title: 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.
abstract: The human reduced folate carrier (hRFC) is the major uptake route for antifolates used in cancer chemotherapy. Here we explored the molecular basis for the decrease or loss of hRFC gene expression in seventeen tumor cell lines with resistance to multiple antifolates due to impaired antifolate transport. We studied the role of various cis-acting elements including CRE/AP-1-like element and GC-box in hRFC promoters A and B, respectively, as well as AP-2, Mzf-1 and E-box that are contained within or near four tandemly repeated sequences upstream of promoter A. Decreased or abolished binding either to [32P]GC-box, Mzf-1, AP-1, E-box, or CRE oligonucleotides was detected in approximately 50-80% of antifolate-resistant cell lines. Strikingly, approximately 80% of the cell lines displayed a simultaneously decreased binding to three or more of these hRFC promoter elements, whereas normal AP-2 binding was retained. The possible contribution of promoter methylation to hRFC gene silencing was also explored. None of the antifolate-resistant cell lines, except for MDA-MB-231 cells, showed hRFC promoter methylation; consistently, MDA-MB-231 was the only cell line that retained binding to all six cis-acting elements. Western blot analysis demonstrated decreased expression of transcriptional activators (pCREB-1, pATF-1, USF-1, c-Fos, c-Jun, Sp1, and Sp3) and/or increased expression of repressors (short Sp3 isoforms), whereas normal AP2alpha levels were retained. Transient expression of the relevant transcription factors restored, at least partially, both promoter binding and hRFC gene expression. This is the first report that transcriptional silencing of the hRFC gene in multiple tumor cell lines with resistance to various novel antifolates is a result of a simultaneous loss of function of multiple transcription factors but not promoter methylation.
authors.affiliation: Department of Biology, The Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Lilah
authors.initials: L
authors.lastname: Rothem
authors.forename: Michal
authors.initials: M
authors.lastname: Stark
authors.forename: Yotam
authors.initials: Y
authors.lastname: Kaufman
authors.forename: Lior
authors.initials: L
authors.lastname: Mayo
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D018388:Codon, Terminator; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000235:genetics; D058973:Folate Receptors, GPI-Anchored; D020868:Gene Silencing; D006801:Humans; D015458:Leukemia, T-Cell; D026901:Membrane Transport Proteins; D009154:Mutation; D020125:Mutation, Missense; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface; D058978:Reduced Folate Carrier Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D018388:Codon, Terminator; D015226:Dinucleoside Phosphates; D058973:Folate Receptors, GPI-Anchored; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D058978:Reduced Folate Carrier Protein; D014157:Transcription Factors; C548699:SLC19A1 protein, human; C015772:CpG dinucleotide