We have studied the molecular basis of drug resistance in human CCRF-CEM
leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable
antifolates that target various
folate-dependent
enzymes. These include the
dihydrofolate reductase (DHFR) inhibitors
edatrexate,
methotrexate and
aminopterin, the
thymidylate synthase (TS) inhibitors
ZD1694 and
GW1843, the
glycinamide ribonucleotide formyltransferase (GARTF) inhibitor
DDATHF as well as the multitargeted
antifolate LY231514 inhibiting both
TS, DHFR and GARTF. Fourteen
antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired
folylpoly-gamma-glutamate synthetase (FPGS) activity as these cell lines: 1) typically lost 90-99% of parental FPGS activity; 2) expressed 1.4-3.3-fold less FPGS
mRNA (only 4 cell lines); 3) displayed up to 10(5)-fold resistance to polyglutamylation-dependent
antifolates including
ZD1694 and
MTA; 4) retained sensitivity to polyglutamylation-independent
antifolates including
ZD9331 and
PT523; 5) were up to 19-fold hypersensitive to the
lipid-soluble
antifolates trimetrexate and AG377; 6) had a normal or a small decrease in [(3)H]MTX transport; and 7) had a 2.1-8.3-fold decreased cellular
folate pools and a consequently increased
folate growth requirement. The remaining 3
antifolate-resistant sublines lost 94-97% of parental [(3)H]MTX transport and thus displayed a high level resistance to all hydrophilic
antifolates. To screen for mutations in the hFPGS gene, we devised an RT-PCR single strand conformational polymorphism (SSCP) assay. RT-PCR-SSCP analysis and
DNA sequencing showed that only a single FPGS-deficient subline harbored an FPGS mutation (Cys346Phe). Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the
enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within alpha-helix
A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for
L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel
antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent
antifolates but in the same time retain parental sensitivity or
hypersensitivity to polyglutamylation-independent
antifolates or lipophilic
antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.