Regulation of carrier-mediated transport of folates and antifolates in methotrexate-sensitive and-resistant leukemia cells.
Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.
Jansen
G
G
Department of Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Mauritz
R M
RM
Assaraf
Y G
YG
Sprecher
H
H
Drori
S
S
Kathmann
I
I
Westerhof
G R
GR
Priest
D G
DG
Bunni
M
M
Pinedo
H M
HM
Schornagel
J H
JH
Peters
G J
GJ
D000241:Adenosine / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D002955:Leucovorin / Q000494:pharmacology; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism* / Q000494:pharmacology*; D058978:Reduced Folate Carrier Protein; D012436:S-Adenosylmethionine / Q000494:pharmacology; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D013763:Tetrahydrofolates; D012436:S-Adenosylmethionine; D005492:Folic Acid; D013940:Thymidylate Synthase; D000241:Adenosine; D002955:Leucovorin; C005984:5-methyltetrahydrofolate; D008727:Methotrexate