ABCMdb

PMID: 12810652

Rajendra R, Gounder MK, Saleem A, Schellens JH, Ross DD, Bates SE, Sinko P, Rubin EH
Differential effects of the breast cancer resistance protein on the cellular accumulation and cytotoxicity of 9-aminocamptothecin and 9-nitrocamptothecin.
Cancer Res. 2003 Jun 15;63(12):3228-33., 2003-06-15 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). Login to comment 7 ABCG2 p.Arg482Thr By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC. Login to comment 8 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptoth- ecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). Login to comment 10 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Login to comment 13 ABCG2 p.Arg482Thr Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Login to comment 24 ABCG2 p.Arg482Thr In this report, we demonstrate that whereas overexpression of Pgp, MRP1, or MRP2 has little effect on the cytotoxicity of either 9-NC or 9-AC, overexpression of either wild-type or mutant R482T BCRP confers resistance to 9-AC, but not to 9-NC. Login to comment 27 ABCG2 p.Arg482Thr In this report, we demonstrate that whereas overexpression of Pgp, MRP1, or MRP2 has little effect on the cytotoxicity of either 9-NC or 9-AC, overexpression of either wild-type or mutant R482T BCRP confers resistance to 9-AC, but not to 9-NC. Login to comment 72 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr HEK293 pcDNA3-10 Human embryonic kidney cells, stably transfected with pcDNA3 control vector 27 HEK293- ABCG2R482-2 HEK293 cells, stably transfected with pcDNA3-BCRP, expressing wild-type BCRP under control of a CMVa promoter 27 HEK293- ABCG2T482-10 HEK293 cells, stably transfected with pcDNA3- BCRPR482T, expressing a mutant form of BCRP with a threonine for arginine substitution at codon 482 27 MDA-MB-231- pcDNA3 Human breast carcinoma cells, stably transfected with pcDNA3 control vector 24 MDA-MB-231- pcDNA3- BCRP(R482T) MDA-MB-231 cells, stably transfected with pcDNA3-BCRP expressing R482T mutant BCRP under control of a CMV promoter 24 and 35 MDCKII MDCK epithelial cells 48 MDCKII-Pgp MDCKII cells, stably transfected with a vector expressing human MDR1 under control of a CMV promoter 49 MDCKII-MRP1 MDCKII cells, stably expressing human MRP1 under control of a CMV promoter 50 MDCKII-MRP2 MDCKII cells, stably expressing human MRP2 under control of a CMV promoter 51 U-937 Human monoblastic leukemia cells 31 U-937-CR U-937 cells selected for resistance to 9-NC 31 U-937-RERC U-937 cells selected for resistance to 9-NC and etoposide 28 RPMI-8402 Human T-cell lymphoblastic leukemia cells 36 CPT-K5 RPMI-8402 cells selected for resistance to irinotecan 36 a CMV, cytomegalovirus. Login to comment 75 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr HEK293 pcDNA3-10 Human embryonic kidney cells, stably transfected with pcDNA3 control vector 27 HEK293- ABCG2R482-2 HEK293 cells, stably transfected with pcDNA3-BCRP, expressing wild-type BCRP under control of a CMVa promoter 27 HEK293- ABCG2T482-10 HEK293 cells, stably transfected with pcDNA3- BCRPR482T, expressing a mutant form of BCRP with a threonine for arginine substitution at codon 482 27 MDA-MB-231- pcDNA3 Human breast carcinoma cells, stably transfected with pcDNA3 control vector 24 MDA-MB-231- pcDNA3- BCRP(R482T) MDA-MB-231 cells, stably transfected with pcDNA3-BCRP expressing R482T mutant BCRP under control of a CMV promoter 24 and 35 MDCKII MDCK epithelial cells 48 MDCKII-Pgp MDCKII cells, stably transfected with a vector expressing human MDR1 under control of a CMV promoter 49 MDCKII-MRP1 MDCKII cells, stably expressing human MRP1 under control of a CMV promoter 50 MDCKII-MRP2 MDCKII cells, stably expressing human MRP2 under control of a CMV promoter 51 U-937 Human monoblastic leukemia cells 31 U-937-CR U-937 cells selected for resistance to 9-NC 31 U-937-RERC U-937 cells selected for resistance to 9-NC and etoposide 28 RPMI-8402 Human T-cell lymphoblastic leukemia cells 36 CPT-K5 RPMI-8402 cells selected for resistance to irinotecan 36 a CMV, cytomegalovirus. Login to comment 78 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Similar studies were done using HEK293 cells overexpressing wild-type BCRP or R482T BCRP and with MDA-MB-231 cells overexpressing R482T BCRP (Table 1). Login to comment 80 ABCG2 p.Arg482Thr As observed previously (27), expression of the R482T protein was slightly lower than that of the wild-type protein in the stable HEK293 transfectants (Fig. 2). Login to comment 81 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Similar to previous results (27, 34, 35), overexpression of wild-type or R482T BCRP conferred resistance to both TPT and SN-38 (Fig. 3; Table 3). Login to comment 83 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr In HEK293 cells expressing wild-type or mutant BCRP, relative resistances to 9-AC were similar to those observed for TPT and SN-38, whereas in MDA-MB-231 cells, R482T BCRP conferred slightly less resistance to 9-AC compared with TPT or SN-38 (Table 3). Login to comment 84 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Furthermore, in HEK293 cells, overexpression of the R482T BCRP mutant was associated with levels of resistance that were similar to those observed with the wild-type protein for TPT, SN-38, and 9-AC (Fig. 3; Table 3). Login to comment 85 ABCG2 p.Arg482Thr By contrast, overexpression of either wild-type or R482T BCRP had no effect on cellular sensitivity to 9-NC (Fig. 3; Table 3). Login to comment 86 ABCG2 p.Arg482Thr In HEK293 cells expressing wild-type or mutant BCRP, relative resistances to 9-AC were similar to those observed for TPT and SN-38, whereas in MDA-MB-231 cells, R482T BCRP conferred slightly less resistance to 9-AC compared with TPT or SN-38 (Table 3). Login to comment 87 ABCG2 p.Arg482Thr Furthermore, in HEK293 cells, overexpression of the R482T BCRP mutant was associated with levels of resistance that were similar to those observed with the wild-type protein for TPT, SN-38, and 9-AC (Fig. 3; Table 3). Login to comment 88 ABCG2 p.Arg482Thr By contrast, overexpression of either wild-type or R482T BCRP had no effect on cellular sensitivity to 9-NC (Fig. 3; Table 3). Login to comment 91 ABCG2 p.Arg482Thr Cells consisting of vector only transfectants (pcDNA) or stably expressing wild-type (WT) or R482T mutant BCRP were subjected to sequential immunoblotting using BCRP and beta-actin antibodies as described in "Materials and Methods." Login to comment 94 ABCG2 p.Arg482Thr Cells consisting of vector only transfectants (pcDNA) or stably expressing wild-type (WT) or R482T mutant BCRP were subjected to sequential immunoblotting using BCRP and beta-actin antibodies as described in "Materials and Methods." Login to comment 97 ABCG2 p.Arg482Thr Effects of overexpression of wild-type or R482T mutant BCRP on the cytotoxicity of 9-NC and 9-AC. HEK293 cells consisting of stable transfectants of vector alone (ࡗ) or vectors expressing wild-type (Œ) or mutant (X) BCRP were incubated for 72 h with various concentrations of the indicated compounds. Login to comment 100 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr 3230 DIFFERENTIAL EFFECTS OF BCRP ON 9-AC AND 9-NC American Association for Cancer ResearchCopyright (c) 2003 on May 3, exposure to 10 ␮M 9-AC, intracellular drug levels were about -fold higher in vector controls compared with wild-type or R482T BCRP transfectants (Fig. 4). Login to comment 102 ABCG2 p.Arg482Thr These results are consistent with those obtained from antiproliferative studies and suggest that wild-type and mutant R482T BCRP mediate cellular efflux of 9-AC, but not 9-NC. Login to comment 103 ABCG2 p.Arg482Thr 3230 DIFFERENTIAL EFFECTS OF BCRP ON 9-AC AND 9-NC exposure to 10 ␮M 9-AC, intracellular drug levels were about 2-fold higher in vector controls compared with wild-type or R482T BCRP transfectants (Fig. 4). Login to comment 105 ABCG2 p.Arg482Thr These results are consistent with those obtained from antiproliferative studies and suggest that wild-type and mutant R482T BCRP mediate cellular efflux of 9-AC, but not 9-NC. Login to comment 117 ABCG2 p.Arg482Thr By contrast, overexpression of wild-type or R482T BCRP confers resistance to 9-AC, associated with reduced intracellular accumulation of this drug. Login to comment 119 ABCG2 p.Arg482Thr Similar to results reported for TPT (26, 27), we found that overexpression of the R482T mutant yielded resistance to 9-AC that was similar to that observed with overexpression of the wild-type protein. Login to comment 120 ABCG2 p.Arg482Thr By contrast, overexpression of wild-type or R482T BCRP confers resistance to 9-AC, associated with reduced intracellular accumulation of this drug. Login to comment 122 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Effects of overexpression of wild-type or R482T mutant BCRP on the intracellular accumulation of 9-NC and 9-AC. HEK293 cells stably transfected with vector alone (pcDNA) or vectors expressing wild-type (WT) or R482T mutant BCRP were incubated with 10 ␮M 9-NC or 9-AC at 37°C for 30 min. Login to comment 125 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Effects of overexpression of wild-type or R482T mutant BCRP on the intracellular accumulation of 9-NC and 9-AC. HEK293 cells stably transfected with vector alone (pcDNA) or vectors expressing wild-type (WT) or R482T mutant BCRP were incubated with 10 ␮M 9-NC or 9-AC at 37°C for 30 min. Login to comment 131 ABCG2 p.Arg482Thr Table 3 Antiproliferative effects of camptothecin analogues in the presence and absence of forced expression of wild-type or mutant BCRP Drug HEK293 HEK293 BCRP RRa HEK293 BCRP R482T RRa MDA-MB-231 MDA-MB-231 BCRP R482T RR TPT 0.034 Ϯ 0.01b 0.39 Ϯ 0.19c 11.5 0.40 Ϯ 0.16c 11.8 0.04 Ϯ 0.01 1.13 Ϯ 0.09c 28 SN-38 0.022 Ϯ 0.00 0.17 Ϯ 0.08c 7.7 0.14 Ϯ 0.02c 6.4 0.10 Ϯ 0.03 3.17 Ϯ 0.15c 32 9-AC 0.033 Ϯ 0.01 0.24 Ϯ 0.13c 7.3 0.40 Ϯ 0.13c 12 0.22 Ϯ 0.07 1.56 Ϯ 0.51c 7.1 9-NC 0.030 Ϯ 0.01 0.03 Ϯ 0.01 1 0.03 Ϯ 0.01 1 0.24 Ϯ 0.02 0.32 Ϯ 0.17 1.4 a RR, relative resistance of BCRP-expressing cell line compared with control cell line. b Results are expressed as mean-SDs (␮M) for calculated IC50s for six replicate experiments. Login to comment 134 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Table 3 Antiproliferative effects of camptothecin analogues in the presence and absence of forced expression of wild-type or mutant BCRP Drug HEK293 HEK293 BCRP RRa HEK293 BCRP R482T RRa MDA-MB-231 MDA-MB-231 BCRP R482T RR TPT 0.034 Ϯ 0.01b 0.39 Ϯ 0.19c 11.5 0.40 Ϯ 0.16c 11.8 0.04 Ϯ 0.01 1.13 Ϯ 0.09c 28 SN-38 0.022 Ϯ 0.00 0.17 Ϯ 0.08c 7.7 0.14 Ϯ 0.02c 6.4 0.10 Ϯ 0.03 3.17 Ϯ 0.15c 32 9-AC 0.033 Ϯ 0.01 0.24 Ϯ 0.13c 7.3 0.40 Ϯ 0.13c 12 0.22 Ϯ 0.07 1.56 Ϯ 0.51c 7.1 9-NC 0.030 Ϯ 0.01 0.03 Ϯ 0.01 1 0.03 Ϯ 0.01 1 0.24 Ϯ 0.02 0.32 Ϯ 0.17 1.4 a RR, relative resistance of BCRP-expressing cell line compared with control cell line. b Results are expressed as mean-SDs (␮M) for calculated IC50s for six replicate experiments. Login to comment