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...

ABSTRACT

Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer reagents such as mitoxantrone, 7-ethyl-10-hydroxycamptothecin, and topotecan. We reported previously that estrone and 17␤-estradiol reverse BCRP-mediated multidrug resistance. In the present study, we demonstrate that BCRP exports estrogen metabolites. First, we generated BCRP-transduced LLC-PK1 (LLC/BCRP) cells, in which exogenous BCRP is expressed in the apical membrane, and investigated transcellular transport of 3 H-labeled compounds using cells plated on microporous filter membranes. The basal-to-apical transport (excretion) of mitoxantrone, estrone, and 17␤-estradiol was greater in LLC/ BCRP cells than in LLC-PK1 cells. Thin-layer chromatography of transported steroids revealed that the transport of estrone and 17␤-estradiol was independent of BCRP expression. Alternatively, increased excretion of estrone sulfate and 17␤-estradiol sulfate was observed in LLC/BCRP cells. BCRP inhibitors completely inhibited the increased excretion of sulfated estrogens across the apical membrane. Conversion of estrogens into their sulfate conjugates was similar between LLC/BCRP and LLC-PK1 cells, suggesting that the increased excretion of estrogen sulfates was attributable to BCRP-mediated transport. Next, the uptake of 3 H-labeled compounds in membrane vesicles from BCRP-transduced K562 (K562/BCRP) cells was investigated.3 H-labeled estrone sulfate, but not 3 H-labeled estrone or 17␤-estradiol, was taken up by membrane vesicles from K562/BCRP cells, and this was ATP-dependent. Additionally, BCRP inhibitors suppressed the transport of estrone sulfate in membrane vesicles from K562/BCRP cells. These results suggest that BCRP does not transport either free estrone or 17␤-estradiol but exports sulfate conjugates of these estrogens.Breast cancer resistance protein (BCRP) is a member of the ATP-binding cassette transporter G family and is also known as ABCG2 or ABCP. BCRP mediates concurrent resistance to such chemotherapeutic agents as mitoxantrone (MXR), SN-38 (an active metabolite of CPT-11), and topotecan, presumably by pumping these reagents out of the cell, thereby resulting in concentrations lower than cytotoxic levels (Allikmets et al., 1998;Doyle et al

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and 17-allylamino-17-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-15 and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH 2 -terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal growth factor and basic fibroblast growth factor enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal growth factor -stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance.

doxorubicin Certain members of ABC transporters such as the MDR1 gene product P-gp and MRP1 are involved in the anticancer drug resistance of tumor cells. 1-3 Such transporters pump out various structurally unrelated antitumor agents in an ATP-dependent manner. BCRP, also called ABCG2, ABCP or MXR, is a member of ABC transporters that has an N-terminal ATP binding domain and a C-terminal TM. 4 -6 BCRP is speculated to function as an S-S homodimer. 7 BCRP overexpression has been reported in various drug-resistant cells selected with mitoxantrone, doxorubicin, topotecan and NB-506. 4 -15 Because these cells acquire resistance to mitoxantrone, SN-38 and topotecan concurrently, BCRP presumably acts as an efflux pump, resulting in decreased intracellular concentrations of these anticancer agents. 4 -15 BCRP-mediated drug resistance could be overcome by fumitremorgin C 16 or estrogens. 17 As shown in mutation analyses of MDR1 and MRP1, amino acid substitutions in the TMs sometimes alter the drug transport specificity of ABC transporters. 18 -22 R482 mutations in TM3 of BCRP were reported in independently isolated drug-resistant cell lines. A doxorubicin-resistant human breast cancer cell line MCF-7 AdVp3000 and a mitoxantrone-resistant human colon carcinoma cell line S1-M1-80 expressed R482T-and R482G-BCRP, respectively and showed high resistance to mitoxantrone and doxorubicin. 5,6,13 Doxorubicin-resistant murine fibroblast cell lines also expressed R482M-or R482S-BCRP and showed high resistance to mitoxantrone and doxorubicin. 14 We recently identified the substitution R482M in a doxorubicin-resistant human T cell line MT-4/DOX 500 . 23 We made 32 mutant BCRP cDNAs with an amino acid substitution in the TMs and examined the effect of the substitutions on cellular drug resistance. Most R482-mutant BCRP transfectants were highly resistant to mitoxantrone and doxorubicin. Some N557-or H630-mutant BCRP transfectants showed different cross resistance patterns. MATERIAL AND METHODS Cell culture and drug sensitivity assayMurine fibroblast PA317 cells were grown in high-glucose Dulbecco's modified Eagle medium supplemented with 10% FBS at 37°C in a humidified incubator with 5% CO 2 . The sensitivity of mutant BCRP-transfected cells to anticancer reagents was evaluated by examining cell growth inhibition after incubation at 37°C for 5 days in the absence or presence of various concentrations of anticancer drugs. In addition, effects of BCRP inhibitors on the sensitivity to anticancer drugs were investigated. Cell numbers were determined with a Coulter counter. IC 50 values (drug dose causing 50% inhibition of cell growth) were determined from growth inhibition curves. Mutant BCRP transfectantsMutant BCRP cDNAs were prepared using the Mutan-Super Express Km site-directed mutagenesis system (Takara, Kyoto, Japan) as directed. Mutant BCRP cDNAs without any other mutations were inserted into pHaL-IRES-DHFR bicistronic retrovirus vector that carries DHFR cDNA. 7 PA317 cells were transfected with the mutant BCRP expression ...