Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization.
Breast cancer resistance protein (BCRP) is a half-molecule ABC transporter highly expressed in mitoxantrone-resistant cells. In our study we established PA317 transfectants expressing Myc-tagged BCRP (MycBCRP) or HA-tagged BCRP (HABCRP). The exogenous BCRP protein migrated as a 70-kDa protein in SDS-PAGE under reducing condition, but migrated as a 140-kDa complex in the absence of reducing agents. The 140-kDa BCRP complex was heat-stable but dissociated into 70-kDa BCRP with the addition of 2-mercaptoethanol. The 140-kDa BCRP complex was immunoprecipitated with anti-Myc antibody from the lysates of PA317 cells double-transfected with MycBCRP and HABCRP. The 140-kDa complex reacted with anti-HA and anti-BCRP antibodies and after the addition of reducing agents, a 70-kDa protein reacting with anti-Myc, anti-HA and anti-BCRP antibodies was detected. These results clearly indicate that BCRP forms a homodimer bridged by disulfide bonds. To assess the possible dominant-negative inhibition of BCRP drug efflux pump, various mutant BCRP cDNAs were isolated by PCR mutagenesis. First, mutant BCRP cDNAs were introduced to parental PA317 cells and tested for their function as drug-resistance genes. Next, inactive BCRP cDNA clones were introduced to MycBCRP-transfected cells and tested for the ability to lower drug resistance. Among the 8 inactive mutant cDNA clones tested, HABCRP cDNA clone 15 with an amino acid change from Leu to Pro at residue 554 in the fifth transmembrane domain of BCRP partially reversed the drug resistance of MycBCRP-transfected cells. These results suggest that homodimer formation is essential for BCRP drug resistance, implicating this dominant-negative inhibition as a new strategy to circumvent drug resistance.
Kage
Kumie
K
Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.
Tsukahara
Satomi
S
Sugiyama
Tomomi
T
Asada
Sakiyo
S
Ishikawa
Etsuko
E
Tsuruo
Takashi
T
Sugimoto
Yoshikazu
Y
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics / Q000494:pharmacology; D019281:Dimerization; D018432:Drug Resistance, Multiple / Q000502:physiology*; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005260:Female; D005799:Genes, Dominant / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins