Cross-reactivity of C219 anti-p170(mdr-1) antibody with p185(c-erbB2) in breast cancer cells: cautions on evaluating p170(mdr-1)
BACKGROUND
Increased expression of the multidrug resistance gene (MDR-1)-encoded P-glycoprotein (p170[mdr-1]) is a major cause of tumor cell multidrug resistance. p170(mdr-1) functions as a drug-efflux pump to reduce the cellular accumulation of specific drugs. MDA-MB-435 human breast cancer cells that have been transfected with oncogene c-erbB2 complementary DNA (435.eb cells) express high levels of the transmembrane glycoprotein p185(c-erbB2) and exhibit increased resistance to the chemotherapeutic agent paclitaxel via p170(mdr-1)-independent mechanisms. We have recently discovered that the widely used monoclonal antibody C219, which is specific for p170(mdr-1), may cross-react with p185(c-erbB2) in 435.eb cells. In this study, we have investigated the nature of this cross-reactivity.


METHODS
Immunoprecipitation experiments involving the use of breast cancer cells that express different levels of p185(c-erbB2) were performed, and C219 was used for western blot analysis of immunoprecipitated proteins. Immunohistochemical analyses were performed on acetone-fixed slides of human breast cancer cells. Peptide sequence comparisons and enzyme-linked immunosorbent assays were performed to determine the molecular basis of C219 cross-reactivity with p185(c-erbB2).


RESULTS
The cross-reactivity of C219 with p185(c-erbB2) was demonstrated by both western blot and immunohistochemical analyses. Peptide sequence comparisons revealed that C219 recognizes an epitope in p170(mdr-1) (C219 epitope) that shares sequence homology with p185(c-erbB2). Enzyme-linked immunosorbent assays demonstrated that C219 recognizes synthetic peptides derived from both the C219 epitope in p170(mdr-1) and the C219 epitope-homologous region in p185(c-erbB2).


CONCLUSIONS
The anti-p170(mdr-1) monoclonal antibody C219 cross-reacts with p185(c-erbB2) through a peptide sequence in p185(c-erbB2) that is homologous to the C219 epitope in p170(mdr-1).
Liu
B
B
Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Sun
D
D
Xia
W
W
Hung
M C
MC
Yu
D
D
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000276:immunology*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008969:Molecular Sequence Data; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D015203:Reproducibility of Results; D016415:Sequence Alignment; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000939:Epitopes; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins