The @entity1 reduced folate carrier ( @entity7177 ) is the major uptake route for antifolates used in @entity5 chemotherapy. Here we explored the molecular basis for the decrease or loss of @entity7177 gene expression in seventeen @entity5 cell lines with resistance to multiple antifolates due to impaired antifolate transport. We studied the role of various cis-acting elements including CRE/ @entity1178 -like element and GC-box in @entity7177 promoters A and B, respectively, as well as @entity18720 , @entity61316 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, @entity61316 , @entity1178 , 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 @entity7177 promoter elements, whereas normal @entity18720 binding was retained. The possible contribution of promoter methylation to @entity7177 gene silencing was also explored. None of the antifolate-resistant cell lines, except for MDA-MB-231 cells, showed @entity7177 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, @entity25576 , @entity1639 , @entity1178 , @entity3419 , and @entity28549 ) and/or increased expression of repressors (short @entity28549 isoforms), whereas normal @entity18720 levels were retained. Transient expression of the relevant transcription factors restored, at least partially, both promoter binding and @entity7177 gene expression. This is the first report that transcriptional silencing of the @entity7177 gene in multiple @entity5 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.

Reduced folate carrier gene silencing in multiple antifolate-resistant XXXX cell lines is due to a simultaneous loss of function of multiple transcription factors but not promoter methylation.

@entity1 :: ('9606', 'Species') :: ['human'] @entity25576 :: ('7391', 'Gene') :: ['USF-1'] @entity5 :: ('MESH:D009369', 'Disease') :: ['tumor', 'cancer'] @entity61316 :: ('7593', 'Gene') :: ['Mzf-1'] @entity1178 :: ('3725', 'Gene') :: ['AP-1', 'c-Jun'] @entity7177 :: ('6573', 'Gene') :: ['hRFC'] @entity1639 :: ('2353', 'Gene') :: ['c-Fos'] @entity3419 :: ('6667', 'Gene') :: ['Sp1'] @entity28549 :: ('6670', 'Gene') :: ['Sp3'] @entity18720 :: ('7020', 'Gene') :: ['AP2alpha', 'AP-2']
@entity5 :: (MESH:D009369,Disease) :: ['tumor']
