Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding

Abstract

ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells. This mutation was shown to modulate the transport of Rh123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482. In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.

Figure 1. Effects of Rh123 on cell growth

MCF-7, MCF-7/AdrVp1000 and MCF-7/Mitox cells were seeded at 1000–2000 cells/well in 24-well plates and grown in the absence or presence of increasing concentrations of Rh123 (0–400 μM). Cell viability was estimated 6 days later by staining the surviving cell colonies with 0.1% Methylene Blue in 50% (v/v) ethanol. Cell growth is expressed as a percentage of the control in the absence of drug. Each point is the mean±S.D. of three independent experiments.

Figure 2. Intracellular uptake of Rh123

Cells (MCF-7, MCF-7/AdrVp1000 and MCF-7/Mitox) were grown in six-well plates, washed with PBS and incubated in the presence or absence of inhibitors of ATP synthesis or cyclosporine A (CSA), as described in the Materials and methods section. Cells were kept for 30 min at 37 °C before adding Rh123 to each well at a final concentration of 1 μM, with or without 2 μM cyclosporine A (as indicated in the Figure). Left panels: cells were washed and subjected to flow cytometry by FACscan using Rh123 excitation at λ=485 nm. Right panels: cells after incubation were viewed at ×400 magnification using a Nikon TE200 inverted microscope equipped with a fluorescence filter.

Figure 3. Photoaffinity labelling of ABCG2 by IAARh123

Total membranes prepared from MCF-7 (−), MCF-7/AdrVp1000 (Mut; expressing mutated ABCG2) and MCF-7/Mitox (WT; expressing wild-type ABCG2) cells were photoaffinity labelled with IAARh123 and resolved on SDS/PAGE (B). Panel (A) shows the Coomassie Blue staining of membrane proteins prepared from the indicated cell lines. (C) Photoaffinity-labelled membrane proteins from MCF-7, MCF-7/AdrVp1000 and MCF-7/Mitox cells immunoprecipitated with an ABCG2-specific monoclonal antibody (BXP-21). (D) Western blot of membranes (10 and 20 μg) prepared from the same cells using monoclonal antibody BXP-21. The position of ABCG2 is indicated by the arrowheads.

Figure 4. Specificity of ABCG2 photolabelling with IAARh123

(A) Membrane proteins (50 μg) from MCF-7/AdrVp1000 cells were photolabelled with increasing concentrations (0–4 μM) of IAARh123. After SDS/PAGE, the bands corresponding to ABCG2 were excised and quantified. (B) Photolabelled ABCG2 from MCF-7/AdrVp1000 cells incubated in the absence or presence of a molar excess (0–1000-fold) of IAARh123. (C) Photolabelling of ABCG2 with IAARh123 in the absence or presence of reserpine at a 300× molar excess.

Figure 5. Effects of diverse drugs on the photoaffinity labelling of wild-type and mutant ABCG2 by IAARh123

Membrane proteins from MCF-7, MCF-7/AdrVp1000 (A) or MCF-7/Mito (B) cells were photoaffinity labelled with IAARh123 in the absence or presence of a molar excess (300–1000-fold) of mitoxantrone (Mitox), vincristine (VCR), doxorubicin (DOX), cisplatin (Cis-pl), VP-16, taxol or methotrexate (MTX). The relative photolabelling signal of wild-type and mutant ABCG2 with IAARh123 in the presence of these drugs is also shown. The photoaffinity labelling results show a representative experiment that was repeated at least three times. The bar graphs show the relative decrease in the photoaffinity labelling of ABCG2 in the presence of increasing molar concentrations of the drugs averaged between three different independent experiments.