title: Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.
abstract: Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.|Department of Natural Sciences, Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt. 25, 10120 Tallinn, Estonia.
authors.forename: K-L
authors.initials: KL
authors.lastname: Habicht
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Singh
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Khadeer
authors.affiliation: Department of Natural Sciences, Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt. 25, 10120 Tallinn, Estonia.
authors.forename: R
authors.initials: R
authors.lastname: Shimmo
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.
authors.forename: I W
authors.initials: IW
authors.lastname: Wainer
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.
authors.forename: R
authors.initials: R
authors.lastname: Moaddel
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004970:Estrone / Q000031:analogs & derivatives / Q000737:chemistry; D005047:Etoposide / Q000737:chemistry; D005909:Glioblastoma / Q000378:metabolism; D006801:Humans; D008024:Ligands; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis* / Q000378:metabolism; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism; D011224:Prazosin / Q000737:chemistry; D011485:Protein Binding; D014700:Verapamil / Q000737:chemistry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D004970:Estrone; D005047:Etoposide; D008024:Ligands; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D011224:Prazosin; D014700:Verapamil; C116614:ABCG2 protein, human; C017296:estrone sulfate; C421109:multidrug resistance-associated protein 1
keywords: Bioaffinity chromatography; Breast cancer resistant protein; Frontal chromatography; Glioblastoma