title: Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis.
abstract: We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.
authors.affiliation: Department of Medical Oncology, VU Medical Center, Amsterdam, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Mauritz
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.forename: David G
authors.initials: DG
authors.lastname: Priest
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.forename: Stavit
authors.initials: S
authors.lastname: Drori
authors.forename: Ietje
authors.initials: I
authors.lastname: Kathmann
authors.forename: Paul
authors.initials: P
authors.lastname: Noordhuis
authors.forename: Marlene A
authors.initials: MA
authors.lastname: Bunni
authors.forename: Andre
authors.initials: A
authors.lastname: Rosowsky
authors.forename: Jan H
authors.initials: JH
authors.lastname: Schornagel
authors.forename: Herbert M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
mesh_terms: D001692:Biological Transport; D018432:Drug Resistance, Multiple / Q000502:physiology*; D004354:Drug Screening Assays, Antitumor; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006706:Homeostasis; D006801:Humans; D007938:Leukemia; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000068437:Pemetrexed; D010453:Peptide Synthases / Q000378:metabolism; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D011622:Pterins / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D013763:Tetrahydrofolates / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism
chemical_list: D005493:Folic Acid Antagonists; D005971:Glutamates; D011622:Pterins; D011799:Quinazolines; D013763:Tetrahydrofolates; D013876:Thiophenes; C094299:ZD 9331; D000068437:Pemetrexed; C085590:N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine; D011099:Polyglutamic Acid; D006147:Guanine; C045894:lometrexol; C014085:methotrexate polyglutamate; D005492:Folic Acid; D009952:Ornithine; D013940:Thymidylate Synthase; D011623:gamma-Glutamyl Hydrolase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; C068874:raltitrexed; D008727:Methotrexate