[CANCER RESEARCH 64, 3313–3319, May 1, 2004] Selective Preservation of Pemetrexed Pharmacological Activity in HeLa Cells Lacking the Reduced Folate Carrier: Association with the Presence of a Secondary Transport Pathway Rongbao Zhao, Marie Hanscom, Shrikanta Chattopadhyay, and I. David Goldman Departments of Medicine and Molecular Pharmacology, the Albert Einstein College of Medicine, and the Einstein Cancer Research Center, Bronx, New York ABSTRACT There have been reports describing cells with primary resistance to PMX that have greater cross-resistance to raltitrexed (ZD1694; To- A methotrexate (MTX)-resistant HeLa subline (R5), developed in this mudex) and cells with primary resistance to ZD1694 with lesser laboratory, with impaired transport due to a genomic deletion of the cross-resistance to PMX (7, 8). This has been observed whether reduced folate carrier (RFC) was only 2-fold resistant to pemetrexed (PMX), but 200- and 400-fold resistant to raltitrexed (ZD1694) and N␣- resistance was due to increased expression of TS or impaired drug (-4-amino-4-deoxypteroyl)-N␦-hemiphthaloyl-1-ornithine (PT523), respec- accumulation (7, 8). This is, however, not always the case (9). tively, compared with parental HeLa cells when grown with 2 ␮M folic Polyglutamation of ZD1694 is also required for activity, and these acid. When folic acid was replaced with the more physiological 25 nM derivatives of both agents have comparable TS inhibition constants 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to (4). Likewise, both drugs are comparable, excellent substrates for PMX but still 40- and 200-fold resistant to ZD1694 and PT523, respec- folypolyglutamate synthase (0.8–1.4 ␮M), and have similar affinities tively. Sensitivity to PT523 and PMX could be completely restored, and for reduced folate carrier (RFC) and folate receptor-␣ (FR-␣; Ref. 10, sensitivity to ZD1694 nearly restored, by transfection of RFC cDNA into 11). One possible explanation for the preservation of PMX activity R5 cells, indicating that the defect in drug transport was the only, or relative to ZD1694 is its inhibitory potential at the level of glycin- major, factor in resistance. The preserved PMX activity in R5 cells could amide ribonucleotide formyltransferase because the site of ZD1694 not be related to the very low expression of folate receptors. Rather, retained PMX activity in R5 cells was associated with residual transport action is solely at TS. -(␮ In a recent report from this laboratory, a methotrexate (MTX ؍ by another process that exhibits good affinity for PMX (Kt 12 M) with ϳ resistant HeLa cell line, R5, was characterized in which RFC was much lower affinities for ZD1694, MTX, and PT523 (Kisof 90, 100, and 250 ␮M, respectively). PMX transported by this route was rapidly con- deleted from the genome (12). This resulted in a large, but incomplete, verted to higher polyglutamates and, when grown with 25 nM 5-formyl- decrease in MTX influx compared with wild-type cells, consistent tetrahydrofolate, the rate of formation of these derivatives and their net with a residual RFC-independent transport pathway. This is unlike accumulation in R5 cells was comparable to that of wild-type cells. These what is observed in murine leukemia cells when RFC activity is lost, data suggest that selective preservation of PMX pharmacological activity when virtually all influx ceases (13). The present report explores the in RFC-null R5 cells is due, in part, to partial preservation of transport by cellular pharmacology of PMX in the R5 cell line, the extent to which secondary process with a higher affinity for PMX than the other antifo- it is transported by the RFC-independent pathway, and how this lates evaluated. impacts on the activity of this agent in comparison to (a) the other INTRODUCTION inhibitors of TS, ZD1694 and AG331 (the latter diffuses into cells and does not undergo polyglutamation), and (b) the potent dihydrofolate Pemetrexed (PMX; Alimta) is a new-generation antifolate with reductase inhibitor N␣-(-4-amino-4-deoxypteroyl)-N␦-hemiphthaloyl- demonstrated clinical activity in Phase II and III studies in the treat- 1-ornithine (PT523), which has a very high affinity for RFC and does ment of mesothelioma, non-small cell lung cancer, and other solid not form polyglutamate derivatives. tumors (1–3). The activity of the drug requires formation of polyglu- tamate derivatives in cells that are potent inhibitors of thymidylate ϭ synthase (TS; Ki 1.3 nM) and to a lesser extent glycinamide MATERIALS AND METHODS ribonucleotide formyltransferase (K ϭ 65 nM; Ref. 4). Despite its i Chemicals. [3Ј,5Ј,7,9-3H]Folic acid was purchased from Amersham Corp. inhibitory potential at two sites, at concentrations in the range of its (Arlington Heights, IL). [3H]PMX (50 Ci/mmol) and unlabeled PMX were IC50, thymidine alone affords full protection from the cytotoxic ef- provided by the Eli Lilly company (Indianapolis, IN). 5-Methyltetrahydrofo- fects of this agent in CCRF-CEM human leukemia cells (4, 5). On the late, 5-formyltetrahydrofolate (5-CHO-THF), and folic acid were obtained other hand, at high PMX concentrations, both thymidine and a purine from Sigma. ZD1694 was provided by AstraZeneca, and AG331 was provided source are required for protection, consistent with inhibition at two by Agouron Pharmaceuticals Inc. PT523 and 5,8-dideazaPT523 (PT632) were sites (4, 5). Furthermore, in cell lines with acquired resistance to PMX provided by Dr. Andre Rosowsky, Dana-Farber Cancer Institute (Boston, due to increased expression of TS, growth inhibition produced by high MA). All radiochemicals were purified by high-pressure liquid chromatogra- concentrations of drug are unaffected by thymidine but are fully phy (HPLC) before use (14). The stability of radiochemicals, once purified, prevented by a purine alone, consistent with inhibition solely at was checked by HPLC on a regular basis, and materials were repurified as glycinamide ribonucleotide formyltransferase under these conditions necessary. Cell Culture Conditions and Growth Inhibition Studies. The HeLa, R5, (6). and RFC-7 (RFC-transfected R5 derivative) cell lines were described in a previous report (12). Cells were maintained in RPMI 1640 (Hyclone) supple- Received 12/17/03; revised 1/29/04; accepted 2/13/04. mented with 10% fetal bovine serum (Gemini Bio-Products), 2 mM glutamine, Grant support: NIH Grant CA-82621 and a grant from the Eli Lilly Company, which 20 ␮M 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 also provided tritiated pemetrexed and other nonlabeled pemetrexed reagents for these ␮ studies. g/ml) at 37°C in a humidified atmosphere of 5% CO2. The RFC-7 transfec- The costs of publication of this article were defrayed in part by the payment of page tant was maintained in 600 ␮g/ml G418. HeLa and R5 cells were also charges. This article must therefore be hereby marked advertisement in accordance with maintained in folate-free RPMI medium (Hyclone) containing 25 nM 5-CHO- 18 U.S.C. Section 1734 solely to indicate this fact. THF in addition to the supplements described above. Mycoplasma adherent to Requests for reprints: I. David Goldman, Departments of Medicine and Molecular Pharmacology, the Albert Einstein College of Medicine, and the Einstein Cancer Research cells produce folate transport activity with very high affinity for PMX and Center, Bronx, NY 10461. other folates (15). This is particularly prominent in mesothelioma cells, but a 3313 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2004 American Association for Cancer Research. PRESERVED PMX ACTIVITY IN CELLS LACKING THE RFC Fig. 1. Growth inhibition by pemetrexed (PMX; A), raltitrexed (ZD1694; B), AG331 (C), and N␣- (-4-amino-4-deoxypteroyl)-N␦-hemiphthaloyl-1-orni- thine (PT 523; D) in wild-type HeLa and reduced folate carrier-null R5 cells maintained in folic acid- containing medium. HeLa and R5 Cells were grown in RPMI medium containing 2.0 ␮M folic acid and exposed continuously to different concentrations of antifolates for 6 days before cell numbers were quan- tified. The growth rate in the absence of drug was set as 100%. The results in each panel represent the mean Ϯ SE (bars) from three separate experiments. low level of activity can also be detected in HeLa cells contaminated with this was used for determination of total PMX accumulation in cells as described organism. (15). Because of this, cell cultures were monitored regularly with a above. In this case, protein was determined with the Bio-Rad protein assay Mycoplasma detection kit (American Type Culture Collection) and were because 2-mercaptoethanol was present. The remaining portion was boiled for shown to be free of this microorganism. 10 min to inactivate enzymes, and the supernatant containing radiolabeled For assessment of growth inhibition by antifolates, cells were transferred to PMX and its metabolites was separated by centrifugation. After nonlabeled 96-well plates (500 cells/well) and exposed continuously to a spectrum of PMX-monoglutamate, -triglutamate, and -pentaglutamate standards were antifolate concentrations for 6 days. G418 was not included in the medium for added, the solution was injected onto a reversed-phase HPLC column (Waters the RFC-7 line in these experiments. Cell growth rate was quantified by Spherisorb; 5 ␮m ODS2; 4.6 ϫ 250 mm) and processed as reported previously sulforhodamine B staining (16). For measurement of total PMX accumulation, (18). The levels of PMX and its polyglutamate derivatives are expressed in cells were grown either in folic acid or 5-CHO-THF medium containing 50 nM units of pmol/mg of protein in the cells. [3H]PMX, 200 ␮M glycine, 100 ␮M adenine, and 10 ␮M thymidine for 1 week. Intracellular tritium was determined as in the transport studies described below. RESULTS Folic Acid-Binding Assay. Cells near confluence were washed with ice- cold acid buffer [10 mM sodium acetate, 150 mM NaCl (pH 3.5)] followed by Resistance and Cross-Resistance Patterns in RFC-null HeLa a wash with ice cold 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, R5 Cells.