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 n m) rather than at ‘standard’ supraphysiological concentrations of 2–10 μ m folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport pro...

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Published in:	Advances in enzyme regulation 1997, Vol.37, p.59-76
Main Authors:	Jansen, Gerrit, Mauritz, Robert M., Assaraf, Yehuda G., Sprecher, Hannah, Drori, Stavit, Kathmann, Ietje, Westerhof, G.Robbin, Priest, David G., Bunni, Marlene, Pinedo, Herbert M., Schornagel, Jan H., Peters, Godefridus J.
Format:	Article
Language:	eng
Subjects:	Adenosine - pharmacology Biological Transport - drug effects Biological Transport - physiology Carrier Proteins - metabolism Cell Division - drug effects Drug Resistance, Neoplasm Enzyme Inhibitors - pharmacology Folic Acid - analogs & derivatives Folic Acid - metabolism Folic Acid - pharmacology Folic Acid Antagonists - metabolism Folic Acid Antagonists - pharmacology Humans Kinetics Leucovorin - pharmacology Membrane Proteins Membrane Transport Proteins Methotrexate - metabolism Methotrexate - pharmacology Reduced Folate Carrier Protein S-Adenosylmethionine - pharmacology Tetrahydrofolates - metabolism Thymidylate Synthase - antagonists & inhibitors Tumor Cells, Cultured
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Summary:	Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1–5 n m) rather than at ‘standard’ supraphysiological concentrations of 2–10 μ m 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 V max 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 over-expression 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 n m LV), purines (100 μ m adenosine) or S-adenosylmethionine (100 μ m), 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 K m for folic acid and a 10-fold decreased K m 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 μ m 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.
ISSN:	0065-2571 1873-2437