Article Figures & Data
Figures
- Fig. 1.
Northern analysis of (A) Bcrp1 and (B) Mrp1 mRNA levels in doxorubicin-resistant fibroblast cell lines during the development of doxorubicin resistance, with mitoxantrone- or topotecan-resistant cell lines and normal mouse tissues for comparison. Names of drug-selected sublines reflect the drug (D, M, or T prefix for doxorubicin, mitoxantrone, or topotecan, respectively) and drug concentration, in nm, to which the cells were adapted. Duplicate blots were hybridized with antisense RNA probes for Bcrp1 or Mrp1. Total RNA loading (C) was checked by rehybridizing one blot with a probe for the 18S rRNA. Signal levels were quantified, corrected for loading, and are presented below the panels in the figure relative to the level of Bcrp1 (or Mrp1) mRNA in the 88.6 cell line.
- Fig. 2.
Southern analysis of Bcrp1 amplification in the doxorubicin-selected cell lines. Parallel blots of EcoRI-digested DNAs were hybridized to either (A) Bcrp1 or (B) Pim-1 probes. Numerical estimates of Bcrp1 amplification relative to the parent cell lines, shown below (A), were obtained by quantifying the Bcrp1 hybridization signals and correcting for loading on the basis of the Pim-1 signals.
- Fig. 3.
Accumulation of (A) mitoxantrone, (B) daunorubicin, and (C) rhodamine 123 in drug-selected mouse cell lines and in Bcrp1-transduced MEF3.8 clone A2, in the absence or presence of 200 nm Ko143, a Bcrp1 inhibitor. Accumulation is shown in terms of relative arbitrary units of drug or dye fluorescence. Experiments were performed in triplicate; bars, ±SD.
- Fig. 4.
Mutations in codon 482 of Bcrp1 from the doxorubicin-selected cell lines. Sequence electropherograms for the vicinity of the mutations are shown for each of the late-passage doxorubicin-selected cell lines. The wild-type example is taken from the KOT52/D80 line but is the same as found in earlier passages of all of the doxorubicin-selected lines, the mitoxantrone- or topotecan-selected lines, the parent cell lines, and mouse liver (Table 2 <$REFLINK> and “Results”).
Tables
- Table 1
Cross-resistance profiles of doxorubicin-resistant fibroblast cell lines
Shown for each cell line and drug combination: IC50 ± SD (nM) with resistance factors in bold below the IC50s.
Drug MEF3.8an = 3 MEF3.8/M32an = 3 MEF3.8/T6400an = 3 KOT52 n = 4 KOT52/D80 n = 2 KOT52/D320 n = 5 88.6 n = 3 88.6/D200-A n = 2 88.6/D800-A n = 3 88.6/D200-B n = 2 88.6/D800-B n = 3 Mitoxantrone 0.81 ± 0.25 142 ± 8 53 ± 6 1.33 ± 0.13 210 ± 70 420 ± 60 2.4 ± 0.3 295 ± 30 600 ± 90 275 ± 20 650 ± 150 174 66 158 318 123 250 115 269 Bisantrene 12.5 ± 3.0 215 ± 22 110 ± 10 9.7 ± 2.5 385 ± 64 1850 ± 400 11 ± 1.1 540 ± 85 2500 ± 560 450 ± 90 4400 ± 600 17 8.8 40 191 48 222 40 394 Doxorubicin 6.4 ± 1.0 56 ± 8 33 ± 9 3.5 ± 0.5 140 ± 32 1100 ± 300 8.3 ± 1.6 260 ± 50 1400 ± 180 230 ± 15 1300 ± 200 8.7 5.2 39 312 31 172 28 156 Daunorubicin 7.4 ± 1.5 14.5 ± 0.5 12.3 ± 1.2 4.0 ± 0.3 34 ± 6 330 ± 20 6.5 ± 0.2 67 ± 4 500 ± 140 51 ± 9 390 ± 60 2.0 1.7 8.6 82 10 77 7.8 60 Topotecan 49 ± 3 6400 ± 2000 16000 ± 5300 166 ± 69 17500 ± 700 23000 ± 5000 98 ± 11 11000 ± 2000 4300 ± 1750 6700 ± 600 3200 ± 1200 132 329 105 137 109 44 69 32 Vincristine 0.38 ± 0.03 0.64 ± 0.07 0.63 ± 0.23 0.82 ± 0.09 0.72 ± 0.18 0.98 ± 0.22 4.1 ± 0.4 6.1 ± 0.4 4.1 ± 0.7 6.2 ± 0.4 7.3 ± 0.7 1.7 1.6 0.87 1.2 1.5 1.0 1.5 1.8 Paclitaxel 4.5 ± 0.3 8.4 ± 1.9 9.1 ± 3.0 2.2 ± 0.4 2.7 ± 0.1 3.4 ± 1.4 5.8 ± 0.4 8.0 ± 0.1 5.9 ± 1.5 3.8 ± 0.6 4.1 ± 0.8 1.9 2.0 1.2 1.7 1.4 1.0 0.7 0.7 -
a These data are taken for comparison from Ref. 3 .
-
- Table 2
Mutations in BCRP/Bcrp1 codon 482 in drug-resistant cell lines
Altered nucleotides are underlined.
cDNA source Selecting drug Codon 482 Encodes Human placentaa – AGG R MCF-7/AdrVpa doxorubicin, 3 μM ACG T S1-M1-80a mitoxantrone, 80 μM GGG G Mouse livera – AGG R MEF3.8 AGG R MEF3.8/M32 mitoxantrone, 32 nM AGG R MEF3.8/T6400 topotecan, 6400 nM AGG R KOT52 – AGG R KOT52/D80 doxorubicin, 80 nM AGG R KOT52/D320 doxorubicin, 320 nM AGG/ATG R/M: 70/30% KOT52/D800 doxorubicin, 800 nM AGG/ATG R/M: 40/60% 88.6 – AGG R 88.6/D200-A doxorubicin, 200 nM AGG R 88.6/D800-A doxorubicin, 800 nM ATG M 88.6/D200-B doxorubicin, 200 nM AGG R 88.6/D800-B doxorubicin, 800 nM AGT S -
a From Refs. (1 2 3 , 12 , 13 , 19) and GenBank records.
-
- Table 3
Drug resistance conferred by ectopic expression of Bcrp1 in MEF3.8 cells
Shown are the IC50s (in nM), determined by n assays and the resistance factors for a representative Bcrp1-transduced clone. Values shown here for the MEF3.8 parent line may differ somewhat from the earlier determinations shown in Table 1 <$REFLINK> because of variations in drug batches and details of the assay procedure. Results of two-tailed heteroscedastic t tests comparing the IC50s of the parent and transduced lines are shown.
Drug Mean IC50 ± SD, nM, and (n) RF t test MEF3.8 MEF3.8/Bcrp1 clone A2 Mitoxantrone 0.40 ± 0.10 (6) 19 ± 2 (5) 48 P < 0.01 Doxorubicin 3.6 ± 0.3 (3) 13 ± 1.2 (3) 3.6 P < 0.01 Topotecan 45 ± 7 (4) 1800 ± 100 (3) 40 P < 0.01 Vincristine 0.25 ± 0.10 (4) 0.26 ± 0.01 (3) 1.0 nsa -
a ns, not significant.
-
- Table 4
Reversal of cross-resistance in doxorubicin-selected cell lines by the BCRP/Bcrp1 inhibitor Ko143
Values shown are the resistance factors in the absence (as per Table 1 <$REFLINK> ) or presence of 200 nM Ko143. Some assays in the presence of Ko143 were performed on two occasions; in these cases both results are shown.
Drug Ko143 Cell line KOT52 KOT52/D320 88.6 88.6/D800-A 88.6/D800-B Mitoxantrone − 1 318 1 250 269 + 0.53 2.1 0.40 1.6 3.3 Bisantrene − 1 191 1 222 394 + 0.48 2.3, 2.4 0.96, 1.1 2.1, 2.3 4.8, 7.6 Doxorubicin − 1 312 1 172 156 + 0.69 6.6, 7.1 0.82, 1.35 2.9, 3.6 3.7, 5.9 Topotecan − 1 137 1 44 32 + 0.30 1.0 0.68 0.60 0.76
Volume 62, Issue 8
