title: Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.
abstract: ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.
authors: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Suneet
authors: S
authors: Shukla
authors: Candice
authors: C
authors: Schwartz
authors: Khyati
authors: K
authors: Kapoor
authors: Abdul
authors: A
authors: Kouanda
authors: Suresh V
authors: SV
authors: Ambudkar
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D016367:Baculoviridae / Q000378:metabolism*; D001692:Biological Transport; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002734:Chlorophyll / Q000031:analogs & derivatives / Q000378:metabolism; D004317:Doxorubicin / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019008:Drug Resistance, Neoplasm; D005822:Genetic Vectors; D006801:Humans; D008322:Mammals; D008942:Mitoxantrone / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D011838:Radiation-Sensitizing Agents; D011994:Recombinant Proteins; D002734:Chlorophyll; D004317:Doxorubicin; D008942:Mitoxantrone; C032623:pheophorbide a