title: Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells.
abstract: In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated. RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells. With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells. Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel. Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells. The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells. The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdr1 promoter activity. NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells. Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells.
authors.affiliation: Department of Histology and Oral Histology, School of Dentistry, The University of Tokushima, 18-15, 3 Kuramoto-cho, Tokushima 770-8504, Japan. okamura@dent.tokushima-u.ac.jp
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Okamura
authors.forename: Kaya
authors.initials: K
authors.lastname: Yoshida
authors.forename: Eiko
authors.initials: E
authors.lastname: Sasaki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morimoto
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Haneji
mesh_terms: D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000502:physiology*; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000032:analysis; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D019450:Genes, MDR; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
chemical_list: D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins