Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Abstract

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Mutational Studies of G553 in TM5 of ABCG2: A Residue Potentially Involved in Dimerization

“…In order to investigate whether this lower molecular weight band was representative of the non-glycosylated protein, isolated membranes from cells bearing wild type or mutant vectors were treated with the PNGase F enzyme to remove N-linked glycans. In case of the G553L mutant, no significant shift in molecular weight was observed after PNGase F treatment (Figure 3) indicating that, indeed, the G553L mutant is underglycosylated, while a clear shift to a lower molecular weight band was seen in membranes extracted from HEK 293 cells transfected with wild type ABCG2 or from flavopiridol-selected MCF-7 cells used as controls (25).…”

Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)

“…This dendrogram was produced using multiple sequence alignments with Clustal_X 1.8 (Thompson et al, 1997), with graphical representation in a tree diagram using TreeView 1.6.6 Table 3 See Litman et al (2002), using polyclonal antibodies directed against peptide epitopes of BCRP. The functional activity of BCRP in transfected insect cells, using a baculovirus-Sf9 cell system, also argues strongly for the activity of the protein as a homodimer (Ozvegy et al, 2001(Ozvegy et al, , 2002.…”

“…The results suggest dominant-negative inhibition as a possible new strategy to circumvent clinical drug resistance. Antibodies directed at BCRP Litman et al (2000Litman et al ( , 2002 developed polyclonal antibodies against several epitopes on BCRP. Rabbit anti-BCRP antisera 84705 was raised against an 18-mer peptide of the ATP-binding region of the protein and could detect BCRP polypeptide at a dilution of 1 : 2000.…”

ABC Transporters in Human Placenta and Their Role in Maternal-Fetal Cholesterol Transfer: ABCA1 Candidate Target

“…Breast cancer resistance protein (BCRP) is an ATP dependent transporter also known as mitoxantrone resistance-associated protein (Allikmets et al, 1998) is highly expressed in the placenta, as well as in the uterus (Langmann et al, 2003) and is localized on the apical surface of the chorionic villi syncytiotrophoblast (Litman et al, 2002). The substrate specificity of BCRP has not been elucidated completely.…”