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. MDAMB-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. Pep- tide 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).