Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss

Yotam Kaufman, Ilan Ifergan, Lilah Rothem, Gerrit Jansen, Yehuda G Assaraf

Research output: Contribution to journalArticleAcademicpeer-review

15 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)3288-94
Number of pages7
Issue number8
Publication statusPublished - 15 Apr 2006


  • Alleles
  • Biological Transport/drug effects
  • Cell Line, Tumor
  • Down-Regulation/drug effects
  • Drug Resistance, Neoplasm/drug effects
  • Enzyme Inhibitors/pharmacology
  • Gene Expression Regulation, Leukemic/drug effects
  • Gene Silencing/drug effects
  • Humans
  • Leukemia/drug therapy
  • Loss of Heterozygosity/genetics
  • Membrane Transport Proteins/genetics
  • Methotrexate/pharmacology
  • Ornithine/analogs & derivatives
  • Pterins/pharmacology
  • Reduced Folate Carrier Protein
  • Response Elements/drug effects
  • Tetrahydrofolate Dehydrogenase/genetics
  • Transcription Factors/genetics

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