has shown more promise than DON as an anti-cancer drug perhaps due to less accumulation of FGAR triphosphate. TABLE II 11379 of amidotransferases in growing leukemia cells by glutamine antagonists Sites of inhibition were determined by comparison of metabolite levels prior to and 4 h after addition of drug to define a metabolic crossover point. While the physiological significance of complementary stimulation is yet to be determined. Increased levels of UTP accumulated in response to acivicin or DON treatment (Fig. corresponding integrated using Nelson to the radioactivity software. 1989). control. or early S phase (Jayaram et al. 1989a) leads to complementary stimulation of purine biosynthesis.. TABLE I Effects of glutamine antagonists on nucleotide levels in leukemia cells Cultures (55 ml) were grown in the presence of [Wlformate (50 pM) and exposed to the indicated glutamine antagonist (25 pM) for 4 h. Blockade of the de nouo pyrimidine pathway by dichloroallyl lawsone (NSC 126771.

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