Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines.
We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97% of parental [(3)H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.
Department of Biology, The Technion, Haifa 32000, Israel.
Lilah
L
Rothem
Ilan
I
Ifergan
Yotam
Y
Kaufman
David G
DG
Priest
Gerrit
G
Jansen
Yehuda G
YG
Assaraf
D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D017931:DNA Primers; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000378:metabolism* / Q000473:pathology; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000493:pharmacokinetics / Q000494:pharmacology; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D058978:Reduced Folate Carrier Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017931:DNA Primers; D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D008727:Methotrexate