Dual Effects of Pyrazofurin and 3-Deazauridine upon ... · novo purine biosynthesis in leukemia cells growing in culture. ... mediates of the de novo purine pathway, ... intermediate,

[CANCER RESEARCH 49, 2645-2650, May 15, 1989]

Dual Effects of Pyrazofurin and 3-Deazauridine upon Pyrimidine and FurineBiosynthesis in Mouse LI 210 Leukemia1

Melissa E. Sant, Stephen D. Lyons, Anthony J. Kemp,2 Linda K. McCIure, Eve Szabados, andRichard I. Christopherson3

Department of Biochemistry, University of Sydney, Sydney, N.S. W., 2006, Australia

ABSTRACT

Pyrazofurin (NSC 143095) as the monophosphate derivative is a potentinhibitor of orotidine S'-monophosphate (OMP) decarboxylase of the

pyrimidine pathway and has been proposed to inhibit 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (EC 2.1.2.3) of thepurine pathway (J. F. Worzalla, and M. J. Sweeney, Pyrazofurin inhibition of purine biosynthesis via S-aminoimidazole^-carboxamide-l-/?-D-ribofuranosyl 5'-monophosphate formyltransferase. Cancer Res., 40:1482-1485, 1980). Measurement of levels of pyrimidine and purineintermediates in cultured mouse 1.1210 leukemia cells has shown that 25MMpyrazofurin induces an 8-fold accumulation of OMP and large accumulations of intermediates proximal to the blockade with abrupt decreases in uridine and cytidine nucleotides. Considerable increases in thecellular concentrations of jV-succino-AICAR (SAICAR), AICAR, 5-for-mamidoimidazole-4-carboxamide ribotide (FAICAR), IMP, XMP, andGMP at later times indicate that AICAR transformylase is not significantly inhibited in cultured cells; rather the purine pathway and the GMPbranch are stimulated. However, addition of 25 MM3-deazauridine (NSC126849) to leukemia cells did result in inhibition of AICAR transformylase: AICAR and SAICAR accumulated, IMP disappeared and therewas a large accumulation of guanosine nucleotides. Blockade of pyrimidine biosynthesis by derivatives of pyrazofurin or 3-deazauridine spares5-phosphoribosyl-l-pyrophosphate and L-glutamine, elevated concentrations of which may stimulate initial reactions of purine biosynthesis andthe reaction XMP -Â»GMP.

INTRODUCTION

Pyrazofurin, first isolated from Streptomyces candidus, is aC-nucleoside readily taken up by mammalian cells and phos-phorylated by adenosine kinase to pyrazofurin S'-monophosphate, a potent inhibitor of OMP4 decarboxylase (EC 4.1.1.23;K,â€¢=5 x 10~9M) (1). A mixture of mono-, di-, and triphosphate

derivatives of pyrazofurin has been found in drug-treatedLSI78Y cells (1). Pyrazofurin exhibits anticancer activity insome leukemia patients but toxicity has limited its general usageas an antitumour agent (2). Blockade of the de novo pyrimidinepathway can be bypassed by increased utilization of exogenousuridine (3). Cadman and Benz (4) proposed that the reductionin uridine and cytidine nucleotide pools following pyrazofurintreatment could result in enhanced intracellular accumulationof antitumour pyrimidine analogues such as S-azacytidine and3-deazauridine. The X-ray crystallographic structure of pyra-

Received 9/23/88; revised 1/18/89; accepted 2/13/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by Grant A08415281 from the Australian Research

Council and Grants AI 183 and AK 123 from the Utah Foundation for HPLCdetectors.

2Supported by a postgraduate scholarship from the Anti-Cancer Council of

Victoria.3To whom requests for reprints should be addressed.'The abbreviations used are: OMP, orotidine S'-monophosphate; PF-MP,

PF-DP, and PF-TP, phosphorylated forms of pyrazofurin; DAU-MP, DAU-DP,and DAU-TP, phosphorylated forms of 3-deazauridine; FGAR, jY-formylglyci-neamide ribotide; FGAM, A'-formylglycineamidine ribotide; CAIR, 4-carboxy-5-aminoimidazole ribotide; SAICAR, Ar-succino-5-aminoimidazole-4-carboxamideribotide; AICAR, S-aminoimidazole-4-carboxamide ribotide; FAICAR, 5-for-mamidoimidazole-4-carboxamide ribotide; sAMP, A"-succino AMP; P-Rib-PP,5-phosphoribosyl-1 -pyrophosphate.

zofurin has been determined and a variety of derivatives havebeen synthesized but without enhanced potency (5). Worzallaand Sweeney (6) demonstrated that pyrazofurin S'-monophos

phate inhibits AICAR transformylase (EC 2.1.2.3) in vitro (KÂ¡= 3 x IO"5M) and administration of pyrazofurin to rats resulted

in excretion of 5-aminoimidazole-4-carboxamide riboside presumably derived from the ribotide derivative (AICAR) of thepurine pathway.

3-Deazauridine was synthesized by Robins and Currie (7)and shown to be converted to the triphosphate derivative bymouse LI210 leukemia cells but with no incorporation intoRNA or DNA (8). 3-Deazauridine 5'-triphosphate is a compet

itive inhibitor of CTP synthetase in mouse LI210 leukemia (KÂ¡= 5.3 x IO"6 M) (9). The growth-inhibitory effects of 3-de

azauridine upon L1210 cells were reversed by cytidine, consistentwith inhibition of CTP synthetase in vivo. A secondary effectof 3-deazauridine may be inhibition of the reduction of CDP todCDP by 3-deazauridine diphosphate (10); 3-deazauridine alsopotentiates the cytotoxicity of cytosine arabinoside and 5-aza-2'-deoxycytidine by depressing cellular levels of dCTP (11, 12).

In addition, thymidine and 3-deazauridine independentlyreduce cellular dCTP and act synergistically against humantumor cells (13). 3-Deazauridine and the S'-monophosphate

inhibit cytidine deaminase and dCMP deaminase, respectively(14). Clinical trials indicate that 3-deazauridine as a singleagent has a limited role in the treatment of patients with acuteleukemia (15). 3-Deazauridine is phosphorylated to the S'-

monophosphate by uridine-cytidine kinase (EC 2.7.1.48) (11).Karle and Cysyk have postulated that 3-deazauridine triphosphate, in addition to inhibiting CTP synthetase (EC 6.3.4.2),acts as an inhibitor of carbamyl phosphate synthetase II (EC6.3.5.5) and uridine-cytidine kinase (16).

New assay procedures for pyrimidine (17) and purine5 intermediates and precursors involving two-dimensional thin-layerchromatography and high-pressure liquid chromatography havebeen developed in our laboratory. These procedures have enabled us to determine the sites in nucleotide metabolism whichare affected in vivo by treatment of leukemia cells with pyrazofurin or 3-deazauridine. Sites of inhibition in vivo have beendetermined by measurement of elevated cellular concentrationsof intermediates before a blockade and depletion of intermediates thereafter, a metabolic cross-over point. Our data confirmprincipal sites of inhibition in the pyrimidine pathway by phosphorylated derivatives of these nucleoside analogues, and wehave found that both analogues have significant effects upon ticnovo purine biosynthesis in leukemia cells growing in culture.

MATERIALS AND METHODS

All chemicals were of analytical reagent grade. 3-Deazauridine waspurchased from the Sigma Chemical Company; pyrazofurin was fromCalbiochem. RPMI 1640 medium (bicarbonate free) with 20 mMK.Hepes as buffer was purchased from Flow Laboratories; mouse

5 M. E. Sant, A. Poiner, M. C. Harsanyi, S. D. Lyons, and R. I. Christopherson,

manuscript submitted for publication.

2645

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IDINE AND PURINE BIOSYNTHESIS

L1210 leukemia cells were provided by Dr. Ian Roos and VirginiaLeopold of the Peter MacCallum Cancer Institute, Melbourne. Sodium[l4C]bicarbonate (35.1 mM, 57.0 Ci/mol) and sodium [14C]formate

(100 mM, 58.0 Ci/mol) were from Amersham (UK). Poly(ethylene)imine-cellulose thin-layer chromatograms were manufactured byMachery-Nagel (Germany); similar chromatograms containing Poly-min SK poly(ethylene)imine were prepared as described previously (17).1640 medium containing 20 mM K.Hepes (pH 7.4) and supplementedwith 13% (v/v) fetal calf serum, 0.35 mM bicarbonate, 2.0 mM L-glutamine, and 50 ng/m\ gentamicin. Cells were inoculated into freshmedium at a density of 4.0 x IO4cells/ml; [14C]bicarbonate (0.35 mM)or [l4C]formate (0.10 HIM)was added at this time and cultures grewwith a doubling time of approximately 11 h after an initial lag. Pyra-zofurin or 3-deazauridine (25 JJM)was added at a density of 5 x 10scells/ml and the culture was sampled within the "window" 2.5-7.5 xIO5cells/ml and processed as previously described (17).

Two-Dimensional Chromatography on Polyethyleneimine-Cellulose.To measure levels of pyrimidine intermediates, leukemia cells weregrown in the presence of 0.35 mM ['4C]bicarbonate (57.0 Ci/mol),

cellular contents were extracted with cold 0.4 MHC1O4, and pyrimidineprecursors and nucleotides were separated by two-dimensional chro-matography on poly(ethylene)imine-cellulose using two different solvent systems as previously described (17). For quantification of intermediates of the de novo purine pathway, leukemia cells were grown inthe presence of 0.1 mM [l4C]formate (58.0 Ci/mol) and cell extracts(50 ill) were subjected to two-dimensional chromatography onpoly(ethylene)imine-cellulose. Briefly, chromatograms were developedin the tirsi dimension with 700 mM ammonium chloride (pH 2.8),desalted by immersion in anhydrous methanol, then developed in thesecond dimension with 0.2 M LiCl, 0.6 M acetic acid (pH 2.5). Radio-labeled spots on chromatograms derived from [14C]bicarbonate or[14C]formate were visualized by autoradiography on Kodak X-Omat RPX-ray film for 3 months. Further details of the chromatography andassignment of the identities of spots are described elsewhere (17).5

Separation of Metabolites by HPLC. A modified version of thegradient solvent system described by Kemp et al. (17) was used with aPartisil 10-SAX anion-exchange column (Whatman) equilibrated initially with Buffer A (7.0 mM KH2PO4, pH 3.0). A 200 ^1 sample of thecell extract was loaded onto the HPLC and eluted at a flow rate of 2.0ml/min with a concave gradient (Waters curve 7) from Buffer A toBuffer B (250 mM KH2PO4, 500 mM KC1, pH 3.8) for 45 min followedby isocratic elution with Buffer B for a further 45 min. Separatedmetabolites were monitored using an LKB Rapid Spectral Detector andan LKB Betacord Radioactivity Monitor. The former records instantaneous whole spectra from 190 nm to 370 nm while the latter monitorsI4C in the eluate. Both data files were stored on the 20 MB hard disk

of an IBM XT microcomputer and subsequently processed using the3000 Series Chromatography Data System software (Version 3.6)written by Nelson Analytical Inc., CA. Elution profiles at 260 nm werestored in Nelson format and peaks of interest were integrated usingthis software to give areas in units of ^v/s which were converted topmol by comparison with appropriate chromatographed standards.Further details of the procedures used and assignment of peaks appearelsewhere.5

Quantification of I4C. Radiolabeled spots on chromatograms were

located by alignment of the autoradiogram film with the chromatogram,spots of interest were cut out and counted in scintillant containing 5.0g 2,5-diphenyloxazole, 0.1 g l,4-bis[2-(5-phenyloxazolyl)]benzene perliter of toluene with an efficiency of 82.1%. Aliquots of HPLC eluatewere counted after addition of 9 volumes of scintillant containing 5.5g 2,5-diphenyloxazole, 0.1 g l,4-bis[2-(5-phenyloxazolyl)]benzene perliter of toluene (two volumes) and Triton X-100 (one volume), countingefficiency was 91.0%.

RESULTS

leukemia cells has been determined by two-dimensional chromatography (17) and the data obtained are shown in Fig. 1. Allintermediates proximal to reaction 6 catalyzed by OMP decar-boxylase accumulate; after 7 h carbamyl aspartate and orotidinereach levels of 12 fmol/cell (8.8 raw) and 13 fmol/cell (9.5mM), respectively. Approximate cellular concentrations inbrackets have been calculated using a specific radioactivity forpyrimidine intermediates of 1.71 Ci/mol (see below) and acellular volume of 1.3 pi has been assumed. AccumulatingOMP, the substrate of the inhibited reaction, is hydrolyzed toorotidine by a phosphatase activity (18, 19); the rates of accumulation and hydrolysis are equal when OMP levels haveincreased 8-fold (Fig. 1Â¿>).By contrast, the level of the nextintermediate, UMP, decreases abruptly (Fig. Ib) defining thesite of inhibition or a metabolic cross-over point at the reactioncatalyzed by OMP decarboxylase. The levels of all subsequentpyrimidine nucleotides rapidly decrease in concert.

Mouse leukemia cells were grown in the presence of [I4C]-

nO

Eo."O

C0)oCoo

50

40

30

20

10

O

1.0

0.8

0.6

0.4

0.2

O

10

8

6

4

2

O

a

-2

The effect of pyrazofurin upon cellular levels of sequentialintermediates of the de novo pyrimidine pathway in mouse

Time (h)Fig. 1. Effects of 25 Â«IMpyrazofurin upon intermediates of de novo pyrimidine

biosynthesis, a: O, carbamyl aspanate; D, dihydroorotate; A, orotate; O, orotidine.A: O, OMP; O, UMP. c: O, UTP; D, UDP-glucose, A, CTP, O, UDP. Mouseleukemia cells were grown in the presence of [14C]bicarbonate, pyrazofurin was

added at zero time, samples were taken at the indicated times and separated bytwo-dimensional chromatography as described in "Materials and Methods."

2646



NUCLEOSIDES AFFECTING PYRIMIDINE AND FURINE BIOSYNTHESIS

formate (0.1 HIM), and '"C-labeled purine intermediates were

separated by two-dimensional chromatography as described in"Materials and Methods." Autoradiograms of chromato-graphed extracts from a "control" culture and cells treated with

pyrazofurin for 8 h are shown in Fig. 2. Spots are numbered asproducts of sequential reactions of the purine pathway; theidentities of those marked alphabetically remain to be positivelyassigned. Comparison of the intensities of the spots for sequential purine intermediates between drug-treated and control cultures shows increases in 8 (AICAR), 9 (FAICAR), 13 (XMP),and 14 (GMP). Spots 10 (IMP), 11 (sAMP), and 12 (AMP)change little while spots A and B decrease substantially. Progress curves were constructed for purine intermediates for whichUV marker compounds were commercially available (Fig. 3).After addition of pyrazofurin, AICAR, IMP, and sAMP showan initial upward spike followed by return toward originalsteady-state levels, AMP and XMP increase, and GMP rapidlyincreases 3.5-fold.

To further investigate the metabolic effects of pyrazofurin onmouse leukemia cells, two cultures were grown in the presenceof [l4C]formate (0.1 HIM), one culture was exposed to pyrazo

furin (25 MM)for 4 h, and extracts were prepared from samples(250 ml) of both cultures. The elution profiles recorded at 260nm (Fig. 4, a and b) show effects upon both pyrimidine andpurine biosynthesis. For the pyrazofurin-treated culture, fourlarge peaks elute between 10 and 20 min (Fig. 4h): orotidine,orotate, IMP and pyrazofurin 5'-monophosphate (PF-MP).

Uridine and cytidine nucleotides have decreased to very lowlevels; radiolabeling cells with [l4C]bicarbonate at the time ofdrug addition showed no incorporation of I4C into UTP and

CTP indicating that the pyrimidine pathway is rapidly andtotally blocked by PF-MP. The UTP and CTP remaining (Fig.4Z>)were not synthesized de novo and may therefore have beensalvaged from recycling of RNA or from disrupted cells. UTPlevels decreased from 0.80 fmol/cell (600 ^M) to 0.046 frnol/cell (35 MM) 4 h after pyrazofurin addition. ATP was notaffected, being 4.9 fmol/cell (3.7 HIM)before and 4.8 fmol/cell(3.6 HIM)after treatment (Fig. 4, a and b).

Effects of pyrazofurin upon intermediates of the purine pathway are shown in the radioactivity elution profiles of Fig. 4, cand d, which were obtained concurrently with the UV absorb-ance profiles of Fig. 4, a and b. Pyrimidine intermediates arenot radiolabeled when cells are grown in the presence of [I4C]-

formate. Comparison of profiles for absorbance at 260 nm withthose for 14Cin the presence and absence of pyrazofurin (Fig.

4) shows accumulation of CAIR, SAICAR, AICAR, FAICAR,IMP and XMP. Thus there is no evidence for a metabolic crossover point between AICAR and FAICAR in leukemia cellsgrowing in culture (both accumulate; Fig. 4, c and d), contraryto the proposal of Worzalla and Sweeney (6). The UV absorbance profile (Fig. 40) shows peaks for the mono- (PF-MP), di-(PF-DP) and triphosphate (PF-TP) derivatives of pyrazofurin

which were identified by three criteria: these metabolites wereonly present in drug-treated cells, they had absorption spectraidentical to pyrazofurin (Xmax= 227,261 nm) and they eluted inthe mono-, di-, and triphosphate regions of the profile (Fig.

4b). The peaks were integrated and converted to fmol/cell andapproximate cellular concentrations by comparison with peaksfor standard amounts of pyrazofurin. Values obtained after a4-h exposure to drug were PF-MP 0.79 fmol/cell (590 MM),PF-DP 0.28 fmol/cell (210 MM),and PF-TP 0.010 fmol/cell (7.8MM).Progress curves for formation of phosphorylated forms ofpyrazofurin are shown in Fig. 5. PF-MP is most abundant withlower concentrations of PF-DP; PF-TP was only detected in

mouse leukemia cells 4 h after drug addition.Addition of 3-deazauridine (25 MM)to leukemia cells also

affects both the pyrimidine and purine pathways. After 4 h,CTP and CDP have decreased to very low levels and UMP,UDP-sugars, UDP and UTP have accumulated (Fig. 6) consistent with potent inhibition of CTP synthetase by 3-deazauridine 5'-triphosphate (DAU-TP) (9). Phosphorylated deriva

tives of 3-deazauridine were identified in the same way as forpyrazofurin; the chromophore of the nucleoside (Xmax= 274nm) had the same absorption spectrum as phosphorylated derivatives. The cellular level of DAU-TP (Fig. 60) is 0.60 fmol/cell equivalent to a cellular concentration of 450 MM.Significantlevels of DAU-DP (0.065 fmol/cell, 49 MM) and DAU-MP(0.12 fmol/cell, 86 MM)are also apparent after 4 h treatmentwith 3-deazauridine, and it is presumably the monophosphatederivative which induces changes in the de novo purine pathway.Comparison of the radioactivity profiles for control and treatedcells grown in the presence of [l4C]formate (Fig. 6, c and d)

shows a marked decrease in IMP and an accumulation inAICAR, but not FAICAR, SAICAR or CAIR as observed forpyrazofurin (Fig. 4). However, as for pyrazofurin, there aresignificant increases in GMP, GDP, and GTP (Fig. 6). Thesedata are consistent with a metabolic cross-over point betweenAICAR and FAICAR, suggesting inhibition of AICAR trans-formylase by a derivative of 3-deazauridine.

Fig. 2. Separation of radiolabeled intermediates from extracts of leukemia cells grown inthe presence of |'*C)formate. The autoradi-

ograms show metabolites from a control culture and a culture treated with 25 JIMpyrazofurin for 8 h. Numbered spots, sequential intermediates of the de novo purine pathway: *,AICAR; 9, FAICAR; 10, IMP; //, sAMP; 12,AMP; 13, XMP; 14, GMP; Ado, adenosine.Spots with letters remain to be assigned.

2647




(0

Time (h)Fig. 3. Effects of 25 I/M pyrazofurin upon intermediates of de novo purine

biosynthesis: O, AICAR; D, IMP; A, sAMP; O, AMP; Â»,XMP; â€¢¿�.GMP. Cellswere grown in the presence of [MC]formate, pyrazofurin was added at zero time,extracts were chromatographed as shown in Fig. 2, marker compounds werevisualized under IV light and excised for scintillation counting as described in"Materials and Methods."

40

Time (min)

80

Fig. 4. Analysis of extracts from control (a, <â€¢)and pyrazof urin-lrealed (h, d)leukemia cells by HPLC. Two cultures were grown in the presence of [I4C]-

formate, one culture was exposed to 25 *iMpyrazofurin for 4 h, extracts wereprepared of both cultures and metabolites were separated by HPLC and monitoredconcurrently by UV (a, b) and MC(c, d) detectors.

DISCUSSION

The inhibitory effects of PF-MP upon OMP decarboxylaseof the pyrimidine pathway are rapid and virtually complete.Intermediates from OMP back to carbamyl aspartate in thepathway accumulate (Fig. 1, a and b); the absence of UTP, acompetitive inhibitor of carbamyl phosphate synthetase and thelikely elevation of P-Rib-PP, an allosteric activator of thisenzyme (20), would promote accumulation of these early intermediates. The less abundant intermediates, OMP, UMP, XMP,and GMP, could only be measured with accuracy using two-dimensional thin-layer chromatography. Separation of intermediates by high-pressure liquid chromatography has enabledidentification and quantification of more abundant leukemiacell metabolites using their retention times, UV absorptionspectra and l4C-labeling patterns from [14C]bicarbonate and['"CJformate. Labeling of FAICAR with ['"Cjformate allowed

detection of this purine intermediate, although large amountsof orotidine were present with the same retention time (Fig.4d). Collection of the entire orotidine peak labeled from [14C]-

bicarbonate followed by scintillation counting allowed a finalspecific radioactivity of 1.71 Ci/mol to be calculated whichshould apply to all the early intermediates of the pyrimidinepathway (Fig. 1, a and b). [l4C]Bicarbonate enters the purine

pathway at the reaction catalyzed by AIR carboxylase (EC

0.8

|

0.4

Time (h)

Fig. 5. Biosynthesis of PF-MP (â€¢)and PF-DP (O) in leukemia cells afteraddition of 25 IÂ¡Mpyrazofurin. The experiment was similar to Fig. 4 with samplestaken at the indicated times and processed to give elution profiles similar to Fig.4e. Peaks for PF-MP and PF-DP were integrated using Nelson software.

4.1.1.21) and thus FAICAR would also be 14C-labeled, but the

amount relative to orotidine is small and FAICAR has nosignificant absorbance at 260 nm (21) where the UV absorbance

2648



NUCLEOSIDES AFFECTING PYR1MIDINE AND FURINE BIOSYNTHESIS

20 40

Time (min)so 80

Fig. 6. Effects of 25 MM3-deazauridine upon intermediates of nucleotidemetabolism. Experimental conditions were otherwise the same as for Fig. 4.

of orotidine was measured. The specific radioactivity of the[l4C]bicarbonate used was 57.0 Ci/rnol, which was reduced to

1.71 Ci/mol in the pyrimidine pathway, presumably due togeneration of unlabeled bicarbonate from oxidation of glucose(17). Cellular levels of pyrimidine intermediates in Fig. 1 expressed as dpm/cell could be converted to approximate cellularconcentrations using the specific radioactivity of 1.71 Ci/moland an assumed cellular volume of 1.3 pi/cell.

Worzalla and Sweeney (6) found that PF-MP inhibitedAICAR transformylase in vitro and rats given pyrazofurin excreted the riboside of AICAR in their urine. They concludedthat AICAR transformylase of the de novo purine pathway wasinhibited in vivo. The data presented here for leukemia cellsgrowing in culture show elevation of the cellular levels ofintermediates before and after the reaction catalyzed by AICARtransformylase in the purine pathway, indicating that pyrazofurin increases the flux through the first 10 reactions of thepurine pathway and the GMP branch from IMP. The almostcomplete blockade of the pyrimidine pathway (Fig. 1) spares P-Rib-PP and L-glutamine, substrates for the first reaction of thepurine pathway, P-Rib-PP â€”¿�Â»5-phosphoribosylamine. Elevatedlevels of cellular L-glutamine could also stimulate the conversion of FGAR -Â»FGAM and XMP -Â»GMP; the latter wouldfavor the GMP branch over the AMP branch of the pathway.The initial increase in levels of AICAR, IMP, sAMP, and AMP(Fig. 3) might be attributed to rapid but transient increases in

P-Rib-PP and L-glutamine, which could also stimulate otherphosphoribosyltransferases and amidotransferases of cellularmetabolism. The upward spike in sAMP levels, similar toAICAR and IMP (Fig. 3), indicates that the rate of the sAMPsynthetase (EC 6.3.4.4) reaction does not increase in responseto increasing sAMP concentrations. By contrast, XMP levelsincrease progressively and GMP increases rapidly without asubsequent decrease (Fig. 3). GMP Ã©lÃ»tesjust before XMPwhen separated by HPLC. GMP was not clearly resolved fromXMP in the elution profile of Fig. 4d. High resolution HPLCexperiments6 showed that the initial levels of GMP and XMP

are comparable and both GMP and XMP increase after addition of pyrazofurin.

Although PF-MP does not significantly inhibit AICAR transformylase in mouse LI210 leukemia cells as proposed by Worzalla and Sweeney (6) for rats, our data suggest that a derivativeof 3-deazauridine, perhaps the monophosphate, does inhibitthis reaction in cultured cells. However, treatment with 3-

deazauridine does not significantly affect levels of adenosinenucleotides, and guanosine nucleotides increase substantially(Fig. 6) indicating that higher levels of AICAR may haverestored the original flux through the AICAR transformylasereaction. Accumulation of substrate to a higher steady stateconcentration can overcome the effect of a competitive inhibitorrestoring the original flux through a metabolic pathway; thisphenomenon has been called "metabolic resistance" (22). In

hibition of CTP synthetase by DAU-TP spares cellular L-glutamine from CTP synthesis and the resultant high levels ofUTP (Fig. 6b) would inhibit carbamyl phosphate synthetase(20), again sparing L-glutamine and subsequent utilization ofP-Rib-PP. Thus, inhibition of de novo pyrimidine biosynthesisby pyrazofurin or 3-deazauridine leads to complementary stimulation of de novo purine biosynthesis and in particular thesynthesis of guanosine nucleotides. This complementary stimulation of guanosine nucleotide biosynthesis has also beenobserved for dichloroallyl lawsone (17), a potent inhibitor ofthe reaction catalyzed by dihydroorotate dehydrogenase (EC1.3.3.1). The imbalances induced in levels of purine deoxynu-cleoside triphosphates should contribute to the cytotoxicitiesagainst cancer cells of inhibitors of the de novo pyrimidinepathway.

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1989;49:2645-2650. Cancer Res Melissa E. Sant, Stephen D. Lyons, Anthony J. Kemp, et al. and Purine Biosynthesis in Mouse L1210 LeukemiaDual Effects of Pyrazofurin and 3-Deazauridine upon Pyrimidine

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Dual Effects of Pyrazofurin and 3-Deazauridine upon ... · novo purine biosynthesis in leukemia cells growing in culture. ... mediates of the de novo purine pathway, ... intermediate,