title: Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.
abstract: BACKGROUND
  Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance.
  
  
  METHODS
  Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR.
  
  
  RESULTS
  Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes encoding glutathione-metabolizing enzymes in 293/B5-11 cells was similar to that in 293/mock cells. The cellular content of Glu, a precursor of glutathione, in 293/B5-11 and 293/mb5-8 cells was higher than that in 293/mock cells.
  
  
  CONCLUSIONS
  ABCB5/Abcb5-transfected cells showed resistance to BSO, which is not a substrate of ABCB5. Our results suggest that ABCB5/Abcb5 upregulates cellular glutathione levels to protect cells from various poisons.
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. shingo-kondo@keio.jp.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kondo
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. ke-ta_1209@keio.jp.
authors.forename: Keita
authors.initials: K
authors.lastname: Hongama
authors.affiliation: Division of Organic and Biocatalytic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. hanaya-kn@pha.keio.ac.jp.
authors.forename: Kengo
authors.initials: K
authors.lastname: Hanaya
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. ryota.yoshida1101@gmail.com.
authors.forename: Ryota
authors.initials: R
authors.lastname: Yoshida
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. kawanobe@kais.kyoto-u.ac.jp.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kawanobe
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. katayama-kz@pha.keio.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Katayama
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. noguchi-kj@pha.keio.ac.jp.
authors.forename: Kohji
authors.initials: K
authors.lastname: Noguchi
authors.affiliation: Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 shibakoen, Minato-ku, 105-8512, Tokyo, Japan. sugimoto-ys@pha.keio.ac.jp.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Sugimoto
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000378:metabolism / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D051549:Glutathione S-Transferase pi / Q000235:genetics / Q000378:metabolism; D005981:Glutathione Synthase / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation
chemical_list: C480744:ABCB5 protein, human; C573563:ABCB5 protein, mouse; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D004791:Enzyme Inhibitors; D019328:Buthionine Sulfoximine; D005980:Glutathione Reductase; D051549:Glutathione S-Transferase pi; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione