Figure 4.

(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.