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

(CANCER RESEARCH 49, 88-92, January 1, 1989] Significance of Furine Salvage in Circumventing the Action of Antimetabolites in Rat 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 negligible dilution of its specific activity. Therefore, the incor poration rates of [I4C]formate into total purine compounds The flux activities of de novo and salvage purine synthesis were were interpreted as the rates of purine de novo synthesis in compared in rat hepatoma 3924A cells in various growth phases. The initial rate assays of [I4C]adenine, ("C]hypoxanthine, and |"C]guanine terms of nmol of purine synthesized per h per cell (12). incorporation yielded Michaelis-Menten kinetics with A'msof 5, 7, and 7 Purine nucleotides are produced by de novo or salvage path #tM,respectively. After replating plateau phase cells in lag and log phases ways and the salvage might play an important role in circum the activity of purine de novo pathway increased 4.5- to 8-fold with a venting the action of antimetabolites targeted against de novo preferential rise in guanylate synthesis, whereas purine salvage activities synthesis in cancer chemotherapy (5, 13-16). The present in increased only 1.6- to 2.1-fold. However, for the syntheses of IMP, AMP, vestigation compares the metabolic fluxes of purine de novo and GMP, the activities of purine salvage pathways were 2- to 7-fold, 5- and salvage pathways in rat hepatoma 3924A cells. The initial to 28-fold, and 2- to 32-fold higher than those of the de novo purine rate kinetics of purine salvage synthesis from adenine, hypo pathway. Treatment of cells with acivicin, an inhibitor of the activity xanthine, and guanine were determined and the metabolic of amidophosphoribosyltransferase, phosphoribosylformylglycinamidine fluxes of purine de novo and salvage pathways were compared synthase, and GMP synthase, inhibited the flux activities of de novo in the hepatoma cells in various growth phases. The response purine, adenylate, and guanylate syntheses to 37, 73, and 3% of the controls and decreased the concentration of GTP to 42%; the concentra of the salvage pathway flux to inhibitors of de novo purine tion of ATP did not change and that of 5-phosphoribosyl 1-pyrophosphate synthesis was elucidated. increased 3.1-fold. Under these conditions the activities of salvage syn thesis from hypoxanthine and guanine were enhanced 2.5-fold. Treatment of hepatoma cells with IMP dehydrogenase inhibitors, tiazofurin, riba- MATERIALS AND METHODS virin, and 4-carbamoylimidazolium 5-olate, to block de novo guanylate Materials. [8-14C]Adenine, [8-14C]hypoxanthine, [8-3H]XMP, [14C] synthesis accelerated the flux activity of guanine salvage pathway. The formate, sodium salt, and L-[i/-'4C]glutamine (40 mCi/mmol) were higher capacity of purine salvage pathway than that of the de novo one purchased from Amersham Corp., Arlington Heights, IL. [14C]Gluta- and the further rise of the activity in response to the drugs targeted mine was purified as described (17). [8-14C]Guanine (55.5 nCi/mmol) against the de novo pathway highlight the important role salvage synthe sis might play in circumventing the impact of antimetabolites of de novo was obtained from ICN Biomedicals, Inc., Costa Mesa, CA. Tissue purine synthesis in cancer chemotherapy. culture 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 Acid INTRODUCTION Research Institute, Costa Mesa, CA, and SM-1085 was supplied by Sumitomo Chemical Co., Takarazuka, Japan. Altered regulation of purine nucleotide synthesis has been an Cell Culture. Rat hepatoma cell cultures were established from trans important factor in human diseases including gout, immuno plantatile hepatoma 3924A and grown in monolayers as reported (12, deficiency, Lesch-Nyhan syndrome, myopathies, skin diseases, 18). In the logarithmic growth phase a doubling time of 14 h was parasitic infections, and neoplasias (1-5). Enzymological stud observed in the standard culture conditions. For measuring purine ies were carried out in purified preparations and crude extracts, nucleotide synthesis the medium was replaced with 1 ml of serum-free and in intact cells the rates of purine de novo synthesis were Eagle's basal medium with Earle's salt, 3 HIML-glutamine, and 25 HIM determined by pulse-labeling assays (6-11). Because of the 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4, as de dilution of the labeled precursors with endogenous substrates scribed (12). the assays provided relative rates of purine de novo synthesis as Assays of Purine Synthesis in Cells. Purine tie novo synthesis was expressed in mol, Ci, or cpm of [14C]precursor incorporated determined with 4 mM [14C]formate as the substrate (12). The overall into purine compounds/h/cells (6-11). Although the changes synthesis of adenylates and guanylates was calculated from the radioac tivities in adenine and guanine after hydrolysis of the purine compounds of the relative rates could be translated into the metabolic (12). For measuring salvage synthesis the cells were pulsed at 37"C for alterations of the pathway flux exerted by biological factors or various time periods (0, 10, and 20 min in the standard assay) with drugs, the relative rates do not provide sufficient information either [8-'4C]adenine, [8-'4C]hypoxanthine, or [8-'4C]guanine at a final comparable with the fluxes of the other metabolic pathways. concentration of 5 to 100 nM (25 mCi/mmol). To terminate the pulse, For measuring purine de novo synthesis we recently reported the medium was suctioned off and 5 ml ice-cold PBS were added. PBS the kinetic properties of [l4C]formate and L-[3-'4C]serine incor was discarded immediately, 0.5 ml of 0.2 N NaOH was added to dissolve poration into total purincs produced in hepatoma cells (12). In the cells, and then 0.5 ml of l N HC1O4 was added. The contents of the the serine-free assay medium excess [14C]formate diluted serine, flasks were filtered onto a glass fiber filter (Grade 934H; 2.4 cm; the major source of one-carbon unit, and labeled purines with Whatman) in a Millipore filter system. Acid-soluble filtrates were neutralized with 0.1 volume of 4.42 N KOH, kept in ice for 30 min, Received 7/13/88; revised 9/22/88; accepted 9/26/88. and centrifuged. The supernatants were then chromatographed in bu- The costs of publication of this article were defrayed in part by the payment tanol:methanol:water:ammonia (60:20:20:1) with adenine, hypoxan of page charges. This article must therefore be hereby marked advertisement in thine, guanine, and nucleotides (a mixture of ATP, ADP, AMP, and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by USPHS Outstanding Investigator Grant CA-42510 to G. W. IMP) as markers on Whatman No. 3MM paper. In this system nucle 2 Present address: Department of Radiology, Yokohama City University otides were clearly separated from bases and nucleosides even when School of Medicine, Yokohama, Japan. 3 Permanent address: Department of Molecular Biology, National Cancer '' The abbreviations used are: SM-108, 4-carbamoylimidazolium S-olate; PBS, Institute of Hungary, Budapest, Hungary. phosphate-buffered saline [containing (g/liter): KC1,0.2; KH2POÂ«,0.2;NaCl, 8.0; ' To whom requests for reprints should be addressed. and Na2HPO4 7H2O, 2.16]; PRPP, 5-phosphoribosyl 1-pyrophosphate. 88

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. PURINE SALVAGE SYNTHESIS IN RAT HEPATOMA CELLS 100 to 300 n\ neutralized samples were applied. The UV-absorbing were distributed to the acid-insoluble fraction after a pulse with spots for nucleotides were cut out and counted in a liquid scintillation [8-'4C]guanine (Fig. 1C). This suggested a smaller functional counting solution (OCS; Amersham). The glass fiber filters with acid- pool and a higher turnover rate of guanylates for nucleic acid insoluble materials were washed 5 times with S ml of 0.4 N HC1O4 and synthesis than those for adenylates. After a pulse with [8-l4C]- twice with 0.5 ml of 95% ethanol, dried, and counted in OCS. The radioactivities of the acid-insoluble precipitates and the acid-soluble hypoxanthine which is converted to IMP and then metabolized to both adenylates and guanylates, the radioactivities were nucleotides for 0 time controls were negligible (30 to 50 cpm, less than distributed between those after the pulse with [8-14C]adenine or 1% of those obtained in the standard assays). after the pulse with [8-'4C]guanine (Fig. \B). The overall purine Assays for Enzyme Activities and Concentrations of Metabolites. For salvage syntheses from [l4C]purine bases were proportionate enzyme assays the cells were collected by scraping, centrifuged, washed twice with cold PBS, and homogenized with 4 volumes of 0.25 M with incubation time up to 20 min (Fig. 1) and with cell density sucrose containing 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesul- from 2.5 to 15 mg cells/ml (0.5 to 3 x IO6 cells/ml) (data not fonic acid, pH 7.6. Enzyme activities were determined in the 100,000 shown). x g supernatants. The effect of the concentrations of purine bases on salvage Amidophosphoribosyltransferase activity was measured by assaying the PRPP-dependent conversion of [uC]glutamine to [14C]glutamicacid synthesis in hepatoma cells is shown in Fig. 2. The overall nucleotide synthesis from adenine, hypoxanthine, and guanine which was separated by high voltage electrophoresis (19). The reaction followed Michaelis-Menten kinetics and the double reciprocal mixture was as described (20). The specific activity determined by plots yielded apparent Kmsof 5,7, and 7 /Â¿M,respectively.These radioassay was similar to that observed by the spectrophotometric assay values were 2- to 6-fold higher than the A"msfor purine bases of method (21). GMP synthase assay was carried out by measuring [8-3H]XMP purine phosphoribosyltransferases determined in the crude ex conversion to [8-3H]GMP. The reaction mixture contained 1.8 mM tracts of solid hepatoma 3924A (14). ATP, 1.8 mM L-glutamine, 130 mM Tris-HCl (pH 7.6), 16 mM MgCl2, From the substrate concentration curves we chose 100 Ã•/M 3 mM creatinine phosphate, 10 mg creatinine phosphokinase (50-100 for the concentration of [l4C]purine bases to perform standard units/mg), 0.13 HIM[8-3H]XMP (5.4 Ci/mmol), and cell extract in a assays. The initial velocities of purine salvage synthesis were total volume of 25 n\. The assay procedure and the separation of the then measured in the hepatoma cells in various growth phases substrate and the product were performed as described (22). Protein concentration was quantitated by a routine method (23) with and compared with those of purine de novo synthesis. bovine serum albumin as standard. Concentrations of nucleotides and Activities of Purine de Novo and Salvage Synthesis in Hepa PRPP were determined by high pressure liquid chromatography and toma Cells. The purine de novo synthetic pathway initially by enzyme assay as reported (24). produces IMP which is shared for production of adenylates and guanylates. Although IMP may be degraded to inosine and hypoxanthine, these metabolites are not converted to adenine RESULTS AND DISCUSSION or guanine compounds at the nucleoside or nucleobase levels. Initial Rate Kinetics of Furine Salvage Synthesis in Hepatoma Since the labeling of either adenine or guanine compound with 3924A Cells. To establish that linear kinetics operate in intact [14C]formate was linear with incubation time, the labeling rates hepatoma cells in measuring purine salvage synthesis, the in of adenine and guanine after hydrolysis were assumed to reflect corporation of [l4C]purine bases into nucleotides and nucleic the rates of IMP utilization for the de novo synthesis of ade acids was investigated under various assay conditions. The time nylates and guanylates (12, 25). courses of labeling with [8-l4C]adenine, [8-l4C]hypoxanthine, The synthetic activities of purine de novo and salvage path and [8-'4C]guanine are shown in Fig. 1. The purine bases were ways in the hepatoma cells are compared in Table 1. When incorporated into acid-soluble nucleotide pool by phosphori- hepatoma 3924A cells in plateau phase were replated in the bosylation and then into acid-insoluble nucleic acids by polym fresh culture medium at a population of 2 x IO4 cells/cm2, the erization. The label distributions to nucleotide and nucleic acid cells were allowed to grow from lag (0-24 h) into log (24-72 h) fractions varied with the substrate. As compared to the labeling and plateau (72-96 h) phases (12, 26). In contrast to the marked with [8-I4C]adenine (Fig. \A ), higher percentages of the label increase of de novo synthetic activities in log phase for total

0.3-

Fig. 1. Time course of incorporation of purine bases into nucleotides and nucleic acids 0.2- in hepatoma 3924A cells. The hepatoma cells in log phase were pulsed with either [8-MCl adenine (A), [8-uC]hypoxanthine (fl), or [8- Z C4('luminine(C) at a concentration of 5 fiMand O labeled acid-soluble nucleotides (O) and acid- insoluble nucleic acids (â€¢)weredetermined as described in "Materials and Methods." To plot Ã–L the total nucleotide synthesis (x) the sums of Â£ the label incorporation into both fractions were OÃ O calculated. O

TIME (min) 89

2.0-

Â« 1.5-1 y

Fig. 2. Dependence of purine salvage syn thesis on concentrations of purine bases. The initial rates of purine salvage synthesis with 1.0- various concentrations of [8-14C]adenine (â€¢), z [8-MC]hypoxanthine (O), or [8-MC]guanine (X) O were measured in intact hepatoma cells as described in "Materials and Methods." Inset, Ã–L double reciprocal plots of the rates and con centrations of the various substrates. 2 te. o 0.5â€” u z

Oâ€”I

0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.10

SUBSTRATE (mivi)

Table 1 Activities of purine de novo and salvage synthesis in hepatoma 3924A cells in various growth phases Activities 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 were significantly higher than those of de novo synthesis in hepatoma 3924 A cells in various growth phases (/*< O.OS). cells)Time Purine nucleotide synthesis (nmol/h/g after plating (h)0 novo89.5 novo63.3 novo14.3 Â±2.5 Â±45 Â±4.4 Â±30 Â±2.6 Â±28 6 94.0 Â±12.1 638 Â±30 42.6 Â±1.8 1210 Â±60 46.4 Â±1.7 497 Â±56 24 399.0 Â±10.0 783 Â±85 129.0 Â±4.0 1510Â± 110 251.0 Â±17.0 541 Â±84 48 720.0 Â±40.0 1390 Â±80 358.0 Â±13.0 1940 Â±160 298.0 Â±25.0 879 Â±61 72 223.0 Â±8.0 635 Â±31 136.0 Â±1.0 1410 Â±80 63.7 Â±6.8 541 Â±26 96IMPJe 89.5 Â±2.5Salvage658 658 Â±45AMPde 63.3 Â±4.4Salvage1250 1250 Â±30GMPde 14.3 Â±2.6Salvage456 456 Â±28 purine (8-fold), adenylates (6-fold), and guanylates (21-fold), Table 2 Effect of acivicin on activities of amidophosphoribosyltransferase and GMP synthase and on concentrations of purine nucleoside triphosphates and the rise of the salvage activities was minor (1.6- to 2.1-fold). PRPP For IMP synthesis, however, the hepatoma cells had 2- to 7- Hepatoma 3924A cells in log phase were treated with 10 *Â¿Macivicinfor 3 h. The enzyme activities and concentrations of nucleotides and PRPP were deter fold higher activity of salvage pathway from hypoxanthine than mined as described in "Materials and Methods." Values are means Â±SE of that of de novo pathway in various growth phases. In addition triplicate assays. to the salvage synthesis of IMP the pathway from adenine could Enzymes provide AMP with 5- to 28-fold higher activity than the de and Acivicin- %of novo adenylate synthesis. The guanine salvage pathway also metabolites Control treated control could contribute to GMP production with a 2- to 32-fold higher Enzyme activities (nmol/h/mg protein) activity than the de novo guanylate pathway. Thus, the activities Amidophosphoribosyl- 134 Â±6 41.5 Â±3.4 31Â° transferase of purine salvage pathways were higher than those of purine de GMP synthase 247 Â±4 10.5 Â±4.4 4" novo pathways for total purine, adenylate, and guanylate syn thesis in hepatoma 3924A cells during the culture growth. Metabolite concentrations Biochemical Impacts of Acivicin in Hepatoma 3924A Cells. (nmol/h/g cells) ATP 1220 Â±30 1190 Â±50 98 Acivicin irreversibly inactivates glutamine-utilizing enzymes GTP 230 Â±4 96.2 Â±3.3 42Â° including amidophosphoribosyltransferase, phosphoribosylfor- PRPP 18.6 Â±1.5 58.2 Â±4.4 313Â° mylglycinamidine synthase, and GMP synthase among purine Â°Significantly different from control (P < 0.05). synthetic enzymes (27). After treatment of hepatoma cells with 10 /iM acivicin for 3 h the activity of amidophosphoribosyltrans metabolic changes are in good agreement with previous in vivo ferase, the rate-limiting enzyme of purine de novo synthesis, studies in solid hepatoma 3924A and other tumors (13, 28, 29). decreased to 31% of the pretreatment value and that of GMP The metabolic fluxes of de novo synthesis of total purities and synthase to 4% (Table 2). Concurrently, the concentration of guanylates were depressed to 37 and 3%, respectively, of the GTP was depressed to 42%, whereas that of ATP did not controls (Table 3). The rate of de novo adenylate synthesis was change significantly. The concentration of PRPP, a shared retained at 73% despite the decreased activity of purine de novo substrate for purine, pyrimidine, and pyridine nucleotide bio synthesis (37%). synthesis, increased 3-fold after treatment (Table 2). These After the acivicin treatment the activities of salvage nucleo- 90

Table 3 Effect of acivicin on purine de novo and salvage synthesis in hepatoma Table 4 Effects of inhibitors of IMP dehydrogenase on de novo and salvage GMP 3924A cells synthesis in hepatoma 3924A cells Hepatoma cells in log phase were treated with 10 fiM acivicin for 3 h. Flux Hepatoma cells in log phase were treated with drugs for 2 h. Activities of de activities of purine de novo and salvage synthesis were then measured after medium novo and salvage guanylate synthesis were measured after medium replacement replacement as outlined in "Materials and Methods." Values are means Â±SE of as described in "Materials and Methods." Values are means Â±SE of 3 or more triplicate assays. assays. Purine nucleotide synthesis GMP synthesis cells)PathwaysIMP (nmol/h/g (nmol/h/g cells) (% of control) change37*253"73 Drugs(pM)Control, novo264 synthesis none Â±14(100) Â±55 (100) de novo' Â±9 Â±49 Tiazofurin, 8 100 Â±7(41)" 1940 Â±90 (232)Â° SalvageAMP 1180Â±110346 2980 Â±220253 SM-108, 10 30 Â±3(12)Â° 1650 Â±110(197)Â° RiiÂ«virili.IOde 82 Â±9(33)Â°Salvage8371540 Â±80(184)Â° synthesis Â°Significantly different from control (/>< 0.05). de novo' Â±25 Â±37 SalvageGMP 1850 Â±80288 2010 Â±809Â± 10934244* lymphoblasts in which the only possible metabolic fate of synthesis de novoÃ¤ Â±32 1 accumulated IMP would be degradation to inosine. The in SalvageControl845 870 Â±63Acivicin-treated3162120 Â±100%of crease of PRPP concentration in those cells was attributed to Â°Total purine de novo synthesis. the enhancement of inosinate cycle consisting of 5'-nucleo tid- * Significantly different from control (P < 0.05). ' de novo synthesis of adenylates. ase, inosine phosphorylase, phosphoribomutase, PRPP syn- d de novo synthesis of guanylates. thase, and hypoxanthine phosphoribosyltransferase (42). Sim ilarly in hepatoma 3924A cells inhibition of IMP dehydrogen- tide synthesis from hypoxanthine and guanine increased 2.5- ase might be able to enhance inosinate cycle and expand PRPP pool. In fact, inosine and hypoxanthine, as well as IMP, the fold (Table 3). This elevation might be attributed to the increase in the concentration of PRPP, the substrate of hypoxanthine- constituents of the inosinate cycle, markedly accumulated in hepatoma 3924A after treatment with tiazofurin (43). Although guanine phosphoribosyltransferase, and to the decrease in the IMP, an inhibitor of hypoxanthine-guanine phosphoribosyl concentrations of guanine nucleotides which are strong com transferase, accumulated in the tumor after tiazofurin treat petitive inhibitors for the salvage enzyme reactions (30, 31). ment, the amount increased in IMP was less than that decreased However, the acivicin treatment did not result in a significant in guanylates (24, 43). Furthermore, the inhibitory effects of increase in the activity of adenine salvage pathway because GTP and GDP were as strong as IMP and that of GMP was there was no change in the concentrations of adenine nucleo much stronger than IMP in the in vitro guanine phosphoribo tides, potent inhibitors of adenine phosphoribosyltransferase syltransferase assays with 30 ^M PRPP (data not shown). (32) (Table 3). Also the concentration of PRPP in the log phase cells (18-25 nmol/g cells) might be nearly saturating for ade Significance of Purine Salvage Synthesis in Cancer Cells. Novel aspects and significant implications of the present study nine phosphoribosyltransferase activity (Km2 ^M) (14). include: (a) establishment of the initial rate kinetics of purine Effects of Inhibitors of IMP Dehydrogenase on Salvage Gua salvage synthesis from adenine, hypoxanthine, and guanine in nylate Synthesis. Early studies in this laboratory identified as rat hepatoma 3924A cells in monolayer culture; (Â¿>)determi essential elements in the expression of the neoplastic prolifer nation of the rates of purine de novo and salvage synthesis as ation program the increased activity of IMP dehydrogenase, measured in nmol of purine nucleotide synthesized per h per g the predominant utilization of IMP for guanylate synthesis, the cells in the hepatoma cell culture in various growth phases; (c) positive correlation of the ratio of the concentrations of gua first direct comparison of the flux rates of purine de novo and nylates to adenylates with the tumor proliferation rates and the salvage pathways in intact cells under the same circumstances; expanded pool of dGTP in cancer cells (12, 26, 33, 34). Tia zofurin and SM-108, inhibitors of IMP dehydrogenase, have (d) demonstration that the activities of the metabolic flux of purine salvage pathways are higher than those of de novo broad antitumor spectra including Lewis lung tumor and rat pathway in rat hepatoma cells; (e) evidence that the flux activity transplantable hepatoma 3924A (35, 36). The oncolytic action of guanine salvage pathway increases after treatment with in of the drugs is linked with the depletion of GTP and dGTP hibitors of de novo guanylate synthesis; (/) attribution of the pools (24, 37). Tiazofurin also is a promising agent in the increase in guanine salvage activity to the decline in guanine treatment of human myelocytic leukemia (38, 39). nucleotide concentrations and to the elevation of PRPP level. However, guanine nucleotides are also produced by the sal The high activity of purine salvage synthesis in cancer cells vage pathway. After treatment of hepatoma cells with tiazo furin, SM-108, or ribavirin, de novo guanylate synthesis was and its further increase in response to inhibition of de novo purine synthesis indicate the important role of the purine inhibited to 41, 12, and 33%, respectively, of controls (Table salvage pathways in circumventing the action of antimetabolites 4). Concurrently, the flux activity of guanine salvage pathway was 1.8- to 2.3-fold enhanced (Table 4). This rise again could of de novo purine synthesis in cancer chemotherapy. These observations provide new evidence for the concept that for an be due to the decline in the concentrations of guanine nucleo effective cancer drug treatment both de novo and salvage path tides and to the elevation of PRPP level as reported previously (24, 26, 36). 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. Significance of Purine Salvage in Circumventing the Action of Antimetabolites in Rat Hepatoma Cells

Yutaka Natsumeda, Tadashi Ikegami, Edith Olah, et al.

Cancer Res 1989;49:88-92.

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