(A) Deduced amino acid sequence of BCRP with display of motifs. The deduced sequence was obtained from the nucleotide sequence of full-length BCRP cDNA by applying the program translate, which is part of the GCG software package. TM 1, TM 2, and TM 3 refer to potential transmembrane regions; Glyc refers to potential sites of N-glycosylation. (B) Phylogram of the evolution of the amino acid sequence of BCRP in relation to certain other members of the ABC family of transport proteins. The phylogram was created by using the multiple-sequence alignment program pileup, which is part of the GCG software package, using a gap weight of 3.0 and a gap length weight of 0.1. The GCG program distances was then applied to create pairwise evolutionary distances between the aligned sequences, by using the Kimura protein-distance correction method. A graphical phylogenetic tree was then created from the distance matrix with the GCG program growtree.

(A) Northern analysis of the expression of BCRP mRNA in subclones of MCF-7/W cells stably transfected with expression vector pcDNA3-BCRP or in MCF-7/W cells transfected with empty vector pcDNA3 (uncloned-vector control) after selection in medium containing G418. The subclones were isolated by plating the transfected cells by limiting dilution in 96-well flat-bottomed culture flasks. The 795-bp PCR product originally obtained from the differential-display studies was radiolabeled and used as probe. Ethidium bromide stains of 1% agarose gel electrophoresis of the total cellular RNA used for the Northern blot demonstrated approximate equivalency of sample loading (results not shown). (B) Daunorubicin (DNR) accumulation and retention of MCF-7 cells transfected with empty pcDNA3 vector (vector control) or with clone 6 or clone 8 isolated from MCF-7/W cells after transfection with pcDNA3-BCRP. The data points are the mean of duplicate determinations; the vertical bars represent the upper or lower range for that data point. The cell volumes, measured by Coulter Channelyzer are 2,515 ± 56, 3,074 ± 112, and 2,459 ± 56 μm³ for MCF-7/BCRP-clone 6, MCF-7/BCRP-clone 8, and MCF-7/pcDNA3 vector control cells, respectively. These values are comparable to our previous measurements of MCF-7 cell volumes (8). (C) Effects of ATP depletion on the retention of rhodamine 123 by transfectant MCF-7/pcDNA3 (empty vector control) or MCF-7/BCRP clone 8 cells. The cells were incubated in complete medium or under ATP-depleting conditions (see Materials and Methods) for 20 min, and rhodamine 123 (0.5 μg/ml final concentration) was added for an additional 30 min. The cells were washed free of rhodamine and returned to culture either in complete medium or under ATP-depleting conditions for an additional 30 min. Rhodamine retention, expressed as arbitrary fluorescence units (FU) per cell, was determined by using flow cytometry (excitation 488 nm, emission 520 nm). The vertical lines over each bar represent the SD of four replicate determinations. Viability (trypan blue dye exclusion) of the cells in each treatment group was >90% at the completion of the study. (D) Representative sulforhodamine-B cytotoxicity (21) studies for mitoxantrone, daunorubicin, doxorubicin, cis-platin, paclitaxel, or vincristine against MCF-7/W (∗) or MCF-7/pcDNA3-BCRP clone 8 (■) cells. These data are typical of those used to obtain LC₅₀ values that comprise the data displayed in Table 1. The vertical bars for each data point represent ± SD of six replicate determinations.

(A) RNA fingerprinting of MCF-7 cells. Total cellular RNA was treated with DNase, reverse-transcribed into cDNA, and amplified by PCR using upstream and downstream primers and radiolabeled [³³P]dATP. The figure depicts a portion of the autoradiograph of a 5% polyacrylamide gel electrophoresis of the PCR mixture by using the primer pair P6 and T9. Lanes 1, 3, and 5 are reaction mixtures in which cDNA diluted 1:10 was added; lanes 2, 4, and 6 represent reaction mixtures in which cDNA diluted 1:40 was added. Lanes 7 and 8 are H₂O controls, in which sterile water was added to the PCR mixture in place of cDNA. Lanes 9, 10, and 11 are RNA controls, in which 0.02 μg of cellular RNA from MCF-7/W, MCF-7/AdrVp, or MCF-7/AdrVpPR cells was added instead of cDNA. These RNA controls served to indicate contamination of the RNA with genomic DNA. The arrow indicates a PCR product representing an mRNA species overexpressed in MCF-7/AdrVp cells, compared with MCF-7/W or MCF-7/AdrVpPR cells. This product was excised from the gel for further amplification by PCR. (B) Northern blot hybridization (Upper) of mRNA from MCF-7/W, MCF-7/AdrVp, or MCF-7/AdrVpPR cells by using the 795-bp PCR product obtained from RNA fingerprinting studies and isolated by TA cloning as a probe after labeling with [³²P]dCTP (Prime-a-Gene labeling kit, Promega). To control for equivalence in sample loading, the blot was stripped and rehybridized with a radiolabeled probe for 18S RNA (Lower).

Tissue distribution of BCRP mRNA. This analysis was performed by using commercially prepared human multiple-tissue Northern blots (MTN Blot and MTN Blot II, CLONTECH) and the manufacturer’s recommended procedure. The 795-bp PCR product obtained from RNA fingerprinting was used as probe after labeling with [³²P]dCTP. Tissues tested were: heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), pancreas (lane 8), spleen (lane 9), thymus (lane 10), prostate (lane 11), testis (lane 12), ovary (lane 13), small intestine (lane 14), colon (lane 15), and peripheral-blood leukocytes (lane 16).