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.

journal_name

Blood

journal_title

Blood

authors

Kaufman Y,Ifergan I,Rothem L,Jansen G,Assaraf YG

doi

10.1182/blood-2005-10-4048

subject

Has Abstract

pub_date

2006-04-15 00:00:00

pages

3288-94

issue

8

eissn

0006-4971

issn

1528-0020

pii

2005-10-4048

journal_volume

107

pub_type

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