Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants.
Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette transporters that has an N-terminal ATP binding domain and a C-terminal transmembrane domain (TM). Expression of wild-type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN-38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446-mutants in TM2, 15 R482-mutants in TM3, 4 N557-mutants in TM5 and 6 H630-mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446-mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2-BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild-type BCRP-transfected cells. Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482-, N557- or H630-mutant BCRP cDNA. Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild-type BCRP-transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.
Miwa
Miyu
M
Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.
Tsukahara
Satomi
S
Ishikawa
Etsuko
E
Asada
Sakiyo
S
Imai
Yasuo
Y
Sugimoto
Yoshikazu
Y
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000493:pharmacokinetics; D001692:Biological Transport; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005347:Fibroblasts; D006801:Humans; D000077146:Irinotecan; D007700:Kinetics; D051379:Mice; D008942:Mitoxantrone / Q000493:pharmacokinetics*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000972:Antineoplastic Agents, Phytogenic; D018076:DNA, Complementary; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D000077146:Irinotecan; D008942:Mitoxantrone; D002166:Camptothecin