Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity

Abstract

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

Figure 1

Expression of ABCG2 in HEK-293 cells transfected with wild-type and mutant ABCG2. (A) Northern blot analysis was performed on 20 μg RNA extracted from six cell lines transfected with ABCG2 (482G-1, 482G-2, 482R-2, 482R-5, 482T-7, 482T-10) and one cell line transfected with empty vector (pcDNA3-10) and probed with a riboprobe generated from the first 662 bp of ABCG2. (B) Immunoblot analysis for ABCG2 using the anti-ABCG2 antibody BXP-21 was performed on membrane protein (30 μg) from the seven transfected cell lines. (C) Immunoblot analysis for ABCG2 with serial dilutions of membranes isolated from same transfected clones (482G-1, 482R-2, 482T-10) subsequently used in cytotoxicity assays. (D) The seven transfected cell lines were incubated for 30 min with either phycoerythrin-labelled negative control antibody (dotted line) or phycoerythrin-labelled anti-ABCG2 antibody (dashed line) and then analysed on a flow cytometer. Representative histograms are shown.

Figure 2

Transport of fluorescent substrates by wild-type and mutant ABCG2. The six transfected cell lines were incubated in rhodamine 123 (0.5 μg ml⁻¹), daunorubicin (5 μg ml⁻¹), mitoxantrone (20 μM), BODIPY-prazosin (250 nM), or LysoTracker Green (250 nM) with or without 10 μM FTC for 30 min at 37°C. Cells were then washed, allowed to efflux for 1 h at 37°C in substrate-free media continuing with (dotted line) or without (solid line) FTC, and analysed on a flow cytometer. Representative results are shown. Mean channel differences from 2 or more experiments are present in Table 1.

Figure 3

Cross-resistance profile conferred by wild-type and mutant ABCG2. The 4-day cytotoxicity assays were performed with mitoxantrone, SN-38, topotecan, daunorubicin, etoposide, and flavopiridol on HEK-293 cells transfected with empty vector (filled squares), or transfected with 482R (hatched squares), 482G (open circles), or 482 T (open triangles) ABCG2. Curves representative of two to five separate experiments are shown.

Figure 4

Novobiocin reverses wild-type ABCG2 but not mutant ABCG2-mediated resistance. (A) The six transfected cell lines were incubated in 250 nM BODIPY-prazosin with or without 50 μM novobiocin for 30 min at 37°C, washed, then allowed to efflux for 1 h at 37°C continuing with (dotted line) or without (solid line) novobiocin and compared to cells incubated in 10 μM FTC (dashed line). (B) The 4-day cytotoxicity assays were performed with topotecan with (filled symbols) or without (open symbols) 50 μM novobiocin on HEK-293 cells expressing wild-type (482R) or mutant (482G, 482T) ABCG2 as detailed in the Materials and Methods section. Representative results are shown. Empty vector transfected cells are shown as squares while ABCG2 transfected cells are denoted by circles. Dose-modifying factor values were obtained for the ABCG2-transfected cells by dividing the IC₅₀ for topotecan without novobiocin by the IC₅₀ value for topotecan in the presence of novobiocin.