Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells
Introduction
Antifolates are competitive inhibitors of their target folate-requiring enzymes within cells. For the classical antifolates, MTX and aminopterin, dihydrofolate that builds up behind the block in DHFR is the basis for the competitive nature of the interaction among these agents, dihydrofolate, and their target enzyme [1]. For new generation antifolate inhibitors of thymidylate synthase and/or GAR transformylase that require formation of polyglutamate derivatives for activity, such as DDATHF, ALIMTA, and ZD1694 [2], [3], [4], the level of endogenous folate pools should modulate the rate and extent of formation of these congeners synthesized by FPGS within cells. Endogenous folate pools also have the potential for diminishing the activity of these agents due to competition at the level of their target enzymes. Low levels of endogenous folates in normal tissues are a determinant of the toxicity of antifolate inhibitors of GAR transformylase, and repleting these pools with added folate in animal models circumvents toxicity and may enhance therapeutic efficacy [5], [6]. Conversely, enhanced accumulation of folate pools within cells that occurs by perturbations of folate transporters can result in markedly diminished antifolate activity [7], [8]. Studies on drug resistance in vitro usually utilize media in which the folate growth substrate is folic acid. This folate has a low affinity for the major folate transporter, RFC1, and enters cells largely by other mechanisms [9]. Recent studies utilizing 5-CHO-THF as the growth substrate demonstrated that ALIMTA or DDATHF activities can be preserved in cell lines resistant to MTX due to impaired RFC1-mediated transport [10], [11], [12]. This was due to selective, partial preservation of transport of these antifolates along with the contraction of the endogenous folate pool that accompanies a decrease in 5-CHO-THF transport [12], [13].
This paper represents the first quantitative analysis of the relationship between levels of cellular folate cofactors and the activities of antifolates, using L1210 murine leukemia cells grown in 5-CHO-THF concentrations that include, and bracket, the physiological blood levels of 5-CH3-THF. These data were then correlated with growth inhibitory activities upon continuous, or brief, exposure to antifolate agents with different enzyme targets, some of which do, while others do not, form polyglutamate derivatives. The data characterize the very critical role that the cellular folate pool can play as a determinant of antifolate activities, findings relevant to the design of clinical regimens in which the co-administration of folate supplements is considered to prevent toxicity.
Section snippets
Chemicals
[3′, 5′, 7-3H]-(6S)-5-CHO-THF was obtained from Moravek Biochemicals, and [3′, 5′, 7-3H]-MTX was purchased from the Amersham Corp. [3H]-ALIMTA (4.1 Ci/mmol), [3H]-DDATHF (3.3 Ci/mmol), ALIMTA, DDATHF, and 6R-2′,5′-thienyl-5,10-dideazatetrahydrofolic acid (LY309887) were provided by Dr. Victor Chen (Eli Lilly). TMQ was a gift from Dr. David Fry (Warner-Lambert). ZD1694 and (2S)-2-{o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)-N
Relationship between the intracellular folate cofactor pool and the extracellular 5-CHO-THF level
L1210 murine leukemia cells were grown with 5-CHO-THF as the folate growth source over extracellular concentrations of from 0.64 to 62.5 nM, a range that encompasses from approximately one-tenth to two times the physiological levels of 5-CH3-THF in humans [16]. As indicated in Fig. 1 , the intracellular folate pool increased in near proportion to the increase in the extracellular folate level. The ratios of the intracellular to extracellular folate concentrations were very high: from 570:1 to
Discussion
This report quantitates the impact that the level of endogenous folate cofactors can have in modulating the activities of antifolate drugs, in particular agents that in their polyglutamyl forms are direct inhibitors of GAR transformylase and/or thymidylate synthase. Increased cellular folate pools were associated with decreased levels of antifolate polyglutamates and diminished drug activities even when the folate pool was modulated within the physiological range. This effect was even more
Acknowledgments
This work was supported by Grants CA-39807 and CA-82621 from the National Cancer Institute.
References (41)
- et al.
Mutations in the reduced folate carrier gene which confer dominant resistance to 5,10-dideazatetrahydrofolate
J Biol Chem
(1998) - et al.
Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells
J Biol Chem
(1997) - et al.
A reduced carrier mutation produces substrate-dependent alterations in carrier mobility in murine leukemia cells and methotrexate resistance with conservation of growth in 5-formyltetrahydrofolate
J Biol Chem
(1998) - et al.
Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier
Biochem Pharmacol
(1999) - et al.
Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells
J Biol Chem
(1997) - et al.
Determinants of the disparate antitumor activities of (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate and methotrexate toward human lymphoblastic leukemia cells, characterized by severely impaired antifolate membrane transport
Biochem Pharmacol
(1993) Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism
Vitam Horm
(1989)- et al.
Biochemical pharmacology of the lipophilic antifolate, trimetrexate
Adv Enzyme Regul
(1984) - et al.
The 6S- and 6R-diastereomers of 5,10-dideaza-5,6,7,8-tetrahydrofolate are equiactive inhibitors of de novo purine synthesis
J Biol Chem
(1989) - et al.
A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis
J Biol Chem
(1989)
Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates
J Biol Chem
Studies with a mathematical model of folate metabolism
Arch Biochem Biophys
Reversal of methotrexate binding to dihydrofolate reductase by dihydrofolate. Studies with pure enzyme and computer modeling using network thermodynamics
J Biol Chem
Cellular folates prevent polyglutamation of 5,10- dideazatetrahydrofolate. A novel mechanism of resistance to folate antimetabolites
J Biol Chem
Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts
Biochem Pharmacol
Mechanism of action of methotrexate. IV. Free intracellular methotrexate required to suppress dihydrofolate reduction to tetrahydrofolate by Ehrlich ascites tumor cells in vitro
Mol Pharmacol
Intracellular metabolism of 5,10-dideazatetrahydrofolic acid in human leukemia cell lines
Mol Pharmacol
LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes
Cancer Res
ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivoa new agent for clinical study
Cancer Res
Augmentation of the therapeutic activity of lometrexol [(6-R)5,10-dideazatetrahydrofolate] by oral folic acid
Cancer Res
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2012, Drug Resistance UpdatesCitation Excerpt :Consequently, several other studies consistently showed that elevated cellular folate pools result in antifolate resistance mediated by diminished polyglutamylation (Assaraf and Goldman, 1997; Assaraf et al., 2003; Drori et al., 2000; Jansen et al., 1998; Tse and Moran, 1998). In this respect, a dedicated study was conducted, aimed at evaluating the impact of the intracellular THF-cofactor pool size on the activity of various polyglutamatable and non-polyglutamatable antifolates (Zhao et al., 2001a). Murine leukemia L1210 cells were grown in different concentrations of leucovorin and it was demonstrated that the intracellular folate pool increased in proportion to the elevation in the extracellular leucovorin concentration.
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