title: Decreased CP-1 (NF-Y) activity results in transcriptional down-regulation of topoisomerase IIalpha in a doxorubicin-resistant variant of human multiple myeloma RPMI 8226.
abstract: Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxorubicin in the absence of MDR1 overexpression or topo II mutations (Futscher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., and Dalton W.S., Int'l. J. Cancer, 66: 520-5, 1996.). Consistent with its drug resistant phenotype, a 2- to 3-fold decrease in topo II expression was identified. To investigate the molecular basis for decreased topo II expression in Dox1V, a semi-quantitative analysis of Topo II activity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level. Southern blot and sequencing experiments revealed wild-type sequence of the topo II promoter in the drug resistant cells. Transient gene expression assays demonstrated that topo II is transcriptionally down-regulated in Dox1V independent of the promoter sequence of the endogenous alleles. Instead, the activity of a ubiquitous transcription factor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with the decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regulation resulted from a reduced CP-1/NF-Y activity is responsible for decreased topo II expression in Dox1V cells.
authors.affiliation: Department of Obstetrics and Gynecology, The Ohio State University College of Medicine and James Cancer Hospital and Research Institute, Columbus, Ohio, 43210, USA.
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: Z g
authors.initials: Zg
authors.lastname: Jiang
authors.forename: Y W
authors.initials: YW
authors.lastname: Wong
authors.forename: W S
authors.initials: WS
authors.lastname: Dalton
authors.forename: B W
authors.initials: BW
authors.lastname: Futscher
authors.forename: V T
authors.initials: VT
authors.lastname: Chan
mesh_terms: D000951:Antigens, Neoplasm; D022762:CCAAT-Enhancer-Binding Proteins; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics*; D009101:Multiple Myeloma / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D004317:Doxorubicin; D004250:DNA Topoisomerases, Type II