Cross-reaction between antibodies raised against the last 20 C-terminal amino acids of BRCA 1 (C-20) and human EGF and EGF-R in MCF 10a human mammary epithelial cell line

Abstract

Cross-reaction between antibodies raised against the last 20 C-terminal amino acids of BRCA 1 (C-20) and human EGF and EGF-R in MCF 10a human mammary epithelial cell lineA recent publication of Wilson et al. (1996) reported on the specificity of rabbit polyclonal antibodies raised against a peptide corresponding to amino acids 1843-1862 mapping at the carboxy terminus of human BRCA 1 (C-20), commercially available from Santa Cruz Biotechnology (Santa Cruz, CA). Surprisingly, this antiserum cross-reacted with the human epidermal growth factor receptor (EGF-R) and HER 2.Here, we confirm the cross-reaction of C-20 antibodies with the human EGF-R (E 2645; Sigma, St Louis, MO) and we report cross-reaction of these antibodies with human EGF (E 1264, Sigma), using a quantitative method for determination of the BRCA 1 protein concentration in the MCF 10a human mammary epithelial cell line, previously developed in our laboratory (Bernard et al., 1985;1986).BRCA 1 proteins were quantified after internal cell protein labeling with [ 35 S]-methionine. All proteins were solubilized with a detergent (Nonidet P 40) and glycoproteins were isolated by affınity chromatography on Lentil-Lectin Sepharose 4B (Pharmacia, Uppsala, Sweden) (Fig. 1a). In this way, BRCA 1, which includes 21 predicted glycosylation sites (Jensen et al., 1996), was eluted with all cell glycoproteins. Specific immunoprecipitation was then performed with anti-BRCA 1 polyclonal antibodies (C-20). The immune complex consisting of BRCA 1 glycoproteins labeled with [ 35 S] bound to antibodies was isolated by affınity chromatography on Protein A-Sepharose CL 4B (Fig. 1b). A final quantification of BRCA 1 proteins via the immune complex was performed by chromatofocusing on PBE 9-4 gel (Pharmacia) because elution along a pH gradient 7.4 to 4 revealed one main labeled peak at pH 7.1, corresponding to immune complex elution, since the g-globulin pHi elutes between 7.3 and 6.3 (Cohn, 1948).Chromatofocusing of the immune complex BRCA 1/anti-BRCA 1 polyclonal antibodies, followed by counting of the radioactivity in the collected fractions, is shown in Figure 1c. A main labeled peak corresponding to [ 35 S]-labeled BRCA 1 proteins bound to anti-BRCA 1 polyclonal antibodies was obtained at pH 7.1 in fractions 12 to 16. In order to validate the technique, we used double-labeling, [ 131 I] for the proteins and [ 125 I] for the antibodies and we observed the main doublelabeled peak at pH 7.1 (data not shown).When the reaction between BRCA 1 proteins and the polyclonal antibodies against BRCA 1 (C-20) was performed in the presence of EGF (Fig. 1d) or EGF-R (Fig. 1e), the elution profile was similar, but the main peak at pH 7.1, meaning elution of labeled BRCA 1 glycoproteins, was markedly reduced in both cases due to cross-reaction of the antibodies with EGF or EGF-R, thus displacing the equilibrium between BRCA 1 and C-20 antibodies.The amount of BRCA 1 proteins obtained after chromatofocusing was expressed as a percentage, and corresponded to the ratio: Amount of labeled gl...

BRCA1 Gene Expression in Breast Cancer: A Correlative Study between Real-time RT-PCR and Immunohistochemistry

“…BRCA1 has been claimed to be an exclusively nuclear protein in both normal and cancer cells (Scully et al 1996;Thomas et al 1996;Thakur et al 1997;Wilson et al 1999), a nuclear protein in normal cells but an aberrantly localized cytoplasmic protein in breast and ovarian tumor cells (Chen et al 1995;Lee et al 1999), and a cytoplasmic protein found in tubelike structures that invaginate the nucleus (Coene et al 1997). The variation in the subcellular localization of BRCA1 might be attribut- able to several causes, including the specificity of the antibodies used to localize the protein, antibody crossreactivity (Smith et al 1996;Bernard-Gallon et al 1997;Wilson et al 1999), and the presence of splice variant isoforms (Thakur et al 1997;Wilson et al 1997). The present study confirms that BRCA1 is expressed as both nuclear and cytoplasmic antigen in breast cancer tissues.…”

The Anti-BRCA1 Peptide Antibody C-20 Recognizes Smooth Muscle Cells

“…BRCA1 protein is thought to be a nuclear protein; however, it is well known that the anti-BRCA1 antibody, C-20, crossreacts with EGFR and HER2 (4,5). Antibody C-20 is raised against residues 1843^1862 of the BRCA1 protein, and because this region is highly conserved between BRCA1 and some members of the tyrosine kinase receptor family, such as EGFR and HER2, the antibody is thus believed to cross-react with them (4).…”

BRCA1 is localized in ductal secretions of normal breast tissues: Detection by antibodies raised against the amino terminus of BRCA1 (K-18)

“…To avoid confusion with C-20 antibodies (raised against a peptide corresponding to amino acids 1843-1862 mapping at the carboxy terminus of BRCA1) and cross reaction with the human epidermal growth factor receptor (EGF-R) and HER 2 (Wilson et al, 1996;Bernard-Gallon et al, 1997)…”

“…To avoid confusion with C-20 antibodies (raised against a peptide corresponding to amino acids 1843-1862 mapping at the carboxy terminus of BRCA1) and cross reaction with the human epidermal growth factor receptor (EGF-R) and HER 2 (Wilson et al, 1996;Bernard-Gallon et al, 1997), we performed an immunochemical analysis of BRCA1 in comparison with a polyclonal antibody against EGF-R (Santa Cruz Biotechnology) on all 24 breast carcinoma samples. With this antibody against EGF-R, we found a very faint staining pattern in the cytoplasm and on the membrane of the mammary cells, but the secretions in the tubes of the normal breast tissue remained unstained (Fig.…”