title: Alterations in the expression of transcription factors and the reduced folate carrier as a novel mechanism of antifolate resistance in human leukemia cells.
abstract: The human reduced folate carrier (hRFC) is the dominant transporter mediating the uptake of reduced folate cofactors and antifolate anticancer drugs. Defective antifolate uptake due to inactivating mutations in the hRFC gene is an established mechanism of drug resistance in various tumor cells. However, while antifolate transport is frequently impaired, either no or only a single hRFC allele is inactivated, suggesting that additional mechanism(s) of resistance are operative. Here we studied the relationship between the expression and function of transcription factors and antifolate resistance in transport-defective leukemia cells that poorly express or completely lack RFC mRNA. Stable transfection with a hRFC expression construct resulted in restoration of normal RFC mRNA expression and nearly wild type drug sensitivity in these antifolate-resistant cells. The loss of RFC gene expression prompted us to explore transcription factor binding to the hRFC promoter. The hRFC promoter contains an upstream GC-box and a downstream cAMP-response element (CRE)/AP-1-like element. Electrophoretic mobility shift assays and oligonucleotide competition revealed a substantial loss of nuclear factor binding to CRE and GC-box in these drug-resistant cell lines. Consistently, antibody-mediated supershift analysis showed a marked decrease in the binding of CRE-binding protein 1 (CREB-1) and specificity protein 1 (Sp1) to CRE and GC-box, respectively. Western blot analysis revealed undetectable expression of CREB-1, decreased ATF-1 levels, parental Sp1 levels, and increased levels of the short Sp3 isoforms, recently shown to repress hRFC gene expression. Transient transfections into these antifolate-resistant cells demonstrated a marked loss of GC-box-dependent, and CRE-driven reporter gene activities and introduction of CREB-1 or Sp1 expression constructs resulted in restoration of hRFC mRNA expression. These results establish a novel mechanism of antifolate resistance that is based on altered expression and function of transcription factors resulting in transcriptional silencing of the hRFC promoter.
authors.affiliation: Department of Biology, the Rappaport Institute for Research in the Medical Sciences and the B. Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa 31096, Israel.
authors.forename: Lilah
authors.initials: L
authors.lastname: Rothem
authors.forename: Ami
authors.initials: A
authors.lastname: Aronheim
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D000483:Alleles; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000378:metabolism; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000494:pharmacology; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D015458:Leukemia, T-Cell / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D026901:Membrane Transport Proteins; D009841:Oligonucleotides / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: C115163:CREB1 protein, human; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D009841:Oligonucleotides; D020033:Protein Isoforms; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D005492:Folic Acid; D008156:Luciferases