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Blood, 15 April 2006, Vol. 107, No. 8, pp. 3288-3294. Prepublished online as a Blood First Edition Paper on December 20, 2005; DOI 10.1182/blood-2005-10-4048. This Article Full Text (PDF) All Versions of this Article: 2005-10-4048v1 107/8/3288    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kaufman, Y. Articles by Assaraf, Y. G Search for Related Content PubMed PubMed Citation Articles by Kaufman, Y. Articles by Assaraf, Y. G Social Bookmarking                   What's this?  Previous Article  |  Next Article  Submitted October 11, 2005 Accepted November 15, 2005 Coexistence of multiple mechanisms of PT523-resistance in human leukemia cells harboring three reduced folate carrier alleles: transcriptional silencing, inactivating mutations and allele loss Yotam Kaufman, Ilan Ifergan, Lilah Rothem, Gerrit Jansen, and Yehuda G Assaraf* Department of Biology, Technion, Haifa, Israel Department of Rheumatology, University Hospital VU, Amsterdam, The Netherlands * Corresponding author; email: assaraf{at}tx.technion.ac.il. 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 µM). Herein we describe the multiple mechanisms of RFC inactivation in human leukemia PT523-resistant cells originally harboring three RFC alleles. Cellular exposure to gradually increasing PT523 concentrations resulted in sublines displaying up to 3,500-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 four 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 (~80-99%) and consequently impaired [3H]MTX transport (87%-99%). This study provides the first evidence that acquisition of PT523 resistance in human leukemia cells harboring three RFC alleles is due to multiple coexisting alterations including transcriptional silencing, inactivating mutations and RFC allele loss. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: K. Balamurugan, B. Ashokkumar, M. Moussaif, J. Y. Sze, and H. M. Said Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans Am J Physiol Cell Physiol, August 1, 2007; 293(2): C670 - C681. [Abstract] [Full Text] [PDF]

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