title: A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2).
abstract: The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.
authors.affiliation: Developmental Therapeutics Department, Medicine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: R W
authors.initials: RW
authors.lastname: Robey
authors.forename: Y
authors.initials: Y
authors.lastname: Honjo
authors.forename: A
authors.initials: A
authors.lastname: van de Laar
authors.forename: K
authors.initials: K
authors.lastname: Miyake
authors.forename: J T
authors.initials: JT
authors.lastname: Regis
authors.forename: T
authors.initials: T
authors.lastname: Litman
authors.forename: S E
authors.initials: SE
authors.lastname: Bates
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001896:Boron Compounds; D001943:Breast Neoplasms; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms; D018432:Drug Resistance, Multiple / Q000235:genetics / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D008942:Mitoxantrone / Q000633:toxicity*; D009363:Neoplasm Proteins; D016133:Polymerase Chain Reaction; D011224:Prazosin / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C116614:ABCG2 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001896:Boron Compounds; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D008942:Mitoxantrone; D014700:Verapamil; D000251:Adenosine Triphosphatases; D011224:Prazosin