Impact of Polyglutamation on Sensitivity to Raltitrexed andMethotrexate in Relation to Drug-induced Inhibition ofde NovoThymidylate and Purine Biosynthesis inCCRF-CEM Cell Lines1

Matthew J. Barnes, Edward J. Estlin,2

Gordon A. Taylor, G. Wynne Aherne,A. Hardcastle, J. J. McGuire, Joanne A. Calvete,John Lunec, Andrew D. J. Pearson, andDavid R. NewellCancer Research Unit, University of Newcastle, Newcastle upon TyneNE2 4HH, United Kingdom [M. J. B., G. A. T., J. A. C., J. L.,D. R. N.]; Department of Child Health, Royal Victoria Infirmary,Newcastle upon Tyne NE1 4LP, United Kingdom [E. J. E.,A. D. J. P.]; CRC Centre for Cancer Therapeutics, Institute of CancerResearch, Sutton, Surrey SM2 5NG, United Kingdom [G. W. A.,A. H.]; and Roswell Park Cancer Institute, New York StateDepartment of Health, Buffalo, New York 14263-0001 [J. J. M.]

ABSTRACTThe aim of this study was to investigate the influence of

folylpolyglutamyl synthetase (FPGS) activity on the cellularpharmacology of the classical antifolates raltitrexed andmethotrexate (MTX) using two human leukemia cell lines,CCRF-CEM and CCRF-CEM:RC2 Tomudex. Cell growth in-hibition and drug-induced inhibition of de novothymidylateand purine biosynthesis were used as measures of the cellu-lar effects of the drugs.

CCRF-CEM:RC2Tomudex cells had<11% of the FPGSactivity of CCRF-CEM cells, whereas MTX uptake and TSactivity were equivalent. In CCRF-CEM:RC2Tomudex cells,MTX polyglutamate formation was undetectable after expo-sure to 1 mM [3H]MTX for 24 h. After exposure to 0.1 mM

raltitrexed, levels of total intracellular raltitrexed-derivedmaterial in CCRF-CEM:RC2 Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM:RC2Tomudex cells were >1000-fold resistant to raltitrexedand 6-fold resistant to lometrexol but sensitive to MTX and

nolatrexed when exposed to these antifolates for 96 h. After6 h of exposure, CCRF-CEM cells retained sensitivity toMTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM:RC2Tomudex cells were markedly insensitive to raltitrexed,lometrexol, and to a lesser degree, MTX. Simultaneousmeasurement ofde novothymidylate and purine biosynthe-sis revealed 90% inhibition of TS activity by 100 nM MTX inboth cell lines, whereas inhibition ofde novopurine synthesiswas only observed in CCRF-CEM cells, and only after ex-posure to 1000 nM MTX. Ten n M raltitrexed induced >90%inhibition of TS activity in CCRF-CEM cells, whereas inCCRF-CEM:RC2Tomudex cells, there was no evidence ofinhibition after exposure to 1000 nM raltitrexed.

These studies demonstrate that polyglutamation is acritical determinant of the cellular pharmacology of bothraltitrexed and MTX, markedly influencing potency in thecase of raltitrexed and locus of action in the case of MTX.

INTRODUCTIONAntitumor antifolates can be classified by their loci of

action,e.g.,as inhibitors of TS,3 DHFR or glycinamide ribonu-cleotide transformylase, and on the basis of the presence (clas-sical antifolates) or absence (nonclassical antifolates) of a glu-tamate moiety. Thea and g glutamate carboxyl groups ofclassical antifolates, such as raltitrexed (an inhibitor of TS) andMTX, are negatively charged at physiological pH, and thusclassical antifolates require carrier-mediated uptake for cell en-try (1, 2). Once inside the cell, classical antifolates, as wellnaturally occurring folates, can undergo polyglutamation in areaction that involves the addition of additional glutamate res-idues at theg-carboxyl position of the glutamate moiety. Poly-glutamation, catalyzed by the enzyme FPGS (3), has beenshown to be an important determinant of the sensitivity of cellsto classical antifolates (4). As a result of polyglutamation,intracellular drug levels can exceed the extracellular concentra-tions (5) and can maintain inhibition of target enzymes afterremoval of extracellular drug (6, 7). In addition, polyglutama-tion can enhance the affinity of classical antifolates for certainfolate-dependent enzymes (8–10). In contrast to classical anti-folates, the nonclassical agents trimetrexate (11), piritrexim(12), and nolatrexed (13) do not require carrier-mediated cellu-

Received 12/21/98; revised 6/15/99; accepted 6/16/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was supported by the Kay Kendall Leukaemia Fund(to M. J. B.), The North of England Children’s Cancer Research Fund(to E. J. E. and A. D. J. P.), the Cancer Research Campaign (toG. W. A., A. H., G. A. T., D. R. N., and J. L.), and by National CancerInstitute Grant CA43500 and Roswell Park Cancer Institute Core GrantCA16056 (both to J. J. M.).2 To whom requests for reprints should be addressed, at Department ofPaediatric Oncology, Royal Hospital for Sick Children, St. Michael’sHill, Bristol BS2 8BJ, United Kingdom. Phone: 44 0117 921 5411; Fax:44 0117 928 5682.

3 The abbreviations used are: TS, thymidylate synthase; DHFR, dihy-drofolate reductase; FPGS, folylpolyglutamate synthetase; HPLC, high-performance liquid chromatography; AICAR, aminoimidazolecar-boxymide; PRPP, 5-phosphorosyl-1-PPi.

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lar uptake and are not substrates for FPGS. As a result, non-classical antifolates are not retained within the cell on removalof extracellular drug and cannot be converted to metaboliteswith enhanced affinities for their target enzyme.

Resistance to antifolates can result from one or more mech-anisms including decreased cellular uptake of the drug (14, 15),reduced polyglutamation (see below), overproduction of thetarget enzyme (14, 16), expression of a mutant form of the targetenzyme (17, 18), or a failure of the cell to appropriately engageapoptosis (19).

Reduced polyglutamation has been described previously asa mechanism of inherent and acquired resistance to classicalantifolates, bothin vitro (14, 20) andin vivo (21, 22). Theunderlying reason for resistance is either reduced FPGS activity(14, 20) or enhanced activity of folylpolyglutamate hydrolase(23), the enzyme that catalyzes the hydrolysis of antifolatepolyglutamate metabolites. The impact of polyglutamation-re-lated resistance on target enzyme inhibition and antifolate-induced cytotoxicity depends on both the antifolate agent used,its target, and the duration of drug exposure. For example,decreased polyglutamation, whichin vitro leads to resistance toshort-term exposures with MTX, can be overcome with moreprolonged drug exposure (20, 24). However, for certain otherclassical antifolates, such as raltitrexed, deficient polyglutama-tion renders cell lines resistant to both short and longer termdrug exposure (14, 25) Indeed, antifolate polyglutamation andFPGS activity have been shown to be important determinants ofboth MTX cytotoxicity in vitro (26, 27) and outcome in clinicalstudies (4, 27).

Work to characterize the sensitivity of a panel of humanleukemic cell lines has identified a CCRF-CEM cell line,CCRF-CEM:RC2Tomudex, which is insensitive to raltitrexed(28). Because studies have shown that resistance to antifolatescan be multifactorial (29, 30), full characterization of the mech-anism of resistance was undertaken and found to be related toreduced FPGS activity. The aim of the studies described herewas to investigate the impact of reduced FPGS activity oncellular sensitivity to a range of antifolates after short term andcontinuous exposure and, in the case of raltitrexed and MTX,the impact of impaired polyglutamation on the inhibition ofdenovo thymidylate and purine biosynthesis.

MATERIALS AND METHODSMaintenance of Cell Cultures. CCRF-CEM (European

Collection of Animal Cell Cultures, Salisbury, United King-dom) and CCRF-CEM:RC2Tomudexhuman leukemic cell lineswere grown as suspension cultures in RPMI 1640 (Life Tech-nologies, Inc., Paisley, United Kingdom) supplemented with 2mM L-glutamine (Life Technologies), 12.5 ml of 7.5% (w/v)sodium bicarbonate solution (Life Technologies), and 10% (v/v)charcoal-dialyzed FCS (Globepharm, Surrey, United Kingdom).Both cell lines were routinely subcultured twice weekly, main-tained in an incubator with 5% CO2 and a humidified atmo-sphere at 37°C, and shown to beMycoplasmanegative at regularintervals.

The CCRF-CEM:RC2Tomudexcell line was derived from aCCRF-CEM cell line in routine use in the Cancer Research Unit,University of Newcastle, United Kingdom. The CCRF-CEM:

RC2Tomudexcell line was found to be relatively insensitive to theantifolate raltitrexed when compared with a CCRF-CEM cellline obtained from the European Collection of Animal CellCultures and was not known to have been exposed to anyantifolate for any period of time prior to use in these studies. Theoriginal raltitrexed-resistant CCRF-CEM cell line was cloned byseeding a cell suspension at a concentration of,1 cell/well intoa 96-well plate (Nunc, Life Technologies, Paisley, UnitedKingdom), and after;4 weeks, the cell line was routinelysubcultured as above. The resulting clone, CCRF-CEM:RC2Tomudex(CEM:RTOM), was characterized by karyotype andimmunophenotype and found to be indistinguishable from theCCRF-CEM cell line obtained from the European Collection ofAnimal Cell Cultures.

Cell Growth Inhibition Studies. All antifolates, i.e.,MTX (Sigma Chemical Co.), nolatrexed (Thymitaq or AG337;Agouron Pharmaceuticals, San Diego, CA), raltitrexed (Tomu-dex or ZD1694; Zeneca Pharmaceuticals, Macclesfield, UnitedKingdom), and lometrexol (DDATHF; Eli Lilly, Indianapolis,U.S.A.) were dissolved in water at 1 mg/ml. Stock solutionswere diluted further in dialyzed RPMI 1640 to the final con-centration required.

For continuous exposure studies, 100-ml aliquots of cellsuspensions at 53 104 cells/ml were seeded into each well of a96-well plate 24 h before drug exposure. One hundredml ofmedium containing drug at twice the concentration requiredwere added to quadruplicate wells and incubated for 96 h,during which time approximately three cell doublings wouldhave occurred in the control (untreated) wells. At the end of theexposure time, the number of cells in each suspension wascounted on a Coulter cell counter.

In short-term exposure experiments, 2.5-ml aliquots of cellsuspensions at 53 104 cells/ml were seeded into Falcon tubes(Becton Dickinson, New Jersey, NJ) 24 h before drug exposure.After this period, 2.5 ml of medium containing drug at twice theconcentration required were added to triplicate tubes and incu-bated at 37°C. After 6 or 24 h exposure to the drug, the cellswere centrifuged at 5003 g for 5 min at room temperature. Thedrug-containing medium was aspirated, and cells were washedwith 5 ml of drug-free medium and then resuspended in 5 ml ofdrug-free medium, followed by incubation for an additional 90or 72 h (96 h total incubation time). At the end of the incubationtime, cells were counted as above.

To determine the IC50s, cell counts for each concentrationof drug were divided by the mean control cell count and mul-tiplied by 100 to give values as a percentage of the controlgrowth. The IC50, i.e., the concentration of drug required toinhibit cell growth by 50%, was calculated by fitting the Hillequation to the data using unweighted, nonlinear least squaresregression analysis (GraphPad Prism, San Diego, CA).

TS Activity in Cell Sonicates. TS activity in exponen-tially growing cell lines was measured in cell sonicates by therelease of [3H]2O from 5-[3H]dUMP using the method de-scribed previously by Estlinet al. (31).

Cellular Methotrexate Transport Kinetics. A total of1–23 107 exponentially growing cells were resuspended in 2.5ml of transport buffer (32) and equilibrated for 5 min at 37°C.Two hundredml of the cell suspension were then added to 200ml of [3H]MTX at twice the concentration required to give final

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concentrations ranging from 0.2mM (specific activity, 910 kBq/mmol) to 18mM (specific activity, 10 kBq/mmol), and the cellsuspension was incubated for an additional 5 min at 37°C. Afterincubation, triplicate 100-ml aliquots of each cell suspensionwere then centrifuged at 67003 g for 1 min through a 200-mllayer of Dow Corning silicone oil [final specific gravity, 1.028;45 ml of Dow Corning 556 silicone oil (specific gravity, 0.98)1 55 ml of Dow Corning 550 silicone oil (specific gravity,1.068)] into 50-ml 3 M potassium hydroxide (BDH, Dorset,United Kingdom; Ref. 33). After 1 h, the tubes were cut in theoil layer, and the potassium hydroxide containing lysed cellswere neutralized with 250ml of 1 M acetic acid (Sigma Chem-ical Co., Poole, Dorset, United Kingdom). Samples were thencounted on a scintillation counter after addition of 10 ml ofOptiphase scintillant (Fisons Chemicals, Loughborough, UnitedKingdom). Parallel experiments using [14C]sucrose (23 kBq/mmol; Amersham, Buckinghamshire, United Kingdom) werecarried out to compensate for any [3H]MTX that passed throughthe oil layer due to trapping in the extracellular space aroundcells.

Uptake of [3H]MTX (pmol/106 cells) was plotted againstthe extracellular concentration of MTX (mM), and a one-sitebinding hyperbolic equation was fitted to the data using un-weighted, nonlinear least squares regression (GraphPad Prism).TheKt (mM), i.e., the concentration of MTX required to achievehalf the maximal uptake rate over a 5-min period, was calculatedand expressed as pmol/106 cells/min, as was theTmax, themaximum rate of uptake.

The uptake of MTX in the presence of raltitrexed was alsostudied as described above with the exception that a constantconcentration of 0.5mM [3H]MTX (specific activity, 364 kBq/mmol) was used with a range of raltitrexed concentrations(0–10mM). Also included in the experiment as a control wereincubations of 0.5mM [3H]MTX (specific activity, 364 kBq/mmol) with a range of MTX concentrations (0–10mM).

Uptake of 0.5mM [3H]MTX (dpm) was plotted against theextracellular raltitrexed or MTX concentration (mM), and aone-site binding competition equation was fitted to the data(GraphPad Prism). The EC50 generated from this equation andthe Kt value generated from the experiments described abovewere used to calculate theKi for inhibition of [3H]MTX uptakeby MTX or raltitrexed from the equation of Cheng and Prusoff(34).

Cellular Methotrexate Polyglutamate Formation. Themethod used was based on that of Whiteheadet al. (27). A totalof 4 3 106 exponentially growing cells was incubated in 5 ml ofdialyzed medium supplemented with 5mM thymidine, 10mM

inosine (Aldrich Chemical Co., Milwaukee, WI), and 1 or 10mM

39,59-79- [3H]MTX (specific activity, 129.5 kBq/mmol;Moravek Biochemicals, Brea, CA) for 24 h at 37°C. After the24-h incubation, the cell suspension was centrifuged at 1203 gfor 5 min, and the medium containing [3H]MTX was removed.The cell pellet was washed twice, in 5 ml and then 2 ml of PBS,and the final cell pellet was lysed with 300ml of ice-cold 0.2M

perchloric acid (Sigma). Samples were vortexed briefly and lefton ice for 5 min before centrifugation at 67003 g for 2 min.The resultant supernatant was removed and pipetted directlyonto ;200 mg of potassium bicarbonate (Sigma) and left tostand on ice for 2 min. The neutralized cell extract was again

centrifuged at 67003 g for 2 min to remove any remainingpotassium bicarbonate and the potassium perchlorate that hadformed, and the final supernatant was removed and stored at220°C.

HPLC analysis of MTX polyglutamate formation involvedthe separation of MTX and MTX polyglutamates on a 10034.6-mm Nucleosil 3mm ODS cartridge column (Jones Chroma-tography, Mid-Glamorgan, United Kingdom) with UV absorb-ance of standard MTX and MTX polyglutamates (Schirks, Jona,Switzerland) being measured at 254 and 304 nm. One hun-dred-ml aliquots, prepared as described above, were injectedonto the column via a 503 2-mm pellicular ODS silica precol-umn (Whatman, Maidstone, Kent, United Kingdom), and[3H]MTX and [3H]MTX polyglutamates were detected andquantitated using an on-line FLO-ONE radiochromatographydetector (Packard, Meriden, CT) with a liquid scintillant cell[flow rate of liquid scintillant (Packard) 1.5 ml/min]. The mo-bile phase used to separate MTX and MTX polyglutamatesconsisted of 0.1M ammonium acetate (ammonia and glacialacetic acid; BDH) pH 4.71 6% (w/w) acetonitrile at a flow rateof 0.5 ml/min with a 30-min analysis time. [3H]MTX and[3H]MTX polyglutamates were quantitated using a standardcurve constructed using [3H]MTX, and results were expressedas pmol of MTX or MTX polyglutamate/109 cells.

Measurement of FPGS Activity in Cell Extracts. Themethod used was based on that of Jansenet al. (35). Exponen-tially growing cell lines (1–23 107) were resuspended in 500mlof ice-cold extraction buffer, 50 mM Tris-HCl, 20 mM potassiumchloride, 10 mM magnesium chloride, and 5 mM DTT at pH 7.6(Sigma; Ref. 29). Cell suspensions on ice were then sonicatedthree times at 10mm for 10 s, with 10-s intervals between sonicbursts. The cell sonicates were then ultracentrifuged at 50,0003g for 30 min at 4°C, and the supernatant containing FPGSprotein was removed and kept on ice.

One hundredml of the above supernatant were added to thereaction buffer, which consisted of 100 mM Tris-HCl, 20 mM

potassium chloride, 20 mM magnesium chloride, 10 mM DTT,and 10 mM ATP (Boehringer, East Sussex, United Kingdom) atpH 8.4; all concentrations were the final working values. Inaddition, the reaction mixture included 250mM MTX, or anequivalent volume of H2O for negative controls, and 4 mM[3H]L-glutamate (specific activity 0.27 kBq/mmol; Amersham).The final volume of the reaction mixture including the proteinextract was 250ml, which was incubated at 37°C for 2 h.

The reaction was terminated by adding 1 ml of ice-cold 5mM sodium glutamate (Sigma Chemical Co.) and placing on ice.Samples were then applied to prewashed (10 ml of methanol),preequilibrated [10 ml of 0.2M sodium acetate (Sigma ChemicalCo.), pH 5.5] Bond Elut LRC bonded phase-C18 columns (Var-ian, Surrey, United Kingdom); all elutions and washes wereaided by a VAC ELUT SPS 24 vacuum manifold (AnalytichemInternational, Bedfordshire, United Kingdom). After the sam-ples had progressed through, the column was washed with 10,2.5, and then 3 ml 0.2M sodium acetate (pH 5.5). Compoundsretained on the column were eluted with 3 ml of methanol,which were subsequently dried down under nitrogen, and resi-dues were kept at 4°C until analysis.

HPLC analysis followed the method described above forthe measurement of whole-cell MTX polyglutamation.

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[3H]MTX polyglutamates were quantitated on the basis of thespecific activity of the [3H]glutamate cosubstrate (0.27 kBq/mmol) used in the initial incubation, and the results were ex-pressed as pmol of MTX polyglutamates/hour/mg of total pro-tein.

Measurement of FPGS Protein. A total of 1–23 107

exponentially growing CCRF-CEM and CEM:RTOM cells wereharvested by centrifugation at 5003 g. To the resulting cellpellet, 150ml of lysis buffer [50 mM Tris/HCl (pH 7.6), 120 mM

NaCl, 10% v/v protease inhibitor cocktail, and 0.5% IGEPAL;Sigma] were added. The mixture was incubated on ice for 30min, followed by centrifugation at 10,0003 g to remove celldebris. The resulting supernatant was removed and stored at280°C before analysis.

SDS-PAGE was performed in 0.75-mm thick mini-gels(Mini-Protean; Bio-Rad, Hercules, CA), and FPGS protein wasdetected with an immunoaffinity-purified rabbit polyclonal an-tibody using essentially standard procedures, as described (36).

Measurement of FPGS mRNA. FPGS mRNA expres-sion was measured by Northern blot hybridization in exponen-tially growing cell lines, using a 700-bp fragment of the 59coding sequence excised from the pX1 plasmid, which containsa 1–2-kb FPGS genomic DNA insert. Northern blot hybridiza-tion was performed as described previously (31), and FPGSmRNA levels were expressed relative to 18S rRNA. The pX1plasmid used in these studies was kindly provided by ProfessorR. Moran (Massey Medical Center, Richmond, VA).

Determination of Total Intracellular Levels of Ralti-trexed-derived Material. Exponentially growing cell lineswere treated with 0.1mM raltitrexed for 4, 24, or for 4 h withfurther incubation in drug-free medium for 20 h. Directly afterthese exposure times, 2–53 106 cells in each case were har-vested, resuspended in 1 ml of PBS, and stored at280°C untilthey were analyzed.

Samples were thawed and sonicated on ice, three times for30 s, and centrifuged, and the supernatant was removed. Totalintracellular levels of raltitrexed-derived material were meas-ured in the resultant crude cell sonicates using a previouslydescribed radioimmunoassay, which detects raltitrexed and ralti-trexed polyglutamates with equal sensitivity (37).

Drug-induced Inhibition of de NovoThymidylate andPurine Biosynthesis. The following method was used tomeasure the inhibition ofin situ TS activity andde novopurinesynthesis induced by exposure to raltitrexed or MTX and wasbased on a combination of the methods Tayloret al. (38) andMassonet al. (39). Estimations ofin situ TS activity involvedthe exposure of cells to 59-[3H]deoxyuridine which is trans-ported and subsequently phosphorylated in the cell by thymidinekinase to 59-[3H]dUMP. TS catalyzes the methylation of the[3H]dUMP at the 59 position, resulting in the release of thetritium atom as [3H]2O, which can be used as a measure of theTS activity. Thede novopurine synthesis assay involves expo-sure of cells to [14C]formate and subsequent incorporation intothe purine ring structure via 10-[14C]formyl tetrahydrofolate,which allows analysis and quantification of purine bases byHPLC with radiochemical detection.

Exponentially growing cells (53 106) were resuspended in2 ml of dialyzed medium supplemented with 5mM thymidine(Sigma) and 10mM inosine (Aldrich). The cell suspension was

then treated with MTX or raltitrexed at concentrations rangingfrom 0 to 1000 nM, and the cells were incubated at 37°C for22 h. At the end of this period, samples were centrifuged (1203g for 5 min), washed, and resuspended in 2 ml of fresh, drug-free medium (without thymidine or inosine) and incubated foran additional 30 min at 37°C. After the 30-min period, the cellsuspensions were added with a mixture of purified [3H]deoxy-uridine (59-[3H] 29-deoxyuridine; Moravek Biochemicals;deoxyuridine, Sigma Chemical Co.), and [14C]formic acid (14Cformic acid sodium salt, Amersham, Buckinghamshire, UnitedKingdom; sodium formate, Sigma Chemical Co.) at final con-centrations of 0.3mM [3H]deoxyuridine (specific activity, 227kBq/mmol) and 0.5 mM formate (specific activity, 0.84 kBq/mmol). Cell suspensions were incubated for an additional 2 h,after which samples were placed on ice for 5 min before ex-traction, as described below.

Estimation of in Situ TS Activity. The supernatant fromthe above centrifugation step, containing the [3H]2O releasedfrom the in situ activity of TS, was removed as three 600-mlaliquots, each of which was pipetted onto 600ml of ice-cold 1M perchloric acid (BDH). The samples were placed on ice for 15min, after which 750ml of activated charcoal suspension (200mg/ml suspended in H2O; Sigma Chemical Co.) in dextran (10mg/ml in H2O; Sigma Chemical Co.) were added to each 1.2-mlsample at 4°C. The activated charcoal solution was constantlystirred prior to and during the pipetting to ensure that no sedi-mentation occurred and, after charcoal addition, samples wereleft for at least 15 min on ice before centrifugation at 18503 gfor 10 min at 4°C. The supernatant containing [3H]2O wasremoved, and 300ml were analyzed by scintillation counting.Results were expressed as a percentage of the release of [3H]2Oin control untreated cells.

Estimation of de NovoPurine Synthesis. The cell pel-lets remaining after removal of the supernatant for [3H]2Oanalysis were resuspended in 2 ml of ice-cold, drug-free dia-lyzed medium (without thymidine or inosine) and centrifuged at120 3 g for 5 min. The cell pellets were washed an additionaltwo times in 2 ml of the same ice-cold medium and finallyresuspended in 500ml of 1 M ice-cold perchloric acid. Aftertransfer to glass tubes (Baxter Healthcare, Thetford, Norfolk,United Kingdom), samples were vortexed briefly and placed ina dry heating block (Techne, Cambridge, United Kingdom) at100°C for 1 h. After acid hydrolysis, samples were removedfrom the heating block and allowed to cool for 15 min. Thesamples were then pipetted into 1.5-ml Eppendorf tubes(Sarstedt, Leicester, United Kingdom) and centrifuged at 67003 g for 5 min at 4°C. The resultant supernatant was then addedto 300 ml of ice-cold 2 M Tris-base and again centrifuged at67003 g for 5 min at 4°C. The supernatant was removed, thepH was checked and if necessary adjusted to pH 7, before thesamples were frozen at220°C for later analysis by HPLC.

HPLC analysis of [14C]formate incorporation into adenineand guanine involved the injection of 100-ml samples via a 5032-mm pellicular ODS silica pre-column (Whatman) onto a250 3 4.6-mm Spherisorb 5m ODS2 cartridge (PhaseSep,Franklin, MA), and UV absorbance of standard (Sigma) andendogenous adenine and guanine was measured at 260 and 280nm. 14C-Labeled material was detected using an on line FLO-

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ONE radiochromatography detector with a liquid scintillant cell(flow rate of liquid scintillant 1.5 ml/min).

Adenine and guanine were resolved using a mobile phaseconsisting of 0.1M sodium acetate adjusted to pH 5.5 withglacial acetic acid (BDH) at a flow rate of 1 ml/min for the first10 min of the analysis, after which a linear gradient from 0.1M

sodium acetate (pH 5.5) to 0.1M sodium acetate (pH 5.5)115% (w/w) acetonitrile (Fisons Chemicals) over 20 min wasinitiated to give a total analysis time of 30 min. Endogenousadenine and guanine were quantitated using linear regressionagainst a standard curve. Standard curves were constructed bythe analysis of standard adenine base and guanine bases (Sigma)as follows: guanine5 0.52, 1.04, 2.08, 4.16, 8.32, 16.64, and33.28 nmol; adenine5 0.44, 0.88, 1.66, 3.32, 6.64, 13.28, and26.56 nmol injected on-column. Newly formed adenine andguanine were quantitated on the basis of the specific activity ofthe [14C]formate (0.84 kBq/mmol), and results were expressedas pmol of newly formed bases/nmol of preexisting or endog-enous bases.

Statistics. Nonweighted linear regression analysis wasused in the calculation of the X coefficients of the standardcurves used to quantifyde novopurine and thymidylate biosyn-thesis. Two-sidedt tests were used to test for differences be-tween the CCRF-CEM and CEM:RTOM cell lines with respect toantifolate sensitivity, TS activity, [3H]MTX transport kinetics,and FPGS activity.

RESULTSCell Growth Inhibition Studies with CCRF-CEM and

CEM:R TOM Cell Lines. The IC50 values for antifolate-in-duced growth inhibition with CCRF-CEM and CCRF-CEM:RC2Tomudex(CEM:RTOM) cell lines, after either continuous orshort-term drug exposure, are shown in Table 1. For continuousexposure, CCRF-CEM cells were markedly more sensitive thanCEM:RTOM cells to both raltitrexed (Fig. 1) and to a lesserextent lometrexol. In contrast, CEM:RTOM cells were somewhatmore sensitive than CCRF-CEM cells to MTX, and there was nosignificant difference between the two cell lines in sensitivity tonolatrexed.

After short-term drug exposures, CCRF-CEM and CEM:RTOM cells were equisensitive to a 24-h exposure to MTX andwere insensitive to a 6-h exposure to nolatrexed. However, fora 6-h drug exposure, CCRF-CEM cells were more sensitive than

CEM:RTOM cells to all three classical antifolates: raltitrexed,methotrexate, and lometrexol.

TS Activity and Methotrexate Transport Kinetics inCCRF-CEM and CEM:R TOM Cell Lines. There was nosignificant difference between the TS activity in cell extractsfrom the CCRF-CEM and CEM:RTOM cell lines (Table 2).Similarly, there was no significant difference between the twocell lines in the transport kinetics of [3H]MTX, as measured bythe Kt value, or in the transport kinetics of [3H]MTX in thepresence of raltitrexed, as measured byKi (Table 2).

In Situ MTX Polyglutamate Formation. After incuba-tion of CCRF-CEM and CEM:RTOM cells with 1mM [3H]MTXfor 24 h, the levels of intracellular MTX and the formation ofMTX polyglutamates were measured by HPLC (Fig. 2). CCRF-CEM cells formed MTX polyglutamates with up to five gluta-mate residues. Mean levels of individual polyglutamate metab-olites in CCEF-CEM cells ranged from 2776 59 pmol/109 cells(diglutamate) to 7456 144 pmol/109 cells (pentaglutamate),with intracellular MTX monoglutamate present at 2756 120pmol/109 cells. In marked contrast, CEM:RTOM cells formed nodetectable MTX polyglutamates (,15 pmol/109 cells) on expo-sure to 1mM [3H]MTX for 24 h. However, the parent MTXmonoglutamate was readily detected (6976 401 pmol/109

Fig. 1 Growth inhibition of CCRF-CEM (f) and CCRF-CEM:RC2Tomudex(Œ) cells exposed to raltitrexed for 96 h. Each point on thegraph represents the mean6 SD of data from three individual experi-ments;bars,SD.

Table 1 Growth-inhibitory activity of antifolates against CCRF-CEM and CCRF-CEM:RC2Tomudexcellsa

Drug exposure (time)

IC50 (nM) (mean6 SD)

Fold-difference PCCRF-CEM CCRF-CEM:RC2Tomudex

Raltitrexed (96 h) 3.06 0.9 3,8766 600 1,292 0.0004Raltitrexed (6 h) 526 26 .200,000 .3846Methotrexate (96 h) 6.06 0.9 3.06 0.2 0.5 0.005Methotrexate (24 h) 426 9.0 776 26 1.8 0.09Methotrexate (6 h) 4966 40 4,5796 788 9 0.001Lometrexol (96 h) 9.06 4.0 576 15 6.3 0.006Lometrexol (6 h) 15,5386 4,356 .200,000 .13Nolatrexed (96 h) 1,0006 170 7876 34 0.8 0.1Nolatrexed (6 h) .175,000 .175,000

a The results are expressed as the mean6 SD of at least three individual experiments.

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cells), at a level 2.5-fold higher than that detected in CCRF-CEM cells (Table 3).

After a 24-h exposure to 10mM [3H]MTX, CCRF-CEMcells again formed MTX polyglutamates up to the pentagluta-mate metabolite. Mean levels of individual polyglutamates fromthree experiments ranged from 17616 536 pmol/109 cells(diglutamate) to 26236 176 pmol/109 cells (pentaglutamate),with intracellular MTX monoglutamate present at 32566 1104pmol/109 cells. The only metabolite detected in CEM:RTOM

cells was MTX diglutamate, with a mean level of only 92pmol/109 cells. The mean level of MTX diglutamate in CEM:RTOM cells was thus 19-fold lower than that formed in CCRF-CEM cells. Intracellular MTX monoglutamate was also de-

tected, with a mean level of 13506 171 pmol/109 cells inCEM:RTOM cells,;2-fold lower than the mean level measuredin CCRF-CEM cells (Table 3).

FPGS Activity, FPGS Protein, and FPGS mRNA inCCRF-CEM and CEM:R TOM Cell Lines. The FPGS activ-ity in extracts prepared from the two cell lines is shown in Table2. HPLC analysis revealed that in the CCRF-CEM cell line, allof the incorporated [3H]glutamate was present as MTX diglu-tamate with no detectable MTX tri-, tetra-, or pentaglutamate. Indirect contrast, FPGS activity in extracts of CEM:RTOM cellscould not be detected. Thus, CEM:RTOM cells were estimated tohave ,11% of the FPGS activity of CCRF-CEM cells. Thisreduction in FPGS activity was associated with a decrease in

Table 2 TS activity, [3H]MTX transport kinetics, and FPGS activity in CCRF-CEM and CCRF-CEM:RC2Tomudexcellsa

CCRF-CEM CCRF-CEM:RC2Tomudex P

TS activity (nmol dUMP/106 cells/h) 2.36 0.6 3.36 0.6 0.106[3H]MTX-Transport kinetics

MTX Kt (mM) 7.96 1.7 7.16 1.0 0.52Vmax (pmol/106 cells) 12.66 1.2 9.96 0.6 0.025RaltitrexedKi (mM) 0.86 0.4 1.26 0.6 0.33

FPGS activity (pmol of [3H]glutamate incorporated/h/mg protein) 1476 14 ,16 ,0.0001a The results represent the mean6 SD of three separate experiments.

Fig. 2 HPLC radiochromatogram illustratingMTX and MTX polyglutamate metabolites inCCRF-CEM and CCRF-CEM: RC2Tomudex cellextracts after exposure of the cells to 1mM

[3H]MTX for 24 h.

Table 3 MTX and MTX polyglutamate levels in CCRF-CEM and CCRF-CEM:RC2Tomudex(CEM:RTOM) cells after exposure to 1mM or 10 mM

[3H]MTX for 24 ha

Methotrexatepolyglutamate

Methotrexate and methotrexate polyglutamate levels (pmol/109 cells)

1 mM [3H]methotrexate 10 mM [3H]methotrexate

CCRF-CEM CEM:RC2 CCRF-CEM CEM:RC2

Pentaglutamate 7456 144 NDb 26236 176 NDTetraglutamate 6196 179 ND 25446 545 NDTriglutamate 5506 142 ND 23336 370 NDDiglutamate 2776 59 ND 17616 536 92 (76,108)Monoglutamate 2756 120 6976 401 32566 1104 13506 171

a The results are expressed as the mean6 SD of three separate experiments.b ND, not detectable (,15 pmol/109 cells).

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FPGS protein expression, with the CEM:RTOM cells expressingapproximately one-third of the FPGS protein levels of theCCRF-CEM cell line (Fig. 3).

FPGS mRNA levels were measured (Fig. 4) and expressedas a ratio of FPGS mRNA to 18S rRNA. There was no signif-icant difference (P 5 0.11) between the FPGS mRNA:18SrRNA ratios for CCRF-CEM and CEM:RTOM with mean (6SD, n 5 3) ratios of 0.0246 0.001 and 0.0306 0.005,respectively.

Intracellular Levels of Total Raltitrexed-derived Mate-rial in CCRF-CEM and CEM:R TOM Cell Lines. As meas-ured by an immunoassay that detects raltitrexed and raltitrexed-polyglutamates with equal sensitivity, CCRF-CEM cellsaccumulated 30-fold higher levels of intracellular raltitrexed-derived material when compared with CEM:RTOM cells, with(mean6 SD) levels of 1146 21 pmol/mg proteinversus4 62 pmol/mg protein, respectively, after a 4-h exposure to 0.1mM

drug (Fig. 5). Similarly, after a 24-h continuous exposure to 0.1mM raltitrexed, CCRF-CEM cells accumulated 50-fold moreintracellular raltitrexed-derived material than CEM:RTOM cells,with levels of 3466 52 pmol/mg protein and 76 2 pmol/mgprotein, respectively. When cells were exposed to 0.1mM ralti-trexed for 4 h, followed by incubation for a further 20 h in drugfree medium, CCRF-CEM cells accumulated and retained.30-

fold more intracellular raltitrexed-derived material than CEM:RTOM cells with mean levels of 756 5 pmol/mg proteinversus2 pmol/mg protein, respectively.

Effect of Raltitrexed and Methotrexate on Inhibition ofin Situ TS in CCRF-CEM and CEM:R TOM Cell Lines. TSactivity in untreated control CCRF-CEM cells and CEM:RTOM

cells was similar, with [3H]2O release rates of 401,3006 36,360dpm/106 (CCRF-CEM) and 334,5006 79,440 dpm/106 (CEM:RTOM).

In contrast to control rates, the two cell lines studiedshowed a striking difference in sensitivity to raltitrexed-inducedinhibition of TS activity (Fig. 6). After exposure to 10 nM

raltitrexed, TS activity was inhibited by.90% in CCRF-CEMcells compared with,5% inhibition in CEM:RTOM cells. More-over, even after exposure of CEM:RTOM cells to 1000 nMraltitrexed, TS activity was not significantly inhibited with re-

Fig. 3 FPGS protein levels as detected by Western blot analysis.Lanes1–3, CCRF-CEM: RC2Tomudex; Lanes 4–6,CCRF-CEM; Lane 7,CEM/C1 crude (a CCRF-CEM cell line in use in the laboratories ofJ. J. M.);Lane 8,CEM/C1 (pure) (a partially purified FPGS extract fromthe CEM/C1 cell line). All lanes were loaded with 20mg of total cellularprotein, exceptLane 8,for which 0.2mg of protein was loaded.

Fig. 4 FPGS mRNA levels in CCRF-CEM and CCRF-CEM:RC2Tomudexcell lines as measured by Northern blot hybridization.

Fig. 5 Total intracellular levels of raltitrexed and raltitrexed polyglu-tamates in CCRF-CEM (p) and CCRF-CEM: RC2Tomudex(f) cells afterexposure to 0.1mM drug. Eachcolumn represents the mean of threeindividual experiments;bars, SD. 4 hr, total intracellular raltitrexedmeasured after a 4-h exposure.24 hr, total intracellular raltitrexedmeasured after a 24-h exposure.4 hr/24 hr,total intracellular raltitrexedmeasured after a 4-h exposure with 20 h in drug-free medium.

Fig. 6 In situ TS activity in CCRF-CEM (squares) and CCRF-CEM:RC2Tomudex(triangles) cells after incubation with MTX (open symbols)or raltitrexed (closed symbols) for 22 h. Each point represents the meanof three individual experiments with data expressed as a percentage ofactivity in control (untreated) cells;bars,SD.

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spect to the untreated controls. In contrast to the effect of 10 nM

raltitrexed, there was no significant difference (P 5 0.18) be-tween TS inhibition in the two cell lines after exposure to 10 nM

MTX, i.e., 35% compared with 61% in CCRF-CEM and CEM:RTOM cells, respectively. However, on exposure to 1000 nM

MTX, residual TS activity in CEM:RTOM cells was significantlyhigher (10% of control) than in CCRF-CEM cells, where activ-ity was undetectable at this MTX concentration (,0.2% control;P 5 0.02).

Effect of Raltitrexed and Methotrexate on de NovoPurine Synthesis in CCRF-CEM and CEM:RTOM Cells.The mean rates ofde novopurine synthesis in untreated controlcells were found to be significantly higher in CEM:RTOM cellsthan in CCRF-CEM cells,i.e., 22 6 2.5 versus 13 6 2.7pmol/nmol preexisting adenine bases/h, respectively (P 50.016).

CCRF-CEM and CEM:RTOM cells showed different pat-terns of sensitivity to both raltitrexed- and MTX-induced inhi-bition of de novopurine synthesis (Fig. 7). In CCRF-CEM cells,de novopurine synthesis was inhibited by increasing concen-trations of raltitrexed. Exposure to 10 nM raltitrexed resulted in15% inhibition ofde novopurine synthesis, which increased to30–50% inhibition after exposure to 1000 nM raltitrexed (P50.0006). In contrast, CEM:RTOM cells showed no evidence ofinhibition of de novopurine synthesis, even after exposure to1000 nM drug.

CCRF-CEM and CEM:RTOM cells also showed differentpatterns of inhibition ofde novopurine synthesis in response toMTX treatment. Exposure of CCRF-CEM cells to MTX onlyproduced inhibition ofde novopurine synthesis after exposureto 1000 nM MTX. In contrast, de novopurine synthesis inCEM:RTOM cells was stimulated by MTX, at 10 and 100 nM, to220% at the latter concentration (P 5 0.002). Even after expo-sure to 1000 nM MTX, a concentration that induced completeinhibition in CCRF-CEM cells, only limited inhibition ofdenovopurine synthesis (20%) was observed in CEM:RTOM cells(P 5 0.03).

DISCUSSIONThe aim of this study was to investigate the influence of

FPGS activity on the sensitivity of two CCRF-CEM cell lines toshort-term and continuous exposures to a range of antifolates. Inaddition, the effect of FPGS activity on raltitrexed- and MTX-induced inhibition ofde novothymidylate and purine biosyn-thesis was studied and related to the antifolate sensitivity of thecell lines. The CCRF-CEM:RC2Tomudex(CEM:RTOM) cell linestudied here was cloned from a CCRF-CEM cell line found tobe insensitive to the antifolate raltitrexed when compared with aCCRF-CEM cell line obtained from the European Collection ofAnimal Cell Cultures. The CEM:RTOM cell line was not knownto have been exposed to any antifolate for any period of timeprior to use in these studies.

Because multifactorial resistance to antifolates had beendemonstrated previously (29, 30), initial studies focused on thecharacterization of the potential mechanism(s) of resistance toraltitrexed in the CEM:RTOM cell line. No significant differencewas found between CEM:RTOM and CCRF-CEM cells for TSactivity in crude cell extracts or cellular [3H]MTX transportkinetics, the latter being used as a surrogate marker for ralti-trexed transport, which, like MTX, uses the reduced folatecarrier (8). However, evidence to support reduced polyglutama-tion as the underlying mechanism of raltitrexed resistance inCEM:RTOM cells came from measurements of the formation ofMTX polyglutamatesin situ, levels of total intracellular ralti-trexed-derived material, and FPGS activity in crude cell ex-tracts.

In contrast to CCRF-CEM cells, CEM:RTOM cells formedno detectable polyglutamates after exposure to 1mM [3H]MTXand only very low levels of MTX diglutamate after exposure to10 mM [3H]MTX. In addition, CCRF-CEM cells were able toaccumulate, and retain, significantly higher concentrations ofraltitrexed-derived material than CEM:RTOM cells, indicatingthat defective polyglutamation was not specific to methotrexate.The finding of reduced FPGS activity and protein expression inCEM:RTOM cells, without a reduction in FPGS mRNA expres-sion, suggests that reduced FPGS activity is the result of aposttranscriptional event. Similar observations have been madein antifolate resistant CCRF-CEM cell clones (40) and L1210variants (41), with reduced FPGS activity. Indeed, for L1210cells, a novel posttranscriptional mechanism of antifolate resist-ance was identified, resulting from a mutationally determineddifference in the secondary structure of mRNA, which preventsthe progression of translation of FPGS protein (42).

In the FPGS-proficient CCRF-CEM cell line, exposure toincreasing concentrations of MTX resulted in the completeinhibition of in situ TS activity at concentrations$100 nM,whereas a higher (1000 nM) concentration of MTX was neededto inhibit de novopurine synthesis. In the FPGS-deficient CEM:RTOM cell line, TS activity was not completely inhibited by even1000 nM MTX, and there was stimulation of, rather than inhi-bition of, de novopurine synthesis. These effects of MTX maybe explained by the cellular pharmacology of the drug, which isknown to directly or indirectly affect at least three folate-dependent enzymes within the cell,i.e., TS (10), AICARformyltransferase, an enzyme required forde novopurine syn-thesis (43), and DHFR (44).

Fig. 7 De novopurine synthesis in CCRF-CEM (squares) and CCRF-CEM: RC2Tomudex (triangles) cells after incubation with MTX (opensymbols)/raltitrexed (closed symbols) for 22 h. Each point represents themean of three individual experiments with data expressed as a percent-age of the rate of adenine synthesis in control cells;bars,SD.

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MTX-mediated TS inhibition can be the result of twoeffects: direct TS inhibition by MTX polyglutamates, and indi-rect inhibition after DHFR inhibition-mediated depletion of5,10-methylene tetrahydrofolate pools. In the studies describedhere, inhibition of whole-cell TS activity at 10 nM MTX wassimilar in the CCRF-CEM and CEM:RTOM cell lines, presum-ably due to a direct effect on DHFR, which is not influenced bypolyglutamation (45). At higher MTX concentrations (100 and1000 nM), the complete inhibition of TS observed in CCRF-CEM cells is consistent with an effect due to MTX polygluta-mates acting directly on TS, because these metabolites were notformed in CEM:RTOM cells, and complete TS inhibition was notobserved.

The relative effects of MTX on purine biosynthesis in thetwo cell lines can also be explained, given the cellular pharma-cology of the drug. The inhibition ofde novopurine biosynthe-sis in CCRF-CEM cells at 1000 nM MTX, but not 10 or 100 nM,suggests that inhibition is due to a direct effect of MTX poly-glutamates on AICAR formyltransferase. The alternative mech-anism, depletion of 10-formyltetrahydrofolate cofactor poolssecondary to DHFR inhibition, can only operate if TS activity ismaintained, which was not the case in CCRF-CEM cells ex-posed to 1000 nM MTX. The data for CEM:RTOM cells are againconsistent with inhibition ofde novopurine biosynthesis beingdue to a direct effect of MTX polyglutamates, because thesemetabolites were not formed in this cell line, and purine bio-synthesis was not markedly inhibited at MTX concentrations upto 1000 nM.

The reason for the apparent stimulation ofde novopurinebiosynthesis in the CEM:RTOM cells at 10 and 100 nM MTX, butnot to any marked extent in the CCRF-CEM cells, may relate tothe availability of PRPP. Low concentrations (20 nM) of MTXhave been shown to elevate PRPP levels in a number of celllines, including Molt-4 human leukemia cells (46) and HCT-8colorectal (47) cell lines. If PRPP levels are rate limiting fordenovopurine synthesis inhibition, and in the absence of AICARformyltransferase inhibition due to a lack of MTX polygluta-mate formation, stimulation of purine biosynthesis could beobserved.

In contrast to the findings with MTX, neitherde novothymidylate or purine biosynthesis was affected by exposure toraltitrexed in CEM:RTOM cells. Because the potency of ralti-trexed as a specific inhibitor of TS is greatly enhanced bypolyglutamation (9) and the intracellular accumulation of theraltitrexed-derived material in the CEM:RTOM cell line wasshown to be markedly reduced in the present study, the lack ofTS inhibition seen is not unexpected. However, the lack ofinhibition of TS is consistent with the requirement for concen-trations above this level to produce cell growth inhibition oncontinuous 96-h exposure (Fig. 1). Together, these data suggestthat the lack of TS inhibition in CEM:RTOM cells after ralti-trexed treatment is due to inadequate polyglutamation.

Although raltitrexed had no effect onde novopurine syn-thesis in the CEM:RTOM cell line, a dose-dependent decrease inde novopurine synthesis was observed in CCRF-CEM cells. Itis possible that in CCRF-CEM cells, there was direct inhibitionof DHFR by raltitrexed (8), an effect that was not observed inthe CEM:RTOM cells because of the much lower accumulationof raltitrexed-derived material (Fig. 5).

The differing cellular biochemical effects of MTX andraltitrexed also offer an explanation for the relative sensitivitiesof CCRF-CEM and CEM:RTOM cells to long- and short-termexposure to a range of antifolates. When exposed to antifolatescontinuously for 96 h, CEM:RTOM cells retained sensitivity toMTX. This finding, which is in keeping with previous reports ofMTX sensitivity in FPGS-deficient human leukemia cell lines(25, 48), can be explained by the reduced TS activity observedin CEM:RTOM cells at higher MTX concentrations. The largedifference in raltitrexed-mediated growth inhibition is also con-sistent with studies in FPGS-deficient human (25) and murine(14) leukemia cell lines. Like raltitrexed, the potency of lome-trexol, a classical antifolate inhibitor of glycinamide ribonucle-otide formyltransferase, is also markedly enhanced by polyglu-tamation (49). However, the CEM:RTOM cell line remainedrelatively sensitive to the growth-inhibitory effects of continu-ous exposure to lometrexol when compared with raltitrexed, aneffect that has also been reported for FPGS-deficient L1210cells (14). This apparent discrepancy between raltitrexed andlometrexol sensitivities may represent a selective effect of re-sidual FPGS activity, because FPGS deficiency has been shownto be associated with differential effects on FPGS substratespecificity with a variety of physiological folates and antifolatedrugs (20).

After short-term exposure to antifolates, both CCRF-CEMand CEM:RTOM cells were insensitive to the growth-inhibitoryeffects of nolatrexed, a nonclassical antifolate TS inhibitor. Inaddition, FPGS deficiency rendered the CEM:RTOM cell lineinsensitive to growth inhibition mediated by short-term expo-sure to raltitrexed, which is in keeping with the markedly loweraccumulation and retention of raltitrexed in this cell line. Theobserved insensitivity of the CEM:RTOM cell line to short-termexposure to lometrexol is also consistent with the presumablyreduced polyglutamation of, and hence intracellular retention of,the drug. In contrast, MTX-mediated growth inhibition of theCEM:RTOM cells was relatively well preserved after short-termexposure, which may reflect the equal affinity of MTX andMTX polyglutamates as tight binding inhibitors of DHFR (44).In the FPGS-proficient CCRF-CEM cell line, the greatergrowth-inhibitory activity after short-term exposure of ralti-trexed when compared with lometrexol may reflect their differ-ent affinities for FPGS (50, 51).

In conclusion, the studies presented herein demonstrate thatpolyglutamation is a critical determinant of the sensitivity ofCCRF-CEM cells to growth inhibition mediated by raltitrexed.Cell growth inhibition data were in keeping with the influenceof reduced polyglutamation onde novopurine and thymidylatesynthesis. In contrast, the influence of reduced polyglutamationwas less pronounced in the case MTX, where growth-inhibitoryactivity was largely preserved after short-term exposure. How-ever, polyglutamation was found to influence the impact ofMTX on thymidylateversus de novopurine biosynthesis. Futurework will examine the effect of more recently developed anti-folates onde novothymidylate and purine biosynthesis and theimpact of various resistance mechanisms on their cellular phar-macology. In addition, the relationship between MTX polyglu-tamation and inhibition ofde novo thymidylate and purinebiosynthesis in lymphoblasts obtained from patients with child-hood acute lymphoblastic leukemia will be investigated.

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ACKNOWLEDGMENTSWe acknowledge Professor R. Moran (Massey Cancer Center,

Richmond, VA) for supplying the pX1 plasmid used in the study ofFPGS mRNA and Dr. W. E. Evans (St. Jude Childrens ResearchHospital, Memphis, TN) and his research group for detailed informationand discussions on the method for measuringde novopurine synthesis.We thank Cynthia Russell for performing the Western blot analysis ofFPGS protein expression.

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2558Impact of Polyglutamation on Sensitivity to Antifolates

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1999;5:2548-2558. Clin Cancer Res   Matthew J. Barnes, Edward J. Estlin, Gordon A. Taylor, et al.   Thymidylate and Purine Biosynthesis in CCRF-CEM Cell Lines

de NovoMethotrexate in Relation to Drug-induced Inhibition of Impact of Polyglutamation on Sensitivity to Raltitrexed and

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