Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis

Biochem Pharmacol. 2002 Jan 15;63(2):105-15. doi: 10.1016/s0006-2952(01)00824-3.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Drug Resistance, Multiple / physiology*
  • Drug Screening Assays, Antitumor
  • Folic Acid / metabolism
  • Folic Acid Antagonists / pharmacology
  • Glutamates / pharmacology
  • Guanine / analogs & derivatives
  • Guanine / pharmacology
  • Homeostasis*
  • Humans
  • Leukemia
  • Methotrexate / analogs & derivatives*
  • Methotrexate / metabolism
  • Methotrexate / pharmacology*
  • Ornithine / analogs & derivatives*
  • Ornithine / pharmacology
  • Pemetrexed
  • Peptide Synthases / metabolism
  • Polyglutamic Acid / analogs & derivatives*
  • Polyglutamic Acid / metabolism
  • Pterins / pharmacology
  • Quinazolines / pharmacology
  • Tetrahydrofolates / pharmacology
  • Thiophenes / pharmacology
  • Thymidylate Synthase / metabolism
  • Tumor Cells, Cultured
  • gamma-Glutamyl Hydrolase / metabolism


  • Folic Acid Antagonists
  • Glutamates
  • Pterins
  • Quinazolines
  • Tetrahydrofolates
  • Thiophenes
  • ZD 9331
  • Pemetrexed
  • N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine
  • Polyglutamic Acid
  • Guanine
  • lometrexol
  • methotrexate polyglutamate
  • Folic Acid
  • Ornithine
  • Thymidylate Synthase
  • gamma-Glutamyl Hydrolase
  • Peptide Synthases
  • folylpolyglutamate synthetase
  • raltitrexed
  • Methotrexate