Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss.
The reduced folate carrier (RFC) is the dominant route for the uptake of various antifolates including PT523, a potent dihydrofolate reductase inhibitor (Ki = 0.35 pM) and an excellent transport substrate of the RFC (Kt = 0.7 microM). Here, we describe the multiple mechanisms of RFC inactivation in human leukemia PT523-resistant cells originally harboring 3 RFC alleles. Cellular exposure to gradually increasing PT523 concentrations resulted in sublines displaying up to 3500-fold resistance to various hydrophilic antifolates that rely on RFC for their cellular uptake. Antifolate-resistant cells lost RFC gene expression (65%-99% loss) due to impaired promoter binding of various transcription factors that regulate RFC gene expression. Additionally, DNA sequencing revealed that PT523-resistant cells contained a cluster of 4 nearly consecutive mutations residing on a single RFC allele including L143P, A147V, R148G, and Q150Stop. Southern blot analysis established the loss of an RFC allele in PT523-resistant cells. These alterations resulted in markedly decreased RFC protein levels (approximately 80%-99% loss) and consequently impaired [3H]methotrexate transport (87%-99% loss). This study provides the first evidence that acquisition of PT523 resistance in human leukemia cells harboring 3 RFC alleles is due to multiple coexisting alterations including transcriptional silencing, inactivating mutations, and RFC allele loss.
Kaufman
Yotam
Y
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Ifergan
Ilan
I
Rothem
Lilah
L
Jansen
Gerrit
G
Assaraf
Yehuda G
YG
D000483:Alleles; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000235:genetics; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D019656:Loss of Heterozygosity / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D011622:Pterins / Q000494:pharmacology / Q000627:therapeutic use; D058978:Reduced Folate Carrier Protein; D020218:Response Elements / Q000187:drug effects / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D011622:Pterins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D014157:Transcription Factors; C085590:N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine; D009952:Ornithine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate