2548 Vol. 5, 2548–2558, September 1999 Clinical Cancer Research Impact of Polyglutamation on Sensitivity to Raltitrexed and Methotrexate in Relation to Drug-induced Inhibition of de Novo Thymidylate and Purine Biosynthesis in CCRF-CEM Cell Lines1 Matthew J. Barnes, Edward J. Estlin,2 nolatrexed when exposed to these antifolates for 96 h. After Gordon A. Taylor, G. Wynne Aherne, 6 h of exposure, CCRF-CEM cells retained sensitivity to A. Hardcastle, J. J. McGuire, Joanne A. Calvete, MTX and raltitrexed but were less sensitive to lometrexol- mediated growth inhibition. In contrast, CCRF-CEM: John Lunec, Andrew D. J. Pearson, and RC2Tomudex cells were markedly insensitive to raltitrexed, David R. Newell lometrexol, and to a lesser degree, MTX. Simultaneous Cancer Research Unit, University of Newcastle, Newcastle upon Tyne measurement of de novo thymidylate and purine biosynthe- NE2 4HH, United Kingdom [M. J. B., G. A. T., J. A. C., J. L., sis revealed 90% inhibition of TS activity by 100 nM MTX in D. R. N.]; Department of Child Health, Royal Victoria Infirmary, both cell lines, whereas inhibition of de novo purine synthesis Newcastle upon Tyne NE1 4LP, United Kingdom [E. J. E., A. D. J. P.]; CRC Centre for Cancer Therapeutics, Institute of Cancer was only observed in CCRF-CEM cells, and only after ex- Research, Sutton, Surrey SM2 5NG, United Kingdom [G. W. A., posure to 1000 nM MTX. Ten nM raltitrexed induced >90% A. H.]; and Roswell Park Cancer Institute, New York State inhibition of TS activity in CCRF-CEM cells, whereas in Department of Health, Buffalo, New York 14263-0001 [J. J. M.] CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. ABSTRACT These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both The aim of this study was to investigate the influence of raltitrexed and MTX, markedly influencing potency in the folylpolyglutamyl synthetase (FPGS) activity on the cellular case of raltitrexed and locus of action in the case of MTX. pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth in- INTRODUCTION hibition and drug-induced inhibition of de novo thymidylate Antitumor antifolates can be classified by their loci of 3 and purine biosynthesis were used as measures of the cellu- action, e.g., as inhibitors of TS, DHFR or glycinamide ribonu- lar effects of the drugs. cleotide transformylase, and on the basis of the presence (clas- CCRF-CEM:RC2Tomudex cells had <11% of the FPGS sical antifolates) or absence (nonclassical antifolates) of a glu- activity of CCRF-CEM cells, whereas MTX uptake and TS tamate moiety. The a and g glutamate carboxyl groups of activity were equivalent. In CCRF-CEM:RC2Tomudex cells, classical antifolates, such as raltitrexed (an inhibitor of TS) and MTX polyglutamate formation was undetectable after expo- MTX, are negatively charged at physiological pH, and thus 3 sure to 1 mM [ H]MTX for 24 h. After exposure to 0.1 mM classical antifolates require carrier-mediated uptake for cell en- raltitrexed, levels of total intracellular raltitrexed-derived try (1, 2). Once inside the cell, classical antifolates, as well material in CCRF-CEM:RC2Tomudex cells were 30- to 50- naturally occurring folates, can undergo polyglutamation in a fold lower than in the CCRF-CEM cell line. CCRF-CEM: reaction that involves the addition of additional glutamate res- Tomudex g RC2 cells were >1000-fold resistant to raltitrexed idues at the -carboxyl position of the glutamate moiety. Poly- and 6-fold resistant to lometrexol but sensitive to MTX and glutamation, 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 concentra- tions (5) and can maintain inhibition of target enzymes after Received 12/21/98; revised 6/15/99; accepted 6/16/99. removal of extracellular drug (6, 7). In addition, polyglutama- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked tion can enhance the affinity of classical antifolates for certain advertisement in accordance with 18 U.S.C. Section 1734 solely to folate-dependent enzymes (8–10). In contrast to classical anti- indicate this fact. folates, the nonclassical agents trimetrexate (11), piritrexim 1 This work was supported by the Kay Kendall Leukaemia Fund (12), and nolatrexed (13) do not require carrier-mediated cellu- (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.). 2 To whom requests for reprints should be addressed, at Department of 3 The abbreviations used are: TS, thymidylate synthase; DHFR, dihy- Paediatric Oncology, Royal Hospital for Sick Children, St. Michael’s drofolate reductase; FPGS, folylpolyglutamate synthetase; HPLC, high- Hill, Bristol BS2 8BJ, United Kingdom. Phone: 44 0117 921 5411; Fax: performance liquid chromatography; AICAR, aminoimidazolecar- 44 0117 928 5682. boxymide; PRPP, 5-phosphorosyl-1-PPi. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 1999 American Association for Cancer Research. Clinical Cancer Research 2549 lar uptake and are not substrates for FPGS. As a result, non- RC2Tomudex cell line was found to be relatively insensitive to the classical antifolates are not retained within the cell on removal antifolate raltitrexed when compared with a CCRF-CEM cell of extracellular drug and cannot be converted to metabolites line obtained from the European Collection of Animal Cell with enhanced affinities for their target enzyme. Cultures and was not known to have been exposed to any Resistance to antifolates can result from one or more mech- antifolate for any period of time prior to use in these studies. The anisms including decreased cellular uptake of the drug (14, 15), original raltitrexed-resistant CCRF-CEM cell line was cloned by reduced polyglutamation (see below), overproduction of the seeding a cell suspension at a concentration of ,1 cell/well into target enzyme (14, 16), expression of a mutant form of the target a 96-well plate (Nunc, Life Technologies, Paisley, United enzyme (17, 18), or a failure of the cell to appropriately engage Kingdom), and after ;4 weeks, the cell line was routinely apoptosis (19). subcultured as above. The resulting clone, CCRF-CEM: Tomudex TOM Reduced polyglutamation has been described previously as RC2 (CEM:R ), was characterized by karyotype and a mechanism of inherent and acquired resistance to classical immunophenotype and found to be indistinguishable from the antifolates, both in vitro (14, 20) and in vivo (21, 22). The CCRF-CEM cell line obtained from the European Collection of underlying reason for resistance is either reduced FPGS activity Animal Cell Cultures. (14, 20) or enhanced activity of folylpolyglutamate hydrolase Cell Growth Inhibition Studies. All antifolates, i.e., (23), the enzyme that catalyzes the hydrolysis of antifolate MTX (Sigma Chemical Co.), nolatrexed (Thymitaq or AG337; polyglutamate metabolites. The impact of polyglutamation-re- Agouron Pharmaceuticals, San Diego, CA), raltitrexed (Tomu- lated resistance on target enzyme inhibition and antifolate- dex or ZD1694; Zeneca Pharmaceuticals, Macclesfield, United induced cytotoxicity depends on both the antifolate agent used, Kingdom), and lometrexol (DDATHF; Eli Lilly, Indianapolis, its target, and the duration of drug exposure. For example, U.S.A.) were dissolved in water at 1 mg/ml. Stock solutions decreased polyglutamation, which in vitro leads to resistance to were diluted further in dialyzed RPMI 1640 to the final con- short-term exposures with MTX, can be overcome with more centration required. prolonged drug exposure (20, 24). However, for certain other For continuous exposure studies, 100-ml aliquots of cell 4 classical antifolates, such as raltitrexed, deficient polyglutama- suspensions at 5 3 10 cells/ml were seeded into each well of a tion renders cell lines resistant to both short and longer term 96-well plate 24 h before drug exposure. One hundred mlof drug exposure (14, 25) Indeed, antifolate polyglutamation and medium containing drug at twice the concentration required FPGS activity have been shown to be important determinants of were added to quadruplicate wells and incubated for 96 h, both MTX cytotoxicity in vitro (26, 27) and outcome in clinical during which time approximately three cell doublings would studies (4, 27). have occurred in the control (untreated) wells. At the end of the Work to characterize the sensitivity of a panel of human exposure time, the number of cells in each suspension was leukemic cell lines has identified a CCRF-CEM cell line, counted on a Coulter cell counter. CCRF-CEM:RC2Tomudex, which is insensitive to raltitrexed In short-term exposure experiments, 2.5-ml aliquots of cell 4 (28). Because studies have shown that resistance to antifolates suspensions at 5 3 10 cells/ml were seeded into Falcon tubes can be multifactorial (29, 30), full characterization of the mech- (Becton Dickinson, New Jersey, NJ) 24 h before drug exposure. anism of resistance was undertaken and found to be related to After this period, 2.5 ml of medium containing drug at twice the reduced FPGS activity. The aim of the studies described here concentration required were added to triplicate tubes and incu- was to investigate the impact of reduced FPGS activity on bated at 37°C. After 6 or 24 h exposure to the drug, the cells cellular sensitivity to a range of antifolates after short term and were centrifuged at 500 3 g for 5 min at room temperature.