significance of furine salvage in circumventing the action...

(CANCER RESEARCH 49, 88-92, January 1, 1989]

Significance of Furine Salvage in Circumventing the Action of Antimetabolites inRat Hepatoma Cells1

Yutaka Natsumeda, Tadashi Ikegami,2 Edith Olah,3 and George Weber4

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223

ABSTRACT

The flux activities of de novo and salvage purine synthesis werecompared in rat hepatoma 3924A cells in various growth phases. Theinitial rate assays of [I4C]adenine, ("C]hypoxanthine, and |"C]guanineincorporation yielded Michaelis-Menten kinetics with A'msof 5, 7, and 7

#tM,respectively. After replating plateau phase cells in lag and log phasesthe activity of purine de novo pathway increased 4.5- to 8-fold with apreferential rise in guanylate synthesis, whereas purine salvage activitiesincreased only 1.6- to 2.1-fold. However, for the syntheses of IMP, AMP,and GMP, the activities of purine salvage pathways were 2- to 7-fold, 5-to 28-fold, and 2- to 32-fold higher than those of the de novo purinepathway. Treatment of cells with acivicin, an inhibitor of the activityof amidophosphoribosyltransferase, phosphoribosylformylglycinamidinesynthase, and GMP synthase, inhibited the flux activities of de novopurine, adenylate, and guanylate syntheses to 37, 73, and 3% of thecontrols and decreased the concentration of GTP to 42%; the concentration of ATP did not change and that of 5-phosphoribosyl 1-pyrophosphateincreased 3.1-fold. Under these conditions the activities of salvage synthesis from hypoxanthine and guanine were enhanced 2.5-fold. Treatmentof hepatoma cells with IMP dehydrogenase inhibitors, tiazofurin, riba-virin, and 4-carbamoylimidazolium 5-olate, to block de novo guanylatesynthesis accelerated the flux activity of guanine salvage pathway. Thehigher capacity of purine salvage pathway than that of the de novo oneand the further rise of the activity in response to the drugs targetedagainst the de novo pathway highlight the important role salvage synthesis might play in circumventing the impact of antimetabolites of de novopurine synthesis in cancer chemotherapy.

INTRODUCTION

Altered regulation of purine nucleotide synthesis has been animportant factor in human diseases including gout, immunodeficiency, Lesch-Nyhan syndrome, myopathies, skin diseases,parasitic infections, and neoplasias (1-5). Enzymological studies were carried out in purified preparations and crude extracts,and in intact cells the rates of purine de novo synthesis weredetermined by pulse-labeling assays (6-11). Because of thedilution of the labeled precursors with endogenous substratesthe assays provided relative rates of purine de novo synthesis asexpressed in mol, Ci, or cpm of [14C]precursor incorporatedinto purine compounds/h/cells (6-11). Although the changesof the relative rates could be translated into the metabolicalterations of the pathway flux exerted by biological factors ordrugs, the relative rates do not provide sufficient informationcomparable with the fluxes of the other metabolic pathways.

For measuring purine de novo synthesis we recently reportedthe kinetic properties of [l4C]formate and L-[3-'4C]serine incor

poration into total purincs produced in hepatoma cells (12). Inthe serine-free assay medium excess [14C]formate diluted serine,the major source of one-carbon unit, and labeled purines with

Received 7/13/88; revised 9/22/88; accepted 9/26/88.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.

1Supported by USPHS Outstanding Investigator Grant CA-42510 to G. W.2 Present address: Department of Radiology, Yokohama City University

School of Medicine, Yokohama, Japan.3 Permanent address: Department of Molecular Biology, National Cancer

Institute of Hungary, Budapest, Hungary.' To whom requests for reprints should be addressed.

negligible dilution of its specific activity. Therefore, the incorporation rates of [I4C]formate into total purine compounds

were interpreted as the rates of purine de novo synthesis interms of nmol of purine synthesized per h per cell (12).

Purine nucleotides are produced by de novo or salvage pathways and the salvage might play an important role in circumventing the action of antimetabolites targeted against de novosynthesis in cancer chemotherapy (5, 13-16). The present investigation compares the metabolic fluxes of purine de novoand salvage pathways in rat hepatoma 3924A cells. The initialrate kinetics of purine salvage synthesis from adenine, hypoxanthine, and guanine were determined and the metabolicfluxes of purine de novo and salvage pathways were comparedin the hepatoma cells in various growth phases. The responseof the salvage pathway flux to inhibitors of de novo purinesynthesis was elucidated.

MATERIALS AND METHODS

Materials. [8-14C]Adenine, [8-14C]hypoxanthine, [8-3H]XMP, [14C]formate, sodium salt, and L-[i/-'4C]glutamine (40 mCi/mmol) werepurchased from Amersham Corp., Arlington Heights, IL. [14C]Gluta-mine was purified as described (17). [8-14C]Guanine (55.5 nCi/mmol)

was obtained from ICN Biomedicals, Inc., Costa Mesa, CA. Tissueculture supplies were from Grand Island Biological Co., Grand Island,NY; Corning Glass Works, Corning, NY; and Sigma Chemical Co., St.Louis, MO. Tiazofurin and ribavirin were provided by Nucleic AcidResearch Institute, Costa Mesa, CA, and SM-1085 was supplied by

Sumitomo Chemical Co., Takarazuka, Japan.Cell Culture. Rat hepatoma cell cultures were established from trans

plantatile hepatoma 3924A and grown in monolayers as reported (12,18). In the logarithmic growth phase a doubling time of 14 h wasobserved in the standard culture conditions. For measuring purinenucleotide synthesis the medium was replaced with 1 ml of serum-freeEagle's basal medium with Earle's salt, 3 HIML-glutamine, and 25 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4, as described (12).

Assays of Purine Synthesis in Cells. Purine tie novo synthesis wasdetermined with 4 mM [14C]formate as the substrate (12). The overall

synthesis of adenylates and guanylates was calculated from the radioactivities in adenine and guanine after hydrolysis of the purine compounds(12). For measuring salvage synthesis the cells were pulsed at 37"C for

various time periods (0, 10, and 20 min in the standard assay) witheither [8-'4C]adenine, [8-'4C]hypoxanthine, or [8-'4C]guanine at a final

concentration of 5 to 100 nM (25 mCi/mmol). To terminate the pulse,the medium was suctioned off and 5 ml ice-cold PBS were added. PBSwas discarded immediately, 0.5 ml of 0.2 N NaOH was added to dissolvethe cells, and then 0.5 ml of l N HC1O4 was added. The contents of theflasks were filtered onto a glass fiber filter (Grade 934H; 2.4 cm;Whatman) in a Millipore filter system. Acid-soluble filtrates wereneutralized with 0.1 volume of 4.42 N KOH, kept in ice for 30 min,and centrifuged. The supernatants were then chromatographed in bu-tanol:methanol:water:ammonia (60:20:20:1) with adenine, hypoxanthine, guanine, and nucleotides (a mixture of ATP, ADP, AMP, andIMP) as markers on Whatman No. 3MM paper. In this system nucleotides were clearly separated from bases and nucleosides even when

'' The abbreviations used are: SM-108, 4-carbamoylimidazolium S-olate; PBS,

phosphate-buffered saline [containing (g/liter): KC1,0.2; KH2POÂ«,0.2; NaCl, 8.0;and Na2HPO4 7H2O, 2.16]; PRPP, 5-phosphoribosyl 1-pyrophosphate.

88

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PURINE SALVAGE SYNTHESIS IN RAT HEPATOMA CELLS

100 to 300 n\ neutralized samples were applied. The UV-absorbingspots for nucleotides were cut out and counted in a liquid scintillationcounting solution (OCS; Amersham). The glass fiber filters with acid-insoluble materials were washed 5 times with S ml of 0.4 N HC1O4 andtwice with 0.5 ml of 95% ethanol, dried, and counted in OCS. Theradioactivities of the acid-insoluble precipitates and the acid-solublenucleotides for 0 time controls were negligible (30 to 50 cpm, less than1% of those obtained in the standard assays).

Assays for Enzyme Activities and Concentrations of Metabolites. Forenzyme assays the cells were collected by scraping, centrifuged, washedtwice with cold PBS, and homogenized with 4 volumes of 0.25 Msucrose containing 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesul-fonic acid, pH 7.6. Enzyme activities were determined in the 100,000x g supernatants.

Amidophosphoribosyltransferase activity was measured by assayingthe PRPP-dependent conversion of [uC]glutamine to [14C]glutamic acid

which was separated by high voltage electrophoresis (19). The reactionmixture was as described (20). The specific activity determined byradioassay was similar to that observed by the spectrophotometric assaymethod (21).

GMP synthase assay was carried out by measuring [8-3H]XMPconversion to [8-3H]GMP. The reaction mixture contained 1.8 mMATP, 1.8 mM L-glutamine, 130 mM Tris-HCl (pH 7.6), 16 mM MgCl2,3 mM creatinine phosphate, 10 mg creatinine phosphokinase (50-100units/mg), 0.13 HIM[8-3H]XMP (5.4 Ci/mmol), and cell extract in a

total volume of 25 n\. The assay procedure and the separation of thesubstrate and the product were performed as described (22).

Protein concentration was quantitated by a routine method (23) withbovine serum albumin as standard. Concentrations of nucleotides andPRPP were determined by high pressure liquid chromatography andby enzyme assay as reported (24).

RESULTS AND DISCUSSION

Initial Rate Kinetics of Furine Salvage Synthesis in Hepatoma3924A Cells. To establish that linear kinetics operate in intacthepatoma cells in measuring purine salvage synthesis, the incorporation of [l4C]purine bases into nucleotides and nucleic

acids was investigated under various assay conditions. The timecourses of labeling with [8-l4C]adenine, [8-l4C]hypoxanthine,and [8-'4C]guanine are shown in Fig. 1. The purine bases wereincorporated into acid-soluble nucleotide pool by phosphori-bosylation and then into acid-insoluble nucleic acids by polymerization. The label distributions to nucleotide and nucleic acidfractions varied with the substrate. As compared to the labelingwith [8-I4C]adenine (Fig. \A ), higher percentages of the label

were distributed to the acid-insoluble fraction after a pulse with[8-'4C]guanine (Fig. 1C). This suggested a smaller functional

pool and a higher turnover rate of guanylates for nucleic acidsynthesis than those for adenylates. After a pulse with [8-l4C]-

hypoxanthine which is converted to IMP and then metabolizedto both adenylates and guanylates, the radioactivities weredistributed between those after the pulse with [8-14C]adenine orafter the pulse with [8-'4C]guanine (Fig. \B). The overall purinesalvage syntheses from [l4C]purine bases were proportionate

with incubation time up to 20 min (Fig. 1) and with cell densityfrom 2.5 to 15 mg cells/ml (0.5 to 3 x IO6 cells/ml) (data not

shown).The effect of the concentrations of purine bases on salvage

synthesis in hepatoma cells is shown in Fig. 2. The overallnucleotide synthesis from adenine, hypoxanthine, and guaninefollowed Michaelis-Menten kinetics and the double reciprocalplots yielded apparent Kmsof 5,7, and 7 /Â¿M,respectively. Thesevalues were 2- to 6-fold higher than the A"msfor purine bases of

purine phosphoribosyltransferases determined in the crude extracts of solid hepatoma 3924A (14).

From the substrate concentration curves we chose 100 Ã•/Mfor the concentration of [l4C]purine bases to perform standard

assays. The initial velocities of purine salvage synthesis werethen measured in the hepatoma cells in various growth phasesand compared with those of purine de novo synthesis.

Activities of Purine de Novo and Salvage Synthesis in Hepatoma Cells. The purine de novo synthetic pathway initiallyproduces IMP which is shared for production of adenylates andguanylates. Although IMP may be degraded to inosine andhypoxanthine, these metabolites are not converted to adenineor guanine compounds at the nucleoside or nucleobase levels.Since the labeling of either adenine or guanine compound with[14C]formate was linear with incubation time, the labeling rates

of adenine and guanine after hydrolysis were assumed to reflectthe rates of IMP utilization for the de novo synthesis of adenylates and guanylates (12, 25).

The synthetic activities of purine de novo and salvage pathways in the hepatoma cells are compared in Table 1. Whenhepatoma 3924A cells in plateau phase were replated in thefresh culture medium at a population of 2 x IO4 cells/cm2, thecells were allowed to grow from lag (0-24 h) into log (24-72 h)and plateau (72-96 h) phases (12, 26). In contrast to the markedincrease of de novo synthetic activities in log phase for total

Fig. 1. Time course of incorporation ofpurine bases into nucleotides and nucleic acidsin hepatoma 3924A cells. The hepatoma cellsin log phase were pulsed with either [8-MCladenine (A), [8-uC]hypoxanthine (fl), or [8-C4('luminine (C) at a concentration of 5 fiMand

labeled acid-soluble nucleotides (O) and acid-insoluble nucleic acids (â€¢)were determined asdescribed in "Materials and Methods." To plot

the total nucleotide synthesis (x) the sums ofthe label incorporation into both fractions werecalculated.

ZO

Ã–L

Â£OÃOO

0.3-

0.2-

TIME (min)89




2.0-

Fig. 2. Dependence of purine salvage synthesis on concentrations of purine bases. Theinitial rates of purine salvage synthesis withvarious concentrations of [8-14C]adenine (â€¢),[8-MC]hypoxanthine (O), or [8-MC]guanine (X)

were measured in intact hepatoma cells asdescribed in "Materials and Methods." Inset,

double reciprocal plots of the rates and concentrations of the various substrates.

Â«y

zO

Ã–L

2te.ouz

1.5-1

1.0-

0.5â€”

Oâ€”I

0.01 0.02 0.03 0.04 0.05 0.06

SUBSTRATE (mivi)

0.07 0.08 0.10

Table 1 Activities of purine de novo and salvage synthesis in hepatoma 3924A cells in various growth phasesActivities of i/c novo and salvage IMP, AMP, and GMP syntheses were determined in hepatoma 3924A cells in various growth phases as described in "Materials

and Methods." IMP synthesis de novo indicates total purine de novo synthesis. Values are means Â±SE of 3 or more samples. The activities of salvage synthesis weresignificantly higher than those of de novo synthesis in hepatoma 3924 A cells in various growth phases (/*< O.OS).

Purine nucleotide synthesis (nmol/h/gcells)Time

afterplating(h)0

624487296IMPJe

novo89.5

Â±2.594.0 Â±12.1

399.0 Â±10.0720.0 Â±40.0223.0 Â±8.0

89.5 Â±2.5Salvage658

Â±45638 Â±30783 Â±85

1390 Â±80635 Â±31658 Â±45AMPde

novo63.3

Â±4.442.6 Â±1.8

129.0 Â±4.0358.0 Â±13.0136.0 Â±1.063.3 Â±4.4Salvage1250

Â±301210 Â±601510Â± 1101940 Â±1601410 Â±801250 Â±30GMPde

novo14.3

Â±2.646.4 Â±1.7

251.0 Â±17.0298.0 Â±25.0

63.7 Â±6.814.3 Â±2.6Salvage456

Â±28497 Â±56541 Â±84879 Â±61541 Â±26456 Â±28

purine (8-fold), adenylates (6-fold), and guanylates (21-fold),the rise of the salvage activities was minor (1.6- to 2.1-fold).For IMP synthesis, however, the hepatoma cells had 2- to 7-fold higher activity of salvage pathway from hypoxanthine thanthat of de novo pathway in various growth phases. In additionto the salvage synthesis of IMP the pathway from adenine couldprovide AMP with 5- to 28-fold higher activity than the de

novo adenylate synthesis. The guanine salvage pathway alsocould contribute to GMP production with a 2- to 32-fold higheractivity than the de novo guanylate pathway. Thus, the activitiesof purine salvage pathways were higher than those of purine denovo pathways for total purine, adenylate, and guanylate synthesis in hepatoma 3924A cells during the culture growth.

Biochemical Impacts of Acivicin in Hepatoma 3924A Cells.Acivicin irreversibly inactivates glutamine-utilizing enzymesincluding amidophosphoribosyltransferase, phosphoribosylfor-mylglycinamidine synthase, and GMP synthase among purinesynthetic enzymes (27). After treatment of hepatoma cells with10 /iM acivicin for 3 h the activity of amidophosphoribosyltransferase, the rate-limiting enzyme of purine de novo synthesis,decreased to 31% of the pretreatment value and that of GMPsynthase to 4% (Table 2). Concurrently, the concentration ofGTP was depressed to 42%, whereas that of ATP did notchange significantly. The concentration of PRPP, a sharedsubstrate for purine, pyrimidine, and pyridine nucleotide biosynthesis, increased 3-fold after treatment (Table 2). These

Table 2 Effect of acivicin on activities of amidophosphoribosyltransferase andGMP synthase and on concentrations of purine nucleoside triphosphates and

PRPPHepatoma 3924A cells in log phase were treated with 10 *Â¿Macivicin for 3 h.

The enzyme activities and concentrations of nucleotides and PRPP were determined as described in "Materials and Methods." Values are means Â±SE of

triplicate assays.

Enzymesand

metabolites ControlAcivicin-treated

%ofcontrol

Enzyme activities (nmol/h/mgprotein)

Amidophosphoribosyl- 134 Â±6 41.5 Â±3.4 31Â°

transferaseGMP synthase 247 Â±4 10.5 Â±4.4 4"

Metabolite concentrations(nmol/h/g cells)

ATP 1220 Â±30 1190 Â±50 98GTP 230 Â±4 96.2 Â±3.3 42Â°PRPP 18.6 Â±1.5 58.2 Â±4.4 313Â°

Â°Significantly different from control (P < 0.05).

metabolic changes are in good agreement with previous in vivostudies in solid hepatoma 3924A and other tumors (13, 28, 29).The metabolic fluxes of de novo synthesis of total purities andguanylates were depressed to 37 and 3%, respectively, of thecontrols (Table 3). The rate of de novo adenylate synthesis wasretained at 73% despite the decreased activity of purine de novosynthesis (37%).

After the acivicin treatment the activities of salvage nucleo-

90




Table 3 Effect of acivicin on purine de novo and salvage synthesis in hepatoma3924A cells

Hepatoma cells in log phase were treated with 10 fiM acivicin for 3 h. Fluxactivities of purine de novo and salvage synthesis were then measured after mediumreplacement as outlined in "Materials and Methods." Values are means Â±SE of

triplicate assays.

Purine nucleotide synthesis(nmol/h/gcells)PathwaysIMP

synthesisde novo'

SalvageAMP

synthesisde novo'

SalvageGMP

synthesisde novoÃ¤

SalvageControl845

Â±91180Â±110346

Â±251850 Â±80288

Â±32870 Â±63Acivicin-treated316

Â±492980 Â±220253

Â±372010 Â±809Â±

12120 Â±100%of

change37*253"73

10934244*

Â°Total purine de novo synthesis.* Significantly different from control (P < 0.05).' de novo synthesis of adenylates.d de novo synthesis of guanylates.

tide synthesis from hypoxanthine and guanine increased 2.5-fold (Table 3). This elevation might be attributed to the increasein the concentration of PRPP, the substrate of hypoxanthine-guanine phosphoribosyltransferase, and to the decrease in theconcentrations of guanine nucleotides which are strong competitive inhibitors for the salvage enzyme reactions (30, 31).However, the acivicin treatment did not result in a significantincrease in the activity of adenine salvage pathway becausethere was no change in the concentrations of adenine nucleotides, potent inhibitors of adenine phosphoribosyltransferase(32) (Table 3). Also the concentration of PRPP in the log phasecells (18-25 nmol/g cells) might be nearly saturating for adenine phosphoribosyltransferase activity (Km2 ^M) (14).

Effects of Inhibitors of IMP Dehydrogenase on Salvage Guanylate Synthesis. Early studies in this laboratory identified asessential elements in the expression of the neoplastic proliferation program the increased activity of IMP dehydrogenase,the predominant utilization of IMP for guanylate synthesis, thepositive correlation of the ratio of the concentrations of guanylates to adenylates with the tumor proliferation rates and theexpanded pool of dGTP in cancer cells (12, 26, 33, 34). Tiazofurin and SM-108, inhibitors of IMP dehydrogenase, havebroad antitumor spectra including Lewis lung tumor and rattransplantable hepatoma 3924A (35, 36). The oncolytic actionof the drugs is linked with the depletion of GTP and dGTPpools (24, 37). Tiazofurin also is a promising agent in thetreatment of human myelocytic leukemia (38, 39).

However, guanine nucleotides are also produced by the salvage pathway. After treatment of hepatoma cells with tiazofurin, SM-108, or ribavirin, de novo guanylate synthesis wasinhibited to 41, 12, and 33%, respectively, of controls (Table4). Concurrently, the flux activity of guanine salvage pathwaywas 1.8- to 2.3-fold enhanced (Table 4). This rise again couldbe due to the decline in the concentrations of guanine nucleotides and to the elevation of PRPP level as reported previously(24, 26, 36). Although SM-108 consumes PRPP on its conversion to bredinin 5'-monophosphate by adenine phosphoribo

syltransferase (40), the concentrations of PRPP increased inhepatoma 3924A cells in culture and in vivo in solid hepatoma3924A after the treatment by SM-108.6 As reported by Pilz etal. (41), inhibition of both IMP dehydrogenase and adenylosuc-cinate synthase activities accumulated PRPP in cultured human

6Y. Natsumeda and G. Weber, unpublished results.

Table 4 Effects of inhibitors of IMP dehydrogenase on de novo and salvage GMPsynthesis in hepatoma 3924A cells

Hepatoma cells in log phase were treated with drugs for 2 h. Activities of denovo and salvage guanylate synthesis were measured after medium replacementas described in "Materials and Methods." Values are means Â±SE of 3 or more

assays.

GMP synthesis(nmol/h/g cells) (% of control)

Drugs(pM)Control,

noneTiazofurin, 8SM-108, 10RiiÂ«virili. IOde

novo264

Â±14(100)100 Â±7(41)"30 Â±3(12)Â°82 Â±9 (33)Â°Salvage837

Â±55 (100)1940 Â±90 (232)Â°1650 Â±110(197)Â°1540 Â±80(184)Â°

Â°Significantly different from control (/>< 0.05).

lymphoblasts in which the only possible metabolic fate ofaccumulated IMP would be degradation to inosine. The increase of PRPP concentration in those cells was attributed tothe enhancement of inosinate cycle consisting of 5'-nucleo tid-

ase, inosine phosphorylase, phosphoribomutase, PRPP syn-thase, and hypoxanthine phosphoribosyltransferase (42). Similarly in hepatoma 3924A cells inhibition of IMP dehydrogen-ase might be able to enhance inosinate cycle and expand PRPPpool. In fact, inosine and hypoxanthine, as well as IMP, theconstituents of the inosinate cycle, markedly accumulated inhepatoma 3924A after treatment with tiazofurin (43). AlthoughIMP, an inhibitor of hypoxanthine-guanine phosphoribosyltransferase, accumulated in the tumor after tiazofurin treatment, the amount increased in IMP was less than that decreasedin guanylates (24, 43). Furthermore, the inhibitory effects ofGTP and GDP were as strong as IMP and that of GMP wasmuch stronger than IMP in the in vitro guanine phosphoribosyltransferase assays with 30 ^M PRPP (data not shown).

Significance of Purine Salvage Synthesis in Cancer Cells.Novel aspects and significant implications of the present studyinclude: (a) establishment of the initial rate kinetics of purinesalvage synthesis from adenine, hypoxanthine, and guanine inrat hepatoma 3924A cells in monolayer culture; (Â¿>)determination of the rates of purine de novo and salvage synthesis asmeasured in nmol of purine nucleotide synthesized per h per gcells in the hepatoma cell culture in various growth phases; (c)first direct comparison of the flux rates of purine de novo andsalvage pathways in intact cells under the same circumstances;(d) demonstration that the activities of the metabolic flux ofpurine salvage pathways are higher than those of de novopathway in rat hepatoma cells; (e) evidence that the flux activityof guanine salvage pathway increases after treatment with inhibitors of de novo guanylate synthesis; (/) attribution of theincrease in guanine salvage activity to the decline in guaninenucleotide concentrations and to the elevation of PRPP level.

The high activity of purine salvage synthesis in cancer cellsand its further increase in response to inhibition of de novopurine synthesis indicate the important role of the purinesalvage pathways in circumventing the action of antimetabolitesof de novo purine synthesis in cancer chemotherapy. Theseobservations provide new evidence for the concept that for aneffective cancer drug treatment both de novo and salvage pathways should be inhibited (5).

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1989;49:88-92. Cancer Res Yutaka Natsumeda, Tadashi Ikegami, Edith Olah, et al. Antimetabolites in Rat Hepatoma CellsSignificance of Purine Salvage in Circumventing the Action of

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