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

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.

• Authors: Yotam Kaufman, Ilan Ifergan, Lilah Rothem, Gerrit Jansen, Yehuda G Assaraf
• Journal: Blood (Blood) Vol. 107 Issue 8 Pg. 3288-94 (Apr 15 2006) ISSN: 0006-4971 [Print] United States
• PMID: 16368880 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Membrane Transport Proteins
• Pterins
• Reduced Folate Carrier Protein
• SLC19A1 protein, human
• Transcription Factors
• N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine
• Ornithine
• Tetrahydrofolate Dehydrogenase
• Methotrexate

Topics

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