Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2
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Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2. / Litman, Thomas; Jensen, Ulla; Hansen, Alastair; Covitz, Kuang-Ming; Zhan, Zhirong; Fetsch, Patricia; Abati, Andrea; Hansen, Paul Robert; Horn, Thomas; Skovsgaard, Torben; Bates, Susan E.
I: BBA General Subjects, Bind 1565, Nr. 1, 20.09.2002, s. 6-16.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2
AU - Litman, Thomas
AU - Jensen, Ulla
AU - Hansen, Alastair
AU - Covitz, Kuang-Ming
AU - Zhan, Zhirong
AU - Fetsch, Patricia
AU - Abati, Andrea
AU - Hansen, Paul Robert
AU - Horn, Thomas
AU - Skovsgaard, Torben
AU - Bates, Susan E
PY - 2002/9/20
Y1 - 2002/9/20
N2 - 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.
AB - 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.
KW - ATP-Binding Cassette Transporters
KW - Amino Acid Sequence
KW - Antibodies
KW - Cell Line
KW - Drug Resistance, Multiple
KW - Epitopes
KW - Fluorescent Antibody Technique
KW - Humans
KW - Immunoblotting
KW - Microscopy, Confocal
KW - Microscopy, Immunoelectron
KW - Mitoxantrone
KW - Molecular Sequence Data
KW - Neoplasm Proteins
KW - Peptides
M3 - Journal article
C2 - 12225847
VL - 1565
SP - 6
EP - 16
JO - B B A - General Subjects
JF - B B A - General Subjects
SN - 0304-4165
IS - 1
ER -
ID: 119646671