Article Figures & Data
Figures
- Fig. 1.
Western blot analysis of BCRP expression in 12 tumor cell lines of the NCI anticancer drug screening. Western blot analysis was performed under a nonreducing condition, so that the dimeric form BCRP was observed as a band at ∼140 kDa. A serial dilution of PA/WT cells (PA317 cells transfected with wild-type BCRP cDNA) were used as a positive control. Six cell lines in the top lane showed considerably high BCRP expression.
- Fig. 2.
Identification of C421A polymorphism in A549 cells. A, wild-type BCRP cDNA. The entire coding region of BCRP mRNA was amplified by RT-PCR, subcloned, and sequenced. The 421st base is cytosine (arrow). B, C421A BCRP cDNA. The 421st base was changed from cytosine to adenine (arrow) in five of eight clones analyzed. C, direct genomic sequencing of the 421st nucleotide. The mutation was confirmed by genomic DNA analysis. Exon 5 covering the 421st nucleotide was amplified by PCR and directly sequenced, demonstrating both the wild-type and C421A alleles (arrow).
- Fig. 3.
Expression analysis of mutant BCRP transfectants. PA317 cells transfected with wild-type, G34A, C421A, and 944–949-deleted BCRP cDNAs were termed PA/WT, PA/V12M, PA/Q141K, and PA/Δ315-6, respectively. A, Western blotting of PA317 cells transfected with mutant BCRP cDNA in pHaL-IRES-DHFR plasmid. Twenty μg of protein was loaded in each lane. Top panel, Western blot analysis processed under a nonreducing condition. BCRP dimer was detected as a band at ∼140 kDa. Bottom panel, Western blot analysis processed under a reducing condition. BCRP monomer was detected as a band at ∼70 kDa. B, top panel, Northern blot analysis of PA317 cells and BCRP transfectants. Twenty μg of total RNA was loaded in each lane of the agarose-formaldehyde gel and transferred to Hybond-N+. The blot was hybridized with a BCRP cDNA probe. Bottom panel, ethidium bromide staining of total RNA after electrophoresis. 28S and 18S indicate 28S and 18S ribosomal RNA, respectively.
- Fig. 4.
Growth inhibition assay of mutant BCRP-transfected PA317 cells. PA317 cells transfected with wild-type, G34A, C421A, and 944–949-deleted BCRP cDNAs were termed PA/WT, PA/V12M, PA/Q141K, and PA/Δ315-6, respectively. A, sensitivity to SN-38 (A-1), mitoxantrone (A-2), and topotecan (A-3) of PA317, PA/WT, PA/V12M, and PA/Q141K cells. B, sensitivity to SN-38 (B-1), mitoxantrone (B-2), and topotecan (B-3) of PA317, PA/WT, and PA/Δ315-6 cells. •, PA317; ○, PA/WT; ▵, PA/V12M; □, PA/Q141K; ⋄, PA/Δ315-6.
- Fig. 5.
Intracellular topotecan uptake of BCRP-transfected PA317 cells. PA317 cells transfected with wild-type, G34A, C421A, and 944–949-deleted BCRP cDNAs were termed PA/WT, PA/V12M, PA/Q141K, and PA/Δ315-6, respectively. Cells were incubated for 30 min with (———) or without (······) 30 μm topotecan, washed, and topotecan uptake was measured by FACS. In parental PA317 cells, a fluorescence peak shift to the right after the incubation with topotecan indicates cellular uptake of topotecan, whereas only marginal shifts occurred in PA/WT, PA/V12M, and PA/Δ315-6 cells. There was a stronger fluorescence peak shift to the right in PA/Q141K cells than the other transfectants (arrow).
Tables
- Table 3
Frequencies of G34A, C376T, and C421A polymorphisms of the BCRP gene in the general Japanese population
Wild-type Heterozygous Homozygous Total G34A 19 (66%) 9 (31%) 1 (3%) 29 C376Ta 121 (98%) 3 (2%) 0 (0%) 124 C421A 67 (54%) 48 (39%) 9 (7%) 124 -
↵a C376T polymorphism in exon 4 substitutes stop codon for Gln-126.
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Volume 1, Issue 8
