Key words: drug resistance; ABC transporter; mitoxantrone; retrovirus vector Tumor cells that acquire resistance to certain chemotherapeutic agents sometimes develop cross-resistance to other structurally unrelated drugs. 1 This phenomenon is known as multi-drug resistance. A family of ATP-binding cassette (ABC) transporters such as MDR1 gene product P-glycoprotein 1-4 and MRP1 5 is involved in multidrug resistance. They pump out various structurally unrelated antitumor agents in an energy-dependent manner.Breast cancer resistance protein (BCRP), also called ABCP or MXR, is a newly discovered ABC transporter, 6 -8 which is a half molecule with a C-terminal transmembrane segment and an Nterminal ATP-binding site. 9 The BCRP gene was first identified as an ABC transporter overexpressed in the placenta. 7 BCRP overexpression is reported in both mitoxantrone-resistant and topotecan-resistant cells. 10 -12 Because resistance to mitoxantrone, SN-38 and topotecan develop concurrently, BCRP presumably acts as an efflux pump, resulting in decreased intracellular concentrations of these anticancer agents. 13,14 Many ABC transporter proteins have two transmembrane segments and two ATP-binding sites. 15 Some half-molecule ABC transporters are known to form homodimers or heterodimers. 16 -18 BCRP is also assumed to act as a functional dimer, however its counterpart has not yet been identified. To determine a protein that forms an ABC complex with BCRP, cells were transfected with HA epitope-or Myc epitope-tagged BCRP cDNAs and cell lysates of the transfectants were used to analyze proteins interacting with BCRP by immunoprecipitation and Western blotting. Our results indicate that BCRP forms an S-S homodimer and therefore it is possible to inhibit the function of BCRP by the introduction of a dominant-negative BCRP mutant. MATERIAL AND METHODS Cell cultures and drug sensitivity assayPA317 amphotropic retrovirus packaging cells were grown in high-glucose Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 . The sensitivity of the cell lines to mitoxantrone and SN-38 was evaluated by the inhibition of cell growth after incubation at 37°C in the presence of various concentrations of the drugs examined. Six days after incubating cells with the drugs, cell numbers were determined by flow cytometry using a Coulter cell counter and drug concentrations that inhibited cell growth by 50% (IC 50 ) were evaluated. 19 Statistical analysis was performed using Student's t-test. Vectors and transfectantsBriefly, complementary DNA for human BCRP was isolated by PCR using Marathone-ready human placenta cDNA library (Clontech, Palo Alto, CA) as a template. Synthetic oligonucleotides corresponding to the Myc epitope peptide (EQKLISEEDL) or HA epitope peptide (YPYDVPDYA) were added upstream from the ATG codon of BCRP cDNA by PCR and termed MycBCRP and HABCRP, respectively. The nucleotide sequences of independently isolated BCRP cDNA clones were analyzed using an automate...

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