Abstract
We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.
Original language | English |
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Pages (from-to) | 30855-63 |
Number of pages | 9 |
Journal | Journal of Biological Chemistry |
Volume | 275 |
Issue number | 40 |
DOIs | |
Publication status | Published - 6 Oct 2000 |
Keywords
- Antimetabolites, Antineoplastic/pharmacology
- Biological Transport
- Blotting, Northern
- Blotting, Southern
- Blotting, Western
- Carrier Proteins/chemistry
- Cell Division/drug effects
- Cell Membrane/metabolism
- Chlorides/pharmacology
- DNA Mutational Analysis
- DNA, Complementary/metabolism
- Dose-Response Relationship, Drug
- Drug Resistance, Neoplasm/genetics
- Enzyme Inhibitors/pharmacology
- Exons
- Folic Acid Antagonists/metabolism
- Folic Acid/analogs & derivatives
- Humans
- Indoles/chemistry
- Inhibitory Concentration 50
- Isoindoles
- Kinetics
- Leucovorin/pharmacology
- Leukemia/genetics
- Membrane Proteins
- Membrane Transport Proteins
- Methotrexate/chemistry
- Mutagenesis, Site-Directed
- Mutation
- Polymorphism, Single-Stranded Conformational
- Protein Structure, Secondary
- Protein Structure, Tertiary
- Quinazolines/chemistry
- Recombinant Proteins/chemistry
- Reduced Folate Carrier Protein
- Thymidylate Synthase/antagonists & inhibitors
- Time Factors
- Transfection
- Trimetrexate/pharmacology
- Tumor Cells, Cultured