Abstract
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 language | English |
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Pages (from-to) | 3288-94 |
Number of pages | 7 |
Journal | Blood |
Volume | 107 |
Issue number | 8 |
DOIs | |
Publication status | Published - 15 Apr 2006 |
Keywords
- 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