Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis
Introduction
The classical antifolate MTX disrupts cellular folate metabolism by potent inhibition of its target enzyme, dihydrofolate reductase (DHFR) [1], [2]. In addition, polyglutamate forms of MTX have also a marked inhibitory effect on TS and enzymes in the de novo biosynthesis of purines [3], [4].
Intrinsic and acquired resistance to MTX has been studied extensively in in vitro and animal model systems [5], [6]. The major causes of resistance to MTX are: (a) qualitative and quantitative alterations in DHFR activity [7], [8], [9]; (b) impaired membrane transport of MTX into the cell via the RFC [10], [11], [12], [13] or increased efflux via members of the multidrug resistance protein family [14], [15]; and (c) decreased polyglutamylation either due to decreased activity of folylpolyglutamate synthetase (FPGS) [16], [17], [18] or increased activity of folylpolyglutamate hydrolase (FPGH) [19], [20]. More recently, MTX resistance has been associated with the loss of functional retinoblastoma protein, which can lead to increased levels of free E2F-1, which is involved in the transcriptional regulation of the DHFR and TS genes [21].
In recent years, the number of studies concerning clinical resistance to MTX has increased steadily [1], [5], [6], [10], [13], [20], [22], [23]. To overcome resistance to MTX, a great deal of effort has been put into the development of novel antifolates that were rationally designed to (a) inhibit crucial DHFR-independent enzymes in folate metabolism, such as TS; (b) exhibit a different route of cellular uptake; and/or (c) display either none or, in contrast, high FPGS substrate affinity [24], [25], [26]. ZD1694 (raltitrexed; Tomudex®) is approved for clinical use [27], [28], whereas other novel antifolates including GW1843U89 [29], [30] and MTA (pemetrexed, LY231514 ALIMTA) [31], [32], [33], [34] are currently being evaluated in advanced clinical trials. ZD1694 and GW1843U89 are potent TS inhibitors that are superior to MTX with respect to transport via the RFC and substrate specificity for FPGS [27], [29], [35]. The pyrrolopyrimidine antifolate MTA has multiple cellular targets, including DHFR, TS and glycinamide ribonucleotide formyltransferase (GARFT) and it is an excellent substrate for FPGS [31]. In contrast, PT523 and ZD9331 are non-polyglutamatable inhibitors of DHFR and TS, respectively; both enter the cell via the RFC [24], [36], [37], [38]. DDATHF is a GARFT inhibitor that is a better substrate for FPGS than MTX [39].
Some of these novel antifolates have shown promising activity in clinical trials [25], [28], [30], [32]. However, limited information is available about how rapidly and by what mechanism(s) resistance to these drugs develops. In the present study, we characterized the mechanisms underlying resistance of human CCRF-CEM leukemia cells that were selected for resistance to this panel of drugs using clinically relevant exposure schedules. Moreover, we analyzed the impact of these antifolate resistance mechanisms on cellular folate homeostasis.
Section snippets
Drugs and biochemicals
RPMI 1640 cell culture medium, with and without folic acid, and (dialyzed) fetal calf serum (FCS) were obtained from Life Technologies, Inc. Folic acid was purchased from Sigma. MTX was a gift from Pharmachemie. ZD1694 (raltitrexed (N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-l-glutamic acid), Tomudex®, property of Zeneca Limited (part of AstraZeneca)) and ZD9331 ((2S)-2-[o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl)methyl)-N
Resistance to intermittent exposures to MTX, ZD1694, MTA, GW1843U89, DDATHF and ZD9331
In order to develop antifolate-resistant sublines, human CCRF-CEM leukemia cells were repeatedly exposed to MTX, ZD1694, MTA, GW1843U89 and DDATHF for 24 hr and to the non-polyglutamatable antifolate ZD9331 for 72 hr (Table 1). We noted a marked difference in the number of drug exposures that was required for the development of resistance; only 3–4 cycles of 24 hr pulse exposures for the polyglutamatable drugs MTX, ZD1694, MTA, GW1843U89 and DDATHF, but 8 cycles of 72 hr pulse exposures for ZD9331 (
Discussion
In this study, we demonstrated that acquired resistance to various novel antifolates may consist of different mechanisms (Table 4) depending on the drug and the method of induction of resistance. Beyond this, these antifolate resistance mechanisms can have a marked impact on cellular folate homeostasis.
In human CCRF-CEM leukemia cell lines that were made resistant to MTX, ZD1694, MTA and DDATHF by intermittent drug exposure, the development of drug resistance was remarkably rapid. The
Acknowledgements
This work was supported by The Netherlands Organization for Scientific Research (NWO 920-03-079) and the Dutch Cancer Society (Grant NKB-VU-2000-2237).
References (76)
- et al.
The clinical pharmacology of methotrexate
Cancer Treat. Rev.
(1983) - et al.
Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates
J. Biol. Chem.
(1985) - et al.
Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate
J. Biol. Chem.
(1987) - et al.
Acquired methotrexate resistance in lymphoblasts resulting from altered kinetic properties of dihydrofoltate reductase
Eur. J. Cancer
(1977) - et al.
Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells
J. Biol. Chem.
(1978) - et al.
Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line
J. Biol. Chem.
(1988) - et al.
The role of impaired transport in (pre)clinical resistance to methotrexate—insights on new antifolates
Drug Resistance Updates
(1998) - et al.
A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates
J. Biol. Chem.
(1984) - et al.
Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines
J. Biol. Chem.
(1991) - et al.
Role of folylpolyglutamate synthetase and folylpolyglutamate hydrolase in methotrexate accumulation and polyglutamylation in childhood leukemia
Blood
(1999)
Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates
Blood
Efficient utilization of the reduced folate carrier in CCRF-CEM human leukemic lymphoblasts by the potent antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) and its B-ring analogues
Biochem. Pharmacol.
A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis
J. Biol. Chem.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
Importance of pharmacodynamics in the in vitro antiproliferative activity of the antifolates methotrexate and 10-ethyl-10-deazaaminopterin against human head and neck squamous cell carcinoma
Biochem. Pharmacol.
Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier: selective effect on carrier-mediated transport of physiological concentrations of reduced folates
J. Biol. Chem.
Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells
J. Biol. Chem.
Impact of dietary folic acid on reduced folates in mouse plasma
Biochem. Pharmacol.
Mechanisms of resistance to N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-l-glutamic acid (ZD1694), a folate-based thymidylate synthase inhibitor, in the HCT-8 human ileocecal adenocarcinoma cell line
Biochem. Pharmacol.
Identification of three key active site residues in the C-terminal domain of human recombinant folylpoly-gamma-glutamate synthetase by site-directed mutagenesis
J. Biol. Chem.
Molecular analysis of murine leukemia cell lines resistant to 5,10-dideazatetrahydrofolate identifies several amino acids critical to the function of folylpolyglutamate synthetase
J. Biol. Chem.
Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines
Biochem. Pharmacol.
A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance
J. Biol. Chem.
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
J. Biol. Chem.
Mutations in the reduced folate carrier gene which confer dominant resistance to 5,10-dideazatetrahydrofolate
J. Biol. Chem.
Cellular folates prevent polyglutamation of 5,10-dideazatetrahydrofolate—a novel mechanism of resistance to folate antimetabolites
J. Biol. Chem.
The effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in L1210 cells: a biochemical basis of the selectivity of leucovorin rescue
J. Biol. Chem.
Karnofsky memorial lecture: ode to methotrexate
J. Clin. Oncol.
Intrinsic and acquired resistance to methotrexate in acute leukemia
N. Engl. J. Med.
Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant L1210 clonal cell lines derived in vivo
Cancer Res.
Characterization of the individual transport routes that mediate the influx and efflux of methotrexate in CCRF-CEM human lymphoblastic cells
Cancer Res.
Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2
Cancer Res.
MRP3, an organic anion transporter able to transport anti-cancer drugs
Proc. Natl. Acad. Sci. U.S.A.
Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug
Cancer Res.
Acquisition of resistance to antifolates caused by enhanced gamma-glutamyl hydrolase activity
Cancer Res.
Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines
Proc. Natl. Acad. Sci. U.S.A.
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2015, Journal of Nutritional BiochemistryCitation Excerpt :The GGH mRNA concentrations were significantly higher in the quercetin-supplemented group compared to controls. The quercetin-induced up-regulation of GGH mRNA concentration might be of importance because increased expression levels of GGH were repeatedly reported to be associated with resistance to methotrexate [42–46]. GGH catalyzes the hydrolysis of (anti)folylpoly-gamma-glutamates by the removal of gamma-linked polyglutamates [37].