ABCMdb

PMID: 15385942

Walter RB, Raden BW, Thompson J, Flowers DA, Kiem HP, Bernstein ID, Linenberger ML
Breast cancer resistance protein (BCRP/ABCG2) does not confer resistance to gemtuzumab ozogamicin and calicheamicin-gamma1 in acute myeloid leukemia cells.
Leukemia. 2004 Nov;18(11):1914-7., [PubMed]

Sentences

No. Mutations Sentence Comment

33 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly Evidence to date suggests that BCRP mainly serves to protect cells from xenobiotic toxins, although additional functions in stem cell populations are discussed.4,5 BCRP confers resistance to certain chemotherapeutic agents; however, the substrate specificity in cells containing a mutation at codon 482 (R482T or R482G) differs somewhat from cells expressing wild-type protein.4,5 BCRP mRNA and protein are variably expressed in AML.5,6 So far, only wild-type BCRP (ie arginine at codon 482) has been Received 27 April 2004; accepted 29 June 2004; Published online 16 September 2004 Correspondence: Dr RB Walter, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D2-373, Seattle, WA 98109-1024, USA; Fax: þ 1 206 667 6084; E-mail: rwalter@fhcrc.org This work has been presented in part at the 45th annual meeting of the American Society of Hematology (December 6-9, 2003; San Diego, CA, USA; Blood 2003; 102: 208b-209b; abstract #4553) Correspondence Leukemia found in primary AML samples7 (and A Suvannasankha et al. Blood 2002; 100: 67a; abstract), whereas R482T and R482G variants have been identified in drug-selected cell lines.4,5 It remains unknown whether mutations at codon 482 might occur in patients after treatment with BCRP substrate drugs. Login to comment 36 ABCG2 p.Arg482Thr Given that BCRP-mediated drug resistance profile partially overlaps those associated with Pgp or MRP, and that additional unidentified mechanisms likely play a role in resistance to GO,3,8 we investigated whether BCRP (wild type or R482T) could confer resistance to GO or to the unconjugated calicheamicin-g1 derivative. Login to comment 37 ABCG2 p.Arg482Thr For this purpose, lentiviral pRRLsin.cPPT.MSCV.BCRP-IRES-EGFP vectors were generated by cloning BCRP cDNA (wild type or R482T; provided by DD Ross, University of Maryland, Baltimore, MD, USA) into pRRLsin.cPPT.MSCV as a BCRP-IRES-EGFP cassette. Login to comment 43 ABCG2 p.Arg482Thr In comparison, lentivirus-mediated transduction of wild-type or R482T BCRP at an MOI of up to 100 yielded stable subpopulations of cells with significant BCRP protein expression and activity (Figure 1). Login to comment 49 ABCG2 p.Arg482Thr As expected,4,5 cells overexpressing either wild-type or R482T BCRP were resistant to mitoxantrone, and coincubation with Ko143 restored sensitivity (Tables 1 and 2). Login to comment 50 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr In contrast, overexpression of either wild-type or R482T BCRP did not alter GOor calicheamicin-g1-induced cytotoxicities, and Ko143 had no effect on drug susceptibilities (Tables 1 and 2; note that R482T-transduced HL-60 cells were not established). Login to comment 53 ABCG2 p.Arg482Thr They further suggest that calicheamicin-g1 is not a substrate for either wild-type or R482T BCRP. Login to comment 56 ABCG2 p.Arg482Thr Figure 1 BCRP wild-type and R482T protein expression and activity in parental and transduced CD33þ hematopoietic cell lines. Login to comment 59 ABCG2 p.Arg482Thr For this assay, cells were incubated for 30 min at 371C in the dark in 20 mM mitoxantrone in the presence or absence of 200 nM Ko143 (for R482T transduced NB4 and ML-1 cells) or 600 nM Ko143 (for all other transduced cells). Login to comment 61 ABCG2 p.Arg482Thr HL-60 cells were not infected with R482T BCRP (ND). Login to comment 90 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Table 2 Effect of BCRP R482T on cytotoxity induced by mitoxantrone, calicheamicin-g1, and GO MSCV-EGFP BCRP/R482T +DMSO +Ko143 +DMSO +Ko143 U937 Mitoxantrone 25 nM 37.2724.1 (5) 51.7724.7 (5) 3.571.8 (5) 33.7719.3 (5) 100 nM 49.6724.0 (5) 61.0722.7 (5) 8.176.1 (5) 41.4722.0 (5) Calicheamicin 2.5 ng/ml 65.5711.2 (3) 70.578.3 (3) 60.577.4 (3) 62.675.2 (3) 10 ng/ml 87.770.8 (3) 89.270.6 (3) 86.872.2 (3) 86.473.3 (3) GO 2.5 ng/ml 13.672.7 (2) 15.073.4 (2) 21.573.5 (2) 20.076.0 (2) 40 ng/ml 27.772.8 (2) 26.070.6 (2) 33.476.1 (2) 35.7710.9 (2) ML-1 Mitoxantrone 2.5 nM 16.078.4 (3) 16.478.6 (3) 1.670.9 (3) 12.577.8 (3) 10 nM 25.275.5 (3) 26.073.9 (3) 6.574.0 (3) 28.272.0 (3) Calicheamicin 1 ng/ml 27.875.4 (2) 18.6715.6 (2) 38.073.6 (2) 34.973.3 (2) 10 ng/ml 69.173.5 (2) 61.377.3 (2) 74.673.0 (2) 73.873.1 (2) GO 0.1 ng/ml 4.672.4 (3) 4.170.8 (2) 12.877.0 (3) 6.871.9 (2) 0.5 ng/ml 33.275.4 (3) 31.273.0 (2) 31.077.7 (3) 27.675.6 (2) NB4 Mitoxantrone 10 nM 27.9713.3 (5) 29.6714.1 (5) 3.271.2 (5) 27.2715.8 (5) 25 nM 52.9716.2 (5) 65.2720.0 (5) 9.473.0 (5) 52.6730.1 (5) Calicheamicin 1 ng/ml 57.2711.5 (3) 54.176.1 (3) 60.077.1 (3) 61.176.9 (3) 10 ng/ml 90.670.6 (3) 91.370.6 (3) 92.770.8 (3) 91.471.5 (3) GO 2.5 ng/ml 9.673.2 (2) 8.971.7 (2) 10.070.7 (2) 9.870.9 (2) 40 ng/ml 29.974.2 (2) 30.373.1 (2) 31.873.8 (2) 31.976.8 (2) Empty-vector-transduced cells (MSCV-EGFP) and BCRP R482T vector-transduced cells (at MOI 10: NB4 cells, or MOI 100: U937 and ML-1 cells) were treated with various concentrations of mitoxantrone, calicheamicin-g1, or GO in the presence or absence of Ko143 at the predetermined optimal concentration (200 nM for NB4 and ML-1 cells; 600 nM for U937 cells) or vehicle (DMSO) for 72 h prior to the determination of cytotoxicity by flow cytometry using PI with duplicate or triplicate measurements. Values represent percentages of PI+ cells compared to corresponding cells treated with DMSO vehicle or Ko143 only. Results for relevant concentrations of the cytotoxic drugs are shown as mean7s.e.m. with the number of independent experiments denoted within parentheses. Login to comment 91 ABCG2 p.Arg482Thr R482T BCRP-transduced HL-60 cells were not established. Login to comment