title: Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2.
abstract: Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.
authors.affiliation: Laboratory of Oncology, Herlev University Hospital, DK-2730 Herlev, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Litman
authors.forename: Ulla
authors.initials: U
authors.lastname: Jensen
authors.forename: Alastair
authors.initials: A
authors.lastname: Hansen
authors.forename: Kuang-Ming
authors.initials: KM
authors.lastname: Covitz
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Zhan
authors.forename: Patricia
authors.initials: P
authors.lastname: Fetsch
authors.forename: Andrea
authors.initials: A
authors.lastname: Abati
authors.forename: Paul Robert
authors.initials: PR
authors.lastname: Hansen
authors.forename: Thomas
authors.initials: T
authors.lastname: Horn
authors.forename: Torben
authors.initials: T
authors.lastname: Skovsgaard
authors.forename: Susan E
authors.initials: SE
authors.lastname: Bates
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D002460:Cell Line; D018432:Drug Resistance, Multiple; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008942:Mitoxantrone / Q000737:chemistry*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D000939:Epitopes; D009363:Neoplasm Proteins; D010455:Peptides; D008942:Mitoxantrone