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Blood, Vol. 113, Issue 18, 4362-4369, April 30, 2009
Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia
Blood Stark et al.
113: 4362
Supplemental materials for: Stark et al
Antifolate growth inhibition assay
Parental cells and their antifolate-resistant sublines as well as FPGS-transfected cells were first grown in antifolate-free growth medium for 5–7 cell doublings. Thereafter, cells were seeded in 96-well plates (3 × 104/well) in growth medium (100µl/well) containing various concentrations of the different antifolates (e.g. MTX, ZD1694 and ZD9331). After 3 days of incubation at 37°C, viable cell numbers were determined using the Cell Proliferation Kit (XTT) (Biological Industries). Percent inhibition of cell growth was calculated relative to untreated controls. Results presented are means of at least three independent experiments. [3H]MTX transport assay
[3H]MTX influx rates were determined as previously described.1 Briefly, cells (2 × 107) were washed with ice-cold HBS (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose, at pH 7.4) and incubated at 37°C for 3 min in HBS containing 2 µM [3′,5′,7-3H]-Methotrexate (0.555 TBq/mmol; 26.7 Ci/mmol; Moravek Biochemicals, Brea, California). Transport controls contained a 500-fold excess (1 mM) of unlabeled MTX. Transport was stopped by the addition of 10 ml of ice-cold HBS. Then, the cell suspension was centrifuged, washed with ice-cold HBS and suspended in water for scintillation counting. FPGS activity assay
The catalytic activity of FPGS was determined as described previously.2 In short, frozen cell pellets (2 × 107 cells) were suspended in 0.4 ml extraction buffer containing: 50 mM Tris-HCl, 20 mM KCl, 10 mM MgCl2 and 5 mM dithiotreitol, at pH 7.5. Crude cell extracts were prepared by sonication (MSE Soniprep, amplitude 6, 3 × 5 sec with 30 sec intervals, at 4°C) followed by centrifugation at 12,000× g for 15 min at 4°C. The activity assay mixture consisted of: 200 µg protein, 4 mM [2,3-3H]-L-glutamic acid (NEN Life Science Products, Boston, MA) and 250 µM MTX in a buffer containing: 0.5 M Tris-HCl, 50 mM ATP, 100 mM MgCl2, 100 mM KCl, and 5 mM dithiotreitol at a pH of 8.5. Following 2 h incubation at 37°C, the reaction was terminated by adding 1 ml of an ice-cold solution containing 5 mM unlabeled L-glutamic acid. Sep-Pak C18 cartridges (Millipore, Waters Associates, Etten-Leur, The Netherlands) were used in order to eliminate free [3H]-L-glutamate. Northern blot analysis
Aliquots (30 µg) of total RNA isolated as described above from parental CCRF-CEM cells and their various antifolate-resistant sublines and transfectants were denatured at 68°C for 10 min in a MOPS-formamide buffer and fractionated by electrophoresis on 1.2% agarose gels containing formaldehyde. Resolved RNA was then capillary blotted onto Zeta-ProbeR-GT nylon membrane (Bio-Rad) and immobilized onto the nylon membrane by UV cross-linking. The nylon membrane was stained with methylene blue in order to verify equal loading and transfer. A 567 bp FPGS cDNA probe prepared by PCR with primers EX3-up and EX10-dw (Table 1) was gel-purified using the Wizard SV Gel and PCR Clean-up kit (Promega) and [32P]labeled using Random Primer DNA Labeling Mix (Biological Industries, Beth Haemek, Israel). The blot was then hybridized over night with the above probe in glass cylinders in EZ-Hybridization Solution (Biological Industries, Beth Haemek, Israel) at 0.1 ml buffer/cm2 at 68°C. Following hybridization, the membrane was washed under high stringency conditions with a final wash in a solution of 0.1 × SSC/0.1%SDS at 65°C for 30 min. The membrane was then visualized by phosphorimaging and the intensity of the FPGS transcript was estimated by scanning densitometry and normalization to the 18S ribosomal RNA band. DNA sequencing
PCR products were first purified using the Wizard SV Gel and PCR Clean-up kit according to the instructions of the manufacturer (Promega). Then, DNA sequencing was performed at HyLabs laboratories (Rehovot, Israel) using BigDye Terminator Cycle Sequencing Kit from ABI. REFERENCES 1. Jansen G, Westerhof GR, Jarmuszewski MJ, Kathmann I, Rijksen G, Schornagel JH. Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates. J Biol Chem. 1990;265:18272–18277.
2. Li WW, Lin JT, Schweitzer BI, Tong WP, Niedzwiecki D, Bertino JR. Intrinsic resistance to methotrexate in human soft tissue sarcoma cell lines. Cancer Res. 1992;52:3908–3913.
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