Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants

Abstract

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 ...

Role of the Breast Cancer Resistance Protein (BCRP/ABCG2) in Drug Transport—an Update

“…Docking calculations indicate that Arg 482 directly interacts with mitoxantrone and Hoechst33342, but not with prazosin and SN-38 (86). This is consistent with previous findings that resistance to mitoxantrone was increased, but resistance to SN-38 or efflux of prazosin was not affected, by mutations of Arg 482 (16,87). This also is in agreement with the studies showing that prazosin binds to a site that does not fully overlap with that for mitoxantrone or Hoechst33342 (88) and the binding of a prazosin derivative to BCRP was relatively unaffected by mutations of Arg 482 (89).…”

Pharmacological interaction with sunitinib is abolished by a germ‐line mutation (1291T>C) of BCRP/ABCG2 gene

“…The R482T and R482G are BCRP variants identified after in vitro selection of culture cells and these variants confer DOX-and MXR-resistances. (34) Before examining the suppressive effect of sunitinib on these BCRP variant-expressing murine fibroblast PA317 cells, cell populations with high BCRP expression were selectively enriched using immunomagnetic beads. The BCRP protein expression levels were confirmed to be comparable between each enriched variant BCRP-expressing PA317 cells (Fig.…”

“…The anti-BCRP polyclonal antibody 3488 was generated as described elsewhere. (34) Cells and drug sensitivity assay. PA317 mouse fibroblast cells, K562 human myelogenous leukemia cells, BCRP-expressing PA317 cells, BCRP-expressing K562 cells (K562 ⁄ BCRP), and P-gp-expressing K562 cells (K562 ⁄ MDR) were established and cultured as previously.…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…s0090 4.2.1 Role of the R482 position p0375 It turned out very early, practically at the cloning of ABCG2 that R482 is a crucial determinant of substrate recognition of ABCG2. In these early studies, it was found that a mutation of R482 to G or T occurred in the cell lines upon anthracycline selection, most probably because these "artificial" (not occurring in vivo) mutant ABCG2 variants results in a "gain-of-function" transporter with increased doxorubicin and rhodamine 123 transport activity (Chen et al, 2003;Honjo et al, 2001;Miwa et al, 2003;Ozvegy, Varadi, & Sarkadi, 2002). Later it turned out that amino acid 482 is also involved in the cholesterol and bile acid sensing of the protein.…”