Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake

S Drori, G Jansen, R Mauritz, G J Peters, Y G Assaraf

Research output: Contribution to journalArticleAcademicpeer-review

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 languageEnglish
Pages (from-to)30855-63
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number40
DOIs
Publication statusPublished - 6 Oct 2000

Cite this

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title = "Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake",
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.",
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/analogs & derivatives, Folic Acid Antagonists/metabolism, 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",
author = "S Drori and G Jansen and R Mauritz and Peters, {G J} and Assaraf, {Y G}",
year = "2000",
month = "10",
day = "6",
doi = "10.1074/jbc.M003988200",
language = "English",
volume = "275",
pages = "30855--63",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "40",

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Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake. / Drori, S; Jansen, G; Mauritz, R; Peters, G J; Assaraf, Y G.

In: Journal of Biological Chemistry, Vol. 275, No. 40, 06.10.2000, p. 30855-63.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake

AU - Drori, S

AU - Jansen, G

AU - Mauritz, R

AU - Peters, G J

AU - Assaraf, Y G

PY - 2000/10/6

Y1 - 2000/10/6

N2 - 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.

AB - 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.

KW - Antimetabolites, Antineoplastic/pharmacology

KW - Biological Transport

KW - Blotting, Northern

KW - Blotting, Southern

KW - Blotting, Western

KW - Carrier Proteins/chemistry

KW - Cell Division/drug effects

KW - Cell Membrane/metabolism

KW - Chlorides/pharmacology

KW - DNA Mutational Analysis

KW - DNA, Complementary/metabolism

KW - Dose-Response Relationship, Drug

KW - Drug Resistance, Neoplasm/genetics

KW - Enzyme Inhibitors/pharmacology

KW - Exons

KW - Folic Acid/analogs & derivatives

KW - Folic Acid Antagonists/metabolism

KW - Humans

KW - Indoles/chemistry

KW - Inhibitory Concentration 50

KW - Isoindoles

KW - Kinetics

KW - Leucovorin/pharmacology

KW - Leukemia/genetics

KW - Membrane Proteins

KW - Membrane Transport Proteins

KW - Methotrexate/chemistry

KW - Mutagenesis, Site-Directed

KW - Mutation

KW - Polymorphism, Single-Stranded Conformational

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Quinazolines/chemistry

KW - Recombinant Proteins/chemistry

KW - Reduced Folate Carrier Protein

KW - Thymidylate Synthase/antagonists & inhibitors

KW - Time Factors

KW - Transfection

KW - Trimetrexate/pharmacology

KW - Tumor Cells, Cultured

U2 - 10.1074/jbc.M003988200

DO - 10.1074/jbc.M003988200

M3 - Article

VL - 275

SP - 30855

EP - 30863

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 40

ER -