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.