HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jansen, G. Articles by Assaraf, Y. G. Search for Related Content PubMed PubMed Citation Articles by Jansen, G. Articles by Assaraf, Y. G.

J Biol Chem, Vol. 273, Issue 46, 30189-30198, November 13, 1998

A Structurally Altered Human Reduced Folate Carrier with Increased Folic Acid Transport Mediates a Novel Mechanism of Antifolate Resistance

Gerrit Jansen, Robert Mauritz, Stavit Drori^¶, Hannah Sprecher^¶, Ietje Kathmann, Marlene Bunni, David G. Priest, Paul Noordhuis, Jan H. Schornagel^**, Herbert M. Pinedo, Godefridus J. Peters, and Yehuda G. Assaraf^¶

From the  Department of Oncology, University Hospital Vrije Universiteit, 1081 HV Amsterdam, The Netherlands, ^¶ Department of Biology, The Technion, Israel Institute of Technology, Haifa 32000, Israel,  Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, 29425, and ^** Department of Internal Medicine, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 µM to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type K_m value for MTX transport but a 31-fold and 9-fold lower K_m values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G  A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 µM folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.

---

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

This article has been cited by other articles:

				Y. Deng, Z. Hou, L. Wang, C. Cherian, J. Wu, A. Gangjee, and L. H. Matherly Role of Lysine 411 in Substrate Carboxyl Group Binding to the Human Reduced Folate Carrier, as Determined by Site-Directed Mutagenesis and Affinity Inhibition Mol. Pharmacol., April 1, 2008; 73(4): 1274 - 1281. [Abstract] [Full Text] [PDF]

				R. C. Cho, P. D. Cole, K.-J. Sohn, G. Gaisano, R. Croxford, B. A. Kamen, and Y.-I. Kim Effects of folate and folylpolyglutamyl synthase modulation on chemosensitivity of breast cancer cells Mol. Cancer Ther., November 1, 2007; 6(11): 2909 - 2920. [Abstract] [Full Text] [PDF]

				Y. Ge, C. L. Haska, K. LaFiura, M. Devidas, S. B. Linda, M. Liu, R. Thomas, J. W. Taub, and L. H. Matherly Prognostic Role of the Reduced Folate Carrier, the Major Membrane Transporter for Methotrexate, in Childhood Acute Lymphoblastic Leukemia: A Report from the Children's Oncology Group Clin. Cancer Res., January 15, 2007; 13(2): 451 - 457. [Abstract] [Full Text] [PDF]

				Z. Hou, J. Ye, C. L. Haska, and L. H. Matherly Transmembrane Domains 4, 5, 7, 8, and 10 of the Human Reduced Folate Carrier Are Important Structural or Functional Components of the Transmembrane Channel for Folate Substrates J. Biol. Chem., November 3, 2006; 281(44): 33588 - 33596. [Abstract] [Full Text] [PDF]

				Y. Kaufman, I. Ifergan, L. Rothem, G. Jansen, and Y. G. Assaraf Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss Blood, April 15, 2006; 107(8): 3288 - 3294. [Abstract] [Full Text] [PDF]

				A. Shafran, I. Ifergan, E. Bram, G. Jansen, I. Kathmann, G. J. Peters, R. W. Robey, S. E. Bates, and Y. G. Assaraf ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates Cancer Res., September 15, 2005; 65(18): 8414 - 8422. [Abstract] [Full Text] [PDF]

				L. Rothem, B. Berman, M. Stark, G. Jansen, and Y. G. Assaraf The Reduced Folate Carrier Gene Is a Novel Selectable Marker for Recombinant Protein Overexpression Mol. Pharmacol., September 1, 2005; 68(3): 616 - 624. [Abstract] [Full Text] [PDF]

				R. de Jonge, J. H. Hooijberg, B. D. van Zelst, G. Jansen, C. H. van Zantwijk, G. J. L. Kaspers, F. G. J. Peters, Y. Ravindranath, R. Pieters, and J. Lindemans Effect of polymorphisms in folate-related genes on in vitro methotrexate sensitivity in pediatric acute lymphoblastic leukemia Blood, July 15, 2005; 106(2): 717 - 720. [Abstract] [Full Text] [PDF]

				K. Fotoohi, G. Jansen, Y. G. Assaraf, L. Rothem, M. Stark, I. Kathmann, J. Gregorczyk, G. J. Peters, and F. Albertioni Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy Blood, December 15, 2004; 104(13): 4194 - 4201. [Abstract] [Full Text] [PDF]

				K-J Sohn, F Smirnakis, D N Moskovitz, P Novakovic, Z Yates, M Lucock, R Croxford, and Y-I Kim Effects of folylpolyglutamate synthetase modulation on chemosensitivity of colon cancer cells to 5-fluorouracil and methotrexate Gut, December 1, 2004; 53(12): 1825 - 1831. [Abstract] [Full Text] [PDF]

				L. Rothem, M. Stark, and Y. G. Assaraf Impaired CREB-1 Phosphorylation in Antifolate-Resistant Cell Lines with Down-Regulation of the Reduced Folate Carrier Gene Mol. Pharmacol., December 1, 2004; 66(6): 1536 - 1543. [Abstract] [Full Text] [PDF]

				J van der Heijden, M C de Jong, B A C Dijkmans, W F Lems, R Oerlemans, I Kathmann, G L Scheffer, R J Scheper, Y G Assaraf, and G Jansen Acquired resistance of human T cells to sulfasalazine: stability of the resistant phenotype and sensitivity to non-related DMARDs Ann Rheum Dis, February 1, 2004; 63(2): 131 - 137. [Abstract] [Full Text] [PDF]

				J van der Heijden, M C de Jong, B A C Dijkmans, W F Lems, R Oerlemans, I Kathmann, C G Schalkwijk, G L Scheffer, R J Scheper, and G Jansen Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNF{alpha} Ann Rheum Dis, February 1, 2004; 63(2): 138 - 143. [Abstract] [Full Text] [PDF]

				R. Zhao, F. Gao, M. Hanscom, and I. D. Goldman A Prominent Low-pH Methotrexate Transport Activity in Human Solid Tumors: Contribution to the Preservation of Methotrexate Pharmacologic Activity in HeLa Cells Lacking the Reduced Folate Carrier Clin. Cancer Res., January 15, 2004; 10(2): 718 - 727. [Abstract] [Full Text] [PDF]

				L. Rothem, M. Stark, Y. Kaufman, L. Mayo, and Y. G. Assaraf Reduced Folate Carrier Gene Silencing in Multiple Antifolate-resistant Tumor Cell Lines Is Due to a Simultaneous Loss of Function of Multiple Transcription Factors but Not Promoter Methylation J. Biol. Chem., January 2, 2004; 279(1): 374 - 384. [Abstract] [Full Text] [PDF]

				W. F. Flintoff, F. M. R. Williams, and H. Sadlish The Region between Transmembrane Domains 1 and 2 of the Reduced Folate Carrier Forms Part of the Substrate-binding Pocket J. Biol. Chem., October 17, 2003; 278(42): 40867 - 40876. [Abstract] [Full Text] [PDF]

				E. L. Volk and E. Schneider Wild-Type Breast Cancer Resistance Protein (BCRP/ABCG2) is a Methotrexate Polyglutamate Transporter Cancer Res., September 1, 2003; 63(17): 5538 - 5543. [Abstract] [Full Text] [PDF]

				K. Wosikowski, E. Biedermann, B. Rattel, N. Breiter, P. Jank, R. Loser, G. Jansen, and G. J. Peters In Vitro and in Vivo Antitumor Activity of Methotrexate Conjugated to Human Serum Albumin in Human Cancer Cells Clin. Cancer Res., May 1, 2003; 9(5): 1917 - 1926. [Abstract] [Full Text] [PDF]

				L. Rothem, A. Aronheim, and Y. G. Assaraf Alterations in the Expression of Transcription Factors and the Reduced Folate Carrier as a Novel Mechanism of Antifolate Resistance in Human Leukemia Cells J. Biol. Chem., March 7, 2003; 278(11): 8935 - 8941. [Abstract] [Full Text] [PDF]

				Y. G. Assaraf, L. Rothem, J. H. Hooijberg, M. Stark, I. Ifergan, I. Kathmann, B. A. C. Dijkmans, G. J. Peters, and G. Jansen Loss of Multidrug Resistance Protein 1 Expression and Folate Efflux Activity Results in a Highly Concentrative Folate Transport in Human Leukemia Cells J. Biol. Chem., February 21, 2003; 278(9): 6680 - 6686. [Abstract] [Full Text] [PDF]

				R. Yang, R. Sowers, B. Mazza, J. H. Healey, A. Huvos, H. Grier, M. Bernstein, G. P. Beardsley, M. D. Krailo, M. Devidas, et al. Sequence Alterations in the Reduced Folate Carrier Are Observed in Osteosarcoma Tumor Samples Clin. Cancer Res., February 1, 2003; 9(2): 837 - 844. [Abstract] [Full Text] [PDF]

				C. Laverdiere, S. Chiasson, I. Costea, A. Moghrabi, and M. Krajinovic Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukemia Blood, November 15, 2002; 100(10): 3832 - 3834. [Abstract] [Full Text] [PDF]

				J. R. Whetstine, T. L. Witt, and L. H. Matherly The Human Reduced Folate Carrier Gene Is Regulated by the AP2 and Sp1 Transcription Factor Families and a Functional 61-Base Pair Polymorphism J. Biol. Chem., November 8, 2002; 277(46): 43873 - 43880. [Abstract] [Full Text] [PDF]

				H. Sadlish, F. M. R. Williams, and W. F. Flintoff Functional Role of Arginine 373 in Substrate Translocation by the Reduced Folate Carrier J. Biol. Chem., October 25, 2002; 277(44): 42105 - 42112. [Abstract] [Full Text] [PDF]

				E. L. Volk, K. M. Farley, Y. Wu, F. Li, R. W. Robey, and E. Schneider Overexpression of Wild-Type Breast Cancer Resistance Protein Mediates Methotrexate Resistance Cancer Res., September 1, 2002; 62(17): 5035 - 5040. [Abstract] [Full Text] [PDF]

				J. R. Whetstine, A. J. Gifford, T. Witt, X. Y. Liu, R. M. Flatley, M. Norris, M. Haber, J. W. Taub, Y. Ravindranath, and L. H. Matherly Single Nucleotide Polymorphisms in the Human Reduced Folate Carrier: Characterization of a High-Frequency G/A Variant at Position 80 and Transport Properties of the His27 and Arg27 Carriers Clin. Cancer Res., November 1, 2001; 7(11): 3416 - 3422. [Abstract] [Full Text] [PDF]

				J. Worm, A. F. Kirkin, K. N. Dzhandzhugazyan, and P. Guldberg Methylation-dependent Silencing of the Reduced Folate Carrier Gene in Inherently Methotrexate-resistant Human Breast Cancer Cells J. Biol. Chem., October 19, 2001; 276(43): 39990 - 40000. [Abstract] [Full Text] [PDF]

				C. L. van der Wilt, H. H. J. Backus, K. Smid, L. Comijn, G. Veerman, D. Wouters, D. A. Voorn, D. G. Priest, M. A. Bunni, F. Mitchell, et al. Modulation of Both Endogenous Folates and Thymidine Enhance the Therapeutic Efficacy of Thymidylate Synthase Inhibitors Cancer Res., May 1, 2001; 61(9): 3675 - 3681. [Abstract] [Full Text]

				I. G. Sharina, R. Zhao, Y. Wang, S. Babani, and I. D. Goldman Mutational Analysis of the Functional Role of Conserved Arginine and Lysine Residues in Transmembrane Domains of the Murine Reduced Folate Carrier Mol. Pharmacol., April 16, 2001; 59(5): 1022 - 1028. [Abstract] [Full Text]

				R. Zhao, F. Gao, S. Babani, and I. D. Goldman Sensitivity to 5,10-Dideazatetrahydrofolate Is Fully Conserved in a Murine Leukemia Cell Line Highly Resistant to Methotrexate Due to Impaired Transport Mediated by the Reduced Folate Carrier Clin. Cancer Res., August 1, 2000; 6(8): 3304 - 3311. [Abstract] [Full Text]

				E. L. Volk, K. Rohde, M. Rhee, J. J. McGuire, L. A. Doyle, D. D. Ross, and E. Schneider Methotrexate Cross-Resistance in a Mitoxantrone-selected Multidrug-resistant MCF7 Breast Cancer Cell Line Is Attributable to Enhanced Energy-dependent Drug Efflux Cancer Res., July 1, 2000; 60(13): 3514 - 3521. [Abstract] [Full Text]

				R. Zhao, F. Gao, P. J. Wang, and I. D. Goldman Role of the Amino Acid 45 Residue in Reduced Folate Carrier Function and Ion-Dependent Transport as Characterized by Site-Directed Mutagenesis Mol. Pharmacol., February 1, 2000; 57(2): 317 - 323. [Abstract] [Full Text]

				M. Aghi, C. M. Kramm, and X. O. Breakefield RESPONSE: Re: Folylpolyglutamyl Synthetase Gene Transfer and Glioma Antifolate Sensitivity in Culture and In Vivo J Natl Cancer Inst, December 1, 1999; 91(23): 2048 - 2049. [Full Text] [PDF]

				F. M. Sirotnak RESPONSE: Re: Folylpolyglutamyl Synthetase Gene Transfer and Glioma Antifolate Sensitivity in Culture and In Vivo J Natl Cancer Inst, December 1, 1999; 91(23): 2049 - 2050. [Full Text] [PDF]

				G. Jansen, G. J. Peters, H. M. Pinedo, D. G. Priest, and Y. G. Assaraf Re: Folylpolyglutamyl Synthetase Gene Transfer and Glioma Antifolate Sensitivity in Culture and In Vivo J Natl Cancer Inst, December 1, 1999; 91(23): 2047a - 2048a. [Full Text] [PDF]

				G. J. Peters, E. Smitskamp-Wilms, K. Smid, H. M. Pinedo, and G. Jansen Determinants of Activity of the Antifolate Thymidylate Synthase Inhibitors Tomudex (ZD1694) and GW1843U89 against Mono- and MultilayeredColon Cancer Cell Lines under Folate-restricted Conditions Cancer Res., November 1, 1999; 59(21): 5529 - 5535. [Abstract] [Full Text] [PDF]

				R. Zhao, I. G. Sharina, and I. D. Goldman Pattern of Mutations that Results in Loss of Reduced Folate Carrier Function under Antifolate Selective Pressure Augmented by Chemical Mutagenesis Mol. Pharmacol., July 1, 1999; 56(1): 68 - 76. [Abstract] [Full Text]

				J. H. Hooijberg, H. J. Broxterman, M. Kool, Y. G. Assaraf, G. J. Peters, P. Noordhuis, R. J. Scheper, P. Borst, H. M. Pinedo, and G. Jansen Antifolate Resistance Mediated by the Multidrug Resistance Proteins MRP1 and MRP2 Cancer Res., June 1, 1999; 59(11): 2532 - 2535. [Abstract] [Full Text] [PDF]

				S. C. Wong, L. Zhang, T. L. Witt, S. A. Proefke, A. Bhushan, and L. H. Matherly Impaired Membrane Transport in Methotrexate-resistant CCRF-CEM Cells Involves Early Translation Termination and Increased Turnover of a Mutant Reduced Folate Carrier J. Biol. Chem., April 9, 1999; 274(15): 10388 - 10394. [Abstract] [Full Text] [PDF]

				G. Jansen, H. Barr, I. Kathmann, M. A. Bunni, D. G. Priest, P. Noordhuis, G. J. Peters, and Y. G. Assaraf Multiple Mechanisms of Resistance to Polyglutamatable and Lipophilic Antifolates in Mammalian Cells: Role of Increased Folylpolyglutamylation, Expanded Folate Pools, and Intralysosomal Drug Sequestration Mol. Pharmacol., April 1, 1999; 55(4): 761 - 769. [Abstract] [Full Text]

				B. van Triest, H. M. Pinedo, Y. van Hensbergen, K. Smid, F. Telleman, P. S. Schoenmakers, C. L. van der Wilt, J. A. M. van Laar, P. Noordhuis, G. Jansen, et al. Thymidylate Synthase Level as the Main Predictive Parameter for Sensitivity to 5-Fluorouracil, but not for Folate-based Thymidylate Synthase Inhibitors, in 13 Nonselected Colon Cancer Cell Lines Clin. Cancer Res., March 1, 1999; 5(3): 643 - 654. [Abstract] [Full Text] [PDF]

				R. Zhao, S. Titus, F. Gao, R. G. Moran, and I. D. Goldman Molecular Analysis of Murine Leukemia Cell Lines Resistant to 5,10-Dideazatetrahydrofolate Identifies Several Amino Acids Critical to the Function of Folylpolyglutamate Synthetase J. Biol. Chem., August 18, 2000; 275(34): 26599 - 26606. [Abstract] [Full Text] [PDF]

				S. Drori, G. Jansen, R. Mauritz, G. J. Peters, and Y. G. Assaraf Clustering of Mutations in the First Transmembrane Domain of the Human Reduced Folate Carrier in GW1843U89-resistant Leukemia Cells with Impaired Antifolate Transport and Augmented Folate Uptake J. Biol. Chem., September 29, 2000; 275(40): 30855 - 30863. [Abstract] [Full Text] [PDF]

				J. R. Whetstine and L. H. Matherly The Basal Promoters for the Human Reduced Folate Carrier Gene Are Regulated by a GC-box and a cAMP-response Element/AP-1-like Element. BASIS FOR TISSUE-SPECIFIC GENE EXPRESSION J. Biol. Chem., February 23, 2001; 276(9): 6350 - 6358. [Abstract] [Full Text] [PDF]