TY - JOUR
T1 - Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles
T2 - transcriptional silencing, inactivating mutations, and allele loss
AU - Kaufman, Yotam
AU - Ifergan, Ilan
AU - Rothem, Lilah
AU - Jansen, Gerrit
AU - Assaraf, Yehuda G
PY - 2006/4/15
Y1 - 2006/4/15
N2 - 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.
AB - 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.
KW - Alleles
KW - Biological Transport/drug effects
KW - Cell Line, Tumor
KW - Down-Regulation/drug effects
KW - Drug Resistance, Neoplasm/drug effects
KW - Enzyme Inhibitors/pharmacology
KW - Gene Expression Regulation, Leukemic/drug effects
KW - Gene Silencing/drug effects
KW - Humans
KW - Leukemia/drug therapy
KW - Loss of Heterozygosity/genetics
KW - Membrane Transport Proteins/genetics
KW - Methotrexate/pharmacology
KW - Ornithine/analogs & derivatives
KW - Pterins/pharmacology
KW - Reduced Folate Carrier Protein
KW - Response Elements/drug effects
KW - Tetrahydrofolate Dehydrogenase/genetics
KW - Transcription Factors/genetics
U2 - 10.1182/blood-2005-10-4048
DO - 10.1182/blood-2005-10-4048
M3 - Article
C2 - 16368880
VL - 107
SP - 3288
EP - 3294
JO - Blood
JF - Blood
SN - 0006-4971
IS - 8
ER -