The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs.

CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 μm [3H]MTX for 24 h. After exposure to 0.1 μm raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM:RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX 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. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nm MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nm MTX. Ten nm raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nm raltitrexed.

These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.

Antitumor antifolates can be classified by their loci of action, e.g., as inhibitors of TS,3 DHFR or glycinamide ribonucleotide transformylase, and on the basis of the presence (classical antifolates) or absence (nonclassical antifolates) of a glutamate moiety. The α and γ glutamate carboxyl groups of classical antifolates, such as raltitrexed (an inhibitor of TS) and MTX, are negatively charged at physiological pH, and thus classical antifolates require carrier-mediated uptake for cell entry (1, 2). Once inside the cell, classical antifolates, as well naturally occurring folates, can undergo polyglutamation in a reaction that involves the addition of additional glutamate residues at the γ-carboxyl position of the glutamate moiety. Polyglutamation, catalyzed by the enzyme FPGS (3), has been shown to be an important determinant of the sensitivity of cells to classical antifolates (4). As a result of polyglutamation, intracellular drug levels can exceed the extracellular concentrations (5) and can maintain inhibition of target enzymes after removal of extracellular drug (6, 7). In addition, polyglutamation can enhance the affinity of classical antifolates for certain folate-dependent enzymes (8, 9, 10). In contrast to classical antifolates, the nonclassical agents trimetrexate (11), piritrexim (12), and nolatrexed (13) do not require carrier-mediated cellular uptake and are not substrates for FPGS. As a result, nonclassical antifolates are not retained within the cell on removal of extracellular drug and cannot be converted to metabolites with enhanced affinities for their target enzyme.

Resistance to antifolates can result from one or more mechanisms including decreased cellular uptake of the drug (14, 15), reduced polyglutamation (see below), overproduction of the target enzyme (14, 16), expression of a mutant form of the target enzyme (17, 18), or a failure of the cell to appropriately engage apoptosis (19).

Reduced polyglutamation has been described previously as a mechanism of inherent and acquired resistance to classical antifolates, both in vitro(14, 20) and in vivo(21, 22). The underlying reason for resistance is either reduced FPGS activity (14, 20) or enhanced activity of folylpolyglutamate hydrolase (23), the enzyme that catalyzes the hydrolysis of antifolate polyglutamate metabolites. The impact of polyglutamation-related 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, which in vitro leads 2to resistance to short-term exposures with MTX, can be overcome with more prolonged drug exposure (20, 24). However, for certain other classical antifolates, such as raltitrexed, deficient polyglutamation renders cell lines resistant to both short and longer term drug exposure (14, 25) Indeed, antifolate polyglutamation and FPGS activity have been shown to be important determinants of both MTX cytotoxicity in vitro(26, 27) and outcome in clinical studies (4, 27).

Work to characterize the sensitivity of a panel of human leukemic 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 antifolates can be multifactorial (29, 30), full characterization of the mechanism of resistance was undertaken and found to be related to reduced FPGS activity. The aim of the studies described here was to investigate the impact of reduced FPGS activity on cellular sensitivity to a range of antifolates after short term and continuous exposure and, in the case of raltitrexed and MTX, the impact of impaired polyglutamation on the inhibition of de novo thymidylate and purine biosynthesis.

Maintenance of Cell Cultures.

CCRF-CEM (European Collection of Animal Cell Cultures, Salisbury, United Kingdom) and CCRF-CEM:RC2Tomudex human leukemic cell lines were grown as suspension cultures in RPMI 1640 (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 2 mml-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, maintained in an incubator with 5% CO2 and a humidified atmosphere at 37°C, and shown to be Mycoplasma negative at regular intervals.

The CCRF-CEM:RC2Tomudex cell line was derived from a CCRF-CEM cell line in routine use in the Cancer Research Unit, University of Newcastle, United Kingdom. The CCRF-CEM:RC2Tomudex cell line was found to be relatively insensitive to the antifolate raltitrexed when compared with a CCRF-CEM cell line obtained from the European Collection of Animal Cell Cultures and was not known to have been exposed to any antifolate for any period of time prior to use in these studies. The original raltitrexed-resistant CCRF-CEM cell line was cloned by seeding a cell suspension at a concentration of <1 cell/well into a 96-well plate (Nunc, Life Technologies, Paisley, United Kingdom), and after ∼4 weeks, the cell line was routinely subcultured as above. The resulting clone, CCRF-CEM:RC2Tomudex (CEM:RTOM), was characterized by karyotype and immunophenotype and found to be indistinguishable from the CCRF-CEM cell line obtained from the European Collection of Animal Cell Cultures.

Cell Growth Inhibition Studies.

All antifolates, i.e., MTX (Sigma Chemical Co.), nolatrexed (Thymitaq or AG337; Agouron Pharmaceuticals, San Diego, CA), raltitrexed (Tomudex or ZD1694; Zeneca Pharmaceuticals, Macclesfield, United Kingdom), and lometrexol (DDATHF; Eli Lilly, Indianapolis, U.S.A.) were dissolved in water at 1 mg/ml. Stock solutions were diluted further in dialyzed RPMI 1640 to the final concentration required.

For continuous exposure studies, 100-μl aliquots of cell suspensions at 5 × 104 cells/ml were seeded into each well of a 96-well plate 24 h before drug exposure. One hundred μl of medium containing drug at twice the concentration required were added to quadruplicate wells and incubated for 96 h, during which time approximately three cell doublings would have occurred in the control (untreated) wells. At the end of the exposure time, the number of cells in each suspension was counted on a Coulter cell counter.

In short-term exposure experiments, 2.5-ml aliquots of cell suspensions at 5 × 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 the concentration required were added to triplicate tubes and incubated at 37°C. After 6 or 24 h exposure to the drug, the cells were centrifuged at 500 × g for 5 min at room temperature. The drug-containing medium was aspirated, and cells were washed with 5 ml of drug-free medium and then resuspended in 5 ml of drug-free medium, followed by incubation for an additional 90 or 72 h (96 h total incubation time). At the end of the incubation time, cells were counted as above.

To determine the IC50s, cell counts for each concentration of drug were divided by the mean control cell count and multiplied by 100 to give values as a percentage of the control growth. The IC50, i.e., the concentration of drug required to inhibit cell growth by 50%, was calculated by fitting the Hill equation to the data using unweighted, nonlinear least squares regression analysis (GraphPad Prism, San Diego, CA).

TS Activity in Cell Sonicates.

TS activity in exponentially growing cell lines was measured in cell sonicates by the release of [3H]2O from 5-[3H]dUMP using the method described previously by Estlin et al.(31).

Cellular Methotrexate Transport Kinetics.

A total of 1–2 × 107 exponentially growing cells were resuspended in 2.5 ml of transport buffer (32) and equilibrated for 5 min at 37°C. Two hundred μl of the cell suspension were then added to 200 μl of [3H]MTX at twice the concentration required to give final concentrations ranging from 0.2 μm (specific activity, 910 kBq/mmol) to 18 μm (specific activity, 10 kBq/mmol), and the cell suspension was incubated for an additional 5 min at 37°C. After incubation, triplicate 100-μl aliquots of each cell suspension were then centrifuged at 6700 × g for 1 min through a 200-μl layer of Dow Corning silicone oil [final specific gravity, 1.028; 45 ml of Dow Corning 556 silicone oil (specific gravity, 0.98) + 55 ml of Dow Corning 550 silicone oil (specific gravity, 1.068)] into 50-μl 3 M potassium hydroxide (BDH, Dorset, United Kingdom; Ref. 33). After 1 h, the tubes were cut in the oil layer, and the potassium hydroxide containing lysed cells were neutralized with 250 μl of 1 m acetic acid (Sigma Chemical Co., Poole, Dorset, United Kingdom). Samples were then counted on a scintillation counter after addition of 10 ml of Optiphase scintillant (Fisons Chemicals, Loughborough, United Kingdom). Parallel experiments using [14C]sucrose (23 kBq/mmol; Amersham, Buckinghamshire, United Kingdom) were carried out to compensate for any [3H]MTX that passed through the oil layer due to trapping in the extracellular space around cells.

Uptake of [3H]MTX (pmol/106 cells) was plotted against the extracellular concentration of MTX (μm), and a one-site binding hyperbolic equation was fitted to the data using unweighted, nonlinear least squares regression (GraphPad Prism). The Ktm), i.e., the concentration of MTX required to achieve half the maximal uptake rate over a 5-min period, was calculated and expressed as pmol/106 cells/min, as was the Tmax, the maximum rate of uptake.

The uptake of MTX in the presence of raltitrexed was also studied as described above with the exception that a constant concentration of 0.5 μm [3H]MTX (specific activity, 364 kBq/mmol) was used with a range of raltitrexed concentrations (0–10 μm). Also included in the experiment as a control were incubations of 0.5 μm [3H]MTX (specific activity, 364 kBq/mmol) with a range of MTX concentrations (0–10 μm).

Uptake of 0.5 μm [3H]MTX (dpm) was plotted against the extracellular raltitrexed or MTX concentration (μm), and a one-site binding competition equation was fitted to the data (GraphPad Prism). The EC50 generated from this equation and the Kt value generated from the experiments described above were used to calculate the Ki for inhibition of [3H]MTX uptake by MTX or raltitrexed from the equation of Cheng and Prusoff (34).

Cellular Methotrexate Polyglutamate Formation.

The method used was based on that of Whitehead et al.(27). A total of 4 × 106 exponentially growing cells was incubated in 5 ml of dialyzed medium supplemented with 5 μm thymidine, 10 μm inosine (Aldrich Chemical Co., Milwaukee, WI), and 1 or 10 μm 3′,5′-7′- [3H]MTX (specific activity, 129.5 kBq/mmol; Moravek Biochemicals, Brea, CA) for 24 h at 37°C. After the 24-h incubation, the cell suspension was centrifuged at 120 × g for 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 300 μl of ice-cold 0.2 m perchloric acid (Sigma). Samples were vortexed briefly and left on ice for 5 min before centrifugation at 6700 × g for 2 min. The resultant supernatant was removed and pipetted directly onto ∼200 mg of potassium bicarbonate (Sigma) and left to stand on ice for 2 min. The neutralized cell extract was again centrifuged at 6700 × g for 2 min to remove any remaining potassium bicarbonate and the potassium perchlorate that had formed, and the final supernatant was removed and stored at −20°C.

HPLC analysis of MTX polyglutamate formation involved the separation of MTX and MTX polyglutamates on a 100 × 4.6-mm Nucleosil 3 μm ODS cartridge column (Jones Chromatography, Mid-Glamorgan, United Kingdom) with UV absorbance of standard MTX and MTX polyglutamates (Schirks, Jona, Switzerland) being measured at 254 and 304 nm. One hundred-μl aliquots, prepared as described above, were injected onto the column via a 50 × 2-mm pellicular ODS silica precolumn (Whatman, Maidstone, Kent, United Kingdom), and [3H]MTX and [3H]MTX polyglutamates were detected and quantitated using an on-line FLO-ONE radiochromatography detector (Packard, Meriden, CT) with a liquid scintillant cell [flow rate of liquid scintillant (Packard) 1.5 ml/min]. The mobile phase used to separate MTX and MTX polyglutamates consisted of 0.1 m ammonium acetate (ammonia and glacial acetic acid; BDH) pH 4.7 + 6% (w/w) acetonitrile at a flow rate of 0.5 ml/min with a 30-min analysis time. [3H]MTX and [3H]MTX polyglutamates were quantitated using a standard curve constructed using [3H]MTX, and results were expressed as pmol of MTX or MTX polyglutamate/109 cells.

Measurement of FPGS Activity in Cell Extracts.

The method used was based on that of Jansen et al.(35). Exponentially growing cell lines (1–2 × 107) were resuspended in 500 μl of ice-cold extraction buffer, 50 mm Tris-HCl, 20 mm potassium chloride, 10 mm magnesium chloride, and 5 mm DTT at pH 7.6 (Sigma; Ref. 29). Cell suspensions on ice were then sonicated three times at 10 μm for 10 s, with 10-s intervals between sonic bursts. The cell sonicates were then ultracentrifuged at 50,000 × g for 30 min at 4°C, and the supernatant containing FPGS protein was removed and kept on ice.

One hundred μl of the above supernatant were added to the reaction 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) at pH 8.4; all concentrations were the final working values. In addition, the reaction mixture included 250 μm MTX, or an equivalent 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 protein extract was 250 μl, which was incubated at 37°C for 2 h.

The reaction was terminated by adding 1 ml of ice-cold 5 mm sodium glutamate (Sigma Chemical Co.) and placing on ice. Samples were then applied to prewashed (10 ml of methanol), preequilibrated [10 ml of 0.2 m sodium acetate (Sigma Chemical Co.), pH 5.5] Bond Elut LRC bonded phase-C18 columns (Varian, Surrey, United Kingdom); all elutions and washes were aided by a VAC ELUT SPS 24 vacuum manifold (Analytichem International, Bedfordshire, United Kingdom). After the samples had progressed through, the column was washed with 10, 2.5, and then 3 ml 0.2 m sodium acetate (pH 5.5). Compounds retained on the column were eluted with 3 ml of methanol, which were subsequently dried down under nitrogen, and residues were kept at 4°C until analysis.

HPLC analysis followed the method described above for the measurement of whole-cell MTX polyglutamation. [3H]MTX polyglutamates were quantitated on the basis of the specific activity of the [3H]glutamate cosubstrate (0.27 kBq/mmol) used in the initial incubation, and the results were expressed as pmol of MTX polyglutamates/hour/mg of total protein.

Measurement of FPGS Protein.

A total of 1–2 × 107 exponentially growing CCRF-CEM and CEM:RTOM cells were harvested by centrifugation at 500 × g. To the resulting cell pellet, 150 μl 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 30 min, followed by centrifugation at 10,000 × g to remove cell debris. The resulting supernatant was removed and stored at −80°C before analysis.

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

Measurement of FPGS mRNA.

FPGS mRNA expression was measured by Northern blot hybridization in exponentially growing cell lines, using a 700-bp fragment of the 5′ coding sequence excised from the pX1 plasmid, which contains a 1–2-kb FPGS genomic DNA insert. Northern blot hybridization was performed as described previously (31), and FPGS mRNA levels were expressed relative to 18S rRNA. The pX1 plasmid used in these studies was kindly provided by Professor R. Moran (Massey Medical Center, Richmond, VA).

Determination of Total Intracellular Levels of Raltitrexed-derived Material.

Exponentially growing cell lines were treated with 0.1 μm raltitrexed for 4, 24, or for 4 h with further incubation in drug-free medium for 20 h. Directly after these exposure times, 2–5 × 106 cells in each case were harvested, resuspended in 1 ml of PBS, and stored at −80°C until they were analyzed.

Samples were thawed and sonicated on ice, three times for 30 s, and centrifuged, and the supernatant was removed. Total intracellular levels of raltitrexed-derived material were measured in the resultant crude cell sonicates using a previously described radioimmunoassay, which detects raltitrexed and raltitrexed polyglutamates with equal sensitivity (37).

Drug-induced Inhibition of de Novo Thymidylate and Purine Biosynthesis.

The following method was used to measure the inhibition of in situ TS activity and de novo purine synthesis induced by exposure to raltitrexed or MTX and was based on a combination of the methods Taylor et al.(38) and Masson et al.(39). Estimations of in situ TS activity involved the exposure of cells to 5′-[3H]deoxyuridine which is transported and subsequently phosphorylated in the cell by thymidine kinase to 5′-[3H]dUMP. TS catalyzes the methylation of the [3H]dUMP at the 5′ position, resulting in the release of the tritium atom as [3H]2O, which can be used as a measure of the TS activity. The de novo purine synthesis assay involves exposure of cells to [14C]formate and subsequent incorporation into the purine ring structure via 10-[14C]formyl tetrahydrofolate, which allows analysis and quantification of purine bases by HPLC with radiochemical detection.

Exponentially growing cells (5 × 106) were resuspended in 2 ml of dialyzed medium supplemented with 5 μm thymidine (Sigma) and 10 μm inosine (Aldrich). The cell suspension was then treated with MTX or raltitrexed at concentrations ranging from 0 to 1000 nm, and the cells were incubated at 37°C for 22 h. At the end of this period, samples were centrifuged (120 × g for 5 min), washed, and resuspended in 2 ml of fresh, drug-free medium (without thymidine or inosine) and incubated for an additional 30 min at 37°C. After the 30-min period, the cell suspensions were added with a mixture of purified [3H]deoxyuridine (5′-[3H] 2′-deoxyuridine; Moravek Biochemicals; deoxyuridine, Sigma Chemical Co.), and [14C]formic acid (14C formic acid sodium salt, Amersham, Buckinghamshire, United Kingdom; sodium formate, Sigma Chemical Co.) at final concentrations of 0.3 μm [3H]deoxyuridine (specific activity, 227 kBq/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 extraction, as described below.

Estimation of in Situ TS Activity.

The supernatant from the above centrifugation step, containing the [3H]2O released from the in situ activity of TS, was removed as three 600-μl aliquots, each of which was pipetted onto 600 μl of ice-cold 1 m perchloric acid (BDH). The samples were placed on ice for 15 min, after which 750 μl of activated charcoal suspension (200 mg/ml suspended in H2O; Sigma Chemical Co.) in dextran (10 mg/ml in H2O; Sigma Chemical Co.) were added to each 1.2-ml sample at 4°C. The activated charcoal solution was constantly stirred prior to and during the pipetting to ensure that no sedimentation occurred and, after charcoal addition, samples were left for at least 15 min on ice before centrifugation at 1850 × g for 10 min at 4°C. The supernatant containing [3H]2O was removed, and 300 μl were analyzed by scintillation counting. Results were expressed as a percentage of the release of [3H]2O in control untreated cells.

Estimation of de Novo Purine Synthesis.

The cell pellets remaining after removal of the supernatant for [3H]2O analysis were resuspended in 2 ml of ice-cold, drug-free dialyzed medium (without thymidine or inosine) and centrifuged at 120 × g for 5 min. The cell pellets were washed an additional two times in 2 ml of the same ice-cold medium and finally resuspended in 500 μl of 1 m ice-cold perchloric acid. After transfer to glass tubes (Baxter Healthcare, Thetford, Norfolk, United Kingdom), samples were vortexed briefly and placed in a dry heating block (Techne, Cambridge, United Kingdom) at 100°C for 1 h. After acid hydrolysis, samples were removed from the heating block and allowed to cool for 15 min. The samples were then pipetted into 1.5-ml Eppendorf tubes (Sarstedt, Leicester, United Kingdom) and centrifuged at 6700 × g for 5 min at 4°C. The resultant supernatant was then added to 300 μl of ice-cold 2 m Tris-base and again centrifuged at 6700 × g for 5 min at 4°C. The supernatant was removed, the pH was checked and if necessary adjusted to pH 7, before the samples were frozen at −20°C for later analysis by HPLC.

HPLC analysis of [14C]formate incorporation into adenine and guanine involved the injection of 100-μl samples via a 50 × 2-mm pellicular ODS silica pre-column (Whatman) onto a 250 × 4.6-mm Spherisorb 5 μ ODS2 cartridge (PhaseSep, Franklin, MA), and UV absorbance of standard (Sigma) and endogenous adenine and guanine was measured at 260 and 280 nm. 14C-Labeled material was detected using an on line FLO-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 phase consisting of 0.1 m sodium acetate adjusted to pH 5.5 with glacial acetic acid (BDH) at a flow rate of 1 ml/min for the first 10 min of the analysis, after which a linear gradient from 0.1 m sodium acetate (pH 5.5) to 0.1 m sodium acetate (pH 5.5) + 15% (w/w) acetonitrile (Fisons Chemicals) over 20 min was initiated to give a total analysis time of 30 min. Endogenous adenine and guanine were quantitated using linear regression against a standard curve. Standard curves were constructed by the analysis of standard adenine base and guanine bases (Sigma) as follows: guanine = 0.52, 1.04, 2.08, 4.16, 8.32, 16.64, and 33.28 nmol; adenine = 0.44, 0.88, 1.66, 3.32, 6.64, 13.28, and 26.56 nmol injected on-column. Newly formed adenine and guanine were quantitated on the basis of the specific activity of the [14C]formate (0.84 kBq/mmol), and results were expressed as pmol of newly formed bases/nmol of preexisting or endogenous bases.

Statistics.

Nonweighted linear regression analysis was used in the calculation of the X coefficients of the standard curves used to quantify de novo purine and thymidylate biosynthesis. Two-sided t tests were used to test for differences between the CCRF-CEM and CEM:RTOM cell lines with respect to antifolate sensitivity, TS activity, [3H]MTX transport kinetics, and FPGS activity.

Cell Growth Inhibition Studies with CCRF-CEM and CEM:RTOM Cell Lines.

The IC50 values for antifolate-induced growth inhibition with CCRF-CEM and CCRF-CEM:RC2Tomudex (CEM:RTOM) cell lines, after either continuous or short-term drug exposure, are shown in Table 1. For continuous exposure, CCRF-CEM cells were markedly more sensitive than CEM:RTOM cells to both raltitrexed (Fig. 1) and to a lesser extent lometrexol. In contrast, CEM:RTOM cells were somewhat more sensitive than CCRF-CEM cells to MTX, and there was no significant difference between the two cell lines in sensitivity to nolatrexed.

After short-term drug exposures, CCRF-CEM and CEM:RTOM cells were equisensitive to a 24-h exposure to MTX and were insensitive to a 6-h exposure to nolatrexed. However, for a 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 in CCRF-CEM and CEM:RTOM Cell Lines.

There was no significant difference between the TS activity in cell extracts from the CCRF-CEM and CEM:RTOM cell lines (Table 2). Similarly, there was no significant difference between the two cell lines in the transport kinetics of [3H]MTX, as measured by the Kt value, or in the transport kinetics of [3H]MTX in the presence of raltitrexed, as measured by Ki (Table 2).

In Situ MTX Polyglutamate Formation.

After incubation of CCRF-CEM and CEM:RTOM cells with 1 μm [3H]MTX for 24 h, the levels of intracellular MTX and the formation of MTX polyglutamates were measured by HPLC (Fig. 2). CCRF-CEM cells formed MTX polyglutamates with up to five glutamate residues. Mean levels of individual polyglutamate metabolites in CCEF-CEM cells ranged from 277 ± 59 pmol/109 cells (diglutamate) to 745 ± 144 pmol/109 cells (pentaglutamate), with intracellular MTX monoglutamate present at 275 ± 120 pmol/109 cells. In marked contrast, CEM:RTOM cells formed no detectable MTX polyglutamates (<15 pmol/109 cells) on exposure to 1 μm [3H]MTX for 24 h. However, the parent MTX monoglutamate was readily detected (697 ± 401 pmol/109 cells), at a level 2.5-fold higher than that detected in CCRF-CEM cells (Table 3).

After a 24-h exposure to 10 μm [3H]MTX, CCRF-CEM cells again formed MTX polyglutamates up to the pentaglutamate metabolite. Mean levels of individual polyglutamates from three experiments ranged from 1761 ± 536 pmol/109 cells (diglutamate) to 2623 ± 176 pmol/109 cells (pentaglutamate), with intracellular MTX monoglutamate present at 3256 ± 1104 pmol/109 cells. The only metabolite detected in CEM:RTOM cells was MTX diglutamate, with a mean level of only 92 pmol/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 detected, with a mean level of 1350 ± 171 pmol/109 cells in CEM:RTOM cells, ∼2-fold lower than the mean level measured in CCRF-CEM cells (Table 3).

FPGS Activity, FPGS Protein, and FPGS mRNA in CCRF-CEM and CEM:RTOM Cell Lines.

The FPGS activity in extracts prepared from the two cell lines is shown in Table 2. HPLC analysis revealed that in the CCRF-CEM cell line, all of the incorporated [3H]glutamate was present as MTX diglutamate with no detectable MTX tri-, tetra-, or pentaglutamate. In direct contrast, FPGS activity in extracts of CEM:RTOM cells could not be detected. Thus, CEM:RTOM cells were estimated to have <11% of the FPGS activity of CCRF-CEM cells. This reduction in FPGS activity was associated with a decrease in FPGS protein expression, with the CEM:RTOM cells expressing approximately one-third of the FPGS protein levels of the CCRF-CEM cell line (Fig. 3).

FPGS mRNA levels were measured (Fig. 4) and expressed as a ratio of FPGS mRNA to 18S rRNA. There was no significant difference (P = 0.11) between the FPGS mRNA:18S rRNA ratios for CCRF-CEM and CEM:RTOM with mean (± SD, n = 3) ratios of 0.024 ± 0.001 and 0.030 ± 0.005, respectively.

Intracellular Levels of Total Raltitrexed-derived Material in CCRF-CEM and CEM:RTOM Cell Lines.

As measured by an immunoassay that detects raltitrexed and raltitrexed-polyglutamates with equal sensitivity, CCRF-CEM cells accumulated 30-fold higher levels of intracellular raltitrexed-derived material when compared with CEM:RTOM cells, with (mean ± SD) levels of 114 ± 21 pmol/mg protein versus 4 ± 2 pmol/mg protein, respectively, after a 4-h exposure to 0.1 μm drug (Fig. 5). Similarly, after a 24-h continuous exposure to 0.1 μm raltitrexed, CCRF-CEM cells accumulated 50-fold more intracellular raltitrexed-derived material than CEM:RTOM cells, with levels of 346 ± 52 pmol/mg protein and 7 ± 2 pmol/mg protein, respectively. When cells were exposed to 0.1 μm raltitrexed for 4 h, followed by incubation for a further 20 h in drug free medium, CCRF-CEM cells accumulated and retained >30-fold more intracellular raltitrexed-derived material than CEM:RTOM cells with mean levels of 75 ± 5 pmol/mg protein versus 2 pmol/mg protein, respectively.

Effect of Raltitrexed and Methotrexate on Inhibition of in Situ TS in CCRF-CEM and CEM:RTOM Cell Lines.

TS activity in untreated control CCRF-CEM cells and CEM:RTOM cells was similar, with [3H]2O release rates of 401,300 ± 36,360 dpm/106 (CCRF-CEM) and 334,500 ± 79,440 dpm/106 (CEM:RTOM).

In contrast to control rates, the two cell lines studied showed a striking difference in sensitivity to raltitrexed-induced inhibition of TS activity (Fig. 6). After exposure to 10 nm raltitrexed, TS activity was inhibited by >90% in CCRF-CEM cells compared with <5% inhibition in CEM:RTOM cells. Moreover, even after exposure of CEM:RTOM cells to 1000 nm raltitrexed, TS activity was not significantly inhibited with respect to the untreated controls. In contrast to the effect of 10 nm raltitrexed, there was no significant difference (P = 0.18) between 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 significantly higher (10% of control) than in CCRF-CEM cells, where activity was undetectable at this MTX concentration (<0.2% control; P = 0.02).

Effect of Raltitrexed and Methotrexate on de Novo Purine Synthesis in CCRF-CEM and CEM:RTOM Cells.

The mean rates of de novo purine synthesis in untreated control cells were found to be significantly higher in CEM:RTOM cells than in CCRF-CEM cells, i.e., 22 ± 2.5 versus 13 ± 2.7 pmol/nmol preexisting adenine bases/h, respectively (P = 0.016).

CCRF-CEM and CEM:RTOM cells showed different patterns of sensitivity to both raltitrexed- and MTX-induced inhibition of de novo purine synthesis (Fig. 7). In CCRF-CEM cells, de novo purine synthesis was inhibited by increasing concentrations of raltitrexed. Exposure to 10 nm raltitrexed resulted in 15% inhibition of de novo purine synthesis, which increased to 30–50% inhibition after exposure to 1000 nm raltitrexed (P = 0.0006). In contrast, CEM:RTOM cells showed no evidence of inhibition of de novo purine synthesis, even after exposure to 1000 nm drug.

CCRF-CEM and CEM:RTOM cells also showed different patterns of inhibition of de novo purine synthesis in response to MTX treatment. Exposure of CCRF-CEM cells to MTX only produced inhibition of de novo purine synthesis after exposure to 1000 nm MTX. In contrast, de novo purine synthesis in CEM:RTOM cells was stimulated by MTX, at 10 and 100 nm, to 220% at the latter concentration (P = 0.002). Even after exposure to 1000 nm MTX, a concentration that induced complete inhibition in CCRF-CEM cells, only limited inhibition of de novo purine synthesis (20%) was observed in CEM:RTOM cells (P = 0.03).

The aim of this study was to investigate the influence of FPGS activity on the sensitivity of two CCRF-CEM cell lines to short-term and continuous exposures to a range of antifolates. In addition, the effect of FPGS activity on raltitrexed- and MTX-induced inhibition of de novo thymidylate and purine biosynthesis was studied and related to the antifolate sensitivity of the cell lines. The CCRF-CEM:RC2Tomudex (CEM:RTOM) cell line studied here was cloned from a CCRF-CEM cell line found to be insensitive to the antifolate raltitrexed when compared with a CCRF-CEM cell line obtained from the European Collection of Animal Cell Cultures. The CEM:RTOM cell line was not known to have been exposed to any antifolate for any period of time prior to use in these studies.

Because multifactorial resistance to antifolates had been demonstrated previously (29, 30), initial studies focused on the characterization of the potential mechanism(s) of resistance to raltitrexed in the CEM:RTOM cell line. No significant difference was found between CEM:RTOM and CCRF-CEM cells for TS activity in crude cell extracts or cellular [3H]MTX transport kinetics, the latter being used as a surrogate marker for raltitrexed transport, which, like MTX, uses the reduced folate carrier (8). However, evidence to support reduced polyglutamation as the underlying mechanism of raltitrexed resistance in CEM:RTOM cells came from measurements of the formation of MTX polyglutamates in situ, levels of total intracellular raltitrexed-derived material, and FPGS activity in crude cell extracts.

In contrast to CCRF-CEM cells, CEM:RTOM cells formed no detectable polyglutamates after exposure to 1 μm [3H]MTX and only very low levels of MTX diglutamate after exposure to 10 μm [3H]MTX. In addition, CCRF-CEM cells were able to accumulate, and retain, significantly higher concentrations of raltitrexed-derived material than CEM:RTOM cells, indicating that defective polyglutamation was not specific to methotrexate. The finding of reduced FPGS activity and protein expression in CEM:RTOM cells, without a reduction in FPGS mRNA expression, suggests that reduced FPGS activity is the result of a posttranscriptional event. Similar observations have been made in antifolate resistant CCRF-CEM cell clones (40) and L1210 variants (41), with reduced FPGS activity. Indeed, for L1210 cells, a novel posttranscriptional mechanism of antifolate resistance was identified, resulting from a mutationally determined difference in the secondary structure of mRNA, which prevents the progression of translation of FPGS protein (42).

In the FPGS-proficient CCRF-CEM cell line, exposure to increasing concentrations of MTX resulted in the complete inhibition of in situ TS activity at concentrations ≥100 nm, whereas a higher (1000 nm) concentration of MTX was needed to inhibit de novo purine synthesis. In the FPGS-deficient CEM:RTOM cell line, TS activity was not completely inhibited by even 1000 nm MTX, and there was stimulation of, rather than inhibition of, de novo purine synthesis. These effects of MTX may be explained by the cellular pharmacology of the drug, which is known to directly or indirectly affect at least three folate-dependent enzymes within the cell, i.e., TS (10), AICAR formyltransferase, an enzyme required for de novo purine synthesis (43), and DHFR (44).

MTX-mediated TS inhibition can be the result of two effects: direct TS inhibition by MTX polyglutamates, and indirect inhibition after DHFR inhibition-mediated depletion of 5,10-methylene tetrahydrofolate pools. In the studies described here, inhibition of whole-cell TS activity at 10 nm MTX was similar in the CCRF-CEM and CEM:RTOM cell lines, presumably due to a direct effect on DHFR, which is not influenced by polyglutamation (45). At higher MTX concentrations (100 and 1000 nm), the complete inhibition of TS observed in CCRF-CEM cells is consistent with an effect due to MTX polyglutamates acting directly on TS, because these metabolites were not formed in CEM:RTOM cells, and complete TS inhibition was not observed.

The relative effects of MTX on purine biosynthesis in the two cell lines can also be explained, given the cellular pharmacology of the drug. The inhibition of de novo purine biosynthesis 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 polyglutamates on AICAR formyltransferase. The alternative mechanism, depletion of 10-formyltetrahydrofolate cofactor pools secondary to DHFR inhibition, can only operate if TS activity is maintained, which was not the case in CCRF-CEM cells exposed to 1000 nm MTX. The data for CEM:RTOM cells are again consistent with inhibition of de novo purine biosynthesis being due to a direct effect of MTX polyglutamates, because these metabolites were not formed in this cell line, and purine biosynthesis was not markedly inhibited at MTX concentrations up to 1000 nm.

The reason for the apparent stimulation of de novo purine biosynthesis in the CEM:RTOM cells at 10 and 100 nm MTX, but not to any marked extent in the CCRF-CEM cells, may relate to the availability of PRPP. Low concentrations (20 nm) of MTX have been shown to elevate PRPP levels in a number of cell lines, including Molt-4 human leukemia cells (46) and HCT-8 colorectal (47) cell lines. If PRPP levels are rate limiting for de novo purine synthesis inhibition, and in the absence of AICAR formyltransferase inhibition due to a lack of MTX polyglutamate formation, stimulation of purine biosynthesis could be observed.

In contrast to the findings with MTX, neither de novo thymidylate or purine biosynthesis was affected by exposure to raltitrexed in CEM:RTOM cells. Because the potency of raltitrexed as a specific inhibitor of TS is greatly enhanced by polyglutamation (9) and the intracellular accumulation of the raltitrexed-derived material in the CEM:RTOM cell line was shown to be markedly reduced in the present study, the lack of TS inhibition seen is not unexpected. However, the lack of inhibition of TS is consistent with the requirement for concentrations above this level to produce cell growth inhibition on continuous 96-h exposure (Fig. 1). Together, these data suggest that the lack of TS inhibition in CEM:RTOM cells after raltitrexed treatment is due to inadequate polyglutamation.

Although raltitrexed had no effect on de novo purine synthesis in the CEM:RTOM cell line, a dose-dependent decrease in de novo purine synthesis was observed in CCRF-CEM cells. It is possible that in CCRF-CEM cells, there was direct inhibition of DHFR by raltitrexed (8), an effect that was not observed in the CEM:RTOM cells because of the much lower accumulation of raltitrexed-derived material (Fig. 5).

The differing cellular biochemical effects of MTX and raltitrexed also offer an explanation for the relative sensitivities of CCRF-CEM and CEM:RTOM cells to long- and short-term exposure to a range of antifolates. When exposed to antifolates continuously for 96 h, CEM:RTOM cells retained sensitivity to MTX. This finding, which is in keeping with previous reports of MTX sensitivity in FPGS-deficient human leukemia cell lines (25, 48), can be explained by the reduced TS activity observed in CEM:RTOM cells at higher MTX concentrations. The large difference in raltitrexed-mediated growth inhibition is also consistent with studies in FPGS-deficient human (25) and murine (14) leukemia cell lines. Like raltitrexed, the potency of lometrexol, a classical antifolate inhibitor of glycinamide ribonucleotide formyltransferase, is also markedly enhanced by polyglutamation (49). However, the CEM:RTOM cell line remained relatively sensitive to the growth-inhibitory effects of continuous exposure to lometrexol when compared with raltitrexed, an effect that has also been reported for FPGS-deficient L1210 cells (14). This apparent discrepancy between raltitrexed and lometrexol sensitivities may represent a selective effect of residual FPGS activity, because FPGS deficiency has been shown to be associated with differential effects on FPGS substrate specificity with a variety of physiological folates and antifolate drugs (20).

After short-term exposure to antifolates, both CCRF-CEM and CEM:RTOM cells were insensitive to the growth-inhibitory effects of nolatrexed, a nonclassical antifolate TS inhibitor. In addition, FPGS deficiency rendered the CEM:RTOM cell line insensitive to growth inhibition mediated by short-term exposure to raltitrexed, which is in keeping with the markedly lower accumulation and retention of raltitrexed in this cell line. The observed insensitivity of the CEM:RTOM cell line to short-term exposure to lometrexol is also consistent with the presumably reduced polyglutamation of, and hence intracellular retention of, the drug. In contrast, MTX-mediated growth inhibition of the CEM:RTOM cells was relatively well preserved after short-term exposure, which may reflect the equal affinity of MTX and MTX polyglutamates as tight binding inhibitors of DHFR (44). In the FPGS-proficient CCRF-CEM cell line, the greater growth-inhibitory activity after short-term exposure of raltitrexed when compared with lometrexol may reflect their different affinities for FPGS (50, 51).

In conclusion, the studies presented herein demonstrate that polyglutamation is a critical determinant of the sensitivity of CCRF-CEM cells to growth inhibition mediated by raltitrexed. Cell growth inhibition data were in keeping with the influence of reduced polyglutamation on de novo purine and thymidylate synthesis. In contrast, the influence of reduced polyglutamation was less pronounced in the case MTX, where growth-inhibitory activity was largely preserved after short-term exposure. However, polyglutamation was found to influence the impact of MTX on thymidylate versusde novo purine biosynthesis. Future work will examine the effect of more recently developed antifolates on de novo thymidylate and purine biosynthesis and the impact of various resistance mechanisms on their cellular pharmacology. In addition, the relationship between MTX polyglutamation and inhibition of de novo thymidylate and purine biosynthesis in lymphoblasts obtained from patients with childhood acute lymphoblastic leukemia will be investigated.

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 in accordance with 18 U.S.C. Section 1734 solely to indicate 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 (to G. W. A., A. H., G. A. T., D. R. N., and J. L.), and by National Cancer Institute Grant CA43500 and Roswell Park Cancer Institute Core Grant CA16056 (both to J. J. M.).

                
3

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

Fig. 1.

Growth inhibition of CCRF-CEM (▪) and CCRF-CEM: RC2Tomudex (▴) cells exposed to raltitrexed for 96 h. Each point on the graph represents the mean ± SD of data from three individual experiments; bars, SD.

Fig. 1.

Growth inhibition of CCRF-CEM (▪) and CCRF-CEM: RC2Tomudex (▴) cells exposed to raltitrexed for 96 h. Each point on the graph represents the mean ± SD of data from three individual experiments; bars, SD.

Close modal
Fig. 2.

HPLC radiochromatogram illustrating MTX and MTX polyglutamate metabolites in CCRF-CEM and CCRF-CEM: RC2Tomudex cell extracts after exposure of the cells to 1 μm [3H]MTX for 24 h.

Fig. 2.

HPLC radiochromatogram illustrating MTX and MTX polyglutamate metabolites in CCRF-CEM and CCRF-CEM: RC2Tomudex cell extracts after exposure of the cells to 1 μm [3H]MTX for 24 h.

Close modal
Fig. 3.

FPGS protein levels as detected by Western blot analysis. Lanes 1–3, CCRF-CEM: RC2Tomudex; Lanes 4–6, CCRF-CEM; Lane 7, CEM/C1 crude (a CCRF-CEM cell line in use in the laboratories of J. J. M.); Lane 8, CEM/C1 (pure) (a partially purified FPGS extract from the CEM/C1 cell line). All lanes were loaded with 20 μg of total cellular protein, except Lane 8, for which 0.2 μg of protein was loaded.

Fig. 3.

FPGS protein levels as detected by Western blot analysis. Lanes 1–3, CCRF-CEM: RC2Tomudex; Lanes 4–6, CCRF-CEM; Lane 7, CEM/C1 crude (a CCRF-CEM cell line in use in the laboratories of J. J. M.); Lane 8, CEM/C1 (pure) (a partially purified FPGS extract from the CEM/C1 cell line). All lanes were loaded with 20 μg of total cellular protein, except Lane 8, for which 0.2 μg of protein was loaded.

Close modal
Fig. 4.

FPGS mRNA levels in CCRF-CEM and CCRF-CEM: RC2Tomudex cell lines as measured by Northern blot hybridization.

Fig. 4.

FPGS mRNA levels in CCRF-CEM and CCRF-CEM: RC2Tomudex cell lines as measured by Northern blot hybridization.

Close modal
Fig. 5.

Total intracellular levels of raltitrexed and raltitrexed polyglutamates in CCRF-CEM (▧) and CCRF-CEM: RC2Tomudex (▪) cells after exposure to 0.1 μm drug. Each column represents the mean of three individual experiments; bars, SD. 4 hr, total intracellular raltitrexed measured after a 4-h exposure. 24 hr, total intracellular raltitrexed measured after a 24-h exposure. 4 hr/24 hr, total intracellular raltitrexed measured after a 4-h exposure with 20 h in drug-free medium.

Fig. 5.

Total intracellular levels of raltitrexed and raltitrexed polyglutamates in CCRF-CEM (▧) and CCRF-CEM: RC2Tomudex (▪) cells after exposure to 0.1 μm drug. Each column represents the mean of three individual experiments; bars, SD. 4 hr, total intracellular raltitrexed measured after a 4-h exposure. 24 hr, total intracellular raltitrexed measured after a 24-h exposure. 4 hr/24 hr, total intracellular raltitrexed measured after a 4-h exposure with 20 h in drug-free medium.

Close modal
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 mean of three individual experiments with data expressed as a percentage of activity in control (untreated) cells; bars, SD.

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 mean of three individual experiments with data expressed as a percentage of activity in control (untreated) cells; bars, SD.

Close modal
Fig. 7.

De novo purine synthesis in CCRF-CEM (squares) and CCRF-CEM: RC2Tomudex (triangles) cells after incubation with MTX (open symbols)/raltitrexed (closed symbols) for 22 h. Each point represents the mean of three individual experiments with data expressed as a percentage of the rate of adenine synthesis in control cells; bars, SD.

Fig. 7.

De novo purine synthesis in CCRF-CEM (squares) and CCRF-CEM: RC2Tomudex (triangles) cells after incubation with MTX (open symbols)/raltitrexed (closed symbols) for 22 h. Each point represents the mean of three individual experiments with data expressed as a percentage of the rate of adenine synthesis in control cells; bars, SD.

Close modal
Table 1

Growth-inhibitory activity of antifolates against CCRF-CEM and CCRF-CEM:RC2Tomudex cellsa

Drug exposure (time)IC50 (nm) (mean ±; SD)Fold-differenceP
CCRF-CEMCCRF-CEM:RC2Tomudex
Raltitrexed (96 h) 3.0 ±; 0.9 3,876 ±; 600 1,292 0.0004 
Raltitrexed (6 h) 52 ±; 26 >200,000 >3846  
Methotrexate (96 h) 6.0 ±; 0.9 3.0 ±; 0.2 0.5 0.005 
Methotrexate (24 h) 42 ±; 9.0 77 ±; 26 1.8 0.09 
Methotrexate (6 h) 496 ±; 40 4,579 ±; 788 0.001 
Lometrexol (96 h) 9.0 ±; 4.0 57 ±; 15 6.3 0.006 
Lometrexol (6 h) 15,538 ±; 4,356 >200,000 >13  
Nolatrexed (96 h) 1,000 ±; 170 787 ±; 34 0.8 0.1 
Nolatrexed (6 h) >175,000 >175,000   
Drug exposure (time)IC50 (nm) (mean ±; SD)Fold-differenceP
CCRF-CEMCCRF-CEM:RC2Tomudex
Raltitrexed (96 h) 3.0 ±; 0.9 3,876 ±; 600 1,292 0.0004 
Raltitrexed (6 h) 52 ±; 26 >200,000 >3846  
Methotrexate (96 h) 6.0 ±; 0.9 3.0 ±; 0.2 0.5 0.005 
Methotrexate (24 h) 42 ±; 9.0 77 ±; 26 1.8 0.09 
Methotrexate (6 h) 496 ±; 40 4,579 ±; 788 0.001 
Lometrexol (96 h) 9.0 ±; 4.0 57 ±; 15 6.3 0.006 
Lometrexol (6 h) 15,538 ±; 4,356 >200,000 >13  
Nolatrexed (96 h) 1,000 ±; 170 787 ±; 34 0.8 0.1 
Nolatrexed (6 h) >175,000 >175,000   
a

The results are expressed as the mean ±; SD of at least three individual experiments.

Table 2

TS activity, [3H]MTX transport kinetics, and FPGS activity in CCRF-CEM and CCRF-CEM:RC2Tomudex cellsa

CCRF-CEMCCRF-CEM:RC2TomudexP
TS activity (nmol dUMP/106 cells/h) 2.3 ±; 0.6 3.3 ±; 0.6 0.106 
[3H]MTX-Transport kinetics    
 MTX Ktm7.9 ±; 1.7 7.1 ±; 1.0 0.52 
Vmax (pmol/106 cells) 12.6 ±; 1.2 9.9 ±; 0.6 0.025 
 Raltitrexed Kim0.8 ±; 0.4 1.2 ±; 0.6 0.33 
FPGS activity (pmol of [3H]glutamate incorporated/h/mg protein) 147 ±; 14 <16 <0.0001 
CCRF-CEMCCRF-CEM:RC2TomudexP
TS activity (nmol dUMP/106 cells/h) 2.3 ±; 0.6 3.3 ±; 0.6 0.106 
[3H]MTX-Transport kinetics    
 MTX Ktm7.9 ±; 1.7 7.1 ±; 1.0 0.52 
Vmax (pmol/106 cells) 12.6 ±; 1.2 9.9 ±; 0.6 0.025 
 Raltitrexed Kim0.8 ±; 0.4 1.2 ±; 0.6 0.33 
FPGS activity (pmol of [3H]glutamate incorporated/h/mg protein) 147 ±; 14 <16 <0.0001 
a

The results represent the mean ±; SD of three separate experiments.

Table 3

MTX and MTX polyglutamate levels in CCRF-CEM and CCRF-CEM:RC2Tomudex (CEM:RTOM) cells after exposure to 1 μm or 10 μm [3H]MTX for 24 ha

Methotrexate polyglutamateMethotrexate and methotrexate polyglutamate levels (pmol/109 cells)
1 μm [3H]methotrexate10 μm [3H]methotrexate
CCRF-CEMCEM:RC2CCRF-CEMCEM:RC2
Pentaglutamate 745 ±; 144 NDb 2623 ±; 176 ND 
Tetraglutamate 619 ±; 179 ND 2544 ±; 545 ND 
Triglutamate 550 ±; 142 ND 2333 ±; 370 ND 
Diglutamate 277 ±; 59 ND 1761 ±; 536 92 (76,108) 
Monoglutamate 275 ±; 120 697 ±; 401 3256 ±; 1104 1350 ±; 171 
Methotrexate polyglutamateMethotrexate and methotrexate polyglutamate levels (pmol/109 cells)
1 μm [3H]methotrexate10 μm [3H]methotrexate
CCRF-CEMCEM:RC2CCRF-CEMCEM:RC2
Pentaglutamate 745 ±; 144 NDb 2623 ±; 176 ND 
Tetraglutamate 619 ±; 179 ND 2544 ±; 545 ND 
Triglutamate 550 ±; 142 ND 2333 ±; 370 ND 
Diglutamate 277 ±; 59 ND 1761 ±; 536 92 (76,108) 
Monoglutamate 275 ±; 120 697 ±; 401 3256 ±; 1104 1350 ±; 171 
a

The results are expressed as the mean ±; SD of three separate experiments.

b

ND, not detectable (<15 pmol/109 cells).

We acknowledge Professor R. Moran (Massey Cancer Center, Richmond, VA) for supplying the pX1 plasmid used in the study of FPGS mRNA and Dr. W. E. Evans (St. Jude Childrens Research Hospital, Memphis, TN) and his research group for detailed information and discussions on the method for measuring de novo purine synthesis. We thank Cynthia Russell for performing the Western blot analysis of FPGS protein expression.

1
Jansen G., Westerhof G. R., Schornagel J. H., Jackman A. L., Boyle F. T. The reduced folate/methotrexate carrier and a membrane-associated folate binding protein as transport routes for novel antifolates: structure-activity relationships.
Adv. Exp. Med. Biol.
,
338
:
767
-770,  
1993
.
2
Goldman I. D., Lichtenstein N. S., Oliverio V. T. Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell.
J. Biol. Chem.
,
243
:
5007
-5017,  
1968
.
3
McGuire J. J., Hsieh P., Coward J. K., Bertino J. R. Enzymatic synthesis of the folylpolyglutamates.
J. Biol. Chem.
,
255
:
5776
-5788,  
1980
.
4
Barredo J., Moran R. G. Determinants of antifolate cytotoxicity: folylpolyglutamate synthetase activity during cellular proliferation and development.
Mol. Pharmacol.
,
42
:
687
-694,  
1992
.
5
Galivan J., Nimec Z., Balinska M. Regulation of methotrexate polyglutamate accumulation in vitro: effects of cellular folate content.
Biochem. Pharmacol.
,
32
:
3244
-3247,  
1983
.
6
Kimbell R., Jackman A. L., Boyle F. T., Hardcastle A., Aherne G. W. The duration of the inhibition of thymidylate synthase in intact L1210 cells exposed to two different classes of quinazoline analogues.
Adv. Exp. Med. Biol.
,
338
:
597
-600,  
1993
.
7
Jolivet J., Schilsky R. L., Bailey B. D., Drake J. C., Chabner B. A. Synthesis,retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells.
J. Clin. Invest.
,
70
:
351
-360,  
1982
.
8
Jackman A. L., Taylor G. A., Gibson W., Kimbell R., Brown M., Calvert A. H., Judson I. R., Hughes L. R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study.
Cancer Res.
,
51
:
5579
-5586,  
1991
.
9
Ward W. H. J., Kimbell R., Jackman A. L. Kinetic characteristics of ICI D1694: a quinazoline antifolate which inhibits thymidylate synthase.
Biochem. Pharmacol.
,
43
:
2029
-2031,  
1992
.
10
Allegra C. J., Chabner B. A., Drake J. C., Lutz R., Rodbard D., Jolivet J. Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates.
J. Biol. Chem.
,
260
:
9720
-9726,  
1985
.
11
Kamen B. A., Eibl B., Cashmore A., Bertino J. Uptake and efficacy of trimetrexate (TMQ, 2, 4-diamino-5-methyl-6-[3, 4, 5-trimethoxyanilino)-methyl]quinazoline), a non-classical antifolate in methotrexate-resistant leukaemia cells in vitro.
Biochem. Pharmacol.
,
33
:
1697
-1984,  
1984
.
12
Duch D. S., Edelstein M. P., Bowers S. W., Nichol C. A. Biochemical and chemotherapeutic studies on 2, 4-diamino-6-(2, 5-dimethoxybenzyl)-5-methyl-pyridol[2, 3-d]pyrimidine (BW301), a novel lipid soluble inhibitor of dihydrofolate reductase.
Cancer Res.
,
42
:
3987
-3994,  
1982
.
13
Webber S., Bartlett C. A., Boritzki T. J., Hilliard J. A., Howland E. F., Johnston A. L., Kosa M., Margosiak S. A., Morse C. A., Shetty B. V. AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies.
Cancer Chemother. Pharmacol.
,
37
:
509
-517,  
1996
.
14
Jackman A. L., Kelland I. R., Kimbell R., Brown M., Gibson W., Aherne G. W., Hardcastle A., Boyle F. T. Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines.
Br. J. Cancer
,
71
:
914
-924,  
1995
.
15
Rosowsky A., Lazarus H., Yuan G. C., Beltz W. R., Mangini L., Abelson H. T., Modest E. J., Frei E. Effects of methotrexate esters and other lipophilic antifolates on methotrexate-resistant human leukaemic lymphoblasts.
Biochem. Pharmacol.
,
29
:
648
-652,  
1980
.
16
Domin B. A., Grill S. P., Cheng Y-C. Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy.
Cancer Res.
,
43
:
2155
-2158,  
1983
.
17
Jackson R. C., Hart L. I., Harrap K. R. Intrinsic resistance to methotrexate of cultured mammalian cells in relation to the inhibition kinetics of their dihydrofolate reductases.
Cancer Res.
,
36
:
1991
-1997,  
1976
.
18
Goldie J. H., Krystal G., Hartley D., Gudauskas G., Dedhar S. A methotrexate insensitive variant of folate reductase present in two lines of methotrexate-resistant L5178Y cells.
Eur. J. Cancer
,
16
:
1539
-1546,  
1980
.
19
Fisher T. C., Milner A. E., Gregory C. D., Jackman A. L., Aherne G. W., Hartley J. A., Dive C., Hickman J. A. bcl-2 Modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways.
Cancer Res.
,
53
:
3321
-3326,  
1993
.
20
McCloskey D. E., McGuire J. J., Russell C. A., Rowan B. G., Bertino J. R., Pizzorno G., Mini E. Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines.
J. Biol. Chem.
,
266
:
6181
-6187,  
1991
.
21
Lin J. T., Tong W. P., Trippett T., Niedzwiecki D., Tao Y., Tan C., Steinherz P., Schweitzer B. I., Bertino J. R. Basis for natural resistance to methotrexate in human acute non-lymphocytic leukemia.
Leuk. Res.
,
15
:
1191
-1196,  
1991
.
22
Goker E., Lin J. T., Trippett T., Elisseyeff Y., Tong W. P., Niedzwiecki D., Tan C., Steinherz P., Schweitzer B. I., Bertino J. R. Decreased polyglutamation of methotrexate in acute lymphoblastic leukemia blasts in adults compared to children with this disease.
Leukemia (Baltimore)
,
7
:
1000
-1004,  
1993
.
23
Rhee M. S., Wang Y., Nair M. G., Galivan J. Acquisition of resistance to antifolates caused by enhanced γ-glutamyl hydrolase activity.
Cancer Res.
,
53
:
2227
-2230,  
1993
.
24
Pizzorno G., Chang Y-M., McGuire J. J., Bertino J. R. Inherent resistance of human squamous carcinoma cell lines to methotrexate as a result of decreased polyglutamation of this drug.
Cancer Res.
,
49
:
5275
-5280,  
1989
.
25
Takemura Y., Kobayashi H., Gibson W., Kimbell R., Miyachi H., Jackman A. L. The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cell lines.
Int. J. Cancer
,
66
:
29
-36,  
1996
.
26
Curt G. A., Jolivet J., Carney D. N., Bailey B. D., Drake J. C., Clendeninn N. J., Chabner B. A. Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.
J. Clin. Invest.
,
76
:
1323
-1329,  
1985
.
27
Whitehead V. M., Rosenblatt D. S., Vuchich M-J., Shuster J. J., Witte A., Beaulieu D. Accumulation of methotrexate and methotrexate polyglutamates in lymphoblasts at diagnosis of childhood acute lymphoblastic leukemia: a pilot prognostic factor analysis.
Blood
,
76
:
44
-49,  
1990
.
28
Barnes M. J., Estlin E. J., Taylor G. A., Calvete J. A., Newell D. R., Lunec J., Hall A. G., Pearson A. D. J., Aherne W., Hardcastle A. Impaired polyglutamation as a mechanism of Tomudex resistance in a CCRF-CEM cell line.
Br. J. Cancer
,
75 (Suppl. 1)
:
32
1997
.
29
Drake J. C., Allegra C. J., Moran R. G., Johnston P. G. Resistance to Tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines.
Biochem. Pharmacol.
,
51
:
1349
-1355,  
1996
.
30
Cowan K. H., Jolivet J. A. Methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates.
J. Biol. Chem.
,
259
:
10793
-10800,  
1984
.
31
Estlin E. J., Balmanno K., Calvert A. H., Hall A. G., Lunec J., Newell D. R., Pearson A. D. J., Taylor G. A. The relationship between intrinsic thymidylate synthase expression and sensitivity to THYMITAQTM in human leukaemia and colorectal carcinoma cell lines.
Br. J. Cancer
,
76
:
1579
-1585,  
1997
.
32
Jansen G., Schornagel J. H., Westerhof G. R., Rijksen G., Newell D. R., Jackman A. L. Multiple membrane transport systems for the uptake of folate-based thymidylate synthase inhibitors.
Cancer Res.
,
50
:
7544
-7548,  
1990
.
33
Wohlhueter R. M., Marz R., Graff J. C., Plagemann P. G. W. A rapid-mixing technique to measure transport in suspended animal cells: applications to nucleoside transport in Novikoff rat hepatoma cells.
Methods Cell Biol.
,
20
:
211
-235,  
1978
.
34
Cheng Y., Prusoff W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction.
Biochem. Pharmacol.
,
22
:
3099
-3108,  
1973
.
35
Jansen G., Schornagel J. H., Kathman I., Westerhof G. R., Hordijk G-J., van der Laan B. F. A. M. Measurement of folylpolyglutamate synthetase activity in head and neck squamous carcinoma cell lines and clinical samples using a new rapid separation procedure.
Oncol. Res.
,
4
:
299
-305,  
1992
.
36
McGuire J. J., Russell C. A. Folylpolyglutamate synthetase expression in antifolate-sensitive and resistant human cell lines.
Oncol Res.
,
10
:
193
-200,  
1998
.
37
Aherne G. W., Ward E., Lawrence N., Dobinson D., Clarke S. J., Musgrove H., Sutcliffe F., Stephens T., Jackman A. L. Comparison of plasma and tissue levels of ZD1694 (Tomudex), a highly polyglutamable quinazoline thymidylate synthase inhibitor, in preclinical models.
Br. J. Cancer
,
77
:
221
-226,  
1998
.
38
Taylor G. A., Jackman A. J., Balmanno K., Hughes L. R., Calvert A. H. Estimation of the in vitro and in vivo inhibitory effects of antifolates upon thymidylate synthase (TS) in whole cells Mikanagnagi K. Nifhioka K. Kelley W. N. eds. .
Purine Metabolism in Man VI
,
:
383
-388, Plenum Publishing Corp. New York  
1988
.
39
Masson E., Relling M. V., Synold T. W., Liu Q., Schuetz J. D., Sandlund J. T., Pui C-H., Evans W. E. Accumulation of methotrexate polyglutamates in lymphoblasts is a determinant of antileukemic effects in vivo.
J. Clin. Invest.
,
97
:
73
-80,  
1994
.
40
McGuire J. J., Haile W. H., Russell C. A., Galvin J. M., Shane B. Evolution of drug resistance in CCRF-CEM human leukemia cells selected by intermittent methotrexate exposure.
Oncol. Res.
,
7
:
535
-543,  
1995
.
41
Roy K., Egan M. G., Sirlin S., Sirotnak F. M. Post-transcriptionally mediated decrease in folylpolyglutamate synthetase gene expression in some folate analogue-resistant variants of the L1210 cell.
J. Biol. Chem.
,
272
:
6903
-6908,  
1997
.
42
Roy K., Sirotnak F. M. A novel posttranscriptional mechanism of antifolate resistance in L1210 cells decreasing synthesis of folylpolyglutamate synthetase.
Proc. Am. Assoc. Cancer Res.
,
38
:
659
1997
.
43
Allegra C. J., Drake J. C., Jolivet J., Chabner B. A. Inhibition of phosphoribosylaminoimadazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates.
Proc. Natl. Acad. Sci. USA
,
82
:
4881
-4885,  
1985
.
44
Whitehead V. W. Synthesis of methotrexate polyglutamates in L1210 murine leukemia cells.
Cancer Res.
,
37
:
408
-412,  
1977
.
45
Drake J. C., Allegra C. J., Baram J., Kaufman B. T., Chabner B. A. Effects of dihydrofolate reductase of methotrexate metabolites and intracellular folates formed following methotrexate exposure of human breast cancer cells.
Biochem. Pharmacol.
,
36
:
2416
-2418,  
1987
.
46
Bokkerink J. P. M., Bakker M. A. H., Hulscher T. W., De Abrew R. R. A., Schretlen E. D. A. M., van Laarhoven J. P. R. M., De Bruyn C. H. M. M. Sequence-, - and dose-dependent synergism of methotrexate and 6-mercaptopurine in malignant human T-lymphoblasts.
Biochem. Pharmacol.
,
35
:
3549
-3555,  
1986
.
47
Benz C., Cadman E. Modulation of 5-fluorouracil metabolism and cytotoxicity by antimetabolites in human colorectal adenocarcinoma HCT-8.
Cancer Res.
,
41
:
994
-999,  
1992
.
48
McGuire J. J., Heitzman K. J., Haile W. H., Russell C. A., McCloskey D. E., Piper J. R. Cross resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines.
Leukemia (Baltimore)
,
7
:
1996
-2003,  
1993
.
49
Baldwin S. W., Tse A., Gossett L. S., Taylor E. C., Rosowsky A., Shih C., Moran R. G. Structural features of 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate that determines inhibition of mammalian glycinamide ribonucleotide formyltransferase.
Biochemistry
,
30
:
1997
-2006,  
1991
.
50
Jackman A. L., Marsham P. R., Moran R. G., Kimbell R., O’Connor B. M., Hughes L. R., Calvert A. H. Thymidylate synthase inhibitors: the in vitro activity of a series of heterocyclic benzoyl ring modified 2-desamino-2-methyl-N10-substituted-5, 8-dideazafolates.
Adv. Enzyme Regul.
,
31
:
13
-27,  
1991
.
51
Beardsley G. P., Moroson B. A., Taylor E. C., Moran R. G. A new folate antimetabolite, 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis.
J. Biol. Chem.
,
264
:
328
-333,  
1989
.