title: Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions.
abstract: Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Finotti
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Bianchi
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Fabbri
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Borgatti
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Breveglieri
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy.
authors.forename: Jessica
authors.initials: J
authors.lastname: Gasparello
authors.affiliation: Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Italy. Electronic address: gam@unife.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Gambari
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle; D002454:Cell Differentiation; D015870:Gene Expression; D006801:Humans; D020014:K562 Cells; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D008926:Plicamycin / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D046912:Multiprotein Complexes; D009842:Oligopeptides; C492571:RICTOR protein, human; D012333:RNA, Messenger; C463659:RPTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; C040758:hemoregulatory peptide 5b; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D008926:Plicamycin; D011761:Pyrrolidonecarboxylic Acid
keywords: Erythroid induction; Fetal hemoglobin; Mithramycin; Mithramycin (PubChem CID: 457831); Raptor; Sp1; mTOR