ANTICANCER RESEARCH 27: 769-774 (2007) Antifolate Pseudo-resistance Due to Elevated Levels of Thymidine and Hypoxanthine in a Commercial Serum Preparation MARCEL SIMON1, JOHANNES BLATTER2 and CHRISTOF GRANZOW1 1Department Molekulare Toxikologie (G120), Deutsches Krebsforschungszentrum, D-69120 Heidelberg; 2Lilly Deutschland GmbH, Medical Oncology, D-61350 Bad Homburg, Germany Abstract. Background: Batch variability of sera used for cell It has long been recognized that extracellular nucleosides culture is of considerable experimental concern. A novel fetal calf allow salvage nucleotide synthesis that circumvents the serum product, FCS Gold, was claimed to be the first defined fetal inhibitory effects of antifolate drugs (3, 4). To eliminate calf serum free of batch variation. Materials and Methods: The extracellular nucleoside sources in vitro, the use of efficacy of methotrexate (MTX) and LY231514 (multitargeted thymidine-less media and of dialyzed serum has been antifolate, MTA) in CCRF-CEM cells and KB cells was proposed (4). Alternatively, in test settings requiring compared using media supplemented with FCS Gold or undialyzed serum, it has been suggested to minimize the conventional fetal bovine serum. Results: IC50 values from tests impact of extracellular nucleosides by increasing cell using conventional serum corresponded to published data. FCS numbers, which may lead to initial, rapid depletion of the Gold fully protected the cells from antifolate drug cytotoxicity. extracellular nucleoside pool (5). Dialysis of FCS Gold restored responsiveness to antifolate drugs. Here, we show the salvage from MTX and MTA action Elevated levels of hypoxanthine and thymidine were present in on two frequently used, drug-sensitive continuous human FCS Gold. They were approximately 10-fold greater than the cell lines by a novel, fractionated and reconstituted fetal calf concentrations required to overcome growth arrest mediated by 2 serum preparation containing elevated levels of thymidine ÌM MTX. Conclusion: FCS Gold or identical products, e.g. FBS and hypoxanthine. Gold, should not be used in studies on antifolate drug action. Materials and Methods Methotrexate (MTX) and LY231514 (multitargeted antifolate, MTA) are antimetabolite drugs inhibiting folate-requiring Chemicals, reagents and cell culture media. If not specified enzymes essential for de novo thymidine and purine synthesis. otherwise, suppliers and manufacturers mentioned resided in Germany. Aqua ad iniectabilia, HEPES (4-(2-hydroxyethyl)-1- The major MTX target is dihydrofolate reductase (1). MTA piperazinethansulfonic acid), trypsin 1:250, bovine serum albumin has been shown to inhibit dihydrofolate reductase, thymidylate fraction V and L-glutamine were products of Braun (Melsungen), synthase and glycinamide ribonucleotide formyltransferase (2). Carl Roth (Karlsruhe), Difco (Detroit, MI, USA), Sigma (Munich) and Biochrom (Berlin), respectively. Hypoxanthine p.a. and thymidine p.a. were purchased from Calbiochem (Schwalbach). Methotrexate (MTX) was obtained as a 5.5 mM pharmaceutical Disclosure: None of the authors have anything to disclose with solution from Lederle Arzneimittel (Münster). LY231514 was respect to the subject matter in the manuscript. provided in powder form by Eli Lilly (Bad Homburg). Minimum essential medium modified for suspension cultures (MEM-S) and Abbreviations: IC50, concentration inhibiting growth by 50 percent; RPMI 1640 medium were prepared, in soluble or powder form, by MEM-S, minimum essential medium for suspension culture; MTA, Biochrom (Berlin). Deviating from the original formulation, multitargeted antifolate; MTX, methotrexate; RPMI 1640, Roswell riboflavin, glutamine and phenol red were omitted. Park Memorial Institute medium 1640. Sera, serum handling and serum metabolite contents. Fetal bovine Correspondence to: Prof. Christof Granzow, DKFZ (G120), Im serum (FBS) was obtained from Integro (Zaandam, The Neuenheimer Feld 280, D-69120 Heidelberg, Germany. e-mail: Netherlands). Here, it is designated FBS Integro [cf. (6)]. The fetal [email protected] calf serum preparation FCS Gold (lot Nos A01120-151, A01120- 650 and A01120-711) was obtained from PAA (Coelbe). An Key Words: Antifolate resistance, antifolate salvage, FBS Gold, identical product is being marketed as FBS Gold by the same FCS Gold, fetal bovine serum, KB cells, CCRF-CEM cells. supplier. Sera were shipped in dry ice and stored at –25ÆC. Prior 0250-7005/2007 $2.00+.40 769 ANTICANCER RESEARCH 27: 769-774 (2007) to use, the contents of individual serum flasks were slowly thawed Table I. Proliferation of CCRF-CEM cells in MEM-S supplemented with at 8ÆC and used within 4 weeks. Normally, undialyzed serum was FBS Integro (n=4). used. For some experiments, sera were dialyzed in the dark at 4ÆC for 24 h against 100 volumes of RPMI 1640 (three changes) using Inocula Duration of Cell numbers Cell numbers Doubling Visking type 20/32 dialysis tubing (molecular weight cut-off 14000 (x104 exponential reached while after time Dalton; Carl Roth, Karlsruhe). Dialyzed sera were used immediately. cells/well) growth (h) growing incubation (h) The concentrations of folate, thymidine and hypoxanthine were 0.022 exponentially for 72 h 4 4 ÌM, 0.8 ÌM and 22.8 ÌM in FBS Integro, and 0.025 ÌM, 63 ÌM and (x10 /well) (x10 /well) 75 ÌM in FCS Gold, as determined by Laborärzte Limbach, Schmidt- 4 84 64.20±1.61 45.33±0.29 21.53 Gayk and Coll., Heidelberg, Germany. 8 84 117.30±0.60 90.89±1.81 21.87 16 72 153.30±5.35 153.30±5.35 22.00 Cell lines, preparation of culture media, cell culture and photochemical precautions. CCRF-CEM T-cell leukemia cells and KB cells were obtained from the American Type Culture Collection (Bethesda, MD, USA) and grown in MEM-S and RPMI indicated. Concentration gradients of drugs were formed by adding 1640 media, respectively. Powder media were processed according 1% (v/v) of 100-fold concentrated drug solutions. In some to (6). No antibiotics were used. The final concentrations of experiments, only one very high drug concentration was tested. Cell sodium bicarbonate and HEPES were 9 mM and 3 mM for MEM- suspension was added to a density of 5x104 cells/well for CCRF- S, and 13.5 mM and 4.5 mM for RPMI 1640, respectively. Media CEM cells and of 3x104 cells/well for KB cells. Cell numbers were with 1 mM L-glutamine were completed by adding, under the determined after incubation under standard conditions for 72 exclusive illumination from a 30 W SOX sodium discharge lamp hours. Control wells produced 5x105 to 8x105 CCRF-CEM cells, (Philips, Hamburg), 10% (v/v) of the serum preparation indicated, and 2x105 to 5x105 KB cells. Growth inhibition was expressed as dispensed to brown glass bottles (Schott, Mainz), kept in the dark at percentage of growth compared to untreated controls. If feasible, 8ÆC and used within 10 days. Cells were subcultivated at alternating the concentration resulting in 50% growth inhibition (IC ) was intervals of 3 and 4 days, using respective inocula of 5x105 and 2x105 50 read from percentage of growth vs. log drug molarity graphs. CCRF-CEM suspension cells and of 3x105 and 2x105 trypsinized KB monolayer cells per 10 mL of culture medium. Cultures were Salvage of CCRF-CEM cells from MTX action by thymidine and incubated under air in sealed 25 cm2 plastic tissue culture flasks hypoxanthine. The interference of added thymidine or (Greiner, Frickenhausen) at 36.5ÆC. Cell numbers were determined hypoxanthine with MTX action was studied by identifying the using a Casy I cell analyser and Casystat software (Schärfe System, concentrations of these metabolites required to abolish MTX- Reutlingen). Only CCRF-CEM cells and KB cells with respective mediated growth inhibition of CCRF-CEM cells. MEM-S diameters between 8 Ìm and 30 Ìm and between 10 Ìm and 30 Ìm supplemented with undialyzed FBS Integro was used. Test were considered. To prevent photochemical artifacts, all concentrations of thymidine and hypoxanthine were chosen procedures involving sera, serum-containing media and/or according to (2). The thymidine concentration required was antifolate drugs were performed under flavin-protecting conditions determined by preparing quadruplicate wells containing MTX (2 (6). Tests were performed in 24-well cell culture plates (Greiner, ÌM), hypoxanthine (100 ÌM) and the thymidine concentrations Frickenhausen) using total volumes per well of 1 mL and 2 mL for indicated. The hypoxanthine concentration required was CCRF-CEM cells and KB cells, respectively. determined using MTX (2 ÌM), thymidine (5 ÌM) and the hypoxanthine concentrations indicated. Cell densities were Determination of proliferation kinetics in CCRF-CEM cells. Inocula determined after incubation for 72 h. of 4x104, 8x104 and 16x104 CCRF-CEM cells/well suspended in MEM-S supplemented with FBS Integro were seeded, in duplicate, into seven cell culture plates and incubated under standard Results conditions (36.5ÆC, 2.5% CO2 in humidified air). Every 12 h, cells of one of the plates were counted. Doubling times were read from Kinetics of CCRF-CEM cell proliferation. From inocula of up log cell number vs. time graphs. to 16x104 cells/well, CCRF-CEM cells grew in FBS Integro- supplemented media exponentially for at least 72 h with Test for the influence of serum preparations on cell proliferation. In population doubling times between 21.5 and 22 h (Table I). duplicate tests, inocula of 5x104 CCRF-CEM cells/well and 3x104 KB cells/well were suspended in the appropriate media supplemented with the serum preparation indicated and incubated Effect of different sera on KB cell growth. As shown in Table II, as above. Cell counts were performed after 72 h. KB cells grew fastest in media supplemented with FBS Integro. With respect to supporting KB cell growth, minor differences Cytotoxicity assessment. Dilutions of the pharmaceutical MTX existed between the three charges of FCS Gold tested. solution or of frozen aliquots of a 1 mM aqueous MTA stock solution were prepared in aqua ad iniectabilia containing 0.2 mg bovine serum albumin fraction V per mL. After appropriate drug Effects of different sera and of serum dialysis on the growth of CCRF-CEM cells.