RFC- and FRα-null MTXRIIOuaR2-4 (R2) Chinese hamster ovary (CHO) cells were a gift from Dr. Wayne Flintoff (University of Western Ontario)30 (link) and were cultured in α-minimal essential medium (MEM) supplemented with 10% bovine calf serum (Invitrogen, Carlsbad, CA), penicillin-streptomycin solution and glutamine at 37°C with 5% CO2. PC43-10 cells are R2 cells transfected with human RFC31 (link) and were cultured in α-MEM plus 1.5 mg/ml G418. FRα- (designated RT16) and FRβ- (designated D4) expressing CHO cells were derived from R2 cells by electroporation with FRα (obtained from Manohar Ratnam, Medical University of Ohio) and FRβ (prepared by RT-PCR; see Supplement ) cDNAs in pcDNA3 vector. Cells were cloned, colonies isolated and expanded for screening by western blots (FRα) or real time RT-PCR (FRβ) (Supplement ). The RT16 and D4 sublines were maintained as for PC43-10 cells. Prior to the cytotoxicity assays (see below), RT16 and D4 cells were cultured in complete folate-free RPMI 1640 (without added folate) for three days. KB human cervical cancer and IGROV1 ovarian cancer cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Cells were routinely cultured in folate-free RPMI 1640 medium, supplemented with 10% fetal bovine serum, penicillin-streptomycin solution, 2 mM glutamine at 37°C with 5% CO2.
For growth inhibition assays, cells (CHO, KB, or IGROV1) were plated in 96 well dishes (∼5000 cells/well, total volume of 200 µl medium) with a range of inhibitors including classical antifolates and the 6-substituted pyrrolo[2,3-d]pyrimidine antifolates1–5 . The sources of the classical antifolate drugs were as follows: MTX, Drug Development Branch, National Cancer Institute (Bethesda, MD); RTX [N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thienoyl)-L-glutamic acid], AstraZeneca Pharmaceuticals (Maccesfield, Cheshire, England); LMX (5,10-dideaza-5,6,7,8-tetrahydrofolate) and PMX [N-{4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid] (Alimta), Eli Lilly and Co. (Indianapolis, IN); and GW1843U89 [(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxo-benzo(f)quinazolin-9-yl) methyl) amino)1-oxo-2-isoindolinyl) glutaric acid], GlaxoWellcome-SmithKline Co. (Research Triangle Park, NC). The culture medium was RPMI 1640 (contains 2.3 µM folic acid) with 10% dialyzed serum and antibiotics for experiments with R2 and PC43-10 cells. For RT-16, D4, KB, and IGROV1 cells, cells were cultured in folate-free RPMI media with 10% dialyzed fetal bovine serum (Invitrogen) and antibiotics supplemented with 2 nM LCV (Drug Development Branch, National Cancer Institute, Bethesda, MD). The requirement for FR-mediated drug uptake in these assays was established in parallel incubations including 200 nM folic acid (Sigma Chemical Co., St. Louis, MO). Cells were routinely incubated for up to 96 h, and metabolically active cells (a measure of cell viability) were assayed with CellTiter-blue Cell Viability Assay (Promega) and fluorescence was measured (590 nm emission, 560 nm excitation) with a fluorescence plate reader. Data were exported from Softmax Pro software to an Excel spreadsheet for analysis and determinations of IC50s, corresponding to the drug concentrations that result in 50% loss of cell growth.
For some of the in vitro growth inhibition studies, drug treatments were performed in the presence of higher concentrations (up to 100 nM) of LCV. In others, the inhibitory effects of the antifolate inhibitors on de novo thymidylate biosynthesis (i.e., TS) and de novo purine biosynthesis (GARFTase and AICARFTase) were tested by co-incubations with thymidine (10 µM) and adenosine (60 µM). For de novo purine biosynthesis, additional protection experiments used AICA (9.6–192 µM), as a means of distinguishing inhibitory effects at GARFTase from those at AICARFTase.32 (link)
For assays of colony formation in the presence of the antifolate drugs, KB cells were harvested in log phase, diluted and 100 cells were plated into 60 mm dishes in folate-free RPMI1640 medium supplemented with 2 nM LCV, 10% dialyzed fetal bovine serum, penicillin-streptomycin, and 2 mM glutamine, in the presence of antifolate drugs. The dishes were incubated at 37°C with 5% CO2 for 10 days. At the end of the incubations, the dishes were rinsed with Dulbecco’s phosphate-buffered saline (DPBS), 5% trichloroacetic acid, borate buffer (10 mM, pH 8.8), followed by 30 min incubation in 1% methylene blue in the borate buffer. The dishes were rinsed with the borate buffer and colonies were enumerated for calculating percent colony-forming efficiency normalized to control.
To test the reversibility of colony-forming inhibition, KB cells were cultured in the presence or absence of 1 µM antifolate compounds for two days before rinsing with saline, trypsining and re-inoculating into 60 mm dishes at low and high density (200 and 400, or 2000 and 4000, cells per dish for cells cultured in absence, or presence of antifolate, respectively). The dishes were incubated for 14 days, and colonies were counted for calculating percent colony-forming efficiency.
For growth inhibition assays, cells (CHO, KB, or IGROV1) were plated in 96 well dishes (∼5000 cells/well, total volume of 200 µl medium) with a range of inhibitors including classical antifolates and the 6-substituted pyrrolo[2,3-d]pyrimidine antifolates
For some of the in vitro growth inhibition studies, drug treatments were performed in the presence of higher concentrations (up to 100 nM) of LCV. In others, the inhibitory effects of the antifolate inhibitors on de novo thymidylate biosynthesis (i.e., TS) and de novo purine biosynthesis (GARFTase and AICARFTase) were tested by co-incubations with thymidine (10 µM) and adenosine (60 µM). For de novo purine biosynthesis, additional protection experiments used AICA (9.6–192 µM), as a means of distinguishing inhibitory effects at GARFTase from those at AICARFTase.32 (link)
For assays of colony formation in the presence of the antifolate drugs, KB cells were harvested in log phase, diluted and 100 cells were plated into 60 mm dishes in folate-free RPMI1640 medium supplemented with 2 nM LCV, 10% dialyzed fetal bovine serum, penicillin-streptomycin, and 2 mM glutamine, in the presence of antifolate drugs. The dishes were incubated at 37°C with 5% CO2 for 10 days. At the end of the incubations, the dishes were rinsed with Dulbecco’s phosphate-buffered saline (DPBS), 5% trichloroacetic acid, borate buffer (10 mM, pH 8.8), followed by 30 min incubation in 1% methylene blue in the borate buffer. The dishes were rinsed with the borate buffer and colonies were enumerated for calculating percent colony-forming efficiency normalized to control.
To test the reversibility of colony-forming inhibition, KB cells were cultured in the presence or absence of 1 µM antifolate compounds for two days before rinsing with saline, trypsining and re-inoculating into 60 mm dishes at low and high density (200 and 400, or 2000 and 4000, cells per dish for cells cultured in absence, or presence of antifolate, respectively). The dishes were incubated for 14 days, and colonies were counted for calculating percent colony-forming efficiency.
