Abstract
CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 microM to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type Km value for MTX transport but a 31-fold and 9-fold lower Km values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G --> A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 microM folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.
Original language | English |
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Pages (from-to) | 30189-98 |
Number of pages | 10 |
Journal | Journal of Biological Chemistry |
Volume | 273 |
Issue number | 46 |
Publication status | Published - 13 Nov 1998 |
Keywords
- Affinity Labels/metabolism
- Biological Transport
- Blotting, Northern
- Carrier Proteins/chemistry
- Drug Resistance, Neoplasm/genetics
- Folic Acid Antagonists/pharmacology
- Folic Acid/metabolism
- Glutamates/pharmacology
- Humans
- Kinetics
- Leukemia/metabolism
- Membrane Proteins
- Membrane Transport Proteins
- Methotrexate/metabolism
- Pyrimethamine/pharmacology
- Pyrimidines/pharmacology
- Reduced Folate Carrier Protein
- Structure-Activity Relationship
- Tetrahydrofolates/pharmacology
- Trimetrexate/pharmacology
- Tumor Cells, Cultured