TY - JOUR
T1 - A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance
AU - Jansen, G
AU - Mauritz, R
AU - Drori, S
AU - Sprecher, H
AU - Kathmann, I
AU - Bunni, M
AU - Priest, D G
AU - Noordhuis, P
AU - Schornagel, J H
AU - Pinedo, H M
AU - Peters, G J
AU - Assaraf, Y G
PY - 1998/11/13
Y1 - 1998/11/13
N2 - 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.
AB - 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.
KW - Affinity Labels/metabolism
KW - Biological Transport
KW - Blotting, Northern
KW - Carrier Proteins/chemistry
KW - Drug Resistance, Neoplasm/genetics
KW - Folic Acid/metabolism
KW - Folic Acid Antagonists/pharmacology
KW - Glutamates/pharmacology
KW - Humans
KW - Kinetics
KW - Leukemia/metabolism
KW - Membrane Proteins
KW - Membrane Transport Proteins
KW - Methotrexate/metabolism
KW - Pyrimethamine/pharmacology
KW - Pyrimidines/pharmacology
KW - Reduced Folate Carrier Protein
KW - Structure-Activity Relationship
KW - Tetrahydrofolates/pharmacology
KW - Trimetrexate/pharmacology
KW - Tumor Cells, Cultured
M3 - Article
C2 - 9804775
VL - 273
SP - 30189
EP - 30198
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 46
ER -